effects of ammonia nitrogen on h2 and ch4 production during anaerobic digestion of dairy cattle...

E�ects of ammonia nitrogen on H2 and CH4 production duringanaerobic digestion of dairy cattle manure

M.C. Sterling Jr., R.E. Lacey a,*, C.R. Engler a, S.C. Ricke b

a Department of Agricultural Engineering, Texas A&M University, College Station, TX 77843, USAb Department of Poultry Science, Texas A&M University, Kleberg Center, Room 101, College Station, TX 77843-2472, USA

Received 28 June 1999; received in revised form 24 August 2000; accepted 8 September 2000

Abstract

A number of researchers have veri®ed the inhibitory e�ects of elevated H2 concentrations on various anaerobic fermentation

processes. The objective of this work was to investigate the potential for using hydrogen gas production to predict upsets in an-

aerobic digesters operating on dairy cattle manure. In an ammonia nitrogen overload experiment, urea was added to the experi-

mental digesters to obtain increased ammonia concentrations (600, 1500, or 3000 mg N/l). An increase in urea concentration resulted

in an initial cessation of H2 production followed by an increase in H2 formation. Additions of 600, 1500, or 3000 mg N/l initially

resulted in the reduction of biogas H2 concentrations. After 24 h, the H2 concentration increased in the 600 and 1500 mg N/l di-

gesters, but production remained inhibited in the 3000 mg N/l digesters. Both methane and total biogas production decreased

following urea addition. Volatile solids reduction also decreased during these periods. The digester e�uent pH and alkalinity in-

creased due to the increased NH�4 formed with added urea. Based on these results, changes in H2 concentration could be a useful

parameter for monitoring changes due to increased NH3 in dairy cattle manure anaerobic digesters. Ó 2001 Elsevier Science Ltd.

All rights reserved.

Keywords: Anaerobic digestion; Hydrogen; Nitrogen; Mesophilic; Dairy manure

1. Introduction

The advantages of anaerobic digestion technology forthe treatment of organic residues have been well docu-mented. Signi®cant advances in reactor design and op-eration have been achieved in recent years. Nevertheless,process-control strategies currently available are thosethat have long been used for anaerobic digesters. Be-cause of the complexity of microbial interactions in-volved, the process can be di�cult to control (Boekhurstet al., 1981). Reasons for digester imbalance includeexcessive change in temperature, a sudden increase inorganic loading, the presence of a toxic material, or achange in feed characteristics (Jeris and Kugelman,1985). Upsets can lead to digester failure causing loss ofproduction for extended periods of time. One means tomonitor the operational health of a digester is to focuson its biochemical state.

The anaerobic digestion process is a natural biolog-ical process in which a community of bacteria cooperateto form a stable, self-regulating fermentation that con-verts waste organic matter into a mixture of carbondioxide and methane gases. A manure digester com-munity generally operates as three interdependentgroups: hydrolytic bacteria, acid-forming bacteria, andmethanogenic bacteria. Hydrolytic bacteria cleavepolymeric carbohydrates and proteins into simplemonomeric sugars and amino acids. Acid formingbacteria are composed of acetogenic bacteria, whichform volatile fatty acids (VFAs) directly; homoaceto-genic bacteria, which form acetate from CO2 and H2;and hydrogenogenic bacteria, which convert largervolatile fatty acids into acetate and H2. Methanogenicbacteria are composed of acetoclastic methanogens,which convert acetate to methane and CO2 and hy-drogen utilizing methanogens, which convert CO2 andH2 to methane.

Because hydrogen is an intermediate for methaneproduction, monitoring it should provide informationon the state of a digester. In a review, Archer andKirsop (1991) outlined the evolution of hydrogen

Bioresource Technology 77 (2001) 9±18

* Corresponding author. Tel.: +1-979-845-3961; fax: +1-979-845-

3932.

E-mail address: [email protected] (R.E. Lacey).

0960-8524/01/$ - see front matter Ó 2001 Elsevier Science Ltd. All rights reserved.

PII: S 0 9 6 0 - 8 5 2 4 ( 0 0 ) 0 0 1 3 8 - 3

monitoring as a potential control parameter for an-aerobic digestion. Mosey (1982) built upon anaerobicmicrobiological studies done by Hungate (1975) andWolin (1976, 1982) to create kinetic models for an-aerobic digesters using hydrogen as a control parame-ter. Other authors have discussed potential advantagesof monitoring hydrogen gas (Robinson and Tiedje,1982, 1984; Bartlett et al., 1980) as well as successfultrials in laboratory and industrial-scale digesters(Mosey and Fernandes, 1984; Archer et al., 1986).Although the potential for using hydrogen gas as anindicator of digester viability has been demonstrated,the feasibility of using hydrogen gas as a control pa-rameter for digesters fed complex substrates is lessclear. Hickey et al. (1987, 1989) found that monitoringhydrogen in anaerobic sludge digesters could providerapid indication of process upsets; conversely, Kidbyand Nedwell (1991) concluded that hydrogen concen-tration in sludge digester biogas could not be used asan indicator of incipient failure resulting from volu-metric overload.

A number of studies have cited the inhibitory e�ectsof free ammonia (NH3) on the metabolism of metha-nogens (Angelidaki and Ahring, 1993; Braun et al.,1981; El-Hag et al., 1982; De Baere et al., 1984; Sprottet al., 1984). As ammonia is added to a digester, the pHincreases until a chemical equilibrium is reached(Georgacakis et al., 1982). However, as ammonia in-hibits methanogen metabolism, VFAs accumulate, re-sulting in a lower pH and a lower concentration of freeammonia. Assuming an adequate digester alkalinity,this mechanism tends to stabilize the digestion process ata certain VFA concentration and pH level (Georgacakiset al., 1982). With an insu�cient alkalinity, the digesterundergoes acidosis, resulting in the cessation of methaneproduction.

The purpose of this study was to determine the fea-sibility of monitoring hydrogen gas as an indicator ofdigester upset resulting from ammonia overloading.Speci®c objectives included determining the e�ects ofadded ammonia nitrogen on digester H2 production,methane production, and volatile solids (VS) removal.

2. Methods

2.1. Experimental apparatus

The reactors used were similar to those used byVartak et al. (1997). The reactors were constructed fromacrylic tubing, with each reactor having an internal di-ameter of 15.2 cm, a height of 30.5 cm, and a nominalworking volume of 5 l (Fig. 1). Separate feed and ef-¯uent ports made of 12.7 and 6.4 mm ID plastic tubingextended into the digester liquor. Separate ports wereused for biogas sampling and collection.

Gas produced from each reactor passed through ahydrogen sensor and into a 4.5-l glass graduated gascollector (Fig. 1). The gas collector was ®lled with waterand used to measure gas production by volume dis-placement. Displaced water from the collector waspassed through an anti-siphon tubing arrangement andcollected in a carboy.

2.2. Hydrogen sensing and recording system

Hydrogen content of the biogas was monitored bystannic oxide sensors (TGS 821, Figaro Engineering,Wilmette, IL) housed in rectangular (3:7� 7:3�10:0 cm3) plastic instrument boxes. Silicone sealant wasused to ®ll any openings in the sensor boxes. Themanufacturer warns that stannic oxide sensors may besensitive to silicone; however, the sensors were cali-brated with gas standards after construction and all 10sensors had virtually the same calibration curve indi-cating that any acute sensitivity either was uniformacross all sensors or was adjusted by the calibration.Additionally, the baseline resistance remained constantover the time of the experiments indicating that gradualsensitivity loss did not occur.

The sensors were heated using a constant voltage(5 V DC) supplied by a power transformer (PL20-10,Microtran, Valley Stream, NY). Current from the sec-ondary side of the transformer was distributed to thesensor heaters through parallel circuits. Sensor resis-tance dropped as it was exposed to hydrogen gas. Sensorresistance was measured by a data acquisition/switchunit (34970A, Hewlett-Packard, Santa Clara, CA) anddownloaded to a personal computer (Packard Bell,Sacramento, CA). Because gas ¯ow rates were extremelylow, convective cooling was considered negligible sothat no temperature-compensation circuitry was re-quired.

2.3. Digester operation

The experiments were conducted in a walk-in envi-ronmental chamber. A set of nine digesters was used,with the temperature maintained at 35� 1°C. The di-gesters initially were inoculated with rumen ¯uid ob-tained from the Dairy Cattle Nutrition Center, TexasA&M University, and had operated continuously ondairy cattle manure for over three years prior to the startof these experiments.

Relatively fresh undiluted dairy cow manure wascollected from the concrete surface of the holding pensat the Texas A&M Dairy Cattle Nutrition Center. Ap-proximately 25 kg (wet basis) of manure were gatheredat one time, thoroughly mixed with tap water to form a5 g VS/l slurry, then placed in a freezer and kept frozenuntil immediately before use.

10 M.C. Sterling Jr. et al. / Bioresource Technology 77 (2001) 9±18

To maintain a 10-day hydraulic retention time(HRT), 500 ml/day of e�uent was removed from eachdigester and replaced with the same volume of feedslurry. This gave an organic loading rate of 0.5 kg VS/m3 d. Before sampling, the digester was isolated fromthe gas collection system and the contents were mixed byrepeated inversion of the reactor. Each digester was in-verted 10 times over a period of about 15 s to obtaincomplete mixing.

After opening both the gas outlet line and liquid ef-¯uent port, 500 ml of digester e�uent was collected in a600-ml beaker. The liquid e�uent port was then closed,the feed inlet port was opened and feed slurry was addedto the digester. After the addition of feed, the feed inletand gas outlet ports were closed and the reactor wasmixed as before sampling. After mixing, the digester wasreconnected to the gas collection system.

Stable operation was achieved before these experi-ments were begun. A 10-day period just prior to col-lecting data was considered su�cient for de®ning stable

operation. The system was considered to be operatingstably when the coe�cient of variation for daily gasproduction was less than 10% (Vartak et al., 1997).

2.4. Nitrogen addition

After the digesters reached stable operation, the di-gesters were switched to batch mode for the ammoniaoverloading experiment. For the experimental digesters,urea was added as a solid to the manure slurry to pro-vide di�erent ammonia nitrogen concentrations asshown in Table 1. Urea was selected as the nitrogensource due to its ease of degradation to ammonia by avariety of microorganisms. After an initial (t � 0 h) re-moval of e�uent and addition of feed slurry (500 mleach), e�uent samples of 125 ml were taken from eachdigester at 3, 6, 9, 12, 24, 48, 72, and 96 h. To maintainconstant volume, 125 ml of tap water was added to eachdigester after each sampling period and concentrationswere adjusted for dilution e�ects. As during continuous

Fig. 1. Experimental apparatus for biogas generation, collection and monitoring. (DAQ, data acquisition unit).

M.C. Sterling Jr. et al. / Bioresource Technology 77 (2001) 9±18 11

operation, digester contents were mixed before samplingand after additions.

2.5. Chemical analysis

Gas volumes were recorded daily. All biogas andmethane production volumes were converted to STP(0°C, 1 atm) conditions. Gas samples were collected ingas-tight syringes and analyzed by gas chromatographfor methane, carbon dioxide and nitrogen composition.Gases were separated using a 2.4 m by 3 mm stainlesssteel column packed with 100/120 mesh HayeSepâ D(Hayes Separations, Bandera, TX). The column wasoperated at 35°C with helium as the carrier gas (30 cc/min).

Hydrogen concentrations were measured on-line withstannic oxide sensors. Sensor voltage was converted tohydrogen concentration using the following calibrationequation:

log�H2 concentration� � ÿ 1

a

� �b

�� log

R0

Rexp

� ��;

�1�where a and b are the correlation coe�cients, R0 thesensor resistance for 100 ppm H2 at 35°C, and Rexp is thesensor resistance measured at experimental conditions.For the sensors used, a, b, and R0 values were ÿ0:9,1.484 and 100 X, respectively.

Gas standards were obtained from Scott SpecialtyGases (Plumstead, PA). H2 standards of 1% in N2 and100 ppm in N2 were used in calibrating selected hydro-gen sensors. Pure (99+%) samples of CH4, CO2, and N2

were used in calibrating the gas chromatograph.Feed and e�uent samples were analyzed for total

solids (TS), VS and alkalinity using standard procedures(APHA, 1991). In addition, biosolids samples were as-sayed for hemicellulose, cellulose, lignin, and silica ashusing Neutral Detergent Fiber (NDF) (Van Soest andWine, 1967), Acid Detergent Fiber (ADF) (Van Soest,1963), and permanganate lignin (Van Soest and Wine,1968) procedures. Colorimetric assays (Hach Company,Loveland, CO) were used to measure chemical oxygendemand (COD), total nitrogen (an analog of TotalKdeldhal Nitrogen), and total ammonia nitrogen. Foreach colorimetric assay, a 5 ml aliquot of e�uent samplewas diluted to 200 ml with deionized water. Free or un-ionized ammonia concentrations were calculated usingthe following equation (McCarty and McKinney, 1961):

NH3� � � 1:13� 10ÿ9 T-NH3� �H�� ; �2�

where [NH3] is the concentration of un-ionized ammo-nia (mg/l), [T-NH3] the concentration of total ammonia(mg/l), and [H�] is the concentration of hydrogen ions(mole/l).

2.6. Statistical analysis

Data reported are averages of the reactors for eachnominal urea concentration. Analysis of variance onchanges in biogas production, methane production, pH,alkalinity, VS, COD, and ®ber content data were con-ducted. Duncans Multiple Range test (Milton and Ar-nold, 1990) was used to do overall comparisons for theexperiments. Duncans multiple comparisons were con-ducted only on analysis of variance main e�ects thatwere signi®cant. For comparisons at speci®c times dur-ing an experiment, t-testing was used to determine sig-ni®cance. Signi®cance was reported at an a of 0.05. TheSAS (Statistical Analysis System, Cary, NC) system forWindows (release 6.11) was used for statistical analyses.

3. Results and discussion

3.1. Total nitrogen and ammonia concentrations

Addition of urea to the experimental digesters causednitrogen and ammonia concentrations to increase(Fig. 2). Total nitrogen content increased after the ad-dition of urea and remained virtually constant through-out the remainder of the experiment. Unanticipated lownitrogen concentration values at 3 and 24 h (Fig. 2)likely resulted from insu�cient heating or reaction timeduring those analyses. This conclusion is consistent withmaterial balance calculations on digester volatile solidsdescribed later. Variations in the nitrogen content valuesin control digesters and in the experimental digestersafter 24 h were attributed to experimental error.

Increases in total ammonia concentration were slowerthan for total nitrogen (Fig. 2). Lagging ammonia pro-duction was expected due to the time required for thedigester micro¯ora to convert urea to ammonia. Theammonia concentration approached the total nitrogenconcentration within 24 h in the 600 mg N/l digestersand within 48 h in the 1500 and 3000 mg N/l digesters.

Table 1

Experimental design for urea addition experiment

Reactor group Digesters in test group Urea concentration in digester (test variable)

Control Reactors 1, 4, 9 Manure slurry only

Group A Reactors 2, 6 Manure slurry� 600 mg N/l

Group B Reactors 5, 7 Manure slurry� 1500 mg N/l

Group C Reactors 3, 8 Manure slurry� 3000 mg N/l


Subsequently, no signi®cant changes in either total ni-trogen or ammonia concentrations in any of the di-gesters occurred, suggesting that equilibrium had beenreached.

The concentration of un-ionized ammonia was re-lated to digester pH and total ammonia concentrationusing Eq. (2). Calculated values of free ammonia aregiven in Table 2. Values for the control and 600 mg N/lgroups remained below 200 mg/l throughout the exper-

iment. Conversely, values for the 1500 and 3000 mg N/lgroups increased to over 200 mg/l by 12 h and the ®nalvalues of 630 and 3143 mg/l, respectively, were aboveinhibitory values reported in the literature.

3.2. Biogas composition

To determine biogas production, gas volumes (re-ported at standard conditions of 1 atm and 273.15 K)were corrected for CO2 produced by urea degradation.Since urea degradation produces 2 mole of ammoniaand 1 mole of carbon dioxide (Eq. (3)), increases inammonia concentration were used to calculate the re-sulting increases in carbon dioxide.

Urea�H2O! CO2 � 2NH3 �3�For a given time interval, volumetric CO2 production(1 atm, 273.15 K) due to urea degradation was calcu-lated using Eq. (4):

DCO2 � 15:821D NH3� �

Dt

� �; �4�

where DCO2 (ml/l d) is the average daily production rateof CO2 during a sampling interval, D�NH3� (mg/l) theincrease in digester ammonia concentration during asampling interval, and (Dt) is the length of the samplinginterval (h).

For the control and 600 mg N/l digesters, biogasproduction followed the expected trend for an anaerobicbatch process (Fig. 3). After addition of feed, the biogasproduction rate increased. Following a peak in the pro-duction rate between 9 and 12 h, the rate of biogas pro-duction decreased through the rest of the experiment.Throughout the experiment, the ratio of gas producedper amount of VS degraded remained within typicalvalues (2.5±4.0 ml/mg) for dairy cattle manure digesters(Loehr, 1984).

Biogas production from the digesters with higheramounts of urea added followed a signi®cantly di�erenttrend. In the 1500 and 3000 mg N/l digesters, biogasproduction rates decreased by approximately 30%and 50%, respectively, during the ®rst 3 h after urea

Table 2

Free ammonia concentrations (mg/l) in digesters after urea addition

Time (h) Control Free NH3 (mg N/l)

600 1500 3000

0 1:8 � 0:15a 4.61 � 0.69 9.19 � 0.74 8.49 � 1.96

3 2:24� 0:71 9.74 � 0.89 31.14 � 2.71 26.65 � 4.04

6 2:66� 0:36 23.47 � 1.93 51.37 � 1.78 72.39 � 6.07

9 2:53� 0:30 28.75 � 1.03 80.55 � 18.08 99.51 � 23.54

12 4:60� 1:28 126.60 � 21.08 248.77 � 21.35 573.44 � 3.20

24 3:99� 0:58 142.58 � 11.11 390.10 � 7.49 1534.75 � 11.45

48 3:33� 0:18 163.02 � 1.98 568.4 � 10.58 2724.68 � 21.88

72 3:94� 0:49 189.98 � 4.06 584.74 � 10.60 3032.03 � 12.64

96 4:10� 0:43 208.96 � 4.14 629.88 � 11.41 3143.30 � 5.87

a Standard deviation of the mean.

Fig. 2. Total nitrogen (closed symbols) and ammonia nitrogen (open

symbols) concentrations in digesters after urea addition ((a) control;

(b) 600 mg N/l; (c) 1500 mg N/l; (d) 3000 mg N/l added).


addition. There was a brief increase in gas productionbetween 12 and 24 h, then the biogas production ratesfor both groups decreased through the end of the ex-periment. The ratio of gas produced per quantity of VSdegraded (ml/mg) was well below typical values fordairy cattle manure throughout the experiment. Thesedi�erences were due to the combined e�ects of increasedsolubility of CO2 in the high ammonia digesters due toincreased pH and inhibited biogas production as high-lighted by decreased methane production.

3.3. Methane production

Methane production trends followed the biogas pro-duction trends for all digesters (Fig. 4). At 24 h, methaneproduction rates had increased over initial productionrates by 175% and 300% in the control and 600 mg N/ldigesters, respectively, as a result of increased VS re-duction rates in these digesters. After 24 h, methaneproduction rates for these digesters decreased to theinitial (t � 0 h) rates as the VS reduction rates decreasedto initial values.

At 24 h, methane production rates in the 1500 and3000 mg N/l digesters decreased to 60% of their original

(t � 0 h) values. During this period, the decrease in VSreduction rates in these digesters contributed to the re-duction in methane production. However, since the VSreduction was only about 10% lower than for the controldigesters, methanogen inhibition likely was the domi-nant contributor to reduced methane production. Ko-ster and Lettinga (1984) provided data showing thatacetogenic methanogens, which produce 70% of the to-tal methane, were inhibited more than the CO2-utilizingmethanogens.

3.4. Hydrogen production

H2 normally is present in digester biogas, with re-searchers reporting normal concentrations ranging from60 to 200 ppm (Harper and Pohland, 1986). Hydrogendata for the control digesters (Fig. 5) shows that therewas little variation in the biogas H2 concentration. Theaddition of urea resulted in a series of changes in biogasH2 concentration for all experimental groups (Fig. 5).For all the experimental groups, the H2 concentrationinitially decreased after the addition of urea. Thesereductions partially resulted from the increased pH,which shifted the equilibrium of the digester system.These reductions also resulted from the inhibition of

Fig. 4. Methane production in digesters after urea addition (j, con-

trol; d, 600 mg N/l; r, 1500 mg N/l; N, 3000 mg N/l).

Fig. 5. Hydrogen concentrations in biogas after urea addition ((a)

control; (b) 600 mg N/l; (c) 1500 mg N/l; (d) 3000 mg N/l added).

Fig. 3. Biogas production in digesters after urea addition (j, control;

d, 600 mg N/l; r, 1500 mg N/l; N, 3000 mg N/l).


hydrolytic and acetogenic bacteria due to increasedammonia concentrations in the digesters.

For the 3000 mg N/l digester group, the excess am-monia resulted in limiting the H2 concentrations to be-low 20% of the control values for the entire experiment.For the 600 and 1500 mg N/l digesters, the biogas H2

concentrations increased after approximately 24 h. At36 h, these increases resulted in peak concentrations of230 and 150 ppm for the 600 and 1500 mg N/l digestergroups, respectively. This indicated that the methano-gens were inhibited more at this time than the hydrolyticand acid-forming bacteria. After these peaks, the H2

concentrations for these digester groups decreased overthe course of the experiment, consistent with the batchoperating mode.

3.5. E�ect on pH

The pattern of pH changes exhibited by the controlgroup was typical of a digester undergoing stable oper-ation (Fig. 6). After feeding, digester pH decreased dueto increased VFA production. However, as VFAs weremetabolized, the pH increased to its normal operatingvalue.

The addition of urea caused an increase in pH in allexperimental groups (Fig. 6). During the ®rst 12 h, di-gester pH values increased proportionally with theamount of urea added. After this period, the rise in pHvalues was signi®cant though small in the 600, 1500 and3000 mg N/l digesters while no signi®cant increase wasobserved in the controls.

As expected, the ®nal pH values for each of thedigesters increased in proportion to the amount ofurea added. The ®nal pH values were 8.2, 8.5, and 9.0for the 600, 1500, and 3000 mg N/l digesters, respec-tively (Fig. 6). These pH values were below the pHlimit of the ammonia bu�er (9.4) (Georgacakis et al.,1982).

3.6. Alkalinity

The control digesters exhibited an expected alkalinitycycle, with the addition of feed causing alkalinity todecrease immediately as VFAs were formed from solu-ble substrates in the manure slurry (Fig. 7). As the newlyformed VFAs were slowly metabolized and removedfrom the digester liquor, the digester alkalinity began toreturn to its stable operating value.

The addition of urea resulted in increased digesteralkalinity due to the increased ammonium ion concen-tration (Kroeker et al., 1979; Georgacakis et al., 1982).While all groups had similar initial alkalinity values(2500±3000 mg CaCO3/l), the initial sample after ureaaddition (3 h) indicated that the alkalinity values for theexperimental digesters remained signi®cantly higherthan for the controls. Between 6 and 24 h, the alkalinityof the controls increased to its original value. However,during that same period, the increase in alkalinity forthe experimental digesters was 3±4 times that of thecontrols. In addition, there were no signi®cant di�er-ences in alkalinity increase among the experimental di-gesters during this period. After 24 h, the alkalinityincreases for the 600 and 1500 mg N/l experimental di-gesters were not signi®cantly di�erent. However, theincrease for each of these was signi®cantly higherthan for the controls and signi®cantly lower than for the3000 mg N/l digesters. Thus, at the end of the experiment,the overall increase in alkalinity was not signi®cantlydi�erent between the 600 and 1500 mg N/l digesters.

Providing excess ammonia contributed to the in-creased alkalinity of the experimental digesters in twoways. As shown in Eqs. (5)±(7), one e�ect of addingammonia was increased bicarbonate concentration inthe digesters through the formation of an ammoniumsalt with bicarbonate taken from dissolved CO2

(Georgacakis et al., 1982).

NH�4 �OHÿNH3 �H2O �5�

Fig. 6. pH in digesters after urea addition (j, control; d, 600 mg N/l;

r, 1500 mg N/l; N, 3000 mg N/l).

Fig. 7. Total alkalinity in digesters after urea addition (j, control; d,

600 mg N/l; r, 1500 mg N/l; N, 3000 mg N/l).


CO2 �H2OH� �HCOÿ3 �6�

NH�4 �OHÿ �H� �HCOÿ3

�NH�4 �HCOÿ3 �salt �H2O �7�Another e�ect of high ammonia concentrations is inhi-bition of both the hydrolytic and acetogenic groups ofbacteria (Hill and Bolte, 1987; Angelidaki et al., 1993),thereby reducing VFA concentrations in the digesters.Lower VFA concentrations would decrease the amountof alkalinity used for acid nutralization in the digesters.

3.7. VS reduction

To analyze changes in manure VS reduction after theaddition of urea, the following assumptions were used indetermining material balances: only urea and ammoniawere measured by the colorimetric total nitrogen anal-ysis, ammonia present in the samples volatilized duringsample drying, and urea present in the samples remainedduring drying and volatilized during ashing. Based onthese assumptions, the following material balanceequations were obtained:

�Urea±N� � �Tot-N� ÿ �NH3±N� �8�

TS�manure� � TS�measured� ÿ 2:15� �Urea±N� �9�

VS�manure� � VS�measured� ÿ 2:15� �Urea±N� �10�where TS�measured� is the experimentally determined con-centration of total solids (mg/l),

TS�manure� is the concentration of total solids due tomanure (mg/l), VS�measured� the experimentally deter-mined concentration of volatile solids (mg/l), VS�manure�the concentration of volatile solids due to manure (mg/l),[Tot-N] the total nitrogen concentration determinedby colorimetry (mg/l), and [NH3±N] was the total am-monia concentration determined by colorimetry (mg/l).A conversion factor (2.15) was included to convert ureanitrogen concentrations to urea concentrations.

Trends in manure VS reduction in the experimentaldigesters were similar to that in the control digesters(Fig. 8). VS concentration initially increased in each setof digesters by approximately 5000 mg/l, the amount offeed added at the beginning of the experiment. Duringthe experimental period, VS decreased rapidly duringthe ®rst 24 h and then more slowly for each set of di-gesters. For the control digesters, the VS reduction ratewas 90:5� 9:5 mg VS/l h. This led to a 29:6� 3:4% VSreduction during the experiment. For the 600 mg N/ldigesters, the VS reduction rate (88:3� 9:7 mg VS/L h)was not signi®cantly di�erent than for the control di-gesters. This led to a 29:4� 2:5% VS reduction. How-ever, for the 1500 and 3000 mg N/l digesters, the rate ofdecrease was lower than for the control digesters,79:4� 6:9 and 74:6� 5:1 mg VS/l h, respectively. The

overall VS reductions for these digesters were25:5� 2:5% and 23:7� 2:9%.

Based on these results, ammonia seemed to havedi�erent impacts on VS reduction by anaerobic diges-tion. At a relatively low concentration (600 mg N/l),ammonia did not a�ect VS reduction and may haveserved as an easily accessible source of nitrogen. How-ever, higher VS concentrations in the digesters withmore urea added suggest that the digester hydrolyticbacteria were inhibited by higher ammonia concentra-tions. In a similar experiment, Angelidaki et al. (1993)proposed that one-half of the loss in methane yield wasattributable to inhibition of the hydrolytic bacteria.However, no mechanism for this inhibition was pre-sented. In a study of anaerobic ruminant bacteria, Rickeand Schaefer (1996) noted that non-limiting growthconcentrations of NH3±N resulted in lower fermenta-tion product formation, increased lactate formation,and less acetate and propionate reduction.

3.8. Fiber composition

An analysis of ®ber composition in the biosolidspresent in the digester e�uent was performed to exam-ine whether any particular group of hydrolytic bacteriawas more impacted by increased ammonia concentra-tions. Biosolid samples taken at 3, 6, 9, 12, and 24 hwere combined and analyzed using ®ber reductionanalysis (Van Soest and Wine, 1967, 1968; Van Soest,1963). These samples were compared to samples takenduring the stable operating period, one week beforeexperimentation. Changes in ®ber composition for thevarious digester groups are shown in Table 3. Weightpercentages for cellulose and hemicellulose were deter-mined analytically, with the remainder of solids cate-gorized as lignin and ash. Pairwise t-tests showed thatthere were signi®cant decreases in the cellulose concen-trations of all digesters after urea addition, while therewere no signi®cant decreases in hemicellulose and ligninconcentrations. However, because the control digesters

Fig. 8. Manure VS concentrations in digesters after urea addition (j,

control; d, 600 mg N/l; r, 1500 mg N/l; N, 3000 mg N/l).


behaved the same as the experimental digesters, it ap-pears that ammonia inhibition was not the cause of thedecrease in cellulose concentrations.

4. Conclusions

The amount of ammonia nitrogen in the digester feedimpacted digester hydrogen production, methane pro-duction, and volatile solids removal. Small increases inammonia nitrogen resulted in increased H2 and methaneproduction. Larger increases caused the inhibition of H2

and methane production. Total biogas production wasuna�ected by small increases in ammonia nitrogen whilehigher increases reduced biogas production to 50% ofthe original rate.

Addition of urea led to nearly immediate increases inpH and alkalinity and a somewhat slower increase infree ammonia concentration. Throughout the experi-ment, the free ammonia concentration remained belowinhibitory levels in the 600 mg N/l digesters but reachedinhibitory levels within 12 h in the 1500 and 3000 mg N/ldigesters.

E�uent VS concentrations were increased by the twolargest increases in ammonia nitrogen concentrationwhile the smallest increase resulted in no signi®cant in-crease in e�uent VS concentration relative to the con-trol digesters.

References

Angelidaki, I., Ahring, B.K., 1993. Thermophilic anaerobic digestion

of livestock waste: the e�ect of ammonia. Appl. Micro. Biotech. 38,

560±564.

Angelidaki, I., Ellegaard, L., Ahring, B.K., 1993. A mathematical

model for dynamic simulation of anaerobic digestion of complex

substrates: focusing on ammonia inhibition. Biotech. Bioeng. 42,

159±166.

APHA, 1991. Standard Method for the Examinations of Water and

Wastewater. American Public Health Association, Washington,

DC.

Archer, D.B., Hilton, M.G., Adams, P., Wiecko, H., 1986. Hydrogen

as a process control index in a pilot scale anaerobic digester.

Biotech. Lett. 8, 197±202.

Archer, D.B., Kirsop, B.H., 1991. The microbiology and control of

anaerobic digestion. In: Wheatley, A. (Ed.), Anaerobic Digestion:

A Waste Treatment Technology, vol. 31. Elsevier, New York, 43±

91.

Bartlett, K., Dobson, J.V., Eastham, E., 1980. A new method for the

detection of hydrogen in breath and its application to acquired and

inborn sugar malabsorption. Clin. Chim. Acta 108, 189±194.

Boekhurst, R.H., Ogilvie, J.R., Pos, J., 1981. An overview of current

simulation models for an anaerobic digester. In: Livestock Waste:

A Renewable Resource. American Society of Agricultural Engi-

neers, St. Joseph, MI, pp. 105±108.

Braun, R., Huber, P., Meyrath, J., 1981. Ammonia toxicity in liquid

piggery manure digestion. Biotech. Lett. 3, 159±164.

De Baere, L.A., Devocht, M., Van Assche, P., Verstraete, W., 1984.

In¯uence of high NaCl and NH4Cl salt levels on methanogenic

associations. Wat. Res. 18, 543±548.

El-Hag, M.G., Vetter, R.L., Kenealy, M.D., 1982. E�ects of silage

additives on fermentation characteristics of corn silage and

performance of feedlot heifers. J. Dairy Sci. 65 (2), 259±266.

Georgacakis, D., Sievers, D.M., Iannotti, E.L., 1982. Bu�er stability in

manure digesters. Agri. Wastes 4, 427±441.

Harper, S.R., Pohland, F.G., 1986. Recent developments in hydrogen

management during anaerobic biological wastewater treatment.

Biotech. Bioeng. 28, 585±602.

Hickey, R.F., Vanderwielen, J., Switzenbaum, M.S., 1987. The e�ects

of organic toxicants on methane production and hydrogen gas

levels during the anaerobic digestion of waste activated sludge.

Wat. Res. 21 (11), 1417±1427.

Hickey, R.F., Vanderwielen, J., Switzenbaum, M.S., 1989. The e�ect

of heavy metals on methane production and hydrogen and carbon

monoxide levels during batch anaerobic sludge digestion. Wat. Res.

23 (2), 207±218.

Hill, D.T., Bolte, J.P., 1987. Modeling fatty acid relationships in

animal waste anaerobic digesters. Trans. ASAE 30 (2), 502±508.

Hungate, R.E., 1975. The rumen microbial ecosystem. Ann. Review

Ecol. Syst. 6, 39±66.

Jeris, J.S., Kugelman, I.J., 1985. Secrets to the success of anaerobic

digestion. Wat./Eng. Man. 132, 32±35.

Kidby, D.W., Nedwell, D.B., 1991. An investigation into the

suitability of biogas hydrogen concentration as a performance

monitor for anaerobic sewage sludge digestion. Wat. Res. 25 (8),

1007±1012.

Koster, I.W., Lettinga, G., 1984. The in¯uence of ammonium±nitrogen

on the speci®c activity of pelletized methanogenic sludge. Agri.

Wastes 9, 205±216.

Kroeker, E.J., Schulte, D.D., Sparling, A.B., Lapp, H.M., 1979.

Anaerobic treatment process stability. J. WPCF 51 (4), 718±727.

Loehr, R.C., 1984. Anaerobic treatment. Pollution Control for

Agriculture. Academic Press, Orlando, FL.

McCarty, P.L., McKinney, R.E., 1961. Volatile acid toxicity anaerobic

digestion. J. WPCF 33 (3), 223±232.

Milton, J.S., Arnold, J.C., 1990. Introduction to Probability and

Statistics: Principles and Applications for Engineering and the

Computing Sciences, second ed. McGraw-Hill series in probability

and statistics, New York, pp. 480±484.

Table 3

Fiber content of digester e�uent before and after urea additiona

Control 600 mg N/l 1500 mg N/l 3000 mg N/l

Cellulose (%) Before 12.11 � 2.36b 11.27 � 2.46 13.12 � 1.51 11.64 � 1.94

After 5.96 � 0.66 10.28 � 1.97 9.37 � 2.29 7.96 � 2.20

Hemicellulose (%) Before 12.06 � 1.89 11.55 � 1.21 8.38 � 1.23 11.61 � 2.62

After 13.16 � 2.13 12.24 � 1.45 8.39 � 1.63 11.89 � 1.65

Lignin�Ash (%) Before 75.84 � 4.25 77.19 � 3.67 78.5 � 2.74 76.76 � 4.56

After 80.88 � 2.79 77.49 � 3.42 82.25 � 3.92 80.16 � 3.85

a n � 6 for control, n � 4 for other experimental groups.b Standard deviation of the mean.


Mosey, F.E., 1982. New developments in the anaerobic treatment of

industrial wastes. Water Poll. Cont. 81, 540±550.

Mosey, F.E., Fernandes, X.A., 1984. Mathematical modelling of

methanogenesis in sewage sludge digestion. In: Grainger, J.M.,

Lynch, J.M. (Eds.), Microbiological Methods for Environmental

Biotechnology. Academic Press, London, pp. 159±168.

Ricke, S.C., Schaefer, D.M., 1996. Nitrogen-limited growth response

of Selenomonas ruminantium to limiting and non-limiting concen-

trations of ammonium chloride. Appl. Microbiol. Biotechnol. 46,

169±175.

Robinson, J.A., Tiedje, J.M., 1982. Kinetics of hydrogen consumption

by rumen ¯uid, anaerobic digester sludge and sediment. Appl.

Environ. Microbiol. 44, 1374±1384.

Robinson, J.A., Tiedje, J.M., 1984. Competition between sulphate-

reducing bacteria and methanogenic bacteria for H2 under resting

and growing conditions. Arch. Microbiol. 137, 26±32.

Sprott, G.D., Shaw, K.M., Jarrell, K.F., 1984. Ammonia/potassium

exchange in methanogenic bacteria. J. Biol. Chem 259, 12602±

12608.

Van Soest, P.J., 1963. Use of detergents in the analysis of ®brous feeds.

II. A rapid method for the determination of ®ber and lignin.

J. Assoc. O�cial Anal. Chem. 46 (5), 829±846.

Van Soest, P.J., Wine, R.H., 1967. Use of detergents in the analysis of

®brous feeds. IV. The determination of plant cell wall constituents.

J. Assoc. O�cial Agr. Chem. 50, 50±62.

Van Soest, P.J., Wine, R.H., 1968. Determination of lignin and

cellulose in acid-detergent ®ber with permanganate. J. Assoc.

O�cial Agr. Chem. 51, 780±792.

Vartak, D.R., Engler, C.R., McFarland, M.J., Ricke, S.C., 1997.

Attached-®lm media performance in psychrophilic anaerobic treat-

ment of dairy cattle wastewater. Bioresource Technol. 62, 79±84.

Wolin, M.J., 1976. Interactions between H2-producing and methane-

producing species. In: Schlegel, G., Gottschalk, G., Pfenning, A.N.

(Eds.), Microbial Production and Utilization of Gases (H2, CH4,

CO). Goltze KG, Gottingen, pp. 141±150.

Wolin, M.J., 1982. Hydrogen transfer in microbial communities. In:

Bull, A.T., Slater, J.H. (Eds.), Microbial Interactions and Com-

munities. Academic Press, London, pp. 323±356.


effects of ammonia nitrogen on h2 and ch4 production during anaerobic digestion of dairy cattle...

Documents