bacterial populations and end products during fermentation of glucose

7/29/2019 Bacterial Populations and End Products During Fermentation of Glucose

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Copyright as part of th e March 1977, Part 1, JOURNAL WATER POLLUTION CONTROLFEDERATION, Washington, D. C. 20016-

Printed in U. S. A.

Bacterial populationsand end products duringanaerobic sludge fermentationof glucoseDavid P. ChynowethUniversity of North Carolina, Chapel HillRobert A. MahUniversity of California, Los Angeles

Waste trea tment of domestic wastewatersludge by anaerobic digestion is effected by amixture of organisms which eventually convertthe organic compounds to methane and carbondioxide. The starting substrates include carbohydrates, fats, long chain organic acids, proteins, and other nitrogenous compounds whichare initially degraded to small molecules oforganic acids, neutral products, hydrogen, andcarbon dioxide. A separa te group of methanogenic bacteria dissimilates certain of these fermentat ion products, yielding methane as amajor product.Although approximately 70 percent of themethane in this fermentation is produced fromacetate,l,2 the actual substrates for acetateformation and organisms responsible for itsproduct ion have not been thoroughly investigated. Formation of acetate from carbohydrates, fats, and proteins was examined byprevious investigators only after prolonged enrichment periods on the appropr ia te substrate.!' 3-5, 11 This procedure leads to selection of organisms which best metabolize thecompound being fed and does not give a trueevaluation of the unaltered fermentation. Toavoid this selection, unenriched sludge must beexamined.

I f a given substrate normally served as animportant source of acetate in the unenrichedsystem, addition of that substrate at high concentrations should result in the accumulationof acetate, provided it is formed at a rate fasterthan its conversion to methane. Determinationof the types of end products (particularly aceta te ) and their rates of formation would permit

an evaluation of the organisms and substratesresponsible for their production.In the following study the anaerobic sludgefermentation of glucose was investigated toexamine changes in bacterial populations and

patterns of fermentation products during adaptation to that substrate.MATERIALS AND METHODSDigester sludge. The source of digestersludge was a laboratory digester inoculatedwith a sample of anaerobic sludge from theprimary digester of a domestic treatment system, Third Fork Sewage Plant, Durham, N. C.I t was maintained on a raw sludge feed underconditions previously described by Mah andSussman. 6Anaerobic culture techniques. Strict anaerobic procedures were employed throughoutthe course of this investigation. Sludge samples were flushed with oxygen-free 70 percentN2-30 percent CO 2 .2 Anaerobic culture methods of Hungate 7 and Mah and Sussman 6 wereused for enumeration and isolation of anaerobicbacteria.Media. The inorganic salts solution contained the following compounds (final percentw/v): NaCI, 0.1; NH4CI, 0.05; MgCI2'6H20,0.005; CaCI2, 0.005; KH2P04, 0.005; CoCI2 '6H20, 0.001; (NH4)6Mo7024'4H20, 0.001;NaHC03, 0.5. Cysteine and sodium sulfidewere added as reducing agents at respectiveconcentrations (w/v) of 0.05 and 0.025 percent. 8 A 33 percent sludge supernatant medium 6 was used in estimating the anaerobic,viable compounds. Selective counts for acid-


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Chynoweth and Mah

o 2 4 6 8 10 12 14 16 18 20 22HOURSFIGURE 1. Fermentation of soluble sugars.Symbols: O-glucose; t::.-cellobiose; X-sucrose; D-endogenous.producing anaerobes were made in indicatormedium containing the basal medium plus 0.5percent glucose, 0.005 percent brom-thymolblue, and 1.0 percent precipitated CaCOa.Aerobic counts were made on plate count agarand eosin methylene blue agar.Manometric procedures. Fermentation ratesof various substrates were measured manometrically by the method of Smith and Mah. 8Speciall00-ml Warburg vessels 8 contained 25ml sludge. Test substrates were added tqdigesting sludge at a final concentration of 0.5percent (w/v) by injection through a side armfitted with a serum cap.Analysis of fermentation products. Volatilefatty acids and ethanol were analyzed by flameionization gas chromatography. A 1.8 m X3.3 mm (6-ft X 0.13-in.) stainless steel columnpacked with 50/80 mesh Porapak Q was used.The column temperature was 185C and theinjector and detector 220C. The carrier gaswas N2 at a flow rate of 20 ml/min. The H 2How rate was 20 ml/min and air 400 ml/min.Prior to analysis, samples were acidified with20 percent metaphosphoric acid (0.15 ml/mlsample) and centrifuged. The injection volume was 2 pl.Glucose assay. Glucose was determinedenzymatically by the Glucostat method. (> All

(> Worthington Biochemical Corp., Freehold,N. J.

700

PRESSUREmm Hg

GLUCOSE

RESULTSFermentation of glucose, cellobiose, andsucrose, was measured manometrically and theresults are shown in Figure 1. The endogenous control represents the rate of gas production without added substrates. All substrateswere metabolized, but vigorous gas productionoccurred only after a 10 to 12-h lag; gas was

400

samples were centrifuged to remove sludgesolids before analysis.Radioisotope measurement of glucose metab-olism. The rates of conversion of glucoseU-l4C to l4C02 were followed after trappingthe l4C02 in hyamine according to the following procedure. An empty vial was placed inthe side arm of the Warburg vessels (asabove); the appropriate sludge sample containing glucose-U_HC was placed in the mainchamber. At appropriate sampling times, 1.5ml of hyamine solution was injected throughthe serum cap into the vial. At the sametime, the vessels were placed in an ice bathand the reaction stopped by injection of 1 mlconcentrated H 2S04, After 60 min, the hyamine solution was transferred to a scintillationvial, 10 ml scintillation fluid 10 added, and thesample counted in a liquid scintillation spectrometer.

o 2 4 6 8 10 12 14 16 18 20 22HOURSFIGURE 2. Fermentation of glucose. Sym-bols: O-glucose; t::.-rawsludge; D-endogenous.

ENOOGENOUS

GLUCOSECELLOBIOSE

SUCROSE

100

200

600PressuremmH9

500

400

700

406 JournalWPCF


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Bacterial Population

ACETATE

10 242 IIHOURS

8

1


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Chynoweth and MahTABLE I. Comparison of Aerobic andAnaerobic Counts in

Endogenous and GlucoseEnriched Sludge.Hours after adding0.5% glucose

depicted in Figure 3. However, the totalaerobic count increased rapidly (1 OOO-fold)after an incubation time corresponding to therapid increase in production of gas and otherproducts indicating that euryoxic organismscould account for the rapid increase in glucosefermentation. Theoretically, the aerobic countshould not exceed the anaerobic count sincethe latter should support growth of facultativeand obligately anaerobic organisms. Thehigher aerobic counts depicted here may becaused by sampling variations. When both anaerobic and anaerobic sample were examinedsimultaneously, the agreement between samples was close at appropriate times.The exponential growth period exhibited bythe aerobic counts corresponded closely to thetime of rapid gas production. Selective anaerobic counts of glucose-fermenting organismswere estimated by inoculation into brom-

Aerobic plate counts(numbers/ml)Selective anaerobic counts(numbers/ml)Total anaerobic counts(numbers/ml)

o 10-126.5 X 10& 4.0 X 1072.0 X 104 2.6 X 1072.7 X 107 3.5 X 107

thymol blue CaCOg indicator medium. Assuming acid-producers were mainly euryoxic,such anaerobic counts should reveal any increase in those organisms capable of producingacid products from glucose and provide another measurement of the increase in euryoxicbacteria. Colonies were selectively counted inthe highest dilution exhibiting growth at thebeginning and during the peak of gas formation. The results (Table I) showed that theincrease in numbers during the enrichmentperiod was similar to that of the aerobic counts(Figure 4). Thus, the total anaerobic count(Figure 4) included the euryoxic bacteria, andthe smaller increase in total anaerobic numberscan be accounted for mainly by an increase inthis acid-producing population.Several organisms were isolated from anaerobic roll tubes (10-8 dilution) inoculatedwith the glucose-enriched sludge. Characteristics of these isolates are shown in Table II.Three isolates (6, 11, 12) were obligate anaerobes and produced acetate and propionatewhen grown on glucose. The remaining eightisolates (2, 3, 4, 5, 7, 8, 9, 10) were euryoxicand produced acetate and ethanol when grownon glucose. These products corresponded tothose produced by the mixed sludge population(Figure 3).A second series of organisms was isolated asfollows:1. From normal sludge which had not received glucose:

Group 1. Isolation of representative colonies from the highest dilution

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TABLE n. Characteristics of Isolates from Glucose-Enriched Sludge.Characteristics

Glucose endproductsIsolate Aerobic EMB* Ethanol Acetate

2 + + + +3 + + + +4 + + + +5 + + + +6 +7 + + + +8 + + + +9 + + + +10 + + + +11 +12 +*EMB-Eosin methylene blue agar selective for coliforms.408 Journal WPCF

Propionate

+

++

,1t


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Bacterial PopulationTABLE m. Number and Designation of Organisms Isolated.

Group 1

Group 2Group 3

Strict anaerobes8: 52, 53, 54, 55,56, 57, 59, 5103: 52-6, R1, R63: G4; G2-6G2-7

Euryoxico

7: R8, RlO, 52-452-5,52-7,52-8,52-99: GI, G3, G5, G7G8, G9, G2-1G2-4, G2-9

Euryoxic, growingbetter anaerobicallyo

I: R26: G2, GIO;G2-3,52-5G2-8, G2-1O

(l0-7) of habitat-simulatingmedium showing growth.Group 2. Acid-producing colonies from

the highest dilution (10-3 ) ofglucose indicator mediumshowing such colonies.2. From a sample of the same sludge incubated with glucose for 14 h:Group 3. Acid-producing colonies from

the highest dilution (10-7) ofindicator medium showingsuch colonies.A summary of the organisms isolated isshown in Table III. Most of the strict anaerobic isolates were identified to the genusBacteroides; fur ther characterization to thespecies level was not made. Some characteristics pertinent to general classification ofeuryoxic organisms are given in Table IV. Allwere gram-negative motile rods of the enterictype. Group A had the general properties ofthe Providence group 9; Group C organismsresembled members of the genus Escherichia.Group B isolates were also apparent ly relatedto the Genus Escherichia with the notable exceptions of a delayed fermentation of lactoseand peptonization of litmus milk.Since addition of soluble sugars to unenriched digesting sludge did not immediatelyresult in manometrically detectable gas production above the endogenous control (Figure1), glucose-U_14C fermentation was measuredas a method of detecting low dissimilationrates. Formation of 14C02 was measured at10-min intervals and the rate of glucose dissimilation was calculated on the assumptionthat two moles of 14C02 were produced permole of glucose dissimilated. The rate ofglucose dissimilation after 12-h enrichment wasalso determined by the same procedure. Aerobic counts and selective (acid-producing)

anaerobic counts were made in unenriched and12-h glucose-enriched sludge. The glucosedissimilation rates and bacterial counts arepresented in Table IV. Glucose was used immediately in normal and glucose-enrichedsludge at rates of 1.3 X 10-3 and 3.4 X 10-2f-LM Imlmin respectively. This represents a20-fold higher dissimilation rate in the glucose-enriched sludge. Aerobic, acid-producinganaerobic, and total anaerobic counts in thisparticular experiment were respectively 160,75, and 48 times higher in the glucose-enrichedsludge.DISCUSSIONAddition of the soluble sugars glucose, cellobiose, and sucrose to actively fermentingdigester sludge initially resulted in a fermentation rate only slightly greater than the endogenous control but less than the normal rawsludge substrate fermenter which receives amixture of lipids, carbohydrates, and proteins.10However, after a 10 to 12-h period, the rate ofsugar fermentation was exponential and greatlyexceeded the endogenous or raw sludge rates.This increase in gas production was accompanied by a corresponding decrease in glucoseand an increase in concentration of acetate,propionate, and ethanol. Since the number ofbacteria present in sludge is approxiinately109Iml,8 either enzyme induction in this population was necessary to metabolize solublesugars, or, more likely, growth and selectionfor sugar-metabolizing organisms took placeduring the lag period. Viable counts of bacteria taken through the experimental perioddisclosed that the latter was the case. Examination of aerobic plate counts for euryoxicorganisms shows an exponential increase innumbers corresponding to the period of maximum gas production. These organisms increased as much as 1 OOO-foid. Anaerobic

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Chynoweth and MahTABLE IV. Character istics of Euryoxic Isolates.

Group CGroup A 52-4; 52-5; 52-7;GI, G3, G5, G7, Group B 52-8; 52-9; G2-1;G8,G9 R8, RIO G2-4; G2-9Litmus milk Acid & peptonization Acid & peptonization Acid coagulationafter 10 days after 10 daysGelatinUreaIndol + -\-Methyl red + + +Voges-ProskauerCitrate + +HtSPhenylalaninedeaminase +Adonitol - ( ) +GCellobiose + +GDulcitolEsculin (+G)Galactose + +G +GGlucose + +G +GGlycerol + +G (+G)Inositol - ( )Lactose + after 2 weeks + after 10 days +GMaltose + +G +GMannose + +G +GMannitol +G +GRaffinose - ( )Rhamnose +G +GSalicin + after 2 weeks + after 10 days (+G)Sorbitol -(:;I::) +G +GSucrose +Starch +Trehalose + +G +G

Note: - = negative; + = growth; G = gas; ( ) = delayed.indicator medium, which permitted identification 'o f glucose-metabolizing acid-producingcolonies, yielded similar results. On the otherhand, the total anaerobic count on non-selectivehabitat-simulating media, which includes euryoxic organisms, yielded only a lO-fold increasein numbers throughout the entire period.

The euryoxic and acid-producing organismshad a generation time of less than 20 min andwere responsible for the sudden increase inaccumulation of fermentation products resulting from glucose dissimilation. They initiallyaccounted for about 2.4 percent of the totalanaerobic count bu t after a 10 to 12-h enrichment period on glucose were equivalent innumbers to the anaerobic counts. This changein composition of the bacterial population alsoshowed that soluble sugars were not in excessduring the normal fermentation since any significant sludge substrates should be quicklymetabolized by resident predominant organisms410 Journal WPCF

and not result in selection for those initiallypresent at low dilutions.Although all isolates examined were obtainedfrom anaerobic roll tubes, 31/41 acid-producing organisms were euryoxic. All 8 isolatesfrom the highest dilution of unenriched sludgewere obligate anaerobes (Table III, Group 1).Acid-producing colonies were not observed atthe highest dilutions of unenriched sludge;however, they were present and isolated fromthe 10-3 dilution (Table II I, Group 2). Ofthese, 8/11 were euryoxic. Colonies isolatedfrom the highest dilution of glucose-enrichedsludge, and selected because of acid-producingproperties (Table II I, Group 3), contained15/18 euryoxic isolates.All of the organisms in Table II I were isolated from the same sludge sample before andafter incubation with glucose. Members of thegenus Escherichia and the Providence group.were among the organisms which predominated


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after glucose enrichment. Organisms of theProvidence group were not isolated prior toglucose enrichment, and members of the genusEscherichia were obtained only from the 10- 3dilution. Organisms of the Providence groupmust also have been present at a concentrationof 103/ml or less. It is clear that the growthrates of members of these two groups weresufficient to permit their establ ishment as oneof the predominating types after only a 10 to12-h enrichment period on glucose. The acidfermentation products produced by these purecultures were the same as those found in thesludge containing glucose which suggests thatenrichment for these organisms during the dissimilation of glucose by the mixed fermentationoccurred.

I f production of volatile acids is not balanced by their conversion to methane andcarbon dioxide, the eventual result is completeinhibition of the overall methane fermentation.When substrates such as glucose enter a digester at concentrations higher than can bereadily metabolized by the resident organisms,a shift in the bacterial population may occur,and the result is an enrichment for organismswhich rapidly convert the substrate to acidend products. The frequent failure of domestic wastewater digesters may be caused bysuch selective pressures which elicit development of unwanted organisms which leads tosuppression of desired organisms. In thisstudy, the rate of conversion of glucose toacetate, propionate, and ethanol by euryoxicbacteria exceeded the rate of conversion ofthese end products to methane and carbondioxide. Organisms responsible for themethanogenic conversion were apparently unable to grow rapidly enough to accommodatethe rapid production of these products. Accumulation of ethanol was, in fact, unexpected;it has not been reported as an intermediate inthe sludge fermentation. Its formation heremay simply reflect its role as a terminal electron acceptor product of the glucose fermentation. In the balanced methane fermentation,methane bacteria serve as mediators of theterminal electron-accepting reaction via reduction of carbon dioxide to methane. Duringglucose enrichment, the rate of reduction ofcarbon dioxide to methane was apparent ly inadequate for the continued dissimilation ofglucose. Other electron-accepting reactions(such as the production of propionate andethanol) replace the methanogenic role of electron removal.

The present findings illustrate that digesters

Bacterial Population"acclimatized" to glucose,l, 4, 11 or other substrates 1, 3, 5, 11 may support a large populat ionof bacteria not present in significant numbersin normal sludge digesters. In the case ofglucose-enriched digesters, a shift in the bacterial population can occur within a 10 to 12-hperiod, and it would be incorrect to assume 4that only slow-growing strict anaerobes werepresent in such "acclimatized" digesters. Although laboratory digesters receiving pure substrates may support a vigorous methane fermentation, some caution should be exercised intheir examination in view of the intimatephysiological association (s) 12 which may existbetween a methane bacterium and its nonmethanogenic partner.Determination of the rate of formation of14C02 from glucose-U_14C permitted a moresensitive measurement of glucose dissimilationduring the initial fermentation period. The20-fold increase in the ra te of 14C02 evolutionafter enrichment corresponded exactly to theperiod of logarithmic growth exhibited by theeuryoxic population. Although the resultsmight also be explained by an increase in thepredominating anaerobic bacteria, it is difficultto establish whether such organisms increasedin numbers sufficient to account for thesechanges during the short enrichment period.The growth rates for such anaerobes may indeed be too slow 6 to effect any significantpopulation changes within this time. However, it should be noted that the 20-fold increase in fermentation rate does not correspond to the 100 to 1 ODD-fold increase ineuryoxic bacteria; that is, these organisms cannot alone account for glucose dissimilation inunenriched sludge. Apparently, strict anaerobes present in the normal sludge (unenriched) also used glucose, but because of theirslow growth rates were unable to compete forglucose with the faster-growing euryoxicorganisms.The anaerobic sludge digestion process maythus be regarded as a heterogenous populationof opportunists each of which can gain predominance because of favorable selective pressures. The balanced activity of these organisms makes the sludge fermentation a practicaltreatment process for disposal of organicsludges via the methane fermentation.SUMMARY AND CONCLUSIONSAddition of glucose to unenriched (unacclimatized) digesting sludge resulted in noapparent response for 8 to 10 h. At this timea sudden and rapid increase in gas production


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Chynoweth and Mahand accumulation of acetate, propionate, andethanol was observed. This shift in the fermentation pattern was shown to result from thesudden selective growth of euryoxic bacterianormally present in low numbers in the unenriched fermentation. Several isolates fromthe enriched digester sludge were characterized to the genus Esoherichia and the Provi-dence group. These isolates produced fermentation products identical to those found in theenriched sludge in high concentrations. Thegrowth and activities observed for these organisms clearly illustrate the imbalance that mayoccur upon altering the substrates added toanaerobic digesters. The continued imbalanceof bacterial populations in the fermentationwould lead to accumulation of toxic fermentation products and the eventual cessation ofdecomposition.This research further demonstrates that acclimatization of digester sludge to pure substrates results in a major change in the inherentbacterial populations. In view of this, knowledge gained in the study of acclimatizeddi-gester sludge may not be applicable in understanding the sludge fermentation receivingwastewater sludge.ACKNOWLEDGMENTSCredits. The technical assistance of LenaMeck and James R. Williams II I is gratefullyacknowledged. This investigation was supported in part by Public Health Service grantWP-00921-03.Authors. At the time of this paper, DavidP. Chynoweth was Graduate Student, Department of Environmental Sciences and Engineer,ing, University of North Carolina, Chapel Hill.He is currently Assistant Professor, Departmentof Environmental and Industrial Health, TheUniversity of Michigan, Ann Arbor. RobertA. Mah is Professor, Division of EnvironmentalScience and Nutritional Biochemistry, University of California at Los Angeles.

412 Journal WPCF

REFERENCES1. Jeris, J. S., and McCarty, P., "The Biochemistry of Methane Fennentation using HCTracers." Jour. Water Poll. Control Fed.,37, 178 (1965).2. Smith, P. H., and Mah, R. A., "Kinetics ofAcetate Metabolism during Sludge Digestion." Appl. Microbiol., 14, 368 (1966).3. Andrews, J. F., and Pearson, E. A., "Kineticsand Characteristics of Volatile Acid Production in Anaerobic Fennentation Processes."Int. Jour. Air Water Pol., 9, 439(1965).4. Jeris, J. S., and Cardenas, P. R., "GluC9se dis-appearance in biological treatment systems."Appl. Microbiol., 14, 857 (1966).5. Kotze, J. P., et al., "A Biological and ChemicalStudy of Several Anaerobic Digesters."Water Res., 2, 195 (1968).6. Mah, Robert A., and Sussman, Carol, "Microbiology of Anaerobic Sludge Fennentation.I. Enumeration of the Nonmethanogenic anaerobic bacteria." Appl. Microbiol., 16, 358(1967).7. Hungate, R. E., "A Roll Tube Method forCultivation of Strict Anaerobes." In Norris,J. R., and Ribbons, D. W. (eds.). "Methodsin Microbiology." Academic Press, NewYork, pp. 117-132. (1969).8. Smith, P. H., "Pure Culture Studies ofMethanogenic Bacteria." Proc. 20th PurdueInd. Waste Cont., Purdue Univ. pp. 583588 (1966).9. Hormaeche, E., Chainnan, EnterobacteriaceaeSubcommittee. "Report of the Nomenclature Committee of the International Association of Microbiological Societies." Int. Bull.Bacteriol. Nom. and Tax, 8, 25 (1958).10. Chynoweth, D. P., and Mah, R. A., "VolatileAcids Fonnation in Sludge Digestion." InPohland, F. G. (ed.) "Anaerobic BiologicalTreatment Processes." Advances in Chern.Ser. 105., Am. Chern. Soc., pp. 41-54,(1971).11. McCarty, P. L., et al., '1ndividual VolatileAcids in Anaerobic Treatment." Jour.Water Poll. Control Fed., 35, 1501 (1963).12. Bryant, M. P., et al., "Methanobacillus ameli-anskii, a symbiotic association of two speciesof bacteria." Arch. Mikrobiol., 59, 20(1967).

bacterial populations and end products during fermentation of glucose

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