APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 1977, p. 298-307Copyright © 1977 American Society for Microbiology

Vol. 33, No. 2Printed in U.S.A.

Thermophilic Methane Production from Cattle WasteV. H. VAREL, H. R. ISAACSON,' AND M. P. BRYANT*

Department ofDairy Science* and Department ofMicrobiology, University ofIllinois, Urbana, Illinois 61801

Received for publication 28 June 1976

Methane production from waste of cattle fed a finishing diet was investigated,using four 3-liter-working volume anaerobic digestors at 60°C. At 550C a start-upculture, in which waste was the only source of bacteria, was generated within 8days and readily adapted to 600C, where efficiency of methanogenesis wasgreater. Increasing the temperature from 60 to 65°C tended to drastically lowerefficiency. When feed concentrations of volatile solids (VS, organic matter) wereincreased in steps of 2% after holding for 1 month at a given concentration, themaximum concentrations for efficient fermentation were 8.2, 10.0, 11.6, and11.6% for the retention times (RT) of 3, 6, 9, and 12 days, respectively. The VSdestructions for these and lower feed concentrations were 31 to 37, 36 to 40, 47 to49, and 51 to 53% for the 3-, 6-, 9-, and 12-day RT digestors, respectively, and thecorresponding methane production rates were about 0.16, 0.18, 0.20, and 0.22liters/day per g ofVS in the feed. Gas contained 52 to 57% methane. At the aboveRT and feed concentrations, alkalinity rose to 5,000 to 7,700 mg of CaCO3 perliter (pH to 7.5 to 7.8), NH3 plus NH4+ to 64 to 90 mM, and total volatile acids to850 to 2,050 mg/liter as acetate. The 3-day RT digestor was quite stable up to8.2% feed VS and at this feed concentration produced methane at the very highrate of 4.5 liters/day per liter of digestor. Increasing the percentage of feed VSbeyond those values indicated above resulted in greatly decreased organic mat-ter destruction and methane production, variable decrease in pH, and increasedalkalinity, ammonia, and total volatile acid concentrations, with propionatebeing the first to accumulate in large amounts. In a second experiment withanother lot of waste, the results were similar. These studies indicate thatloading rates can be much higher than those previously thought useful formaximizing methanogenesis from cattle waste.

Livestock production has changed rapidlyover the past decade, in particular, the concen-tration of cattle into large feedlots. The solidwaste annually produced by farm animals inthe United States is estimated at two billiontons. Roughly one-half of this waste is producedby intensive animal production systems (3, 9).This has created significant waste disposalproblems along with problems of stream pollu-tion and odor control (16). Anaerobic bacterialconversion of this waste with methane produc-tion may offer a partial solution to these prob-lems and also serve to supplement the supply ofnatural gas. In an economic analysis of meth-ane production from municipal solid waste in-volving mesophilic anaerobic microbial diges-tion, Kispert et al. (12) found that the cost ofproducing methane is economically acceptablewhen compared with projected costs of naturalgas. Studies of Pfeffer (17) showed that meth-ane production from municipal organic refusemay be economical if carried out at 600C. The

I Present address: Argonne National Laboratory, 9700 S.Cass Avenue, Argonne, IL 60439.

cost of methane was estimated to be 7.8 centsper 1,000 ft at 60°C compared with 11.0 centsfor optimum conditions at 350C. Higher rates ofdigestion and, thus, less capital cost, greaterconversion of waste organics to gas, faster solid-liquid separation, and minimization of bacte-rial and viral pathogens are some benefits ob-tained with the higher temperature (7, 17). Al-though some work has been done on methano-genesis from cattle waste (16), little has beendone at the optimum thermophilic temperatureor at high loading rates (19). Methane produc-tion from cattle waste could be economicallymore feasible than from urban refuse. Cattlewaste would not require the shredding and sep-aration process or the supplementation of nitro-gen and other minerals, which could increasethe cost for the municipal refuse digestion. Ourstudies were initiated to investigate the biologi-cal efficiency of thermophilic methane produc-tion at long to very short retention times (RT)and from low to high feed concentrations, usingwaste of cattle fed a finishing diet. Very highrates of methanogenesis were demonstrated.

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THERMOPHILIC METHANE PRODUCTION 299

MATERIALS AND METHODSReactors. Figure 1 shows the basic design of the

3-liter working volume reactors used in this study.The reactors were 4-liter Pyrex aspirator bottlesequipped with a side arm (13-mm ID) to which wasattached flexible, thin-walled black rubber tubing;this served as the effluent removal port which wasclosed with a screw clamp. The top of the bottle wassecured with a no. 10 black rubber stopper thatcontained three insertions: a thermometer, a gasoutlet of glass tubing (5-mm ID) connected to thick-walled butyl rubber tubing, and a feed inlet port ofglass tubing (13-mm ID) connected to a polypropyl-ene funnel by thin-walled flexible, black rubber tub-ing, closed with a screw clamp. Gas-impermeableevacuated bags of appropriate size to collect thedaily gas production were attached to the gas outlettube. The reactors were placed on a rotary platformshaker (2.4 cm in diameter, rotation at 140 rpm) in a37°C constant temperature room. Thermophilic tem-peratures were maintained, using a heating tapesecured to the outside of each reactor with maskingtape, and adjusted by a variable transformer. Thereactors were fed once per day, and effluent of anequal volume was removed just prior to feeding.The gas-impermeable bags were made by the pro-

cedure ofJohnson et al. (11) from '/2-mil (ca. 0.001 m)thick Scotchpak no. 20 (Film and Allied ProductsDiv., 3 M Co., St. Paul, Minn.). This is heat-sealablematerial that has a polyolefin coating on each side ofa very thin sheet of aluminum. A polyethylene tubewith a one-way stopcock is sealed into one corner ofthe bag to allow controlled entrance and exit ofreactor gas.When the effect of temperature was studied by

using a 50-day acclimation period for 65°C, a 5-literMicroferm fermentor (New Brunswick ScientificCo., New Brunswick, N.J.) was used with a 3-literworking volume. The heat control system was modi-fied to allow the temperature to be raised as high as80°C. Effluent was removed with a gentle suction to

gascollectionbag

feed inlet

thermometer

gasoutlet

heating tape

effluentoutlet\

ReactorFIG. 1. Schematic drawing of the reactor contain-

ing a 3-liter working volume. Four to six reactorswere maintained on a rotary shaker with a rotation of2.4 cm in diameter and 140 rpm.

initiate a siphon. The impellers were operated con-tinuously at 300 rpm.

Substrate. Approximately 100 kg of dairy-beefsteer waste (feces and urine without bedding) wascollected during the month of June 1974 from a 2.5-day accumulation on an open concrete surface. Thesteers (about 270 kg) were fed the following rationad libitum (percentage): 69.5 corn, 20.0 oats, 4.0alfalfa leaf meal, 5.0 soybean oil meal, 0.7 lime-stone, 0.4 trace mineralized salt, 0.3 K2CO3, 0.1Quadrex. In addition, each animal received 1.4 kg ofdry matter per day as alfalfa haylage. The wastewas approximately 45% total solids (TS) and 35%volatile solids (VS). After collection it was slurriedand diluted to approximately 15% VS with tap waterin a Waring blender. This slurry was then dispensedinto screw-cap plastic bottles, each to provide thedesired daily VS input for each reactor. It was storedat - 25°C until 1 day before use when it was placed at4°C. Just prior to the daily feeding, hot tap waterwas added to bring the influent substrate to the de-sired volume and temperature. The shaker was inoperation while the effluent was removed andturned off when the substrate was added.

The substrate for the second experiment was col-lected from the same area in February 1975 andtreated in the same manner. This substrate con-tained 25% TS and 20% VS.

Experimental design for studies on loading rate.The experimental design shown in Table 1 was usedto find RT (RT refers to both hydraulic and solid RT)and feed concenrations which allowed the most effi-cient methanogenesis. After cultures were estab-lished (see below), the four reactors were fed onceper day concentrations of VS from about 2 to 14%,increasing in steps of 2%. The reactors were held ata particular concentration for 3 weeks, after whichtime four to five daily samplings were obtained andanalyzed. After the samplings were completed, thepercent VS in the feed was raised to the next levelover 2 days and the equilibration time and samplingwere repeated. The process was continued until thereactors failed due to excess concentration of waste.

In experiment 2 the time before sampling wasslightly modified. The reactors were considered to bein a steady-state condition for each change in per-cent VS as follows: 27 days for the 9-day RT reactor,18 days for the 6-day reactor, and 9 days for the 3-day reactor. This is equivalent to 3-volume turn-overs in each case. After this equilibration period,five consecutive daily samples were obtained.The effect of various thermophilic temperatures

on the efficiency of the fermentation was studied byusing the New Brunswick fermentor operating at60°C with daily feed (4% VS) and 6-day RT. Thetemperature was increased 1°C per week until 65°Cwas reached. A 50-day acclimation period was per-mitted, after which time the various parameterswere measured from five daily samplings. Thetemperature was then lowered to 60°C overnightfollowed by a 12-day acclimation period, after whichtime the parameters were again measured. Thetemperature was then lowered to 55°C overnight,and the parameters were again measured after a

12-day acclimation period.

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300 VAREL, ISAACSON, AND BRYANT

TABLE 1. Experimental design for study of feedlotcattle waste at 60°C

Loading rateb in reactor:Time VS inperioda feed (%) A B C D

(12) (9) (6) (3)

1 1.90 0.16 0.21 0.32 0.632 3.87 0.32 0.43 0.65 1.293 5.90 0.49 0.65 0.98 1.974 8.20 0.68 0.91 1.37 2.735 9.98 0.83 1.11 1.66 3.336 11.56 0.96 1.28 1.93 3.857 14.10 1.18 1.57

a See text.b Daily VS in feed as percentage of reactor volumes.c RT in days.

Analytical procedures. Once each day the gas bagattached to each reactor was replaced with an evac-uated bag. The gas volume in the bag was thendetermined by fluid volume displacement from a 10-liter jar. The jar was filled with 20% NaCl and 0.5%citric acid solution and closed with a rubber stoppercontaining a tube for gas inlet and a glass syphontube for solution outflow. The syphon tube containeda rubber tube and pinch clamp on the external endso that the flow of solution could be controlled. Tomeasure the gas volume, the bag was attached tothe inlet tube and the gas was transferred to the jarby syphoning out the solution. The solution volumedisplaced by the gas was measured in a graduatedcylinder, and the volume was corrected to 760 mm ofHg pressure and 0°C. As the gas was being siphonedfrom the bags, 0.5-ml samples were drawn with asyringe from the rubber tube attached to the inlet,and the gas composition was analyzed with an Aero-graph gas chromatograph with a silica gel columnand a thermal conductivity detector (20). Heliumserved as the carrier gas for CH4 and CO2, whilenitrogen was the carrier gas for the detection of H2.Identification and percentage of CH4, CO2, and H2were based on a comparison of RT and peak heightsof unknowns with those of steaidard amounts of thethree gases.TS and VS were determined by a previously pub-

lished procedure (1). After centrifuging a sample at18,000 x g for 10 min, alkalinity was determined asdescribed previously (1) by titrating to pH 4.5.

Total volatile acids were determined by the previ-ously published procedure (1). Sintered-glass col-umns (20-mm ID and 20 cm long) were used insteadof sintered-glass crucibles. Individual volatile acidswere determined in some samples by gas chromatog-raphy as described by Iannotti et al. (10). The ef-fluent sample was centrifuged at 27,000 x g for 20min prior to being acidified and analyzed.Ammonia nitrogen was determined with the

phenol-hypochlorite method of Chaney and Marbach(6). Samples were first centrifuged as described forthe alkalinity determinations. The assays in experi-ment 1 were determined immediately after sampleswere collected, whereas in experiment 2 the sampleswere acidified to pH 5.5 and stored at -25°C andassayed at a later date. Total nitrogen was deter-

mined by the Kjeldahl method and ether extractswere determined by previously published methods(2).

Cellulose, hemicellulose, and lignin were deter-mined by the methods of Goering and van Soest (8).Due to the lack of precision in determination of

VS destruction at the higher feed concentrations byusing the direct gravimetric determination, a sec-ond method was employed whereby VS destructionwas calculated from the amount of CO2 and CH4produced. It was assumed that all of the carbon inthe VS destroyed was evolved as CO2 and CH4 andalso that the VS destroyed was 40% carbon; thus,(moles of CO2 and CH4 x 12)/0.4 = grams of VSdestroyed. The carbon content of many low-fat or-ganic materials containing mainly carbohydrateand protein is near 40%.

RESULTS

Substrate compositions. In Table 2 are listedsome constituents of the cattle waste used. Thewaste was low in VS (72%), probably due tosand obtained while collecting the waste from aconcrete surface. Cellulose and hemicelluloseaccounted for approximately 35% of the TS and50% of the VS. Total nitrogen was high, andabout 20% of it was in the form of ammonia(either NH3 or NH4+).Inocula for reactors. Figure 2 shows some

results of an initial experiment done to deter-mine the ease with which a culture could begenerated that would effectively produce meth-ane at thermophilic temperatures. In this ex-periment cattle waste plus a nitrogen and sul-fur-free mineral solution (4) was added to afinal concentration of the following (milligramsper liter): KH2PO4, 900; NaCl, 900; CeC12,20; MgC12 . 6H20, 20; MnCl2 - 4H20, 10; CoCl.26H20, 1, to each of four rectors set at 55'C.A control reactor had no inoculum otherthan the waste itself, whereas the others re-ceived 10% (vol/vol) of either sewage sludge,rumen fluid, or a culture from a 60'C thermo-philic reactor fed municipal refuse (obtained

TABLE 2. Composition of dry matter of waste fromcattle fed a high grain finishing diet"

Constituent %

VS ................. 72.0Ether extract ....... ......... 3.5Cellulose ................ 17.0Hemicellulose ....... ......... 19.0Lignin.6.8Total N (CP)b.3.0(19)Ammonia-N.0.55Non-ammonia CP.15.0Volatile acid (as acetic).1.2

a See the text for methods of determination.b CP, Crude protein (N x 6.25).

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THERMOPHILIC METHANE PRODUCTION 301

Time (days)

FIG. 2. Relationship of inoculum source to start-up of reactors at 55°C. Reactors were batch fed cattlewaste at 1% volatile solids, and after 9 days,feeding at 2% volatile solids and 15-day RT was

initiated.

through the courtesy of John Pfeffer, Univer-sity of Illinois). Each reactor was gassed withN2 for 15 min to remove air before being sealed.The reactor inoculated with thermophilic mu-

nicipal refuse effluent initiated methane pro-

duction immediately. After a delay of about 6days, the other reactors initiated rapid meth-ane production. On day 3 no H2 was detected inthe reactor with the thermophilic refuse inocu-lum, whereas approximately 200 ml was pres-

ent in each of the others. Only a trace amountof H2 was detected on days 4 and 5 and none was

detected later. A few pH adjustments usingapproximately 5 ml of 5 N sodium hydroxide, as

noted by the arrows, were necessary to main-tain the digesta above 6.8 in these three reac-

tors. Methane production peaked at 9 days, afterwhich time new substrate was fed once per dayat a feed concentration of 2% VS and a 15-dayRT. These results show that thermophilicmethanogenesis was easily initiated, andtherefore the bacteria necessary were appar-

ently readily available. The contents of the fourdigestors were mixed to ensure that similarorganisms were present in each before furtherexperiments were done.

Effect of changes in temperature and RT.Table 3 shows data on total daily gas produc-tion during transitions shortly after startingthe cultures. Results, especially with reactors 1and 4, show that temperature can be changedabruptly from 55 to 60°C without adverse effecton gas production; the low gas production in allreactors on day 34 was due to a failure of theshaker. Although the experiment was not de-signed to precisely compare gassing rates at 55and 60°C, comparison of data for days 13 to 25 at55°C and that for the reactors after changing to60°C but before changing the RT suggests a 10to 15S% increase in gas at 60°C; e.g., the mean

gas production was 0.55 liters/liter of reactor fordays 30 to 35 for reactor 1 after the changeversus a mean of 0.47 liters for days 13 to 25.The results show that, at least with this lowfeed concentration (2% VS), a rapid change inRT can be made without adverse effect. Noteespecially reactor 4, which was changed at onetime from an RT of 9 to 3 days. The pH re-mained stable at 6.9 to 7.1 during all of thesetransitions, and, although the proportion ofmethane in the gas phase was measured onlyon 24 of the 44 days, it also remained between50 and 59% of the gas phase.When a Microferm reactor run at 6-day RT

with 4% VS in the feed was changed from 60 to62.5°C on 1 day and to 65°C the next day, meth-ane production was strongly inhibited. In sixdeterminations before the change, methaneproduction averaged 0.17 + 0.01 liters/g of VSfed. After the change the production droppedradically on day 2 and did not return to produc-tion near that obtained at 60°C. The averageproduction from six daily samples taken after15 days at 65°C was only 0.054 + 0.003 liter. Onreturn to 60°C the production immediately in-creased. In a separate experiment increasingthe temperature from 60 to 65°C at 1°C per weekplus a 50-day acclimation period finally re-sulted in some adaptation although methaneproduction was still less than at 60°C (Table 4).Methane production was also less at 55°C thanat 60°C. Thus, as previously shown (17) 60°Cseems to be near optimum, even after long-term adaptation for the necessary mixture ofthermophilic organisms ubiquitous to centralIllinois.Efficiency of the fermentation. Figure 3

shows the VS destruction based on gravimetricdeterminations at the various feed concentra-tions and RT. As expected, the destruction wasless at the shorter RT. The data indicate ap-proximate VS destructions of 33, 38, 47, and50% at the respective RT of 3, 6, 9, and 12 dayswhen up to 9.98 VS were fed. VS destructiondata for feed concentrations above 9.98% werenot obtained in experiment 1. In experiment 2with another lot of waste (Table 5), the resultsindicate that the destruction remained aboutthe same when 11.7% VS were fed with RT of 6and 9 days. However, destruction greatly de-creased with a 3-day RT, where only 7% of theVS were destroyed. At the 14.12% feed concen-tration digestion was severely retarded at allRT tested. Some reservations on the precisionof the gravimetric analysis due to samplingproblems led us to calculate the amount of VSdestroyed as explained above. These data areshown in Fig. 4 and indicate an approximatedestruction of 38, 44, 48, and 50% at the respec-

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TABLE 3. Daily liters ofgas produced after culture initiation and during transitions from 55 to 60°C and from15- to 3-day RT in the reactors receiving 2% VS in the influent

Daily liters of gas per liter of reactor:Day

1 2 3 4

13-25 0.47 ± 0.05" 0.49 ± 0.06 0.48 ± 0.05 0.46 + 0.0326 0.55 0.51 0.56 0.5229 0.68 0.54c 0.57c 0.5030 0.61 0.55 0.58 0.5431 0.58 0.60 0.62 0.5932 0.63 0.58 0.59 0.6133 0.51 0.58 0.62 0.62b34 0.44 0.44 0.43 0.4235 0.53 0.59 0.54 0.5336 0.46d 0.47d 0.53d 0.51d37 0.52 0.53 0.51 0.5338 0.62e 0.60 0.61e 0.60'39 0.63 0.68 0.54 0.6640 - _ _ -f42 0.71 0.59 0.671 1.5343 0.68 0.66 0.91 1.8344 0.73 0.77 0.93 1.8345 0.71 0.55 0.96 1.85

46-57 0.71 ± 0.06", 0.57 ± 0.02 0.98 ± 0.03 1.74 ± 0.06

"Mean and standard error of nine determinations while the reactors were at 15-day RT and 55°C. No datawere available for days 27, 28, 40, and 41.

Temperature abruptly changed from 55 to 60°C.These reactors were raised from 55°C, 10C per day between days 29 and 33, to 60°C."RT was changed from 15 to 12 days.RT was changed from 12 to 9 days.

f RT was changed from 9 to 3 days."RT was changed from 9 to 6 days.h Mean and standard error of seven determinations while the reactors were at 60°C, 2% VS in the feed,

and 9-, 12-, 6-, and 3-day RT for reactors 1 to 4, respectively.

TABLE 4. Effect of temperature on the fermentation of cattle waste fed at a feed concentration of4% volatilesolids with a 6-day RT"

CH4 production VS destruction

Temp OH4 (% in Alkalinity(,IC) gas phase Liters/g of VS Liters/day

bpH (mg of CaCO3,Ili-

Temp per liter of Gravimetric Calculated p ter)reactor

65 52.9 + 0.9X 0.186 + 0.004 1.24 + 0.03 38.9 + 1.3 45.6 ± 1.3 7.19 ± 0.02 2,600 ± 3060 56.7 + 0.9 0.225 + 0.003 1.51 + 0.02 43.6 ± 0.9 52.2 ± 0.7 7.32 ± 0.04 3,250 ± 8055 57.5 + 1.4 0.217 t 0.007 1.45 + 0.05 44.6 ± 1.0 50.6 ± 1.6 7.14 ± 0.05 2,790 + 100

See text for details of the experiment.b As given in text.e The data represent the average of five consecutive daily samples.

tive RT of 3, 6, 9, and 12 days when up to 8.2%VS were fed. The destruction greatly decreasedas the feed concentration was increased above8.2% in the 3-day RT reactor, above 9.98% inthe 6-day RT reactor, and above 11.56% in the9- and 12-day RT reactors.

Figure 4 also presents data on the efficiencyof methane production on the basis of liters ofmethane produced per gram of VS fed. Theamount of methane produced averaged 0.17 li-ters/g of VS fed up to a feed concentration of8.2% in the 3-day RT reactor, 0.19 liters/g in the

6-day RT reactor up to the feed concentration of9.98% VS, and 0.20 and 0.21 liters/g for the 9-and 12-day reactors up to a feed concentrationof 11.56% VS.

Figure 5 shows data on efficiency of methaneproduction from the same experiment but froma different viewpoint, i.e., liters of methaneproduced per day per liter of reactor. Note thatthe 3-day RT reactor at 8.2% VS produced 4.5liters of methane per day per liter of reactor.This is the most rapid rate of methane produc-tion that we have found in review of the litera-

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THERMOPHILIC METHANE PRODUCTION 303

ture on methanogenesis from mixed organicmaterial. Only the work of Buswell (5) withthermophilic digestion of distillery waste fromwhich he obtained 3.8 liters/day per liter ap-

proached this level. At a given feed concentra-tion up to about 8.2%, the methane productionwas an inverse function of the RT; that is, if theRT was doubled, the methane production was

almost halved.As expected in a thermophilic fermentation

of common organic matter, methane was foundto be 52 to 57% of the total gas produced, withthe balance being mainly carbon dioxide, ex-

cept at the highest feed concentration such as

the reactor set with a 6-day RT and 11.7% VS inthe feed, where a shift to less methane (48%)and more carbon dioxide was observed.Ammonia concentrations. Figure 6 shows

the relationship ofRT and feed concentration tothe level ofammonia nitrogen. Very high levelsof ammonia were found at the higher feed con-

centrations. For example, at 8.2% VS, ammo-

nia ranged from 70 to 91 mM in the effluent. Inexperiment 2 (Table 4), even higher concentra-tions were obtained (70 to 100 mM at 7.8% VS)and values always continued to increase at feedconcentrations above 7.8% VS. The reason forthe lack of increase in ammonia concentrationat each increment of increase in VS in experi-ment 1 (Fig. 6) is not known.Organic acid concentrations. Figure 7

shows data on the levels of total volatile acidspresent in the reactors stabilized at differentfeed concentrations. As is the case with many

mesophilic systems (15), the levels of acid were

usually below 2,000 mg/liter until feed concen-

trations that caused major decreases in meth-ane production (Fig. 4) were reached. Thesecritical feed levels; i.e., those above which

8 60

a 50

9' 40

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F-

RT (days)

* 12o 9

O 6 0a 3 ,

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FIG. 3. Relationship of RT and feed concentra-tion to volatile solids destruction.

methane was greatly reduced and acids greatlyincreased, were about 8.2, 11.6, and 11.6% VSfor the reactors at 3-, 9-, and 12-day RT, respec-tively, and 8.2 to 10% VS for the 6-day RTreactor.Of interest is the fact that even at the higher

acid levels the pH remained high, presumablydue to the high levels of ammonia produced.Only small changes in pH occurred, and it in-creased froin about 7.0 at the low feed concen-trations to 7.8 at the higher feed concentra-tions.Table 6 shows some values for concentrations

of individual volatile acids. These determina-tions were done on only a few of the samples inwhich total volatile acids were at a high level.As the feed concentration was increased in areactor at a given RT, propionate was the firstacid to increase to a high level, i.e., 10 mM orhigher; then at the higher feed concentrationsboth acetate and propionate were found in largeamounts. Butyrate was detected in more thantrace amounts in only one of the reactors, i.e.,the one set for 3-day RT with 10% VS in thefeed, and was present in only a moderateamount, 5.5 mM.Experiment 2. Table 5 shows the results

from experiment 2 in which a different lot ofwaste, collected in the winter rather than sum-mer, was studied. The results are similar tothose of experiment 1, except that the pHdropped, but only at the highest feed concentra-tions studied. The ammonia levels differed asalready indicated, and the value from methaneproduction at 6-day RT and feed concentrationof 9.71% VS seems to be quite low as comparedwith the expected value.

DISCUSSION

These results indicate that methane fermen-tation of cattle feedlot waste at thermophilictemperature is maximum at about 60°C and isvery easily and rapidly initiated. It is highlystable to temperature changes between 55 and60°C, to changes in RT between 15 and 3 days,and to increases in the amount ofVS in the feedfrom 2% to about 8 to 12%, depending on the RTand lot of waste. The study indicates morerapid methanogenesis with higher loadingrates and shorter RT than previously thoughtpossible. Smaller reactors with greater stabilitymay be possible with the thermophilic processas suggested by Pfeffer (17) and Pfeffer andLiebman (18), reducing capital and, possibly,overall costs of methane fuel production. Evengreater efficiencies might be obtained with con-tinuous feeding rather than the once-per-dayfeeding used in these experiments. Under the

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THERMOPHILIC METHANE PRODUCTION 305

-60

40 ,,

iz

b

20e

*

RT (days)

* 12o 90 6a 3

2 4 6 8 10 12 14

Feed Concentrotion (% VS)

FIG. 4. Relationship ofRT and feed concentrationto efficiency of methane production based on litersproduced per gram of volatile solids fed and effect onvolatile solids destruction calculated as given in thetext.

RT (days)

* 12o 9O 6a 3

5), all reactors were held at a given feed for atime period equal to three times the RT beforedaily samples for analysis were collected. Thus,continuous culture theory indicates that theconcentration of materials in the reactor wouldhave reached about 95% of the final concentra-tion, i.e., the concentration after an infinitenumber of RT, and the change from period toperiod was only a small one, i.e., 2% of VS,before any samples were taken for analysis.The small values for standard error (Table 5),except at the point of reactor failure, also sug-gest long-term stability of the reactors at agiven loading rate. In experiment 1 on loadingrates, the reactors were all run at a given ratefor 3 weeks before any samples were taken.Thus, the 3-day RT reactor, which received thehighest loading rates (Table 1), was held for atime equal to at least seven RT (materials theo-retically reached over 99.9% of the final concen-tration) before samples were collected. Anotherindication of long-term stability is the fact that,-1

8000

S- 6000

0 2 4 6 8 10 12 14

Feed Concentration (M/ VS)

FIG. 5. Effect of RT and feed concentration on

efficiency of methane production based on liters ofmethane produced per day per liter of reactor.

120

100

'- 80

E 60

R 40

20

RT (doys)

* 12o 90 6a 3

2 4 6 10 12 14

Feed Concentrotion (% VS)

FIG. 6. Effect ofRT and feed concentration on theammonia (NH. plus NH41) concentration.

latter scheme huge variations in rates of pro-

duction and utilization of materials occur inrelationship to time of feeding (unpublisheddata).The probable long-term stability of methano-

genesis at high feed rates should be empha-sized. In experiment 2 on loading rates (Table

a4000-

2000

* It_w#

* 12o 90 6a 3

0~ ~ ~ ~ ~~~

0 2 4 E 8 10 12 14

Feed Concentration (% VS)

FIG. 7. Effect ofRT and feed concentration on thetotal volatile acid concentration.

TABLE 6. Concentration of acetate, propionate, andbutyrate in some 60°C reactors fed high levels of

cattle waste

Concn (mM)Feed concn RT% VS) (days) Acetate Propio- Butyrate

nate

8.2 6 4.821' 6.69 Trace8.2 3 2.41 22.32 0b9.98 12 3.21 2.23 09.98 9 3.21 5.58 09.98 6 4.82 26.22 09.98 3 20.90 29.57 5.4811.56 9 4.82 10.04 014.1 12 36.18 28.46 014.1 9 32.96 34.60 Trace

Values represent the average of two samplescollected on different days during the steady-stateperiod.

b None detected with a sensitivity of 1.0 mM.

.1

VOL. 33, 1977

RT (dovs)

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306 VAREL, ISAACSON, AND BRYANT

in connection with other more fundamental mi-crobiological studies, we have maintained onereactor (a Microferm fermentor) for approxi-mately 2 years with waste identical to that ofexperiment 2 and with 7% VS in the feed and a6-day RT. The methane production has re-mained essentially identical to that expectedfrom the experiments reported here.The reason for the decreased efficiency at the

highest loading rates is not known, but one

might suggest that inhibition is caused by a

general overabundance of solutes such as min-erals, ammonia, and fatty acids. It seemshighly improbable that acids alone are involvedbecause pH usually remained high and fattyacids exert their inhibitory effects chiefly whenin the unionized form. The high ammonia leveltogether with high pH, as well as alkalinity,could be factors. In mesophilic systems, Mc-Carty (14) indicates that, if the concentration ofammonia is between 100 and 200 mM and thepH is greater than 7.4, the system may beinhibited. At concentrations above 200 mM theammonium ion itself becomes quite toxic re-

gardless of pH. The values we obtained athigher feed concentrations fall into this inhibi-tory range. McCarty (13) indicates alkalinityconcentrations up to 6,000 mg of CaCO:3 perliter can be tolerated without producing anyadverse or toxic effects in mesophilic systems.No upper limit has been established.Because of the high content of ammonia and

other minerals, the cattle waste could be fer-mented in combination with wastes, such as

municipal refuse, which is deficient in nitrogenand phosphorus (17). Alternatively, because oflowered rapidly degradable organic matter, theeffluent should have better value as a soil fertil-izer than the original waste. Another possibil-ity of utilization of the effluent is removal ofbacterial cells, an excellent source of protein,before disposal of the fluids. The bacterial cellscould be fed to nonruminant livestock as a

source of protein. The 60°C temperature usedshould kill most nonsporing pathogenic orga-nisms, if these are present in the waste.

Preliminary studies show, as expected, thatlittle or no nitrogen is lost from the system andmost is passed out in the effluent as microbialcells or ammonia. For example, effluent fromthe system set at a medium loading rate (6-dayRT and 7% VS in the feed; waste of experiment2, loading rate = 1.9% or 0.74 lb/ft3) contains166 mM total nitrogen, about 80 mM ammonia-nitrogen, and about 53 mM bacterial nitrogen(about 0.5% bacterial protein in the effluent, W.Maeng, University of Illinois, unpublisheddata). At higher feed concentrations and

shorter RT, more ammonia and higher bacterialnitrogen and protein values are expected.We are continuing studies on kinetics of bac-

terial growth and protein production as well aschemical composition in relationship to the effi-ciency of methane production in this systemand with a wide variety of organic materials.These studies indicate the relevant biological

values and efficiencies of the methanogenic fer-mentation of cattle waste. If large-scale facili-ties fed chemically similar materials do notoperate with similar efficiencies, then one canassume that engineering problems, rather thanbiological potential, are involved.

ACKNOWLEDGMENTS

This study was supported by a Granite City Steel Corp.Environmental Science Scholarship to Vincent Varel, byU.S. Department of Agriculture grants 35-331 and 35-352,by National Science Foundation grant ENG 74-20777, andby the Agricultural Experiment Station of the University ofIllinois.

The technical assistance of Jack Althaus and the aid ofJimmy Clark and Alice Griffith in the Kjeldahl and etherextract determinations, of Carl Davis in determining indi-vidual fatty acids, of Sidney Spahr and Wayne Siefert inobtaining well-controlled lots of waste, and of VelmaRoughton in organizing and typing the manuscript is grate-fully acknowledged.

LITERATURE CITED

1. American Public Health Association. 1971. Standardmethods for the examination of water and sewage,13th ed. American Health Association Inc., Washing-ton, D.C.

2. Association of Official Agricultural Chemists. 1965.Official methods of analysis, 10th ed. Association ofOfficial Agricultural Chemists, Washington, D.C.

3. Bhattacharya, A. N., and J. C. Taylor. 1975. Recyclinganimal waste as a feedstuff: a review. J. Anim. Sci.41:1438-1457.

4. Bryant, M. P., and I. M. Robinson. 1961. Some nutri-tional requirements of the genus Ruminococcus.Appl. Microbiol. 9:91-95.

5. Buswell, A. M. 1939. The treatment of "beer slop" andsimilar wastes. Ill. State Water Surv. Bull. 32:165-172.

6. Chaney, A. L., and E. P. Marbach. 1962. Modifiedreagents for determination of urea and ammonia.Clin. Chem. 8:130-132.

7. Cooney, C. L., and D. L. Wise. 1975. Thermophilicanaerobic digestion of solid waste for fuel gas produc-tion. Biotechnol. Bioeng. 17:1119-1135.

8. Goering, H. K., and P. J. van Soest. 1970. Forage fiberanalysis. Agriculture handbook no. 379. U.S. Depart-ment of Agriculture, Washington, D.C.

9. Humphrey, A. E. 1975. Economic factors in the assess-ment of various cellulosic substances as chemical andenergy resources. Biotechnol. Bioeng. Symp. 5:49-65.

10. lannotti, E. L., D. Kafkewitz, M. J. Wolin, and M. P.Bryant. 1973. Glucose fermentation products ofRum-inococcus albus grown in continuous culture withVibrio succinogenes: changes caused by interspeciestransfer of H2. J. Bacteriol. 114:1231-1240.

11. Johnson, R. E., F. Robbins, R. Schilke, P. Mole, J.Harris, and D. Wakat. 1967. A versatile system formeasuring oxygen consumption in man. J. Appl.Physiol. 22:377-379.

APPL. ENVIRON. MICROBIOL.

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THERMOPHILIC METHANE PRODUCTION 307

12. Kispert, R. G., S. E. Sadek, and D. L. Wise. 1975. Aneconomic analysis of fuel gas production from solidwaste. Resource Recov. Conserv. 1:95-109.

13. McCarty, P. L. 1964. Anaerobic waste treatment funda-mentals. II. Environmental requirements and con-

trol. Public Works 95:123-126.14. McCarty, P. L. 1964. Anaerobic waste treatment funda-

mentals. III. Toxic materials and their control. PublicWorks 95:91-94.

15. McCarty, P. L., and R. E. McKinney. 1961. Volatileacid toxicity in anaerobic digestion. J. Water Pollut.Control Fed. 33:223-232.

16. Miner, J. R., and R. J. Smith. 1975. Livestock wastemanagement with pollution control (North CentralRegional Research publication 222). Midwest PlanService Handbook, Iowa State University, Ames,Iowa.

17. Pfeffer, J. T. 1974. Reclamation of energy from organic

refuse. Final report EPA-R-800776. Department ofCivil Engineering University of Illinois, Urbana.

18. Pfeffer, J. T., and J. C. Liebman. 1975. Biological con-version of organic refuse to methane. Annual reportNSF/RANN/SE/GI-39191/PR/75/2. Report UILU-ENG-75-2019. Department of Civil Engineering, Univer-sity of Illinois, Urbana.

19. Turk, M., and W. B. Coe. 1972. Production of power fuelby anaerobic digestion of feedlot waste. Phase I. Finalreport, contract no. 12-14-100-109-98(71) for U.S. De-partment of Agriculture, Fermentation Laboratory,Northern Marketing and Nutrition Research Divi-sion, Peoria, Ill.

20. Wolin, E. A., R. S. Wolfe, and M. J. Wolin. 1964.Viologen dye inhibition of methane formation byMethanobacillus omelianskii. J. Bacteriol. 87:993-998.

VOL. 33, 1977

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