APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1985, p. 1472-14770099-2240/85/061472-06$02.00/0Copyright © 1985, American Society for Microbiology

Methanogenesis in an Upflow Anaerobic Sludge Blanket Reactor atpH 6 on an Acetate-Propionate Mixture

ERIK TEN BRUMMELER,' LOOK W. HULSHOFF POL,1* JAN DOLFING,2t GATZE LETTINGA,' ANDALEXANDER J. B. ZEHNDER2

Department of Water Pollution Control' and Department of Microbiology,2 Agricultural University, 6703 BC Wageningen,The Netherlands

Received 13 November 1984/Accepted 13 March 1985

High-rate anaerobic digestion can be applied in upflow anaerobic sludge blanket reactors for the treatmentof various wastewaters. In upflow anaerobic sludge blanket reactors, sludge retention time is increased by anatural immobilization mechanism (viz. the formation of a granular type of sludge). When this sludge iscultivated on acid-containing wastewater, the granules mainly consist of an acetoclastic methanogen resemblingMethanothrix soehngenii. This organism grows either in rods or in long filaments. Attempts to cultivate a stablesludge consisting predominantly of Methanosarcina sp. on an acetate-propionate mixture as substrate bylowering the pH from 7.5 during the start-up to approximately 6 failed. After 140 days of continuous operationof the reactor a filamentous organism resembling Methanothrix soehngenii prevailed in the sludge. The specificmethanogenic activity of this sludge on acetate-propionate was optimal at pH 6.6 to 6.8 and 7.0 to 7.2,respectively.

In recent years anaerobic wastewater treatment has metan increasing interest. This is mainly a result of the positiveenergy balance of anaerobic treatment processes and thedevelopment of inexpensive, high-rate treatment systems.Various reactor types for anaerobic wastewater treatmenthave been developed. They are the anaerobic filter, thefluidized and expanded bed reactors, the upflow anaerobicsludge blanket (UASB) reactor, and the fixed film reactorswhich can be operated either as a down- or an upflowreactor. Results of studies of these reactor types have beenpresented by Henze and Harremoes (5), Speece (18), andvan den Berg (19). The UASB process, as described byLettinga and co-workers (12, 14), so far has been the mostfrequently applied treatment process.At present, full-scale plants are successfully treating a

variety of wastewaters like wastewaters from sugar beets,potato processing, and corn and potato starch (17). Inconventional, completely mixed anaerobic digestors thesludge retention time is controlled by the hydraulic loadingrate and the organic matter (volatile suspended solids [VSS])content of the primary and secondary sludge. In high-rateanaerobic wastewater treatment systems, however, sludgernust be immobilized by some mechanism, causing thesludge retention time to be almost independent of the flowrate in the system. In UASB reactors imnmobilization isachieved by a natural mechanism, i.e., the formation ofhighly settleable aggregates of microorganisms. In specificcases this phenomenon can be indicated as granulation, viz.when the aggregates are growing in the form of the distinctgranules (7, 8, 9). These granules frequently are high in VSScontent (up to 90%), show a high specific activity (2.2 kg ofCH4 chemical oxygen demand [COD] VSS-1 day-' at 30°C),and are 1 to 5 mm in size (8). The predominant organism inthe granules, which develop in UASB reactors in which a

volatile fatty acids-containing wastewater is being treated,appears to be a bacterium resembling Methanothrix soehn-

* Corresponding author.t Present address: Department of Microbiology and Public Health,

Michigan State University, East Lansing, MI 48824.

genii, an acetoclactic methanogen described previously byHuser (10). In pure culture Methanothrix soehngenii growsin two different shapes: rod-like units of 2 to 10 cells and longfilaments of several hundred cells (4). Granules grown infull-scale UASB reactors mainly consist of Methanothrix-like organisms growing as rods. A minority of the granulescontains filamentous organisms.

During the start-up of a UASB reactor a selective washoutof dispersed growing sludge is considered to be essential inthe granulation process (7, 9). As a result of washout, onlybacteria which grow in the heavier particles remain in thereactor. Occasionally, the washout of biomass may be fairlyhigh, leading to undesirable prolonged start-up periods.Certain conditions, such as long periods of underloading,lead to the formation of anaerobic bulking sludge (7) inwhich little formation of granules occurs. An important rolein this bulking is played by Methanothrix sp. because itgrows in long filaments. It was thought previously that theformation of anaerobic bulking sludge, apart from specificstart-up procedures, can be overcome by creating conditionsthat favor the development of Methanosarcina sp., anotheracetoclastic methanogen. Methanosarcina sp. readily formsagglomerates in pure culture (2, 15, 23). These agglomeratessettle very well, are spherical in shape, and are up to 2 to 3mm in size. Consequently, by stimulating the growth ofMethanosarcina sp., i.e., making it the predominant organ-ism in the anaerobic sludge of a UASB reactor, the danger ofa massive washout can be reduced during the start-upperiod. In Table 1 the most important kinetic and physiolog-ical characteristics of Methanothrix sp. and Methanosarcinasp. obtained in pure culture are summarized. In digestingsludge Methanothrix sp. predominates under conditions oflow acetate concentrations. However, at high acetate con-centration Methanosarcina sp. prevails. This phenomenoncan be explained on the basis of a considerable difference ofthe Ks for acetate of these organisms. The two organismsalso differ in their optimal pH growth on acetate. Accordingto Huser (Ph.D. thesis, Swiss Federal Institute of Technol-ogy, Zurich, Switzerland, 1981), Methanothrix soehngeniihas its optimum at pH 7.8 and shows no activity below pH

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TABLE 1. Characteristics of the acetoclastic methanogens Methanosarcina sp. and Methanothrix soehngenii grown in pure culture onacetate

Organism (no. tested) (h-1) K, (mM) Y (g g_ I)b Optimum OptimuM Other substrates Morphology Habitat(temp)a pH temp (TC) OtesusatsorhogHbit

Methanothrix 0.0032 (33°C) 0.72 2.1 7.8 37 Rods (2 to 10 cells), Digestionsoehngenii (10) filaments (100 to sludge

300 cells)Methanosarcina 0.02-0.03 5.0 1.4 6-8 40-45 Methanol, H2, Packets of cocci Acetate-rich

sp. (23) methylamines growing in anaerobicclumps environments

a ILmax, Maximum specific growth rate.b Y, Growth yield.

6.8. Methanosarcina sp. forms methane in a much wider pHrange, namely 5 to 8 (22). UASB reactors are generallyoperated at pH 6.9 to 7.5. When considered in combinationwith the low acetate concentrations generally pursued inUASB reactors, the reason for the predominance of Meth-anothrix sp. in the sludge becomes obvious. In view of whathas been mentioned above, it is expected that little, if any,growth of Methanothrix-like organisms will occur when aUASB reactor is started up at pH 6; instead, Methanosar-cina sp. will prevail. The purpose of this was to prevent theformation of anaerobic bulking sludge. Anaerobic wastewa-ter treatment can proceed well at a pH range of 6.6 to 7.6(16). Reactor failure is often caused by decreases in pH (10).This is especially true for propionate degradation. Theoxidation of this acid is strongly inhibited for a long periodafter a pH shock, although adaption is possible (11). Presum-ably, the main reason for the inhibition at low pHs is the hightoxicity of the undissociated fatty acids which becomeabundant below pH 6 (3). If a methanogenic populationcould develop at pH 6, it would undoubtedly represent asignificant and beneficial feature of anaerobic treatmentbecause this would extend the application of the process tofairly acidic wastewaters without the need for a supply ofalkali.The purpose of this study was (i) to investigate whether a

reactor start-up at a low pH could be accomplished andwhether it would be feasible; and (ii) to confirm that inducedMethanosarcina sp. prevail in the cultivated sludge under areactor pH of 6 and that this is accompanied by an improvedsettleability of the sludge as compared with the start-up atnormal pH (7.5).

MATERIALS AND METHODS

Medium, seed sludgea and experimental conditions. Twoidentical UASB reactors (Fig. 1), each with a volume of 2.5liters, were used in the experiments. The cylindrical reactorswere made of plexiglass, and each had a diameter of 12 cm.The lower part of the reactors was conically shaped toenhance contact of the substrate with the biomass. In theupper part of the reactors a three-phase separator was situ-ated to capture evolved gas and to allow settling. They werefed with a medium consisting of the following (in milligramsper liter): acetic acid (1.250), propionic acid (1.000), H3BO3(0.5), FeCl3 3H20 (20), ZnCl2 (0.5), MnCl2 * 4H20 (5),NH4Mo7 * 4H20 (0.5), CoCl2 * 6H20 (0.5), NiCl (0.5), EDTA(5), NaSeO3 (1), AlCl3 (0.5), resazurin (5), HCl (36% solu-tion, 10-2 mi/liter of medium), yeast extract (100), NH4Cl(60), (NH4)2SO4 (15), KH2PO4 (15). The seed sludge wasobtained from the municipal sewage sludge digester in Ede,The Netherlands. The volatile suspended solids (VSS) con-tent of the sludge was 62% and the maximum specific activ-

ity, as determined in a batch-fed experiment, amounted to0.12 kg of CH4 COD kg of VSS-1 day-'.Averaged over the total reactor volume 10 g of VSS/liter

was supplied to the reactors. The start-up procedure appliedwas identical to that described previously by de Zeeuw andLettinga (4). The space loading rate (in terms of kg of CODm-3 day-') was increased for 75% once the COD reductionof the system exceeded 85% of the influent concentration(2,850 mg of COD liter-').To prevent channeling of the medium in the sludge bed

during the initial stages of start-up, the reactors were me-chanically stirred (30 rpm) every 30 min for 5 s. Once themethane production exceeded 1 liter per reactor volume aday, stirring was terminated because beyond that point thenatural mnixing of the system appeared to be sufficient,according to our observations. One reactor, which acted asa reference, was operated at pH 7.5, whereas in the secondreactor the pH was kept at approximately 6. The pH of thefeed was adjusted to values of respiratioh of 6.5 and 4.5 to 6with 6 N HCL and 6 N NaOH, respectively. The pH in thereactors was measured several timnes a day. This was doneby taking a sample from the reactor fluid just above thesludge bed. The pH of the sample was measured in a smallvessel containing the pH electrode (Knick mV meter; Berlin,Federal Republic of Germany). In this way the release ofCO2 from the oversaturated fluid could be prevented. Theexperiments were performed in a temperature-controlledroom at 30°C (+i°C).VFA analyses. Samples (one sample per day per reactor)

were taken fromn the settler compartment of the reactors and

e: -Effluent

Gas

FIG. 1. UASB reactors (2.5 liter) applied in the start-upexperiments.

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1 otime (days)

VFA (mg COD/ l)14001

time (days)

FIG. 2. (a) Course of gas production rate (VG), space loadingrate (VL), and pH during the start-up experiment with the UASBreactor operated at pH 7.5. (b) Levels of acetate (0) and propionate(0) expressed as milligrams of COD per liter in the effluent of theUASB reactor operated at pH 7.5.

analyzed for acetate and propionate with a gas chromato-graph (model 417; Becker; flame ionization detector [FID]200°C chromosorb column [200 by 0.2 cm ]; carrier gas, N2)equipped with a computing integrator (Spectra Physics 4100).Peak areas were measured and compared with a standardvolatile fatty acid (VFA) mixture (precision, ±3%).Methane measurement. (i) Volume. The total volumetric

methane production was measured every 24 h, after thebiogas (CH4-CO2 mixture) was put through a 3 N NaOHsolution to scrub the C02, with a wet gas meter (DordrechtMeterfabrieken, The Netherlands).

(ii) Concentration. The methane concentration was deter-mined with a gas chromatograph (Packard-Becker 406)equipped with a thermal conductivity detector and a molec-ular sieve column, operated at 50°C. The carrier gas wasargon and was used at a flow rate of 20 ml min-'.

Activity tests. Serum vials with a volume of 130 ml were

made anaerobic by flushing with O2-free nitrogen gas, andthey were subsequently filled with 40 ml of an anaerobicbuffer solution. The nitrogen gas was made free from resid-ual 02 by passing it over hot copper coils. The buffer wasmade anaerobic by boiling and then by cooling to roomtemperature under continuous gassing with O2-free nitrogen.The buffer solution (pH values as indicated) was a 0.2 MKH2PO4-K2HPO4 buffer containing 0.5 g of NH4Cl per literof demineralized water.

Sludge was anaerobically distributed over the vials inportions of 100 to 1,000 mg. Substrate was added to themixed liquor from concentrated stock solutions to reach finalconcentrations of 20 to 50 mM. The vials were closed withserum bottle caps and incubated in a shaking water bath at30°C. The sludge was stored at 4°C and was reacclimatizedby incubating it overnight in the presence of small amountsof substrate. After an overnight reacclimatization, the headspace of the vials was flushed with O2-free nitrogen beforethe methane production rate was determined. At the end ofthe activity tests, the pH was checked, and the amount ofsludge present in the vials was determined by weighing thepellet after the mixed liquor was centrifuged and the super-natant was discarded. The pellet was dried to constantweight overnight at 105°C. For methane analysis sampleswere taken from the head space with a gas pressure locksyringe.

RESULTSReactor run at pH 7.5. Figures 2A and B show the

concentrations of acetate and propionate in the effluentsolution, the course of the rate of gas production, and thereactor pH in relation to the space loading rate that wasapplied. The conversion of propionate was poor until day 50.At day 50 the pH of the medium was increased from 5.8 to6.5 to prevent the occurrence of pH shocks after the loadingincrements. Granules of 0.5 to 1 mm were detected in thesludge bed after 80 days of operation. The predominantorganism in the washed out sludge was similar to Methano-thrix soehngenii. The mean VSS content of the reactordecreased from 10 g liter-' (day 0) to 1.1 g liter-1 at day 80and then gradually increased to 1.8 g liter-' at day 150. Afterthe experiments were terminated, the sludge was character-ized by the measurement of its specific activity and scanningelectron microscopy. The characteristics of the sludge aregiven in Table 2. The scanning electron micrographs areshown in Fig. 3 and Fig. 4.

Reactor run at pH 6. The results of the reactor run at pH6 are shown in Fig. 5A and B. The reactor feed wasinterrupted 7 days after the start of the experiment, becauseno gas could be detected. However, during the feed inter-ruption a slow but distinct increase in gas production oc-curred. This was noticed by observing the evolution of smallgas bubbles. Therefore, at day 17, it was decided to resumethe feeding of the reactor. During the experiment it appearedto be difficult to maintain the pH constant at 6 because of the

TABLE 2. Characteristics of sludges obtained in UASB -reactors run at pH 7.5 and after 150 days of operation

Biomass concn Sp act (g of Yield (g of PredominantReactor pH (g of VSS CH4of g VSS g of organism Avg granule

liter-') dayo-) COD-')a morphologyb diameter (mm)

7.5 1.8 1.8 0.031 Filaments 1.56 3.5 1.3 0.034 Filaments 1.0

a Calculated from the total converted COD during the experiment.b Organisms resembling Methanothrix soehngenii.

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VFA (mg COD!))

1000-8 0 100

VL ,V (9COD. 1 day1) b pH200

FIG. 3. Scanning electron micrograph of a part of a granulecultivated in a UASB reactor operated at pH 7.5. Bar, 10.0 ,um.

low pH of the influent and the absence of a sufficientbuffering capacity. This resulted in pH fluctuations and a pHdrop at day 120, which caused a strong inhibition of theprocess. Moreover, the flow rate was doubled, despite thefact that only 85 to 90% of the acetate was converted andthat propionate conversion was negligible. After day 50 adistinct attachment of biomass to the reactor wall becamevisible. This process of attachment and subsequent growthof the attached biomass continued until at the termination ofthe experiment at day 140, roughly 50% of the biomass wasattached to the reactor wall and 50% was present in thesludge bed. The attached biomass was grey in color, andwhite, dispersed aggregates (ca. 0.5 mm) could be distin-guished. The propionate conversion started after day 50,which coincided with an increase in wall growth. The sludgewashed out from the reactor during the period from days 0 to50 consisted mainly of Methanosarcina-like organisms,whereas after day 50 filamentous organisms predominated inthe biomass that was present in the effluent. Granulation ofthe sludge became apparent 80 days after the start of theexperiment. The VSS content of the reactor decreased from10 g liter -1 at day 0 to 2.0 g liter-' at day 80, but then itgradually increased to 3.5 g liter-' at day 140. The maincharacteristics of the sludge after termination of the experi-ments are shown in Table 2. It appeared that the biomass on

460

-40

time (days)

FIG. 5. (a) Course of the gas production (VG), space loading rate(VL) and pH during the start up experiment with the UASB-reactoroperated at pH 6. (b) Levels of acetate (0) and propionate (0)expressed as milligrams of COD per liter of effluent of the UASBreactor operated at pH 6.

the reactor wall was very loosely attached, so it wasimpossible to analyze it independently of the biomass of thesludge bed. The predominant organism in the cultivatedsludge was a filamentous bacterium (Fig. 6). Only a fewgranules consisted of Methanosarcina-like organisms (Fig.7). To assess the effect of pH on the acetate and propionateconversion rates of the sludge, the specific activity foracetate and propionate degradation was measured at several

I L-- FIG. 6. Scanning electron micrograph showing the filamentousFIG. 4. Scanning electron micrograph of the Methanothrix-like nature of the granules obtained in a UASB-reactor operated at pH 6.

organisms of Fig. 3 shown in more detail. Bar, 1.0 p.m. Bar, 10.0 p.m.

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FIG. 7. Scanning electron micrograph of a part of a Methanosar-cina granule cultivated in the UASB reactor operated at pH 6. Bar,10.0 Jim.

pHs in batch experiments. The results are shown in Fig. 8and 9. For acetate degradation an optimum was found at pH6.6 to 6.8. The sludge continued to show a slight but distinctactivity at pH 5. For propionate degradation one distinctoptimum was found. Activity measurements at pH 6.6indicated that the sludge might have a second optimum inthis range (Fig. 9).

DISCUSSIONThe predominance of a filamentous bacterium in the

sludge cultivated at pH 6 indicates that Methanosarcina sp.cannot compete successfully for acetate with this organism.Consequently, the washout during the start-up period, aswas intended originally, cannot be minimized significantly,in comparison with a start-up period at pH 7.5. The differ-ence that was found, i.e., the difference in the lowestcalculated VSS content during the experiments of 0.9 gliter-', in favor of the reactor operated at pH 6, can beexplained by an improved attachment of biomass in the

specitic activity (gmol CH, .gVSS 1min')41

3-

2-

0

pH6 7

FIG. 8. Specific activity on acetate in relation to the pH of thesludge cultivated at pH 6.

reactor operated at pH 6. This phenomenon was not ob-served in the reactor run at pH 7.5. During the period fromdays 0 to 50 there was no substantial acetoclastic activity.As a result the acetate concentration prevailing in thereactor was high (1,000 to 1,200 mg of COD liter-'), andconsequently preferential growth of Methanosarcina sp.occurred. Once the acetate degradation proceeded satisfac-torily, the kinetic circumstances gradually seemed to changein favor of the filamentous organisms.

Indeed, beyond day 50 a gradual shift occurred in thebacterial composition of the sludge, viz. the filamentousorganisms became predominant instead of Methanosarcinasp. Apart from the kinetic advantage of the filamentousorganisms, another mechanism favored the population shift.Methanosarcina sp. tend to form relatively small aggregatesof less than 0.5 mm, in comparison with the sludge aggre-gates, in which the filamentous organisms dominate (1.0 to1.5 mm). These small Methanosarcina sp. aggregates washout more readily from the reactor, at comparable hydraulicretention times, than the conglomerates formed by thefilamentous organisms. The Methanothrix-like organismsshow a strong tendency to attach to either the reactor wall orthe inert particles that originate in the inoculum. Thisphenomenon leads to improved retention of this type ofsludge, and consequently Methanosarcina sp. is increas-ingly outcompeted. The low methanogenic activity fromdays 0 to 50 of the experiment at pH 6 indicates that bothMethanosarcina sp. and the filamentous organism werepresent in the inoculum in relatively low numbers.Two possible explanations for the development of the

filamentous organisms at this pH level can be given: (i) thebacterium was already present in the inoculum in very lownumbers and represents a Methanothrix strain with a lowerpH optimum on acetate; and (ii) Methanothrix soehngeniiadapted to the lower pH. van den Berg et al. (20) havedescribed an acetate-utilizing methanogenic culture with asimilar optimal pH range for the specific activity at pH 6.6 to6.9. The predominant organism in this culture also appearedto be a filamentous bacterium (filaments of 100 to 200 ,um),which is possibly the same organisms as described in ourexperiments.The results of the experiment at pH 6 show that propio-

:activity (Amol CH,, gVSS mnin '1)

0

FIG. 9. Specific activity on propionate in relation to the pH ofthe sludge cultivated at pH 6.

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METHANOGENESIS AT pH 6 1477

nate degradation is possible in a UASB reactor operated atrelative low pHs. This may be a result of the formation ofmicroenvironments, in which higher pHs might exist withinthe granules or the biofilm attached to the reactor wall.According to the investigations of Arvin and Kristensen (1),higher pH values prevail in denitrifying biofilms comparedwith the pH in bulk solutions. The maximum differencemeasured amounted to 0.5 to 2 pH units. Arvin and Kris-tensen have assumed that this phenomenon is a result oflower diffusion coefficients of H+ and HCO3 ions inside thebiofilm. In the case of methanogenic biofilms the existenceof higher pH values inside the biofilm (granules) is fairlylikely because VFAs are being degraded here as a result ofthe high bioactivity which necessarily causes a rise in the pHof the entrapped solution. According to Dolfing (J. Dolfing,Ph.D. thesis, Agricultural University, Wageningen, TheNetherlands, in press), on the other hand, a high pH gap isnot likely, because mass transfer resistance is very limited inthe granules.Another possible explanation for the propionate degrada-

tion at pH 6 may be the existence of a second group ofpropionate utilizers, as suggested by Heyes and Hall (6),which is faster growing and less sensitive to pH shocks incomparison with the propionate degraders normally found inanaerobic digesters.From our experiments it can be concluded that high rate of

anaerobic digestion in a UASB reactor in which an acetate-propionate mixture is treated is possible at pH 6. In a periodof 4 months of continuous operation at pH 6, a space load ofalmost 10 kg COD m-3 day -1 could be reached. The start-upperiod of a pH 6 reactor can possibly be shortened byapplying a more proper seed material, i.e., a sludge adaptedto low pH conditions. As reported previously by Williams(21), methanogenic activity in some acid peatlands is foundto be optimal at pH 6. For the treatment in UASB reactors ofacid wastewater, complete neutralization of the influent isnot a prerequisite. This fact has economic implicationsbecause fewer chemicals for neutralization are needed. Theimportance and the effect of different pHs in microenviron-ments and bulk solutions need to be investigated further.

ACKNOWLEDGMENTThis work was supported by a grant from the Dutch Ministry of

Housing, Physical Planning and the Environment.

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on the pH in biofilms. Water Sci. Technol. 14:838-848.2. Bochem, H. P., S. M. Schoberth, B. Sprey, and P. Wengler.

1982. Thermophilic biomethanation of acetic acid: morphologyand ultrastructure of a granular consortium. Can. J. Microbiol.28:500-510.

3. Buhr, H. O., and J. F. Andrews. 1977. The thermophilicanaerobic digestion process. Water Res. 11:129-143.

4. de Zeeuw, W., and G. Lettinga. 1983. Start-up of UASB-reac-tors, p. 348-369. In W. J. van den Brink (ed.), Proceedings ofthe European Symposium on Anaerobic Wastewater Treat-ment. TNO Corporate Communication Department, the Hague,The Netherlands.

5. Henze, M., and P. Harremoes. 1983. Anaerobic treatment of

waste water in fixed film reactors-a literature review. WaterSci. Technol. 15:1-101.

6. Heyes, R. H., and R. J. Hall. 1983. Kinetics of two subgroups ofpropionate-using organisms in anaerobic digestion. Appl. Envi-ron. Microbiol. 46:710-715.

7. HulshoffPol, L. W., W. J. de Zeeuw, C. T. M. Veizeboer, and G.Lettinga. 1983. Granulation in UASB-reactors. Water Sci. Tech-nol. 15:291-305.

8. Hulshoff Pol, L. W., J. Dolfing, W. de Zeeuw, and G. Lettinga.1982. Cultivation of well adapted pelltized methanogenic sludge.Biotechnol. Lett. 5:329-332.

9. HulshoffPol, L. W., J. Dolfing, K. van Straten, W. J. de Zeeuw,and G. Lettinga. 1984. Pelletization of anaerobic sludge inupflow anaerobic sludge bed reactors on sucrose containingsubstrates, p. 636-642. In M. J. Klug and C. A. Reddy (ed.),Current perspectives in microbial ecology. American Societyfor Microbiology, Washington, D.C.

10. Huser, B. A., K. Wuhrmann, and A. J. B. Zehnder. 1982.Methanothrix soengenii gen. nov. sp. nov. a new acetotropicnon-hydrogen oxidizing methanobacterium. Arch. Microbiol.132:1-9.

11. Kaspar, H. F., and K. Wuhrmann. 1978. Product inhibition insludge digestion. Microbiol. Ecol. 4:241-248.

12. Lettinga, G., S. W. Hobma, L. W. HulshoffPol, W. de Zeeuw, P.de Jong, P. Grin, and R. Roersma. 1982. Design operation andeconomy of anaerobic treatment. Water Sci. Technol. 15:177-197.

13. Lettinga, G., A. T. van der Geest, S. Hobma, and J. van derLaan. 1979. Anaerobic treatment of methanolic wastes. WaterRes. 13:725-737.

14. Lettinga, G., A. F. M. van Velsen, S. W. Hobma, W. de Zeeuw,and A. KlapwUk. 1980. Use of the upflow sludge blanket (USB)reactor concept for biological wastewater treatment, especiallyfor anaerobic treatment. Biotechnol. Bioeng. 22:699-734.

15. Mah, R. A., M. R. Smith, and L. Baresi. 1978. Studies on aacetate-fermenting strain of Methanosarcina. Appl. Environ.Microbiol. 35:1174-1184.

16. McCarty, P. L. 1964. Anaerobic waste treatment fundamentals.Part two, Environmental requirements and control, p. 123-126.Public works, October 1964.

17. Pette, K. C., and A. I. Versprille. 1982. Application of theUASB-concept for wastewater treatment, p. 121-137. In D. E.Hughes (ed.), Anaerobic digestion 1981. Elsevier/North-Holland Biomedical Press, Amsterdam.

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20. van den Berg, L., G. B. Patel, D. G. Clark, and C. P. Lentz.1976. Factors affecting rate of methane formation from aceticacid by enriched methanogenic cultures. Can. J. Microbiol.22:1312-1319.

21. Williams, R. T., and R. L. Crawford. 1984. Methane productionin Minnesota Peatlands. Appl. Environ. Microbiol. 47:1266-1271.

22. Zehnder, A. J. B., B. A. Huser, T. D. Brock, and K. Wuhrmann.1980. Charactization of an acetate-decarboxylating, non-hydro-gen-oxidizing methane bacterium. Arch. Microbiol. 124:1-11.

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24. Zhilina, T. N. 1976. Biotypes of Methanosarcina. Mikrobiolo-gya 45:481-489.

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