anaerobic purification of waste water from a potato-starch producing factory

War. Res. Vol. 20, No. 1, pp. 97-103, 1986 0043-1354/86 $3.00+0.00 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

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 rom the (valuable) protein moiety over 80% is removed by heat-coagulation followed by ultra-filtration. The remaining organic matter is degraded 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 organic and inorganic intermediates. Besides carbon containing products, also some reduced inorganic compounds are being produced. Ammonium is released 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.

MATERIALS AND METHODS 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 immediately 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

97

98 HENK J. NANNINGA and JAN C. GOTTSCHAL

inflOW of waS_] anaerobic purification .h *r,c~n{ -~ oulftc>w of

sir,~ factory [ i

sc~

i ctmmonla /

mM 3o1 f 20~

tO,

~04

-]:'< ~car bohyrJr ares

acetate - / ' ~ "

30 ~ -

mM

i

L actate - ~

1 ~ " ~ o×atate 5

~ < -4'meth anc~ ~

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 isovalerate may therefore represent either of these two compounds. 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 analyzed 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 enzymatically (Kun and Kearney, 1974) and colorimetrically (Richterich, 1965) after nitrogenous compounds in the

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 sampling. 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 centrifugation for 20min at 5000rpm. In hydrolyzed samples. 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 tryptophan concentration in potatoes was assumed to be similar to the concentration of threonine and serine (Davies, 1977).

Sulfide and sulfite were both determined, immediately after sampling, by methods of Pachmayer as described by Triiper and Schlegel (1964). Elemental sulfur, thiosulfate, tetrathionate, sulfate and total inorganic sulfur were determined 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 suspension with oxygen free nitrogen gas. Subsequently 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 concent ra t ions of various compounds in the waste water as it passed th rough the purification plant while in full opera t ion in December 1983. The concent ra t ions of isobutyrate, formate and succinate are not shown in this figure. I sobutyra te appeared in the same concent ra t ions and

followed the same pat tern as isovalerate. At all sample points the formate concent ra t ion varied between 0.4 and 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 concent ra t ions of formate and succinate these compounds may yet be impor t an t intermediates in the b reakdown of organic matter . Their tu rnover rate was not determined but could be quite high as was

Anaerobic waste water purification

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

99

demons t ra t ed for some other anaerobic env i ronments (Blackburn and Hungate , 1963; Chynoweth and Mah , 1970; Hunga te et al., 1970; Strayer and Tiedje, 1978).

Degradation of nitrogenous organic compounds

The mos t i m p o r t a n t n i t rogen-conta in ing compounds in the waste water were: coagula ted protein, dissolved protein, peptides, f ree-amino acids, bacterial b iomass and free a m m o n i u m . In the purif icat ion p lant these compounds were expected to be degraded pr imari ly in the first reactor and to a lesser extent in the second (methane) reactor. However, as may be concluded f rom the da ta presented in Fig. 1 and Table 1, a considerable ammonif ica t ion did take place already in the sed imenta t ion pond preceding the first anaerobic reactor. The rat io between a m m o n i u m - N tota l K j e l d a h l - N (KNT) may serve as a measure o f the degrada t ion of n i t rogenous organic compounds . As the total K j e i d a h l - N includes ni tro- gen present in the bacteria, a correct ion for this a m o u n t of n i t rogen has been made in the calculat ion of the NH2- /KNT ratio. This correct ion was based on an est imate of the bacterial cell mass as derived f rom total cell numbers de termined in a count ing chamber (Luria, 1960; Van Veen and Paul, 1979). More de-

tailed examina t ion of the ammonif ica t ion process taking place in the sedimenta t ion pond revealed tha t the p roduc t ion of free a m m o n i u m was mainly caused by the degrada t ion of f ree-amino acids and smaller peptides (Tables 1 and 2). O f these, aspartate , glutamate, and their amides make up the bulk of ammo- n ium conta in ing organic mat te r (Table 2). The rat io of aspar ta te /asparagine and of g lu tamate /g lu tamine in the influent of the sedimenta t ion pond was abou t 1/2 and 1/1, respectively, as de termined by compar ing the amino acid analyses of non-hydrolyzed (results no t shown) and hydrolyzed samples. Longer peptides and proteins were degraded in the first reactor and methane reactor. The pool of f ree-amino acids also decreased as the waste water passed th rough these two reactors.

Reduction of inorganic sulfur compounds

The concent ra t ions of the quant i ta t ively most im- po r t an t inorganic sulfur compounds were de termined at several sampling points. Sulfite was present in low concent ra t ions only in the influent of the sedimenta t ion pond (0.1-0.3 mM). The concen t ra t ion of ele- menta l sulfur, thiosulfate and te t ra th iona te did not exceed 0.1 m M at any sampl ing point . F r o m Table 3 it can be seen tha t some sulfate reduct ion took 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


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 COD data were obtained from the AVEBE

Influent lnfluent Effluent Influent Effluent sedimentation first first methane methane


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 methane 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 hydrogen 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 sedimentation 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 <10h, too short for (acetate-metabolizing) methanogens 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

and methane were produced (Table 5), the organic carbon content decreased further. The overall decrease 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 anaerobic conditions will have been balanced by the increase 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 pattern.

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

Component Firs t reac tor ~ Methane reactor + ammonium, biomass and small amounts of various

H 2 (%) 0.10 0.01 organic compounds such as acetate, propionate and H2S (~o) 1.48 0.49 c02 (%) 48.0 27.z oxalate. These components were removed during a CH+ (%) 6.3 72.0 final aerobic treatment.

Anaerobic waste water purification 101

DISCUSSION

Anaerobic purification of the waste water produced 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 dioxide) 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 acetate, propionate and oxalate (Fig. 1). The concentration 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 anaerobic 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 during 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 operation.

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

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 concentrations 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 during 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 ammonium 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 concentration would cause significant inhibition of methanogenesis, 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 4mM sulfate, this electron acceptor is not completely reduced. A de- tailed study of this particular problem will be published elsewhere (Nanninga and Gottschal, in preparation) 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 retention) to cope with the relatively short hydraulic retention time (about 9.5 h) of the fluid in the first

102 HENK J. NANNINGA and 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 populat ion 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 port ion 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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anaerobic purification of waste water from a potato-starch producing factory

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