Bioresource Technology 245 (2017) 615624

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Bioresource Technology
journal homepage: www.elsevier.com/locate/biortech

Recovery of phosphorus and volatile fatty acids from wastewater and food MARK
waste with an iron-occulation sequencing batch reactor and acidogenic co-
fermentation

Ruo-hong Li, Xiao-yan Li
Environmental Engineering Research Centre, Department of Civil Engineering, The University of Hong Kong, Pokfulam, Hong Kong, China

G RA P H I C A L AB S T R A C T

A R T I C L E I N F O A B S T R A C T

Keywords: A sequencing batch reactor-based system was developed for enhanced phosphorus (P) removal and recovery
Phosphorus recovery from municipal wastewater. The system consists of an iron-dosing SBR for P precipitation and a side-stream
Chemical phosphorus removal anaerobic reactor for sludge co-fermentation with food waste. During co-fermentation, sludge and food waste
Acidogenic fermentation undergo acidogenesis, releasing phosphates under acidic conditions and producing volatile fatty acids (VFAs)
Vivianite
into the supernatant. A few types of typical food waste were investigated for their eectiveness in acidogenesis
Food waste
Sequencing batch reactor
and related enzymatic activities. The results show that approximately 96.4% of total P in wastewater was re-
tained in activated sludge. Food waste with a high starch content favoured acidogenic fermentation. Around
55.7% of P from wastewater was recovered as vivianite, and around 66% of food waste loading was converted
into VFAs. The new integration formed an eective system for wastewater treatment, food waste processing and
simultaneous recovery of P and VFAs.

1. Introduction fertilisers for food production. The global mineral phosphate reserves
are limited and are predicted to be depleted by the end of this century
Phosphorus (P) is a non-renewable and irreplaceable resource of (Cordell et al., 2009). The market price of phosphate rock increased by

Corresponding author.
E-mail address: xlia@hku.hk (X.-y. Li).

http://dx.doi.org/10.1016/j.biortech.2017.08.199
Received 17 July 2017; Received in revised form 29 August 2017; Accepted 30 August 2017
0960-8524/ 2017 Elsevier Ltd. All rights reserved.
R.-h. Li, X.-y. Li Bioresource Technology 245 (2017) 615624

approximately four times in the past 10 years, and the demand for Table 1
phosphate fertiliser continues to increase due to the growth of the Summary of the reactor and process parameters of the aerobic SBR and anaerobic fer-
mentation system for simultaneous P and VFAs recovery.
global population and living standards (Mew, 2016). At the same time,
phosphorus is a main pollutant in wastewater that causes eutrophica- Aerobic SBR Anaerobic fermenter
tion in natural waters. Chemical precipitation, such as by adding FeCl3,
has been commonly applied to remove P from wastewater Temperature (C) 25 25
Working volume (L) 4 1.6
(Nancharaiah et al., 2016). Every year, approximately 1.3 million tons
DO (mg/L) 45 0
of phosphorus are removed and lost in wastewater treatment globally. Alkalinity (mg-CaCO3/L) 275 304
The recovery of phosphorus from wastewater should address the needs ORP (mV) -105
of water pollution control and resource reuse. It is anticipated that P Flow rate (L/d) 8 0.4
recovery from wastewater will eventually be driven by both economics HRT (d) 0.5 4
SRT (d) 10 4
and legislations. For instance, in Germany, a new policy is formed that
SS (g/L) 3.2 0.3 5.6 0.4
will require P recovery by all large wastewater treatment plants (BMUB, VSS (g/L) 2.4 0.2 4.5 0.4
2017). Inuent COD (mg/L) 171 8 2500
Chemical acidication extraction is the current method that can be Inuent TN (mg/L) 25.0 0.8
Inuent TP (mg/L) 7.19 0.19 69.4
used to recover P from wastewater sludge or sludge ash (e.g., the
COD: N: P 24:3:1
Stuttgart process (Egle et al., 2016) and RecoPhos (Weigand et al., Organic loading rate (g COD/L/d) 0.342 0.625
2013)). This method adjusts the pH of sludge or sludge ash below 2 via F/M (g COD/g SS/d) 0.107 0.112
the addition of strong acids, such as HCl or H2SO4, to dissolve metal Euent COD (mg/L) 17.5 0.9 1991 36
salts and release phosphates into the acid leachate. However, the Euent TN 6.4 0.2
Euent TP (mg/L) 0.51 0.04 106.5 1.4
drawbacks of this method, such as the use of large amounts of strong
Euent SS (mg/L) 33 3
acids, high operating costs and secondary pollution, limit the applica- Circulation sludge ratio (%) 20
tion of P recovery (Sartorius et al., 2012). In this study, we introduce a
process of biological acidogenesis instead of chemical acidication to
extract phosphorus from sludge. Acidogenic fermentation connes treatment plant, Stanley Sewage Treatment Works (SSTW), Hong Kong,
anaerobic organic digestion to the acidogenic stage and makes volatile for the experimental study. The raw wastewater was typical domestic
fatty acids (VFAs) the main products. VFAs are considered to be value- sewage in nature, and the main water quality parameters of the was-
added carbon sources for uses such as denitrication in wastewater tewater inuent are summarised in Table 1. If the wastewater collected
treatment (Kim et al., 2016) and biosynthesis of polyhydroxyalkanoates was not used on the day it was collected, it was stored for no more than
(PHAs) (Frison et al., 2015). Suitable organics must be provided to 6 d in a refrigerator at 4 C for later use.
achieve stable acidogenesis in fermentation. Food waste, with its high
biodegradability, has been proved to be a desirable carbon source for
acidogenic fermentation (Shen et al., 2017). 2.2. The Fe-coagulation sequencing batch reactor and sludge fermentation
Approximately one third of global food is lost or wasted along the system
food supply chain to human consumption (Aschemann-Witzel, 2016). It
is estimated that more than 97% of food waste in the United States is The integrated units of the system consisted of an aerobic SBR for
disposed by landlling, which causes additional environmental pro- biological wastewater treatment and a side-stream anaerobic reactor for
blems, such as leachate contamination (Levis et al., 2010). Another sludge fermentation (Fig. 1). The aerobic SBR was a cylindrical Plex-
disposal method is incineration (You et al., 2016), which wastes both iglass column with inner dimensions of 50 800 mm (diame-
energy and resources and emits air pollutants and greenhouse gases. ter height) and a working volume of 4 L. The SBR column was lled
There is a growing interest in the development of more sensible and with seed activated sludge obtained from the SSTW, and the mixed-
environmentally friendly strategies for food waste processing and use. liquor suspended solids concentration was maintained at around 3.2 g/
Unlike other types of waste, food waste consists of easily biodegradable L in the SBR. For enhanced one-step P removal from wastewater by the
organic matter, mainly carbohydrates and proteins, with few hazardous SBR, FeCl3 was dosed into the wastewater for pre-occulation, and the
materials (Oh and Logan, 2005), so it can be readily processed by occulated inuent without sedimentation was pumped into the SBR
biological means of material and energy recovery, such as anaerobic
FeCl3
digestion (Jiang et al., 2013, Thyberg and Tonjes, 2016). Aerobic Tank
In this study, a sequencing batch reactor (SBR) was used with Fe(III)
dosing for enhanced P removal in municipal wastewater treatment. A Influent Effluent
side-stream anaerobic reactor was integrated with the aerobic SBR for
co-fermentation of the activated sludge with food waste. Instead of
chemical acidication, acidogenic fermentation was realised to create a
reduced and acidic condition to extract P from the solid phase of the Sludge
recirculation
sludge into the supernatant for recovery, and VFAs were produced in
the solution for potential use. Various types of food waste were selected
Food waste
to investigate their eects on acidogenic co-fermentation. Continuous
operation of the new system Fe-occulation SBR and side-stream co-
fermentation with food waste was conducted to evaluate its eec- Supernatant VFAs-rich
Air
tiveness in wastewater treatment, food waste processing and simulta- liquor
Return Anaerobic
neous recovery of P and VFAs.
sludge Fermenter
P precipitation
2. Materials and methods
Waste sludge

2.1. Raw wastewater Fig. 1. Schematic ow chart of the SBR-based system with Fe(III)-dosing and side-stream
anaerobic sludge co-fermentation with food waste for simultaneous recovery of P and
VFAs.
Municipal wastewater was collected from a local wastewater

616
R.-h. Li, X.-y. Li
for biological treatment. The aerobic SBR was operated for four cycles a
day, and each sequencing cycle consisted of 5.5 h of aeration, 28 min of
settlement, 1 min of euent withdrawal and 1 min of inuent feeding.
By the end of each cycle, 2 L of treated euent, or 50% of the SBR
working volume, was withdrawn, resulting in a hydraulic retention
time (HRT) of 12 h.
A 2-L glass bottle was used as the anaerobic reactor for sludge fer-
mentation (Fig. 1).
The seed sludge was formed by co-fermentation of the activated
sludge from the SBR together with the food waste addition. The fer-
menters were operated in a semi-continuous mode for more than
2 months with a SRT of 4 days. The acidogenic fermentation was then
performed with the seed sludge under the same anaerobic condition.
For continuous system operation, coupled with the aerobic SBR, a
portion of the settled activated sludge from the SBR was circulated
through the anaerobic reactors for fermentation. Specically, 400 mL of
the SBR sludge mixture, or 20% of the activated sludge after sedi-
mentation, was withdrawn from the SBR each day and fed into the
sludge fermenter. Meanwhile, a predetermined amount of selected food
waste was also added with an organic content of 800 mg-COD into the
fermenter for co-fermentation. The sludge and food waste mixture was
maintained at a nal volume of 1.6 L, which was mixed by magnetic
stirring at room temperature (25 C) in anaerobic conditions. After
24 h of fermentation, 400 mL of the mixture was withdrawn from the
fermenter before the next daily feed. Thus, the anaerobic fermenter had
an HRT and a sludge retention time (SRT) of 4 d.
After the fermented sludge mixture was allowed to settle for 30 min,
50 mL of the settled sludge was returned to the aerobic SBR, 50 mL of
the settled sludge was discharged, and 300 mL of the supernatant was
retrieved for resource (P and VFAs) recovery. To recover P, the super-
natant was adjusted to pH 8.0 with NaOH to induce the re-precipitation
of orthophosphate with ferrous iron. After 30 min of precipitation, the
Fe(II)-P precipitate was collected by sedimentation or centrifugation.
The supernatant was then analysed for organic and VFA contents.
The aerobic SBR was rst operated for biological wastewater
treatment for about 60 d for enhanced P removal, without the side-
stream sludge circulation and fermentation. During this stage, occu-
lant FeCl3 was added to the wastewater inuent at increasing doses,
from 10 to 15, 20 and 25 mg-Fe/L each for 15 d. About half of the
activated sludge was then collected from the SBR for batch studies on
its co-fermentation with various types of food waste. The Fe-occula-
tion SBR was then integrated with the side-stream sludge fermenter to
form the system as shown in Fig. 1. The experiment was operated
continuously at room temperature for more than 60 d. The Fe dose for P
removal was 25 mg/L based on the previous test results, and rice was
added as a model food waste for co-fermentation. During the experi-
mental study, the wastewater inuent and euent, activated sludge,
anaerobic supernatant and sludge were sampled every 2 d to determine
the concentrations of total phosphorus (TP), soluble orthophosphate
(SP), total nitrogen (TN), total iron (TFe), soluble ferrous ion (SFe(II)),
chemical oxygen demand (COD), total organic carbon (TOC), VFAs,
suspended solids (SS) and volatile suspended solids (VSS). To in-
vestigate pollutant removal and resource recovery performance, the
material balance of C, P and Fe was also analysed for the integrated
system.
2.3. Batch tests on acidogenic co-fermentation of activated sludge with
various food wastes
Five typical food waste materials rice, wheat, corn, bean and
chicken meat were tested for acidogenic co-fermentation with the SBR
sludge. The composition of carbohydrates and proteins in the model
food waste was measured. The food waste materials were crushed and
homogenised in a blender and boiled in deionised water for 30 min.
Each food waste slurry (around 50 mL) was put into a 500-mL glass
bottle as an anaerobic fermenter for a nal organic loading of 4 g-COD/
617
Bioresource Technology 245 (2017) 615624
L. A control reactor was lled with 50 mL of boiled deionised water
instead of food waste slurry. Each fermenter was then lled with
400 mL of the P-rich activated sludge from the aerobic SBR and 50 mL
of anaerobic seed sludge. Batch fermentation experiments were con-
ducted to investigate the co-fermentation performance and results with
the addition of dierent types of food waste, including the concentra-
tion of P release from the solid phase of the sludge and the yield of VFAs
from the food waste. The mixtures were mixed by magnetic stirring in
the bottle fermenters for 96 h at room temperature (25 C). Throughout
the course, the supernatant from each fermenter was sampled every day
to measure the solution pH and the concentrations of SP, TP, SFe(II),
TFe, COD, VFAs, SS and VSS. In addition, enzymatic assays of -glu-
cosidase, -glucosidase, protease and acid phosphatase were conducted
for the sludge mixtures after fermentation to indicate the organic hy-
drolysis activities.
2.4. Analytical methods
The concentrations of COD, SP, TP, SFe(II), TFe, TN, SS and VSS
were determined according to Standard Methods (APHA, 1998). The
protein content was determined by Coomassie brilliant blue protein
assay using bovine serum albumin as the standard (Bradford, 1976).
The carbohydrate content was determined with the phenolsulphuric
acid method using glucose as the standard (DuBois et al., 1956). The
TOC concentration was measured with a TOC analyser (Aurora 1030,
OI Analytical). The dissolved oxygen (DO) concentration was measured
with a digital DO meter (HI 2030, HANNA), the solution pH was
measured with a pH meter (Starter 2100, OHAUS) and the solution
oxidation-reduction potential (ORP) was monitored with an ORP tester
(HI 98201, HANNA). VFAs, including acetic acid, propionic acid, bu-
tyric acid, isobutyric acid, valeric acid, isovaleric acid, isocaproic acid,
caproic acid and hexanoic acid, were quantied with a gas chromato-
graph (Agilent 6890) equipped with a capillary column (Agilent
19095F-123) and a ame ionisation detector, following the methods
described by Lin et al. (2017). For gas chromatography measurement,
the samples were pre-ltered with a 0.45-m syringe polyvinylidene
uoride membrane (Millipore), and all of the samples were analysed in
triplicate.
To evaluate the potential and activity of hydrolysis of food wastes,
enzymatic activity was analysed according to the methods described by
Dick (2011). The procedures for assaying -glucosidase, -glucosidase,
protease and acid phosphatase are summarised in Table 2. Modied
universal buer and Tris (hydroxymethyl) aminomethane buer of the
desired pH was prepared as described by Eivazi and Tabatabai (1988).
The solutions of p-nitrophenyl -D-glucopyanoside, p-nitrophenyl -D-
glucopyanoside and p-nitrophenyl phosphate disodium salt (50 mM)
were prepared by dissolving 2.5 mmol of the appropriate compound in
50 ml of modied universal buer (pH 6.0). One unit of the specic
enzyme activity was dened as 1 mol substrate catalysed by the en-
zyme per minute per gram of VSS (EU/g-VSS). For proteases, one en-
zyme unit was dened to hydrolyse 1 mg of azocasein per min per gram
VSS (EU/g-VSS). Each assay was performed in triplicate.
3. Results and discussion
3.1. Pollutant removal in aerobic SBR system
The aerobic SBR with the ferric iron dosing was rst operated
without the anaerobic SBR for biological wastewater treatment. The
reactor performed well in removing both the TP and organic pollutants,
as shown by the Supplementary data. During this stage, the P removal
eciency increased as the Fe dose increased. At an Fe dose of 25 mg/L,
satisfactory P removal of around 92.2% was achieved. At this dose, the
Fe to P molar ratio was around 1.9, near the optimal Fe/P ratio of 2.0 or
higher reported by others for P removal by Fe-based chemical pre-
cipitation means (Hauduc et al., 2015). The COD removal eciency by
R.-h. Li, X.-y. Li Bioresource Technology 245 (2017) 615624

the SBR was maintained at around 90%, which was not aected by the

1 mL CaCl2 (0.5 M) and 4 mL THAM buer

1 mL CaCl2 (0.5 M) and 4 mL THAM buer

Fe dosing into the inuent. Towards the end of this stage, the total P

1 mL CaCl2 (0.5 M) and 4 mL NaOH

and Fe contents in the activated sludge mixture in the SBR grew to
179.8 and 590.8 mg/L, respectively, and the VSS/SS ratio of the sludge
decreased to 64.6%. Whilst the Fe dosing resulted in sucient P re-
moval by the SBR, a considerable quantity of inorganic contents accu-
mulated in the reactor; this accumulation would reduce the biomass
2 ml TCA (10%) fraction and aect the bioactivity of the activated sludge (Fan et al.,
(0.1 M, pH 12)

(0.1 M, pH 12)

2015). Continuous removal of P and Fe from the SBR sludge would not
Termination

only allow recovery of the material but also benet the activated sludge
(0.5 M)

for biological wastewater treatment.

3.2. Batch acidogenic co-fermentation of sludge with various food wastes

0.1 mL sample, 0.2 mL toluene, 4 mL MUB (pH

0.1 mL sample, 0.2 mL toluene, 4 mL MUB (pH

0.1 mL sample, 0.2 mL toluene, 4 mL MUB (pH

Activated sludge from the SBR was processed by anaerobic fer-

mentation with food waste. During co-fermentation, organic solids
undergo acidogenesis, producing VFAs. Meanwhile, in anaerobic and
3 ml sample and 1 mL substrate

acidogenic conditions, ferric iron is reduced to ferrous iron, releasing

both Fe2+ and PO43-P from the solid phase into the supernatant at the
6.0) and 1 mL substrate

6.0) and 1 mL substrate

6.0) and 1 mL substrate

lower pH. Batch acidogenic sludge fermentation was conducted with

Mixture composition

various types of food waste to determine their eectiveness in co-fer-

mentation for P extraction and VFA production.

3.2.1. Food waste compositions

Five typical types of food waste were chosen for the study based on
main groups of food supplies, including cereals (rice and wheat), ve-
getables (corn and bean) and meat (chicken) (Galanakis, 2012). In
Incubation time: 60 min, temperature: 37 C, centrifuge after assay and retain the

Incubation time: 60 min, temperature: 37 C, centrifuge after assay and retain the

Incubation time: 60 min, temperature: 37 C, centrifuge after assay and retain the

Incubation time: 90 min, temperature: 37 C, centrifuge after assay and mix 2 ml

particular, rice is the staple food in Asia, as wheat is in Europe and the
United States. The compositions of those food materials are given in
Table 3. The main organic components included carbohydrates and
proteins, and their fractions varied greatly. For rice, wheat and corn,
carbohydrates were the main fraction, accounting for 80.5%, 77.6%
and 86.0%, respectively. Chicken meat had a much higher protein
content (48.0%) than the others. Bean contained 46.5% carbohydrates
and 23.3% proteins. The variations in the composition of the model
supernatant in 2 ml NaOH (2 M), absorbance at 440

food waste types had a signicant eect on the performance and results
of co-fermentation with the SBR sludge.

3.3.2. Acidogenic fermentation of food waste and VFAs production

The model food waste was digested by hydrolysis and acidogenesis,
supernatant absorbance at 405

supernatant absorbance at 405

supernatant absorbance at 405

and the resulting changes in pH and chemical concentrations in the

supernatants are presented in Fig. 2. At the beginning, with the hy-
drolysis of carbohydrates and proteins, an increasing amount of soluble
Incubation conditions

organic material dissolved into the supernatant, of which some was

present as soluble carbohydrates and proteins and some was already
converted further into VFAs. During the rst 24 h of fermentation, the
performance of organic hydrolysis was as follows: wheat (86.2%), rice
(73.3%), corn (62.9%), chicken meat (53.9%), and bean (37.6%). The
main hydrolysed organics were carbohydrates from rice, wheat and
corn and proteins from chicken meat. From 24 to 96 h, the concentra-
Summary of the enzyme assay techniques and conditions.

tions of soluble carbohydrates and proteins decreased greatly in the

50 mM p-nitrophenyl phosphate

supernatant, along with the increase in VFA production and the de-
crease in the solution pH. For wheat, the soluble carbohydrates de-
50 mM p-nitrophenyl -D-

50 mM p-nitrophenyl -D-

creased from 2470 to 186 mg-COD/L, whilst for chicken meat, soluble
proteins decreased from 1145 to 210 mg-COD/L.
After co-fermentation with food waste, the VFA concentrations in-
glucopyanoside

glucopyanoside

0.5% azocasein
disodium salt

creased signicantly in the sludge mixtures. Especially after 4 d of

Substrate

fermentation with rice, wheat and corn, VFAs were produced at 3584,
4447 and 4295 mg-COD/L, respectively, in the solutions; these levels
were much higher than those from bean and chicken meat (1940 and
2572 mg-COD/L, respectively). According to the analysis of VFAs,
Acid phosphatase

acetic and propionic acids were the dominant fermentation products

Enzyme assay

-glucosidase

-glucosidase

(Fig. 3). For wheat, the acetic and propionic acid concentrations
Protease

reached 1394 and 2331 mg-COD/L, respectively, which accounted for

Table 2

31.3% and 52.4% of total VFAs. Other species, including butyric, iso-
valeric and valeric acid, accounted for approximately only 16.2%. As

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R.-h. Li, X.-y. Li Bioresource Technology 245 (2017) 615624

Table 3
Analysis of the compositions of the selected types of food wastes.

(mg/g dry weight) Rice Wheat Corn Bean Chicken

Carbohydrates 805 53 776 41 860 45 465 38 145 32

Protein 10 5 90 8 <1 233 12 480 71
P 7 1 7 1 10 1 34 2 77 2
COD 1045 86 828 76 671 70 877 79 1259 102

VFAs with more than three carbon atoms can be biodegraded to form
acetic acid during anaerobic fermentation (Feng et al., 2009), there was
less accumulation of these VFAs in the solution.
3.2.3. Hydrolysis and acidogenesis of the food wastes
Higher VFA yields were obtained from rice, wheat and corn that is,
carbohydrate-based food waste than from bean and chicken meat.
VFAs were produced for solid organics via hydrolysis and acidogenesis.
Hydrolysis of particulate organic matter to soluble organic monomers is
commonly considered as the rate-limiting step for anaerobic organic
digestion (Lee et al., 2014). As shown in Fig. 2, the hydrolysis e-
ciencies of rice, wheat and corn were much higher than those of bean
and chicken meat. The main composition of rice, wheat and corn is
carbohydrates, or starch. Bean also contained 46.5% carbohydrates;
however, the carbohydrates in bean were reported as mainly dietary
bre, unlike cereals (Bednar et al., 2001). The main composition of
chicken meat is protein.
The hydrolysis of particulate organic matter requires a number of

Control Rice Wheat Corn Bean Chicken

7.0 5000
(a) (b)
6.5
4000

6.0

(mg-COD/L)
3000
pH

VFAs
5.5

2000
5.0

4.5 1000

4.0 0
2500 1200
(c) (d)
1000
2000

800

(mg-COD/L)
Carbohydraes

1500
(mg-COD/L)

Protein
600
1000
400

500
200

0 0
120
600
(e) (f)
100 500

80 400
Solubel Fe(II)
Solubel P
(mg/L)

(mg/L)

60 300

40 200

20 100

0
0

0 1 2 3 4 0 1 2 3 4

Time (d) Time (d)

Fig. 2. Batch fermentation performance of sludge with selected types of food waste: (a) pH, (b) VFAs, (c) soluble carbohydrates, (d) soluble proteins, (e) SP and (f) SFe(II) concentrations
in the supernatants during the co-fermentation process.

619
R.-h. Li, X.-y. Li Bioresource Technology 245 (2017) 615624

Acetic acid Propionic acid Isobutyric acid

Butyric acid Isovaleric acid Valeric acid
2500 2500
(a) Control (b) Rice
2000 2000

1500 1500
(mg-COD/L)

(mg-COD/L)
VFAs

VFAs
1000 1000

500 500

0 0
2500 2500
(c) Wheat (d) Corn
2000 2000

1500 1500
(mg-COD/L)

(mg-COD/L)
VFAs

VFAs
1000 1000

500 500

0 0
2500 2500
(e) Bean (f) Chicken meat
2000 2000

1500 1500
(mg-COD/L)

(mg-COD/L)
VFAs

VFAs
1000 1000

500 500

0 0
0 1 2 3 4 0 1 2 3 4
Time (d) Time (d)
Fig. 3. Species and amount of VFAs produced during fermentation of types of food waste indicated.

enzymes to cleave polymers to monomers (Dick, 2011). To characterise
the dierence in hydrolysis of starch, bres and proteins, the related
enzyme assays were further analysed. -Glucosidase and -glucosidase
are related to the last step of production of glucose or maltose. Usually,
-glucosidase is involved in the hydrolysis of polysaccharides
composed of -1,4-glycosidic bonds such as starch (Eq. (1)), and -
glucosidase is involved in the hydrolysis of polysaccharides composed
of -1,4-glycosidic bonds such as cellulose (Eq. (2)) (Zechel and
Withers, 2000). Protease is involved in the hydrolysis of peptide bonds
in proteins to produce amino acids (Eq. (3)) (Alvarez et al., 2006).

620
R.-h. Li, X.-y. Li
The results of the hydrolytic enzyme activity assays are shown in
Fig. 4. Compared with the control reactor without the addition of food
waste, the activities of -glucosidase and -glucosidase were greatly
enhanced in the batch sludge fermenters dosed with model food waste.
The addition of rice, wheat and corn led to increased activity of -
glucosidase and -glucosidase, even in low-pH conditions. In particular,
the activity of -glucosidase was more than 12 times that of the control
reactor and 6 times that of the reactor with bean. Bean induced a higher
activity of -glucosidase only because of its higher cellulose content.
However, the activity of -glucosidase with bean was still lower than
that with rice, wheat and corn, suggesting that cellulose had a lower
hydrolysis eciency. Chicken meat caused somewhat higher activity in
protease than other types of food waste. Overall, the hydrolysis
pathway via -glucosidase had greater activity than those via -glu-
cosidase and protease. It is apparent that organic degradation via the -
glucosidase pathway was resistant to the decrease in pH and hence was
not aected by the accumulation of VFAs in the fermenters.
3.2.4. P recovery from the sludge after acidogenic fermentation
The TP concentration in the Fe-occulation SBR sludge reached
179.8 mg/L, mainly in the form of chemical precipitates. Fe(III)-P
precipitates that formed via chemical P precipitation are reported to be
of Fe-PO4 minerals and FeOOH-adsorbed P (Wu et al., 2015). To release
P from the solid phase of the sludge into the solution for P recovery,
anaerobic and acidic conditions were required. The production of VFAs
during acidogenic fermentation greatly lowered the solution pH. Spe-
cically, co-fermentation with rice, wheat and corn led to pH values as
low as 4.5, with high VFA concentrations of about 4000 mg/L.
Meanwhile, Fe(III) in activated sludge was reduced to Fe(II) in
anaerobic fermentation conditions. During the process called dissim-
ilatory iron reduction, Fe(III) functioned as a terminal electron acceptor
for the iron-reducing microorganisms (Weber et al., 2006). At the
(a) -glucosidase
0.08
Specific enzyme activity
0.06
(EU/g-VSS)
0.04
0.02
0.00
(c) Protease
0.08
Specific enzyme activity
0.06
(EU/g-VSS)
0.04
0.02
0.00
Control Rice Wheat Corn Bean Chicken Control Rice
Bioresource Technology 245 (2017) 615624
beginning of sludge fermentation, little Fe(II) was found in the sludge
solution. After fermentation, the total Fe(II) fraction in the batch sludge
fermenters had increased to 26.5% (control), 77.9% (rice), 82.6%
(wheat), 84.0% (corn), 70.5% (bean) and 88.7% (chicken). With the
addition of food waste, the reduction of Fe(III) was greatly enhanced
via dissimilatory iron reduction. As a result, Fe(III)-P in sludge was
largely reduced to Fe(II)-P, including Fe3(PO4)2 and Fe(OH)2-adsorbed
P. The solubility of Fe(II)-based chemical compounds is much higher
than that of Fe(III)-based compounds. For instance, the solubility of
FePO4 and Fe3(PO4)2 at pH 5 are around 0.01 and 1 mmol/L, respec-
tively (Stumm and Morgan, 2012).
With the dissimilatory iron reduction and VFA production, the Fe
(II)-P precipitates dissolved into ferrous ion and orthophosphate in the
solution. The SP and SFe(II) concentrations were found to greatly in-
crease during the rst 48 h of co-fermentation with rice, wheat and
corn; they then remained relatively stable (Fig. 2). For instance, with
the addition of wheat, the orthophosphate concentration increased
from 0.1 to 88.8 mg/L and the SFe(II) increased from 0.5 to 503.0 mg/
L. The fractions of phosphorus release from the sludge were 58.6%
(rice), 58.0% (wheat), 56.4% (corn), 20.0% (bean) and 34.0% (meat).
Dissolution of Fe(II)-P precipitates is believed to be the main cause of P
release from the solid phase of SBR sludge. Thus, the eciency of P
extraction from the sludge was expected to be further increased with a
larger food waste load for co-fermentation. Moreover, the detected high
enzyme activity of acid phosphatase suggests the possible release of P
from the hydrolysis of organic-P compounds (Fig. 4).
With the SP and SFe(II) released into the fermented sludge liquor, P
could be readily recovered from the supernatant. The supernatant was
withdrawn from the fermenters, and P was simply recovered by re-
precipitation. After adjustment of the pH to 8.0, more than 99.5% of the
SP precipitated with Fe(II) to form Fe(II)-P solids that could be readily
separated from the solution by sedimentation or centrifugation.
According to the X-ray diraction analysis given in Supplementary
Fig. 4. Comparison of the enzyme activities
(b) -glucosidase
Specific enzyme activity
0.08 amongst various types of food waste after 4 d of
acidogenesis and fermentation: (a) -glucosi-
(EU/g-VSS)
dase, (b) -glucosidase, (c) protease and (d) acid
0.06
phosphatase.
0.04
0.02
0.00
(d) Acid Phospatease
Specific enzyme activity
0.08
(EU/g-VSS)
0.06
0.04
0.02
0.00
Wheat Corn Bean Chicken
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R.-h. Li, X.-y. Li Bioresource Technology 245 (2017) 615624

Chemical P Removal Dissimilatory iron reduction Fig. 5. Illustration of the main mechanisms involved in the new pro-
cess for simultaneous recovery of P and VFAs, including chemical
precipitation for enhanced P removal, organic acidogenesis for VFA
Fe3+ FePO4 Fe3(PO4)2 Fe2+
PO43- production, Fe(III) reduction and P release for eective recovery.
FeOOH-P Fe(OH)2-P PO43-
e- H+

Polysaccharide Monosaccharide Acetic acid

Protein Amino acids Propionic acid

Hydrolysis Acidogenesis

data, the recovered Fe(II)-P solids consisted mainly of vivianite
(Fe3(PO4)2 8H2O), a valuable material for P fertilisers. Moreover, it is
also possible to recover P in the form of struvite from the fermented
sludge supernatant, as reported in other studies (Guney et al., 2008).
The results of the batch experiments show that acidogenic co-fer-
mentation can be used to recover P from the Fe-occulated activated
sludge and to produce VFAs from food waste. The starch-rich food
waste, such as rice, a staple food in Asia, is particularly eective for co-
fermentation and subsequent P and organic C recovery. The major
mechanisms involved in chemical transformation during the co-fer-
mentation process and related resource recovery are illustrated in
Fig. 5. The activated sludge, with greatly reduced P and Fe content after
fermentation, can be returned to the SBR for biological wastewater
treatment. It is known that P can be extracted and recovered from the
excess sludge by means of chemical acidication, such as the Stuttgart
process (Egle et al., 2016) and RecoPhos (Weigand et al., 2013).
However, chemical acidication methods require a large amount of
chemical acids to adjust the sludge pH to 2 or lower to dissolve P into
the solution. For example, around 0.35 g/g-SS of H2SO4 was required to
extract 5075% P from the sludge by the chemical acidication method
(Egle et al., 2015). In this study, only 0.71 g/g-SS of food waste was
used to achieve around 60% P extraction from the sludge. The chemical
processes not only are costly but also produce dangerous wastes that
cause potential secondary pollution. The method of acidogenic co-fer-
mentation developed in this study can achieve the same P recovery
results using low-cost food wastes in relatively simple and mild op-
eration. In addition, VFAs are produced as valuable organic resources
for benecial uses, such as the carbon source for denitrication or or-
ganic substrates for biosynthesis of PHAs, whilst biomass sludge can be
returned to the aerobic bioreactors. Thus, Fe-occulation-activated
sludge coupled with side-stream acidogenic co-fermentation of food
waste appears to be a more suitable and eective process to replace
chemical acidication for P recovery from wastewater, together with
signicant production of VFAs.
3.3. Continuous operation of the SBR and co-fermentation system for
simultaneous P recovery and VFA production
3.3.1. Long-term performance
The Fe-occulation SBR, together with the side-stream acidogenic
sludge fermentation, was operated for more than 60 d for simultaneous
recovery of P and VFAs from municipal wastewater and food waste.
Rice, a staple Asian food, was added as the model food waste for co-
fermentation. The wastewater treatment performance of the aerobic
SBR was similar to the previous stages without the side-stream sludge
fermentation (Fig. 6). With the Fe3+ occulant addition at 25 mg/L, the
average COD and TP removal eciencies were 89.8% and 93.1%, re-
spectively. The COD and TP concentrations in the euent were mostly
below 20.0 and 0.5 mg/L, respectively, with the one-step SBR treat-
ment. The removed P was retained in activated sludge for recovery.
In the anaerobic fermenter, the P-rich sludge from the aerobic SBR
was mixed with the model food waste (rice) for co-fermentation. The
fermenter had a daily feed exchange ratio of 25%, corresponding to an
overall HRT and SRT of 4 d. Via natural acidogenic fermentation under
anaerobic conditions, orthophosphate and VFAs were produced in the
supernatant that was recovered every day (Fig. 7). The average VFA
concentration in the solution was around 1991 mg-COD/L, accounting
for 79.6% of the initial food organic loading. The solution pH was

Influent COD Effluent COD COD Removal Efficiency Fig. 6. Performance of the aerobic SBR with Fe(III) dosing for was-
tewater treatment: (a) chemical oxygen demand and (b) total P re-
Influent TP Effluent TP TP Removal Efficiency
(a) 300 100 moval.

250
Efficiency (%)

80
200
COD (mg/L)

60
150
40
100
20
50
0 0
(b) 10 100
80
Efficiency (%)

8
Total P (mg/L)

6 60

4 40

2 20
0 0
0 10 20 30 40 50 60
Time (d)

622
 R.-h. Li, X.-y. Li Bioresource Technology 245 (2017) 615624

Solubel Organics VFAs SFe(II) SP pH Fig. 7. Performance of co-fermentation in the anaerobic reactor for
organic acidogenesis, and SFe(II) and SP release into the supernatant.
3000 450 6.0

400
Soluble Organics & VFAs (mg-COD/L)

2500
350

Soluble P & Fe(II) (mg/L)

2000 300 5.5

250
1500

pH
200

1000 150 5.0

100
500
50

0 0 4.5
0 10 20 30 40 50 60
Time (d)

maintained at a low level of around 5.0. At this pH, the average SP
concentration in the supernatant reached 106.5 mg/L. More than 45%
of the precipitated P in the SBR sludge entering the fermenter was re-
leased and dissolved into the liquid phase after co-fermentation.
In addition to P recovery facilitated by acidogenic fermentation, the
side-stream anaerobic sludge processing successfully reduced the in-
organic content in the activated sludge in the aerobic SBR. As P and Fe
dissolved and were removed after fermentation before the sludge was
returned to the SBR, the TP and Fe concentrations in the aerobic SBR
sludge mixture were reduced considerably from to 179.8 to 87.8 mg/L
and from 590.8 to 339.2 mg/L, respectively. Compared with the single
SBR without the side-stream process, the VSS/SS ratio in the aerobic
SBR also increased from 64.6% to 75.0%. Thus, the use of anaerobic
sludge fermentation not only helped recover valuable resources from
wastewater and food waste, but also improved the bioactivity of acti-
vated sludge in the SBR system for biological wastewater treatment.
The microbial community of the sludge in the iron-dosing aerobic
bioreactor and anaerobic fermentation system was also analyzed during
the experimental study. Some of the results about the microbial popu-
lation structure in the stabilized sludge in comparison to the seed sludge
are given as supplementary data. It was found that the iron-dosing and
sludge acidogenesis largely reduced the species richness and diversity
of the microbial community, but enriched phylum Proteobacteria. In the
anaerobic reactor, the genus of Prevotella responsible for acidogenic
organic fermentation, and the family of Clostridiaceae important to
dissimilatory iron reduction were greatly enriched. Such changes in

Fig. 8. Mass balance analysis of organic C, P and Fe throughout the

Influent Aerobic SBR Effluent Fe-occulation SBR and side-stream co-fermentation system. FC,W: C
VA: 4 L mass ow rate in wastewater; FC,FW: C mass ow rate in food waste;
FC,W: 808.0 mg/d (100%) FC,W: 78.4 mg/d (9.7%)
FP: P mass ow rate; FFe: Fe mass ow rate; Q: water or liquid ow
FP: 57.6 mg/d (100%) CP,A: 87.8 mg/L FP: 4.0 mg/d (6.9%) rate; CP: total P concentration; CFe: total Fe concentration.
FFe: 200.0 mg/d (100%) CFe,A: 339.2 mg/L FFe: <0.01 mg/d (0.0%)
Q: 8.15 L/d Q: 8.00 L/d

Circulation Sludge Return Sludge

FC,W: 1.9 mg/d (0.0%) FC,FW: 39.4 mg/d (11.0%)
FP: 70.2 mg/d (122%) FP: 19.1 mg/d (33.2%)
FFe: 271.4 mg/d (136%) FFe 85.5 mg/d (42.8%)
Q: 0.20 L/d Q: 0.05 L/d

Supernatant
FC,FW: 236.0 mg/d (66.1%)
Food Waste Anaerobic SBR FP: 32.1 mg/d (55.7%)
FC,FW: 357.0 mg/d (100%) VAn: 1.6 L QFe: 111.6 mg/d (55.8%)
FP: <0.01 mg/d (0.0%) CP,An: 176.1 mg/L Q: 0.30 L/d
FFe: <0.01 mg/d (0.0%) CFe,An: 695.2mg/L
Q: 0.20 L/d
Waste sludge
FC,F: 39.4 mg/d (11.0%)
FP: 19.1 mg/d (33.2%)
FFe: 85.5 mg/d (42.8%)
Q: 0.05 L/d

623
R.-h. Li, X.-y. Li
microbial diversity were expected for the treatment system with iron-
dosing and acidogenic co-fermentation.
3.3.2. Mass balance analysis
Mass balance analysis was performed on the transformation of P, Fe
and organic C throughout the aerobic SBR, anaerobic fermentation and
P and VFAs recovery process; the results are shown in Fig. 8. Ap-
proximately 96.4% of TP in the wastewater inuent was retained by
chemical precipitation and SBR. In the side-stream anaerobic fermenter,
approximately 45.6% of the P in the sludge ow was released as or-
thophosphate into the solution, which was then recovered as vivianite
by pH adjustment and re-precipitation. The overall P recovery e-
ciency from the wastewater inuent was about 55.7%, whilst 33.2% of
the P was disposed with the sludge waste. Meanwhile, approximately
66.1% of the food waste was converted to VFAs and recovered in the
supernatant of side-stream anaerobic co-fermentation. It should be
noticed that the waste sludge from common P removal processes would
contain more than 90% of the P from the wastewater inuent. Thus,
relatively, what was discharged from the fermenters was not P-rich
sludge. Owing to the low P content, it may not worth further extraction
and other processing for P recovery from the sludge. As the sludge al-
ready underwent co-fermentation and acidogenesis for P extraction and
recovery, the residual P should be rather stable in the waste sludge for
disposal.
4. Conclusions
Food waste with a high starch content was more favourable for
acidogenic fermentation. The batch co-fermentation results show
that the types of food waste performed as follows for acidogenesis
and P extraction: wheat > rice > corn > chicken meat > bean.
By co-fermentation with rice for continuous operation, the solution
pH could drop to 5.0 in the fermenter, around 45.6% of P in the
activated sludge from the SBR was released into the supernatant,
and around 66% of the model food waste was converted into VFAs.
An overall P recovery eciency of 55.7% from the wastewater in-
uent was achieved.
Acknowledgements
This research was supported by grants 17204914 and C7044-14G
from the Research Grants Council (RGC) of the Hong Kong SAR
Government and funding KQJSCX20160226190815 from Shenzhen
Municipal Science and Technology Innovation Council of Shenzhen
Government, China. The technical assistance of Mr. Keith C. H. Wong is
greatly appreciated.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, in the
online version, at http://dx.doi.org/10.1016/j.biortech.2017.08.199.
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