War. Res. Vol. 20, No. 1, pp. 97-103, 1986 0043-1354/86 $3.00+0.

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Printed in Great Britain. All fights reserved Copyright © 1986 Pergamon Press Ltd

ANAEROBIC PURIFICATION OF WASTE

WATER FROM A POTATO-STARCH
PRODUCING FACTORY

HENK J. NANNINGA and JAN C. GOTTSCHAL

Department of Microbiology, University of Groningen, Kerklaan 30, 9751 NN HAREN,
The Netherlands

(Received June 1985)

Abstract--The waste water of the potato-starch factory of the AVEBE in De Krim (The Netherlands)
passed, during the anaerobic purification, a sedimentation pond, a first upflow reactor (in which there was
practically no sludge retention) and a UASB methane reactor. The fermentation of free-amino acids and
smaller peptides occurred in the sedimentation pond and first reactor. Proteins and longer peptides were
degraded in the first reactor and in the methane reactor. The decrease in COD and TOC content of the
waste water between influent sedimentation pond and effluent methane reactor was 83 and 71%,
respectively. In the effluent of the first reactor, 60% of the inorganic sulfur was present as sulfide.

Key words--waste water, amino acid fermentation, sulfate reduction, UASB methane reactor

INTRODUCTION MATERIALS AND METHODS

At the potato-starch factory in De Krim (AVEBE,
The Netherlands) large amounts of waste water are
discharged. The most important organic compounds
in this effluent stream are soluble carbohydrates
(7 g l-t), protein (15 g 1-1), amino acids (15 g 1-t) and
citrate (5 g l-t). F r o m the (valuable) protein moiety
over 80% is removed by heat-coagulation followed by
ultra-filtration. The remaining organic matter is de-
graded into carbon dioxide and methane in an upflow
anaerobic sludge blanket (UASB) methane reactor
(Lettinga et al., 1980; Van Bellegem, 1980). This
reactor is preceded by an upflow reactor with a fairly
short hydraulic retention time ( < 1 0 h ) in which
organic matter is degraded to relatively simple or-
ganic and inorganic intermediates. Besides carbon
containing products, also some reduced inorganic
compounds are being produced. A m m o n i u m is re-
leased as a result o f the fermentation of nitrogenous
compounds. Sulfide, on the other hand, originates
primarily from the anaerobic respiratory processes in
which oxidized sulfur-containing compounds (mainly
sulfite and sulfate) serve as electron acceptors. These
oxidized sulfur compounds are present in the waste
water as a result of their use in the process o f starch
extraction from the potatoes and the subsequent
protein recovery.
In this presentation, attention will be focused on
the reduction of inorganic sulfur compounds and on
the extent and nature of the conversions of organic
(nitrogen containing) compounds present in the waste
water as it passes through the different phases of the
purification process.
97
Short description of the purification plant
During the anaerobic treatment the waste water passes
successively a sedimentation pond which is exposed to air,
an upflow reactor with practically no sludge retention, a
sulfide stripper and a UASB methane reactor (see Fig. l).
At the time the samples were taken the sulfide stripper was
not operative and the influent of the methane reactor was
diluted by a factor of 1.4. In the sedimentation pond, first
upflow reactor and methane reactor, with volumes of 700,
1700 and 5000 m 3, respectively, the temperatures were 33, 33
and 35°C, the pH values were 6.2, 6.2 and 7.5, and the
hydraulic retention times 3.25, 9.5 and 20h, respectively.
The methane reactor contained 50 tons volatile suspended
solids (VSS).
Sampling
The samples taken from the purification plant were
collected in serum bottles. The biological activity was
inhibited either by putting small quantities (40 ml) on ice
(determination of fatty acids, formate, glucose, alcohols,
organic carbon content and nitrogen content) or by adding
concentrated formic acid to 1% (v/v) final concentration
(determination of ammonium, amino acids, carbohydrates
and citrate). After transport to the laboratory part of these
samples was stored in a deep-freezer after separation of
biomass and fluid by centrifugation (8 rain at 10,000 rpm).
The remaining samples were stored in the freezer immedi-
ately after transport.
When samples were taken at the different points in the
purification plant they were drawn within a period of I h.
Although at times variations in the composition of the
inflowing waste water stream did occur, care was taken that
for a period of 48 h before sampling no such changes had
taken place. Furthermore, the results obtained with samples
taken on other occasions were comparable with those
presented in this paper.
Chemical analysis
Both volatile and non-volatile short chain fatty acids were
analyzed with a Packard 437 gaschromatograph equipped
98
inflOW of waS_] anaerobic purification .h
*r,c~n{
sir,~ factory [ i
sc~
i ctmmonla /
mM 3o1 f gation for 20min at 5000rpm. In hydrolyzed samples.
20~
tO, -]:'< ~carbohyrJrares
~04
acetate -/'~ "
30 ~-
mM
i 1977).
L actate
5 tetrathionate, sulfate and total inorganic sulfur were deter-
1 ~" ~ o×atate
~< -4'meth anc~ ~ suspension with oxygen free nitrogen gas. Subsequently the
Fig. 1. Concentrations of various compounds present in the
waste water stream as it passes the purification process: (1)
sedimentation pond, (2) first reactor, (3) sulfide stripper, (4)
dilution point, (5) methane reactor. Arrows indicate the
sampling points. The carbohydrate content is expressed as
glucose-units (mM).
with a flame ionization detector, connected to a Packard 604
integrator. Glass columns (2 m long; 2 mm i.d.) were filled
with Chromosorb WAW, 100-120 mesh, coated with 10%
SP-1000 and 3[/0 H3PO4 (Chrompack Nederland B.V.,
middelburg, The Netherlands). The flow rate of the carrier
gas (nitrogen) was 50 ml min-L The temperature of injec-
tion port, column, and detector were 175, 120 and 175°C,
respectively. The flow rates of H 2 and air were 30 and
200 ml min i, respectively. Volatile fatty acids were
determined after diethyl ether extraction as described by
Laanbroek et aL (1983). Isovalerate (3-methylbutyrate) was
not separated from 2-methylbutyrate. In the results iso-
valerate may therefore represent either of these two com-
pounds. Lactate, oxalate and succinate were determined
after methylation and chloroform extraction using malonic
acid as internal standard (Laanbroek et al., 1983). Formate
was determined in the same way as these non volatile acids
but with a column temperature of 50°C.
H 2, CH4 and CO 2 in the gas phase of the cultures were
analyzed on a Pye Unicam 104 gaschromatograph equipped
with a katharometer detector (Laanbroek et al., 1983). The
detection limit for H 2, CH 4 and CO 2 was 0.001, 0.05 and
0.1% (v/v), respectively. Methanol and ethanol were ana-
lyzed with a Packard 427 gaschromatograph (Laanbroek et
al., 1984), using 2 m glass columns (2 mm i.d.), packed with
Poropack Q (Waters Associates Inc., Milford, Mass.),
100-120 mesh.
Carbohydrates were determined as glucose equivalents by
the anthrone method (Herbert et al., 1971). Glucose was
determined enzymatically with glucose oxidase (Boehringer
Test-combination glucose) and citrate with citrate lyase
(Daigly, 1974).
Total nitrogen content was determined both en-
zymatically (Kun and Kearney, 1974) and colorimetrically
(Richterich, 1965) after nitrogenous compounds in the
HENK J. NANNINGA and JAN C. GOTTSCHAL
-~ oulftc>w of
samples had been converted into ammonium (Bailey, 1967).
Free-amino acids were analyzed in hydrolyzed and non-
hydrolyzed samples on a Kontron Liquimat III analyzer
according to Vereyken et al. (1980). The analyses were
carried out on the supernatant of samples which had been
centrifugated (8 min at 10,000 rpm) immediately after sam-
pling. Before analysis the supernatants were deproteinized
by the addition of an equal volume of 6°.0 (w/v) sulfosalicylic
acid. incubation for 15 rain at - 2 f f C and finally centrifu-
asparagine and glutamine were converted into aspartate and
glutamate, respectively. Peptides were, as far as they had not
been removed by the deproteinization, converted into free-
amino acids resulting in an increase of the concentration of
the individual amino acids by less than a factor of 2. Cystine
was not determined separately but measured as cysteine
equivalents. Tryptophan was not determined. The tryp-
tophan concentration in potatoes was assumed to be similar
to the concentration of threonine and serine (Davies,
Sulfide and sulfite were both determined, immediately
after sampling, by methods of Pachmayer as described by
- ~ Triiper and Schlegel (1964). Elemental sulfur, thiosulfate,
mined in samples from which previously the sulfide had been
removed by adding concentrated formic acid [2% (v/v) final
concentration] under anaerobic conditions and flushing the
samples were centrifuged for 8 min at 10,000 rpm. Elemental
sulfur was extracted from the pellet with 99% ethanol and
measured as sulfide after reduction with dithioerythrytol
(Krauss et ul., 1984). In the supernatant the concentration
of thiosulfate and tetrathionate was determined according
to S6rbo (1957t. Sulfate was determined chro-
matographically (HPLC) (Brunt, 1983). Total inorganic
sulfur was determined as sulfate after conversion of the
inorganic sulfur moiety into sulfate, using the method
described by Thorpe (1980).
Both inorganic and organic carbon were determined using
a Beckman Total Carbon Analyser (Model 915A). The
chemical oxygen demand (COD) was determined according
to NEN 3235-5.3, a standard method published by the
Dutch Normalization Institute in Rijswijk.
Chemicals
Glucose oxidase, citrate lyase, lactate dehydrogenase and
glutamate dehydrogenase were purchased from Boehringer
(Mannheim, F.R.G.). Malate dehydrogenase was obtained
from Merck (Darmstadt, F.R.G.). All other chemicals were
of analytical grade.
RESULTS
I n t e r m e d i a t e s in the a n a e r o b i c d e g r a d a t i o n
Figure 1 summarizes data on the c o n c e n t r a t i o n s o f
various c o m p o u n d s in the waste water as it passed
t h r o u g h the purification plant while in full operation
in December 1983. The c o n c e n t r a t i o n s o f isobutyrate,
formate a n d succinate are not s h o w n in this figure.
Isobutyrate appeared in the same c o n c e n t r a t i o n s a n d
followed the same pattern as isovalerate. A t all
sample points the formate c o n c e n t r a t i o n varied be-
tween 0.4 a n d 0.8 m M whereas succinate was only
detectable at sampling point A and B (0.243.6 mM).
It must be noted that despite the presence of only low
c o n c e n t r a t i o n s of formate a n d succinate these com-
p o u n d s may yet be i m p o r t a n t intermediates in the
b r e a k d o w n of organic matter. Their t u r n o v e r rate
was not determined but could be quite high as was
 Anaerobic waste water purification 99

Table 1. Amount of nitrogen (expressed as mM N) present in various fractions of five

different sample points of the anaerobic waste water treatment plant in De Krim
Influent Influent Effluent Influent Effluent
sedimentation first first methane methane
pond reactor reactor reactor reactor
Kjeldahl-N pellet 15 17 11 8 4
Kjeldahl-N dissolved
compounds 60 57 58 39 39
Kjeldahl-N total (KNT) 75 77 74 52 46
Ammonium (NH~) 3 29 47 33 42
(NH~-/KNT).I00% 4.1-4.5 38.6-42.0 65.3-71.2 66.0-75.0 95.5-100

d e m o n s t r a t e d for some o t h e r a n a e r o b i c e n v i r o n m e n t s tailed e x a m i n a t i o n o f the a m m o n i f i c a t i o n process

( B l a c k b u r n a n d H u n g a t e , 1963; C h y n o w e t h a n d
M a h , 1970; H u n g a t e et al., 1970; Strayer a n d Tiedje,
1978).
Degradation of nitrogenous organic compounds
T h e m o s t i m p o r t a n t n i t r o g e n - c o n t a i n i n g com-
p o u n d s in the waste water were: coagulated protein,
dissolved protein, peptides, free-amino acids, bacte-
rial b i o m a s s a n d free a m m o n i u m . In the purification
p l a n t these c o m p o u n d s were expected to be degraded
primarily in the first reactor a n d to a lesser extent in
the second ( m e t h a n e ) reactor. However, as m a y be
concluded from the d a t a presented in Fig. 1 a n d
T a b l e 1, a considerable a m m o n i f i c a t i o n did take
place already in the s e d i m e n t a t i o n p o n d preceding
the first a n a e r o b i c reactor. The ratio between
a m m o n i u m - N total K j e l d a h l - N ( K N T ) m a y serve as
a measure o f the d e g r a d a t i o n o f n i t r o g e n o u s organic
c o m p o u n d s . As the total K j e i d a h l - N includes nitro-
gen present in the bacteria, a correction for this
a m o u n t of nitrogen has been m a d e in the calculation
o f the N H 2 - / K N T ratio. This correction was based on
a n estimate o f the bacterial cell mass as derived f r o m
total cell n u m b e r s determined in a c o u n t i n g c h a m b e r
(Luria, 1960; V a n Veen a n d Paul, 1979). M o r e de-
taking place in the s e d i m e n t a t i o n p o n d revealed t h a t
the p r o d u c t i o n of free a m m o n i u m was mainly caused
by the d e g r a d a t i o n o f free-amino acids a n d smaller
peptides (Tables 1 a n d 2). O f these, aspartate, glu-
tamate, a n d their amides m a k e u p the bulk of a m m o -
n i u m c o n t a i n i n g organic m a t t e r (Table 2). T h e ratio
of aspartate/asparagine and of glutamate/glutamine
in the influent o f the s e d i m e n t a t i o n p o n d was a b o u t
1/2 a n d 1/1, respectively, as determined by c o m p a r i n g
the a m i n o acid analyses of n o n - h y d r o l y z e d (results
n o t shown) a n d hydrolyzed samples. L o n g e r peptides
a n d proteins were degraded in the first reactor a n d
m e t h a n e reactor. T h e pool o f free-amino acids also
decreased as the waste water passed t h r o u g h these
two reactors.
Reduction of inorganic sulfur compounds
T h e c o n c e n t r a t i o n s o f the quantitatively most im-
p o r t a n t inorganic sulfur c o m p o u n d s were determined
at several sampling points. Sulfite was present in low
c o n c e n t r a t i o n s only in the influent o f the sedimen-
tation p o n d (0.1-0.3 m M ) . T h e c o n c e n t r a t i o n o f ele-
m e n t a l sulfur, thiosulfate a n d t e t r a t h i o n a t e did not
exceed 0.1 m M at any sampling point. F r o m Table 3
it can be seen t h a t some sulfate reduction t o o k place

Table 2. Concentration of amino acids (mM) in deproteinized hydrolyzed samples from the
anaerobic waste water treatment plant in De Krim
Influent lnfluent Effluent Effluent
sedimentation first first methane
pond reactor reactor reactor
Aspartate "~ 8.02 0.352 0.102 0.010
Asparagine J
Threonine 0.35 0.119 0.032 < 0.01
Serine 0.64 0.152 0.034 < 0.01
Glutamate )
Glutamine 5.38 1.967 0.131 0.010
Proline 0.77 0.469 < 0.01 < 0.01
Hydroxyproline 0.02 < 0.01 < 0.01 <0.01
Glycine 0.50 0.328 0.112 < 0.01
Alanine 1.75 1.613 0.076 < 0.01
Cysteine 0.33 0.306 0.015 <0.01
Valine 0.81 0.384 0.038 < 0.01
Methionine 0.24 0.088 0.022 < 0.01
Isoleucine 0.26 0.096 0.026 0.010
Leucine 0.23 0.161 0.045 <0.01
Tyrosine 0.26 0.140 0.020 0.010
Phenylalanine 0.28 0.159 0.045 < 0.01
?-Aminobutyrate 2.52 0.465 0.021 <0.01
Lysine 0.59 0.182 0.049 <0.01
Histidine 0.28 0.043 0.017 < 0.01
Arginine 0.91 0.064 0.018 < 0.01
Total nitrogen in mM N 35.93 8.761 1.115 <0,2
100 HENK J, NANNINGA a n d JAN C. GOTTSCFIAL

Table 3. Concentration of various inorganic sulfur compounds (mM) in the waste water
in various stages of the purification process. The sulfide stripper was not operative
Influent Influent Effluent Infiuent Effluent
sedimentation first first methane methane
pond reactor reactor reactor reactor
Sulfate 3.3 3.0 1.3 0.7 < 0. l
Sulfide < 0. l 0. l 1.9 1.3 1.4
Other inorg. S 0.4 0.1 0. I <0.1 <0.1
Total inorg. S 3.7 3.2 3.3 2.0 1.4

Table 4. Organic carbon content (mg 1+J) and chemical oxygen demand (COD; mg O21-~)
in various fractions of the waste water sampled at five different locations of the purification
plant in De Krim. The C O D data were obtained from the AVEBE
Influent lnfluent Effluent Influent Effluent
sedimentation first first methane methane
pond reactor reactor reactor reactor
Organic carbon
(dissolved) 6030 4430 3700 2590 1400
Organic carbon
(total) 7200 6350 4990 34t0 2060
COD (total) 17,500-18,000 17,800 16,400 11,700 3000
T
Waste water
dilution

already in the sedimentation pond, though most of
the sulfide formed will have escaped to the atmo-
sphere, as the pH in this pond was 6.2. The major
part of the more oxidized sulfur compounds was
reduced in the first anaerobic reactor, whereas the
remainder was eventually reduced in the methane
reactor. The pH of the effluent of the first reactor
(6.2) was suitable for a proper removal of sulfide in
the sulfide stripper. The presence of sulfide in the
methane reactor is undesirable because this will lead
to sulfide-containing biogas and effluent of the meth-
ane reactor. As the pH of the effluent of the methane
reactor is usually 7.5 the sulfide cannot readily be
removed by means of a stripping process. The hydro-
gen sulfide content of the biogas produced in the
methane reactor was 6.5 g m -3 biogas (0.49~ v/v).
Carbon removal
The decrease of organic carbon in the sedimen-
tation pond and in the first reactor (Table 4) as a
result of microbial activity, is due to the evolution of
carbon dioxide rather than methane (Table 5). This
was not unexpected as in both stages the pH was
below 6.3 which is considerably below the optimum
value for methanogenic bacteria, with reported pH
optima for growth between 6.8 and 8.0 (Balch et al.,
1979). Moreover, the hydraulic retention time was
< 1 0 h , too short for (acetate-metabolizing) meth-
anogens to sustain themselves in the absence of
appreciable sludge retention.
In the methane reactor, where both carbon dioxide
Table 5. Composition of the gas phase above the first reactor and
the methane reactor
Component First r e a c t o r~ Methanereactor
H 2 (%)
H2S (~o)
0.10
1.48
0.01
0.49
c02 (%) 48.0 27.z
CH+ (%) 6.3 72.0
and methane were produced (Table 5), the organic
carbon content decreased further. The overall de-
crease of total organic carbon amounted to
5140mgl ~ when the influent of the sedimentation
pond and the effluent of the methane reactor are
compared (Table 4). This decrease was due to waste
water dilution (1580mgl -t) and to the release of
carbon dioxide and methane (3560 mg 1-t). From the
latter quantity 69~ was released as carbon dioxide
and 31~ as methane, as can be calculated from the
data presented in Tables 4 and 5.
The chemical oxygen demand (COD) showed a
pattern which differed from that of the total organic
carbon content (Table 4). In the sedimentation pond
virtually no decrease of the COD was observed. In
the first anaerobic reactor only a small drop in COD
occurred. This is to be expected as the loss of COD
as a result of carbon dioxide production under anaer-
obic conditions will have been balanced by the in-
crease of the COD caused by the production of
reduced organic (e.g. propionate, butyrate, valerate
and cell-mass) and inorganic (e.g. ammonium and
sulfide) compounds. The drop in COD in the effluent
of the first reactor is probably due to (temporary)
accumulation of particulate organic matter in this
reactor, which was observed to take place to some
extent, following a very irregular, unpredictable pat-
tern.
In the methane reactor, organic matter was mainly
converted to carbon dioxide and methane. The exten-
sive release of methane from the waste water was
primarily responsible for the drop in COD in this
reactor. The COD in the effluent of the methane
reactor was mainly the result of the presence of
+ ammonium, biomass and small amounts of various
organic compounds such as acetate, propionate and
oxalate. These components were removed during a
final aerobic treatment.
 Anaerobic waste water purification
DISCUSSION
Anaerobic purification of the waste water pro-
duced by the potato processing factory of the
AVEBE in De Krim serves three distinct purposes:
the removal of organic matter, the reduction and
subsequent removal of sulfate and the generation of
"biogas" (predominantly methane and carbon diox-
ide) to be used as a fuel.
Regarding the removal of organic material, it can
be concluded that indeed a considerable quantity is
removed and converted into methane (Tables 4 and
5). Yet about 29~ of the organic carbon entering the
purification plant remains in the effluent of the
methane reactor. Of this remaining fraction only a
small part (~<20~) can be accounted for by well
known, readily identifiable compounds such as ace-
tate, propionate and oxalate (Fig. 1). The concen-
tration of acetate (3.6 mM) in the effluent of the
methane reactor was subject to some variation and
ranged from 2.8 to 5.2 mM on other occasions. The
limited conversion of propionate has been reported
for other purification plants as well (Kaspar and
Wuhrmann, 1978; Zehnder and Koch, 1983). With
respect to oxalate complete degradation under anaer-
obic conditions in continuous culture at hydraulic
retention times longer than 12.8 h has been reported
(Dawson et al., 1980). Apparently the conditions for
oxalate metabolism in the methane reactor were
sub-optimal despite the presence of granular sludge
and a hydraulic retention time of 20 h. Another
fraction may be accounted for by biomass and
undefined particulate organic matter (<38~). The
identity of the larger part remains unknown. Proba-
bly this fraction is composed of relatively refractory
substances already present in the potato juice as it
enters the plant, together with products formed dur-
ing the degradation of amino acids, particularly the
aromatic ones (Barker, 1961; Elsden et al., 1976), of
which some are known to be degraded only slowly.
Especially phenolic compounds, though in principle
degradable anaerobically (Healey and Young, 1978,
1979; Balba and Evans, 1980; Boyd et al., 1983),
might require much longer hydraulic retention times
in the methane reactor than currently employed
(20 h). A possible alternative for the economically
unattractive solution of lower feed rates or much
larger reactor volumes would of course be the appli-
cation of much larger amounts of active sludge
granules. Indeed during the course of this in-
vestigation sludge granules occupied only 1.0-1.5~ of
the available reactor volume, but this amount may
increase considerably upon prolonged plant oper-
ation.
In addition to the problems associated with the
slow degradation of "recalcitrant" material in the
potato juice, it seems likely that breakdown of several
compounds gives rise to potentially toxic products.
Well known examples of such compounds are sulfur-
containing amino acids which have been shown to be
101
converted, under anaerobic conditions, into toxic
volatile sulfur compounds (Barker, 1961; Zinder and
Brock, 1978). Methyl mercaptan, for example, a
product of methionine degradation, was shown to
inhibit nitrification in soil at concentrations from 0.21
to 1.05#molg-t soil (Bremner and Bundy, 1974).
Volatile organic sulfur compounds may also interfere
with anaerobic purification processes. At low concen-
trations no such inhibitions have been reported,
however. Zinder and Brock (1978) demonstrated that
methyl mercaptan present in the gasphase
(2.2nmol/10ml gas) of a vial containing 1 ml lake
sediment was converted into methane, carbon dioxide
and hydrogen sulfide. Hydrogen sulfide, formed dur-
ing sulfate reduction and in the fermentation of
sulfur-containing amino acids, was also shown to
considerably impair proper functioning of methane
reactors in some purification plants (Butlin et al.,
1956; Callander and Barford, 1983; Kroiss and Plahl-
Wabnegg, 1983). However, in the methane reactor
described here the sulfide concentration in the reactor
fluid did not exceed 2 mM, which has repeatedly been
shown not to affect adversely methane production
(Mountfort and Ascher, 1979; Scherer and Sahm,
1981; Kroiss and Pfabi-Wabnegg, 1983). Still another
potentially inhibiting fermentation product is ammo-
nium which was found up to concentrations of nearly
50 mM in the methane reactor. Yet, it is rather
unlikely that even this fairly high ammonium concen-
tration would cause significant inhibition of meth-
anogenesis, as it has been demonstrated (Koster and
Lettinga, 1983; Zeeman et al., 1983; De Baere et al.,
1984) that adaptation to ammonium concentrations
of 120mM and higher (at a pH of 7.0-8.0) could
occur. Nevertheless, such data should be viewed with
some care as it has also been shown that ammonium
concentrations of 70 mM and higher did strongly
impair the formation of active granular sludge in
UASB-reactors (Hulshoff Pol et al., 1983). It would
therefore, perhaps, be worthwhile to investigate pos-
sibilities of trapping ammonium before it enters the
methane reactor or even better, before it reaches the
first reactor. This brings us back to the problems
associated with the complete removal of sulfate sup-
posed to take place in the first reactor. As a matter
of fact only about 60~ (Table 3) of the sulfate load
of the first reactor is being reduced to sulfide. The
question is, why in the presence of large quantities of
organic matter and only about 4 m M sulfate, this
electron acceptor is not completely reduced. A de-
tailed study of this particular problem will be pub-
lished elsewhere (Nanninga and Gottschal, in prepa-
ration) but preliminary results appear to indicate a
combination of at least two major causes. Firstly,
acetate, propionate, butyrate and several other fatty
acids, all present in considerable quantities in the first
reactor, do not allow sulfate reducers to grow fast
enough (in the absence of appreciable sludge reten-
tion) to cope with the relatively short hydraulic
retention time (about 9.5 h) of the fluid in the first
102 HENK J. NANNINGAand JAN C. GOTTSCHAL
reactor (Widdel, 1980; Widdel and Pfennig, 1981a,b).
Secondly, substrates which do permit sulfate reducing
species, isolated from the first reactor (Nanninga and
Gottschal, in preparation), like hydrogen, formate,
ethanol and lactate are absent or occur at growth-
limiting concentrations. The reason that these sulfate
reducers, usually very successful competitors for such
substrates (Abram and Nedwell, 1978; Sorensen et
al., 1981; Kristjansson et al., 1982; Laanbroek et al.,
1983; Lovley and Klug, 1983; Robinson and Tiedje,
1984) do not outcompete their competitors in this
case probably lies in the presence of a vast spectrum
of other substrates which sustains a large population
of fermentative bacteria. These non-sulfate reducing
populations, though being poorer competitors for
single, sulfate reduction sustaining electron donors,
probably do consume by their sheer abundance a
significant portion of these potential substrates. In
fact this would be a clear illustration of the enormous
impact on population kinetics of the presence of
other substrates in addition to the ones competed for
(Gottschal et al., 1979; Laanbroek et al., 1979; Rob-
inson and Tiedje, 1984).
With these considerations in mind, it would seem
logical to try and investigate whether sulfate reducing
bacteria, capable of using fatty acids as electron
donors, could be maintained in the first reactor by
applying sludge retention. Experiments along these
lines, using small scale laboratory equipment, are well
underway.
Acknowledgements--For free-amino acid analyses the au-
thors acknowledge H. J. Bak and J. J. Beintema (De-
partment of Biochemistry, University of Groningen, The
Netherlands); for sulfate analyses we acknowledge K. Brunt
(Potato Processing Research Institute T.N.O., Groningen,
The Netherlands). We wish to thank H. Veldkamp for
valuable discussions and for reading the manuscript. The
co-operation of J. B. M. Meiberg (AVEBE, Foxhol, The
Netherlands) and D. J. Wijbenga (Potato Processing Re-
search Institute TNO, Groningen, The Netherlands) is
gratefully acknowledged. We are grateful to M. Th. Broens-
Erenstein and M. Pras for preparing the manuscript.
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