BIOLOGICAL TREATMENT OF HIGH SALINITY WASTEWATER USING YEAST

AND BACTERIAL SYSTEMS

by

Nguyen Phuoc Dan

A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of
Engineering

Examination Committee:

Prof. C. Visvanathan (Chairman)

Prof. Chongrak Polprasert (Co-chairman)
Prof. Nguyen Cong Thanh
Dr. Josef Trankler
Dr. Sudip K. Rakshit

External Examiner:

Prof. Ronald E. Simard

Nationality:
Previous Degree:

Vietnamese
Bachelor of Engineering (Civil)
Hochiminh City University of Technology (HUT)
Hochiminh City, Vietnam
Master of Engineering (Environmental Engineering)
AIT, Thailand

Scholarship Donor:

Swiss Development Cooperation (SDC)

Asian Institute of Technology

School of Environment, Resources and Development
Bangkok, Thailand
December 2001

Acknowledgements

The author wishes to deeply express his gratitude to his advisor, Prof. C. Visvanathan
for kindly giving valuable guidance, suggestions and encouragement through his study in
AIT. He would like to express his appreciation to his co-advisor, Prof. Chongrak Polprasert
for his valuable comments and suggestions provided throughout the research work. The
author wishes to express deepest sincere thanks to Prof. Nguyen Cong Thanh, Dr. Josef
Trankler, Dr. Sudip K. Rakshit and Dr. A. Sathasivan for their valuable comments, critical
ideas and serving as members of examination committee.
A special thank is addressed to Prof. Ronald E. Simard for kindly accepting to serve as
External Examiner His constructive and professional comments are highly appreciated.
The author gratefully acknowledges Swiss Development Cooperation (SDC)EPFL,IGE/GS for his financial support. Grateful acknowledgement is also extended to
Nishihara ERSC. for supporting partially experimental equipment. Also acknowledgement is
given to the SERD school for financial support on attendance of Conference in Malaysia.
The author is very grateful to Mdm. Visvanathan, Mr. Jonathan Shaw and Mr. Basu
for providing comments and editing in English language.
A special thank is extended to Lab Supervisors, Mr. Suwat, Ms. Salaya, Mr. Peter and
Mr. Chai, technicians Khun Verin, Khun Tam and others. The author would like to thank his
friend, Master student, M.M. Cho, for co-operation of thesis works.
The author is most grateful to his family and CEFINEAs Director, Prof. L.M. Triet,
for mental support during study in AIT.

ii

Abstract

This study aimed to compare the performance of aerobic treatment using wild mixed
yeast and bacterial culture for high salinity wastewater. The operating conditions of yeast
treatment under high salinity such as pH, sludge retention time (SRT) and dissolved oxygen
(DO) were examined. The comparative evaluation is based on determination of biokinetic
coefficients using the respirometric method and treatment efficiency of long-term operation of
two laboratory-scale membrane bioreactor systems.
The biokinetic experiments reveal that yeast culture has a lower observed maximum
specific grow rate (Pobs) at low salt content (20g/L) than that of bacteria. But Pobs of yeasts at
higher salt contents (above 30 g/L) did not decline dramatically and had higher value than that
of bacteria. The osmotolerant yeast mixture was able to tolerate a wider pH range than
bacterial culture. The chemical oxygen demand (COD) removal rate of the yeast mixture was
highest at pH values 5.0-5.5.
Two laboratory-scale membrane bioreactor systems were investigated to treat high
salinity wastewater containing high organic (5,000 mg/L COD) and salt content (32 g/L
NaCl), namely: the Yeast Membrane Bioreactor (YMBR), and Yeast pretreatment followed
by Bacterial Membrane Bioreactor (BMBR). In the YMBR system, experimental runs were
conducted with a mean biomass concentration of 12 g MLSS/L. Here, the maximum COD
removal rate of 0.93 g COD/g MLSS.day was obtained at F/M of 1.5 g COD /g MLSS.d,
whereas the BMBR system was operated with a biomass concentration of up to 25 g
MLSS/L, resulting in maximum COD removal rate of 0.32 kg COD /kg MLSS.day at F/M
ratio of 0.4. In comparison the BMBR, the YMBR could obtain higher COD removal rate at
higher organic loading, indicating the potential of the yeast reactor system to treat high
salinity wastewater containing high organic concentration.
Transmembrane pressure in the BMBR was progressively increased from 2 to 60 kPa
after 12d, 6 d and 2 d at hydraulic retention time (HRT) of 14h, 9 h and 4h, with average
biomass concentration of 6.1, 15 and 20 g MLSS/L respectively. By contrast, the
transmembrane pressure in YMBR was only increased from 2 to 60 kPa only after 76 days of
operation, with an average biomass concentration of 12 MLSS/L and an operating HRT range
of 5 - 32 h.
The comparative evaluation of treatment performance of both YMBR and BMBR with
the low organic-feed wastewater (1,000 mg/L COD and 32 g/L NaCl) was examined. COD
removal of both processes were above 90% at HRT of 5 h. Under the same operating
conditions, the YMBR could run under transmembrane pressure 10 times lower than the
BMBR with a significantly reduced membrane fouling rate. This may be due to low
production of adhesive extracellular polymers (ECP) and the secondary filtration layer formed
from large free yeast cells. ECP production of bacterial sludge was increased considerably at
high salt contents and high sludge retention time (SRT). For the bacterial sludge, the increase
salinity led to increase in ECP value, whereas the ECP content of the yeast sludge was
relatively very small.

iii

Table of Contents
Chapter

Title

Page

Title page
Acknowledgements
Abstract
Table of Contents
List of Figures
List of Tables
Abbreviations
1

i
ii
iii
iv
vii
x
xii

Introduction
1.1 Background
1.1.1 Environmental Concerns
1.1.2 Effects of High Salinity on Biological Treatment Processes
1.1.3 Salt-Tolerant or Halophilic Microorganisms
1.1.4 Membrane Bioreactor Process
1.2 Objectives of the Study
1.3 Scope of the Study

1
1
2
3
3
4
4

Literature Review
2.1 Introduction
2.1.1 The Seafood Processing Industry
2.1.2 Pickled Vegetable Processing
2.1.3 Other Saline Wastewaters
2.2 Effects of High Salinity on Biological Waste Treatment Process
2.2.1 Aerobic Treatment
2.2.2 Anaerobic Treatment
2.2.3 Nutrient Removal
2.3 Application of Halophilic Bacteria for Saline Wastewater Treatment
2.4 Yeasts
2.4.1 General
2.4.2 Applications of Yeasts for Wastewater Treatment
2.5 Theoretical Modeling Consideration
2.5.1 Growth without Inhibition
2.5.2 Growth with Inhibition
2.6 Respirometric Method
2.6.1 Respirometer
2.6.2 Experimental Procedure
2.6.3 Determination of Kinetic Constants
2.7 Membrane Bioreactor (MBR)
2.7.1 Advantage of the MBR Process
2.7.2 Main Design Parameters
2.7.3 Membrane Fouling

6
6
6
9
13
13
14
17
18
19
22
22
24
33
33
35
38
38
38
40
41
42
42
45

Methodology
3.1 Biokinetic Study
3.1.1 Seed Sludge
3.1.2 Acclimation
3.1.3 Biokinetic Experiments
3.2 Parametric Study

49
51
52
52
54
iv

3.3

3.4
3.5
4

3.2.1 pH values
3.2.2 Sludge Retention Time (SRT)
Biomembrane Study
3.3.1 High COD loading
3.3.2 Low COD loading
Sludge Characterization Study
Analytical Methods

Results and Discussion

4.1 Biokinetic Study
4.1.1 Enrichment and Acclimation of Yeast and Mixed Bacterial Sludge
4.1.2 Evaluation and Comparison of Biokinetic Coefficients
4.2 Parametric Study
4.2.1 DO and pH
4.2.2 Nitrogen Variation in Mixed Yeast and Bacterial Cultures
4.2.3 Effect of SRT on COD and Nitrogen Removal
4.3 Biomembrane Study
4.3.1 High COD loading
4.3.2 Low COD loading
4.4 Sludge Characterization Study
4.4.1 Culture Study
4.4.2 YMBR and BMBR
4.4.3 Microscopic Observations of Mixed Yeast Sludge
4.4.4 Nutrient Uptake
Conclusions and Recommendations
5.1 Conclusions
5.2 Recommendations
Appendix A: Pictures of Experiments
Appendix B: Experimental Data of Acclimation
Appendix C: Experimental Data of Biokinetic Study
Appendix D: Experimental Data of Parametric Study
Appendix E: Experimental Data of Biomembrane Study

54
55
56
56
59
60
60

62
62
69
73
74
78
80
81
82
87
93
94
96
97
98

100
101
A-1
B-1
C-1
D-1
E-1

List of Figures
Figure

2.1
2.2
2.3
2.4
2.5
2.6
2.7

Title

Page

2.29

Flow diagram of steamed canned shrimp processing

Flow diagram of Dried and Salted fish processing
Flow diagram of kim chi pickles processing
Variation of COD removal rate with salt contents (Kargi and Uygur, 1996)
Diagram of a nitrogen treatment system
Schematic diagram of percolation reactor (Kargi and Uygur, 1996)
Variation of COD removal rate (R) as function of salt content (Kargi and Dincer,
2000)
Schematic diagram of the biofilter and trickling filter treatment system
(Yang et al., 2000)
Diagram of yeast cell (Salle, 1961)
Budding is a common reproductive process in yeasts
True mycelium (formed by fission) and pseudomycelium (formed by budding)
Specific growth rate of Candida ingens vs DO and VFA concentration
(Anciaux et al., 1989)
Traditional carbon and nitrogen removal system can be altered with anaerobic
and yeast treatment system (Ortiz et al. 1997)
Schematic diagram of the Yeast Cycle System (YCS)
Comparison between Yeast Cycle System (YCS) and complete mixed activated
sludge (AS) (Nishihara ESRC Ltd., 2001)
SCP from confectionery effluent (Gray, 1989)
The Symba process (Gray, 1989)
Growth curve of microorganisms in a culture
The effects of a limiting substrate on the specific growth rate (Monod model)
Curves of inhibition growth models (n =1: Ghose and Tyagi; n= 0.5: Bazua and
Wilke model)
Curves of substrate inhibition growth models
pH and DO models
Schematic diagram of respirometer
Recorder chart with a typical respirogram (Cech et al., 1984)
OUR response in respirometer (Ekama, et al., 1986)
Diagram of membrane bioreactor processes
Diagram of fouling mechanisms (adsorption and deposition)
Schematic illustration of membrane biofouling process (Ridgway and Flemming,
1996).
Schematic diagram of biofloc or biofilm

3.1
3.2
3.3
3.4
3.5
3.6

Flowchart of different phases of experimental study

Flowchart of biokinetic experiments
Schematic diagram of enrichment procedure
Respirometer set-up
Membrane reactor systems in the high COD loading
Schematic diagram of biomembrane reactor

49
51
51
53
58
59

4.1
4.2

Appearance of yeast cells predominantly grown in glucose-feed wastewater

Acclimation of yeast sludge cultured with glucose at high salt contents

62
63

2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.16
2.17
2.18
2.19
2.20
2.21
2.22
2.23
2.24
2.25
2.26
2.27
2.28

vi

7
9
11
15
18
20
20
21
22
23
23
25
27
28
29
31
31
33
34
35
36
37
38
39
40
42
45
46
47

4.3
4.4
4.5
4.6
4.7
4.8
4.9
4.10
4.11
4.12
4.13
4.14
4.15
4.16
4.17
4.18
4.19
4.20

4.21
4.22
4.23
4.24
4.25
4.26
4.27
4.28
4.29

Acclimation of microbial mixed culture with glucose-feed wastewater as

function of salt
64
Typical COD and COD removal profile of mixed yeast batch in glucose-feed
wastewater at 32 g salt/L
65
Variation in COD removal rate versus salt contents in acclimatized yeast and
bacterial mixed cultures
66
Acclimation of yeast and bacterial sludges to fish-protein-feed wastewater
containing 32 g/L salt
67
Predominance of wild yeast strains in the cultures fed with fish-protein wastewater
(at 32 g/L salt)
68
OUR curves of mixed yeast and bacterial sludges feed with 50 mg/L COD and
32 g/L salt (glucose-feed wastewater)
70
OUR curves of mixed yeast and bacterial sludges feed with 100 mg/L COD and
32 g/L salt (protein-feed wastewater)
70
Variation in specific growth rate of yeast sludge as function of COD at different
salt contents for glucose-feed wastewater
71
Variation in specific growth rate of bacterial culture as function of COD
concentration at different salt contents for glucose-feed wastewater
71
Inhibition effect of salt contents on mixed yeast and bacterial cultures on glucosefeed wastewater
73
Inhibition effect of salt contents on mixed yeast and bacterial cultures on proteinfeed wastewater
73
DO and COD changes of yeast batch fed with glucose and protein wastewater at
32 g salt/L
74
DO and COD changes of mixed bacterial batch fed with glucose and protein
wastewater at salt content of 32 g/L
75
pH changes of yeast culturefed with glucose and protein wastewater at
32 g salt/L
75
pH changes of mixed bacterial batch fed with glucose and protein wastewaters at
32 g salt/L
76
Variation in OUR as funtion of initial pHs for mixed yeast fed with protein
wastewater at 32 g salt/L
77
Variation in OUR as funtion of initial pHs for mixed bacterial fed with glucose
wastewater at 32 g salt/L
77
Variation in nitrogen components as funtion of time in the mixed yeast at 32 g
salt/L NaCl
(Nitrite and nitrate concentration of both feed wastewaters were not
79
dectected)
Variation in nitrogen components vs. time in the mixed bacterial culture at
32 g salt/L NaCl
79
Variation in MLSS as funtion of SRT
80
Variation in COD, nitrogen removal and MLSS in funtion of SRT in mixed yeast
culture at VLR of 5 kg COD/m3.d (32 g salt/L)
81
Variation in flux as function of membrane transmembrane pressure (Viscosity of
water at 26oC = 8.70 x 10-4 kg/m.sec)
82
Variation in COD, biomass and transmembrane pressure in the YMBR as
function of volumetric loading
83
Variation in COD, biomass and transmembrane pressure in the BMBR as
function of volumetric loading
84
Variation in COD removal in function of volumetric loading rate
86
Variation in COD removal rate in function of F/M ratio (initial COD = 5,000 mg/L) 86
Variation in COD, biomass and transmembrane pressure in the YMBR as
function of volumetric loading
88
vii

4.30
4.31
4.32
4.33
4.34
4.35
4.36

Variation in COD, biomass and transmembrane pressure in the BMBR as

function of volumetric loading
Variation in COD removal as function of HRTs in YMBR and BMBR
Variation in specific growth rate of yeast and bacteria at 32 g salt/L in function
of COD
Possible mechanisms for flux enhancement by yeast cells
Variation in ECP and CST in function of salt content
Variation in SVI, SS, ECP and viscosity with salt content in mixed bacterial
cultures
ECP contents of mixed yeast and bacterial sludges in YMBR and BMBR

viii

89
89
90
93
95
96
97

List of Tables
Table

Title

1.1

Comparison of pollutant loads from seafood processing and other industries in the
Saigon-Dong Nai river catchment area (DOSTE-HCMC and CEFINEA, 1998) 2

2.1
2.2
2.3
2.4
2.5

Characteristics of herring brine waste (Balslev-Olesen et al., 1990)

7
Characteristics of wastewater from the dried salted fish plant (Dan, 2000)
8
Composition of brines used for canning vegetables (Joslyn and Timmons, 1967) 9
Raw waste loads and quality of wastewater from some pickling industries
10
Characteristics of the waste brine from four different kim chi factories located in
Suwon city and Kyunggi province, Korea (Park and Choi, 1999)
10
Wastewater characteristics of from various fishery product and vegetable
pickling industries
12
Characteristics of oil field brine (Dalmacija et al., 1996)
13
Characteristics of leachates (Pirbazari, 1996)
13
Adverse effects of high salinity in activated sludge process
16
Adverse effects of high salinity in anaerobic treatment processes
18
Summary of adverse effects of high salinity in nutrient removal processes
19
Effects of using halophilic bateria for high salinity wastewater treatment
22
Basic composition of Candida utilis yeast biomass (Defrance, 1993)
24
A comparison between yeast and anaerobic treatment process (Defrance, 1993) 27
Operating conditions of YCS (Nishihara ESRC Ltd., 2001)
28
Quality of treated water and efficiency of the YCS for seafood processing
wastewater treatment (Nishihara ESRC Ltd., 2001)
29
Summary of studies on yeast treatment of high salinity wastewater
30
Kinetic models for inhibition growth (Han and Levenspiel, 1988)
35
Comparison between biological performances of MBR process and conventional
AS process
44

2.6
2.7
2.8
2.9
2.10
2.11
2.12
2.13
2.14
2.15
2.16
2.17
2.18
2.19

Page

3.1
3.2
3.3
3.4
3.5
3.6
3.7
3.8
3.9
3.10
3.11
3.12

Composition of glucose-feed wastewater (Defrance, 1993)

Composition of protein-feed wastewater
Operating conditions for high salinity acclimation
Operating conditions for the respirometric experiments
Operating conditions for the pH effect experiments
Operating conditions of the experiments on SRT effect
Difference between the high COD loading and low COD loading
Experimental operating conditions of YMBR and BMBR systems
Composition of the low COD wastewater
Effects of different HRTs and SRTs on yeast and bacterial membrane reactors
Operating conditions for the sludge characterization study
Parameters and their analytical method

4.1

Performance of mixed yeast and bacterial batches adapted to glucose-feed

wastewater with high salt
64
Performance of mixed yeast and bacterial sludges adapted to protein-feed
wastewater with high salt contents (Initial COD cof 5,000 mg/L).
69
Biokinetic coefficients of the yeast and bacterial sludges at different salt contents
for glucose and protein-feed wastewaters
72
Variation of parameters during various SRTs (Initial COD of 5000 mg/L)
81

4.2
4.3
4.4

ix

50
50
52
53
54
55
56
57
59
60
60
61

4.5
4.6
4.7
4.8
4.9

Operating parameters of the YMBR, BMBR, some yeast treatments, MBR

processes treating different wastewaters and conventional AS system
Operating parameters and performance of YMBR and BMBR in high COD
loading phase
Values of different parameters during YMBR and BMBR filtration cycle
Yeast and bacterial sludges characterization
Composition of mixed bacterial and mixed yeast sludge

85
91
92
94
98

List of Abbreviations
AF
AS
BOD
BMBR
COD
CST
DO
DOSTE
ECP
EPS
ESRC
F/M
HCMC
HRT
J
MBR
MF
MLSS
MLVSS
N
NH3-N
NO2-N
NO3-N
OUR
P
SBR
SCP
SRT
SS
SSL
SVI
TDS
TOC
TKN
TS
TVS
U
UASB
UNEP
UF
VFA
VLR
VOC
VSS
Y
YCS
YR
YMBR
'P

Anaerobic Filter
Activated Sludge
Biochemical Oxygen Demand
Bacterial Membrane Bioreactor
Chemical Oxygen Demand
Capillary Suction Time
Dissolved Oxygen
Department of Science, Technology and Environment
Extracellular Polymers
Extracellular Polymer Substances
Environmental Sanitation Research Center
Food/Microorganism ratio
Hochiminh City
Hydraulic Retention Time
Permeate flux
Membrane Bioreactor
Microfiltration
Mixed Liquor Suspended Solids
Mixed Liquor Volatile Suspended Solids
Nitrogen
Ammonia Nitrogen
Nitrite Nitrogen
Nitrate Nitrogen
Oxygen Uptake Rate
Phosphorus
Sequencing Batch Reactor
Single-Cell-protein Production
Sludge Retention Time
Suspended Solids
Spent Sulphite Liquor
Sludge Volume Index
Total Dissolve Solid
Total Organic Carbon
Total Kjedahl Nitrogen
Total Solids
Total Volatile Solids
Substrate Utilization Rate
Upflow Anaerobic Sludge Blanket
United Nations Environment Programme
Ultrafiltration
Volatile Fatty Acid
Volumetric Loading Rate
Volatile Organic Carbon
Volatile Suspended Solids
Yield coefficient
Yeast Cycle System
Yeast Reactor
Yeast Membrane Bioreactor
Transmembrane Pressure
xi

Chapter 1
Introduction

1.1

Background

High salinity wastewater containing high inorganic salt content is mostly generated
from industries such as seafood processing, vegetable canning, pickling and cheese
processing. Among these, the seafood processing industry is an industrial sector that produces
large volumes of saline wastewater with high organic and nutrient concentration. Therefore it
causes heavy pollution to receiving waters. At present, the seafood processing industry plays
an important role in South East Asias economy. Under stringent environmental regulations,
this industry is now facing both high treatment costs and problems in the operation of
conventional wastewater treatment plant. These operational problems are linked to high
organic loading, high salt content and very large seasonal variation leading to change in waste
characteristics.
1.1.1 Environmental Concerns
In seafood processing, the main environmental concern is the use of large amounts of
fresh water for processing, including washing raw material and products, for cleaning of
machines, containers or flushing the working floor, for de-icing, thawing and salt soaking. In
general, 90-95% of water consumed is converted into highly polluting wastewater. Frozen
seafood processing consumes particularly large volumes of water, ranging from 70 to 120
m3/ton of product, the equivalent of 32-60 m3/ton of raw fish (DOSTE-HCMC and
CEFINEA, 1998). The wastewater generated by fish processing factories has high loads of
organic and nutrients. This waste is commonly discharged directly into coastal areas. Another
important aspect of this industrial waste is its high salinity (Na+, Cl-, SO42-), caused both by
the raw materials and seawater used in various processes. Here, using pre-filtered seawater for
processing leads to high salinity in the wastewater, which reduces the biodegradation rate in
effluent treatment units (Mendez et al., 1992).
Because factories process a broad range of products with large seasonal variation,
pollution characteristics vary significantly both from plant to plant, and even within the same
plant. In Ho Chi Minh City (HCMC), the seafood processing sector is one of the major
industrial contributors to the heavy pollution to receiving waters. The average BOD5 generally
ranges from 1,200 to 1,800 mg/L (COD of 1,600 - 2,300 mg/L) (DOSTE, 1994). In addition,
the wastewater contains high levels of suspended solids (150-200 mg/L), and is rich in
nutrients with total nitrogen ranging from 70 to 110 mg/L. The pollutant loads from the
seafood processing industry and other industries in the Saigon-Dong Nai river catchment area
is shown in Table 1.1. These data indicate that seafood processing is a sector that causes
considerable pollution to the environment in this part of Viet Nam.

Table 1.1 Comparison of pollutant loads from seafood processing and other industries in the
Saigon-Dong Nai river catchment area (DOSTE-HCMC and CEFINEA, 1998)
Industry

Flow rate
m3/day

SS
Seafood processing
Pulp and paper
Cassava
Textiles & dyeing
Beverages
Latex processing
Meat processing and slaughterhouse

Sugar (sugar cane)

Vegetable canning

18,900
49,200
47,100
32,500
15,600
11,600
6,400
5,520
3,700

4,200
54,900
30,600
5,600
4,400
2,500
4,000
6,900
520

Pollution load, kg/day

BOD5
TKN
28,400
1,700
104,800
340
590,000
NA
17,300
NA
19,000
630
86,600
2,800
13,300
1,020
32,000
72
2,700
70

SS suspended solids
NA None available

1.1.2 Effects of High Salinity on Biological Treatment Processes

Past studies on saline wastewater treatment reveal that salinity decreases BOD5 removal
efficiencies, increases effluent turbidity due to sludge settling in the secondary sedimentation
unit, solid losses, and changes in the mixed liquor floc protozoan population in an activated
sludge system (Dalmacija et al., 1996; Woolard and Irvine, 1995; Kargi and Dincer, 1998).
Kargi and Uygur (1996) reported many adverse effects of salt on aerobic attach growth such
as trickling filter and rotating biological contactors. The efficiency of COD removal decreased
significantly with increases in salt contents over 20g/L.
The anaerobic digester were much more sensitive to chlorides than activated sludge
processes (Burnett, 1974). Biogas production and COD removal of anaerobic treatment
processes such as anaerobic filter, UASB and batch reactor were inhibited significantly at salt
content above 30g NaCl/L (Baere et al., 1984; Feijoo et al., 1995). In addition, high salt
content also depressed the treatment ability of nitrifying and denitrifying bacteria, even
though pre-acclimation had been done (Dahl et al., 1997; Panswad and Anan, 1999).
The adverse effects of high salinity on conventional biological processes can be
attributed to high osmotic stress or inhibition of the reaction pathways in the organic
degradation process. In addition, high salt content induces cell lysis, which increases effluent
solids. The population of protozoa for proper flocculation is also significantly reduced at high
salt contents. Here, although salt acclimation can be expected from conventional processes,
the extent of adaptation is limited, and thus conventional processes can not be used to treat
wastewaters containing more than 3% salt (Woolard and Irvine, 1995).
Currently many saline wastewater treatment plants are able to overcome the technical
problems associated with high salinity by diluting the saline waste stream with fresh water.
Nevertheless, this practice is unsustainable, due to continuous pressure on the industries to
reduce fresh water consumption.

1.1.3 Salt-Tolerant or Halophilic Microorganisms

In order to improve organic and nitrogen removal efficiency, application of salt-tolerant
microorganisms in biological treatment of saline wastewater has been investigated
experimentally by several researchers (Nishihara ESRC Ltd., 2001; Woolard and Irvine,
1995; Hinteregger and Streichsbier, 1997; Park and Choi, 1999; Kargi and Dincer, 2000). Salt
tolerant microorganisms are those which can tolerate high salt content during their growth.
This utilization of halophilic microorganisms (e.g. Halobacter halobium) along with activated
sludge culture resulted in better treatment performances at salt contents above 2% (Kargi and
Dincer, 2000). Woolard and Irvine (1995) studied the treatment of hypersaline wastewater by
a moderate halophilic bacterial mixture isolated from soil of a saltern and fed in sequencing
batch reactor. They found that over 99% phenol removal was possible from 15% saline
wastewater.
In investigating the application of yeast in the food processing wastewater treatment,
researchers have investigated this potential in the treatment and reuse of wastes containing
solids and high concentrations of salt, fat and antibiotics. Park and Choi (1999) studied the
possibility of culturing an osmotolerant yeast, Pichia guilliermondii A9, using waste brine
from Kim Chi factory. The growth of Pichia guilliermondii A9 in waste brine was not
inhibited by NaCl concentrations of up to 60 g/L. In the Yeast Cycle System, wild yeasts were
utilized for treatment of wastewater, and the recovered excess sludge could be reused
(Nishihara ESRC Ltd., 2001). This yeast system is used as a pre-treatment to reduce the
organic pollutants, followed by a conventional activated sludge process. Primary treatment
plays an important role in removal of high organic and nutrient loadings. Moreover, excess
yeast sludge that had high protein, vitamins, lipid content could be used as animal feedstuff,
mushroom growing or as fertilizer.
1.1.4 Membrane Bioreactor Process
Application of the membrane bioreactor (MBR) concept in high salinity wastewater
treatment offers the possibility of overcoming low biodegradation rate and poor sludge
settling in the secondary sedimentation tank. MBR process can be operated at high MLSS and
thus organic removal can be improved. This results in sludge wastage and plant size reduction
(Visvanathan et al., 2000). Moreover, the selection of microorganisms present in the
membrane bioreactor is no more dependent on their ability to form biological flocs and
settling characteristics.
However, the membrane fouling problems lead to rapid flux reduction in MBR. This
secondary effect results in increase in energy consumption, and more frequent chemical
cleaning is required. These major problems hinder the widespread application of MBR in
effluent treatment processes. The membrane fouling might be the result of (a) the biofilm
growth or attachment of bacterial flocs on the upper surface of the membrane, and (b) the
deposition of macromolecules at the pore entrances or within the internal pore structure of the
membrane. The macromolecules can be protein from wastewater, extracellular polymers
(ECP) or long chain organic by-products generated during the biodegradation process.

1.2

Objectives of the Study

The overall objectives of this research were:

(1)

To evaluate variation of biokinetic coefficients of salt-tolerant yeast mixture and

bacterial mixture with high salt contents.

(2)

To find out suitable operating parameters for membrane bioreactor systems using salttolerant yeast and bacterial mixture to treat saline seafood processing wastewater.

(3)

To investigate membrane fouling of both microorganisms in terms of sludge

characteristics and ECP production.

1.3

Scope of the Study

To accomplish the above objectives, four studies were carried out:

(1)

Biokinetic study. Biokinetic coefficients of mixed yeast and mixed bacterial treatment at
high salt contents were evaluated using respirometric experiments. The three salt
contents examined were 20, 32 and 45 g/L NaCl. Two feed wastewaters were used,
namely glucose-feed wastewater and fish-protein-feed wastewater. In the fish-proteinfeed wastewater, commercial tuna fish protein extract was mixed to obtain wastewater
composition similar to tuna fish processing wastewater. Whereas, glucose-feed
wastewater was composed of glucose as carbon source and inorganic ammonia as the
nitrogen source.

(2)

Parametric study (optimization of operating conditions). The fish-protein wastewater

with salt content of 32 g/L was used in this study.
a.

Optimum pH for mixed yeast and bacterial treatment at a salt content of 32 g/L was
evaluated in terms of oxygen uptake rate (OUR) by respirometric experiments.
Based on theresponse of maximum OUR at different pH values, the optimum pH
range was determined.

b.

The variation of COD, DO and nitrogen (organic nitrogen, ammonia, nitrite and
nitrate) with aeration time in acclimatized mixed yeast and bacterial cultures was
monitored. Based on these COD, nitrogen profile data, suitable hydraulic retention
time (HRT), organic removal rate and nutrient uptake or nitrogen removal were
evaluated.

c.

Five sludge retention times (5, 7, 10, 20 and 45 days) were investigated for mixed
yeast treatment. Every the sludge retention time (SRT) experiment was conducted
in two-liter batch reactors with fill-and-draw operation. Based on the COD and
nitrogen removal, optimum SRT was suggested.

(3) Biomembrane study. This study consisted of two phases:

a.

High COD loading: Two parallel experimental set-ups were carried out, namely (1)
Yeast pretreatment followed by Bacterial Membrane Bioreactor (BMBR), and (2)
the Yeast Membrane Bioreactor (YMBR). Fish-protein-extract wastewater with
feed COD of 5,000 mg/L and salt of 32 g/L was used. The experimental
investigations were carried out by step-wise increase in volumetric loading at SRT
of 50 d.

b.

Low COD loading: The fish-protein wastewater with 1,000 mg/L COD and 32 g/L
NaCl was used in this phase. Two experimental set-ups were conducted (1) YMBR
and (2) BMBR. Treatment performance of both reactors was investigated at
different HRTs and SRTs (5 and 10 days).
4

The process efficiency was evaluated in terms of organic removal and membrane
filtration flux for various volumetric loading rates vis--vis HRTs.
(4)

Sludge characterization study: Variation of sludge characteristics with different salt

contents (0.5, 15, 32 and 45 g/L) was investigated by using two-liter batch reactors with
the fill-and-draw operation. Sludge properties were evaluated in terms of extracellular
polymer content, CST, SVI, viscosity and nutrient contents.

Chapter 2
2
Literature Review

2.1

Introduction

High salinity wastewaters are usually generated from industries such as seafood
processing, vegetable canning, pickling, tanning and chemical manufacturing. Seafood
processing factories located in arid zones use treated seawater or reused or recycled water in
processing steps such as defrosting, butchering and washing raw materials. Thus, the effluent
from these industries contains high salinity, which is approximately the same as that of
seawater. In addition, the adoption of waste minimization techniques within these industries
has led to reductions in waste volume, while waste concentration has been increased
(Woolard and Irvine, 1995). These wastewaters are often difficult to treat with conventional
treatment processes such as activated sludge, trickling filter and anaerobic processes. High
salinity can cause osmotic stress or inhibit the reaction pathways in the organic degradation
process. This results in a significant decrease in biological treatment efficiency or
biodegradation kinetics. In addition, high salt content induces cell lysis which causes
increased effluent solids. The populations of protozoa and filamentous organisms required for
proper flocculation are also significantly reduced at elevated salt content (Burnett, 1974).
Therefore, conventional treatment process can hardly meet the effluent standards for high
salinity wastewater.
2.1.1

The Seafood Processing Industry

The seafood processing sector contributes serious organic pollution loads and high
salinity to receiving waters. This feature leads to difficulty in biological treatment processes
(Mendez et al., 1992). The fish processing industry with cooking and brine filling operations
normally produces high strength organic matter, high level of oil and grease, and high salt
content. Typical water consumption ranges from 1874 m3/ton of fish processed (Battistoni
and Fava, 1994).
Process for canning shrimp is shown in Fig 2.1. In this process, receiving, peeling and
washing discharge large quantities of wastewater containing 90% of total COD. High salinity
wastewater is generated from precooking or brine treatment. In the precooking operation,
shrimp is boiled in brine solution for 3-5 minutes, or it is steamed. These operations curl the
meat, extract moisture and develop the pink or red color of the finished product. The salt
content of precooking wastewater is in range of 2 - 3% (UNEP, 1999).
Sardine and herring are classified as small, oily fish. These fishes contain a considerable
amount of oil or fat located between the skin and the flesh. The hot brine separates the oil
from the sardines. The oil rising to the surface of the brine forms a thick oil film on the top
which is then skimmed off. After cooking, the cans are taken out the brine and the remaining
brine in the cans is drained off. After cooling in a drying chamber, the cans are sealed, washed
down and packed.
The sardine and herring processing industry regenerates two kinds of wastewater: (1) sardine
and herring brine and (2) wastewater from cleaning and rinsing operations. The flow rate of
wastewater from these processes is as large as 6 times that of sardine or herring brine.
However, herring brine is a very concentrated wastewater consisting of a mixture of acetic
6

acid, sugar, fish protein and fish oil, as well as a number of spices and salt. The characteristics
of the herring brine is shown in Table 2.1.
water

water

water, salt

water

water

water

RECIEVING

THAWING

ice, water
debris, shells
water, debris

COOKING

shells

water, salt
SHELLING

WASHING

INSPECTION

water, salt

water, salt

water, debris

SALTING

CANNING

steam

meat particles

water

water

water, salt

water, salt

hot water
RETORTING

COOLING

to wastewater
treatment plant
Product

Figure 2.1 Flow diagram of steamed canned shrimp processing

Table 2.1 Characteristics of herring brine waste (Balslev-Olesen et al., 1990)
Parameter
COD
BOD5
Oil/fat
Ntotal
Ptotal
SS
VSS
Chloride
TDS
pH

Units
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
g/L
g/L

Average value
90,000
78,000
4,000
3,000
1,000
10,000
7,000
65
110
3.8

The canning process for mollusks such as mussel, oysters, clams or scallops also
generates large quantities of wastewater with salt content above 2%. The mollusks are shelled
and washed with 3 to 6% salt solution. Then they are drained and steamed or cooked for 10 to
15 minutes at 100qC. After inspection and grading, the cooked mollusks are packed in cans
7

with 1 to 2% brine. Mendez et al. (1992) reported that wastewater from processing mollusks
contained very high organic, nitrogen and salt content (18.5 g COD/L, 4.0 g N/L and above
2% salt).
The typical processing of dried salted fish is schematized in Figure 2.2. Slime, blood
and other contaminating substances of raw fish are washed off using a 3% solution of clean
salt in water. This reduces bacterial loads on the fish during subsequent salting. Large fish like
mackerel are split open at the ventral side from the head down. All visceral matter and blood
are removed. The fish is then cut into large pieces (1.5-2 cm thick, 10 cm wide and 20 cm
long). Fishes have an odor of ammonia, the dressed fish, or fish fillets, are soaked in mild
brine (10%) and crushed iced for six to 10 hours. This may be followed by salting. After
washing in clean brine solution, the eviscerated fish is salted in 21% brine for about 15 hours.
Salted fish is placed on bamboo trays and sun dried for two to three days in full sunshine,
depending on the size of the fish. Salted fish can also be dried in ovens. Fishes are then
packed and stored.
The characteristics of wastewater from the dried salted fish plant are shown in Table
2.2. This wastewater contains very high salt contents, ranging from 17g to 46g NaCl/L. A
large volume of wastewater is produced from soaking and washing operations. The volume
ranges from 10 to 12 m3/ton of preprocessed fish, and 20-30 m3/ton of iced or fresh fish. The
preprocessed fish, namely, is eviscerated or beheaded and cleaning at fishing boats or villages
in coastal zone before it is transported to sea-food processing plant.
Table 2.2 Characteristics of wastewater from the dried salted fish plant (Dan, 2000)
Parameter

Unit

COD
SS
TDS
ClSO42TKN
Total P

mg/L
mg/L
g NaCl/L
g/L
mg/L
mg N/L
mg P/L

(*)

Concentration
Washing + soaking tank

5,250
371
46
27
1,240
747
5

Except brine waste from soaking tank

Combine Wastewater(*)

873
119
17
10
164
128
5

water

wastewater

RECEIVING
THAWING

ice, debris

energy

wastewater, blood

water, ice

EVISCERATION

eviscera, skin

eviscera

water, ice
wastewater

Energy

FILLETING

meat particles

meat particles

wastewater, salt

water, ice

WASHING

meat particles

Offal recovery
(animal feed)
wastewater, salt

water

SOAKING

10% salt

organics

wastewater, salt

water

SALTING

21% salt

Energy

DRYING

organics

WWTP
for high salinity
wastewater

GRADING

Energy

PACKING

Energy

COOLING

WWTP(*)
for combined
wastewater

Product

(*)

WWTP Waste water treatment plant

Figure 2.2 Flow diagram of Dried and Salted fish processing

2.1.2

Pickled Vegetable Processing

Salt is used widely in vegetable canning processing to enhance flavor, to preserve, or for
conditioning. Therefore this industry in general produce wastewater containing high salt
content. The composition of the brines commonly used in canning vegetables is presented in
Table 2.3.
Table 2.3 Composition of brines used for canning vegetables (Joslyn and Timmons, 1967)
Product
Asparagus
Green bean
Cabbage
Beets
Peas

Brine, g/L
21.5 24.0 salt
19.2 27.5 salt
15.6 25.2 salt
24.0 salt, 18 24 sugar may be added
21.5 salt and 36 48 sugar

Brine waste from fermenting pickles contains high salt content (3 to 20%) and
extremely high organic concentrations. This is due to extraction from pickled vegetable tissue
during fermentation process. In addition, large amounts of saline wastewater are also
9

generated from washing or rinsing pickles after fermentation and rinsing equipment, soaking
or fermenting tanks. Table 2.4 presents waste loads in some pickling industries. EPA (1975)
reported that the BOD:N:P ratio of pickles and sauerkraut wastewaters were 100:1:0.2 and
100:4:0.5, respectively. These ratio show that nutrient concentrations in the pickling
wastewater are low for microorganism growth (BOD:N:P for bacteria growth is 100:5:1).
Table 2.4 Raw waste loads and quality of wastewater from some pickling industries

Category

Flow
m3/ton(*)

Raw waste loads

SS
BOD5
kg/ton
kg/ton

Pickles: (EPA, 1975)

- Fresh packed
- Process packed
- Salting stations
Total

7.76
8.70
0.96
17.4

8.61
16.7
7.21
32.5

1.72
2.97
0.38
5.07

Sauerkraut: (EPA, 1975)

- Canning
- Trimming
Total

3.19
0.39
3.58

3.18
1.13
4.31

0.55
0.17
0.72

19
15

6.8
6.4

1.36
0.45

Sauerkraut:
- (Woodroof, 1975)
- (NCA, 1971)
(*)

BOD5
mg/L

Concentration
SS
mg/L

pH

1,5005,800
(3,280)

135-825
(400)

4.3 6.3
(5.3)

1,400
6,300

60 - 630

4.3 6.3

Ton of raw material

Kim chi pickle which is pickled celery cabbage is a well-known food in Korea, Japan
and Vietnam. Figure 2.3 illustrates kim chi processing. Park and Choi (1999) reported that the
volume of waste brine produced from a kim chi factory is approximately 0.53 - 0.67 m3/ton of
product. Typically, the composition of waste brine contains sugars and other nutrients
extracted from the vegetables during fermentation, as well as a high salt content
(approximately 10%). Table 2.5 describes characteristics of the waste brine from four
different Kim chi factories located in Suwon city and Kyunggi province in Korea.
Table 2.5 Characteristics of the waste brine from four different kim chi factories located in
Suwon city and Kyunggi province, Korea (Park and Choi, 1999)
Parameter
pH
NaCl, g/L
BOD5 , mg/L
COD, mg/L
TKN, mg/L

Factory A
5.36
116
1,100
1,300
25

Factory B
4.91
95
1,200
1,790
28

10

Factory C
5.48
84
1,060
1,550
20

Factory D
5.80
70
1,040
1,250
25

Fresh celery
Cabbage

water

water, debris

RINSING

spoiled leaves/bases
CUTTING

Outer leaves
and bases
brine

Squares

brine

FERMENTATION

water, salt
FERMENTATION

discarded leaves
and bases

water

BOILING

onion, chili

CANNING

water, spices

water

water
citric acid

ginger
Animal feeds
recovery

debris

Liquid

water, salt
WASHING

SEALING

water

water, salt
RINSING

CAN FILLING

water, acid

water, salt
WASHING

SEALING

Kim Chee Juice

to WWTP

Kim Chee Nappa

to WWTP

Figure 2.3 Flow diagram of kim chi pickles processing

The characteristics of high salinity wastewater generated from seafood processing and
vegetable pickling industries is shown in Table 2.6. In seafood processing, the main
environmental issue concerns the use of large amounts of fresh water for processing, and its
emission as wastewater. The volume of wastewater discharged depending on the type of
products or raw materials range from 10 to 120 m3/ton of product. In comparison with the
vegetable pickling process, a ten-fold increase in organic loading (COD or BOD5) is
discharged by the seafood processing industry. However, the salt content of wastewater from
vegetable pickling is normally very high, possibly as much as 200 g/L.

11

kg/ton

kg/ton

kg/ton

BOD5 load

SS load

Oil & Grease

g NaCl/L

TDS of saline
wastewater

(1) ton of raw material

(2) Soderquist, 1971
(3) Estimated for waste brine only

Sources

% of total
volume

Saline wastewater
volume

Operation units
generating saline
wastewater

m3/ton

Unit

Water & wastewater

volume

Type of product

UNEP, 1999

30-35

(seawater)

39

Off-load,
sauce
filling/can
washing

27

5-6

Canned
sardine

(2)

(2)

UNEP, 1999

20 30

2 2.5

Brine filling,
cooking,
sealing, can
washing

42

54

120

60

Canned
shrimp(2)

(3)

UNEP, 1999

21(3)

cooking,
washing

60

20-120

Canned
mussel/oyster

11

15

22

(3)

(3)

12

UNEP, 1999

23

cooking,
sauce
filling/sealin
g/ can
washing

Tuna

UNEP, 1999

(seawater)

95

Off-load,
centrifuging,
storage

194

97

Fish meal

Choi &
Park,1999

100

100

Fermenting

0.7

0.6

Kim chi
pickles (3)

Table 2.6 Wastewater characteristics of from various fishery product and vegetable pickling industries

Middlebrook
1979

1979

30 200

89

Fermenting,
pickle washing

0.7

Sauerkraut(1)

Middlebrook

30 200

56

Fermenting,
pickle washing

33

17

Cucumber
pickles (1)

2.1.3

Other Saline Wastewaters

Wastewater generated from oil field exploitation contains high salt content and is refered to as
oil-field brine. Its characteristics are presented in Table 2.7.
Table 2.7 Characteristics of oil field brine (Dalmacija et al., 1996)
Parameter

Max
1,200
7.6
34.6
0.14
315
17.9
17.7

COD, mg/L
pH
TS, g/L
Phenol, mg/L
Oil, mg/L
Cl-, g/L
SO4-, mg/L

Value
Min
200
7.3
29.3
0.01
139
17.4
9.2

Average
400
7.5
32.3
0.05
237
17.6
11.9

In coastal areas, when subsurface water rises, infiltration of saline water into sewers can
result in high concentrations of chloride and sulfate in wastewater. Therefore, large variation
of salinity in domestic wastewater occurs normally in this areas. This can cause salt shocks or
adverse effects on conventional biological treatment methods.
Hypersaline wastes are produced in significant quantities in chemical industries such as
oil and gas production. These wastes contain organic compounds and high concentrations of
salt (>3.5%). High salinity is also found in landfill leachates. Pirbazari (1996) reported that
the leachates from domestic waste landfill (Los Angeles) and hazardous waste landfill for
chemical and petroleum waste (Niagara) had high strength organic matters and high total
dissolved solids (TDS). The characteristics of two leachates are described in Table 2.8.
Table 2.8 Characteristics of leachates (Pirbazari, 1996)
Parameters

Unit

COD
BOD5
TOC
SS
TDS
TKN
Oil and grease
pH

mg/L
mg/L
mg/L
mg/L
mg/L
mg/L
mg/L

2.2

Leachate
Domestic waste landfill
3,050 3,450
1,505 1,710
905 965
460 565
5,800 6,250
75 - 84
60 80
-

Hazardous waste landfill

9,000 10,500
6,950 7,500
3,040 3,500
862 946
22,600 25,900
160 180
4.3 6.0

Effects of High Salinity on Biological Waste Treatment Process

In wastewater treatment, there are conflicting reports on the influence of salt on the
biological processes. Some reports have indicated adverse effects of high salinity, or shocks
of NaCl on organic removal efficiency and sludge settleability (Burnett, 1974). Others have
reported that constant application of NaCl to biological treatment systems does not upset the
organic removal efficiency, and results in good flocculation of the biomass. This shows that
acclimation of the biomass and level of salt are important factors that may explain these
different observations (Hamoda and Al-Attar, 1995).
13

2.2.1

Aerobic Treatment

Previous studies reported that operation of activated sludge process at salt contents
higher than 20 g/L is characterized by poor flocculation, high effluent solids, and a severe
decrease in substrate utilization rate (Burnett, 1974).
Microscopic observations of the mixed liquor flocs (Burnett, 1974) showed that
alterations in saline wastewater caused alterations in the mixed liquor floc ecology. There was
a rapid die-off for rotifers, stalked protozoa and motile ciliate protozoa coincided with a
decrease in BOD removal and disruption in clarifier performance. After a few days, motile
ciliated protozoa were again observed, but rotifers and stalked ciliata were absent.
Tokuz and Eckenfelder (1979) estimated the effects of inorganic salts (NaCl and
Na2SO4) on continuous flow activated sludge with low F/M. The results indicated that the
relative high concentration of NaCl (up to 35 g/L) had only a slight effect on the performance
of the activated sludge process and the effluent SS did not increase. This was probably due to
a decrease in the F/M ratio. A further increase of NaCl over 35 g/L caused sudden increases in
effluent SS. The effect of sodium sulfate on the system was not significant. They also
observed that the protozoa population decreased gradually and disappeared at salt contents
above 35 g/L. The disappearance of the protozoa coincided with the sudden increase in
effluent turbidity or SS. Likewise the effects of high sodium chloride concentrations in an
activated sludge process was studied by Hamoda and Al-Attar (1995). The results showed that
the organic removal efficiency, and the treated effluent quality of activated sludge process did
not deteriorate as constant application of NaCl up to 30 g/L. COD removal efficiency ranged
from 93 to 99%. However, in order to obtain equivalent substrate removal, three-fold-lower
F/M ratios were applied in conventional AS at salt content of 30 g/L compared to those
applied in AS at salt-free wastewater (at the same SRT). Thus the substrate removal rate
decreases at high salinity. The MLVSS in the activated sludge reactor increased at salt content
up to 30 g/L. This result differs from the research conducted by Burnett (1974). An
explanation for this may be the long acclimation of microorganisms to the saline wastewater
might result in the growth of halophilic microorganisms in the system. Burnett (1974) also
noted that, although the substrate utilization rate decreased, the biomass yield obtained was
increased at higher NaCl concentrations. This may be due to a change in the efficiency of
microbial metabolism and the selection of salt tolerant species in the system. The salt tolerant
species may be halophilic micro-organisms such as Zooglea ramugera or Halobacteriaceae
which are aerobic heterotrophs.
Dalmacija et al. (1996) reported that the nature of pollutants and the high salinity (about
29 g/L) of oil-field brine has an unfavorable effect on the activated sludge process. High
hydraulic loadings (above 2.5 m3/m3.day) increased the wash-out of the activated sludge from
the reactor. The addition of PAC improved the sludge volume index and increased the rate of
biodegradation. This is due to the ability of biofilm formation on the activated carbon surface.
Kargi and Uygur (1996) investigated the effects of high salinity on the Rotating Biodisc
Contactor (RBC). The results indicated that the rate and efficiency of COD removal decreased
significantly with increases in salt content above 10g/L. COD removal efficiency with salt
free wastewater was 95%. Due to the adverse effect of salt on microorganisms, the COD
removal was down to 60% at 5% salt content. The increase in salt content causes a linear
reduction in COD removal rate as shown in Fig. 2.4.

14

COD removal rate, mg/m2.h

5000

4500

4000

3500

3000

2500
0.0

1.0

2.0

3.0

4.0

5.0

Salt concentration, %

Figure 2.4 Variation of COD removal rate with salt contents (Kargi and Uygur, 1996)
A review of the literature (Table 2.9) confirms the presence of adverse effects of the high
salinity on the conventional activated sludge systems. Major problems encountered in the
biological treatment of saline wastewater were summarized by Kargi and Dincer (2000). They
are:
x
Limited extent of adaptation: Conventional cultures cannot be effectively used to treat
saline wastewaters with salt contents above 3%.
x

Sensitivity to changes in ionic strength: Shifts in salt content from 0.5 to 2% usually
cause disruptions in system performance. Rapid change in salt contents causes more
adverse effects than gradual change. Equalization to constant salt content is necessary
before biological treatment.

Reduced degradation kinetics: Biological degradation rates decrease with increasing salt
content. Therefore, saline wastewaters should be treated at lower F/M ratios.

High effluent SS: Salt content in wastewater reduces the population of protozoa,
resulting in low settlability. Salt content in wastewater increases the buoyancy forces,
causing low sedimentation efficiencies.

15

Changing TDS up to 35,5 g NaCL/L

Operating continuous flow activated sludge with low F/M

ratio at d 35 g NaCl/L
If salt content > 35g/L

Increasing salt content to 10 g/L and 30 g NaCL/L

Oil-field brine with salt content of 29 g/L

Increasing in influent salt contents over 1% for RBC

Burnett (1974)

Tokuz & Eckenfelder

(1979)

Hamoda & Al-Attar (1995)

Dalmacija et al. (1996)

Kargi & Uygur (1996)

16

Experiment
Increasing influent from 100 mg Cl/L 20,000 mg Cl/L (|
33 g NaCL/L) over 2 to 3 weeks

Authors
Ludzack and Noran (1965)

Table 2.9 Adverse effects of high salinity in activated sludge process

Results
Solid losses disrupting clarifier
10% loss in BOD5 removal
Inhibiting nitrification
Decreasing BOD5 removal from 97% to 25% for 6 days after
Rapid die-off of rotifers & stalked/mobile ciliata protozoa
Turbid in effluent
Slight effect on BOD removal
Effluent SS did not increased due to low F/M
Decreasing the population of protozoa and then disappeared
Increasing effluent SS
Decrease in substrate utilization rate
But increasing biomass yield due to selecting salt tolerant species (halophilic
bacteria such as Zooglea ramugera, Halobacteriaceace etc.)
Increasing wash-out of activated sludge as hydraulic loadings > 2.5m3/m3.day
Decreasing COD removal rate & efficiency.
COD removal was down to 60% at 5% salt content.
Increasing salt content caused linear reduction in COD removal efficiency.

2.2.2

Anaerobic Treatment

Anaerobic process has become one of the most interesting treatment for highly organic
polluted wastewaters. However, the presence of salts may cause inhibition and toxicity
problems in the methanogenic activity. High salt levels can dehydrate anaerobic bacterial cells
because of osmotic pressure.
Anaerobic digester was much more sensitive to chlorides than activated sludge (Burnett,
1974). Baere et al. (1984) examined influence of high NaCl on methanogenic activity on
anaerobic filter (AF) process. The AF reactor was filled with ether-based polyurethane foam
with a specific surface area of 600 m2/m3. The results showed that initial inhibition occurred
at 30g NaCL/L. A shock treatment with 35g/L had a sharp decrease in gas production, which
dropped by 65%, and TOC removal efficiency, which decreased from 98% to 70%. The pH
dropped significantly after each shock treatment, from about 6.8 in the influent to a pH of 5.4.
When Methanosarcina, a halophilic anaerobic bacteria strain, was predominant (>99% of the
methanogenic biomass) in the reactor, TOC removal was improved. The methanogenic
activity of these bacteria was inhibited at 60 g NaCl/L. TOC removal was less than 20% and
the gas production dropped below 15% at 50 g/L.
An anaerobic and aerobic system consisting of an aerobic contactor followed by
activated sludge was tested for the biological treatment of high salinity wastewater (Belkin et
al. 1993). This wastewater generated from several chemical factories. The mean salt and COD
concentration were 32 g/L and 4900 mg COD/L respectively. Low COD removal efficiency
(55%) of the whole system was obtained at F/M ratio of 0.56 g COD/gMLSS.d for anaerobic
process and 0.28 for aerobic process. The COD efficiency increased to 74% at very low F/M
ratios (0.02 and 0.04 for the anaerobic and aerobic process, respectively).
Feijoo et al. (1995) examined the continuous exposure of high salinity in pilot scale
UASB and AF reactors. The results shows that the methanogenic activity of both anaerobic
processes was reduced by 50% at sodium concentrations above 20g/L. For unadapted sludge,
the anaerobic reactors could be shocked in the concentrations ranging from 6 to 13 g/L. In
addition, sodium inhibition in anaerobic digestion process was conducted with batch assays
(Feijoo et al., 1995). At low concentrations, sodium is essential for methanogens. The
optimum concentrations were reported to be about 0.23 0.35 g Na/L. The effect of NaCl on
the methanogenic activity depends on the type of sludge. When the sodium concentration was
increased by 4 to 10g/L (10 to 25 g NaCl/L), methanogenic activity reduced to 50%. After 40
days of digestion, the relative methanogenic activity of the sludge increased from 0% to 45%.
The sludge pregrown in the presence of high salt content showed a higher tolerance to
sodium, probably due to the adaptation of methanogenic bacteria to sodium. However, the
treatment efficiency after recovery was still low (45%).

17

Table 2.10 Adverse effects of high salinity in anaerobic treatment processes

Authors
Baere et al. (1984)

Experiment
AF with surface area of 600m2/m3
at 30 g NaCl/L
at salt content of 60 g/L

Feijoo et al. (1995)

UASB and AF

Belkin et al. (1993)

Anaerobic and aerobic system at

32 g salt/L

Feijoo et al. (1995)

Anaerobic batch digestion

2.2.3

Results
Decrease in gas production (dropped 65%)
TOC removal was decreased from 98% to 70%
Decrease in pH from 6.8 to 5.4
Gas production dropped below 15%
TOC removal < 20%
Reducing 50% methanogenic activity at salt
content > 33 g NaCl /L
Shocked at concentrations ranging from 10-21 g
NaCl/L for unadapted sludge
Low COD removal for whole system (50%)
COD removal (70%) could be improved at very
low F/M ratio (0.02 for anaerobic and 0.04 for
aerobic process
Decreasing 50% of methane activity as increasing
TDS by 10-25 g NaCl/L.

Nutrient Removal

In the nitrogen removal processes (Fig. 2.5), the oxidation of ammonia to nitrite and
then nitrite to nitrate (nitrification process) takes place under aerobic conditions (autotrophic
bacteria) and reduction of nitrate to nitrogen gas (denitrification process) occurs under anoxic
condition (hetetrophic bacteria). Dahl et al. (1997) found inhibition of the nitrifiers in the case
of a rapid increase of chloride concentration. The decrease in nitrification activity resulting
from increasing salt content from 16 g NaCl/L to 32 g/L, was approximately 30%.

Figure 2.5 Diagram of a nitrogen treatment system

Panswad and Anan (1999) investigated the effects of various salinity levels on ammonia
and
nitrate
uptake
rates
of
the
biological
nutrient
removal
systems
(Anaerobic/anoxic/aerobic). In the steady state, the specific ammonia and nitrate uptake rates
decreased with increase in chloride concentrations. The total nitrogen removal dropped from
85% to 70% at high salt contents (20 and 30 g NaCl/L). Concurrently, COD removal of the
system also was dropped from 90% at 5 g NaCl/L to 71% at 30 g/L. This indicated that the
nitrifying and denitrifying bacteria are very sensitive to sudden high salt content even with a
high degree of pre-acclimation. Similarly in conventional activated sludge process,
acclimation was clearly proven to be an important factor in improving the nitrification and
denitrification performance of the system. The phosphorous removal of this system decreased
from 38 to 10% with gradually increase in salt content from 0 to 30 g NaCl/L. This indicates
that poly-P bacteria have intense sensitivity to high salinity condition.
18

Dincer and Kargi (1999) reported that the salt content reduced the rate and the
efficiency of nitrification and denitrification at salt contents above 2% and 1% respectively.
Nitrobacter was more adversely affected by high salinity than Nitrosomonas resulting in
accumulation of nitrite in the effluent at salt contents above 2%. The denitrification rate
seemed to be more sensitive to salt inhibition than nitrification is. A summary of adverse
effects of high salinity in nutrient removal is shown in Table 2.11.
Table 2.11 Summary of adverse effects of high salinity in nutrient removal processes
Authors
Dahl et al. (1997)

Dincer and Kargi

(1999)
Panswad and
Anan (1999)

2.3

Experiment
Synthetic wastewater, combined
biological nitrification and
denitrification lab-scale experiment
Activated sludge for nitrification and
downflow packed column for
denitrification
Lab-scale anaerobic/anoxic/aerobic
with synthetic wastewater

Results
- Nitrification and denitrification rates were
reduced with increase in salt content (32 g/L)
- Salt concencentrations > 3% resulted in
significant reductions in performance of both
nitrification & denitrification.
- Shocked at 70 g NaCl/L.
- Specific nitrate and ammonia decreased as
increasing Chloride concentration
- Nitrifying and denitrifying bacteria were very
sensitive to sudden salt content.
- More intense sensitivity of P-bacteria to high
salinity

Application of Halophilic Bacteria for Saline Wastewater Treatment

Removal of salt content from wastewaters by reverse osmosis, electrodialysis before

biological treatment, is normally expensive. However, because of salt inhibition of bacteria
growth, application of conventional treatment processes does not obtain acceptable efficiency.
In recent years, several studies have shown that, in utilization of salt-tolerant microorganisms
in biological treatment such as halophilic bacteria, yeasts could be a reasonable approach for
treatment of high salinity wastewater.
Non-halophilic bacteria grow well in media which contain less than 1% salt content.
True halophilic bacteria require salt for survival. These bacteria can be divided into two
groups, namely moderate and extreme halophiles. The moderate halophiles are
microorganisms which grow best in medium containing 3-15% NaCl (0.5-2.5M). While
extreme halophiles exhibit optimum growth in media containing 15 30% NaCl (Woolard
and Irvine, 1995). To tolerate the osmotic forces present in saline environments, halophilic
microorganisms accumulate compatible solutes to equalize the ionic strength of the cytoplasm
with external environment. Moderate halophiles accumulate a mixture of inorganic cations
(K+, Na+) and organic compounds (amino acids, glycerol) for osmosis regulation.
Kargi and Dincer (1996) examined the treatment ability of Zooglea ramigera, a
moderate halophilic bacteria strain, at different salt contents using a fed-batch reactor. This is
different from sequencing batch reactor (SBR). The fed batch operation involves slow
addition of highly concentrated or wastewater into an aeration reactor until the tank is full.
With slow feeding, concentrated/toxic wastewater gets diluted inside the reactor, resulting in
less inhibition and higher BOD removal rates. COD removal efficiency for salt-free
wastewater was about 85%. This was not effected at salt content of 0.5%. However, the
efficiency dropped quite significantly with increasing salt contents above 1%, and attained
nearly 60% at 5% salt content.

19

In order to estimate the removal efficiency of salt tolerant microorganisms, Kargi and
Uygur (1996) used different types of microbial flora, namely Zooglea ramigera and
Halobacter halobium. The experiments were conducted using an aerated percolation reactor
with 1% salt content (Fig. 2.6). The percolator column was filled with crushed ceramic
particles (I = 4 mm) on which microorganisms were immobilized on the medium surface
(fixed biofilm).
air pump

Ceramic
particles
air
diffusor

Feed
tank
Effluent
tank

Percolate
reactor

Figure 2.6 Schematic diagram of percolation reactor (Kargi and Uygur, 1996)
The highest COD removal efficiency obtained (90%) corresponded to the mixed culture
of activated sludge and Halobacter halobium. Kargi and Dincer (2000) conducted a further
study with Halobacter halobium. This species was cultured along with activated sludge in the
fed-batch reactor. The organic removal rate was significantly improved (Fig. 2.7). COD
removal of 85% was obtained within 9 hours at high salt contents (3-5%).

COD removal rate, g/ m3h

400

300

200

100
0.0

1.0

2.0

3.0

4.0

5.0

Salt concentration, %
Halobacter halobium+activated sludge
Only activated sludge

Figure 2.7 Variation of COD removal rate (R) as function of salt content (Kargi and Dincer,
2000)
Woolard and Irvine (1995) investigated the treatment of phenolic wastewater with
extremely high salinity. A moderate halophilic bacteria was seeded in a sequencing batch
reactor. Over 99% phenol removal was achieved from 15% saline wastewater. Tellez et al.
(1995) evaluated biokinetic coefficients in biodegradation of oil field produced wastewater. It
is generated during recovery of natural gas and crude oil from onshore and offshore
20

operations. A commercial bacterium sp. (Petrobac-S) was used in this study. This is a
hydrocarbon degrader specially formulated for degrading crude or refined hydrocarbons in
moderated saline environments. The result indicated that when TDS was increased from 50 to
100 g/L, the maximum specific growth rate reduced from 0.137 to 0.047 h-1. There was a
slight increase in the half-velocity-constant (Ks) at higher salt contents. Ks is substrate
concentration at one-half the maximum growth rate. The affinity level for subtrate can be
evaluated in terms of KS .
The phenol removal capacity of a new moderate halophilic bacterium, Halomonas sp.
was investigated by Hinteregger and Streichsbier (1997). This bacteria consumed phenol as a
source of carbon at NaCl concentrations between 10 and 140 g/L. Under optimum conditions,
the degradation of 0.1g phenol/L in the aerated reactor was completed after 13 hours at salt
contents in the range of 30 and 50 g NaCl/L.
Dincer and Kargi (1999) reported that biological treatment of pickling industry
wastewater usually resulted in low COD removal efficiencies because of plasmolysis of cells
caused by high salt content (3-5%). Utilization of halophilic microorganisms (e.g. Halobacter
halobium) along with the activated sludge culture usually resulted in a better treatment
performance. COD removal of 97% was obtained at HRT of 30 hrs and sludge age of 10 days.
An aerobic, submerged biofilter, coupled with a trickling filter was investigated to treat
emulsified diesel fuel wastewater with high salinity (2% salt) (Yang et al., 2000). Figure 2.8
illustrates the schematic diagram of this system. The biofilter was randomly packed with
plastic media particles. The salt-tolerant-bacteria were isolated from the sediments on an
estuary. This system could give high removal efficiency (TOC removal > 90%) at volumetric
loading of 1.5 kg TOC/m3.d. The biodegradation of captured VOCs in the trickling filter was
effective (68% removal). The adsorption of VOCs was accomplished by countercurrent flow
of the gas and liquid phases through media bed. Based on the biodegradability tests at high
salt contents of 3.4% and 4.0%, the authors postulated that the bacterial mixture could
undergo high salinity up to 4.0%.
DO meter
pH meter

Biofilter

Trickling fliter

Feed tank
Air blower

Figure 2.8 Schematic diagram of the biofilter and trickling filter treatment system (Yang et
al., 2000)
21

Table 2.12 Effects of using halophilic bateria for high salinity wastewater treatment
Authors
Kargi and Dincer
(1996)

Experiment
Feed batch reactor with Zooglea
ramigena

Kargi & Uygur

(1996)

Percolator with Zooglea ramigena,

Halobacter halobium

Woolard and
Irvine (1995)
Tellez et al.
(1995)

SBR with moderate halophilic

bacteria
Biokinetic experiments for oil field
produced wastewater

Hinteregger and
Streichsbier
(1997)
Dincer and Kargi
(1999)
Yang (2000)

Using moderate halophilic

bacterium, Halomonas sp. to treat
phenolic wastewater
Using Halobacter halobium to
treat pickling wastewater
Aerobic-submerged biofilter
coupled with trickling filter
cultured with salt-tolerant-bacteria

2.4
2.4.1

Results
- Not affected at salt content of 0.5% (| 5 g/L)
- The efficiency dropped fast at increasing salt
contents above 1%.
- Zooglea ramigera culture obtained COD efficiency
of about 77% at 1% salt.
- Halobacter alone obtained lowest efficiency
- Mixed culture of activated sludge and Halobacter
obtained the highest efficiency at 1% salt and COD
removal of 70- 80% at 4 5% salt
- 99% phenol removal was obtained at 15% salt
- Maximum specific growth rate reduced from 0.14 to
0.05 h-1 when TDS was increased from 50 to 100
g/L
- 0.1 g/L phenol was completely degraded after 13 h
at 30 g/L salt
- 97% COD removal was obtained at HRT of 30 hrs
and salt content of 3-5%
- TOC of above 90% was obtained at VLR of 1.5 kg
TOC/m3.d at salt content of 3.4 %.

Yeasts
General

Yeasts are eucaryotic, heterotrophic, unicellular microorganisms with a variety of

shapes ranging from spherical to egg-shaped (common shape) and ellipsoidal, and from
cylindrical to considerably elongated and even filamentous (mycelium). Yeast have no
flagella or other organs of locomotion. In general, yeast cells are larger than bacteria, ranging
from 1 to 5 Pm in width and from 5 to 30 Pm or more in length. Yeasts have a complex
internal structure as shown in Figure 2.9. The vegetative budding yeast cell, in the log growth
phase, contains a very large vacuole and has rigid walls.
Yeasts multiply as single cells, which divide by budding or direct division (fission
which is similar to that by bacteria reproduce), or sporulation (sexual reproduction takes place
by means of ascospores) (Fig 2.10). In some unicellular varieties, large numbers of cells
attach themselves after budding, to form a pseudomycelium. In other cases, true mycelia are
formed by fission (Fig. 2.11).
Plasma membrane
Centrosome
Centrochromatin
Cell wall
Cytoplasm

Mitochondrium
Nuclear membrane
Vacuole

Figure 2.9 Diagram of yeast cell (Salle, 1961)

22

Budding

Fission

Sporulation
Figure 2.10 Budding is a common reproductive process in yeasts

a. Pseudomycelium

b. True mycelium

Figure 2.11 True mycelium (formed by fission) and pseudomycelium (formed by budding)
The dissimilation of organics may occur anaerobically (fermentation) or aerobically
(oxidation). The most typical yeast process applied in food or beverage industries is
anaerobic, also known as alcoholic fermentation. The end products of a fermentation can be
alcohols, acids, esters, glycerol and aldehydes. Prior to fermentation, polymeric substances
(carbohydrates, lipids, proteins) are hydrolyzed by enzymes (hydrolases). A typical reaction
of sugar fermentation by yeasts is shown in the following reaction:
yeasts, nutrients

Carbohydrate

C2H5OH + CO2 + new yeast cells

Under aerobic process (assimilation), complete oxidation of organics yields carbon

dioxide and water. Abundant supply of oxygen enhances considerable yeast growth. When
yeast are supplied with both sugar and oxygen, the colonies grow up to 20 times faster
through cell division than without oxygen. However, incomplete oxidation may generate acids
and other intermediary products. There are differences in the compounds which can be
assimilated by various species of yeasts. Some can degrade pentoses, polysaccharides
(starch), sugars, alcohols, organic acids (lactic, acetic, citric) and other organic substrates.
yeasts, nutrients

Organics + O2

CO2 + H2O + new yeast cells+ end products

23

Yeasts may utilize the nitrogen required in their metabolism for the synthesis of protein
from organic (amino acids, urea, vitamins, peptone, aliphatic amines, etc.) and inorganic
sources (ammonia, nitrite and nitrate). Most species can utilize the ammonium ion. Other
nutrients required for yeast growth include phosphorous, sulfur (organic sulfur and sulphate),
minerals (potassium, magnesium, sodium and calcium). Trace amounts of boron, copper, zinc,
manganese, iron, iodine, molybdenum are required to obtain optimum yields in synthetic
culture media. Basic components of Candida utilis are showed in Table 2.13.
Table 2.13 Basic composition of Candida utilis yeast biomass (Defrance, 1993)
Element
Value (%)

C
43.7

O
32

N
10.2

H
6.7

P
2.4

S
0.6

Mg
0.2

Ash
7.4

Based on these components, the chemical composition of Candida utilis yeast strain is
formulated as follows:
C3.64H6.7N0.73P0.07S0.02.
The nutrient demands can also be found from this formula. The C:N:P ratio of Candida
utilis biomass is 100:20:5, corresponding to BOD5:N:P ratio of 100:7.5:2. Therefore, nutrient
demands of yeasts are higher than that of bacteria whose BOD5:N:P ratio is 100:5:1
(Defrance, 1996).
Yeasts can grow in temperatures ranging from 0 to 470C. The optimum temperature for
most yeasts is 20 to 30oC. It is noted here that osmophilic yeasts are cable of growing in high
osmotic pressure habitats such as high concentrations of salt or sugar which restrict the
availability of moisture. On the other hand, yeasts can grow in a wide pH range (from 2.2 to
8.0). In general, yeasts grow well on media with acid reactions (3.8-4.0), whereas optimum
pH values for bacteria growth range from 7.5 to 8.5.
Fungi or yeasts may be found wherever nonliving organic matter exists. Unpolluted
stream water generally has relatively large numbers of species. Therefore, because of the
relation between fungi and yeasts densities and organic loading, it is suggested that fungi and
yeasts may be useful indicators of pollution. A survey of yeast populations along the St
Lawrence River that received domestic wastewater from Quebec Province (Simard 1971;
Simard and Blackwood, 1971). The results indicated that the blooms of pink yeasts
(Rhodotorula spp.) and black yeasts (Candida, Crytococcus, Torulopsis and Pullularia spp.)
occurred after bacteria had utilized the easily degradable components of the raw sewage.
Their density can be used as indicator of pollution.
2.4.2
a.

Applications of Yeasts for Wastewater Treatment

Domestic and Industrial Wastewater Treatment

The use of yeasts in biological treatment of domestic and industrial waste has been
studied since 1970. Thanh and Simard (1971) studied the biological treatment of domestic
wastewater with different yeast strains. All the tests were carried out with shaken 500mLflasks at 26-28oC for 3 days. The initial pH was adjusted to 5.0. The result indicated that the
yeast strains which gave high ammonia-nitrogen and COD removal efficiency were
Rhodotorula marina (85% NH3-N and 67% COD removals) and Candida krusei (91% NH3-N
and 72% COD removals). Especially, yeast strain Rhodotorula glutinis and Trichothecium
roseum could completely remove phosphorous compounds in domestic wastewater (Simard
24

and Thanh, 1973). However, COD reduction was not as high as had initially been expected.
The authors analyzed the cause to be the result of rapid uptake of phosphorous and nitrogen
compounds before the organics could be assimilated. Deocadiz (1977) studied yeast treatment
of mixture of domestic and paper mill white wastewater. Two yeast strains Candida utilis and
Rhodotorula glutinis were cultured in shaking flasks. Approximately 80% of COD, 50% of N
and 62% of P were removed after 24h. Rhodotorula yeast strain also gave the highest removal
efficiency for the biological treatment of potato chips wastewater. The COD, N and P
removals were 80%, 96% and 57%, respectively (Simard et al., 1973). The yeast sludge
contained high protein content (53%).
Henry and Thomson (1979) observed that Candida ingens yeast spontaneously grew
and formed a thick film on the supernatant of anaerobic piggery waste digesters. Based on this
observation, the authors investigated treatment ability of C. ingens for the effluent from these
digesters. The yeast was cultured in the stirring batch reactor. The results indicates that C.
ingens could utilize almost all the VFA up to a concentration of 0.09 mol/L after 24h growth
period. C.ingens grew well at pH ranged from 4.8 to 5.0 and at temperature of 29-32oC.
Miskiewicz et al. (1982) developed further yeast treatment of fresh piggery wastes by
adding carbon source (beet molasses or sucrose). Four yeast strains, Candida tropicalis,
Candida tropicalis, Candida robusta and Candida utilis were cultured in a batch aerated
reactor. The study shows that the use of raw piggery waste without carbon supplement leads
to low biomass yield and low treatment efficiency, even though the nutrients (N, P) are high.
It is found that molasses are the most appropriate carbon source. The culture of C. utilis on
molasses-enriched piggery waste (5570 mg COD/L) could obtain high treatment efficiencies.
76% TKN, 60% COD, 84% total P were removed at HRT of 7 hours and F/M ratio of 1.73 g
COD/g MLSS.d. The maximum specific growth rate of C. utilis was 0.19 h-1.
Anciaux et al. (1989) investigated the influence of DO, substrate concentration, type of
VFA on the growth of C. ingens in the aerated and stirred batch reactor. The result shows that
higher the DO concentration, the shorter the lag phase and Pmax increased with the DO
concentration according to the trend of the Monod model curve. Thus DO becomes a ratelimiting factor at a very low concentration (Figure 2.12a). The effect of substrate
concentration is shown in Figure 2.12b. This figure shows inhibition of growth by the
substrate concentration. At VFA concentration of 0.25 g /L (approximately 270 mg COD/L),
the best growth rate (P) and yields obtained were 7.5 d-1 and 0.6 g DS/ g acid consumed,
respectively.
8.00
Specific Growth Rate P day-1

Specific Growth Rate P day-1

7.20

6.80

6.40

6.00

5.60

5.20

10

20

30

40

50

60

70

6.00

4.00

2.00

80

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

VFA concentration (g/L)

% DO (% saturation)

a. Specific growth rate vs. DO

b. Specific growth rate vs. VFA concentration

Figure 2.12 Specific growth rate of Candida ingens vs DO and VFA concentration (Anciaux
et al., 1989)
25

Katayama-Hirayama et al. (1994) cultured the yeast strain of Rhodotorula glutinis with
phenolic wastewater. The cultures were propagated in shaking flasks and incubated at 30oC.
Phenol and monochlorophenols were completely degraded after 2 days. COD removal
obtained ranged from 79% to 83%. When compared with cell yields on the glucose (0.66 g/g)
and acetate (0.39 g/g) media, phenol is an excellent carbon source (y = 0.61 g/g). None of the
studies reviewed above evaluated the settling ability of yeasts.
Hu (1989) used ten different yeast strains in cultures to treat vermicelli wastewater
which contains high concentration of starch, lactic acid and protein with BOD ranging from
24,000 to 44,000 mg/L. Based on the ability of starch degradation, protein hydrolysis and
lactic acid tolerance, these yeast strains were screened from 391 colonies isolated from soil
samples. Most could grow well within pH range of 3.0-5.0, with pH 4.0 being the optimum.
The results shows that the two strains could reduce soluble COD by 92% at HRT of 7 days,
F/M ratio of 0.48 g COD/g MLSS.d and VLR of 1.03 kg COD/m3.d. The long HRT in this
process is due to the poor settling ability of yeasts. The yeasts could not be flocculated or
settled as in a conventional activated sludge process, and were easily washed out with the
effluent. Therefore, the HRT and SRT were kept constant. The author postulated that the
fungi contamination prevented the formation of yeast flocs.
Similarly, Chigusa et al. (1996) used nine different strains of yeasts capable of
decomposing the oil to treat wastewater from oil manufacturing plants. A pilot scale yeast
treatment system had been run for one year. The results showed that 10,000 mg/L of hexane
extracts in the raw wastewater were reduced by the yeast mixture to about 100 mg/L.
Also, Elmaleh et al.(1996) investigated the yeast treatment of highly concentrated acidic
wastewater from the food processing industry. The strain Candida utilis was cultured in
continuously completed mixing reactors. This system did not have a separate settling tank; the
SRT and HRT of the system are identical. The carbon source of feed wastewater was a
mixture of acetic acid, propionic and butyric acid. The pH was maintained at 3.5 to prevent
any bacterial contamination. The TOC removal obtained was 97% at high loading rates (30 kg
TOC/m3.day). The growth yield and maximum specific growth rate of yeasts were similar to
those for conventional activated sludge (Pmax = 0.5 h-; Y = 0.85-1.05 kg SS/kg TOC for acetic
acid). In this study, the authors only evaluated biokinetic constants, but did not focus on the
settling ability of yeasts.
Olive mill wastewater normally contains high concentration of fats, sugars, phenols,
volatile fatty acids which contribute to a very high COD concentration (100-200 g/L). Scioli
and Vollaro (1997) reported that Yarrowia lipolytica cultured in the 3.5L-aerated fermenter
was capable of reducing the COD level of olive oil processing wastewater by 80% in 24 hrs.
The effluent had a pleasant smell due to the presence of methanol and ethanol, while fats and
sugars were completely assimilated. The authors postulated that using membrane to filter
effluent before discharging into the sewage system might be a feasible approach for pollution
reduction in olive-oil-producing countries. Useful biomass (40% protein) and valuable lipase
enzyme could also be obtained in this process.
Silage is produced by the controlled fermentation of a crop with high moisture content,
such as grass or maize. This silage can be used as an animal feedstock. Its effluent is
extremely polluting, having very high BOD (30 80 g/L) and low pH (3.0-4.5). Arnold et al.
(2000) investigated the ability of selected yeast strains (C. utilis and Galactomyces
geotrichum) to purify silage effluent on the shaker-flask scale. High removal efficiencies of
COD (74-95%), VFA (85-99%) and phosphate (82-99%) were obtained after 24 hrs. Some
26

ammonia was also removed. pH rose during treatment to 8.5-9.0 from initial values of 3.7-5.8.
This was presumably due to removal of lactic acid and VFAs. The dramatic decrease in P
(resulting in extreme P removal) may be attributed to the shortage of phosphorus.
In general, carbon and nitrogen removal from high organic-strength wastewater can be
conducted with different processes, namely, anaerobic and aerobic processes, nitrification and
denitrification. Ortiz et al (1997) proposed an effective and economic alternative process in
which it is possible to achieve both carbon and nitrogen removal in two stages: anaerobic
bacterial treatment and yeast treatment process. The fermentative bacteria transforms the
organic nitrogen and the carbonaceous substrates into ammonia and volatile fatty acids (VFA)
which are degradable substrates for the yeast growth (Fig. 2.13). A comparison between yeast
and anaerobic treatment process is presented in Table 2.14.
Anaerobic
process

Nitrification
process

Dinitrification
process

a. Traditional coupling for carbon and nitrogen removals

Yeast
process

Acidogenesis
process

b. Coupling for Anaerobic acidogenesis and Yeast treatment

Figure 2.13 Traditional carbon and nitrogen removal system can be altered with anaerobic and
yeast treatment system (Ortiz et al. 1997)
Table 2.14 A comparison between yeast and anaerobic treatment process (Defrance, 1993)
Yeast process

Anaerobic process

Cannot degrade complex organic compounds

Degrades cellulose

High nutrient requirement (BOD5:N:P)

Low nutrient requirement

Need for oxygen and agitation

Slight agitation

Exothermal reaction need for cooling

Producing methane as valuable bio-fuel

Sensitive to variation of temperature

Can consume VFAs produced from

acidogenesis

Dependent on two phases: liquefaction and

gasification

High organic loadings

Low organic loadings (< 3 kg COD/m3.d)

Short HRT

High HRT (minimum 10 days)

Valuable biomass

Poor sludge production

Generally, dairy industry effluents contain large quantity of milk constituents such as
casein, lactose, fat and high inorganic salts. Marwaha et al.(1999) investigated the effect of
nitrogen supplements (urea and yeast extract) on the treatment ability of two yeast strains
Candida parapsilosis and Candida haemulonii isolated from the dairy effluents. All tests
were conducted with shaker-flasks and incubated at 30oC for 24 hrs. The pH of the medium
was adjusted to 5.5. The result indicated that maximum BOD (90%) and COD (82%)
27

removals could be obtained when 0.6% yeast extract was supplemented. The relation between
biomass growth and organic removal was not determined in this study.
b.

High salinity wasterwater treatment

Choi and Park (1999) studied the treatment ability of an osmotolerant yeast, Pichia
guilliermondii A9, for waste brine from a kim chi factory using a shaker-flask scale. The
growth of Pichia guilliermondii A9 in waste brine was not inhibited by NaCl concentrations
up to 100 g/L. However, it was affected at concentrations above 120 g/L. Approximately 90%
of BOD was removed from the waste brine after 24 hrs. The maximum cell yield was 0.69 g
of dry cells per liter, containing 40% of protein. Cell growth was highest at pH 4, and
declined slightly when pH increased to 8.
Nishihara ESRC Ltd. (2001) studied the effect of the Yeast Cycle System on marine
products processing wastewater. In this system, yeasts are used for wastewater, where the
excess yeast from the treatment process is recovered and reused. The system consists of
pretreatment by yeast and secondary treatment by activated sludge process (Fig. 2.14). Table
2.15 and Figure 2.15 give a comparison with conventional complete mixing activated sludge
in terms of the operating conditions. Marine products processing wastewater has BOD5 and
SS concentrations ranging from 3,550-8,850 mg O2/L and 680-940 mg/L respectively. The
chloride concentration was 5,160 mg/L. Some yeast strains, Candida edax, Candida
valdivana and Candida emobii, were predominantly grown during enrichment with this raw
wastewater. The predominance of yeast strains with the enrichment culture technique is based
on free competition among different organisms in real wastewater. It was found that the yeast
treatment process can obtain high efficiency at a higher volumetric loading (5 6 times), F/M
ratio (2 3 times) when compared with the AS process.
Yeast reactor

Settling tank I

yeast sludge return

AS reactor

Settling tank II

AS sludge return

excess yeast sludge

excess AS

Figure 2.14 Schematic diagram of the Yeast Cycle System (YCS)

Table 2.15 Operating conditions of YCS (Nishihara ESRC Ltd., 2001)
Parameter
Influent BOD5
Salt content
BOD5 volumetric loading
Yeast concentration
BOD5 sludge loading (F/M)
Water temperature
pH
DO
SVI
Y

Unit
mg/L
g/L NaCl
kg/m3.day
mg/L
kgBOD5
/kgVSS.day
qC
mg/L
ml/g
kgVSS/kgBOD5

YCS
Range
3,550 8,650
5-8
4.5 10.4
8,000 10,000
0.56 1.04

Mean
6,100
6
7.5
9,000
0.9

AS(*)
Range
110-400
0.05-0.45
0.8 1.9
2,500 4,000
0.2 0.6

23 30
4.3 5.2
0.51 0.95
60 72
-

26
4.8
0.7
66
0.16

23 30
6.5 8.5
t2
100 120
0.4 0.8

(*) Complete mixed activated sludge (Metcalf and Eddy, 1991)

28

6
Activated sludge

No. times higher than value

of activated sludge process

Yeast Cycle System

4

MLSS/L

F/M

SVI

Note:
L
MLSS/L
F/M
Y
SVI

=
=
=
=
=

BOD5 volumetric loading (kg/m3.day)

Concentration of microorganism (mg/L)
BOD5 sludge loading (kgBOD5/kgSS.day)
Sludge yield (kg VSS/kg BOD5)
Sludge Volume Index (ml/g)

Figure 2.15 Comparison between Yeast Cycle System (YCS) and complete mixed activated
sludge (AS) (Nishihara ESRC Ltd., 2001)
Large flocs formed in the yeast treatment system were able to settle quickly. Thus the
MLSS could be maintained at high concentration of about 10,000 mg/L. The yeast sludge has
low SVI of 50-60 mL/gram, which was equal to half the SVI value for activated sludge. This
makes reducing the size of the sedimentation tank, and thickening of yeast sludge or chemical
conditioning for dewatering are not necessary. The efficiency of this system is presented in
Table 2.15 and Figure 2.23.
Table 2.16 Quality of treated water and efficiency of the YCS for seafood processing
wastewater treatment (Nishihara ESRC Ltd., 2001)
Parameter

BOD5 , mg/L
SS, mg/L
T-N, mg/L
T-P, mg/L
Cl-, mg/L

Influent

After pretreatment
by yeast

5,450
798
153
33
5,160

150
113
72
18
5,080

E%

97%
86%
53%
46%

After
activated sludge

4
15
10
15
5.080

E, %

99%
87%
86%
17 %

BOD5 and SS removal by yeast were more than 95% and 87% respectively. The
nitrogen (52%) and phosphorous removal (46%) during the yeast treatment were equal to
those found in the components of excess yeast sludge. The company postulated that the
structure of yeast flocs facilitated oxygen diffusion. Therefore energy could be saved through
the reduction of the supplied air flow. Moreover, the excess yeast with high protein, vitamins,
and lipid content could be used for animal feedstuff, mushroom growing or fertilizer.

29

Table 2.17 Summary of studies on yeast treatment of high salinity wastewater

Authors
Nishihara ESRC
Ltd. (2001)

Experiment
Yeast treatment system for
marine products process
wastewater

Results
- BOD5 and SS removal were more than 95, 97%
respectively
- High BOD5 volumetric loading (7.5 kg/m3.day)
- High BOD5 sludge loading (0.9 kg BOD5/kgVSS.day)
- Low excess yeast, 0.16 kg VSS/kg BOD5

Choi and Park

(1999)

Yeast treatment for Kim Chi

waste brine

- Pichia guilliermondii can tolerate NaCl up to 100 g/L

- 90% BOD removal obtained for 24h

c.

Waste recycle

Single-cell protein production (SCP)

Linkage between biomass for food production and waste and wastewater treatment has
been widely developed. A number of organisms are utilized for biomass and protein
production. These include: (1) protein-rich algae, fish, duckweed and water hyacinth in
oxidation/stabilization ponds; (2) bulrush, cattails and other plants in constructed wetlands;
(3) worms from composting waste and sludge and (4) yeasts and fungi cultured from
carbohydrate-rich wastewater. Therefore, utilization of food-processing wastewater as
substrates for biomass production or single-cell protein production (SCP). SCP results in
purification of effluent. This application can also obtain savings from decrease in disposal and
treatment costs. Single-cell protein production (SCP) is defined as microbial biomass
produced by some biological process and it can be used as food or food additives. Industries
that produce large volumes of carbohydrate-rich-containing wastewaters free of toxic
materials are most promising substrates for SCP. Such industries include milk, cheeseprocessing, confectionery manufacturing, and food canning. Effluents from these industries
which contain high concentrations of COD and nutrients are costly to treat. Thus their
utilization for SCP is an attractive alternative.
Yeasts such as Saccharomyces cerevisiae, Candida utilis and most fungi, are quite
acceptable to animals and man. Whereas algal and bacterial biomass are less pleasant and
contain undesirable levels of certain cellular materials (such as high nucleic acid content,
toxic or carcinogenic substances absorbed from the growth substrate). In addition, due to
abundance of valuable nutritious substances such as proteins and vitamins, yeasts are the most
feasible and acceptable microorganisms in the production of SCP. In their simplest
processing, yeasts can be cultured in a suitable substrate, normally carbohydrates, such as
molasses, whey or starch, and under suitable conditions. The yeast biomass is harvested from
the fermentation or assimilation followed by separation (settling, filtration, centrifugation,
membrane), washed and dried to produce a free-flowing powder, rich in protein (Gray, 1989).
For example, 50 thousand tons of yeasts per year are produced in North America from
sulphide liquor (paper mill waste) containing high pentoses and hexoses.
A novel SCP process developed by George Bassett Co. in Sheffield is shown in Fig.
2.16. In this process, Candida utilis is cultured with confectionery wastewater and then
harvested by centrifuging and drying. The SCP is packed and sold as a high-protein additive
for animal feed. The present output of this plant is about 140 tons/year. It is able to remove
65% COD of the wastewater. Therefore, the wastewater after the SCP process contains
remaining COD concentration, which is low enough to be discharged directly to the sewer
(Gray, 1989).
30

Sterilizer
Centrifuger

Spray dryer

Crude effluent

Inocula reactor Main reactor

Package

Equalization tank

Figure 2.16 SCP from confectionery effluent (Gray, 1989)

The Symba process developed by Swedish Sugar Company is based on the symbiotic
culture of yeasts Endomycopsis fibuliger and Candida utilis with potato processing waste and
wastewater (Figure 2.17). The basic substrate for SCP production in wastewater is starch,
which is not easily assimilated by Candida utilis. However, the starch can be hydrolyzed to
low molecular weight sugars (glucose, maltose) by the enzyme amylase. This enzyme is
produced in large quantity by E. fibuliger. These hydrolyzed products (sugars) are then easily
degradable substrates for the growth of C. utilis which has high nutritional value. In the
process, the wastewater is strained in order to remove any large particles and then sterilized
by heating to destroy any microbial contamination. The sterilized substrate is introduced to a
preliminary reactor, in which E. fibuliger is grown to provide its population in the main
reactor, as its growth rate is much lower than that of C. utilis. These reactors are maintained in
aerobic condition. A large quantity of heat is generated from yeast assimilation activity and
removed by cooling towers or heat exchangers. Excess biomass is purified by sieving and
then concentrated by centrifuge and dried by spray drier. The Symba process can achieve
BOD removal of 90% (BOD of wastewater is reduced from 15,000 mg/L to 1,500 mg/L),
both N and P removals of about 50%. The yeast contains about 45% protein with less nucleic
acids but rich vitamins, especially vitamin B (Gray, 1989).
Endomycopis
reactor

Symbiosis
reactor

Cooling tower
Sterilizer
Storage tank

Storage tank

Air blower

Spray drier

Separater

Separater
Sewer

Sewer

Figure 2.17 The Symba process (Gray, 1989)

31

Packing

Simard and Cameron (1974) evaluated the growth of Candida utilis on dilutions of
spent sulphite liquor (SSL) with addition of different nitrogen sources. These were urea,
ammonia sulphate and ammonium hydroxide. The result shows that the dilution of SSL (2
water:1 SSL) increased significantly the dried biomass. Urea, ammonia sulphate gave high
conversion efficiency (70%).
Candida utilis yeast can be used to purify and uptake effectively ammonia present in the
anaerobic digester supernatant supplemented with molasses as a source of carbohydrate (Irgen
and Clark, 1976). The result shows that 100 g of molasses could yield 41 g of dried Candida
utilis yeast cells and 20 g of protein.
Likewise, Barker et al. (1982) cultured Hansenula anomala, Candida krusei and
Geotrichum candidum with whisky distillery spent waste. The influent COD of this waste
ranges from 15 g/L to 58 g/L. These yeast strains were isolated from whisky distillery
effluent. The results indicated that the yeasts could give high yield of protein biomass and
COD removal of 55% .
Rashad et al.( 1990) found that mango peel waste from drink processing industries can
be used for SCP production in which Pichia pinus is cultivated under optimum conditions (pH
= 4.8-5.0; temperature = 30oC). The maximum yield obtained after 3 days of growth was 6.2
g/ biomass/L of wastewater and dried yeast biomass contained high crude protein (62%) and
low nucleic acid (12%).
Liquid waste (deproteinized leaf juices) is generated from vegetable protein production.
Chanda and Chakrabarti (1996) reported that depended on type of vegetable, BOD5 and COD
of the waste can range from 12.9 to 19.0 g/L and 20.2 to 28.5 g/L respectively. This liquid
waste can be a good substrate for the cultivation of S. cerevisiae, T. utilis, C. lipolytica. The
yeast biomass obtained was rich in protein and vitamins. BOD of wastewater reduced
significantly (74 97%) by the growth of these yeasts. The shrimp shell waste could also be
converted into proteins by using the yeast Scharomyces cerevisiae KIV-1116 (Ferrer et al.,
1996).
Bio-fuel
Fuel-alcohol production by yeast/fungi fermentation of the industrial or agricultural
wastes has received considerable attention in Brazil and India in early 1990. Development of
such technologies could reduce the oil imports and a partial solution to disposal of wastes.
Nigam (1999) reported that the culture of Saccharomyces cerevisiae on pineapple cannery
waste had high ethanol productivity (0.98g ethanol /g yeast.h) and sugar uptake rate (2.3 g
sugar/g of yeast. h).
Yu et al. (1987) used the yeast Candida shehatae to ferment the spent sulphite pulp and
paper mill. The ethanol yield gained was 0.46 g ethanol/g initial sugar at HRT of 18 h. This
corresponds to the sugar removal of 95.5%. In general, the municipal primary wastewater
solids contains about 10% cellulose and 26% lignin. These cellulose components can be
effectively converted to ethanol by fungal cellulase and yeast Sacharomyces cerevisiae
(Cheung and Anderson, 1997).
Lark et al. (1997) studied on the reuse of recycled paper sludge. At least 72% of
cellulose in the sludge was converted into ethanol by the yeast Kluyveromyces marxianus.
The paper sludge volume was reduced to 30 35% of the original volume after 72h of
32

fermentation. The ethanol production by yeasts from cassava grate waste was also studied
(Agu et al.,1998) where 60% of cellulose and lignin materials was hydrolyzed and converted
to ethanol.
The cheese waste normally contains very high content of lactose which can be suitable
substrate for ethanol production. Its concentration can be up to 50 g lastose/L. Ghaly and ElTaweel (1997) reused this waste for continuous ethanol production by yeast Candida
pseudotropicalis. The results shows that high ethanol concentration (58 g/L) could be
achieved at HRT of 42 hours.
2.5

Theoretical Modeling Consideration

2.5.1
a.

Growth without Inhibition

Specific growth rate (P)

Biomass concentration

Jackson and Edwards (1975) estimated specific growth rates of microorganisms in a

culture by the following expressions:
dX
PX
(2-1)
dt
X X o e P ( t to )
(2-2)
ln X P (t  t o )  ln X o
(2-3)
ln X  ln X o
P
(2-4)
t  to
Where X
=
Biomass concentration at the time t (mg/L)
Xo
=
Biomass concentration at the time to (mg/L)
P
=
Specific growth rate (1/h).

Ln(Xt)

P
Ln(Xo)

to

Time

Figure 2.18 Growth curve of microorganisms in a culture

Generally, Monod's model is used to estimate different biokinetic reactions between
microorganisms and the substrate in a continuous culture (Metcalf and Eddy, 1991).
According to this model, specific growth can be related to substrate by the following
relations:
S
P Pm
(2-5)
S  KS
Where
= Specific growth rate of microorganism (d-1)
P
33

Pm
S
KS

=
=
=

Maximum specific growth rate (d-1)

Substrate concentration (mg/L)
Half-velocity constant or Monod constant (mg/L).

Specific growth rate ( P)

time-1

Pmax

Pmax
2

Ks

Substrate concentration, mg/ L

Figure 2.19 The effects of a limiting substrate on the specific growth rate (Monod model)

b. Substrate Utilization rate (U)

Substrate utilization rate(U) vis-a-vis COD removal rate, can be expressed as:
(S o  S )
U
T .X
Where
= Substrate utilization rate, (mg substrate removed/ mg MLSS.day)
U
So = Initial substrate concentration (mg/L)
X
= Biomass concentration (mg/L)
= Final substrate concentration (mg/L)
S
= Hydraulic retention time (day).
T

c.

(2-6)

Growth Yield Coefficient (Y)

The relation between new cell production and soluble substrate consumption can be
represented as follows:
dX
dS
Y*
(2-7)
dt
dt
Where
= true growth yield coefficient (g SS/ g substrate removed.day)
Y
X
= Biomass concentration (mg/L)
= Substrate concentration (mg/L)
S

For a given microorganism and essential nutrient/substrate under the same environmental
conditions, the weight of microbial cells produced per weight of nutrient/substrate consumed
is constant. This relationship is expressed as:
Y = Weight of organisms produced/Weight of substrate utilized

34

2.5.2

Growth with Inhibition

Han and Levenspiel (1988) proposed generalization of the Monod expression which
takes into account inhibition effects caused by high concentration of substrate, product or
toxics, ammonia, ion strength (salts) and other inhibitory substances.

P m 1 

Where
I
KI
n

=
=
=

I
KI

S  K S 1 
K I

(2-8)

concentration of inhibitor
the critical inhibitor concentration above which reaction stops
constants

Some kinetic models for growth with inhibitory substances are shown in Table 2-18.
Table 2.18 Kinetic models for inhibition growth (Han and Levenspiel, 1988)
Equation

I S

P P m 1 
K I S  K S

P m 1 

I
KI

0.5

Model name

P obs

S
S  KS

S
S  KS

P obs

I
S

P m 1 
KI S  KS

P obs

(2-9) Ghose and Tyagi

S
S  KS

(2-10) Bazua and Wilke

S
S  KS

(2-11)

Han and Levenspiel

Where
Pm = Maximum specific growth rate at inhibitor concentration of zero (I = 0).
Pobs = Observed maximum specific growth rate at certain inhibitor concentration (I).

(Pobs ), time-1

Observed specific growthrate

Pmax

n = 0.3

n = 1.0

n = 0.5

Inhibitor concentration ( I), mass/ vol

KI

Figure 2.20 Curves of inhibition growth models (n =1: Ghose and Tyagi; n= 0.5: Bazua and
Wilke model)
Eq.2-11 is a generalized form of Eq.2-9 and Equ.2-10, in which the constant n is 1.0 and
0.5 respectively.
.
35

a.

Substrate Inhibition

Some authors have suggested models for the growth inhibition of Candida utilis on
acetic acid as substrate. These are:

Pm

Pm

S
S
1 

KS  S
S c
S

( K S  S )1 
K i

Pm

KS  S 

S2
Ki

(2-12)

Defrance (1993)

(2-13)

Haldane (Ortiz et al. 1997)

(2-14)

Andrew (Ortiz et al. 1997)

Where Sc = The critical substrate concentration above which reaction stops

Ki = Inhibition constant
Defrances model is also a modification of Han & Levenspiels model (Eq. 2-11) in which KI
=Sc and n = 1.

Observed specific growthrate

( Pobs ) ,time-1

Pmax
Monod

Andrew

Defrance

Haldane

Substrate concentration ( S) , mass/ vol

Sc

Figure 2.21 Curves of substrate inhibition growth models

b. Salt/ions Strength Inhibition

Webbs model presents ion-strength inhibition for microorganism growth:

P
Where V =

P max

S  K S 1  V

K
I

Ion strength

exp(1.17V )

(2-15)

Dincer and Kargi (1999) proposed expression to estimate salt inhibition for nitrification
and denitrification.
No  N
K TN
RN
RON
(2-16)
T
K TN  C s
Where RN = Rate of nitrification and denitrification, kg/m3.h
KTN = Salt inhibition constant for nitrification and denitrification, g/L
36

Cs = Salt content, g/L

RON = Nitrification and denitrification rates for salt-free wastewater.

c.

Other Inhibition Factors

The effect of pH, ammonia on the aerobic growth of Candida utilis at constant
temperature (30oC) are illustrated by the following models:
Effect of
ammonia

>NH 3 @

Pm

K >NH 3 @  >NH 3 @ 

Effect of pH

>NH 3 @2

(2-17)

(Ortiz et al., 1997)

(2-18)

(Jackson and Edards, 1975)

K i>NH 3 @

Pm
1

K2


H
Where K1 , K2 = pH constants

H
K1

Henze et al. (1997) described effects of pH, temperature and DO on aerobic

heterotrophic micro-organisms by the following kinetic models:
Effect of pH:

P m ( pH ) P m ( pH opt )

Effect of temperature:

KpH

D
SO2
KS,O2

(2-19)

K pH  I

P m (t o C ) P m (20 o C ) * exp[D (t o C  20 o C )]

Effect of DO:

K pH

Pm

(2-20)

S O2
S
K S  S K O2  S O2

(2-21)

Where
= 10 ( pH opt  pH )  1
= pH constant
= Constant
= DO in the mixed liquor
= Saturated constant for oxygen
P(DOopt)
Observed specific growth rate
P( DO) , time-1

Observed specific growth rate

time-1

PpHopt)

10

pH

DO concentration, mg/L

Figure 2.22 pH and DO models

37

2.6

Respirometric Method

The respirometry measurement technique is used to measure the biochemical oxygen

uptake rate (OUR) under well-defined experimental conditions. The respirometers are based
on measuring the rate at which biomass takes up dissolved oxygen from the liquid phase
(Vanrolleghem et al., 1999). Assessment of wastewater components is often referred to as
wastewater characterization. The procedures for characterization involve a combination of
physic-chemical and biodegradation tests. Using this method, the biodegradable components
in the wastewater can be quantified (Vanrolleghem et al., 1999).

2.6.1

Respirometer

In principle, the respirometer consists of an oxygen electrode, DO meter, recorder,

respirometric reactor and water jacket vessel to maintain a constant temperature. It is placed
on a magnetic mixer in order to obtain a complete mixing of the reactor volume. A ceiling of
the respirometric cell is oblique, so that the air bubbles can easily escape from the cell. The
expansion funnel is used for adding the substrate solution and for escaping air bubbles during
periods of aeration. A cross-sectional area of the funnel stalk is small enough to minimize
oxygen absorption during the measurement. (Fig.2.23).

Figure 2.23 Schematic diagram of respirometer

1. Respirometric cell
2. Water jacket
3. DO probe
4. Air diffuser
5. Magnetic bar
6. Expansion funnel
7. DO meter
8. Recorder

2.6.2

Experimental Procedure

An expected concentration of endogenous activated sludge is transferred into the respirometry

and aerated to increase the dissolved oxygen concentration to 6-8 mg/L. When these
concentrations are reached, the aeration is stopped. A slow decrease in oxygen concentration
is due to heterotrophic endogenous respiration. A typical respirogram is shown in Fig. 2.24,
and can be interpreted as follows (Cech et al., 1984):

38

Adding substrate

A
DO, mg/L

C
OC

OURx,e
OURx,t

Endogenous phase

Time, minute

Figure 2.24 Recorder chart with a typical respirogram (Cech et al., 1984)
During the endogenous phase of respiration, heterotrophic microorganisms utilize
oxygen at a constant rate over a relatively long period of time, as demonstrated by the line AB-C. At time B, a small volume of concentrated substrate solution is injected into the cell by
means of a hypodermic syringe. Addition of a limited amount of substrate to the respirometry
reactor causes a temporary increasing respiration rate, as shown by the line B-D. This line is a
maximum-value tangent to the curve B-E. It represents the constant total respiration rate at the
substrate concentration S added. When the substrate concentration decreases with time, the
respiration rate also decreases. When the substrate has been removed (at point E) the
respiration rate returns to a value (line D-E), which is equal to, or perhaps slightly different
from, the original endogenous rate.
When the measurement of one concentration is finished, a new dose of substrate can be
injected into the cell and a next respirogram is recorded (Cech et al.,1984). In order to
evaluate a respirogram, the endogenous respiration rate (OURx,e), the total respiration rate
(OURt) and net oxygen consumption (OC) are calculated. The line section CE is equal to net
oxygen consumption (Fig. 2.25). If the OC value is higher than 4 mg/L O2, the determination
of OC is conducted using Ekama et al. (1986) method (Fig. 2.25). The high OC value occurs
when a high substrate concentration is introduced. This method is normally used for
determination of COD fraction (i.e. biodegradable COD/total COD).
In this test a preselected volume of wastewater of known total COD is mixed with a
preselected volume of mixed liquor of known MLVSS concentration in a batch reactor. After
mixing, the OUR is measured approximately every 5 to 10 minutes until OUR attains to a
constant value that is approximate or equal to OUR in the endogenous phase (Ekama et al.
1986). The respirogram is obtained by plotting the curve of OUR versus time (Fig. 2.25).

39

D
OUR, mg/L.h

A
f

B
C

Time, minute

Figure 2.25 OUR response in respirometer (Ekama, et al., 1986)

Where

Area A:

This area gives the concentration of Readily Biodegradable COD

(RBCOD) oxidized by the biomass. This is useful for assessing the
amount of volatile fatty acids (VFA) that needs to be added in a
biological phosphorus removal plant.

Area B:

This area represents the amount of less readily biodegradable material

being oxidized.

Area C:

This area shows the amount of oxygen being used to convert ammonia
into oxidized nitrate (nitrification).

Area D:

The area under the whole curve shows the total oxygen demand of the
liquor. This is the total amount of oxygen which must be supplied to the
sludge to achieve full treatment.

OUR at line e: The respiration rate at the end of the curve, when at least 95% of the
organic waste has been treated, is the endogenous respiration rate. This
rate is proportional to the activity of the biomass.
OUR at line f: This rate is termed the Average Viability, and it is the average
respiration rate for the period where nitrification and the breakdown of
less readily biodegradable substrates are occurring.
OUR at line g: This is the maximum respiration rate observed at the start-up of the
respiration cycles. At this point all oxidative reactions take place,
including the oxidation of carbon and nitrogen compounds and the
uptake of phosphates.
Time T:

2.6.3

The time for the sample to reach an endogenous respiration rate. This is
a direct method to determine the minimum HRT required to achieve at
least 95% treatment efficiency.

Determination of Kinetic Constants

Specific OUR of substrate oxidation at a substrate concentration S (OURx,ox) is given

by:
40

OUR X ,ox

OUR X ,t  OUR X ,e

(2-22)

Where:

OURx,t =
OURx,e =

Total respiration rate (mg O2/mg VSS.h)

Endogenous respiration rate (mg O2/mg VSS .h)

Further specific substrate removal rate at a substrate concentration S (RX) is given by:
RX

Where
=
RX
OC =
=
S

OURX ,ox
OC / S

(2-23)

Substrate removal rate (mg COD removed/mg VSS.h)

Net oxygen consumption (mg O2/L)
Substrate concentration (mg COD/L)

OC is then equal to the area between the OUR curve and the second plateau level where
the OUR decreases rapidly and levels off (OC = Area A+area B) (Figure 2.25)
Biomass yield coefficient (Y) is expressed as:
1 OC
Y
1 

f
S
and the specific growth rate (P ) as:
P Y.R X
Where
= Specific growth rate (h-1)
P
= COD/VSS ratio of the sludge (mg COD/mg VSS)
f
= Yield coefficient (mg VSS/mg COD removed)
Y
2.7

(2-24)
(2-25)

Membrane Bioreactor (MBR)

MBR is the combination of two basic processes: (1) biological degradation and (2)
membrane seperation into a single process where suspended solids and microorganisms
responsible for biodegradation are seperated from the treated water by a membrane filtration
unit (Manem and Sanderson, 1996).
MBR systems have two principal configurations: (1) The submerged MBR (or
integrated MBR) in which outer-skinned membrane is submerged inside the bioreator and
permeate is extracted by suction or by pressuring the bioreactor; (2) The external circuit MBR
(or recirculated MBR) in which the mixed liquor is recirculated at high pressure through a
membrane module placed outside the bioreactor (Fig. 2.26). The permeate is extracted by
high cross-flow velocity through the membrane and the concentrated mixed liquor at the feed
side is returned to the bioreactor. Excess sludge is withdrawn in order to maintain constant
sludge age and the membrane is regularly cleaned by air or water backwash and chemical
cleaning (Visvanathan et al. 2000).

41

Recirculation
Inffluent

Bioreactor
Inffluent

Air
Inffluent

Membrane filtration

Bioreactor

Air
Membrane module
Air diffuser

Effluent

a. Recirculated MBR

b. Surbmerge MBR process

Figure 2.26 Diagram of membrane bioreactor processes

2.7.1

Advantage of the MBR Process

The main advantages of MBR process can be listed as follows:

High quality of treated water: Biological treatment using the MBR process can obtain
extreme high removal efficiency of SS, COD, BOD and pathogen concentration.
Therefore the treated water can be discharged directly into the surface water, or reused
for cooling, toilet flushing and lawn watering.

Flexible in the operation: SRT is independent on HRT and can be controlled completely.
Long SRT can be maintained to allow the development of slow-growing microorganisms,
such as nitrifying bacteria.

Compact plant size:Due to the high biomass concentration that can be maintained in
MBR, a high volumetric loading rate can be applied which results in the reduced size of
bioreactor. In addition, secondary settling tank, sludge thickener or post treatment for
further BOD and SS removal are not necessary in the MBR process, and thus the plant
becomes more compact.

Independence of settling ability: the selection of microorganisms present in the

membrane bioreactor is no longer dependent on either their ability to form biological
flocs or the settling characteristics (Manem and Sanderson, 1996).

Low sludge production: Maintaining a low F/M ratio results in minimum sludge waste.

High degradation rate: High tangential velocities limit floc size and lead to an increase in
mass transfer rates of microorganisms.

2.7.2

Main Design Parameters

In order to have an optimal MBR process from the economic point of view, many
parameters should be considered. These can involve membrane selection, membrane
42

performance (permeate flux, transmembrane pressure, viscosity) and biological performance

(MLSS, SRT, HRT, F/M ratio) and economic considerations (energy consumption, sludge
treatment and disposal cost). These parameters can influence each other, and are mutually
dependent. For example, high MLSS can be maintained by controlling long SRT and thereby
increasing the volumetric loading (reducing HRT) and reducing the investment costs.
However, high MLSS requires high maintenance energy consumption due to increases in
viscosity that results in flux decline, high oxygen demand for aerobic organic degradation and
cell growth (Manem and Sanderson, 1996; Visvanathan et al. 2000).
Table 2.19 presents a comparison between biological performances of MBR and a
conventional AS process. This table shows that the mixed-liquor volatile suspended solid
concentrations (MLVSS) in aerobic MBR process are much higher than those in conventional
AS processes, reaching concentrations of 30 g/L. Based on particle size distribution tests,
Nazim et al. (1999) indicated the AS sludge contained large flocs, while the MBR sludge
contained small flocs. The high mass loads applied to aerobic MBR can be explained by high
biomass concentrations and high specific substrate removal rate (Manem and Sanderson,
1996). HRTs of 2-4 hrs and F/M ratio of 0.1 g COD/g MLSS.d are normally applied to
domestic wastewater treatment (Table 2.19). A combination of carbonaceous BOD removal
and nitrification can achieved high efficiency in MBRs. Muller et al. (1995) reported using
MBR process for domestic wastewater treatment could obtain 90% carbon removal and 100%
nitrification efficiency at COD loading rates of 0.9-2.0 kg/m3.d. (HRT of 2.0 h - 7.5 h). Thus
slow-growing nitrifying bacteria are retained by the membrane in the reactor. The aerobic
MBRs are capable of high-strength industrial wastewater treatment. High organic loading
rates (1.7- 8.6 kg COD/m3.d) can also applied.

43

Source

VLR, kg BOD/m3.d
kg COD/m3.d
HRT
F/M ratio, g/g.d
Sludge concentration, g/L
SRT
COD removal, %
DO
Y

Type of membrane

Parameters

Yamamoto
et al. (1989)

1.35
1.5
4h
0.1
14
50-100
>95%
Buisson et
al. (1998)

1.2
0.1
11
50
96
0.5-1.5
0.2
Trouve et al.
(1994)

0.47
24 h
0.2
2.5-3.0
25
96
>3
0.2

44

Fuchs and
Scholz
(2000)

97
-

99
6.3
Krauth
and Staab
(1993)

8.6
13 h
0.6-0.8
15-25

5.4
0.5
11

Scott and
Smith, 1977

97
-

12.8
6.2
5.8 d
16

Combined
domestic
Domestic
Domestic
Vegetable
Oily
wastewater+
Ice cream
wastewater wastewater
canning wastewater
industrial
wastewater
MF
MF
Tubular UF
MF 0.1Pm MF 0.1Pm MF 0.1Pm

Table 2.19 Comparison between biological performances of MBR process and conventional AS process

Lu et al.
(1999)

92-98
2.0-3.5

0.8-1.7
2.7 d
0.4
10-15

UF

Metcalf and
Eddy (1991)

0.8-1.9
6-8 h
0.2-0.6
2.5-4.0
5-10
99
t 2.0

Completed
mixing

Fermentation conventional
wastewater
AS

2.7.3

Membrane Fouling

Membrane fouling or flux decline which leads to high-energy consumption and a large
cleaning chemical requirement is a major problem hindering the widespread application of
biomembrane reactor process. Membrane clogging in the MBR process might be the result of
(a) the biofilm growth, or adsorption or deposition of foulants on the top surface of the
membrane (external fouling) and (b) at the pore entrances or within the internal pore structure
of the membrane (internal fouling). Fig. 2.27 schematises the fouling mechanisms.
Adsorption is used here to mean an interaction between foulants and membrane. The
adsorption arises from physical forces which involve electrostatic forces (surface electric
charges), Van Der Waals forces (attractive forces in close proximity), solvation forces
(hydrogen bonds) and steric forces (attachment of polymers on the surfaces).
macromolecule

particle

Concentrate

Fluid flow

membrane

pore

Permeate

Figure 2.27 Diagram of fouling mechanisms (adsorption and deposition)

The phenomenon of fouling is very complex and depends on physical chemical
parameters such as concentration, pH, ion strength and surface properties of particles and
membrane such as electrical charges, hydrophilicity or hydrophobicity. For example, a more
hydrophilic membrane can decrease the ability of adsorption and fouling rate.
Macromolecules (proteins, EPS) which are normally hydrophobic adsorb easily on
hydrophobic membranes. The adsorption layer is also more difficult to wash out from a
hydrophobic surface than from a hydrophilic one. Surface charges can also have an effect on
fouling. If there is electrostatic repulsion between the membrane surface and bioflocs or
macromolecules, fouling is decreased. Stability of macromolecules is influenced by the pH of
mix-liquor. Macromolecules are more compact at their isoelectric point, pI, where
intramolecular electrostatics repulsion is minimum. Thus rapid flux decline occurs at this pH
value due to an increase in the amount of deposited macromolecules.
a.

Foulants

Three main types of foulant can be differentiated (Mulder, 1996):

x

Organic precipitates (biological substances, macromolecules, etc.): Macromolecules can

be protein molecules in wastewater, extracellular polymers (ECP) or long chain organic
by-products generated from biodegradation process.

Inorganic precipitates (metal hydroxides, calcium salts, etc.): Changes in environmental

conditions (pH, solute or ion strength) due to microorganism actions in MBR can form
precipitates. Gelatinous precipitates (such as hydrated complex of calcium phosphate and
citrate) can seriously foul membranes (Howell and Nystrom, 1993).
45

Particulates (cells, debris, microbial flocs, etc.): Particulates in the mixed liquor build-up
the solid cake on the surface of membrane, which results in a decline in flux.

b. Biofouling

Biofouling can be defined as adsorption/adhesion and growth of microorganisms which

forms biofilm on the membrane surfaces. Adhesion can be due to bonding interactions
between the membrane surface and adhesive structures such as flangella, fimbria, or
macromolecules (proteins, extracellular polymers) on the cell surface. Once attached, cells
may grow and multiply by using substrates and nutrients from the bulk solution (Fig. 2.28).
Harry et al.(1996) postulated flux decline can be significantly attributed to extracellular
polymers (EPS) rather than to the colloidal nature of bacterial cells.
cell # 1
cell # 1

EPS

primary
adhesion

surface EPS charges

secondary
adhesion

Growth

Growth

membrane

Figure 2.28 Schematic illustration of membrane biofouling process (Ridgway and Flemming,
1996).
Extracellular polymers (EPS)

The production of EPS is a general property of micro-organisms in natural

environments, and occurs in bacteria, algae, yeast and fungi (Flemming and Wingender,
2001). EPS are major component of activated sludge (AS) matrix and biofilms. They play an
important role in bioflocculation, settling and dewatering of AS process and in biofilms
development in attached-growth process. They consist largely of proteins, polysaccharides
and humid substances. EPS act as a bridge between cell surface and therefore initiate floc or
biofilm formation (Bura et al. 1998). However, the presence of high EPS concentration may
result in poor settling (bulking phenomenon) or dewatering condition (i.e. increasing sludge
volume index SVI, or capillary suction time CST) in conventional AS process. In addition,
high EPS concentration can increase the specific hydraulic resistance (R) of the filtration cake
in MBR process (Manem and Sanderson, 1996). Fig. 2.29 presents the schematic diagram of
the matrix of biofloc or biofilm.

46

Divalent
cation

PO43-

COO-

bacteria cell
EPS

Figure 2.29 Schematic diagram of biofloc or biofilm

Various experimental studies have demonstrated the important role of macromolecules
in fouling and flux decline. Hodgson et al.(1993) investigated the role of the bacterial EPS in
cake resistance of MF system (0.2 Pm membranes at 100 kPa in batch filtration cells). The
gram-negative marine bacterium SW8 was used in this study. The role of the EPS in resistance
was confirmed by changes in flux through treated and untreated bacterial cakes. The treated
bacterial suspensions here means bacterial cells whose EPS were removed by proteolytic
enzyme and chelating agent (EDTA). Whereas the untreated cake consists of particular cells
with the void spaces filled with EPS proteins and polysaccharide The authors found that the
untreated cake had more higher resistance than the treated cakes. This confirmed that the
major cause of resistance was not the bacterial particles themselves, but the EPS associated
with those bacteria.
Likewise, Nagaoka et al. (1996) carried out a study on the influence of bacterial EPS on the
membrane separation AS process. Loop-type hollow fiber membrane modules with pore size
of 0.1 Pm were used. The experiments were intermittently operated with a cycle of 10minutes-run and 5-minutes-off. Feed wastewater was a mixture of acetic acid (as carbon
source) and necessary nutrients. The results indicate that EPS which was accumulated in the
aerations and also on the membrane caused an increase of viscosity of the mixed liquor and an
increase in the filtration resistance. There was a linear relationship between the filtration
resistance and viscosity of the mixed liquor, which is caused by rapid attachment of the
suspended EPS.
Mukai et al.(2000) estimated flux decline of ultrafiltration membrane in different
cultural growth phases, different EPS and metabolic product concentrations in AS process.
The authors reported that flux decline was effected by protein to sugar ratio of EPS and
metabolic products. Lower permeate flux occurred with higher retention of protein and greater
amounts of retained protein during filtration.
The other influences

Autolysis occurring at high SRT or starving conditions (low F/M ratio) can lead to
increase in concentrations of cell debris and soluble microbial by-products (such as humid
substances and proteins). Absorption of these substances on the membrane surface can boost
biofouling (mainly internal fouling). In addition, high hydraulic stresses may also enhance
cell and floc breakage that releases metabolites and cell debris.
47

The effects of MLSS, soluble COD and viscosity on membrane fouling was estimated
by Sato and Ishiis model as follows (Manem and Sanderson, 1996):
(2-26)
R 842.7 * 'P * ( MLSS ) 0.926 * (COD )1.368 * ( P ) 0.326
Where
= Filtration resistance, m-1
R
= Transmembrane pressure, Pa
'P
= Viscosity, Pa.s
P
MLSS = mixed liquor suspended solid, mg/L
COD = Soluble chemical oxygen demand, mg/L
= Total resistance for filtration, m-1
Rt
It can be seen that soluble COD concentration can contribute significantly to increase in
filtration resistance (Eq. 2.26).

48

Chapter 3
3
Methodology

This research comprises four main studies: (1) biokinetic study; (2) parametric study
(optimization of operating conditions); (3) biomembrane study, and (4) sludge
characterization study. Flowchart of the different phases of the experimental studies is shown
in Fig. 3.1.
Aclimatized yeast and
bacterial sludge

DO

Glucose
(1) Biokinetic
experiments

SRT

pH

Protein extract
(2) Parametric study
(Optimization of
operating conditions)

(4) Sludge
characteristics
(3) MBR study

High influent COD

5,000 mg/L

Low influent COD

1,000 mg/L

VLR

SRT

(4) Sludge
characteristics

Figure 3.1 Flowchart of different phases of experimental study

3.1

Biokinetic Study

The objective of this study was to evaluate the biokinetic coefficients of mixed yeast
and mixed bacterial treatment of high salinity wastewater by means of respirometric
techniques. Two feed synthetic wastewaters were used, namely: glucose-feed wastewater and
protein-feed wastewater. In the protein-feed wastewater, commercial tuna fish protein extract
(T.C. Union Agrotech Co., Thailand) was mixed with tap water to obtain wastewater whose
composition was similar to that of tuna fish processing wastewater. The composition of two
feed wastewaters is presented in Tables 3.1 and 3.2. The flowchart of biokinetic study is
shown in Fig. 3.2.

49

Table 3.1 Composition of glucose-feed wastewater (Defrance, 1993)

Concentration (*) (mg/L)

Component
Glucose

4,673 (**)
1,870
94
235
467
0.5
1.0
1.0
1.0
0.2
0.2
0.2

(NH4)2SO4
Yeast extract
KH2PO4
MgSO4.7H2O
ZnSO4.7H2O
CaCl2
MnCl2
FeCl2
(NH4)6Mo24.4H2O
CuSO4
CoCl2
(*)

(**)

Composition of synthetic wastewater used for bacterial system was similar to that for yeast culture, but the
concentration of each component was five times lower than that of the yeast culture.
Corresponding to COD concentration of 5,000 mg/L and BOD20 concentration of 4,440 mg/L
(Biodegradability of glucose is 0.95 g BOD20/g glucose (Henze et al., 1997).

Table 3.2 Composition of protein-feed wastewater

Parameters
COD
BOD5
Organic-nitrogen
Ammonia-nitrogen
Total phosphorous

Concentration (mg/L)
5,000
3,850
690
26
45
Seed yeast sludge

Seed bacterial sludge

Enrichment

Acclimation of
high salinity

- Glucose as substrate
- up to 45 g/L NaCl

Acclimation of
protein extract

Biokinetic
experiments

Glucose

Protein extract

50

Figure 3.2 Flowchart of biokinetic experiments

3.1.1
a.

Seed Sludge

Yeast sludge and enrichment

The term mixed yeast sludge implies the mixture of all wild yeasts which exist in the
raw wastewater and then quantitatively propagate under proper enrichment conditions. The
procedure of enrichment for yeasts was carried out according to the Standard Methods for the
examination of water and wastewater (APHA et al., 1995). The yeast strains were selected by
the enrichment culture technique based on free competition among different organisms in
wastewater (Nishihara Ltd., 2001). Figure 3.3 shows the procedure of the enrichment process.
The osmotolerant yeast sludge was enriched from the bottom sediments of an
equalization tank of a fish sauce factory located in Rayong province, Thailand. This tank
received wastewater containing high salt and organic content (30.2 g/L NaCl and 800 mg/L
COD). The enrichment was conducted using two-liter container and the fill-and-draw
operation. In the first batch, the raw sediment was added to two containers containing two
liters of feed wastewater with 20 g/L and 32 g/L NaCl. MLSS concentration of mixed liquors
obtained were around 1,000 mg/L. The feed wastewater (glucose as substrate) was mixed
using a diffused aeration system, and pH was adjusted to 3.5 in order to optimize yeast growth
and limit the bacterial contamination (Pelczar and Reid, 1972; Elmaleh, et al., 1996). After
eight hours of aeration, the biomass suspension was allowed to settle for 12 hours. Yeast cells,
normally, settle to the bottom; acid-tolerant bacteria and filamentous fungi remain in
suspension. The bacteria and fungi are removed by decanting supernatant. 1.5 liters of
supernatant was decanted and a fresh medium was added for next batch. When yeast biomass
(MLSS) exceeded 3,000 mg/L, the enrichment process was stopped.
Seed yeast sludge
(sediments)

Feed-glucosewastewater

Filling

Drawing

Aeration
(32 h)

Settling
(10 h)

no
MLSS>3000 mg/L
yes
Completion

Figure 3.3 Schematic diagram of enrichment procedure

51

b. Bacterial sludge

The bacterial seed sludge was obtained from the activated sludge process of the same
fish sauce processing wastewater treatment plant. This plant treats combined wastewater with
salt content of approximate to 1g/L NaCl and mean COD concentration of 540 mg/L.
3.1.2

Acclimation

Acclimation was carried out to obtain mixed bacterial and yeast sludges that can
tolerate salt contents (32 and 45 g /L NaCl). The two-litre-batch-reactors with fill-and-draw
operation were used in the acclimatization stage. Table 3.3 shows the operating conditions. In
order to obtain operating conditions similar to saline seafood processing wastewater treatment
using yeast treatment followed by mixed bacterial system, the initial COD concentrations of
5,000 mg/L for yeast and 1000 mg/L for mixed bacterial culture were selected.
Table 3.3 Operating conditions for high salinity acclimation
Operating conditions
Initial COD (mg/L)
pH
Temperature ( oC)
MLSS (mg/L)
HRT (h)

Yeast sludge
5,000
3.5
25 32
5,000
36

Bacterial sludge
1,000
7.5
25 32
4,000
24

After aeration (24 h for bacterial sludge and 32 h for yeast sludge), the biomass suspension
was allowed to settle for 12 hours, and the supernatant was sampled and centrifuged at 4,000
rpm for 15 min. COD of the supernatant was analyzed. When the COD removal efficiency
was less than 80%, the experiment was repeated at the same operating conditions. When the
COD removal exceeded 80%, the NaCl concentration was increased by 3 g/L. Acclimation to
high salt contents (32 and 45 g NaCl/L) was assumed to be completed when 80% COD
removal was attained.
3.1.3

Biokinetic Experiments

The kinetic coefficients of the acclimated yeast and bacterial cultures at different salt
contents with two substrates (glucose and protein extract) were assessed using a closed 0.9
liter batch respirometer, equipped with a recorder, DO meter and water jacket vessel to
maintain a constant temperature as shown in Figure 3.4. Table 3.4 presents the operating
conditions of the respirometric experiments for yeast and bacterial culture. Initially, glucose
was used as a substrate complemented with necessary nutrients. The So/Xo ratio (initial
substrate concentration/biomass concentration) governs the quality of the batch respirometric
tests (Cech et al., 1984). In order to obtain So/Xo ratio ranging from 0.01 to 0.20 (Mathieu and
Etienne, 2000), the Xo and So values should be ranged from 1,500 to 2,000 mg VSS/L and 20500 mg COD/L, respectively.

52

2
3
1

5
9
6

1. Respirometric cell
4. DO probe
7. Expansion funnel

2. Water jacket
5. Magnetic bar
8. DO meter

3. Air diffuser
6. Magnetic mixer
9. Recorder

Figure 3.4 Respirometer set-up

Table 3.4 Operating conditions for the respirometric experiments
Operating conditions
Initial pH
Temperature (oC)
Xo (mg MLSS/L)
Substrate (mg COD/L)
So/Xo ratio
Suppressing nitrification

Yeast sludge
3.5
30 r 0.5
1,500
20 500
0.01 0.35
None

Bacterial culture
7.5
30 r 0.5
1,500 (1)
20 200

0.01 0.15 (2)

Adding 70 mg N-ammonia/L (3)

Sources:
(1)

Cech et al., (1984)

Chudoba. et al., (1992)
(3)
Liebeskind, (1999)
(2)

The experimental procedure for OUR determination are summarized below:

1)

Obtaining endogenous sludge: The respirometer was filled with fresh sludge without
substrate and aerated at least for 2 hours.

2)

Suppressing nitrification: NH4Cl was used with the concentration of 70 mg Nammonia/L. Liebeskind (1999) postulated that if ammonia was present in wastewater,
organic oxidation and nitrification simultaneously occurred. At high enough ammonia
concentration (70 mg/L N), OUR of nitrification is constant during organic oxidation.
When this ammonia dose is added to the endogenous sludge, nitrification OUR will be
determined. Thus, OUR of organic oxidation will be the difference between total OUR
and the sum of endogenous OUR and nitrification OUR.

3)

Recording endogenous OUR: After suppressing the nitrification process, the mixture
was aerated at least half an hour before measuring endogenous OUR.

53

4)

Adding substrate: An accurate amount of substrate was added to the respirometer and
total OUR was recorded by respirogram. New reaeration was necessary when the
dissolved oxygen concentration dropped below 2 mg/L.

The results of the respirometric experiments provided OUR data that are used for
calculating specific growth rates (P) using Equations 2.22 to 2.25 in Chapter II. By using
OUR values and specific growth rates (P) with respect to corresponding substrate
concentrations (S), maximum oxygen utilization rate (OURmax), observed specific growth rate
(Pobs) and half velocity constant (Ks) at the selected salt content were determined by
regression analysis based on Monod kinetics.
By using observed maximum specific growth rates (Pobs) at the corresponding salt
contents (I), the critical salt content above which reaction stops (KI) and maximum specific
growth rate of free-salt solution (d-1) were evaluated by linear regression analysis based on the
Ghose and Tyagi model (2-9). The regression analysis was done by Grapher sofware.
3.2
3.2.1

Parametric Study
pH values

The effects of pH values on bacterial and yeast treatment systems were also evaluated in
terms of OUR by respirometer. The protein-feed wastewater with 32 g salt/L was used. The
experiments were done at constant temperature of 30r0.5 oC. The operating conditions are
presented in Table 3.5.
Table 3.5 Operating conditions for the pH effect experiments
Operating Condition
COD
MLSS
Temperature
Salt
pH values:
+ For mixed yeast sludge
+ For mixed bacterial sludge

Units
mg/L
mg/L
o
C
g/L NaCl

Values
50
2,000
30 r 0.5
32
2.5 9.0
4.0 10.5

The main procedure of the experiment can be described as follows:

x

Obtaining endogenous sludge: The respirometer was filled with fresh sludge without
substrate and then aerated for at least 2 hours.

Suppressing nitrification (for mixed bacterial sludge): NH4Cl was used with a
concentration of 70 mg N-ammonia/L

Adjusting pH value: H2SO4 solution 0.1N or NaOH 0.1N solution was added into the
mixture until the desired pH value.

Recording endogenous OUR: After 10 minutes of aeration, the endogenous OUR was
recorded.

Adding substrate: Stock solution of fish extract (25,000 mg/L) had been prepared and
adjusted to the desire pH value before adding into the mixture. An accurate amount of
54

substrate (300 mg/L COD) was injected into the respirometer and then total OUR was
recorded. New reaeration was necessary when the DO dropped below 2 mg/L.
3.2.2

Sludge Retention Time (SRT)

Each of the SRT variation experiments was conducted in a two-liter batch reactor using
the fill-and-draw operation. Here, the MLSS variation was monitored for a minimum period
of 3 weeks. The steady state condition was reached when the MLSS values remain constant
for at least 5 days. The experimental operating conditions are presented in Table 3.6. To avoid
the effects of protein precipitation (at low pH) on the nitrogen uptake ability of yeast sludge,
removal of protein precipitates prior to feeding is necessary. Raw wastewater was acidified to
pH 3.5 and left to settle at least for 12 hours at 40C.
Table 3.6 Operating conditions of the experiments on SRT effect
Operating conditions
COD
pH
MLSS
HRT
Salt
SRT

Unit
mg/L

Value
5,000
3.5-4.0
7,000
24
0.5, 15, 32 and 42
5, 7, 10, 20 and 45

mg/L
h
g/L
d

Sludge retention time is defined as the average time for which a unit of biomass remains
in the system. For a completely mixed process with a sludge return arrangement, SRT can be
expressed mathematically as follows:

Tc

Vr X
Q w X  (Q  Q w ) X e

(3-1)

Where
Tc
Qw
Q
Vr
Xe
X

=
=
=
=
=
=

Sludge retention time, d

Sludge waste rate, L/d
Influent flow rate, L/d
Volume of aeration tank, L
Volatile suspended solids in effluent
Mixed liquor volatile suspended solids in the aeration tank, mg/L

To facilitate operation of lab-scale experiment, it is assumed that solids lost in the effluent can
be neglected (Xe = 0). Equation 3-1 can be rewritten as:

Tc

Vr
Qw
Vr

Qw

Tc

(3-2)

Equation 3-2 provides the calculation for sludge volume to be wasted daily from the
reactors. After 24 hours of aeration, waste sludge was withdrawn. Due to poor settling ability
of mixed yeast sludge, settling step in a typical batch process was replaced with
55

centrifugation. The remaining of suspended biomass was centrifuged at 3,000 rpm for 15 min.
The centrifuged sludge was returned to the reactor for the next batch.
3.3

Biomembrane Study

The biomembrane study was conducted with the fish-protein-feed wastewater at 32 g/L
NaCl. This study consisted of two phases, namely: (1) high COD loading, and (2) low COD
loading. The difference between the two phases is shown in Table 3.7.
Table 3.7 Difference between the high COD loading and low COD loading
Parameter

High COD loading

Low COD loading

Influent COD (mg/L)

Experimental
set-ups

5,000

1,000
Feed wastewater
(1,000 mg/L COD)

Yeast
reactor

SRT

3.3.1

YMBR

YMBR
Feed wastewater
(5,000 mg/L COD)

50 d for both YMBR and BMBR;

15 d for yeast reactor

BMBR

BMBR

50 d and 10 d were
investigated for both YMBR
and BMBR

High COD loading

The fish-protein-feed wastewater used in this phase was similar to that used for
biokinetic study (Table 3.2). Both yeast and bacterial sludges were excess sludges obtained
from the biokinetic study. Two parallel experimental set-ups were conducted, namely: (1)
yeast pretreatment followed by the Bacterial Membrane Reactor (BMBR) as schematized in
Figure 3.5, and (2) the Yeast Membrane Reactor (YMBR).
Both YMBR and BMBR tanks were made of transparent acrylic tube of 10 cm in
diameter with working volumes of 8 L and 3.6 L respectively. In order to provide enough
effluent volume to BMBR at different HRTs, the working volume of the yeast reactor (YR)
was made adjustable by changing outlet points installed along the height of the column. The
total volume of the yeast reactor was 21 L. These reactors were continuously aerated through
the stone diffusers placed at the bottom, and equipped to monitor pH and DO. For the YMBR
system, through an external pH dosing pump, the reactor pH values were maintained at the
required level. In each reactor, a polyethylene 0.1 Pm hollow fiber membrane module with a
surface area of 0.42 m2 was fixed on the upper end.
Speed-controlled roller pumps were used to withdraw the permeate from these
membrane modules. Both bioreactors were operated with periodic air backwashing (20
minutes filtration and 2 minutes of air backwashing at a pressure of 400 kPa arrangement. The
alternative operations of filtration and air injection was controlled by an intermittent
controller and solenoid valves. The transmembrane pressure was measured using a mercury
manometer.
For the BMBR system the feed wastewater was pretreated with the yeast reactor with
continuous aeration through diffusers with a mean HRT of 32 h. The pH was maintained at
3.5. The treated effluent from this reactor was continuously withdrawn and sent to the
sedimentation unit where the yeast sludge is separated then returned to the yeast reactor. The
hydraulic retention time of the settling tank was 5 h, which corresponds to the shortest HRT
of BMBR (4.5 h). From the yeast reactor, excess sludge was periodically withdrawn to
56

maintain a mean sludge retention time (SRT) of 15 days and mean biomass concentration of
4,500 mg/L MLSS. The settled effluent was stored in an intermittent storage effluent tank
and fed into the BMBR reactor system. In the BMBR system, the pH was maintained at above
7.0, whereas in the YMR, the feed water did not undergo this pre-treatment, but was fed
directly into the YMBR tank where the pH was maintained at 3.5.
The investigations were carried out by step-wise increase in volumetric loading. The
different loading steps used is summarized in Table 3.8. Each of volumetric loading rate
(VLR) variation was maintained for at least 7 days. The mean influent COD concentration fed
into the BMBR was the effluent COD from the yeast reactor.
Table 3.8 Experimental operating conditions of YMBR and BMBR systems
YMBR
Stage
I
II
III
IV
V
VI
VII
(*)

Time
days

1-22
22-29
30-40
41-51
52-62
63-78

VLR
kg
COD/m3.d

5.0
3.4
6.6
9.9
16.3
23.0

BMBR

mg/L

Mean
HRT
h

5,000
5,000
5,000
5,000
5,000
5,000

24.0
36.0
18.2
12.2
7.1
5.1

Mean influent
COD

Effluent from the yeast reactor

57

Time
days

111
11-21
22-31
32-41
42-51
52-75
77-90

VLR
kg
COD/m3.d

2.1
3.4
7.9
5.3
1.7
2.4
3.6

Mean
influent
COD(*)

mg/L

Mean
HRT
h

1,200
1,280
1,450
1,080
1,170
1,140
1.450

13.7
9.1
4.5
4.9
16.1
11.7
3.6

Compressed air

Bacterial
Membrane reactor

BMR effluent tank

Hg-manometer

Bacterial excess sludge

Yeast excess sludge

Effluent tank 1

58

Level water
tank

Air backwash

Timer

Sulphuric acid sol.

(pH 3.5)

Yeast effluent tank

Hg-manometer

YEAST MEMBRANE REACTOR

Yeast excess sludge

Compressed air

to feed tank

Figure 3.5 Membrane reactor systems in the high COD loading

BACTERIAL MEMBRANE REACTOR SYSTEM

Yeast excess sludge

Compressed air

Feed tank

Sulphuric acid sol.

(pH 3.5-3.8)

YEAST REACTOR

NaOH sol.
(to pH 6.8-7.5)

Level water
tank

Overflow

Outlet point

Air backwash

Timer

Overflow

3.3.2

Low COD loading

This phase was a sequence of the high COD loading. Therefore, yeast and bacterial
sludges were acclimated. Both YMBR and BMBR had the same working volume and influent
wastewater with COD of 1,000 mg/L. The experimental set-up is schematized in Fig. 3.6.
Both YMBR and BMBR tanks were made of transparent acrylic tube of 15 cm diameter with
working volume of 10 L. Likewise, in the high COD loading, the reactor pH values were
maintained to 3.5 in the YMBR set-up. In each reactor a 0.1 Pm hollow fiber membrane
module with a surface area of 0.42 m2 was fixed on the upper end. The air-backwash
operation of both set-ups in this phase was similar to that of the high COD loading.
The composition of the low COD wastewater is presented in Table 3.9. The experiments
were conducted using a step-wise increase in the flux rate (i.e. increase in the volumetric
loading) at sludge retention times of 10 and 50 d. During the transition period (between SRT
of 10d and 50 d), no sludge was wasted from the reactors except for sampling. The operating
conditions for this phase is shown in Table 3.10.
Table 3.9 Composition of the low COD wastewater
Parameters
COD
BOD5
Organic-nitrogen
Ammonia-nitrogen
Total phosphorous
Salt content

Unit
mg/L
mg/L
mg/L
mg/L
mg/L
g/L NaCl

Concentration
1,000
780
120
25
12
32
Timer

Air backwash
Hg manometer

Feed tank
Bioreactor
Level water
tank
Membrane
Effluent tank
(H2SO4 solution only for YMR)

Compressed air
Excess sludge

Figure 3.6 Schematic diagram of membrane bioreactor

59

Table 3.10 Effects of different HRTs and SRTs on yeast and bacterial membrane reactors

SRT
days

10
10
10
10
Transition
Transition
50
50
3.4

YMBR
VLR

BMBR
VLR

kg COD/m3.d

Mean HRT
h

SRT
days

kg COD/m3.d

Mean HRT
h

2.66
2.95
3.66
4.59
3.58
4.28
4.93
6.55

8.8
7.7
6.1
5.0
7.2
6.1
5.3
4.0

10
10
10
Transition
Transition
50
50

2.97
3.57
4.30
3.63
4.08
5.56
6.35

8.1
6.3
5.2
7.1
6.0
4.7
4.0

Sludge Characterization Study

To investigate variation of sludge characteristics with salt contents and simultaneously

obtain a comparison between membrane bioreactor and batch systems, yeast and bacterial
batch reactors were operated at different salt contents (0.5, 15, 32 and 45 g/L). Two-literbatch reactors with fill-and-draw operation were used. The initial mixed yeast and bacterial
sludges were withdrawn from the YMBR and BMBR run at SRT of 10 d and 32 g salt/L.
These sludges were later acclimatized at salt contents by gradual increases or decreases. Table
3.11 presents the experimental operating conditions. The sludge was sampled when each
batch reached the steady state condition (i.e. COD removal was above 80% with a stable
MLSS value). The sludge was examined for ECP content, dewatering property (CST) and
sludge settleability (SVI). In addition, N and P contents of both yeast an bacterial sludge were
evaluated.
Table 3.11 Operating conditions for the sludge characterization study
Operating conditions
Initial COD (mg/L)
pH:
- Mixed yeast sludge
- Mixed bacterial sludge
Initial MLSS (mg/L)
SRT (d)
HRT (h)
Salt (g/L)
3.5

Value
1,000
3.5-4.0
7.0-7.5
7,000
10
8
0.5, 15, 32 and 42

Analytical Methods

All analyses were conducted according to Standard Methods (APHA et al., 1995). COD
was analyzed by the potassium dichromate close reflux method with correction for chloride
interference.
The extraction of extracellular polymers (ECP) is based on the thermal extraction and
ethanol precipitation method (Brown and Lester, 1980). The sludge was separated by
centrifugation (2,000 g for 15 minutes), then washed and resuspended in distilled water. A
portion was taken for measurement of suspended solids and the remaining part was heated at
60

800C for 1h. The extracted polymers were collected by removing the sludge by centrifugation
(9,500 g for 15 minutes). The extracellular polymers in supernatant were precipitated by
adding two volumes of solvent mixture (1:1 acetone and ethyl alcohol) to one volume of
supernatant. It was then left overnight at 4oC. The ECP content was measured by means of
suspended solid analysis. Viscosity of the mixed liquor was measured using a rotating torque
cylinder. Table 3.12 listed parameters and their analytical methods used in this study.
Table 3.12 Parameters and their analytical method
Parameter

Analytical method

Analytical
equipment

Interference
of salt

Treatment

Source

pH

pH meter

pH meter

None

APHA et al., 1995

DO

DO meter

DO meter

None

APHA et al., 1995

COD

Dichromate Reflux

Titration

Yes

Ammonia

Distillation

UV-vis
Spectro.

None

APHA et al., 1995

Nitrite

Colorimetric

UV-vis
Spectro.

None

APHA et al., 1995

Nitrate

Cadmium reduction

UV-vis
Spectro.

None

APHA et al., 1995

TKN

Macro-Kjeldahl

Titration

Yes

Phosphate

Ascorbic acid

UV-vis
Spectro.

None

APHA et al., 1995

TS

Dried at 103-105oC

Oven

None

APHA et al., 1995

Adding HgSO4
according to the
10:1 ratio of HgSO4:
Cl.

Adding conc. H2SO4

APHA et al., 1995

APHA et al., 1995

VS

Ignited at 550 C

Furnace

None

APHA et al., 1995

CST

Capillary time

CST
apparatus

None

APHA et al., 1995

SVI

Settled sludge
volume after 30
minutes

1000mL
cylinder

None

APHA et al., 1995

Viscosity

Rotating torque
cylinder

None

EPS

Thermal and
centrifugation
method

None

Brown and Lester, 1980

SS

Dryed at 103-105oC

None

APHA et al., 1995

Filter/Oven

61

Chapter 4
4
Results and Discussions

4.1

Biokinetic Study

Respirometric experiments were used in this study to evaluate biokinetic coefficients in

yeast and mixed bacterial treatment of saline wastewaters containing 20, 32 and 45 g/L NaCl.
Two carbon sources were investigated, namely (1) Glucose-feed wastewater (glucose as
carbon source) and (2) Fish-protein-feed wastewater (fish protein extract as carbon and
nitrogen sources).
4.1.1

Enrichment and Acclimation of Yeast and Mixed Bacterial Sludge

Prior to the biokinetic study, enrichment and acclimation were carried out to obtain a
mixed yeast sludge and mixed bacterial sludge able to tolerate high salt contents (32 and 45 g
NaCl/L). In order to propagate all wild yeasts present in the raw sediments taken from a fish
sauce factory, the enrichment technique was applied prior to the acclimation. pH was adjusted
to 3.5 in order to limit bacterial contamination. The enrichment was completed when the yeast
concentration reached to 3000 mg/L. The enrichment and acclimation were conducted with
two-litre batch reactors using the fill-and-draw operationes.
a.

Yeast Enrichment with Glucose-feed Wastewater

During enrichment and acclimation of the yeast culture, it was found that the color of
the sludge changed from black to brown and finally to white. A change in color is a typical
indication of the change in the proportion of different species in any microbiological culture.
Further, microscopic observations revealed that the yeast sludge contained predominantly
spherical yeast cells with few egg-shaped cells and few hyphal filaments. However round
cells budding multilaterally, bipolarly and unipolarly could be easily recognized in the culture
(Fig. 4.1).
Cultural characteristics were estimated by differences of yeast colonies in terms of
shape, texture and margin. To evaluate cultural characteristics, isolation was done for three
yeast mixtures. Isolation was carried out on the yeast-glucose-peptone agar (APHA et al.,
1995). The results of isolation for yeast mixtures at different salt contents show that there
were two predominant colony types. The majority of colonies were round shape, smooth
surface, opaque in color and round edge. The second colony had irregular shape, rough
surface and curled edges (Appendix A).
20 Pm
x 500

10 Pm

x 1500

Figure 4.1 Appearance of yeast cells predominantly grown in glucose-feed wastewater

62

b. Acclimation of Mixed Yeast and bacterial sludges to High Salt

The acclimation of mixed yeast and bacterial sludges to high salt was conducted with
the glucose-feed and fish-protein wastewaters. In order to obtain feed wastewater composition
similar to saline seafood processing wastewater using yeast treatment followed by mixed
bacterial system, the initial COD of 5,000 mg/L for yeast and 1000 mg/L for mixed bacterial
culture were used. When COD removal reached more than 80% (after 24 hours of aeration),
the NaCl concentration was increased by 3 g/L.

Biomass
The time required for yeast acclimation was about 16 days for an initial F/M ratio of
1.12 g COD/g MLSS.day compared to 26 days for bacterial culture for an initial F/M ratio of
0.5 g COD/g MLSS.day, at 45 g salt/L. Acclimation was assumed to be complete when COD
removal exceeded 80 %. Fig. 4.2 shows variation in COD removal with time at 45 g salt /L
for mixed yeast culture. Similar trend curves were obtained for the mixed bacterial culture
(Fig. 4.3). The asymptotic nature of the curves indicates that COD removal efficiency was
stable after a certain time, which marks the completion of acclimation.

MLS S, mg /L

16000
12000
8000
4000
60

50
80

40

70
MLSS

60

Salt concentration (g/ L NaCl)

COD removal ( %)

90

COD%

30

Salt concentration

50
0

10

20

30

40

50

Time (days)

Figure 4.2 Acclimation of yeast sludge cultured with glucose at high salt contents
During the acclimation, the biomass increased 4.5 times that of the initial biomass
concentration for yeast, as compared to 1.7 times for bacterial culture. Yeast biomass
concentration increased 10,700 mg/L after 40 days, whereas bacterial sludge concentration
increased from 2,040 to 3,400 mg/L. Differences in biomass concentration can be attributed to
the application of higher volumetric loading for yeast sludge (3.3 kg COD/m3.d for yeast and
1.0 kg COD/m3.d for bacterial sludge). Therefore, a large quantity of substrate consumed was
converted to yeast biomass. Even at a higher volumetric loading, the time required for yeast
acclimation was about 60 % that of the bacterial sludge showing a far better adaptability of
yeast at high salt. A better acclimation influences the start-up time of a wastewater treatment
63

plant, and also indicates the tolerance of the culture to occasional salt variation in the glucosefeed wastewater.

MLS S, mg /L

3600
3200
2800
2400
2000

COD removal ( %)

90

40

80
30
70
20
MLSS

60

Salt concentration (g/ L NaCl)

50

COD%
Salt concentration

10

50
0

10

20

30

40

Time (days)

Figure 4.3

Acclimation of microbial mixed culture with glucose-feed wastewater as

function of salt

Organic removals
In order to estimate organic removal rates, the COD profiles of acclimatized yeast and
bacteria batches were examined. The COD profile is defined as the quantity of COD varies
with aeration time in a culture batch. The typical COD profiles of a mixed yeast batch at 32 g
salt /L is shown in Fig 4.4. All COD profile data of yeast and bacterial batches are presented
in Appendix B. Table 4.1 summarizes operating conditions and COD removal of yeast and
bacterial sludges acclimatized to glucose-feed wastewater at high salt contents. Optimum
HRT referred to aeration time at which COD removal exceeded 90%.
Table 4.1

Performance of mixed yeast and bacterial batches adapted to glucose-feed

wastewater with high salt

Parameters
Mean MLSS
Optimum HRT
F/M
Effluent COD
%COD
COD removal rate

Unit
mg/L
h
g/g.d
mg/L
%
g /g.d

Yeast batch
Salt content
20 g/L
32 g/L
8700
9500
5
9
2.77
1.39
220
255
95.6
94.9
2.65
1.32

64

45 g/L
9750
13
0.96
290
94.2
0.90

Bacterial batch
Salt content
20 g/L
32 g/L
45 g/L
3050
3650
3200
2.5
8
17
3.27
0.84
0.44
20
30
70
98
97
93
3.20
0.81
0.41

100

5000
COD%

80

3000

60

2000

40

1000

COD removal ( %)

COD (mg/L)

COD

4000

20

0
0

8 HRTopt 10

12

14

Time (days)

Figure 4.4

Typical COD and COD removal profile of mixed yeast batch in glucose-feed
wastewater at 32 g salt/L

The COD removal efficiency for acclimated yeast and bacterial cultures at 25, 32 and
45 g salt/L was studied with respect to substrate utilization rate U as shown in Figure 4.5.
Since the F/M ratio for the two cultures differs significantly, the effect of the variation has to
be taken into account for determining COD removal efficiency. COD removal efficiency is
therefore expressed by Equation (4-1) (Metcalf and Eddy, 1991).

F /M *E
* 24
HRTopt . X

(4-1)

Where
U
F/M

=
=

Substrate utilization rate (g COD removed/g MLSS.day)

Food:microorganism ratio (g COD applied/g MLSS.day)
CODinf
* 24
F/M
HRTopt . X

CODinf
HRTopt
X
E

=
=
=
=

Initial COD concentration (mg/L)

Optimum hydraulic retention time (h)
Biomass concentration (mg/L MLSS)
COD removal efficiency (%)

It could be observed that, while increasing the salt content, U is significantly decreased
for both cultures due to salt inhibition. However, the rate of decrease is found to be much
higher for bacterial culture than for yeast, indicating that the bacterial culture is much more
sensitive to changes in high salt content. When salt content increased from 20 to 45 g/L, U
decreased from 3.26 to 0.40 g COD/g MLSS.d for the bacterial culture, while U decreased
from 2.65 to 0.88 g COD/g MLSS.d for the yeast culture.

65

4.0
COD removal rate ( g COD/ g MLSS.d)

Mixed bacterial sludge

Mixed yeast sludge

3.0

6.890 u e  0.0485 X

Y
2.0

R = 0.978

1.0

25.48 u e 0.104 X

R2 = 0.989

0.0
20

30

40

50

NaCl (g/L)

Figure 4.5

Variation in COD removal rate versus salt contents in acclimatized yeast and
bacterial mixed cultures

It can be concluded that wastewater containing high salt is better for yeast culture, it
may be better to opt for yeast culture while at low salt bacterial culture is preferred. The
intersection point was found to be at 25 g salt/L, which indicates that below this value
bacterial culture may be a better solution and vice versa.
As indicated in Section 4.1.1.b, the yeast culture is subjected to a higher F/M ratio than
the mixed bacterial culture in order to obtain a comparable substrate removal efficiency. This
can be considered a specific advantage of the mixed yeast culture over bacterial culture.
Normally, for aerobic treatment systems, higher F/M ratio vis--vis higher organic loading
puts greater stress on the system. This generally results in lower efficiency of substrate
removal and oxygen utilization. Therefore, in practice, aerobic systems are not subject to
volumetric loading exceeding 1.2 kg COD/m3.d. The normal range is 0.3 to 0.8 kg COD/m3.d,
which avoids low efficiency and higher O2 requirement (because of low utilization
efficiency). Thus, by allowing a higher F/M ratio on the yeast culture without sacrificing
efficiency, the problem of higher organic loading on an aerobic treatment system is to some
degree addressed.
However, higher organic loading allows downsizing the treatment reactors, which
improves the overall economy of the system. In this study, the mixed bacterial culture was
loaded at 1.0 kg COD/m3.d (corresponding to a F/M ratio of 0.50 g COD/g MLSS.d) as
compared to 5.0 kg COD/m3.d (F/M ratio of 1.12 g COD/g MLSS.d) for yeast culture. This
range of loadings (for yeast) is generally acceptable for anaerobic treatment systems having a
low efficiencies normally in the range of 60 - 70% at best. Moreover, it has also been found
(Feijoo et al., 1995) that anaerobic microorganisms are highly sensitive to salt and total
inhibition could be noted for some treatment systems at a salt exceeding 20 g/L, whereas a
COD removal efficiency of more than 80 % (for 45 g/L) and 90 % (for 20 g/L) can be easily
obtained for yeast culture at HRT of 24 h. Thus, the yeast system is more useful than an
anaerobic system at high salt, and can be considered a better substitute for anaerobic systems
in terms of COD removal efficiency.

66

Protein-feed wastewater
Prior to the estimation of biokinetic constants for protein-feed wastewater, acclimation
of yeast and bacteria sludges to this new substrate was necessary. Both yeast and bacterial
sludges were the ones that had been adapted previously to glucose-feed wastewater at high
salt contents (20, 32 and 45 g/L). This acclimation lasted two weeks. The COD removal of
yeast batches at high salt contents was higher than 80% after 12 days of acclimation (Fig.
4.6). Whereas, COD removal of bacterial batches was higher than 90% after a few days. Thus,
the mixed bacterial sludge was able to acclimatize faster to the substrate with high protein
content. Unlike composition of glucose feed wastewater, the protein-feed wastewater contains
a large complex of organics such as proteins, colloidals and polysaccharides. These substance
can be slowly degraded by most yeast strains grown predominantly in the previous glucose
culture (Defrance, 1993). During acclimation to protein-feed wastewater, yeast strains could
be inhibited due to limitation of glucose, while other strains which are able to degrade the
complex organics grow rapidly and become predominant after acclimation. In general,
complex organics must be hydrolyzed by enzymes (hydrolases) before yeasts can degrade
them. The type of hydrolases produced by yeasts are dependent on the species (Pelczar and
Reid, 1972). This reveals that utilization of mixed yeast culture, based on a symbiotic process,
for treatment of wastewater having complex composition is more efficient than pure yeast
culture.

MLS S, mg /L

10000
8000
6000
4000
2000

COD removal ( %)

90

80

70
Yeast sludge

60

Bacterial sludge

50
0

10

12

14

16

18

Time (days)

Figure 4.6 Acclimation of yeast and bacterial sludges to fish-protein-feed wastewater

containing 32 g/L salt
Sludge characteristics
The change in color of the sludge and microscopic observations strengthen the fact that
acclimation of yeast and bacterial culture is completed. The yeast sludge that was fed with
glucose wastewater was milky white and became dark brown to black often acclimation to
protein wastewater. Microscopic observation detailed the changes by predominant yeast
67

strains when substrate was changed. The yeast sludge fed with glucose mainly contained
spherical yeast cells with multilateral or bipolar budding, whereas mycelia (hypha filament)
and large size egg-shaped cells with monopolar budding were predominant in the yeast
mixture fed with protein wastewater (Fig. 4.7).

20 Pm
x 500

a. Predominance of egg-shaped cells with monopolar budding in suspension

20 Pm

x 800

b. Predominance of mycelial yeast (hypha filament) in settled sludge

Figure 4.7

Predominance of wild yeast strains in the cultures fed with fish-protein

wastewater (at 32 g/L salt)

Similarly, the bacterial sludge changed in color and settling properties. Its color changed
from light brown or orange to brown or dark brown when glucose-feed wastewater was
altered with protein-feed wastewater. Its flocs were larger and easily trapped fine particles
during settling. Therefore, supernatants of bacterial batches fed with protein wastewater were
clearer than those from glucose wastewater after 30 minutes of settling. SS of supernatant and
SVI in the batch fed with glucose wastewater (at 32g/L salt) were 275 mg/L and 16 mL/g,
respectively, while SS and SVI in the protein-feed wastewater were 230 mg/L and 81 mL/g.
The result revealed that there is a large difference in SVI but not in supernatant SS. Thus, the
bacterial sludge fed with protein wastewater may have poor thickening or dewatering ability;
details are discussed in Section 4.4. These can be explained by proteins in the wastewater
enhancing formation of extracellular polymers (ECP) which have a great influence on
bacterial floc structure, settling and dewatering ability (Dignac et al. 1998).

68

Organic removals
All COD profile data of yeast and bacterial cultures fed with protein wastewater were
presented in Appendix B. Table 4.2 summarizes operating conditions and COD removal
efficiency of yeast and bacteria acclimatized to protein-feed wastewater at high salt contents.
Here, optimum HRT refers to aeration time at which COD removal efficiency obtained was
higher 80%.
Table 4.2 Performance of mixed yeast and bacterial sludges adapted to protein-feed
wastewater with high salt contents (Initial COD cof 5,000 mg/L).

Parameters

Unit

Mean MLSS
HRT
F/M
CODeff
%COD
COD removal rate (U):
+ Protein ww
+ Glucose ww

mg/L
h
g/g.d
mg/L
%
g /g.d

Yeast
Salt content
20 g/L 32 g/L 45 g/L
6050
5810
6430
31
34
37
0.64
0.61
0.51
790
830
950
84.2
83.4
81
0.54
2.65

0.51
1.32

0.41
0.90

Bacteria
Salt content
20 g/L 32 g/L 45 g/L
4300
3720
4120
9
21
28
0.64
0.31
0.21
40
50
90
96
95
91
0.61
3.20

0.29
0.81

0.19
0.41

The COD removal rate of both acclimatized yeast and bacterial sludges were
significantly reduced when glucose-feed wastewater was added protein-feed wastewater as
shown in Table 4.2. However, the COD removal rates of mixed yeast sludge at higher salt (32
and 45 g/L NaCl) were still higher than those of bacterial sludge when protein wastewater was
used. The difference in U of yeast sludge with salt increases was relatively minor. In addition,
in order to obtain equivalent COD removal efficiency, lower F/M ratios were required at
higher salt contents for both yeast and bacterial sludges. However, F/M reduction for the
mixed yeast sludge was small. This enhanced the advantages of the yeast system for seafood
processing wastewater having high organic strength and high salinity.
4.1.2

Evaluation and Comparison of Biokinetic Coefficients

Specific growth rates (P) were obtained through OUR measurement by respirometric
method. The P values of different initial CODs (20 to 500 mg/L) at 20, 32 and 45 g salt/L
were determined. Observed maximum specific growth rate (Pobs) and the half-velocity
constant (KS) were determined from regression analysis.
Figures 4.8 and 4.9 show typical OUR curves of yeast and bacterial sludges fed with
glucose and protein wastewater at 32 g salt/L. OUR curves for a specific COD concentration
can represent maximum oxygen uptake rate of microorganisms for a given substrate, knowing
its biodegradability. For example, Fig. 4.8 indicates that for glucose-feed wastewater, OURs
of the mixed yeast sludge was higher than that of the bacterial sludge. Thus, yeast culture was
able to degrade glucose more efficiently at 50 mg/L COD and high salinity (32 g/L salt),
while the degradation ability of the mixed bacterial culture fed with protein-feed wastewater
was better at COD lower than 100 mg/L. Discussion is presented in Section 4.3.2.a (Fig.
4.32). The fraction of readily biodegradable matter is represented by the area A in these
figures. By comparison between the area A of the two substrates, it is noted that glucose-feed
wastewater mainly contains readily biodegradable matter, whereas the fraction of readily
biodegradable matter was lower for protein-feed wastewater.
69

50

OUR ( mg O2/ mg VSS.h)

40

A
30

bacterial sludge

20

yeast sludge

A
10

0
0

10

15

20

25

30

Time ( minutes)

Figure 4.8

OUR curves of mixed yeast and bacterial sludges feed with 50 mg/L COD and
32 g/L salt (glucose-feed wastewater)
30

OUR ( mg O2/ mg VSS.h)

25

20

A
15
bacterial sludge

10

yeast sludge

B
B

0
0

20

40

60

80

100

120

Time (days)

Figure 4.9

OUR curves of mixed yeast and bacterial sludges feed with 100 mg/L COD and
32 g/L salt (protein-feed wastewater)

A given microorganism will survive in the system if it is able to reproduce at a faster

rate than the rate at which it was removed from the system. The mechanisms to remove the
microorganism can involve predation or wash-out in the effluent. Therefore the growth rate is
important in the biological treatment process. It is normally used to estimate the effects of
toxic substances, inhibitors or overloads on performance of the process. Figures 4.10 and 4.11
show the variation in specific growth rate with COD concentration (glucose-feed wastewater)
at different salt contents, for yeast and bacterial cultures, respectively. It can be seen that the
specific growth rate progresses in according to the Monods model to reach a maximum
value, then this value decreases as the salt content increases. Table 4.3 summarizes values of
Pobs, KS, and Y of yeast and bacterial sludges at different salt contents for glucose and proteinfeed wastewater.

70

5.0
20 g NaCl/L

Specific Growth Rate Pday-1

32 g NaCl/L

4.0

45 g NaCl/L

3.0

2.0

1.0

0.0
0

100

200

300

400

500

COD (mg/L)

32 g/L NaCl:

20 g/L NaCl:

S
P 5.60
158  S
R2 = 0.971

45 g/L NaCl:

S
P 4.74
118  S
R2 = 0.975

S
129  S
R2 = 0.967
2.70

Figure 4.10 Variation in specific growth rate of yeast sludge as function of COD
concentration at different salt contents for glucose-feed wastewater
20 g NaCl/L

Specific Growth Rate Pday-1

8.0

32 g NaCl/L
45 g NaCl/L

6.0

4.0

2.0

0.0
0

40

80

120

160

200

COD (mg/L)

20 g/L NaCl:
S
P 9.95
45  S
R2 = 0.965

32 g/L NaCl:
S
P 2.80
55  S
R2 = 0.974

45 g/L NaCl:
S
53  S
R2 = 0.950

P 1.15

Figure 4.11 Variation in specific growth rate of bacterial culture as function of COD
concentration at different salt contents for glucose-feed wastewater
For both substrates, the specific growth rate of mixed yeast culture is higher than the
bacterial one at high salt contents, while the opposite is observed for lower salt contents. This
observation is in line with the nature of the COD removal rate (Fig. 4.5). It was also found
that irrespective of the salt content, the yield constant Y of mixed yeast batch fed with glucose
is lower than that of the bacterial culture, whereas there was no considerable difference
between the Y constants of yeast and bacterial systems fed with protein at any salt content.
This may be due to change in the predominant species or changes in the carbon assimilation
metabolism as substrates change. The yield constants for both yeasts and bacteria grown on
71

protein-feed wastewater were slightly lower than for glucose-feed wastewater. The yield
constant estimated by the respirometric method is considered as the maximum yield
coefficent (Ymax) under certain environmental conditions such as temperature and type of
wastewater. In a biological treatment system, the observed yield constant Yobs may vary from
0 and upto Ymax. The observed yield constant Yobs values depend on design of the system such
as F/M ratio and sludge age (Henze et al., 1997). Therefore, unlike specific growth rate, Y
constant should not be used to evaluate the effects of inhibitors or to compare performance of
two systems with different operating conditions.
Table 4.3 Biokinetic coefficients of the yeast and bacterial sludges at different salt contents
for glucose and protein-feed wastewaters

Pobs

S
g /L
NaCl

Substrate

day-1
Yeasts Bacteria

Y
g VSS/g COD
Yeasts Bacteria

KS
mg COD/L
Yeasts Bacteria

Glucose feed wastewater

20
32
45

5.60
4.74
2.70

9.95
2.80
1.15

0.46
0.48
0.41

0.57
0.58
0.53

158
118
130

45
55
53

4.69
3.66
2.46

5.65
1.95
1.11

0.43
0.44
0.40

0.40
0.47
0.40

201
329
396

54
93
96

Protein feed wastewater

15
32
45
Candida ingens in culture
using VFAs (Anciaux et
al., 1989)
C.utilis in culture using
acetic acid
Jackson and Edward, 1975

<1.0

Domestic wastewater
(Henze et al., 1997)

6.8-7.2

0.56

N/A

7.2-9.6

0.38

N/A

N/A

4-8

N/A

0.35-0.50

N/A

5-30

Ks values of bacterial cultures (both substrates) at high salt contents were found to be
higher than Ks of normal activated sludge. This indicates that heterotrophic aerobic
microorganisms living at high salinity show lower affinity for the substrate than those at low
salinity, probably because of reduced functioning and multiplication. Whereas Ks values of
yeasts were found to be 3-6 times higher than those of bacteria. Thus maximum growth rate of
yeast culture is only obtained at high organic substrate concentration.
Figures 4.12 and 4.13 compare maximum specific growth rate of mixed yeast and
bacterial cultures with glucose and protein-feed wastewaters at different salt contents. At
higher salt contents, the bacterial growth is severely inhibited, while the growth rate of yeast
mixture is sustained. The inhibition effect of high salt contents on yeasts and bacteria based
on the Ghose and Tyagi model is also shown. Salt inhibition constants (KI) have been
calculated from the linear relationship. It was 70 g/L (80g/L) for yeast and 46 g/L (51g/L) for
the bacterial culture with glucose (protein). This indicates that the inhibitory salt content is
much lower for bacterial culture compared to the yeast which is in line with the previous
observations. However, actual critical salt limits may be higher than the values derived, found
from the Ghose and Tyagi model. This can be recognized from studies concerning
osmotolerant microorganisms by Choi and Park (1999). They reported that growth of Pichia
guilliermondii on Kimchi brine waste is still sustained at up to 120 g NaCl/L. Similarly,
Dincher and Kargi (2000) also reported that Halobacter sp. in activated sludge culture could
72

continue removing COD even at 50 g salt/L. Therefore, the limits obtained in this study may
only be used to compare the relative performance.
16.0

Observed Specific Growth Rate

Pday-1 )

P 15.9 * 1 
12.0

Bacterial sludge

46

Yeast sludge

R = 0.959
8.0

P
4.0

8.1* 1 
70
2

R = 0.870

0.0
0

10

20

30

40

50

60

70

80

Salt (g/L NaCl)

Figure 4.12 Inhibition effect of salt contents on mixed yeast and bacterial cultures on
glucose-feed wastewater
16.0
Bacterial sludge

Observed Specific Growth Rate

Pday-1)

Yeast sludge

12.0

P
8.0

7.65 * 1 
49
2

R = 0.928

5.86 * 1 
80

4.0

R = 0.985

0.0
0

10

20

30

40

50

60

70

80

Salt (g/L NaCl)

Figure 4.13 Inhibition effect of salt contents on mixed yeast and bacterial cultures on
protein-feed wastewater
4.2

Parametric Study

This study focused on several operating parameters such as pH, DO, SRT and nitrogen
content for the acclimatized mixed yeast and bacterial cultures. In fact, the effect of pH on
bacterial and yeast systems were evaluated in terms of OUR by respirometric assays. The
optimum pH values for bacterial and yeast growth under high salinity conditions were
evaluated. Variation in DO and nutrient components in the yeast and bacterial batches was
monitored for the two feed wastewaters (glucose and fish-protein) with 32 g/L salt. The SRT
variation experiments were conducted in mixed yeast cultures using the fill-and-draw
operation. Based on nutrient and COD removals, a suitable SRT value for yeast treatment was
determined.

73

4.2.1
a.

DO and pH

DO

Figures 4.14 and 4.15 compare DO profiles of mixed yeast and bacterial cultures fed
with glucose and protein wastewaters at 32 g salt/L. These figures also show that there is a
link between DO of mixed liquor and remaining COD (COD profile). DO of the mixed-liquor
in the yeast batch fed with glucose was low (0.7 mg/L) in the first 5 hours. DO then sharply
increased to saturated value (6.3 mg/L), whereas low DO level of 0.7 mg/L in the batch fed
with protein was steady for longer duration (28 h of aeration). DO then was also sharply
raised to 5.6 mg/L. DO values were lower for both substrates during the first hours of
aeration. This can be attributed to the presence of high initial concentration of organics (5,000
mg/L COD) after filling. The oxygen uptake rate of yeasts exceeded oxygen diffusion rate
from aeration at the first hours. Based on COD profiles, DO increased to saturated value when
most of the organic matters in the system was completely degraded.
5000

COD-protein-yeast

4000

DO-protein-yeast

COD-glucose-yeast

6.0

3000

4.0

2000

DO (mg/ L)

COD (mg/L)

DO-glucose-yeast

2.0
1000

10

20
Time ( h)

30

40

0.0

Figure 4.14 DO and COD changes of yeast batch fed with glucose and protein wastewater at
32 g salt/L
Similarly, DO in mixed bacterial cultures for both glucose and protein wastewater was
low during the first hour of aeration. It then increased sharply to the saturated concentration of
6.3 mg/L. This suggests that COD loading (influent COD of 1000 mg/L) applied to bacterial
batches was not as high as for the yeast batch. Oxygen consumption rate of bacterial sludge
did not exceed oxygen supply rate through aeration.

74

1000
6.0

4.0

600
COD-protein-bacteria

400

DO (mg/ L)

COD (mg/L)

800

DO-protein-bacteria

2.0
COD-glucose-bacteria

200

DO-glucose-bacteria

10

15
Time ( h)

20

0.0
30

25

Figure 4.15 DO and COD changes of mixed bacterial batch fed with glucose and protein
wastewater at salt content of 32 g/L
b. pH

A comparison of pH profiles of mixed yeast and bacterial batches fed with glucose and
protein wastewater (at 32 g/L salt) is presented in Figures 4.16 and 4.17. In the yeast batch fed
with protein, pH increased from 3.5 to above 4.0 after 2 hours of aeration. In order to maintain
pH at 3.5, 0.1N H2SO4 was added after every two or three hours. Inversely, pH of yeast fed
with glucose decreased pH to 2.6 after 8 h of aeration. Then it slightly increased to 2.9. Based
on the COD profile of the yeast batch fed with glucose-feed wastewater (Fig. 4.14), pH
started to raise when carbon supply was limited (about 250 mg/L COD).

5.0

Adjust pH to 3.5

pH

4.0

3.0
yeast-protein
yeast-glucose

2.0

10

20

30

40

Time ( h)

Figure 4.16 pH changes of yeast culturefed with glucose and protein wastewater at 32 g
salt/L
Thus, there is a significant difference in pH variation between glucose and protein-feed
wastewaters. This may be attributed to difference in substrates involving carbon and nitrogen
sources. The main components of glucose wastewater are the glucose as carbon source and
inorganic nitrogen (ammonium sulphate) as nitrogen source, while organic acids or complex
organics such as lipids and polysaccharides may be predominant carbon sources in protein75

feed wastewater. The amount of organic nitrogen (such as protein, amino acids) is very high
in the protein-feed wastewater (690 mg /L organic-N; 26 mg/L ammonia-N).
The increase in pH for the protein wastewater has been previously observed by Lu
(1983) who used yeast mixture to treat vermicelli wastewater. pH increased from 4.0 to 8.5.
Arnold et al. (2000) examined silage wastewater treatment using Candida utilis and
filamentous yeast Galactomycetes geotrichum. The initial pH was 3.65, rising to 8.8 after
treatment. The authors postulated that the increase in pH was due to lactic acids removal,
VFAs or consumption of H+ during oxidation of organic N into ammonia. Lu (1983) also
suggested that the degradation of protein and release of ammonia caused increase of pH.
By contrasts, the decrease in pH for the yeast fed with glucose wastewater can be due to
the production of organic acids or the use of basic compounds such as ammonia by the cells
or the absorption by the medium of CO2 produced by yeasts (Thanh and Simard, 1973).
9.0

8.5

pH

8.0

7.5

7.0
Bacteria-glucose
Bacteria-protein

6.5

6.0

10

15

20

25

30

Time ( h)

Figure 4.17 pH changes of mixed bacterial batch fed with glucose and protein wastewaters at
32 g salt /L
Unlike yeast growth, variation in pH in the mixed bacterial batch was not much
dependent on type of substrates. Both wastewaters had same initial pH of 7.5 and after
treatment, pH of both batches were increased. The difference in pH variation between yeast
and bacteria culture on glucose substrate may be explained by the difference in pathways of
nitrogen assimilation under respiratory conditions with no contribution by carbon metabolism
(Vicente, et al. 1998). Yeasts liberate H+ ion during ammonia uptake. In general, bacteria
produce alkalinity during ammonification and consume alkalinity in nitrification. However at
high salinity, nitrification is inhibited. Nitrite and nitrate concentrations of treated wastewater
in bacterial batches were lower than 2.5 mg/L N at 32 and 45 g/L salt. Fig. 4.17 shows that pH
of the mixed bacteria fed with protein wastewater (8.5) was higher than that fed with glucose
(7.8). This might be due to the increase of alkalinity during conversion of protein to ammonia.
c.

Evaluation of optimum pH

Optimum pH of mixed yeast and bacterial cultures were evaluated in terms of OUR
using respirometric experiments. These were conducted with the protein-feed wastewater at
50 mg/L COD and 32 g salt /L. The bacterial and yeast sludges used in these experiments
were from the biomembrane reactors operated with the protein-feed wastewater at 32 g salt/L.
76

Figures 4.18 and 4.19 show suitable range of pH values for yeast and bacteria growth in high
salinity.
18

OURmax ( mgO 2/ gVSS.h)

16
14
12
10

Total OUR

Endogenous OUR

6
4
2
0
2.0

4.0

6.0

8.0

10.0

pH

Figure 4.18 Variation in OUR as funtion of initial pHs for mixed yeast fed with protein
wastewater at 32 g salt/L
25
Total OUR
Oxidation OUR

OURmax ( mgO 2/ gVSS.h)

20

15

10

0
4.0

5.0

6.0

7.0

8.0

9.0

10.0

11.0

pH

Figure 4.19 Variation in OUR as function of initial pHs for mixed bacterial fed with glucose
wastewater at 32 g salt/L
OUR obtained in the mixed yeast culture was highest at pH values of 5.0 - 5.5 and
declined slightly as pH increased to 8.0 or decreased to 3.0. The respiration rate of yeasts was
inhibited at pH 2.5 and above 9.0, whereas bacterial culture attained the highest OUR at pH
range of 7.5-9.0 where OUR declined slightly when pH was increased to 9.7, or decreased to
6.3. The respiration rate of bacteria was inhibited at pH below 5.3 or pH above 10.0. Thus, the
osmotolerant yeasts were able to tolerate a wider pH range than bacterial culture. Choi and
Park (1999) obtained similar results for Pichia guilliermondii, an osmotolerant yeast used to
treat kim chi waste brine with 80 g/L salt. Cell growth was not affected at pH ranging 4.0 to
8.0. By scanning electron micrographs, they showed that yeast cells only shrunk in size, but
did not rupture at the high osmotic shock pressure or high ion strength. This appears to be a
typical advantage of using yeasts to treat industrial effluents having large pH fluctuation.
However, at neutral pH (6.5 - 8.5), bacteria do multiply in significant numbers. Bacterial
growth should be inhibited by pH control in the yeast treatment process for the following
reasons:

77

a.

If the yeast biomass is used as animal feed, large numbers of bacteria or pathogens will
reduce the quality of the yeast biomass product;

b.

Excessive bacterial growth leads to operating problems, especially membrane clogging.

Previous studies have reported that bacterial contamination could be inhibited in pH

range of 3.5 - 3.8 (Peczar and Reid, 1972; Elmaleh, et al., 1966). The results of this study
shows that there was not a significant difference between OUR at 3.5 and OUR at the
optimum pH (5.0 - 5.5). The difference was 9%. Therefore, maintenance of pH 3.5 in the
reactor cannot reduce considerably COD removal rate.
4.2.2

Nitrogen Variation in Mixed Yeast and Bacterial Cultures

Figure 4.20 shows the variation in nitrogen components in the mixed yeast cultures fed
with glucose and protein wastewater. In the yeast fed with glucose wastewater, nitrogen
source was an inorganic salt (NH4)2SO4 with initial concentration of 365 mg N/L
(corresponding to COD:N = 100:7.2). Ammonia-nitrogen removal of 65% was obtained after
eight hours. Total nitrite and nitrate-N concentration of treated wastewater was very low
(around 2.0 mg N/L). This indicated that nitrification did not occur in the yeast reactor.
Likewise, total nitrite and nitrate concentration was not important in the yeast batch fed with
protein wastewater. The initial total nitrogen of the protein-feed wastewater was 790 mg N/L
mostly organic-N (745 mg/L organic-N). The total-N concentration was reduced to 446 mg/L
after 32 h, whereas ammonia-N increased from 45 mg/L to 420 mg/L. In comparison to the
yeast culture with glucose-feed wastewater, ammonia content and ammonia removal was
dependent to the availability of nitrogen sources (e.g. proteins, amino acids or ammonium
salt) in the feed wastewater and the BOD:N ratio. Due to the high BOD:N ratio (100:18) of
the protein-feed wastewater, the total nitrogen concentration of treated wastewater was still
high and mainly in the form of ammonia. If reuse of yeast biomass for single-cell-protein
production vis--vis further removal of nitrogen is considered, the combination with
carbohydrate-rich wastes (lack of nitrogen) such as molasses or pulp and paper wastewater
would be possible.
Moreover, the increase in ammonia may be related to the increase in the accumulated
acid volume used for adjusting pH to 3.5 as shown in Fig. 4.21. The link between H+ ion
consumption and ammonia release was discussed in Section 4.2. The amount of acid
consumed per gram ammonia-N released was about 26 meq H+.

78

800
Accumulated acid volume

NH4-N (protein ww)

NH4-N (glucose ww)

16

600
N (mg /L N)

14

12

400

10
200
8

Accumulated 0.1N H2SO4 volume (mL)

18
Total N (protein ww)

6
0

10

20

30

40

Time (hours)

Figure 4.20 Variation in nitrogen components as funtion of time in the mixed yeast at 32 g
salt/L NaCl (Nitrite and nitrate concentration of both feed wastewaters were not
dectected)

The total-N removal obtained in the batch fed with protein wastewater after 32 h was
45%. Because nitrification did not occur in the yeast treatment, all nitrogen removed was
uptaken in the yeast sludge. Based on biomass produced in the batches and the amount of
nitrogen removed, the nitrogen content of the yeast sludge was estimated to be about 7.5% of
dried solids for glucose wastewater, and 13.8% for protein wastewater. The high nitrogen
content of the yeast sludge fed with protein wastewater can be attributed to precipitation of
protein at low pH (3.5). This can be confirmed through the sudden decrease in total nitrogen
concentration during the first hour of aeration (Fig. 4.20). The nitrogen uptake ability of
yeasts will be further discussed in Section 4.4.
200

Nitrogen ( mg /L N)

160

120
Total-N (protein ww)
Ammonia (protein ww)

80

NO2+NO3 (protein ww)

Ammonia (glucose ww)

40

NO2+NO3 (glucose ww)

< 1.5 mg/L N

0
0

10

20

30

Time (hours)

Figure 4.21 Variation in nitrogen components vs. time in the mixed bacterial culture at 32 g
salt/L NaCl
In the bacterial culture fed with protein wastewater, 24% of total nitrogen was removed
at HRT of 21 hrs. Fig. 4.21 shows that the oxidation of organic-N (ammonification) results in
an increase from 49 to 137 mg N/L after 21 hrs, while in the culture fed with glucose
wastewater, the initial ammonia concentration of 53 mg N/L was reduced to 14 mg/L (about
72% N removal) after 9 hrs. Thus the variation of ammonia during the bacterial cultures was
similar to that of mixed yeast cultures. Nitrite+nitrate-N concentration was lower than 2.5
79

mg/L and 1.2 mg/L for the cultures fed with protein and glucose, respectively. Whereas nitrite
and nitrate-N (fed with protein) at 15 g salt/L was 12.3 mg/L after 21 hours (Appendix A).
Thus nitrification was inhibited at 32 g salt/L. Lower reduction at higher salt (32g/L) is
consistent with previous studies (Dincer and Kargi, 1999; Panswad and Anan, 1999). In fact,
Dincer and Kargi (1999) reported that nitrification efficiency dropped quite sharply at salt
content above 3%. Depending on nitrogen balance, nitrogen uptake in the mixed bacterial
sludge for both feed wastewaters was reached to 4.5 % of dry solids. This result will be
confirmed by nutrient analysis of sludge in the sludge characterization study.
4.2.3

Effect of SRT on COD and Nitrogen Removal

The optimum SRT was evaluated on the basis of COD and nitrogen removal. Five
mixed yeast batch experiments corresponding to SRT of 5, 7, 10, 20 and 45 days were
conducted at the same organic loading of 5 kg COD/m3.d (HRT of 24 h) for 25 days. The seed
sludge used was taken from the yeast membrane bioreactor (YMBR) operated with SRT of 50
days. As the degradation of protein and the release of ammonia caused an increase in pH
during the culture, pH was adjusted to 3.5 - 4.0 using 0.1 N H2SO4 during aeration. Figure
4.22 shows that all the runs reached steady state after 15 days.
The mixed yeast culture run at higher SRT reached higher biomass concentration. At the
VLR of 5.0 kg COD/m3.d, when SRT increased from 5 d to 45 d, the MLSS increased from
2,400 to 10,300 mg/L. Thus a long SRT implies low F/M ratio. This resulted in high organic
removal efficiency at long SRT. The results are shown in Table 4.4 and Fig 4.23. COD
removal increased from 43 to 85% when SRT increased from 5 d to 45 d. Furthermore, the
sludge production (yield constant) was also minimized at long SRT (Table 4.4). Therefore,
SRT has a mutual relationship with the net specific growth rate of the mixed yeast sludge. The
net specific growth rate (P) was the difference between the specific growth rate (P) and the
endogenous decay rate (kd), which includes endogenous respiration, death and subsequent
lysis. At long SRT, the system is operated in the endogenous phase. Thus kd will have a
significant effect on the net amount of biomass produced. This means that large fraction of the
substrate removal is oxidized for energy required for cell maintenance rather than for
synthesis of new cells.
12000

MLSS ( mg/L)

10000

8000

6000

4000

2000
0

10

15

20

25

Time (days)
45 d

10 d

20 d

7d

Figure 4.22 Variation in MLSS as funtion of SRT

80

5d

Table 4.4 Variation of parameters during various SRTs (Initial COD of 5000 mg/L)

SRT
(d)
5
7
10
20
45
(*)

CODeff
(mg/L)

COD
removal
(%)

TKNeff
(mg/L)

2850
1950
1050
900
950

524
515
509
535
550

N
removal(*)
(%)

43
61
79
82
85

7.3
8.9
9.8
5.2
2.7

Biomass
production
rate

Mean
MLSS
(mg/L)

(mg SS
produced/d)

3245
5351
8150
9455
10335

Yield
(g SS/g COD
removed)

657
765
810
475
228

0.305
0.251
0.205
0.116
0.053

Influent TKN concentration was 565 mg/L N.

The data are average values of at least three steady state batches.

Figure 4.23 indicates that if the highest COD removal was obtained at SRT of 45 d,
maximum nitrogen removal was achieved at SRT of 10 d. Uptake of nitrogen by the biomass
is a major nitrogen reduction mechanism in yeast culture as discussed in Section 4.2.2. Hence,
the nitrogen removal efficiency will depend on the biomass production rate. Table 4.4 shows
that the highest biomass production was obtained at SRT of 10 d. It can be suggested that
selection of optimum SRT should be based on the purpose of yeast application. SRT of 10 d is
optimum for single-cell-protein production, while longer SRT is suitable for enhancing
treatment efficiency.
100

14000

12000

10000
60
8000
COD removal

40

Nitrogen removal

MLSS ( mg/L)

Removal efficiency)

80

6000

MLSS

20

4000

2000
4

8 9 10

20

30

40

50

SRT (days)

Figure 4.23 Variation in COD, nitrogen removal and MLSS in funtion of SRT in mixed
yeast culture at VLR of 5 kg COD/m3.d (32 g salt/L)
4.3

Biomembrane Study

The objective of this study was to examine the potential for development of membrane
bioreactor systems using wild salt-tolerant yeast mixture and bacteria mixture to treat high
salinity wastewater (32 g/L NaCl). Based on COD of the feed-wastewater, this study was
divided into two phases, namely (1) high COD loading with 5000 mg COD/L and (2) low
COD loading with 1000 mg COD/L. The process efficiency was investigated in terms of
organic removal and membrane filtration flux for various volumetric loading rates, F/M ratio
and SRT values.

81

Two membrane modules having 0.1 Pm pore size and 0.42 m2 area were used for
YMBR and BMBR. When the pressure reached a value of 70 kPa, the membrane was
removed from the reactor, and chemical cleaning was conducted. During the chemical
cleaning, the external sludge cake layer was initially washed with water and the attached
biomass (on the membrane surface) was collected and analysed. After removal of the sludge
cake, the membrane was washed with tap water and backwashed with 2.5% sodium hydroxide
for 15 minutes followed by 1% nitric acid for 5 minutes before reuse.

P (kPa)

P (kPa)

After every chemical cleaning, the initial membrane resistance was measured, to verify
the cleaning efficiency. The initial Rm were determined by filtering the tap water through a
new or chemically cleaned membrane. Here, linear flux variation with applied pressure was
obtained. This variation for two fresh membrane modules is presented in Fig. 4.24. The
obtained initial Rm of two cleaned membrane modules were quasi equivalent (7.11x1011 and
7.18x1011 m-1).

2
1

2
1

Y = 0.172 * X - 0.1372

Y = 0.174* X - 0.1472

0
0

10

15

20

25

30

35

J (L/ m2.h)

10

15

20

25

30

35

J (L/ m2.h)

a. First membrane
b. Second membrane
Figure 4.24 Variation in flux as function of membrane transmembrane pressure (Viscosity of
water at 26oC = 8.70 x 10-4 kg/m.sec)
4.3.1

High COD loading

In this phase, fish-protein wastewater with 5,000 mg COD/L and 32 g salt/L was used.
Two experimental set-ups were investigated: (1) Yeast pretreatment followed by BMBR and
(2) YMBR. These were run at different HRTs at SRT of 50 d (as presented in Fig. 3.5).
a.

Organic removal

The performance of YMBM and BMBR systems for various volumetric loading are
shown in Figures 4.25 and 4.26. Here it can be note that after 11 days of acclimation (stage I),
yeast biomass increased from 3,700 to 14,500 mg/L at a volumetric loading of 5.0 kg
COD/m3.d (average HRT of 24 h). COD and BOD removal obtained were above 76% and
85%, respectively.
In contrast, the BMBR system which involved the yeast reactor (YR) and BMBR
reached the steady state after 11 days. The YR reached the mean biomass of 6,500 mg/L and
COD removal of 76% at SRT of 15 days and average HRT of 36 h. The bacterial biomass in
BMBR increased from 4,000 to 11,000 mg/L at a volumetric loading of 2.1 kg COD/m3.d
(HRT of 13.7 h). COD and BOD removal obtained was above 85% and 97%, respectively.
These results demonstrate the rapid adaptability of the mixed yeast and bacterial cultures to
degrade the high salinity-organic wastewater.

82

80

30

60

40

20
Mean HRT

20

Transmembrane Pressure

10
6000

Transmembrane ( kPa)

Mean HRT (h)

40

20000
Stage I

Stage II

Stage III

Stage IV

Stage V

Stage VI

16000

12000

8000

MLSS ( mg/L)

COD ( mg/L)

4000

2000
Effluent COD

4000

Influent COD
MLSS

0
0

10

20

30

40

50

60

70

80

Time (days)

Figure 4.25 Variation in COD, biomass and transmembrane pressure in the YMBR as
function of volumetric loading
In the YMBR, as the loading rate was progressively increased through different stages
(3.4 - 16.3 kg/m3.d), the COD removal efficiency decreased from 85 to 60%, with the COD in
the effluent increasing from 870 to 2,300 mg/L. For the BMBR process, when the VLR was
increased from 2.1 to 7.9 kg COD/m3.d (F/M of 0.08 - 0.41), COD removal efficiency
decreased from 91 to 76 % (Fig. 4.7). Effluent BOD5 of the BMBR ranged from 45 - 60 mg/L
at low F/M ratio. Thus, the low BOD5:COD ratio (0.12 - 0.17) indicates that BMBR effluent
also contains a high proportion of non-degradable organic compounds due to the presence of
these products from yeast pretreatment.
YMBR could attain a COD removal efficiency higher than 60% at VLR ranging from 5
- 15 kg COD/m3.d as shown in Figure 4.27. These VLRs are generally within the acceptable
range for anaerobic treatment systems, which normally have low efficiency in the range of 60
- 70%. Moreover, it has also been found (Feijoo et al. 1995) that anaerobic microorganisms
are highly sensitive to salt content and total inhibition could be noted for some treatment
systems at a salt content above 20 g/L. Thus the yeast system is more useful than an anaerobic
system at a high salt content, and can be considered a better substitute for anaerobic systems
in terms of COD removal efficiency.
In addition, YMBR attained a lower COD removal rate at F/M ratios lower than 0.34
g/g.d (the corresponding average VLRs less than 7 kg/m3.d), compared to BMBR as shown in
Fig.4.28. However, YMBR achieved higher specific COD removal rate at F/M ratios higher
than 0.34 g/g.d. Thus, it can be concluded that the YMBR is subjected to a higher F/M ratio
and higher VLR than the BMBR to obtain a comparable COD removal efficiency. This can be
considered as an advantage for the yeast sludge compared to bacterial sludge.

83

Mean HRT

80

Mean HRT (h)

Pressure

15
60
10

40

20

Transmembrane Pressure ( kPa)

20

1600
Stage II

Stage IV

Stage V

Stage VI

Stage III

COD ( mg/L)

30000

Stage VII

1200

20000

800

MLSS ( mg/L)

Stage I

Effluent COD

400

10000

Influent COD
MLSS

0
0

10

20

30

40

50

60

70

80

90

Time (days)

Figure 4.26 Variation in COD, biomass and transmembrane pressure in the BMBR as
function of volumetric loading
The mean biomass concentration of the yeast reactor was 6,500 mg/L after steady state.
77% of BOD5 removal was achieved at VLR of 2.6 - 3.1 kg BOD5/m3.d and F/M ratio of 0.39
- 0.47 d-1, while the Yeast Cycle System (YCS) for seafood processing wastewater treatment
(with 8 g salt/L) reached 97% BOD removal at higher VLR (4.5-10.4 kg BOD5/m3.d) and
higher F/M ratio (0.6 - 1.0 d-1) (Nishihara ESRC Ltd., 2001). This may be due to yeast ability
to grow in relative low salinity environment with higher specific degradation rate. Similar
results were found in the biokenetic study (Section 4.1). In comparison to the yeast reactor,
the YMBR that was run at higher biomass concentration (11,000 mg SS/L) enables the
enhancement of volumetric loading to 5.0 kg BOD5/m3.d with higher BOD removal efficiency
(81%).
Table 4.5 compares operating conditions between YMBR, BMBR and few yeast
treatments, MBR process treating different wastewaters. Krauth and Staab (1993) found that
using BMBR for treatment of vegetable canning wastewater could achieve COD removal
efficiency exceeding 99% at high F/M ratio (0.5 g/g.d) and high VLR (5.4 kg/m3.d). BMBR
could also efficiently treat oily wastewater at mean F/M ratio of 0.7 g/g.d and VLR between
8.6 and 12.9 kg/m3.d (Scholz and Fuchs, 2000). Whereas the BMBR in this experiment can
only be subjected to lower VLR (3.4 - 5.0 kg/m3.d) and lower F/M ratio (0.1 - 0.3 g/g.d) to
obtain a comparable COD efficiency. It is important to note that all the above mentioned
BMBR systems were operated with salt contents lower than 1.0 g/L NaCl. However, this
current work was carried out at 32 g/L, the high salinity could be the major cause of the drop
in specific organic removal rate of bacterial sludge. Thus high salinity also reduces the
specific organic removal rate of bacterial sludge. However, compared to conventional
activated sludge systems for low salinity wastewater treatment (maximum VLR of 1.2
kg/m3.d), the BMBR could be operated efficiently at higher VLRs (3.4 - 6.0 kg/m3.d).
84

(*)
MBR
MF
UF
YCS
AS

:
:
:
:
:
:

<1
Hu (1989)

2.6
92
3.0-4.0

0.5

Mixture of
10 yeast
strains
1.03

shaking
culture

Vermicelli

Nishihara ESRC,
Ltd. (2001)

8 10
97
4.3 5.2
0.5-0.9

0.6 1.0 (*)

4.5 10.4(*)

Yeast
mixture

(Continuous
completed
mixing)

YCS

Seafood
processing

Yeast

kg BOD5/m3.d
Membrane Bioreactor
Microfiltration
Ultrafiltration
Yeast Cycle System
Activated sludge

This study

Reference

Note:

32

Salt, g/L

15
86-91
3.5-3.8
1.5

MLSS, g/L
COD removal, %
pH
DO, mg/L

4.9-6.5

VLR, kgCOD/m3.d

0.4

Yeast
mixture

Microorganisms

F/M ratio, g/g.d

MBR
with MF

Protein
extract

Process

Wastewater

Parameter

This study

32

16-20
91-97
7.5-8.0
2.1

0.1-0.3

3.4-6.0

AS

MBR
with MF

Protein
extract

85

Krauth and
Staab (1993)

<1

11
99
>6.5
6.3

0.5

5.4

AS

MBR with
MF

Vegetable
canning

Scholz and
Fuchs (2000)

<1

15-25
97
7.0-7.8
-

0.6-0.8

8.6-12.9

AS

MBR with
MF

Oily
wastewater

Lu et al. (1999)

<1

10
92
6.8-7.2
2.0-3.5

1.73

AS

MBR with UF

Fermentation
wastewater

Bacterial membrane bioreactor process

Hamoda and
Al-Attar
(1995)

> 2.0
30
(salt free)

99

0.3-0.6
(0.8-2.1)

AS

Continuous
completed
mixing

Glucose

Metcalf and
Eddy (1991)

<1

2.5-4.0
99
6.5-8.5
t 2.0

0.2-0.6

0.8-1.9

AS

Completed
mixing

Domestic
wastewater

Conventional AS

Table 4.5 Operating parameters of the YMBR, BMBR, some yeast treatments, MBR processes treating different wastewaters and
conventional AS system

This can be explained by high sludge concentration and high substrate removal rate.
Similar results have been reported by Manem and Sanderson (1996), who found that VLR for
dairy wastewater was six times greater than for conventional activated sludge process,
although the biomass concentration was only twice as high. Moreover, the effluent suspended
solids of both membrane reactors were less than 5 mg/L and was almost constant throughout
all experiments.
100
YMBR

90

BMBR

COD removal ( %)

80
70
60
50
2

YMBR: Y = -0.044*X -1.045*X+88.0

2
R = 0.929
2
BMBR: Y = -0.572*X + 3.568*X + 82.7
2
R = 0.836

40
30
20
0

10

12

14

16

18

20

22

24

Volumetric Loading ( kg COD/ m3.d)

Figure 4.27 Variation in COD removal in function of volumetric loading rate

COD removal rate ( g COD/ g MLSS.d)

1.0

0.8

0.6

0.4

YMBR: Y = -0.337*X + 1.156*X 0.1041*X

2
R = 0.951
2
BMBR: Y = -1.285*X + 1.331*X - 0.0323
2
R = 0.938

0.2

BMBR
YMBR

0.0
0.0

0.5

1.0

1.5

2.0

2.5

F/M ratio ( d-1)

Figure 4.28 Variation in COD removal rate in function of F/M ratio (initial COD = 5,000 mg/L)
b. Transmembrane pressure and membrane clogging

Variation in transmembrane pressure in YMBR and BMBR at different operating stages

is shown in Figures 4.25 and 4.26. The pressure ('P) in YMBR was almost constant
throughout the various stages of VLR for a total duration of 72 days. It then increased rapidly
after the 76th day (63 kPa), indicating rapid membrane clogging. This may be due to the
increase in high filtration flux (89.6 L/d.m2), corresponding to HRT of 5 h in the last stage.
However, when YMBR was run at short HRT (5h), uncompleted biodegradation by yeast
results in high soluble COD and accumulation of fine particles (from the influent) retained in
the reactor which may cause a rapid fouling rate. The high soluble COD and fine particles in
the reactor could increase the filtration resistance (Manem and Sanderson, 1996). Whereas 'P
in BMBR sharply increased from 2 to 60 kPa after 12d, 6 d and 2 d at hydraulic retention time
86

(HRT) of 14h, 9 h and 4h, with average biomass concentrations of 6.1, 15 and 20 g MLSS/L
in stage I, II and III, respectively.
Values of different parameters during YMBR and BMBR filtration cycle in both phases
are presented in Table 4.7. As noted, increasing biomass concentration promotes the
membrane clogging, and difference between bacterial and yeast sludge results in different
filtration performances. In fact, characteristics of yeast mixture in the YMBR could prolong
the filtration cycle period. These characteristics are responsible for reducing membrane
clogging rate and can result in large yeast cells, low operating pH, poor adhesion capacity,
inhibiting biofilm formation, low net negative surface charge, low viscosity and low
production of the adhesive extracellular polymers (ECP) that play an important role in floc or
biofilm formation. These characteristics will be discussed in the next section (low COD
loading) and in the sludge characteristics study (Section 4.4).
4.3.2

Low COD loading

Fish-protein wastewater with 1,000 mg COD/L and 32 g salt/L was used in this phase.
Two experiments were conducted: (1) YMBR and (2) BMBR. The objective of this phase was
to obtain a comparative evaluation of treatment performance of YMBR and BMBR at
different HRTs and SRTs of 10 and 50 days (as presented in Fig. 3.6).
a.

COD removals

Figures 4.29 and 4.30 present the overall performance of YMBR and BMBR process for
various HRTs in this phase. The mean influent COD concentration was maintained at 1,000
mg/L, and the VLR was increased from 2.7 - 6.5 kg COD/m3.d with a corresponding decrease
in HRT from 9 to 5 hours. The MLSS concentration for SRT of 10 days ranged from 4,500 5,100 mg/L and 5200 - 5500 mg/L for yeast and bacterial membrane reactors respectively. By
contrast, when the SRT was increased to 50 days, the MLSS concentration increased to within
the range of 13,600 - 15,200 mg/L in the YMBR and 15,200 - 16,300 mg/L in the BMBR. In
all experiments, the DO was maintained above 2.0 mg/L with salt content of 32 g/L. The
effect of changing VLR (vis--vis HRT) on COD removal at SRT of 10 days and 50 days is
shown in Fig. 4.31.
It was observed that at SRT of 10 days, the COD removal efficiency of the YMBR
remained low (about 76%) at lower HRTs (5h), but increased to 94% with the increase in
HRT (> 8 h). Whereas for the BMBR, the COD removal efficiency remained constant within
the range of 92 - 97% when HRT ranged from 5 to 8 h. Thus the COD removal efficiency of
the YMBR is lower than that of the BMBR at short HRTs, but converged at longer HRTs. In
general, the removal efficiency of a biological system increases with HRT (until a certain
limit), this was also observed for both systems.

87

Mean HRT

Mean HRT (h)

60

Tran.. pressure

40

7
6

20

5
4

Transmembrane Pressure ( kPa)

10

3
20000

1200

16000

800
12000

SRT = 10 d
Effluent COD

600

Influent COD

400

MLSS ( mg/L)

COD ( mg/L)

1000

8000

MLSS

SRT = 50 d
200

4000

0
0

10

20

30

40

50

60

70

80

90

Time (days)

Figure 4.29 Variation in COD, biomass and transmembrane pressure in the YMBR as
function of volumetric loading
However, due to lower MLSS vis--vis the higher F/M ratio (1.02 g/g.d) in the YMBR
or possibly lower specific growth rate of yeast, the efficiency was low at lower HRT. Indeed,
the difference in the specific growth rates of yeast and bacteria at 32 g salt/L can be found in
the biokinetic study. Figure 4.32 reveals that although yeasts had higher maximum specific
growth rate (Pmax) at 32 g salt/L. Its specific growth rate (P) at low substrate concentrations
(less than 180 mg/L) was lower than that of bacteria. Thus, the yeast growth was more
inhibited at low COD in the membrane reactor.
Higher HRT vis--vis low F/M ratio (0.5 d-1) enabled better conversion of organic
matters with higher yeast mass available. The MLSS concentration in BMBR was relatively
high at SRT of 10 d. This might be due to higher specific growth rate of bacteria in a
substrate-limiting condition (COD < 200 mg/L) as shown in Fig. 4.9. Therefore, the COD
removal efficiency of BMBR remained high through out HRTs. A peak efficiency of 97%
was obtained for BMBR at a HRT of 7 - 8 hours, which represents the best range of operating
conditions.

88

Mean HRT (h)

80

7
60
6
40
5
4

20

Mean HRT

Transmembrane Pressure ( KPa)

100

Trans.Pressure

3
20000

1200

800
Effluent COD

SRT = 10 d

12000

Influent COD

MLSS ( mg/L)

COD ( mg/L)

16000

8000

MLSS

400
SRT = 50 d

4000
0
0

20

40

60

80

Time (days)

Figure 4.30 Variation in COD, biomass and transmembrane pressure in the BMBR as
function of volumetric loading
100

COD removal (%)

90
80
2

YMBR: Y = -0.683 * X + 10. 824 * X + 53.67

2
R = 0.885
2
BMBR: Y = -0.502 * X + 7.815*X + 67.01
2
R = 0.701

70
60

YMBR

SRT of 50 d

BMBR

100

COD removal ( %)

90
80
2

YMBR: Y = -1.491 * X + 26.0218 * X - 18.294

2
R = 0.885
2
BMBR: Y = -1.149 * X + 16.744*X + 36.636
2
R = 0.767

70
60

YMBR

SRT of 10 d

BMBR

50
4.0

5.0

6.0

7.0

8.0

9.0

10.0

HRT ( h)

Figure 4.31 Variation in COD removal as function of HRTs in YMBR and BMBR

89

Specific Growth Rate Pday-1)

2.50

2.00

1.50

1.00

0.50

Bacterial sludge
Mixed yeast sludge

0.00
0

50

100

150

200

250

300

350

400

450

500

COD (mg/L)

Figure 4.32 Variation in specific growth rate of yeast and bacteria at 32 g salt/L in function
of COD
At SRT of 50 d, there was no significant difference between the COD removal
efficiency of YMBR (86-91%), and BMBR (9193%) probably due to the lower F/M ratio
(0.35 - 0.40) in both reactors. As mentioned earlier, yeast growth is limited at low substrate
concentration. Thus, this result shows that maintenance of high MLSS (long SRT) in the
MBR can significantly enhance treatment efficiency for substrate limited growth. Moreover,
there was no appreciable change in the COD removal efficiency in the transition phase (Table
4.6). In this phase, low F/M ratios were maintained within the range of 0.35 - 0.55 by
controlling high HRT. High efficiency (95%) could be obtained at these low F/M ratios for
both YMBR and BMBR.
However, a conventional activated sludge system can be operated at a maximum VLR
of 1.2 kg/m3.d and F/M of 0.6 d-1, and degradation rates reduced considerably with an
increase in salinity. Therefore, this high salinity wastewater should be treated at lower F/M
ratios (Kargi and Dincer, 2000). Three-fold-lower F/M ratios were applied in conventional
activated sludge at 30 g salt/L compared to those applied in salt-free wastewater (at the same
SRT) in order to obtain equivalent substrate removal.
In comparison, a comparable performance is obtained from the membrane bioreactors at
a very high salinity and VLR (3.0 - 5.0 kg/m3.d). Similar results have been reported by
Manem and Sanderson (1996) who found that a six-fold higher VLR could be applied for
dairy wastewater compared to conventional activated sludge system without deteriorating
performance. The results show that both YMBR and BMBR can be effectively used to treat
high salinity wastewater such as pickling and seafood processing wastewater to conform to
effluent standards of COD lower than 120 mg/L and BOD lower 20 mg/L.

90

(*)

(*)

Transition phase

50
50

10-50

10-50

2.66
2.95
3.66
4.59
3.58
4.28
4.93
6.55

10
10
10
10

(*)

VLR
(kg
COD/m3.d)

SRT
(days)

8.8
7.7
6.1
5.0
7.2
6.1
5.3
4.0

Mean
HRT
(h)

5050
5030
4440
4530
5300
11500
14500
15000

MLSS
(mg/L)

0.49
0.56
0.83
1.02
0.58
0.37
0.35
0.44

F/M
(g/g.d)

YMBR

94
94
84
76
96
95
91
86

COD
removal
(%)

50
50
150
230
45
50
90
150

Effluent
COD
(mg/L)

10
15
90
150
15
15
40
90

Effluent
BOD
(mg/L)

91

50
50

10-50(*)

10-50(*)

10
10
10

SRT
(days)

2.97
3.57
4.30
3.63
4.08
5.56
6.35

VLR
(kg
COD/m3.d)

8.1
6.3
5.2
7.1
6.0
4.7
4.0

Mean
HRT
(h)

Table 4.6 Operating parameters and performance of YMBR and BMBR in high COD loading phase

4800
4600
5730
6430
10600
13100
16300

0.38
0.78
0.76
0.57
0.39
0.43
0.39

F/M
(g/g.d)

BMBR
MLSS
(mg/L)

97
97
92
97
95
93
91

COD
removal
(%)

25
25
70
30
55
70
100

Effluent
COD
(mg/L)

<5
<5
30
5
15
30
55

Effluent
BOD
(mg/L)

b. Membrane clogging

Membrane systems are often subjected to clogging, and this poses serious problems for
operation and maintenance. In order to investigate membrane clogging, experiments were
carried out in a continuous operational mode. Figures 4.29 and 4.30 show variations in
transmembrane pressure with time for YMBR and BMBR. The trend of pressure variation in
the YMBR and BMBR was similar to that in the high COD loading. Table 4.7 shows the
mean flux, transmembrane pressure and accumulated permeate volume during the filtration
cycle of the BMBR and the YMBR in both phases. In the high COD loading, it was observed
that the transmembrane pressure ('P) of the YMBR remained almost constant for
approximately 72 days before rising sharply. Whereas, the 'P increased sharply after 32 days
for the low COD loading in which the mean flux was three times higher than that in the high
COD loading. Total operating time (before 'P reaching to 70 kPa) for a SRT of 10 days is
higher than 50 days. For YMBR, there was no significant difference in the volume of the
permeate collected, although mean MLSS concentration at SRT of 50 days was 2.5 times that
at SRT of 10 days. This indicates that the clogging rate of the membrane is not entirely
dependent on the concentration of MLSS.
Table 4.7 Values of different parameters during YMBR and BMBR filtration cycle
SRT
(days)

Reactor

Influent
COD

Mean
MLSS
(mg/L)

Mean flux
(L/m2.d)

Accumulated
permeate
(L/cycle)

Filtration
cycle
(d)

High COD loading:

50
YMBR
BMBR

5,000
1,200

11000
20000

34
60

2550
63

76
1.0

Low COD loading:

10
YMBR
BMBR
50
YMBR
BMBR

1,000
1,000
1,000
1,000

4,500
4,600
11,000
10,600

95
91
98
95

3,100
320
2,910
120

38
3.5
32
1.5

Membrane clogging in the BMBR was much more severe than in the YMBR. The
average filtration time for the BMBR, at SRT of 10 days was 3.5 days. This decreased to 1.5
days for SRT of 50 days, requiring frequent membrane washing. The rapid development of
biofilm for BMBR was in relation to the MLSS concentration in the reactor. At higher MLSS
concentrations (10,600 mg/L) for 50-day SRT, the rate of clogging was much higher than in
the YMBR. The difference in the performance between both YMBR and the BMBR is
probably due to the mechanism by which the biofilm develops on the membrane surface.
For bacterial system, biofilm develops by two mechanisms; first, by attachment of cells
on the membrane surface, which promotes further growth of bacteria; secondly by capturing
more cells (from the mixed liquor) in the already developed matrix formed by the first
process. The formation of biofilm on the membrane surface could be related to the production
of adhesive extracellular polymers (ECP) by bacteria. ECP is partly soluble in water and goes
into a colloidal suspension after production, which results in ECP accumulation in the mixed
liquor. During filtration, the macromolecular ECP compounds accumulate on the membrane
surface, and play the key role of binding the cells on the membrane surface and entrapping
larger organic particles in the slimy matrix. The availability of organic substance in the ECP
matrix promotes growth of new bacteria. This is further aided by the entrapment of more cells
92

from the mixed liquor, which seriously impairs membrane performance and module life.
Thus, at higher concentration of microorganisms in the mixed liquor, production of ECP
increases and biofilm develops at a much higher rate, which leads to rapid development of
transmembrane pressure.
The mechanism of biofilm development in the YMBR is different to that in the BMBR.
In the YMBR the yeasts attached physically to the membrane surface during filtration instead
of getting trapped in a matrix. Though ECP is produced in the YMBR, the quantity is much
less than that of the BMBR where a dense gel matrix is formed. The yeast cells are attached
together by physical interwinding of mycelia or pseudomycelia (Nishihara ESRC Ltd., 2001).
In addition larger yeast cells (2.5 - 3.9 Pm) in the YMBR also forms a secondary layer on the
membrane surface that acts like a second barrier to fouling particles and aggregates (Guell et
al. 1998). However, with increase in thickness of the cake, it gets heavier and starts sloughing
off by uplifting air flow or air-backwash, as soon as the cake is unable to sustain its own
weight. Thus the cake thickness cannot enlarge indefinitely and thus reduce the problem of
frequent membrane clogging. Mechanisms of flux enhancement by yeast sludge are shown in
Fig. 4.33.
Partial detachment

Hydrogel
(high resistance)

biofilm
(ECP matrix)
membrane

Complete detachment

Porous cake
(secondary filter layer)

Yeast cells
Air bubble
(from aeration)
Backwash

Filtration

Figure 4.33 Possible mechanisms for flux enhancement by yeast cells

The membrane clogging depends upon the MLSS concentration of bacteria in the
BMBR, but to a much lesser extent for YMBR, primarily due to the difference in the
mechanisms of clogging. Due to this difference between BMBR and YMBR, a prolonged
filtration cycle (about 10 times higher compared to BMBR) could be obtained for the YMBR
without much problem with membrane clogging.
4.4

Sludge Characterization Study

In the membrane bioreactor system, fouling problem can be linked to the sludge
characteristics as discussed in Section 4.3.2.b. In order to investigate the difference in fouling
phenomenon in both YMBR and BMBR, sludge characterization was carried out. Series of
yeast and bacterial cultures were run at various salt contents. The sludge was examined for
ECP content, dewatering property (CST), viscosity as well as sludge settleability (SVI). These
results were also compared with sludge obtained from YMBR and BMBR systems operated at
32 g salt/L. Furthermore, in both these bioreactors, prior to chemical cleaning, the
characteristics of the sludge cake formed on the membrane surface was also analyzed.
93

4.4.1

Culture Study

Results of ECP, CST and viscosity of the mixed yeast and bacterial sludges at different
salt contents are presented in Table 4.8 and Figure 4.34. ECP content in the mixed yeast
sludge was very low at all salt contents compared to that in bacterial sludge. While the ECP
concentration of bacterial sludge rises considerably with salt content. Similar phenomena
have been reported for on flocculation of marine bacteria (Watanabe et al., 1998). They
suggested that the ECPs did not originate from autolysis, but seem to be excreted by living
marine bacterial cells. Unlike ECP production of bacteria living in salt-free water, which only
occurs during the stationary phase, marine bacteria can produce large amount of ECP in the
exponential growth phase (Flemming and Wingender, 2001). This leads to significant
accumulation of ECP in a high salinity environment. In addition, due to alteration of genetic
structure under high osmotic stress by NaCl concentration, bacteria can synthesize more
specific proteins that may contribute to increase in the ECP production (Vijaranakul et al.,
1997). Eikelboom (2000) found that Zoogloea bulking problems were due to high production
of ECP in the bacterial flocs. This phenomenon could explain the acceleration of biofouling
problem in BMBR.
In contrast, the production of ECP in yeasts is much lower and its concentration remained
low. Reeslev et al. (1996) reported that Aureobasidium pullulans, a mycelial yeast, can only
synthesized ECP when growth was nitrogen-limited while no ECP was produced when the
culture was carbon-substrate-limited. The feed wastewater was rich nitrogen (COD:N =
100:15), and therefore growth of yeast is carbon-substrate-limited. This may result in less
ECP production, saving it from frequent clogging.
Table 4.8 Yeast and bacterial sludges characterization

Sludge

Salt
(g/L)

MLSS
(mg/L)

SS (*)
(mg/L)

SVI
(mL/g)

ECP
(mg/g)

CST
(s/g) (**)

0.5
15
32
45
32

3320
3860
3200
3840
6320

80
152
232
220
N/A

72
63
81
108
159

28.4
29.0
34.6
48
57.8

1.48
1.22
8.41
14.9
9.8

5.04
5.14
5.60
7.14
7.26

0.5
15
32
45
32

3260
4020
4480
4580
4300

1373
1767
1440
1530
N/A

N/A
N/A
N/A
N/A
N/A

<1
l<1
5
7.5
11.4

1.19
1.12
0.94
0.94
1.44

5.01
5.28
6.00
6.34
4.86

Viscosity
centipoises

Bacteria

Bacterial MBR
Yeast

Yeast MBR
(*)

Suspended solids concentration of supernatant after 2 h of settling

s/g = second/g MLSS

(**)

94

50

16
Mixed yeast sludge

CST ( s/g MLSS)

ECP (g / mg)

40
30
20
10
0

Mixed bacterial sludge

12

0
0

10

20

30

40

50

Salt (g/L)

10

20

30

40

50

Salt (g/L)

a) ECP vs. salt

b) CST vs. salt

Figure 4.34 Variation in ECP and CST in function of salt content

Sludge dewaterability is measured by CST, which is used as a relative indicator to
characterize the performance of sludge dewatering. While SVI is used as a field measure of
sludge settleability. CST of bacterial sludge increased from 1.5 to 15 s/g with the increase of
salt content from 15 to 45 g/L. Similarly, SVI of the bacterial sludge also increased with an
increase in salt content, although the SVI values remained lower than 150 (a value
corresponding to bulky sludge) even for a salt content of 45 g/L. However, the bacterial
sludge at high salinity (above 30 g/L) was not compacted and remained fluffy compared to the
sludge at salt contents of 15 g/L or lower. Similarly, it was that increasing viscosity trend with
salt content was comparable to CST and ECP. In general, viscosity is dependent on MLSS.
MLSSs of cultures at different salt contents were maintained relatively stable in this study.
Thus, increase in viscosity with salt increase may be due to the ECP content of the sludge.
Microscopic examination did not show any filamentous bacteria or fungi in the bacterial
sludge even at high salt contents, and relationships could be established between the increase
in the SVI, CST and ECP with salt content for the bacterial sludge. The linear correlation
between SVI and ECP was found in some previous studies (Goodwin and Foster, 1985;
Urbain et al., 1993). The increase in CST and SVI as function of salt content in bacterial
sludge may be attributed to excess sodium which can cause a deterioration in the floc
structure, and an increase in ECP. This results in weak flocs, poor settling (increase in SVI)
and dewatering (increase in CST). Likewise, Urbain et al. (1993) reported that ECP content
was correlated linearly with SVI, high polysaccharides and proteins resulting in a worsening
of sludge settleablity. In addition, the increase of ECP can be explained by the structure of the
three-dimensional ECP matrix kept together by divalent cations such as Ca2+, Mg2+. An
exchange of divalent cations (Ca2+, Mg2+) with Na+ will take place when the ratio of Na+ to
divalent cation (Na+:M2+) exceeds two. In general, the Na+:M2+ ratio is very high in saline
environment. In such conditions, large amount of polymers produced resulted in weaken the
floc strength (Bruus et al., 1992). These ECP molecules extend out from cell surfaces and
form a dense gel that retains water in gel pores (Liao et al., 2001). Liss et al. (1996) also
reported that these complexes and hydrated ECPs within the floc matrix have a large capacity
to retain water, which decreases dewaterability. Furthermore, suspended solids in the
supernatant of bacterial batches appear to increase when the CST or SVI (vis--vis ECP)
increases (Fig. 4.35). This may be due to disruption of weakened floc structure at high salinity
(by shearing forces from aeration) which forms pin-point flocs and thus results in high
effluent suspended solids.

95

SVI (mL/ g)

Viscosity ( centipois)

ECP (mg/ g)
SVI

140

8.0

50

SS of supernatant ( mg/L)
250

SS of supernatant

ECP

Viscosity

40

120

200

7.0
150

100

30
6.0

80

100

20

50

5.0

60
10
0

10

20

30

40

50

Salt (g/L)

10

20

30

40

50

Salt (g/L)

Figure 4.35 Variation in SVI, SS, ECP and viscosity with salt content in mixed bacterial
cultures
For mixed yeast sludge, CST remained relatively constant with salt increases. This can
be understood in terms of the negligeable amount of ECP produced and indicates that the
yeast sludge has better dewatering or thickening ability than bacterial sludge at high salinity,
which is a specific advantage of the yeast system. The viscosity of yeast mix liquor, was
slightly higher than that of bacteria. This can be attributed to the difference between MLSS of
mixed yeast (4500 mg/L) and bacterial sludges (3500 mg/L). Thus, unlike bacterial sludge,
viscosity of yeast sludge is mainly effected by biomass concentration, but not by ECP.
Table 4.8 shows that SS in the supernatant of the mixed yeast sludge was high even
after 2 hours of settling. SS removal efficiency was only around 65%. The SS in supernatant
were mainly dispersed as small yeast cells, which had not been captured by mycelia or
pseudomycelial yeast matrix as shown by microscopic examination. Settled solids after two
hours of settling were found to be compacted, and mainly consisted of yeasts interwind with
each other and large yeast cells. Unlike activated sludge flocs, yeast flocs could not trap all
fine yeast cells or fine particles, which led to high SS in the supernatant even after prolonged
settling time. Conflicting results were obtained by different studies concerning the floc
characteristic of yeast sludge. Nishihara ESRC Ltd. (2001) obtained large yeast flocs that
settled quickly while other authors (Hu,1989; Arnold et al., 2000) obtained poor settling
sludge when dealing with different yeast strains. This suggests that the predominance of
mycelial yeasts may depend on the free competition among different yeast strains in
wastewater.
4.4.2

YMBR and BMBR

Large differences of ECP obtained from both YMBR and BMBR and the batch reactors
(run at 32 g salt/L) are shown in Figure 4.36. This may be due to of washing-out of
macromolecules (ECPs and proteins) with fine particles in supernatant during the decantation
stage, while most of these substances were retained by membrane module with spore size of
0.1 Pm. However, in comparison between YMBR and BMBR, the ECP concentration of
mixed bacterial sludge was higher than that of yeast sludge at both SRTs. This observation
was similar to that obtained in batch studies.

96

600
SRT of 10 d

mg ECP/g dried biomass

500

400

490

SRT of 50 d
Sludge cake
(attached to the membrane surface)

300
232
200

147

100
11

58

39

YMBR

BMBR

Figure 4.36 ECP contents of mixed yeast and bacterial sludges in YMBR and BMBR
Meanwhile, visual observation of the clogged membrane revealed that large amount of
extremely viscous and gelatinous sludge cakes was attached to the BMBR (Appendix A).
However, the YMBR has a very thin layer of sludge cake, which could be easily washed out
with tap water. This difference in the nature of the attached sludge contributes to the
improvement of the membrane performance in the YMBR process.
In both the YMBR and BMBR, the ECP content seems to vary with the SRT, as shown
in Fig. 4.36. It was found that ECP concentration increased with increase in SRT for both
BMBR and YMBR. This can be explained by the variation in ECP production rate at different
growth phases. Low SRT corresponds to the stationery phase, while long SRT (50 d)
corresponds to the decay phase in which cell lysis takes place. Similarly, Pavoni et al. (1972)
reported that during the early decay phase, the rate of ECP production was maximum.
Sheintuch et al. (1986) reported that ECP content is a function of SRT in continuous
bioreactors and it increased linearly with SRT.
4.4.3

Microscopic Observations of Mixed Yeast Sludge

From microscopic observations, it was noted that there were changes in predominant
yeast strains and presence of other microorganisms (protozoa, rotifers) when operating
conditions was changed. The operating conditions consisted of influent COD concentration,
organic loading, SRT and the type of substrate (glucose and protein extract as protein source)
and salt content. Photographs of mixed yeasts and bacteria flocs are shown in Appendix A.
Most yeast cells in the cultures were larger than those in the YMBR, even though both
reactors was run at the same operating conditions (Protein-feed wastewater, SRT of 50 days,
COD of 5,000 mg/L). Mycelia (hypha filament) and large size egg-shaped cells with
monopolar budding were predominant in the cultures. While round or smaller egg-shaped
cells, which may be different yeast strains from the cultures, were predominant in the YMBR
(mean size of 2.4 Pm). The mean size of the mother cells was 3.9 Pm. This might be due to
membrane with pore sizes of 0.1 Pm retaining most fine size cells among which minority of
acid-tolerant bacteria was involved. Fine cells suspended in supernatant were washed out by a
decanting step after more than 2 hours of settling in batch operation.

97

For substrate as glucose, most yeast cells are round with multilateral budding and mean
size of 5.5 Pm. In the YMBR, predominant yeast strains were also changed as organic loading
rate varied. The white or light brown color of yeast mixture gradually changed to dark brown
when the high COD loading (5,000 mg COD/L) changed to low COD loading (1,000 mg
COD/L). The majority of yeast colonies were black, orange and yellow growing on the yeastglucose-peptone agar when growing in the low COD loading (influent COD of 100mg/L).
Most yeast cells were egg-shaped and mycelia. It could be easily observed that free-living
ciliates (protozoa) grow well in this yeast mixture. These ciliates move freely and rapidly in
the mixed liquor. This change may be due to the increase in DO concentration. DO of all runs
in the low COD loading is above 4.0 mg/L, whereas DO in that with COD of 5,000 mg/L was
less than 2.0 mg/L.
4.4.4

Nutrient Uptake

Nutrients of mixed yeast and bacterial sludges are presented in Table 4.9. The volatile
solids of both yeast and bacterial sludges were not significantly different, ranging from 89 to
95% of dried solids. The nitrogen content of the mixed yeasts sludge fed with glucose
wastewater was found to be 7.1% on average compared to 3.2 % for bacterial sludge.
Similarly, the phosphorus uptake ability of the yeasts was approximately twice as high as that
of bacterial sludge. For fish-protein wastewater, nitrogen content of the mixed yeast cultures
was more than 15%. In general, nitrogen content of the yeast and fungi biomasses was in the
range of 7-12% (Westhuizen and Pretorius, 1998; Defrance, 1993). Thus the amount of
nitrogen uptaken for protein-feed wastewater is too high (above 15%) compared to normal
values or values of yeast sludge fed with glucose wastewater (7.1%). This may be attributed
to the precipitation of a part of influent protein at low pH (3.5). The nitrogen content of yeast
sludge in the YMBR was not excessive (7.64%) and was in the normal range. Thus, it is
postulated that in the YMBR process with a low F/M ratio, yeast can produce enzyme to
hydrolyze protein aggregates and can assimilate them.
Table 4.9 Composition of mixed bacterial and mixed yeast sludge

Microorganisms
Yeast sludge:
+ 20 g /L:
+ 32 g /L:
+ 45 g /L:

Volatile solids
(%)
Glucose
Protein
94.1
92.3
95.0

YMBR sludge
(low-COD loading)
Bacterial sludge
+ 20 g /L:
+ 32 g /L:
+ 45 g /L:
BMBR sludge
(High COD loading)

Nitrogen content
(% of dries solids)
Glucose
Protein

90.3
93.4
90.5

6.79
7.28
7.19

91.5

89.5
92.0
94.2

15.2
17.1
15.7

Phosphorous content
(% of dried solids)
Glucose
Protein
2.23
1.79
1.70

7.64

91.6
92.9
90.2

2.70
3.05
3.74

89.7

5.61
5.02
5.32
5.21

3.61
3.43
3.53
3.52

0.61
0.86
0.90

1.56
2.37
2.23
1.51

The nitrogen and phosphorous contents of the bacterial biomass fed with protein-feed
wastewater were approximately twice higher than that fed with glucose wastewater as shown
in Table 4.9. By exception of nutrient uptake into real bacterial cells, the remaining of these
98

nutrients may entered into composition of floc structure such as ECP and phosphate bonds
which can enhance ECP production.
Even when different wastewaters were used, the mixed yeast sludge from the cultures or
the YMBR contained higher nutrients than the bacterial sludge. The average crude protein
content obtained was 45% (corresponding to 7.2% N). This value was similar to the protein
content obtained from the well-known Symba process which is a single-cell-protein
production (SCP) for human food consumption by using potato processing waste to culture
yeasts Endomycopsis fibuliger and Candida utilis. In general, algal and bacterial biomasses
are less pleasant to taste because they contain undesirable levels of certain cellular materials
such as high nucleic acid content, toxic or carcinogenic substances absorbed from the growth
substrate. By contrast, yeasts and most fungi are quite acceptable to animals and man due to
the abundance of valuable nutritious substances such as proteins and vitamins. Thus, it is
suggested that a combination of yeast treatment and SCP production can be a cost-effective
approach for seafood processing industries which, at present, face difficulties in treatment
efficiency and high costs.

99

Chapter 5
5
Conclusions and Recommendations

This study investigated biological processes in using wild salt-tolerant yeast and
bacteria for treatment of saline seafood processing wastewater. Basic studies on biokinetic
coefficients and optimum operating parameters of yeast and bacterial treatment were
conducted. The effects of high salt contents (20, 32 and 45 g/L NaCl) on the biokinetic
coefficients were evaluated using respirometric method. Then the optimum operating
parameters for the yeast and bacterial treatments were found from the parametric study using
the acclimatized mixed yeast and bacterial cultures.
The main part of this study focused on the membrane bioreactor. The potential for
developing membrane bioreactor systems using salt-tolerant yeast and bacteria to treat saline
seafood processing wastewater was examined. A comparative evaluation of treatment
performance of both systems was done. The last section focused on sludge characteristics
concerning membrane clogging. The relationship between sludge properties and membrane
flux decline was investigated. The conclusions drawn from these results are presented below.
5.1

Conclusions

From the biokinetic study, it can be concluded that the yeast is more efficient for
treating wastewater containing high organic load and high salt content. It can be reasoned that
they would be more suitable for varying salt loads because they require lower acclimation
time than does bacterial culture. This was attributed to their better osmotolerant properties.
The salt inhibition was found to be much higher for yeast culture. The maximum specific
growth rate for yeast is higher than for bacterial culture at high salt contents. However, yeast
growth was more inhibited at low CODs.
The results of the parametric study indicate that the osmotolerant yeasts were able to
tolerate wider pH range than bacterial culture. Total OUR of yeast sludge was highest for pHs
5.0 5.5. The respiration rate of yeasts was inhibited at pH 2.5 or pH above 9.0. The OUR of
yeast at pH 3.5 was slightly lower than that at pH 5.0 - 5.5. However, pH of YMBR was
maintained at pH 3.5 in order to limit bacterial contamination. The results of SRT study show
that the highest nitrogen removal by uptake into yeast biomass was obtained at SRT of 10 d,
whereas the maximum COD removal efficiency was obtained at SRT above 45 d.
In the high COD loading of membrane bioreactor study, the COD removal rate for
BMBR was lower than the YMBR at high VLRs at high salt contents (32 g/L). Thus, the
mixed yeast system could be subjected to higher F/M ratio. Even though DO concentration of
yeast mixed liquor was lower than 1.0 mg/L at the high F/M ratio, the treatment efficiency of
the yeast system does not decline. This may be due to the structure of yeast flocs facilitating
oxygen diffusion. It is suggested that the yeast system represent a better substitute for an
anaerobic system in terms of COD removal rate at high salinity.
The low COD loading phase revealed that both YMBR and BMBR give high COD
removal efficiency (>90%) at high salt content, low F/M ratio and high SRT. Both reactors
generated good effluent quality (COD < 120 mg/L, BOD < 20 mg/L and SS < 5 mg/L).

100

Yeast sludge in fact achieve significantly better reduction in the membrane clogging
rate than bacterial sludge. BMBR is highly prone to membrane clogging, whereas YMBR can
be operated at a relatively low pressure for prolonged filtration cycle. The filtrate cycle of
YMBR was approximately 10 times higher than BMBR. Thus using yeasts in the
biomembrane reactor can enhance membrane performance and has the potential to improve
the economics of treatment system due to reducing operating and maintenance costs.
Several factors such as mechanisms of biofilm formation, concentration of ECP and size
of cells contributed to the better filtration cycle of the YMBR. Reduction of problems
associated with membrane clogging supports the use of YMBR in practice. Variation of ECP
content that is responsible for biofilm as well as floc formation was found to vary with salt
content for bacterial sludge, whereas for yeast sludge, ECP concentration remained practically
constant even at high salt contents. Along with increases in ECP, CST of bacterial sludge was
increased at high salinity, while CST of yeast sludge remained practically constant, indicating
that dewatering would be easy for high salinity wastewater. Thus, using yeasts in membrane
bioreactor can enhance membrane performance and reduce the operational problems
associated with sludge dewatering and disposal.
Nitrogen and phosphorous contents of the yeast sludge were approximately twice higher
than those of bacterial sludge. This suggests that yeasts have high nutritional values. In
addition, yeasts and most fungi, are quite acceptable to animals and man due to the abundance
of other valuable nutritious substances (vitamins). Thus, a combination of yeast treatment and
SCP production can be a cost-effective approach for seafood processing industries.
5.2

Recommendations

Based on the extensive experimental data obtained, several recommendations for future
studies can be outlined:

High Salinity Wastewater

1.

Due to flux-enhancing ability of yeast sludge, operation modes for yeast membrane
bioreactor can be examined to shorten backwash time. This may results in reduction of
operation costs and increase in total permeate flux.

2.

This study has not evaluated in depth the yeast sludge properties at different salt
contents which may be related to membrane flux. These properties can consist of
specific filtration resistance, hydrophobicity, surface charge, bound water, cell size and
composition of ECP. In order to understand thoroughly the fouling inhibition
mechanism of yeast sludge, a detailed study of the sludge properties at various salt
content should be undertaken.

3.

ECP production of mixed yeast sludge or activated sludge under varying nutrient
compositions or cation concentrations of influent wastewater may be effected. A
balance of nutrient contents or cations (divalent cation: monovalent cation) may lead to
significant decrease in the membrane clogging rate. A study on the effects of nutrient
and cation contents on ECP production would perhaps be useful.

4.

Since seafood factories process a large range of products with important seasonal
variations, changing in pollution characteristics vary significantly from plant to plant,
and even within the same plant. Large variations in salt content can therefore be
expected. Thus, a study on the effects of salt shock loading on yeast sludge may be
101

necessary. The shock salt loading experiment for bacterial sludge may be conducted in
parallel to obtain a comparative evaluation.
5.

In order to confirm the effectiveness of yeast treatment, pilot-scale yeast membrane

bioreactors for long-term treatment of real saline seafood processing wastewater should
be developed.

Biomembrane process and Membrane clogging

1.

The wastewater pH of the environment influences surface charges, protein deposition

and deflocculation. Low pH can cause an increase in surface charges, protein aggregates
and deflocculation, which may enhance membrane filtration water flux. However, low
pH values will also inhibit bacterial growth. Therefore, optimum pH values to control
membrane clogging in the bacterial system should be considered.

2.

A low pH environment may result in predominance of acid-tolerant microorganisms

such as fungi, yeasts and acidogenic bacteria. Thus, under suitable operating conditions
(such as pH, DO, organic loading and SRT) there may be a symbiotic relationship
between these acid-tolerant microorganisms. For example, acidogenic bacteria enable to
hydrolyze and convert quickly organic complexes (such as protein, lipids, and
polysaccharides) to lower molecular-weight intermediate compounds (such as VFAs,
amino acids and short-chain carbohydrates) which may be suitable substrates for yeast
and fungal growth in a low pH environment. Therefore, using a mixture of acid-tolerant
microorganisms in the biomembrane process for treatment of certain wastewaters can be
investigated.

3.

In this study, it was observed that ECP production increases with salt content. Thus,
combination of activated carbon (AC) adsorption and the bacterial membrane process
should be examined. Activated carbon addition to the BMR process can enhance
permeate flux by forming porous cakes and can remove refractory organic matters by
AC adsorption. Therefore, this method may be applied successfully for TOC removal as
a pre-treatment of raw water in the RO process.

Using Yeasts and Fungi for treatment of toxic wastewaters

Based on tolerance ability of yeasts or fungi in extremely strict conditions, further
investigation are proposed:
1.

A comparative study of bacterial and yeast sludge response to acute and chronic heavy
metal stress using respirometric method is recommended. The results from such a study
might provide information on quantitative toxicity evaluation of the heavy metals for
both yeast and bacterial treatment systems. Resting and growing cells (biomass) can
demonstrate completely different mechanisms of resistance to acute and chronic heavy
metal stress. Thus, mechanisms of heavy metal biosorption by yeasts and bacterial
sludge may also be investigated.

2.

Using yeast or fungi biomembrane process with low F/M ratio for treatment of high
strength hazardous organic wastewater may be a feasible biological approach. Toxic
wastes or chemicals suggested include pesticides, herbicides, phenolic derivatives,
aromatic compounds, cyanides and tannery wastewater.

3.

Identification of the various yeast or bacteria present at high salt conditions.

102

References

Agu R.C., Amadife A.E., Ude C.M., Onyia A., Ogu E. O., Okafor M. and Ezejiofor E. (1998).
Combined Heat Treatment and Acid Hydrolysis of Cassava Grate Waste Biomass for Ethanol
Production. Waste Management, 17 (1) 91-96.
Anciaux C.M., Meyer M.D., Levert J.M. and Vanthournhout M. (1989). Influence of several
parameters on the growth of Candida ingens in controlled culture using volatile fatty acids as
substrates. Biological Wastes, 30, 21-34.
APHA-AWWA-WPCF (1995). Standard Methods for the Examination of Water and
Wastewater.19th Edition.
Arnold J.D., Knapp J.S. and Johnson C.L. (2000). The use of yeasts to reduce the polluting
potential of silage effluent. Wat.Res., 34 (15) 3699-3708.
Baere D., L.A., Devocht, M., Van Assche, P., and Verstraete, W. (1984). Influence of high
NaCl and NH4Cl salt levels on methanogenic associations. Water Res.,18 (5) 543-548.
Balslev-Olesen P. Lynggaard-Jensen A. and Nickelsen C. (1990). Pilot-scale experiments on
anaerobic treatment of wastewater from a fish processing plant. Wat. Sci.Tech., 22 (1/2) 463474.
Barker T.W., Quinn J.P. and Matchant R. (1982). The use of a mixed culture of Geotrichum
candidum, Candida krusei and Hansennula anmola for microbial protein production from
whiskey distillery spent waste. Euro. Appl. Micro. Bio., 14, 247-253.
Battistoni P. and Fava G (1994). Fish processing wastewater treatment requirements by line
production changes. Wat. Sci. Tech., 29 (9) 111119.
Battistoni P., Fava G. and Gatto A. (1994). Fish processing wastewater: emission factors and
high load trickling filters evaluation. Wat. Sci. Tech. 25 (1) 1-8.
Belkin S, Brenner A. and Abeliovich A. (1993). Biological treatment of a high salinity
chemical industrial wastewater. Wat. Sci. Tech., 27 (7-8) 105-112.
Brown M.J. and Lester J.N. (1980). Metal removal in activated sludge: the role of bacteria
extracellular polymers. Water Res., 33, 817-838.
Bruus J.H., Nielsen P. H. and Keiding K. (1992). On the stability of activated sludge flocs
with implications to dewatering. Wat. Res. 26 (12) 1597-1604.
Buisson H., Cote P., Praderie M. and Paillard H. (1998). The use of immersed membranes for
upgrading wastewater treatment plants. Wat. Sci. Tech., 37 (9) 89-95.
Bura R., Cheung M., Liao B., Finlayson J., Lee B.C., Droppo I.G., Leppard G.G. and Liss
S.N. (1998). Composition of extracellular polymeric substances in the activated sludge floc
matrix. Wat. Sci. Tech. 37 (4-5) 325-333.
Burnett W.E. (1974). The effect of salinity variations on the activated sludge process. Water
& Sewage Works, 37-55.
Cech J.S., Chudoba J. and Grau P. (1984). Determination of kinetic constants of activated
sludge microorganisms. Wat. Sci. Tech.,17, 259-272.
Chanda S. and Chakrabarti S. (1996). Plant Origin Liquid Waste: A resource for single-cell
protein production by yeast. Bioresource Technology, 57 (1966) 51-54.
Cheung S. W. and Anderson B.C. (1997). Laboratory Investigation of Ethanol Production
from Municipal Primary Wastewater Solids. Bioresource Technology, 59 (1) 81-96.
103

Chigusa K., Hasegawa T, Yamamoto N., and Watanabe Y. (1996). Treatment of Wastewater
From Oil Manufacturing Plant By Yeasts. Wat. Sci.Tech., 34 (11) 51-58.
Choi M.H. and Park Y.H. (1999). Growth of Pichia guilliermondii A9, an Osmotolerant
Yeast, in Waste Brine Generated From Kimchi Production. Bioresource Technology,70
(1999) 231-236.
Chudoba P., Capdeville B. and Chudoba J. (1992). Explanation of biological meaning of the
So/Xo ratio in batch cultivation. Wat. Sci. Tech., 26 (3-4) 743-751.
Dahl C., Sund C., Kristensen G.H. and Vredenbregt L. (1997). Combined Biological
Nitrification and Denitrification of High Salinity Wastewater. Wat. Sci. Tech.,36 (2-3) 345352.
Dalmacija B., Karlovic V., Tamas Z. and Miskovic D. (1996). Purification of high salinity
wastewater by activated sludge process. Wat. Res., 30 (2) 295-298.
Dan N.P. (2000). Special Study: Saline wastewater survey and trends of study on biological
treatment. AIT, Bangkok, Thai Land.
Defrance M.B. (1993). Etude dun reacteur levurien pour le traitement des effluents
dindustries alimentairs. Doctoral Thesis, University of Montpellier II-Sciences et Techniques
du Languedoc, France.
Deocadiz E.S. (1977). Joint biological treatment of paper mill effluent with sewage by yeasts.
AIT Thesis. Bangkok, Thailand.
Dincer A.R. and Kargi F. (1999). Salt Inhibition of Nitrification and Denitrification in Saline
Wastewater. Environ. Tech., 20, 1147-1153.
Dignac M. F., Urbain V., Rybacki D., Bruchet A., Snidaro D. and Scribe P. (1998). Chemical
description of extracellular polymers: Implication on activated sludge floc structure. Wat. Sci.
Tech.,38 (8-9) 45-53.
Dincer. A.R. and Kargi F. (2000). Use of Halophilic Bacteria in Biological Treatment of
Saline wastewater by fed-batch operation. Wat. Env. Res., 72 (2) 170-174.
DOSTE-HCMC and CEFINEA (1998). Survey and set-up of list and evaluation of pollution
loads from industrial and domestic wastewater in Saigon-Dong Nai river catchment area. Ho
Chi Minh City, Department of Science, Technology and the Environment.
DOSTE-HCMC (1994). Survey on major industrial pollution sources and set-up of pollution
management and control in Ho Chi Minh City. Ho Chi Minh City, Department of Science,
Technology and the Environment.
Eikelboom D.H. (2000). Process control of activated sludge plants by microscopic
investigation. First Edition. IWA Publishing.
Ekama G. A., Dold P. L. and Marais G. V. R. (1986). Procedures for determining influent
COD fractions and the maximum specific growth rate of heterotrophs in activated sludge
systems. Wat. Sci. Tech., 18 (6) 91-114.
Elmaleh S., Defrance M.B., Ghommidh C. and Navarro J.M. (1996). Technical note:
Acidogenic effluents treatment in a yeast reactor. Wat.Res., 30 (10) 2526-2529.
EPA USA (1975). Development Document for Interim Final and Proposed Effluent
Limitations Guidelines and New Source Performance Standards for the Fruits, Vegetables and
Specialties Segment of the Canned and Preserved Fruits and Vegetable Point Source
Category. EPA-440/1-75/046. Washington D.C.

104

Feijoo G., Soto M., Mndez R. and Lema, J. M. (1995). Sodium inhibition in the anaerobic
digestion process: Antagonism and adaptation phenomena. Enzyme and Microbial
Technology, 17, 180-188.
Ferrer J. et al., (1996). Acid hydrolysis of shrimp-shell wastes and the Production of Single
Cell Protein from the hydrolysate. Bioresource Technology, 57 (1) 55-60.
Flemming H.C. and Wingender J. (2001). Relevant of microbial extracellular polymeric
substances-Part II: Technical aspects. Wat. Sci. and Tech., 43 (6) 9-16.
Ghaly A.E. and El-Taweel A.A. (1997). Continuous Ethanol Production from Cheese Whey
Fermentaion by Candida pseudotropicalis. Energy Sources, 9 (10) 1043.
Guell C., Czekaj P. and Davis R.H. (1998). Microfiltration of protein mixtures and the effects
of yeast on membrane fouling. Journ. Mem. Sci., 155, 113-122.
Hamoda M.F., and Al-Attar I.M.S. (1995). Effects of high sodium chloride concentrations on
activated sludge treatment. Wat. Sci. Tech., 31 (9) 61-72
Han K. and Levenspiel O. (1988). Extended Monod Kinetics for Substrate, Product and, Cell
Inhibition. Biotechnology and Bioengineering, 32, 430-47.
Henry D.P. and Thomson R.H. (1979). Growth of Candida ingens on supernatant from
Anaerobically fermented pig waste: Effects of temperature and pH. Appl. Env. Microbio., 37
(6) 1132-1136.
Henze M., Harremoes P., Jansen J.C. and Arvin E. (1997). Wastewater treatment- Biological
and chemical process. Second Edition, Springer-Verlag. Germany.
Hinteregger C. and Streichsbier F. (1997). Halomonas sp., a moderately halophilic strain, for
biotreatment of saline phenolic wastewater. Biotechnology Letter, 19 (11) 1099-1102.
Hodgson P.H., Leslie G.L., Schneider R.P., Fane A.G., Fell C.J.D. and Marshall K.C. (1993).
Cake resistance and solute rejection in bacterial microfiltration: The role of the extracellular
matrix. Journal of Membrane Science, 79, 35-53.
Howell J.A. and Nystrom M. (1993). Chapter 6: Fouling phenomena, In: Membranes in
Bioprocessing-Theory and Applications. Edited by Howell J.A., Sanchez V. and Field R.W.
Blackie Academic & Proffessional.
Hu T. (1989). Treatment of Vermicelli Wastewater by an acid-tolerant, starch degrading
yeast. Biological Waste, 28 (3) 163-174.
Jackson J.V. and Edwards V.H. (1975). Kinetics of substrate inhibition of exponential yeast
growth. Biotechnol. Bioeng., 17, 943-983.
Joslyn and Timmons (1967). Chapter 4: Salt-Use in Food Processing. Fundamentals of Food
Processing Operations: Ingredients, Methods and Packaging. HEID, J.L. and JOSLYN, M.A.
The Avi Publishing Co., England 1967.
Kargi F. and Dincer A.R. (1996). Effect of salt content on biological treatment of saline
wastewater by fed-batch operation. Enzyme & Microbial Technology, 19, 529-537.
Kargi F. and Uygur A. (1996). Biological Treatment of Saline Wastewater in an Aerated
Percolator Unit Utilizing Halophilic Bacteria. Env. Tech., 17, 325-320.
Kargi F. and Dincer A.R. (1998). Saline wastewater treatment by halophile-supplemented
activated sludge culture in an aerated rotating biodisc contactor. Enzyme and Microbial
Technology. 22, 427-433.
Kargi F. and Dincer A.R. (2000). Use of Halophilic bacteria in biological treatment of saline
wastewater by fed-batch operation. Wat. Environ.Res., 72 (2) 170-174.
105

Katayama-Hirayama K., Tobita S. and Hirayama K. (1994). Biodegradation of phenol and

monochlorophenols by yeast Rhodotorula glutinis. Wat. Sci. Tech., 30 (9) 59-66.
Krauth K. and Staab K.F. (1993). Pressurized bioreactor with membrane filtration for
wastewater treatment. Wat. Res., 27 (3) 405-411.
Lark N., Xia Y., Qin C., Gong C.S. and Tsao G.T. (1997). Production of Ethanol from
Recycled Paper Sludge Using Cellulase and Yeast Kluveromyces marxianus. Biomass and
Bioenergy, 12 (2) 135-143.
Liao B.Q., Allen D.G., Droppo I.G., Leppard G.G and Liss S.N. (2001). Surface properties of
sludge and their role in bioflocculation and settleability. Wat. Res., 35 (2) 339-350.
Liebeskind M. (1999). Parameter for Dynamic Simulate of Municipal Sewage Treatment
Plant (German), Gewaessersghutz Wasser Abwasser No 171, Aachen Technical University.
Liss S.N., Droppo I.G., Flannigan D.T. and Leppard G.G. (1966). Floc architecture in
wastewater and natural riverine systems. Environ. Sci.Technol. 30 (2) 680-686.
Lu S.G., Imai T., Ukita M., Sekine M., Fukagawa M. and Nakanishi H. (1999). Fermentation
wastewater treatment in a membrane bioreactor. Environmental Technolgy, 20, 431-436.
Ludzack F.J. and Noran D.K. (1965). Tolerance of high salinity by conventional wastewater
treatment process. Journal WPCF, 37 (10) 1404-1416.
Manem and Sanderson (1996). Chapter 17: Membrane bioreactors in Water Treatment
Membrane Processes. AWWA. Lyonnaise des Eaux. Water Research Commission of South
Africa. McGraw-Hill.
Marwaha S.S., Panesar P.S. and Singh B. (1999). Effect of supplementation on the efficiency
of yeast isolates for the treatment of dairy industry effluents. Jr. of Industrial Pollution
Control, 15 (1) 1-7.
Mathieu S. and Etienne P. (2000). Estimation of wastewater biodegradable COD fractions by
combining respirometric experiments in various So/Xo ratios. Wat.Re., 34 (4) 1233-1246.
Mendez R., Omil F., Soto M. and Lema J.M. (1992). Pilot plant studies on the anaerobic
treatment of different wastewaters from a fish-canning factory. Wat. Sci. Tech. 25 (1) 37-44.
Metcalf & Eddy (1991). Wastewater Engineering-Treatment, Disposal, Reuse. Third Edition.
McGRAW-HILL.
Miskiewicz T., Oleszkiewicz J. A., Kosinska K., Koziarski S., Kramarz M. and Ziobrowski J.
(1982). Dynamic tests on yeast production from piggery effluents. Agriculture Wastes, 4, 315.
Mukai T, Takimono K., Kohno T. and Okada M. (2000). Ultrafiltration behaviour of
extracellular and metabolic products in activated sludge system with UF separation process.
Wat. Res., 34 (3) 902-908,
Mulder M. (1966). Basic principle of membrane technology. Second Edition, Kluwer
Academic Publisher, The Nertherlands.
Muller E.B., Stouthamber A.H., Verseveld H.W. and Eikelboom D.H., 1995. Aerobic
domestic wastewater treatment in a pilot plant with complete sludge retention by cross-flow
filtration. Wat. Res. 29 (4), 1179-1189.
Nagaoka H., Ueda S. and Miya A. (1996). Influence of bacterial extracellular polymers on
the membrane seperation activatd sludge process. Wat. Sci. Tech., 34 (9) 165-172.
Nazim C., Franco J.P., Suidan M.T. Urbain V., and Manem J. (1999). Characterization and
comparison of a membrane bioreactor and a conventional activated sludge system in the
106

treatment of wastewater containing high-molecular weight compounds. Wat. Env. Res., 71 (1)
64-70.
NCA (National Cannery Association Western Research Laboratory, 1971), Liquid Wastes
from Canning & Freezing Fruits and Vegetables. US EPA. Berkeley California.
Nigam J.N. (1999). Continuous ethanol production from pineapple cannery waste. Journal of
Biotechnology. 72, 197-202.
Nishihara ESRC (2001). Technical details of the yeast cycle system, Personal Communication
& Company Process Catalog.
Omil F., Mndez R and Lema J.M. (1996). Anaerobic treatment of seafood processing
wastewaters in an industrial anaerobic pilot plant. Water SA., 22 (2) 173-181.
Ortiz C.P., Steyer J.P, and Bories A. (1997). Carbon and nitrogen removal from wastewater
by Candida utilis: Kinetics aspects and mathematical modelling. Process Biochemistry, 32 (3)
179-189.
Panswad T. and Anan C. (1999). Specific Oxygen, ammonia and Nitrate Uptake Rates of a
Biological Nutrient Removal Process Treating Elevated Salinity Wastewater. Biosource
Technolog, 70, 237-243.
Park Y.H. and Choi M.H. (1999). Growth of Pichia guilliermondii A9, an osmotolerant yeast,
in waste brine generated from Kim Chi production. Bioresource Technolog,. 70, 231-236.
Pavoni J.L., Tenny M.W. and Chelberger W.F. (1972). Bacterial exocellular polymers and
biological flocculation. Journal WPCF, 44 (3) 414-431.
Pelczar M.J. and Reid R.D. (1972). Chapter 15: The yeasts. In Microbiology. McGraw-Hill
Co. Newyork, USA.
Pirbazari (1996). Hybrid membrane filtration process for leachate treatment. Wat. Res., 30,
2691-2706.
Rashad M.M., Moharib S.A. and Jwanny E.W. (1990). Yeast Conversion of Mango Waste or
Methanol to Single Cell Protein and other metabolites. Biological Wastes, 32, 277-284.
Reeslev M., Jorgensen B.B. and Jogensen O.B. (1996). Exopolysaccharide production and
morphology of Aureobasidium pullulans grown in continuous cultivation with varying
ammonium-glucose ratio in the growth medium. Journal of Biotechnology, 51, 131-135.
Ridgway H.F. and Flemming H.C. (1996). Chapter 6: Membrane biofouling in Water
Treatment Membrane Processes. AWWA. Lyonnaise de Eaux. Water Research Commission
of South Africa. McGraw-Hill.
Sall A.J. (1961). Fundamental principles of bacteriology. McGraw-Hill. Newyork.
Scholz W. and Fuchs W. (2000). Treatment of oil contaminated wastewater in a membrane
bioreactor. Wat. Res., 34 (14) 3621-3629.
Scioli C. and Vollaro L. (1997). Use of Yarrowia lipolytica to Reduce Pollution in Olive Mill
Wastewaters. Wat. Res., 31 (10) 2520 2524.
Scott J.A. and Smith K.L (1997). A bioreactor coupled to a membrane to provide aeration and
filtration in ice-cream factory wastewater remediation. Wat. Res., 31 (1) 69-74.
Sheintuch M., Lev Q., Einav and Rubin E. (1986). Role of exocellular polymer in the design
of activated sludge. Biotech. and Bioeng., 18, 1564-1576.
Simard R.E. (1971). Yeasts as a indicator of pollution. Marine Pollution Bulletin, 2 (8) 5-10.
107

Simard R.E. and Blackwood A.C. (1971). Ecological studies on yeasts in the St. Lawrence
River. Can. Microbio., 17, 353-358.
Simard R.E. and Cameron A. (1974). Fermentation of spent sulphite liquor by Candida utilis.
Pulp & Paper Magazine of Canada, 75 (3) 117-120.
Simard R.E. and Thanh N. C. (1973). Biological treatment of domestic sewage by yeast and
fungi. AlChE Symposium Series.
Simard R.E., Busque G. and Riel R.R. (1973). Traitement Biologique des Eaux Uses de
Croustilles par les Levures. Can. Inst. Food Sci. Technol. J., 6 (1) 33-37.
Soderquist, M.R.(1971). Current Practice in Seafood Processing Waste Treatment.
Department of Food Science and Technology, Oregon State University. EPA, Water Quality
Office.
Tellez G. T., Nirmalakhandan N. and Gardea-Torresdey J. L. (1995). Evaluation of biokinetic
coefficients in degradation of oilfield produced water under varying salt concentrations. Wat.
Res., 29 (7) 1711-1718.
Thanh, N.C. and Simard, R.E., (1971). Biological Treatment of Wastewater by Yeasts.
Journal WPCF, 45 (4) 674-680.
Tokuz R.Y. and Eckenfelder W.W. (1979). The effect of inorganic salts on the activated
sludge process performance. Wat. Res., 13, 99-104.
Trouve E, Urbain V. and Manem J. (1994). Treatment of municipal wastewater by a
membrane bioreator: Results of a semi-industrial pilot-scale study. Wat. Sci. Tech. 40 (4) 151157.
UNEP (1999). Industrial Sector Guide. Cleaner Production Assessment in Fish Processing
Industry. Danish Environmental Protection Agency in Co-operation with COWI Consulting
Engineering and Planners AS.
Urbain V., Block J.C. and Manem J. (1993). Bioflocculation in activated sludge: An analytic
approach. Wat. Res., 27 (5) 829-839.
Vanrolleghem P.A., Spanjers H., Petersen B., Ginestet P. and Takacs, I. (1999). Estimating
(combinations of) activated sludge model No.1 parameters and components by respirometry.
Wat.Sci.Tech., 39 (1), 195-214.
Vijaranakul U, Nadakavukaren M.J., Bayles D.O., Wilkinson B.J. and Jayaswal R.K. (1997).
Characterization of an NaCl-sensitive phenotype by Glycine Betain but not by other
compatible solutes. Appl. Environ. Micro., 63 (5) 1889-1897.
Vicente A., Castrillo J.I., Teixeira J.A. and Ugalde U. (1998). On-line estimation of biomass
through pH contron analysis in aerobic yeast fermentation systems. Biotech. Bioeng., 58 (4)
445-450.
Visvanathan C., Aim B. R. and Parameshwaran (2000). Membrane separation bioreactors for
wastewater treatment. Critical Reviews in Environmental Science and Technolog, 30 (1) 1-48.
Visvanathan C., Yang S.B., Muttamara S. and Maythanukhraw R. (1997). Application of air
backflushing technique in membrane bioreactor. Wat. Sci. Tech., 36 (12) 259-266.
Watanabe W., Sasaki K., Nakashimada Y., Kakizono T., Noparatnaraporn N. and Nishio N.
(1998). Growth and flocculation of a marine photosynthetic bacterium Rhodovulum sp. Apl.
Microbiol. Biotechnol., 50, 682-691.

108

Westhuizen T.H. and Pretorius W.A. (1998. Use of filamentous Fungi for the purification of
industrial effluents. Report to the Water Research Commission by the Department of
Chemical Engineering-University of Pretoria. No. 535/1/98/. South Africa.
Woodroof J.G. (1975). Chapter 14: Plant Sanitation and Waste Disposal in Commercial
Vegetable Processing. Avitextbook.
Woolard C.R. and Irvine R..L. (1995). Response of a periodically operated halophilic biofilm
reactor to changes in salt content. Wat. Sci. Tech., 31(1) 41-50.
Woolard C.R. and Irvine R.L. (1995). Treatment of hypersaline wastewater in the sequencing
batch reactor. Wat. Res., 29 (4) 1159-1168.
Yamamoto K., Hiasa M., Mahmood T., and Matsuo T. (1989). Direct solid-separation using
hollow fiber membrane in an activated sludge aeration tank. Wat. Sci. Tech., 21, 43-54.
Yang L., Lai C.T. and Shieh W.K. (2000). Biodegradation of dispersed diesel fuel under high
salinity conditions. Wat. Res., 34 (13) 3303-3314.
Yu S., Wayman M. and Parekh S.K. (1987). Fermentation to ethanol of Pentose containing
spent sulphite liquor. Biotechnology and Bioengineering,16, 1144-1150.

109

Fig. A-1 Y
east colonies cultured with glucose-feed wastewater containing high salt concentrations

Round, smooth
colony
Irregular, rough
colony

Fig. A-2 Two predominant yeast colonies cultured with glucose-feed wastewater

10 Pm

10 Pm

Fig. A-3 rPedominant yeasts in the batch culture with glucose-feed wastewater
containing 32 g/L salt (x 1500)

A-1

20 Pm

Fig. A-4 rPedominant yeasts in the batch culture with protein-feed wastewater
containing 32 g/L salt (x 500)

20 Pm

Fig. A-5 rPedominant yeasts in the batch culture with protein-feed wastewater
containing 45 g/L salt (G
rowth of mycelia yeasts occurred)( x 500)

A-2

20 Pm

Fig. A-6 Mycelia yeasts c( ultured with fish-protein wastewater)( x 800)

20 Pm

Fig. A-7 eYast flocs formed by interwinding of mycelia yeasts

s( ettled sludge in theY
MBR)( x 500)

A-3

20 Pm

Fig. A-8 Fine yeast cells suspended in supernatant of M

Y BR

10 Pm

Protozoa

Fig. A-9 Free-living ciliates (protozoa)grew well in the yeast mixture of Y

MBR
i(n COD loading) (x 250)

A-4

10 Pm

Fig. A-10 Bacterial flocs in the batch culture with protein-feed wastewater at 15 g/L salt
r(ounded and compacted sludge flocs) (x250)

10 Pm

Fig. A-11 Bacterial flocs in the batch culture with protein-feed wastewater at 32 g/L salt
O
( pen and weakflocs)( x 250)

10 Pm

Fig. A-12 Bacterial flocs in the BMBR

F
( ine and weakflocs) (x 250)

A-5

Fig. A-13 Respirometer system (Respirometer, recorder, DO meter and thermostat)

Fig. A-14 Respirometer

A-6

Fig. A-15 Respirogram (low COD dose)

Fig. A-16 Respirogram (high COD dose)

A-7

Fig. A-17 Y
east (Y
MBR)and bacterial membrane
B
( MBR)reactors

A-8

Fig. A-18 Hg-U tube p( ressure measurement)

Fig. A-19 eYast reactor and settling tank

Fig. A-20 Full clogged membrane of Y

MBR

A-9

Fig. A-23 Membrane clogging

Fig. A-21 Fu ll clogged membrane of BMBR

Fig. A-22 Clean membrane

Table B-1

Time
day

Salt
20.5
19.0
18.9
19.6
20.0
20.9
20.6
21.3
21.3
21.3
20.0
20.5
20.5
20.5
20.5

Table B-2

day

0
2
4
5
7
9
11
13
15
17
19
28
30
38
40
42
44
46

Cl

g/L NaCl g/L Cl

2
4
5
7
9
11
13
15
17
19
28
40
42
44
46

Time

Acclimation of mixed bacterial sludge to 20 g salt/L with the glucose-feed

wastewater

12.9
12.9
12.2
12.4
12.4
12.0
12.4

Ecod

MLSS

mg/L

mg/L

mg/L

24
23
22
24
24
29
28
25
25
26
24
22
15
14
17

952
931
940
930
930
1,010
870
915
915
915
1,090
1,070
1,070
1,070
1,070

175
143
143
154
136
32
51
35
60
55
52
102
127
24
71

81.6
84.6
84.8
83.4
85.4
96.8
94.1
96.2
93.4
94.0
95.2
90.5
88.1
97.8
93.4

2,116
2,120
2,132
2,274
2,488
2,640
2,516
2,668
2,672
2,888
2,750
2,724
2,760
2,837
2,993

HRT

CODin CODeff

pHo

pHt

SVI
mL/g

6.79
7.17
6.76
6.99
7.61
7.80
7.75
7.84
7.69
7.32
7.80
7.47
7.75
6.97
7.64

6.79
7.47
6.35
6.11
7.01
7.10
6.85
6.40
6.33
6.22
6.26
6.28
6.52
6.16
6.53

88
83
81
71
70
80
87
47
45
43
36
34
35
34
32

Acclimation of mixed bacterial sludge to 32 g salt/L with the glucose-feed

wastewater
Salt

Cl

g/L NaCl g/L Cl

20.5
19.8
20.9
24.9
25.0
26
26.6
29
31.8
32.6
34
33.5
33.9
32.6
33.6
32.2
32.2
32

20.3
20.6
19.8
20.4
19.6
19.6
20.0

HRT

CODin CODeff

Ecod

MLSS

mg/L

mg/L

mg/L

24
23
22
24
24
24
24
26
26
25
25
25
26
24
24
15
14
17

952
931
940
930
930
950
950
950
870
870
915
915
915
1,090
1,090
1,090
1,090
1,090

181
145
134
185
121
145
95
100
120
31
12
88
61
169
118
125
23
29

81.0
84.4
85.7
80.1
87.0
84.7
90.0
89.5
86.2
96.4
98.7
90.4
93.3
84.5
89.2
88.5
97.9
97.3

2,072
1,948
2,152
2,324
2,292
2,208
2,190
2,176
2,328
2,576
2,852
3,260
3,000
2,300
2,164
2,253
2,337
2,501

B-1

pHo

pHt

SVI
mL/g

6.79
7.11
6.89
6.86
7.44
7.51
7.5
7.85
7.73
7.84
7.85
7.28
7.31
7.42
7.43
7.44
7.22
7.46

6.88
7.65
6.34
6.06
6.64
7.33
6.88
7.67
7.61
6.75
7.19
7.93
7.93
7.24
7.41
7.72
7.23
7.99

91
79
81
62
71
76
78
82
82
74
49
40
43
24
25
23
29
16

Table B-3

Time
day

Salt
20.5
19.3
21.4
24.8
24.9
25.5
27.4
30.5
33.5
33.5
37.4
40.0
42.0
42.2
46.2
45.3
43.5
44.3
47.0
44.4

Table B-4

day

0
2
4
6
8
10
21
23
33
35
37
39

Cl

g/L NaCl g/L Cl

0
2
4
5
7
9
11
13
15
17
19
21
23
25
26
28
30
38
40
42

Time

Acclimation of mixed bacterial sludge to 45 g salt/L with the glucose-feed

wastewater

27.5
26.4
26.9
28.6
27.0

HRT

CODin CODeff

Ecod

MLSS

mg/L

mg/L

mg/L

24.0
23.0
22.0
24.0
24.0
24.0
24.0
24.0
25.0
25.0
26.0
25.0
26.0
25.0
24.0
24.0
24.0
24.5
24.0
24.0

952
931
940
930
930
1,010
1,010
1,010
870
870
875
915
1,031
1,031
1,031
1,031
1,031
1,090
1,090
1,090

415
420
431
445
395
375
310
325
215
201
215
197
220
231
205
157
120
135
148
167

56
55
54
52
58
63
65
70
67
77
75
78
79
81
80
85
88
88
86
89

2,040
1,888
2,100
1,738
2,408
2,344
2,143
2,048
2,200
2,412
2,792
2,748
2,848
2,936
3,252
3,270
3,310
3,100
3,400
3,230

pHo

pHt

SVI
mL/g

6.8
6.93
6.78
6.89
7.51
7.33
7.2
7.7
7.85
7.87
7.85
7.85
7.14
7.85
7.26
7.38
7.42
7.48
7.54
7.56

6.81
7.5
6.37
6.47
7.71
7.51
6.68
6.64
7.72
6.89
7.31
7.41
7.86
7.2
7.75
7.93
7.92
7.79
7.98
7.98

86.5
79.4
71.8
81.7
62.3
64.0
70.0
75.0
70.5
71.3
58.7
62.2
54.1
48.7
44.9
35.9
33.1
21.0
18.0
18.0

Acclimation of mixed yeast at 20 g salt/L with the glucose-feed wastewater

Salt

Cl

g/L NaCl g/L Cl

20.5
21.5
20.8
21.1
20.6
20.1
20.1
20.9
20.2
20.0
20.0
20.6

15.9
15.9
14.5
23.1
14.5
14.6
15.5

HRT

CODin

CODeff

Ecod

MLSS

mg/L

mg/L

mg/L

24
24
26
26
25
27
26
27
22
15
21
15

5,100
5,100
5,100
4,980
7,400
7,400
7,400
5,350
7,490
7,490
7,490
7,490

325
305
370
272
363
1,230
1,270
447
352
352
331
444

93.6
94.0
92.7
94.5
95.1
83.4
82.8
91.6
95.3
95.3
95.6
94.1

3,700
5,420
6,732
7,916
6,676
6,416
5,028
6,596
7,756
9,340
10,367
12,216

B-2

pHo

pHt

5.61
5.87
5.97
5.54
5.64
5.72
5.66
3.50
3.57
5.00
4.79
4.07

3.05
2.86
2.92
2.91
2.54
3.25
4.22
2.93
2.37
2.40
2.51
2.32

Table B-5
Time
day

Acclimation of mixed yeast at 32 g salt/L with the glucose-feed wastewater

Salt

Cl

0
2
4
5
7
9
11
13
15
17
28
30
38
40
42
44
46
Table B-6

32.8
33.0
32.3
32.3
31.3
32.6
33.5
32.2
32.6
33.8
31.9
31.7
32.0
31.7
32.0
33.0
31.8

CODin

CODeff

Ecod

MLSS

mg/L

mg/L

mg/L

24
24
25
24
24
24
27
27
25
26
24
24
24
23
24
21
24

4,980
4,980
4,980
4,980
4,980
5,100
4,980
4,980
7,400
7,400
7,400
7,400
7,500
7,400
7,400
7,400
7,400

920
1,010
415
428
351
368
413
409
355
382
478
423
369
449
396
278
411

81.5
79.7
91.7
91.4
93.0
92.8
91.7
91.8
95.2
94.8
93.5
94.3
95.1
93.9
94.6
96.2
94.4

4,679
4,710
5,704
5,828
3,804
4,242
6,596
8,040
7,496
10,168
9,312
10,152
13,944
14,324
14,713
14,557
15,550

23.0
23.5
22.5
21.7
22.5
22.5
21.1

pHo

pHt

4.09
5.52
5.32
5.51
5.49
5.84
5.92
5.56
5.64
5.46
5.78
5.08
4.46
5.14
4.56
5.36
4.57

2.78
2.83
2.83
3.13
3.07
2.86
2.98
2.84
2.50
3.34
2.78
2.52
2.64
2.65
2.59
2.58
2.53

Acllimation of mixed yeast to 45 g salt/L with the glucose-feed wastewater

Time
Salt
Cl
day g/L NaCl g/L Cl

0
2
4
5
7
9
11
13
15
17
30
40
42
44
46

HRT

g/L NaCl g/L Cl

32.0
32.0
32.0
35.0
38.0
40.8
43.3
45.3
44.1
43.1
44.9
45.0
44.1
45.0
45.0

31.7
31.6
29.1
30.7
30.1
31.7
32.2

HRT
h

F/M
d

CODin
mg/L

CODeff
mg/L

Ecod
%

MLSS
mg/L

pHo

pHt

23
24
24
26
25
24
23
24
24
25
27
24
26
24
24

1.12
1.15
0.82
0.63
0.58
0.56
0.55
0.51
0.50
0.46
0.38
0.36
0.32
0.33
0.32

5,100
5,100
4,980
4,980
4,980
5,050
5,100
5,100
5,100
5,050
5,100
5,200
5,200
5,200
5,100

1,720
1,560
1,150
1,095
950
765
680
719
539
471
369
345
362
411
342

66.3
69.4
76.9
78.0
80.9
84.9
86.7
85.9
89.4
90.7
92.8
93.4
93.0
92.1
93.3

3,768
3,984
6,100
7,920
8,652
8,950
9,240
10,060
10,144
10,984
13,480
14,456
16,047
15,733
15,967

5.54
5.88
5.97
5.66
5.84
5.68
5.66
5.43
5.79
5.26
4.99
5.00
5.23
5.37
4.88

3.12
2.92
2.96
2.92
2.77
2.87
2.83
2.73
2.84
2.53
2.54
2.4
2.53
2.49
2.54

B-3

Table B-7

Acclimation of mixed yeasts to protein-feed wastewater at 20 g/L salt

Time

CODin

CODeff

days

mg/L

mg/L

0
2
4
6
8
10
12
14
16

4930
5120
5210
5020
4970
4760
5010
5150
5090

1587
1667
1456
1243
809
752
567
458

Table B-8

COD%

69
68
71
75
83
85
89
91

MLSS

F/M

mg/L

d-1

5450
5400
5870
6780
7920
9250
9350
10850
10750

0.63
0.59
0.49
0.42
0.34
0.36
0.32
0.32

Acclimation of mixed yeasts to protein-feed wastewater at 32 g/L salt

Time

CODin

CODeff

MLSS

F/M

days

mg/L

mg/L

mg/L

d-1

0
2
4
6
8
10
12
14
16

5010
4850
5120
4950
4760
5230
5100
5010
5120

1795
1640
1730
1190
1200
870
701
770

5700
5400
5990
5930
6950
7050
8345
9050
9710

0.60
0.57
0.56
0.46
0.49
0.41
0.37
0.35

Table B-9

COD%

63
68
65
75
77
83
86
85

Acclimation of mixed yeasts to protein-feed wastewater at 45 g/L salt

Time

CODin

CODeff

days

mg/L

mg/L

0
2
4
6
8
10
12
14
16

4790
5100
5020
4970
5120
5070
5025
4790
4990

2091
1640
1730
1587
1200
870
701
770

COD%

59
61
61
69
72
83
84
83

B-4

MLSS

F/M

mg/L

d-1

5500
5200
5350
5320
6240
8705
8956
9310
9420

0.65
0.63
0.62
0.55
0.39
0.37
0.34
0.35

Table B-10 Acclimation of mixed bacterial sludge to protein-feed wastewater at 20 g/L salt
Time

CODin

CODeff

days

mg/L

mg/L

0
2
4
6
8
10
12
14
16

1020
990
1070
1120
1010
980
990
1130

92
79
86
56
51
29
50
45

COD%

91
92
92
95
95
97
95
96

MLSS

F/M

mg/L

d-1

3120
3650
4270
4750
5120
5250
5430
5910
5670

0.28
0.23
0.23
0.22
0.19
0.18
0.17
0.20

Table B-11 Acclimation of mixed bacterial sludge to protein-feed wastewater at 32 g/L salt
Time

CODin

CODeff

MLSS

F/M

days

mg/L

mg/L

mg/L

d-1

165
78
90
51
73
38
78
55

3560
3750
4340
4950
5120
5010
5700
5600
6310

0.29
0.23
0.20
0.20
0.21
0.17
0.17
0.18

0
2
4
6
8
10
12
14
16

1100
980
995
1020
1050
950
970
1105

COD%

85
92
91
95
93
96
92
95

Table B-12 Acclimation of mixed bacterial sludge to protein-feed wastewater at 45 g/L salt
Time

CODin

CODeff

days

mg/L

mg/L

0
2
4
6
8
10
12
14
16

1010
1200
1105
1030
970
990
1025
990

222
228
221
175
175
109
113
89

COD%

78
81
80
83
82
89
89
91

B-5

MLSS

F/M

mg/L

d-1

3950
3650
3750
4210
4760
4790
5700
5970
6105

0.28
0.32
0.26
0.22
0.20
0.17
0.17
0.16

Table B-13 COD and DO profile data of the mixed yeast batch culture with glucose-feed
wastewater at 20g/L salt
Time

COD

DO

mg/L

mg/L

0.0
2.5
5.0
7.5
9.0
10.5

5020
1250
220
230
205
220

1.2
6.4
6.3
6.4
6.4

COD%

MLSS

g/g.d

mg/L

8250
75
95.6
95.4
95.9
95.6

4.15
2.65
1.76
1.48
1.26
Average

9130
8700

Table B-14 COD and DO profile data of the mixed yeast batch culture with glucose-feed
wastewater at 32 g/L salt
Time

COD

DO

mg/L

mg/L

0.0
2.5
5.0
8
9.0
10.5
13

4950
2750
1505
540
255
210
245

0.7
0.9
4.2
6.3
6.2
6.3

COD%

MLSS

g/g.d

mg/L

9350
45
69.9
89.2
94.9
95.8
95.1

2.25
1.75
1.49
1.32
1.14
0.91
Average

9645
9500

Table B-15 COD and DO profile data of the mixed yeast batch culture with glucose-feed
wastewater at 45 g/L salt
Time

COD

DO

mg/L

mg/L

0.0
2.5
5.0
7.5
9.0
10.5
13

5050
4200
2740
2000
1560
790
290

0.7
0.9
4.2
6.3
6.2
6.3

COD%

MLSS

g/g.d

mg/L

9360
16
45
60
69
84
94

B-6

0.80
1.12
0.99
0.95
1.00
0.90
Average

10130
9750

Table B-16 COD and DO profile data of the mixed bacterial batch culture with glucose-feed
wastewater at 20 g/L salt
Time

COD

DO

mg/L

mg/L

0
0.25
1.25
2.5
3.5
6.5

1036
821
146
20
26
45

1.97
6.3
6.3
6.5
6.4

COD%

MLSS

g/g.d

mg/L

2752
18
85
98
97
96

5.84
5.57
3.20
2.27
1.20
Average

3351
3050

Table B-17 COD and DO profile data of the mixed bacterial batch culture with glucose-feed
wastewater at 32 g/L salt
Time

COD

DO

mg/L

mg/L

0
0.25
1.25
2.25
3.5
6.5
8
11.5
14.5

1020
760
320
235
160
45
30
35
25

2.1
4.95
5.7
6.2
6.3
6.4
6.3
6.4

COD%

MLSS

g/g.d

mg/L

3245
24
68
77
84
96
97
97
98

6.44
3.65
2.28
1.61
0.99
0.81
0.56
0.45
Average

4053

3650

Table B-18 COD and DO profile data of the mixed bacterial batch culture with glucose-feed
wastewater at 45 g/L salt
Time

COD

DO

mg/L

mg/L

0.25
1.25
2.25
3.5
6.5
8.5
11.5
14.5
17

945
836
769
578
405
259
218
113
70

0.9
1.2
2.3
3.4
4.7
5.4
5.9
6.1
6.2

COD%
6
16
23
42
60
74
78
89
93

B-7

MLSS

g/g.d

mg/L

1.65
0.98
0.77
0.90
0.69
0.65
0.51
0.46
0.41
Average

3450

3200

Table B-19 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed
wastewater at 20 g/L salt
Time

COD

DO

mg/L

mg/L

0.0
2.5
5.5
7.5
10.5
16.0
18.5
20.5
26.0
29.0
31.0
34.0
37.0
42.0

5010
3180
2800
2490
2191
1650
1500
1010
790
680
490
540

COD%

U
g/g.d

MLSS
mg/L

5432
0.6
0.7
0.7
0.6
0.7
0.6
0.6
0.6
2.2
4.5
4.9
5.1
5.6

36.5
44.1
50.3
56.3
67.1
70.1

1.32
1.17
0.95
0.70
0.72
0.68

79.8
84.2
86.4
90.2
89.2

0.55
0.54
0.51
0.48
0.42
Average

6670

6050

Table B-20 COD and DO profile data of the mixed yeast batch culture with protein-feed
wastewater at 32 g/L salt
Time

COD

DO

mg/L

mg/L

0.0
5.5
7.5
10.5
16.0
18.5
20.5
26.0
29.0
32.0
34.0
37.0
42.0

5010
3234
2707
2392
2191
2031
1913
968
772
830
584
541

COD%

MLSS

g/g.d

mg/L

5325
0.7
0.7
0.6
0.7
0.6
0.6
0.6
2.2
4.0
4.2
4.3
5.6

B-8

35.4
46.0
52.3
56.3
59.5
61.8

1.33
1.27
1.03
0.73
0.67
0.62

80.7
84.6
83.4
88.3
89.2

0.58
0.55
0.51
0.49
0.44
Average

6302

5810

Table B-21 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed
wastewater at 45 g/L salt
Time

COD

DO

mg/L

mg/L

0.0
2.5
5.5
7.5
10.5
16.0
18.5
20.5
26.0
29.0
32.0
34.0
37.0
42.0

5010
4030
3830
3690
3230
2700
2590
1500
1330
1240
950
920

COD%

U
g/g.d

MLSS
mg/L

5915
0.3
0.2
0.4
0.4
0.5
0.4
0.5
0.5
0.8
1.1
2.5
3.9
4.1

19.6
23.6
26.3
35.5
46.1
48.3

0.67
0.59
0.47
0.42
0.47
0.44

70.1
73.5
75.2
81.0
81.6

0.45
0.43
0.41
0.41
0.36
Average

6946

6430

Table B-22 COD and DO profile data of the mixed bacterial batch culture with fish-proteinfeed wastewater at 20 g/L salt
Time

COD

DO

mg/L

mg/L

0
0.25
1.25
2.25
3.5
6.5
9.0
11.5
14.5

1020
805
619
416
300
143
40
25
35

1.9
2.1
4.2
4.5
5.9
6.5
6.4
6.5

COD%

21
39
59
71
86
96
98
97

B-9

MLSS

g/g.d

mg/L

4.80
1.79
1.50
1.15
0.75
0.61
0.48
0.38
Average

3730

4870

4300

Table B-23 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed
wastewater at 32 g/L salt
Time

COD

DO

mg/L

mg/L

0
0.25
1.25
2.25
3.5
6.5
9
11.5
14.5
18.5
21.0
26.0
28.0

976
870
645
568
509
383
320
200
110
65
50
45
20

1.6
1.8
2.5
4.0
5.7
6.0
6.2
6.3
6.2
6.3
6.2
6.2

COD%

MLSS

g/g.d

mg/L

3270
11
34
42
48
61
67
80
89
93
95
95
98

2.80
1.75
1.20
0.88
0.60
0.48
0.45
0.39
0.33
0.29
0.24
0.23
Average

4172

3721

Table B-24 COD and DO profile data of the mixed yeast batch culture with fish-protein-feed
wastewater at 45 g/L salt
Time

COD

DO

mg/L

mg/L

0
0.25
1.25
2.25
3.5
6.5
9.0
11.5
14.5
18.5
21.0
26.0
28.0
30.0

985
935
941
888
760
691
534
473
337
298
206
127
90
75

0.7
0.9
1.5
1.5
1.6
2.4
3.8
5.5
5.7
6
5.7
6.2
6.3

COD%

MLSS

g/g.d

mg/L

3650
5
4
10
23
30
46
52
66
70
79
87
91
92

1.17
0.21
0.25
0.37
0.26
0.29
0.26
0.26
0.22
0.22
0.19
0.19
0.21

4589
4120

B-10

Salt 20
Yeast
Time
0
2
4
6
8
10
12
14
16

Bacteria
Time
0
2
4
6
8
10
12
14
16

CODin
CODeff
COD%y
MLSSy
F/M
4930
5450
5120
1587
69
5400
5210
1667
68
5870
5020
1456
71
6780
4970
1243
75
7920
4760
809
83
9250
5010
752
85
9350
5150
567
89
10850
5090
458
91
10750

CODin

1020
990
1070
1120
1010
980
990
1130

CODeff

COD%
92
79
86
56
51
29
50
45

91
92
92
95
95
97
95
96

MLSS
F/M
3120
3650
4270
4750
5120
5250
5430
5910
5670

0.63
0.59
0.49
0.42
0.34
0.36
0.32
0.32

0.28
0.23
0.23
0.22
0.19
0.18
0.17
0.20

Salt 32
Yeast
Time
0
2
4
6
8
10
12
14
16

Bacteria
Time
0
2
4
6
8
10
12
14
16

CODin
CODeff
COD%y
MLSSy
F/M
5010
5700
4850
1795
63
5400
5120
1640
68
5990
4950
1730
65
5930
4760
1190
75
6950
5230
1200
77
7050
5100
870
83
8345
5010
701
86
9050
5120
770
85
9710

CODin

1100
980
995
1020
1050
950
970
1105

CODeff
165
78
90
51
73
38
78
55

COD%
85
92
91
95
93
96
92
95

MLSS
F/M
3560
3750
4340
4950
5120
5010
5700
5600
6310

0.60
0.57
0.56
0.46
0.49
0.41
0.37
0.35

0.29
0.23
0.20
0.20
0.21
0.17
0.17
0.18

Salt 45
Yeast
Time
0
2
4
6
8
10
12
14
16

Bacteria
Time
0
2
4
6
8
10
12
14
16

CODin
CODeff
COD%y
MLSSy
F/M
4790
5500
5100
2091
59
5200
5020
1640
61
5350
4970
1730
61
5320
5120
1587
69
6240
5070
1200
72
8705
5025
870
83
8956
4790
701
84
9310
4990
770
83
9420

CODin

1010
1200
1105
1030
970
990
1025
990

CODeff
222
228
221
175
175
109
113
89

COD%
78
81
80
83
82
89
89
91

MLSS
F/M
3950
3650
3750
4210
4760
4790
5700
5970
6105

0.65
0.63
0.62
0.55
0.39
0.37
0.34
0.35

0.28
0.32
0.26
0.22
0.20
0.17
0.17
0.16

200

400

600

800

1000

1200

10

15

CODBf

20

25

30

35

CODBf

S32
Time5000 CODy-pr DOy-pr
COD%
U
0.0
5010
2.5
0.6
35.4
5.5
3234
0.7
46.0
7.5
2707
0.7
9.0
52.3
10.5
2392
0.6
12.5
56.3
16.0
2191
0.7
59.5
18.5
2031
0.6
61.8
20.5
1913
0.6
26.0
0.6
80.7
29.0
968
2.2
84.6
32.0
772
4.0
83.4
34.0
830
4.2
88.3
37.0
584
4.3
89.2
42.0
541
5.6

S20
Time5000 CODy-pr DOy-pr
%COD
U
0.0
5010
2.5
0.6
36.5
5.5
3180
0.7
44.1
7.5
2800
0.7
9.0
50.3
10.5
2490
0.6
12.5
56.3
16.0
2191
0.7
67.1
18.5
1650
0.6
70.1
20.5
1500
0.6
26.0
0.6
79.8
29.0
1010
2.2
84.2
31.0
790
4.5
86.4
34.0
680
4.9
90.2
37.0
490
5.1
89.2
42.0
540
5.6

0.58
0.55
0.51
0.49
0.44

0.73
0.67
0.62

1.03

1.33
1.27

0.55
0.54
0.51
0.48
0.42

0.70
0.72
0.68

0.95

1.32
1.17

5810

6302

MLSS
5325

6050

6670

MLSS
5432

S45
Time5000 CODy-pr DOy-pr
COD%
U
0.0
5010
2.5
0.3
19.6
5.5
4030
0.2
23.6
7.5
3830
0.4
9.0
26.3
10.5
3690
0.4
12.5
35.5
16.0
3230
0.5
46.1
18.5
2700
0.4
48.3
20.5
2590
0.5
26.0
0.5
70.1
29.0
1500
0.8
73.5
32.0
1330
1.1
75.2
34.0
1240
2.5
81.0
37.0
950
3.9
81.6
42.0
920
4.1
0.45
0.43
0.41
0.41
0.36

0.42
0.47
0.44

0.47

0.67
0.59

6430

6946

MLSS
5915

S32
Time5000 CODy-pr DOy-pr
COD%
U
0.0
5010
2.5
0.6
35.4
5.5
3234
0.7
46.0
7.5
2707
0.7
9.0
52.3
10.5
2392
0.6
12.5
56.3
16.0
2191
0.7
59.5
18.5
2031
0.6
61.8
20.5
1913
0.6
26.0
0.6
80.7
29.0
968
2.2
84.6
32.0
772
4.0
83.4
34.0
830
4.2
88.3
37.0
584
4.3
89.2
42.0
541
5.6

S20
Time5000 CODy-pr DOy-pr
%COD
U
0.0
5010
2.5
0.6
36.5
5.5
3180
0.7
44.1
7.5
2800
0.7
9.0
50.3
10.5
2490
0.6
12.5
56.3
16.0
2191
0.7
67.1
18.5
1650
0.6
70.1
20.5
1500
0.6
26.0
0.6
79.8
29.0
1010
2.2
84.2
31.0
790
4.5
86.4
34.0
680
4.9
90.2
37.0
490
5.1
89.2
42.0
540
5.6

0.58
0.55
0.51
0.49
0.44

0.73
0.67
0.62

1.03

1.33
1.27

0.55
0.54
0.51
0.48
0.42

0.70
0.72
0.68

0.95

1.32
1.17

5810

6302

MLSS
5325

6050

6670

MLSS
5432

S45
Time5000 CODy-pr DOy-pr
COD%
U
0.0
5010
2.5
0.3
19.6
5.5
4030
0.2
23.6
7.5
3830
0.4
9.0
26.3
10.5
3690
0.4
12.5
35.5
16.0
3230
0.5
46.1
18.5
2700
0.4
48.3
20.5
2590
0.5
26.0
0.5
70.1
29.0
1500
0.8
73.5
32.0
1330
1.1
75.2
34.0
1240
2.5
81.0
37.0
950
3.9
81.6
42.0
920
4.1
0.45
0.43
0.41
0.41
0.36

0.42
0.47
0.44

0.47

0.67
0.59

6430

6946

MLSS
5915

Time

S20
Time

0
0.25
1.25
2.25
3.5
6.5
9
11.5
14.5
18.5
21.0
26.0
28.0

0
0.25
1.25
2.25
3.5
6.5
9
11.5
14.5

CODBf
DOBf
COD%
U
976
870
1.6
11
645
1.8
34
568
2.5
42
509
4.0
48
383
5.7
61
320
6.0
67
200
6.2
80
110
6.3
89
65
6.2
93
50
6.3
95
45
6.2
95
20
6.2
98

CODBf
DOBf
COD%
U
1020
805
1.9
21
619
2.1
39
416
4.2
59
300
4.5
71
143
5.9
86
40
6.5
96
25
6.4
98
35
6.5
97

2.80
1.75
1.20
0.88
0.60
0.48
0.45
0.39
0.33
0.29
0.24
0.23

4.80
1.79
1.50
1.15
0.75
0.61
0.48
0.38

3721

4172

MLSS
3270

4300

4870

3730

MLSS

4300

S45
Time
0
0.25
1.25
2.25
3.5
6.5
9
11.5
14.5
18.5
21.0
26.0
28.0
30.0

CODBf
DOBf
COD%
U
985
935
0.7
5
4
941
0.9
10
888
1.5
23
760
1.5
30
691
1.6
534
2.4
46
52
473
3.8
66
337
5.5
298
5.7
70
79
206
6
87
127
5.7
91
90
6.2
75
6.3
92
1.17
0.21
0.25
0.37
0.26
0.29
0.26
0.26
0.22
0.22
0.19
0.19
0.21

4120

4589

MLSS
3650

45 g/L
Time

32 g/L
Time

20 g/L
Time

0
0.25
1.25
2.25
3.5
6.5
8.5
11.5
14.5
17

0
0.25
1.25
2.25
3.5
6.5
8
11.5
14.5

0
0.25
1.25
2.5
3.5
6.5
8.5

CODB2
DOB2
COD%
1000
945
0.9
6
16
836
1.2
23
769
2.3
42
578
3.4
60
405
4.7
74
259
5.4
78
218
5.9
89
113
6.1
93
70
6.2

CODB2
DOB2
COD%
1020
760
2.1
24
68
320
4.95
77
235
5.7
84
160
6.2
96
45
6.3
97
30
6.4
97
35
6.3
98
25
6.4

CODB2
DOB2
COD%
1036
821
1.97
18
146
6.3
85
20
6.3
98
26
6.5
97
45
6.4
96
15
6.4
99

1.65
0.98
0.77
0.90
0.69
0.65
0.51
0.46
0.41

6.44
3.65
2.28
1.61
0.99
0.81
0.56
0.45

5.84
5.57
3.20
2.27
1.20
0.94

3200

3450

MLSS
2950

3650

4053

MLSS
3245

3351
3050

MLSS
2752

20 g/L
32 g/L
45 g/L
3050
3650
3200
2.5
8
17
3.27
0.84
0.44
20
30
70
98
97
93
3.2
0.81
0.41

10.4
16.1
24.3
38.5
57.0
60.7
98.0
131.4
136.1

10
15
20
30
50
100
200
300
500

2.74
3.09
2.73
3.88
3.99
5.16
4.47
3.55
4.43

mg O2/gVSS. h

Rx,e
3.33
5.27
7.17
10.2
16.9
35.6

mg/L

OC
7.61
13
22
35
53
56
94
128
132

mg O2/g VSS.d

Rx,ox
0.336
0.355
0.362
0.342
0.341
0.360

OC/S
Rx
21.8
37.3
61.8
99.1
152
159
268
366
377

mg COD/g VSS.d

Rx,t

mg O2/g VSS.h

8.1
12.8
23.1
25.1
42.9
95
103
102

mg/L COD

10
15
20
30
50
200
300
500

Rx,e

2.81
2.89
2.72
2.79
5.12
5.43
5.29
3.60

mg O2/gVSS. h

OC
3.10
4.83
6.12
9.50
15.69
63.56

mg/L

Rx,ox
5.2
9.9
20
22
38
90
98
98

mg O2/g VSS.d

C-1

0.313
0.325
0.309
0.320
0.317
0.321

OC/S

Rx
16
31
64
70
119
282
308
309

mg COD/g VSS.d

YCOD

Yvss

0.484
0.475
0.487
0.479
0.481
0.478

0.480

Average Y

g VS/g COD

0.687
0.675
0.691
0.680
0.683
0.679

g COD/g COD

Yvss

0.457

Average Y

YCOD

0.468
0.454
0.449
0.463
0.464
0.451

g VS/g COD

0.664
0.645
0.638
0.658
0.659
0.640

g COD/g COD

Biokinetic experimental data of mixed yeast sludge with glucose-feed wastewater at 32 g/L salt

mg O2/g VSS.h

mg/L COD

Table C-2

Rx,t

Biokinetic experimental data of mixed yeast sludge with glucose-feed wastewater at 20 g/L salt

Table C-1

0.19
0.36
0.74
0.81
1.37
3.25
3.55
3.56

day-1

0.24
0.41
0.68
1.09
1.67
1.75
2.95
4.03
4.15

day-1

9.2
28.1
28.4
57.5
64.2
90.1
91.8

10
30
50
100
200
300
500

Fig. C-1

Rx,t

mg O2/g VSS.h

100

200

300

COD concentration S (mg/ L COD)

45 g NaCl/L

32 g NaCl/L

20 g NaCl/L

400

0.427
0.425
0.404
0.419
0.415

OC/S
Rx

500

15.2
61.0
63.0
131.1
147.4
209.4
214.5

mg COD/g VSS.d

C-2

Variation of specific growth rate of yeast sludge versus COD concentration

at different salt contents for glucose-feed wastewater

0.0

1.0

2.0

3.0

4.0

5.0

4.23
12.62
20.00
41.48
82.17

2.82
2.63
2.05
2.73
2.62
2.57
2.11

6.4
25
26
55
62
88
90

Rx,ox
mg O2/g VSS.d

OC
mg/L

Rx,e

mg O2/gVSS. h

YCOD

Yvss

0.411

Average Y

S
158  S
R2 = 0.971

5.60

S
118  S
R2 = 0.982

4.74

S
129  S
R2 = 0.967
2.70

45 g/L NaCl:

32 g/L NaCl:

20 g/L NaCl:

0.404
0.405
0.420
0.409
0.412

g VS/g COD

0.573
0.575
0.596
0.581
0.585

g COD/g COD

Biokinetic experimental data of mixed yeast sludge with glucose-feed wastewater at 45 g/L salt

mg/L COD

Table C-3

Specific Growth Rate Pday-1

0.15
0.60
0.62
1.29
1.45
2.06
2.11

day-1

17
22
24
48
84
109
109

5
7
10
30
50
100
200

2.64
2.49
2.48
2.88
3.15
3.79
3.69

mg O2/gVSS. h

Rx,e

OC
1.04
1.42
1.79
5.05
8.91
18.91

mg/L

14
19
21
46
81
105
105

mg O2/g VSS.d

Rx,ox
0.210
0.205
0.181
0.170
0.180
0.191

OC/S
Rx
75
101
112
240
427
555
554

mg COD/g VSS.d

YCOD

Rx,t

mg O2/g VSS.h

7.2
10.9
11.6
12.9
14.7
22.0
30.5
31.1

mg/L COD

5
10
15
20
30
50
100
200

Rx,e

4.79
4.14
3.82
3.72
3.64
3.90
4.20
4.62

mg O2/gVSS. h

OC
0.92
1.79
2.61
3.50
5.11
8.37
0.00

mg/L

Rx,ox
2.42
6.74
7.76
9.16
11.07
18.07
26.34
26.47

mg O2/g VSS.d

C-3

0.185
0.181
0.176
0.177
0.172
0.169

OC/S

Rx
13.7
38.1
43.9
51.8
62.5
102.1
148.8
149.5

mg COD/g VSS.d

Yvss

0.570

Average Y

YCOD

0.574
0.577
0.580
0.580
0.583
0.585
0.704
0.583

Average Y

g VS/g COD

0.815
0.819
0.824
0.823
0.828
0.831
1.000

g COD/g COD

Yvss

0.556
0.560
0.577
0.585
0.577
0.570

g VS/g COD

0.790
0.795
0.819
0.830
0.820
0.809

g COD/g COD

Biokinetic experimental data of mixed bacterial sludge with glucose-feed wastewater at 32 g/L salt

mg O2/g VSS.h

Table C-5

Rx,t

Biokinetic experimental data of mixed bacterial sludge with glucose-feed wastewater at 20 g/L salt

mg/L COD

Table C-4

0.19
0.53
0.61
0.72
0.87
1.42
2.07
2.08

day-1

1.03
1.38
1.54
3.28
5.85
7.60
7.58

day-1

mg O2/g VSS.h

6.8
10.9
12.2
13.6
20.5
20.8

10
20
30
50
100
200

Fig. C-2

Rx,t

40

80

120

COD concentration ( mg/ L COD)

45 g NaCl/L

32 g NaCl/L

160

0.261
0.257
0.232
0.241
0.245

Rx

200

11.8
29.1
30.6
38.5
66.8
67.6

mg COD/g VSS.d

C-4

YCOD

Yvss

0.531

Average Y

S
44  S

S
52  S

R2 = 0.969

2.80

P 1.14

S
53  S
R2 = 0.947

45 g/L NaCl:

32 g/L NaCl:

R2 = 0.965

P 9.95

20 g/L NaCl:

0.520
0.523
0.541
0.535
0.532

g VS/g COD

0.739
0.743
0.768
0.759
0.755

g COD/g COD

Variation of specific growth rate of mixed bacterial sludge versus COD

concentration at different salt contents for glucose-feed wastewater

0.0

2.0

4.0

6.0

8.0

2.58
5.09
6.89
11.93
24.26

3.85
3.75
4.61
4.05
4.00
4.10

20 g NaCl/L

mg O2/g VSS.d

mg O2/gVSS. h

2.91
7.18
7.57
9.51
16.50
16.69

Rx,ox

OC
mg/L

Rx,e

OC/S

Biokinetic experimental data of mixed bacterial sludge with glucose-feed wastewater at 45 g/L salt

mg/L COD

Table C-6

Specific Growth Rate Pday-1

0.15
0.37
0.39
0.49
0.85
0.86

day-1

Rx,e

1.30
1.26
1.46
1.30
1.46

mg O2/gVSS. h

OC
6.8
16.0
28.9
80.7

mg/L

Rx,ox
16
43
47
116
121

mg O2/g VSS.d

0.435
0.410
0.371
0.345

OC/S
Rx
41.8
110.4
121.1
298.3
311.4

mg COD/g VSS.d

YCOD
0.565
0.590
0.629
0.655

g COD/g COD

Rx,t

mg O2/g VSS.h

14
37
47
75
77

mg/L COD

45
100
200
300
500

3.81
4.21
3.10
4.20
4.10

mg O2/gVSS. h

Rx,e
14.9
30.5
54.8
74.6

mg/L

OC
10
32
44
71
73

mg O2/g VSS.d

Rx,ox

C-5

0.425
0.391
0.351
0.319

OC/S

26.3
87.1
117.7
189.6
196.9

mg COD/g VSS.d

Rx

0.575
0.609
0.649
0.681

g COD/g COD

YCOD

Biokinetic experimental data of mixed yeast sludge with fish-protein-feed wastewater at 32 g/L salt

18
44
49
118
123

20
50
100
300
500

Table C-8

Rx,t

mg O2/g VSS.h

Biokinetic experimental data of mixed yeast sludge with fish-protein-feed wastewater at 20 g/L salt

mg/L COD

Table C-7
Yvss

0.405
0.429
0.457
0.480

g VS/g COD

Yvss

0.398
0.415
0.443
0.461

g VS/g COD

0.28
0.92
1.25
2.01
2.09

day-1

0.43
1.14
1.25
3.08
3.21

day-1

mg O2/g VSS.h

11
13
41
73
70

20
50
100
300
500

Fig. C-3

Rx,t

100

300

COD concentration S (mg/ L COD)

200

400

0.472
0.452
0.405
0.391

OC/S
Rx

500

16.6
21.8
88.2
163.0
163.0

mg COD/g VSS.d

C-6

YCOD

Yvss

0.403

Average Y

S
201  S
R2 = 0.964

4.69

S
322  S

S
228  S
R2 = 0.943
2.46

45 g/L NaCl:

R2 = 0.942

P 3.62

32 g/L NaCl:

15 g/L NaCl:

0.372
0.386
0.419
0.429

g VS/g COD

0.528
0.548
0.595
0.609

g COD/g COD

Variation of specific growth rate of yeast sludge versus COD concentration

at different salt contents for fish-protein-feed wastewater

0.0

1.0

2.0

3.0

45 g NaCl/L

32 g NaCl/L

20 g NaCl/L

7.4
17.6
31.6
91.5

3.51
3.51
3.29
3.29

4.0

mg O2/g VSS.d

mg O2/gVSS. h

7
9
38
70
70

Rx,ox

OC
mg/L

Rx,e

Biokinetic experimental data of mixed yeast sludge with fish-protein-feed wastewater at 45 g/L salt

mg/L COD

Table C-9

Specific Growth Rate Pday-1

0.16
0.21
0.85
1.57
1.57

day-1

mg O2/g VSS.h

100
148
196
221
220

20
50
100
150
300

3.73
3.43
4.16
4.26

mg O2/gVSS. h

Rx,e

OC
7.2
17.4
32.1
47.0

mg/L

96
145
192
216
220

mg O2/g VSS.d

Rx,ox
0.461
0.445
0.411
0.402

OC/S
Rx
223.2
337.4
446.3
503.4
510.7

mg COD/g VSS.d

YCOD

Rx,t

mg O2/g VSS.h

14.1
18.9
34.7
37.8
50.2

mg/L COD

20
36
100
200
300

3.81
2.82
3.66
2.70
4.3

mg O2/gVSS. h

Rx,e
5.5
9.7
25.0
47.0

mg/L

OC
10
16
31
35
46

mg O2/g VSS.d

Rx,ox

C-7

0.352
0.345
0.321
0.301

OC/S

Rx
31.0
48.6
93.7
106.1
138.9

mg COD/g VSS.d

Yvss

0.402

Average Y

YCOD

0.456
0.461
0.478
0.492
0.471

Average Y

g VS/g COD

0.648
0.655
0.679
0.699

g COD/g COD

Yvss

0.380
0.391
0.415
0.421

g VS/g COD

0.539
0.555
0.589
0.598

g COD/g COD

Table C-11 Biokinetic experimental data of mixed bacterial sludge with fish-protein-feed wastewater at 32 g/L salt

Rx,t

mg/L COD

Table C-10 Biokinetic experimental data of mixed bacterial sludge with fish-protein-feed wastewater at 20 g/L salt

0.35
0.55
1.06
1.20
1.57

day-1

2.15
3.25
4.30
4.85
4.92

day-1

mg O2/g VSS.h

14.8
23.7
33.3
43.2
41.8

20
50
100
150
300

Fig. C-4

Rx,t

mg/L COD

100

200

COD concentration S (mg/ L COD)

45 g NaCl/L

32 g NaCl/L

20 g NaCl/L

0.449
0.438
0.415
0.418

Rx

300

24.9
46.7
70.6
91.3
88.2

mg COD/g VSS.d

C-8

YCOD

Yvss

0.401

Average Y

S
33  S

S
93  S

S
64  S
R2 = 0.944

P 1.11

45 g/L NaCl:

R2 = 0.973

P 1.95

32 g/L NaCl:

R2 = 0.982

P 5.65

15 g/L NaCl:

0.388
0.396
0.412
0.410

g VS/g COD

0.551
0.562
0.585
0.582

g COD/g COD

Variation of specific growth rate of mixed bacterial sludge versus COD

concentration at different salt contents for fish-protein-feed wastewater

0.0

1.0

2.0

3.0

4.0

7.0
17.1
32.4
48.9

4.06
3.65
2.94
3.93
3.9

5.0

mg O2/g VSS.d

mg O2/gVSS. h

11
20
30
39
38

Rx,ox

OC
mg/L

Rx,e

OC/S

Table C-12 Biokinetic experimental data of mixed bacterial sludge with fish-protein-feed wastewater at 45 g/L salt

Specific Growth Rate Pday-1

0.24
0.45
0.68
0.88
0.85

day-1

Table D-1

Time
h
0
0.25
1.25
2.50
3.50
6.50
9.00
11.50
14.50
17.00
Table D-2

Time
h
0
0.25
1.25
2.25
3.50
6.50
8.00
11.50
14.50

Profile data of pH and nitrogen components of mixed yeast culture with glucosefeed wastewater at 32 g salt/L (mean MLSS = 9500 mg/L)
pH

3.50
2.88
2.72
2.64
2.60
2.65
2.71
2.80
2.85

COD
mg/L
4950

H
N 3-N
mg/L
365

1200
2750
N
/A
950
255
210
245
N
/A

317
267
237
172
139
127
117
112

O
N

N
+
3O

2-

mg/L

N
/A
2.13
2.00
2.01
2.05
2.00
2.20
N
/A

Total-N Nremoval
mg/L
%
365
317
269
239
174
141
129
119
112

13.2
26.3
34.5
52.3
61.4
64.7
67.3
69.3

Profile data of pH and nitrogen components of mixed bacterial culture with

glucose-feed wastewater at 32 g salt/L (mean MLSS = 3650 mg/L)
pH

7.50
7.76
8.10
8.26
8.28
8.31
8.28
8.25

COD
mg/L
1020
760
320
235
160
45
30
35
25

N
H 3-N
mg/L
53

43
32
20
17
14
14

D-1

O
N

N
+
3O

2-

mg/L

0.70
1.74
1.72
1.85
1.90
2.50

Total-N Nremoval
mg/L
%
53

44
34
21
19
16
17

17.5
36.3
59.8
64.8
70.0
68.9

Table D-3

Profile data of pH and nitrogen components of mixed yeast culture with fishprotein-feed wastewater at 32 g salt/L (mean MLSS = 5810 mg/L)

Time

COD

h
0.0
2.3
5.5
7.5
10.5
16.0
18.5
20.5
26.0
29.0
32.0
34.0
38.0
42.0

mg/L
5010
3234
2707
2392
2191
2031
1913
968
772
830
584
541

Adjusted
Organic-N H
N
pH

pH

mg/L
745
3.57
4.26
3.78
4.06
4.79
4.00
4.14
4.37
4.09
4.21
4.63
4.47
3.99

3.50
3.47
3.45
3.52
3.51
3.46
3.50
3.56
3.50
3.52
3.50
3.41
3.46

3-N

mg/L
45

O
+
3N

O
N

mg/L
N
D

2-

Total N Nremoval

mg/L
790
560

554

37.8

554

37.8

524

41.3

N
D

290

63
97
127
161
209
234

109
31

329
396

1.20
1.50

438
427

50.9
52.1

16

424
430

2.10
2.30

446

50.0

458
393

N
D
N
D

D
N- oNne detected

Table D-4

Profile data of pH and nitrogen components of mixed bacterial culture with fishprotein-feed wastewater at 32 g salt/L (mean MLSS = 3720 mg/L)

Time

COD

h
0
0.25
1.25
2.25
3.5
6.5
8.5
11.5
14.5
18.5
21.0
26.0
28.7

mg/L
976
870
645
568
509
383
320
200
110
65
30
45
20

pH

7.50
7.93
8.20
8.40
8.55
8.54
8.53
8.54
8.52
8.53

Organic-N

H
N

3-N

O
N

N
+
3O
N

2-

Total N Nremoval

mg/L
150

mg/L
49

mg/L
0

mg/L
199

120

52
67
75
97
107
110
126
143
142
137
142

0.5
1.09
1.51
1.5
2.5
2.40
3.10
2.6
4.9
4.3
4.9

172

13.6

161

19.1

149

25.1

152

23.6

63

2.1
5.1

D-2

Table D-5

H2SO4 amount consumed to maintain pH 3.5 of the mixed yeast culture with
fish-protein-wastewater at 32 g salt/L

Time

pH

Adjusted pH

h
0.0
2.3
5.5
7.5
10.5
16.0
18.5
20.5
26.0
28.7
31.7
34.8
38.3
41.8

3.57
4.26
3.78
4.06
4.79
4.00
4.14
4.37
4.09
4.21
4.63
4.47
3.99

3.50
3.52
3.47
3.54
3.50
3.46
3.47
3.49
3.55
3.51
3.50
3.41
3.46

1NH

olume Accumulated acid

2SO4 v
consumed
ovlume
mL
mL
0
6.6
0.9
0.7
0.9
1.7
0.7
0.7
0.9
0.5
0.6
0.8
0.9
0.5

7.5
8.2
9.2
10.8
11.5
12.3
13.2
13.7
14.3
15.2
16.1
16.6

Table D-6 Results of optimum pH experiment for mixed yeast sludge cultured with fishprotein-wastewater at 32 g/L salt (COD dose = 50 mg/L; MLV
SS = 1190 mg/L)
pH

OU
R

OU
R

total

mgO2/gV
SS.h

2.5
3.0
4.0
5.1
5.5
6.6
7.5
7.9
8.7
9.1

mgO

4.2
13.9
14.9
16.1
16.3
14.7
14.9
13.4
11.7
8.1

2.3
4.2
4.2
3.8
3.8
2.9
2.7
3.0
2.5
2.2

D-3

OU
R

endo

SS.h
2/gV

mgO

ox

SS.h
2/gV

2.0
9.7
10.7
12.4
12.5
11.8
12.1
10.4
9.2
5.8

Table D-7 Optimum pH experiment for mixed bacterial sludge cultured with fish-proteinwastewater at 32 g/L salt (COD dose = 50 mg/L; MLV
SS = 1380 mg/L)
pH

OU
R

OU
R

total

mgO2/gV
SS.h

4.5
5.3
6.3
7.6
8.1
8.9
9.7
10.1
10.8
10.9
Table D-8

mgO

2.8
7.8
14.8
21.3
21.0
21.0
17.7
12.0
7.1
7.5

OU
R

endo

SS.h
2/gV

mgO

1.10
2.31
4.14
5.45
5.23
5.88
4.8
3.4
2.2
2.3

ox

SS.h
2/gV

1.7
5.5
10.7
15.8
15.3
15.6
12.9
8.6
4.9
5.2

V
ariation of MLSS during SRT experiments
(U
nit:mg/L)

Time
day
0
3
5
7
9
11
13
15
17
19
21
25

5d
9000
6200
4140
3640
3920
2490
2770
2820
2950
3230
3450
3380

SRT
10d
9000
8400
8300
8000
8500
8700
7640
8330
7770
8150
7950
8250

7d
9000
7950
7570
6800
6500
6200
6550
5770
5820
5110
5360
5210

D-4

20d
9000
9100
8800
9300
8910
9140
9400
8900
9440
9740
9300
9520

45d
9000
9150
10130
9600
9860
9550
10270
10710
10230
10320
10510
10130

Table D-9

Experimental data of SRT avriation runs for mixed yeast batch with fish-proteinfeed wastewater at 32 g/L salt (Inititial COD = 5000 mg/L, HRT = 24 h)
SRT = 5 d

Parameter

CODeff

Batch 1
Batch 2
Batch 3
Average

mg/L
3120
2655
2776
2850

TK
N

eff

mg/L
467
534
572
524

SRT = 7 d
Mean
MLSS
mg/L
3230
3450
3380
3340

CODeff

TK
N

mg/L
2075
1811
1964
1950

eff

mg/L
532
502
512
515

SRT = 10 d
Mean
MLSS
mg/L
5115
5355
5210
5330

CODeff

TK
N

mg/L
1054
1145
952
1050

eff

mg/L
495
497
534
509

Mean
MLSS
mg/L
8150
7950
8250
8117

(continuous)
SRT = 20 d
Parameter CODeff
Bach 1
Bach 2
Bach 3
Average

mg/L
854
1023
845
907

TK
N

eff

mg/L
517
572
515
535

SRT = 45 d
Mean
MLSS
mg/L
9740
9295
9525
9520

CODeff

TK
N

mg/L
937
925
988
950

eff

mg/L
521
576
553
550

Mean
MLSS
mg/L
10320
10520
10130
10320

Table D-10 Result of SRT experiment

SRT
Vlume of wasted sludge, mL/d
o
F/M, g COD/g MLSS.d
COD removal, %
N remov
al, %
Amount of SS produced, mg/d
Amount COD removed, mg/d
Observed yield coefficient

5
400
1.54
43
7.24
649
2150
0.302

7
286
0.93
61
8.90
765
3050
0.251

D-5

10
200
0.62
77
9.83
809
3850
0.210

20
100
0.53
82
5.22
475
4100
0.116

45
44
0.49
85
2.67
228
4250
0.054

Table E-1

Experimental data for determination of initial membrane resistance of two

membrane modules (A = 0.42 m2; pore size = 0.1 Pm, temperature = 31.7oC )

a. Module 1

b. Module 2

Flowrate
Flux
(L/h)
(L/m2.h)

12.8
10.4
8.9
7.3
5.9
4.3
2.7
1.2

30.5
24.6
21.1
17.4
14.1
10.2
6.3
2.8

Pressure
(mmHg)

(kPa)

37
31
27
22
18
12
6
2.5

4.93
4.13
3.60
2.93
2.40
1.60
0.80
0.33

Flowrate
(L/h)

Flux
(L/m2.h)

2.6
4.2
7.1
10.0
12.7

6.1
10.1
17.0
23.9
30.2

Pressure
(mmHg)

(kPa)

6
12
22
31
37

0.80
1.60
2.93
4.13
4.93

Table E-2 Experimental data of YMBR in high COD loading

Time
(day)
0
5
11
13
16
17
19
22
25
29
31
35
38
40
43
45
47
51
54
56
59
62
65
73
75
78

HRT
(h)
23.7
24.2
24.0

F/M
g/g.d

23.8
24.4
23.5
32.3
31.7
32.2
18.3
17.9
18.2
18.4
12.0
12.2
12.4
12.2
8.0
7.1
7.2
7.1
5.0
5.1
5.2

0.39
0.34
0.23
0.23
0.25
0.53
0.66
0.69
0.68
0.93
0.95
0.88
0.87
1.43
1.49
1.59
1.47
2.22
2.04
2.15
2.15

0.60
0.32

L
MLSS
3
(kg/m .d) (mg/L)
3650
4.8
8100
4.8
14200
14100
12900
5.3
13500
5.0
12000
3.4
13600
3.4
11700
3.3
11000
6.3
10500
6.5
9890
7.0
10120
6.4
9530
9.7
10440
10.1
10610
9.9
11250
9.8
11200
14.7
10260
16.9
11290
17.1
10710
16.8
11450
23.3
10530
21.8
10730
23.7
11050
10050

CODinf
(mg/L)
4830
4780
4870
4950
4750
5200
5100
5120
4780
4900
4780
4850
5290
4940
4882
5128
5100
4970
4872
5000
5120
4972
4870
4670
5120
5230

E-1

CODeff
(mg/L)
1691
1162
998
1105
1050
1456
969
870
884
784
1147
1261
1323
1136
1528
1436
1683
1541
1705
1909
2340
1756
3120
2870
3215
3430

COD%
(%)
80
81

83
85
86
86
84
87
76
74
75
77
71
69
68
69
62
54
65
39
37
41

Time
(day)
0
4
8

Pressure
(kPa)
0.3
0.5
0.9

13
19
25
30
33
37
39
43
46
48
51
53
54
59
62
64
67
70
73
75
76
79

0.0
0.8
0.9
0.8
1.0
0.9
0.9
1.2
1.2
1.6
2.0
2.0
2.3
3.3
4.3
4.3
15.0
45.0
54.0
62.0
65.0
65.0

Table E-3 Experimental data of BMBR in high COD loading

Time
(day)
0
2
4
5
6
9
11
11
12
14
16
19
21
21
24
26
31
31
32
34
39
40
41
41
44
46
49
51
51
54
60
63
66
70
72
75

HRT
(h)
12.4
12.7
12.7
13.8
13.9
15.1
15.3
9.7
9.2
8.3
8.2
10.0
4.8
4.0
5.0
4.0
5.0
4.2
4.5
5.8
15.5
16.1
16.5
16.3
12.3
12.5
12.1
13.2
10.5
10.3
10.7
3.2
3.7
4.1
3.2
3.6

F/M
g/g.d

2.11
2.11
2.00
1.92
2.97
3.17
3.64
3.78
3.44
6.98
8.52
6.98
9.24
4.90
5.80
5.46
5.21
1.87
1.52
1.92
1.65
2.61
1.99
2.01
2.45
2.81
2.37
2.20
9.38
9.41
8.78
10.50
9.00

L
MLSS
(kg/m3.d) (mg/L)
4005
5200
6200
0.35
5970
0.32
6700
0.23
8600
0.18
10950
0.24
12500
0.27
11540
0.22
16500
0.22
17450
0.20
17400
0.35
19800
0.42
20100
0.35
19850
0.50
18500
0.25
19400
0.32
18200
0.27
20050
0.23
22400
0.09
20150
0.07
22500
0.10
18950
0.08
21750
0.13
19970
0.10
19750
0.11
18769
0.12
19700
0.13
21720
0.11
21434
0.11
20352
0.48
19500
0.45
21000
0.43
20500
0.53
19700
0.42
21200

CODinf
(mg/L)
1115
1097
1176
1216
1224
1258
1224
1200
1215
1260
1290
1435
1395
1420
1455
1540
1020
1015
1023
1260
1210
1020
1320
1120
1340
1035
1012
1347
1230
1015
980
1250
1450
1500
1400
1350

E-2

CODeff
(mg/L)
321
307
312
216
160
198
132
228
172
142
232
202
272
450
520
550
126
158
99
158
120
95
158
97
154
123
117
145
110
134
85
320
420
570
630
790

COD%
(%)
71.2
72.0
73.5
82.2
86.9
84.3
89.2
81.0
85.8
88.7
82.0
85.9
80.5
68.3
64.3
64.3
87.6
84.4
90.3
87.5
90.1
90.7
88.0
91.3
88.5
88.1
88.4
89.2
91.1
86.8
91.3
74.4
71.0
62.0
55.0
41.5

Time
(day)
1
3
5
6
7
8
9
10
11
12
16
17
18
19
21
22
23
23
24
23
25
26
28
29
30
32
33
35
37
38
41
43
46
48
49
50
51

Pressure
(kPa)
3
5
6
8
10
14
32
43
61
1
4
5
8
60
13
18
23
47
73
1
9
18
39
57
65
17
36
74
17
27
2.0
2.7
5.0
7.0
15
35
65.0

Table E-4 Experimental data of YMBR in low COD loading

Time
(day)
1
3
5
7
9
11
12
13
15
17
18
19
21
22
23
24
25
27
29
31
33
35
37
38
41
46
50
52
54
56
59
62
64
66
67
70
71
72
73
75
77
80
82
84
86
89

HRT
(h)
9.7
9.0
8.3
8.1
8.2
9.1
9.2
8.7
8.7
9.0
7.5
7.8
7.8
7.5
7.5
8.1
6.1
6.5
5.9
5.8
4.9
5.1
5.2
5.1
4.8
7.5
7.0
7.0
6.9
6.8
6.3
6.2
6.2
5.8
5.3
5.4
5.0
5.3
5.3
5.3
4.0
4.2
4.0
4.0
4.0
3.9

F/M
g/g.d
2.60
2.59
2.66
2.98
3.22
2.24
2.48
2.39
2.61
2.69
3.17
2.99
2.74
3.23
2.86
2.71
3.44
3.58
3.46
4.18
4.46
4.00
4.57
4.64
5.28
3.42
3.60
4.01
3.51
3.35
4.03
4.53
3.91
4.63
5.00
5.00
5.09
4.51
5.07
4.94
6.75
6.05
6.36
7.00
6.27
6.89

L
MLSS
(kg/m3.d) (mg/L)
0.51
5120
0.43
6040
0.44
6100
0.49
6100
0.56
5700
0.42
5400
0.52
4750
0.52
4600
0.47
5500
0.54
4970
0.57
5520
0.58
5200
0.55
4950
0.67
4790
0.58
4950
0.56
4800
0.72
4750
0.87
4133
0.80
4300
0.92
4560
1.00
4450
0.86
4670
0.98
4670
1.03
4520
1.21
4350
0.70
4900
0.74
4850
0.78
5120
0.63
5600
0.54
6200
0.39
10400
0.42
10900
0.34
11500
0.35
13200
0.38
13500
0.35
14200
0.36
14300
0.32
14000
0.35
14700
0.33
15050
0.47
14500
0.40
15150
0.43
14750
0.46
15200
0.40
15500
0.46
15100

CODinf
(mg/L)
1050
970
920
1005
1100
850
950
866
945
1009
990
972
892
1010
893
913
874
970
850
1010
910
850
990
985
1056
1070
1050
1170
1009
950
1059
1170
1009
1120
1120
1125
1060
995
1120
1090
1125
1059
1060
1170
1050
1120

E-3

CODeff
(mg/L)
263
233
288
185
150
45
24
25
80
62
40
35
45
24
95
80
110
170
140
170
240
180
270
240
240
51
20
45
65
45
70
45
20
65
75
65
120
110
95
95
148
190
135
190
150
101

COD%
(%)
75
76
69
82
86
94.7
97.5
97.1
91.5
93.9
96.0
96.4
95.0
97.6
89.4
91.2
87.4
82.5
83.5
83.2
73.6
78.8
73
75.6
77.3
95.2
98.1
96.2
93.6
95.3
93.4
96.2
98.0
94.2
93.3
94.2
88.7
88.9
91.5
91.3
86.8
82.1
87.3
83.8
85.7
91.0

Time
(day)
1
3
5
7
9
9
12
13
15
17
17
19
21
22
23
24
24
27
29
31
31
35
36
38
45
45
49
50
56
56
60
61
62
62
66
66
70
71
72
73
75

Pressure
(kPa)
0.67
0.93
1.20
0.93
0.80
0.80
0.93
0.93
0.93
2.00
2.00
2.00
1.86
2.13
2.13
6.00
6.00
6.00
6.00
9.0
9.0
25.0
45.0
62.0
65.0
0.52
0.67
0.67
0.67
0.67
1.20
2.30
2.30
2.30
5.00
5.00
12.0
25.0
32
63
65

76
76
79
80
81

1.5
1.5
1.7
2.3
2.5

Table E-5
Time
(day)
1
3
6
9
12
15
17
19
21
25
27
29
31
33
35
37
41
42
44
46
48
50
52
54
56
59
62
64
66
67
70
71
72
73
75
77
80
82

Experimental data of BMBR in low COD loading

HRT
(h)
7.8
8.1
8.4
7.9
8.2
5.9
6.5
6.0
6.5
6.8
4.9
5.1
5.5
4.9
5.2
5.6
6.8
6.8
7.1
7.3
7.4
5.7
5.9
6.3
5.9
4.4
4.7
5.2
4.6
3.9
4.1
4.3
3.8
4.1
4.3
4.5
4.0
4.2

F/M
g/g.d
3.23
2.87
2.63
3.34
2.78
3.84
3.73
3.89
3.29
3.08
4.75
4.00
4.41
4.46
3.92
4.24
3.73
4.13
3.21
3.52
3.58
4.42
4.76
3.84
3.29
5.78
5.97
4.66
5.84
6.89
6.59
5.92
6.28
6.56
6.08
6.13
6.35
6.06

L
MLSS
(kg/m3.d) (mg/L)
0.29
5500
0.28
4750
0.28
4500
0.46
5200
0.51
4650
0.82
4700
0.76
4920
0.86
4530
0.77
4300
0.68
4550
0.96
4950
0.78
5120
0.82
5400
0.69
6500
0.62
6300
0.70
6100
0.61
6100
0.70
5900
0.59
5450
0.53
6700
0.44
8100
0.47
9500
0.45
10600
0.37
10500
0.28
11700
0.48
12050
0.46
12950
0.35
13200
0.41
14300
0.46
15070
0.45
14700
0.38
15700
0.38
16500
0.40
16300
0.35
17400
0.35
17100
0.38
16900
0.35
17200

CODinf
(mg/L)
1050
970
920
1100
950
945
1009
972
892
874
970
850
1010
910
850
990
1056
1170
950
1070
1103
1050
1170
1009
810
1059
1170
1009
1120
1120
1125
1060
995
1120
1090
1150
1059
1060

E-4

CODeff
(mg/L)
50
20
45
25
15
45
45
25
20
20
110
45
90
75
45
70
20
45
15
20
45
110
65
25
20
45
95
65
76
155
120
45
120
105
110
76
76
110

COD%
(%)
95.2
97.9
95.1
97.0
98.0
95.2
95.5
97.4
97.8
97.7
88.7
94.7
91.1
91.8
94.7
92.9
98.1
96.2
98.4
98.1
95.9
89.5
94.4
97.5
97.5
95.8
91.9
93.6
93.2
86.2
89.3
95.8
87.9
90.6
89.9
93.4
92.8
89.6

Time
(day)
1
2
3
4
5
7
8
9
10
12
13
15
16
17
19
20
21
22
23
24
25
27
29
30
31
34
35
36
37
42
43
44
45
46
47
48
49
50
51
52
53

Pressure
(kPa)
20.0
33.3
79.8
82.5
85.1
20.0
33.3
59.9
79.8
86.5
86.5
27.9
46.6
85.0
23.9
59.9
86.0
86.5
27.9
53.2
86.5
33.3
85.0
85.1
86.5
58.5
82.0
85.1
86.5
20.0
32.0
78.0
81.0
86.0
86.5
86.0
25.0
42.0
65.0
86.5
86

100
YMBR

90

BMBR

COD removal ( %)

80
70
60
50
40
30
20
0

10

12

14

16

18

20

22

24

Volumetric Loading ( kg COD/ m3.d)

Table F-1 Regression analysis of Fig.

YMBR
Equation Y = 88.00264 - 1.04453 * X - 0.04398 * X2

BMBR
Equation Y = 82.7394 + 3.5684* X - 0.5722 * X2

Degree = 2
Number of data points used = 22
Average X = 10.45
Average Y = 70.3889

Degree = 2
Number of data points used = 34
Average X = 4.71309
Average Y = 81.5303

Degree: 0
Residual sum of squares = 4949.26
Coef of determination, R-squared = 0

Coefficients:
Degree 0 = 82.7394
Degree 1 = 3.5683
Degree 2 = -0.57221

Degree: 1
Residual sum of squares = 403.182
Coef of determination, R-squared = 0.918537

Orthogonal Polynomial Factors:

X Shift = 6.010248447204969
X Scale = 0.4454589472227987

Degree: 2
Residual sum of squares = 350.384
Coef of determination, R-squared = 0.929205

Degree: 1
Residual sum of squares = 1091.09
Coef of determination, R-squared = 0.732687
Degree: 2
Residual sum of squares = 668.19
Coef of determination, R-squared = 0.836296

F-4

COD removal rate ( g COD/ g MLSS.d)

1.0

0.8

0.6

0.4

0.2

BMBR
YMBR

0.0
0.0

0.5

1.0

1.5

2.0

2.5

F/M ratio ( d-1)

Table F-2 Regression analysis of Fig.

YMBR
Equation Y = -0.10406 + 1.1556 * X - 0.33699 * X2

BMBR
Equation Y = -0.03234 + 1.3306 * X - 1.2847 * X2

Degree = 2
Number of data points used = 22
Average X = 0.951382
Average Y = 0.559181

Degree = 2
Number of data points used = 34
Average X = 0.264056
Average Y = 0.202548

Coefficients:
Degree 0 = -0.104058111
Degree 1 = 1.155606227
Degree 2 = -0.3369879819

Coefficients:
Degree 0 = -0.03233949848
Degree 1 = 1.330610919
Degree 2 = -1.284738775

Degree: 1
Residual sum of squares = 0.345839
Coef of determination, R-squared = 0.76943

Degree: 0
Residual sum of squares = 0.273731
Coef of determination, R-squared = 0

Degree: 2
Residual sum of squares = 0.0467869
Coef of determination, R-squared = 0.968807

Degree: 1
Residual sum of squares = 0.0350683
Coef of determination, R-squared = 0.871888
Degree: 2
Residual sum of squares = 0.0169625
Coef of determination, R-squared = 0.9380

F-4

100

COD removal (%)

90
80
70
60

YMBR

SRT of 50 d

BMBR

100

COD removal ( %)

90
80
70
60

YMBR

SRT of 10 d

BMBR

50
4.0

5.0

6.0

7.0

8.0

9.0

10.0

HRT ( h)

Table F-3 Regression analysis of Fig.

YMBR
SRT 10 days
Equation Y = -18.2938 + 26.02184 * X - 1.4905 * X2

Y = 91.219 - 9.232 * X + 2.896 * X2 - 0.2059 * X3

Degree = 2
Number of data points used = 19
Average X = 7.13158
Average Y = 88.2956

Degree = 3
Number of data points used = 14
Average X = 6.24286
Average Y = 94.8309

Coefficients:
Degree 0 = -18.29383197
Degree 1 = 26.02183739
Degree 2 = -1.490548568

Coefficients:
Degree 0 = 91.21936071
Degree 1 = -9.231993703
Degree 2 = 2.895593329
Degree 3 = -0.2059391806

Degree: 1
Residual sum of squares = 260.884
Coef of determination, R-squared = 0.799519
Degree: 2
Residual sum of squares = 149.022
Coef of determination, R-squared = 0.885482

BMBR

Degree: 2
Residual sum of squares = 36.227
Coef of determination, R-squared = 0.652589
Degree: 3
Residual sum of squares = 35.8103
Coef of determination, R-squared = 0.656585

F-4

(Continuous)
YMBR
SRT 50 days
Equation Y = 53.6689 + 10.8237 * X - 0.6825 * X2

BMBR
Equation Y = 67.0097 + 7.8151 * X - 0.50207 * X2

Degree = 2
Number of data points used = 21
Average X = 5.49667
Average Y = 91.6494

Degree = 2
Number of data points used = 22
Average X = 5.24091
Average Y = 93.4416

Coefficients:
Degree 0 = 53.66891328
Degree 1 = 10.82366557
Degree 2 = -0.6825361179

Coefficients:
Degree 0 = 67.00973409
Degree 1 = 7.815103512
Degree 2 = -0.5020654975

Degree: 0
Residual sum of squares = 419.6
Coef of determination, R-squared = -2.22045E-016

Degree: 1
Residual sum of squares = 118.846
Coef of determination, R-squared = 0.576309

Degree: 1
Residual sum of squares = 123.575
Coef of determination, R-squared = 0.705495

Degree: 2
Residual sum of squares = 113.208
Coef of determination, R-squared = 0.59641

Degree: 2
Residual sum of squares = 110.933
Coef of determination, R-squared = 0.735623

F-4