Bioresource Technology 63 (1998) 221-229

1998 Elsevier Science Ltd. All rights reserved

Printed in Great Britain
0960-8524/98 $19.00
ELSEVIER PII:S0960-8524(97)00140-5

PROTEIN R E C O V E R Y D U R I N G THE OVERALL TREATMENT

OF WASTEWATERS FROM FISH-MEAL FACTORIES

L. Guerrerofl F. Omil, b* R. M 6 n d e z b & J. M. L e m a ~'

"Department of Chemical Processes, FedericoSanta Mafia Technical University, Casilla l 1O-V,Valparaiso, Chile
bDepartment of Chemical Engineering, Universityof Santiago de Compostela, Avda. das Ciencias s/n, 15706 Santiago de
Compostela, Spain

(Received 24 May 1997; revised version received 12 September 1997; accepted 17 September 1997)

Abstract discharge of these effluents can cause important

The suspended solids contained in the wastewaters
generated by fish-meal industries (5-40g VSS/I) are
composed mostly of protein, which could be recovered
to optimise the manufacturing process. In this work,
settling, centrifugation and coagulation-flocculation
were studied as alternative treatment processes to
attain this objective. Settling experiments showed that
the flotation of a high fraction of suspended solids
occurred (up to 45%), even with previous alkalinisa-
tion or the addition of a coagulant such as chitosan.
Centrifugation at lO000rpm for 12"5min gave
optimum results, TSS and soluble COD removal
efficiencies of 45-82% and 10%, respectively, being
obtained. The amino acid profile of the solids was
quite similar to fish meal, indicating the feasibility of
solids reuse. Results obtained after coagulation-floccu-
lation followed by centrifugation showed that among
the different compounds assayed sodium polyacrylate
and chitosan produced the best results, operating at pH
4 and 7"2-7"8, respectively, and giving TSS removal
efficiencies up to 97 :and 75%, respectively. 1998
Elsevier Science Lid. All rights reserved.
Key words: Centrifugation, coagulation-flocculation,
fish-meal processing wastewaters, protein, reuse,
suspended solids.
INTRODUCTION
Depending on the raw material used, there are
basically two types of fish-meal manufacturing
processes: those that use fish wastes, such as heads,
bones or other residues, and those that use the
whole fish. Wastewaters generated by these factories
are characterised by their high organic content
(30-120g COD/I), an important part of it being
suspended solids (5-40 g VSS/1). The direct
*Author to whom correspondence should be addressed.
221
environmental problems, as found in some areas like
Galicia (northwest of Spain) and region VIII of
Chile (Omil et al., 1994; Roeckel et al., 1996).
The first process, using fish wastes, is the
commonest used in the European industries, such as
those in Galicia, and such processes add value to the
wastes, which are mainly constituted of residues
from tuna and sardines. The first step is the
compression and crushing of the raw material, which
is then cooked with steam, and the liquid effluent is
filtered off in a filter press. The solids obtained are
introduced to a rotating drier and finally cut and
crumbled to obtain the commercial fish-meal
product. The liquid stream contains oils and
suspended solids (fats, proteins). This stream is
passed through two centrifugation steps in order to
recover a high fraction of the suspended solids,
which are fed back into the drier, and an effluent
rich in oils, mainly constituted by polyunsaturated
fatty acids (PUFA) of high commercial value. In
addition, an aqueous stream is generated from the
last centrifugation step with a high content of
organic suspended solids. This effluent, together
with the periodical washing purges, constitutes the
main source of the wastewaters generated by these
factories.
The production of the wastewaters is around
5.4 m 3 per tonne of fish processed, with a variable
content in organic matter and suspended solids,
ranging from 30 to 120gCOD/1 and from 5 to
40 g VSS/1. The final temperature is between 65 and
70C. The composition of this stream depends on
the nature of the raw material processed in the
factory (Table 1), being mainly constituted by
proteins, around 72%, with fats and carbohydrates
content around 21 and 7%, respectively. Another
specific characteristic of these wastewaters is their
high salinity, around 7-8 g C1 /1.
The second group of processes are the ones that
use the whole fish. These are widely developed in
222 L. Guerrero et al.
Table 1. Characteristics of the wastewaters (i) generated by the factory based on the raw material processed and (ii) used
in this work (concentrations in g/I)
Tuna bones Tuna heads Sardine
heads
TSS 13.0 29.9 6-6
VSS 12.8 29.5 6-5
CODt 46"7 81"3 38"2
CODs 22"8 24"3 23"4
pH 6"4 6"3 6"5
Chile, one of the main fish-meal producers in the
world. In Chile more than 90% of the volume of fish
captured is used for fish-meal production. The
mackerel is the most common fish used, but ancho-
vies and hake are also utilised. These processes
involve the hydraulic transport of fish from ships to
factories, and the discharge, cooking and evapora-
tion operations. Three polluting streams arise from
these processes, corresponding to the hydraulic
transport of fishes (86% of total COD), the effluents
generated during the fish reduction process by the
evaporation and deodorisation steps (8"5%) and the
ones resulting from the washing of the process
equipment and the floors of the plant (5.5%). A
complete characterisation of these wastewaters has
been previously reported (Roeckel et al., 1996).
The high protein content of the suspended solids
in all these wastewaters adds interest to the imple-
mentation of a process to recover the solids in order
to optimise fish-meal production (Marti et al., 1994)
or to use them directly as an additive for animal
feedstuffs. In addition, the separation of these solids
can be of interest if high-rate anaerobic reactors are
selected to treat these wastewaters, as anaerobic
filter or UASB reactors (Young, 1991; Lettinga and
Hulshoff Pol, 1991). Separation would avoid opera-
tional problems (clogging, bulking, etc.) and
minimise the possible inhibitory effects on the
methanogenic biomass of ammonia (Omil et al.,
1995).
The aim of the present work was to study the
feasibility of removing by physico-chemical
processes, such as settling, centrifugation and coagu-
lation-flocculation, the suspended solids present in
the fish-meal processing wastewaters. The character-
istics of the recovered solids were determined in
order to study their potential reuse in fish-meal
production.
METHODS
Wastewaters
The wastewaters were collected from a factory
located in Galicia (Spain). Their complete character-
isation has been previously reported (Veiga et aL,
1994). Due to the high variability of their composi-
Type I Type II Type III Type IV
21.7 21-8 25.7 13.0
20.0 21.7 24.3 12.8
107"2 88"9 76"2 53"7
77"2 56"8 39"8 38"4
6"8 6"2 6"0 6"7
tion, it was not possible to operate always with the
same effluents and different wastewaters were used
for the settling, centrifugation and coagulation-
flocculation assays as shown in Table 1 and
described in the following sections.
Experimental operation
Three alternative operations were studied to
maximise the suspended solids recovery: (a) settling,
(b) centrifugation and (c) the addition to coagulants/
flocculants followed by centrifugation (Fig. 1).
Results obtained from these three sets of experi-
ments were compared in terms of suspended solids
and soluble COD removed from the raw
wastewaters.
Settling assays
These experiments were carried out in a 'jar-test'
conventional set-up, with four 400ml beakers
provided with stirring (300 rpm maximum) and time
control. The operational procedure was based on
preliminary results and comprised the following
steps: (1) stirring at 100 rpm for 10 min and (2)
settling for 48 h. The characteristics of the waste-
waters used for these experiments are shown in
Table 1 (Type I). These experiments were carried
out with: (a) the raw influent, (b) after alkalinisation
of pH 8 and (c) after alkalinisation to pH 8 and
addition of chitosan (Fig. 1).
Centrifugation
A Beckman centrifuge (model J2-21) with tempera-
ture, speed and time controls was used. The influ-
ences of speed (5000-15 000 rpm) and centrifugation
time (5-15 min) were studied in order to optimise
solids removal, after which the clarified effluents and
solids were characterised. Temperature was
maintained at 4C. The experiments were carried
out at 3000, 5000, 10000, 12000 and 15000rpm,
which corresponded to relative centrifugation veloci-
ties of 988, 2744, 10976, 15805 and 24696 g, respec-
tively. Once the optimum speed had been
determined, the performance of the centrifugation
was determined after 5, 7.5, 10, 12.5 and 15 min.
Three types of wastewaters were used for these
experiments and their characteristics are shown in
 Protein recoveryfrom fish-meal wastewaters 223

/
Raw w a s~:t e w~ ~:~ '~~s

Settling experiments pH change

pH change
Addition of:
Raw Addition + NaOH - Coagulants
effluent NaOH (pH 8) - Flocculants
(pH 6.8) (pH 8) + Chitosan Centrifugation
Centrifugation

,-. . . . . . . . . . . . . . . _~. . . . . . . . . . . . . . . . . . . . . . . . . . ~.................. Y_. . . . . . . .

i
i

,, Comparison of results
I (TSS, VSS and soluble COD removal efficiencies)
i
. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Fig. 1. Experimental design.

T a b l e 1 (Types II, I I I and IV). T y p e s II and I I I were Addition of coagulants/flocculants

collected f r o m the e f f l u e n t of the factory w h e n Different c o m p o u n d s w e r e used as coagulants:
washing was being u n d e r t a k e n , thus explaining their ferrous and a l u m i n i u m salts (FeCI3, FeSO4,
higher content of s u s p e n d e d solids and C O D in Fe2(504)3, A12(SO4)3), as well as an organic cationic
c o m p a r i s o n to T y p e IV. polymer, chitosan. F o r flocculation, the following

Table 2. Experimental design of coagnlation/flocculation assays (concentrations in mg/l)

Experiment Wastewater Coagulant Concentration Flocculant Concentration

AI Type III A12(804)3 100 S-2515 10

Fel Type III FeSO4 200 BL-507 10
Fe2 Type III Fe2(SO4)3 200 BL-514 10
Fe3 Type III FeCI3 200 BL-507 10
C1 Type III Chitosan 100 None --
C2 Type III Chitosan 100 S-611 10
C3 Type III Chitosan 100 S-2515 10
C4 Type III Chitosan 100 BL-514 10
C5 Type III Chitosan 100 BL-507 10
PI Type IV None -- PA-F 10
P2 Type IV None -- PA-CH 10

Table 3. Results obtained with the settling experiments (concentrations in g/I, volumes in percentage of original sample)

Raw wastewater Wastewater + NaOH Wastewater + NaOH + chitosan

Initial After settling Initial After settling Initial After settling

Top Liquid Bottom Top Liquid Bottom Top Liquid Bottom

pH 6"8 6"8 6"8 6"8 8"0 8"0 8"0 8"0 8"0 8"0 8"0 8"0
TSS 21 '7 39"6 13"7 40"9 18"8 30-6 8'7 41"7 18"7 32"9 8"3 42"8
VSS 20'0 36"6 12"2 37"4 17"5 28"5 7"6 38-5 16"8 29"6 7-5 38"5
CODt 107'2 133"3 95"3 136"3 107"2 128"3 91'5 142"1 107"7 131"8 92"8 141"8
CODs 77'2 78"4 77"1 80"2 81.1 85-5 80"1 84.5 82"5 87"3 81.6 84'1
Volume 100 24 70 6 100 15 64 21 100 14 65 21
224 L. Guerrero et al.

100%
[ ] T S S O V S S CODs
.....................
80% ..........

60% - -- -
if!i! i!i!i~ ~
:i:!:~ !ilili
40% iiii! i:::i ::::~

>
O
E 20% -- :::::I -
:::::l
r
o% iiii!il I= iiiiiiI

-20%
6.2 6.5 6.8 7.0 7.2 7.5 7,8 8.0 8.2 8.5 8.7 9.0

pH
Fig. 2. Removal efficiencies obtained after pH modification followed by centrifugation (pH of raw wastewaters was 6.2).

substances were used: S-611, S-2515, BL-514 and the optimum conditions previously determined and
BL-507 (Buckman Laboratories, Gent, Belgium). the solids and supernatants obtained were then
The combination of coagulants and flocculants used characterised. The wastewaters employed were the
in the different experiments, as well as their concen- same as those described in the previous section
trations, are shown in Table 2. In addition, sodium (Types II, III and IV).
polyacrylate (Nippon Kayaku Co. Ltd.) was used
without previous addition of coagulants. Sodium Analytical techniques
polyacrylate was used in two different forms: fine Total suspended solids (TSS), volatile suspended
particles, PA-F (Panakayaku-F sodium polyacrylate), solids (VSS), chloride and phosphate were analysed
and large particles, PA-CH (Panakayaku-CH sodium according to Standard Methods (APHA-
polyacrylate). AWA-WPLF, 1985). A semi-micro method (Soto et
The experimental procedure comprised pH al., 1989) was used to determine accurately the total
correction, addition of coagulant and stirring at and soluble COD (CODt and CODs, respectively) in
10 rpm for 10 min. When flocculants were used, the samples with high chloride concentrations. Soluble
samples were stirred after their addition at 50 rpm protein was analysed by Lowry's method (Lowry et
for 12min. After coagulant and/or flocculant al., 1951) and soluble carbohydrates were analysed
addition and stirring, samples were centrifuged at by means of the hydrazine sulphate/sulphuric

Lysine r--'m i
Fish meal
Phenylalanine

Leucine Solids recovered

Isoleucine r~ :o

Cysteine D :o

Methionine o:

Valine

Tyrosine

Alanine

Threonine

Arginine

Histidine i
Glycine

Serine
Glutamic acid

Aspartic acid

0 4 6 10 12 14 16 18 20

Composition (%)

Fig. 3. Amino acid composition (%) present in normal fish meal and in the solids obtained after centrifugation.
 Protein recoveryfrom fish-meal wastewaters
100%
F 1~ T S S ( 3 V S S C O D s
80/ L "-
il-I
'~
o%i11:
i i_l_If60%
20%
- -
--
0% ~ - , ~
Fel Fe2 Fe3
Fig. 4. Removal efficiencies obtained after the addition of coagulants/ttocculants followed by centrifugation at the pH of
the raw wastewaters (detailed description of each experiment in Table 2).
method (Miller, 1959). Ammonium and redox
potential were determined by selective electrodes.
Amino acids were determined with a hydrolysis
apparatus (PICO-TAG) and HPLC (Waters, Model
510).
RESULTS AND DISCUSSION
Settling assays
The results obtained during the settling experiments
showed that the formation of three different zones
occurred: a floating solids layer, the clarified liquid
and a bottom layer of solids. The characterisation of
these zones is shown in Table 3. It can be seen that
the clarified liquid ranged from 64 to 70% of the
total volume of the original samples. A TSS reduc-
tion of 39% of the raw wastewater was obtained and
it was further increased to 62% when a previous
alkalinisation was carried out. The addition of
chitosan produced little improvement in these
results. In all experiments the formation of a super-
ficial layer with a high content of suspended solids
(mainly composed of fats) occurred, although the
initial alkalinisation caused a decrease of its volume
from 24 to 15%.
These results show that, even after 48h of
settling, it is not possible to accomplish a successful
separation of the suspended solids due to the flota-
tion of a large fraction of the solids (45% of TSS at
raw pH and 24% when alkalinisation was carried
out).
Centrifugation
Initial experiments showed that optimum centrifuga-
tion velocity and operational time were 10000 rpm
225
I'- r-- i
i iii~
I :i
~ ,- . . . .
AI C1 C2 C3 C4 C5 P1 P2
Coagulant / Flocculant
and 12-5 min, respectively. The operation at higher
velocities and/or longer periods of operation did not
improve the removal efficiencies attained. The
reduction of TSS and VSS ranged between 45 and
49% for the Type II wastewaters, and around
74-75% and 82-87% for Types III and IV, respec-
tively. However, the reduction of soluble COD was
much lower, from 7 to 11% for all wastewaters.
Influence of pH
In order to determine the influence of pH, NaOH
was added to the Type II wastewaters before centri-
fugation. As it can be observed in Fig. 2, the
maximum TSS and VSS removal efficiencies were
obtained at pH 8. It can be also observed that the
pH change promoted a slight soluble COD removal
(5-18%) except at pH 8, when an increase of
around 11% in soluble COD was observed, possibly
as a result of a greater partial hydrolysis of solids
under these conditions.
Amino acid profile
As an important characteristic in determining the
potential reuse of the recovered solids in the fish-
meal manufacturing process, the amino acid profile
of the solids was compared to the values of fish
meal. As can be seen in Fig. 3, the characteristics of
the recovered solids are compatible with the require-
ments for fish meal. Although there appear to be
some significant differences in the concentrations of
particular amino acids, such as glycine or phenyl-
alanine, it would not be expected that the amount of
solids added would significantly alter the composi-
tion of the fish-meal product. Based on the average
values of the wastewaters flow and VSS concentra-
226 L. Guerreroet al.

tions, and supposing a complete suspended solids the removal efficiencies obtained with each
recovery, solids would account for only up to 8% of combination of coagulants/flocculants used. The
the total amount of fish meal produced. highest TSS and VSS removal efficiencies were
achieved when sodium polyacrylate and chitosan
Addition of coagulants/flocculants (87-92%) were used, whereas when metallic salts
Different compounds were used as coagulating and were dosed maximum removal efficiencies ranged
flocculating reagents to maximise suspended solids only from 75 to 82%. However, metallic salts
removal. The optimum pH was determined for each allowed a higher soluble COD removal efficiency
substance. (32% versus 26% with chitosan), whereas sodium
polyacrylate again produced the best results (up to
52%).
Use of coagulants/flocculants without pH modification
These experiments were carried out at the usual pH
of the raw wastewaters (6.2 for the experiments with Evaluation of optimum pH
metallic salts and chitosan, and 6.7 for the ones Based on preliminary experiments, the selected pH
carried out with sodium polyacrylate). Fig. 4 shows ranges were 4-7 for metallic salts, 4-8 for chitosan

1 0 0 % ~. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

t A

It)
80% -~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ~ TSS .......
.2 QVSS
f-
.2 60% CODs .......
F ...............
m

tg 40%
0
E
I
Ig 20% ~ ....
i
m

0% .~ I i r --I I
3.0 4.0 5.0 6.2 6.5 6.8 7.0 7.2 7.5 7.8 . 8.0

pH

100% ~ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

B
TSS
80% __ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
I OVSS
t,- F
r

60% t . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . CODs . . . . . .
E: l
1> 40,, . . . . . . . .
. . . . . . . . . . . . . . . . . . . . . . . . . .

0
I=:

0%
0o,o
,
oI
I ~ ::il
I, ii ...... ,..... i :!|, i:

, , ,

3.0 4.0 5.0 6.2 6.5 6.8 7.0 7.2 7.5 7.8 8.0

pH
Fig. 5. Removal efficiencies obtained after the addition of metallic salts followed by centrifugation at different pH: (a)
A12(SO4)3+S-2515; (b) FeCl3.
 Protein recovery from fish-meal wastewaters
100%
l~ TSS [] VSS
80%
, M
o 60% . . . . . . . . . . . . . . . . .
._~
--'
400/0 - . .
'
E 20% . . . .
0% ', ',
I
1
-20%
3.0 4.0
Fig. 6. Removal efficiencies obtained after the addition of chitosan followed by centrifugation at different pH.
and 3-6.7 for sodium polyacrylate. Figures 5-7
summarise the results obtained with metallic salts,
chitosan and sodium polyacrylate, respectively.
The effect of pH appears to be significant in all
cases, the optimal range being different for each
substance: 5-6 for metallic salts, 7-8 for chitosan
and 3-4 for sodium polyacrylate. This fact has to be
considered for an overall economic balance since the
pH of the raw wastewaters usually ranges from 6 to
7.
The highest removal efficiencies were obtained
with sodium polyacrylate, either in its PA-F or
PA-CH form. With this compound the reductions of
suspended solids and soluble COD were around 97
and 25-35%, respectively (Fig. 7a and b). Chitosan
also provided high efficiencies (74-80% of TSS and
VSS) but in this case the highest reduction of
suspended solids was obtained at pH 8 when an
increase of soluble COD was observed. It was not
possible to determine if there was a solids reduction
because of the coagulation process or if it was
merely due to a hydrolysis (Fig. 6). However, at pH
7.2-7.8 the TSS and VSS removal efficiencies were
remarkable (around 75%) but with only a slight
soluble COD removal, around 2-9%. In contrast,
the results obtained with metallic salts were lower
than those obtained without pH modification and
very different from those obtained with chitosan or
sodium polyacrylate, ranging from 45 to 50% for
TSS and VSS. However, these substances are
indicated for soluble COD removal, which ranged
from 13 to 20% (Fig. 5a and b).
This work showed the feasibility of using both
centrifugation and coagulation-flocculation as
operations to reduce significantly the content of
organic solids present in the wastewaters from fish-
meal factories. The use of settling as an alternative
227
CODs
!
i ~i!ii
.
i!~ii
', r
5.0 6.2 6.5 6.8 7.0 7.2 7.5 7.8 8.0
pH
process did not give good results, even when pH was
previously modified or chitosan was added, due to
the flotation of a fraction of the suspended solids
(up to 45%). TSS removal efficiencies obtained by
direct centrifugation for 12-5 min at 10000 rpm were
45-85% and these solids could be incorporated in
the manufacturing process to optimise fish-meal
production because of their similar composition to
the normal product. Treatment processes based on
coagulation-flocculation followed by centrifugation
were adequate for TSS, VSS and soluble COD
removal. Among the different substances employed
(metallic salts, chitosan, sodium polyacrylate and
other flocculants), the best results were achieved
with sodium polyacrylate (up to 97% TSS at pH 4)
and chitosan (up to 74% TSS at pH 7.2).
Although sodium polyacrylate gave the best
results both in suspended solids recovery and in the
removal of soluble COD from the wastewaters, it
was necessary to operate at low pH, which requires
the implementation of two steps of pre-acidification
and subsequent neutralisation. This synthetic
polymer has been reported to be widely used in
some countries, such as Japan, although there is so
far no report focused on its digestibility, which is
essential when considering the direct use of the
sludges obtained for feeding purposes. In Europe its
use is also very common, especially for the purposes
that have been described in this paper, and it is
expected that the use of this compound will be
officially regulated in the near future.
Chitosan also gave good results, with the
additional advantage that the optimum operation
was achieved at neutral pH, i.e. 7-8. Chitosan is a
derivative of chitin, the biopolymer that forms the
exoskeleton of insects and crustaceans, and is the
second most abundant natural polymer after cellu-
228 L. Guerrero et al.

100%
A
I/) 80%
: r-,
................. t~TSS .....

t-. VSS
I1)
60% I~, C O D s --

40%
O
E i
i ,iii~i
20% ~ lii~ii
iili!i
:iii~
I Ii!iii:
0%
3.0 4.0 5.0 6.2 6.5 6.8 7.0 7.2 7.5 7,8 8.0
pH

100%
B
............. ~TSS .......
I/) 80%
o DVSS
P
I1}
"5 60% ............... CODs . . . . . .

N> 40%
0
E
20%

0% ..... i .....t t I I i

3,0 4.0 5.0 6.2 6.5 6.8 7.0 7.2 7,5 7,8 8.0
pH
Fig. 7. Removal efficiencies obtained after the addition of sodium polyacrylate followed by centrifugation at different pH:
(a) PA-F; (b) PA-CH.

lose. Its use as cattle feedstuff has been suggested,
among other potential applications (Rawis, 1984).
Buelna et al. (1990) reported the use of chitosan for
the recovery of algal biomass grown on pig excre-
ment for its use as animal feedstuff, both alone or in
combination with synthetic polymers. They reported
removal efficiencies of 85% with 10 mg/l of chitosan
at pH 7, which could be increased with the use of
synthetic polymers. The potential use of these
sludges for feeding purposes clearly indicates the
'clean' nature of these process.
Apart from the feasibility of both centrifugation
and coagulation-flocculation operations for the
reduction of suspended solids, this treatment makes
the further treatment of the remaining clarified
liquid streams possible in high-rate anaerobic
reactors, such as anaerobic filters or UASB reactors,
in which the soluble organic matter would be effect-
ively removed.
ACKNOWLEDGEMENT
This work was supported by the Spanish Commis-
sion of Science and Technology (CICYT), Project
AMB95-0365.
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APHA-AWA-WPCF (1985). Standard Methods for the
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Buelna, G., Bhattarai, K. T., de la Noue, J. & Taiganides,
E. P. (1990). Evaluation of various flocculants for the
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Lettinga, G. & Hulshoff Pol, L. W. (1991). UASB-process
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Lowry, O. H., Rosenbrough, N., Farr, A. L. & Randall, R.
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Marti, C., Roeckel, M., Aspe, E. & Kanda, H. (1994).
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Rawis, R. L. (1984). Prospects brighten for converting
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