ENVIRONMENTAL ENGINEERING SCIENCEVolume 25, Number 10, 2008© Mary Ann Liebert, Inc.DOI: 10.1089/ees.2007.0282

Ammonia Inhibition of Methanogenesis and Identification of ProcessIndicators during Anaerobic Digestion

Ryoh Nakakubo,1,2,* Henrik B. Møller,2 Anders M. Nielsen,2 and Juzo Matsuda3

1Graduate School of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.2Institute of Agricultural Engineering, Aarhus University Faculty of Agricultural Sciences,

Research Center Bygholm, 8700 Horsens, Denmark.3Hokkaido University, Sapporo 060-8589, Japan.

Received: October 19, 2007 Accepted in revised form: January 11, 2008

Abstract

In continuously stirred tank reactor experiments using pig manure codigested with solid fractions separatedfrom pig manure (a mass mixture ratio of 40:60) with an organic loading rate of 9.4 gVS/ldigester/day at a ther-mophilic temperature of 51°C, we investigated acute inhibitory effects of ammonia on methanogenesis by us-ing pulses of ammonium chloride, and assessed whether volatile fatty acids (VFAs) could be used as processindicators. Codigestion with the solid fractions seemed to result in a slight ammonia inhibition effect, thoughthe digestion process was stable and the methanogens were adapted to a high ammonia concentration. Wefound a strong negative correlation (R2 � 0.91) between total ammonia concentration and methane gas yield.However, free ammonia concentration did not reflect the acute ammonia inhibition significantly. Compared toa control reactor which was at an ammonia concentration of 4.6 gNH4–N/l, without pulsing ammonium chlo-ride, a 50% decrease in methane gas yield in terms of loaded volatile solids was observed at an ammonia con-centration of 11.0 gNH4–N/l (1.45 gNH3–N/l). Acetic acid and propionic acid, the two VFAs produced mostabundantly during digestion of animal manure, did not accumulate with increased ammonia concentration;thus total VFA concentration was not a clear indicator of ammonia inhibition. However, we found that usingisobutyric, butyric, and isovaleric acids individually as process indicators was useful. Isobutyric acid, in par-ticular, accumulated initially by ammonia inhibition even before the yield of methane gas was affected.

Key words: volatile fatty acids; methane; codigestion; ammonium chloride; methanogens; inhibition; processindicators

1487

Introduction

CENTRALIZED BIOGAS plants in Denmark codigest mainlymanure, together with other organic waste products

such as industrial organic waste, source-sorted householdwaste, and sewage sludge. Currently, 22 large-scale central-ized biogas plants are in operation throughout Denmark,treating approximately 1.2 million tons of manure togetherwith approximately 300,000 tons of organic industrial waste(Angelidaki and Ellegaard, 2003). The biogas productionfrom manure (in terms of yield per volume) is low, and thebiogas plants are dependent on easily degradable wasteproducts to make them economically viable. However, sup-ply of these products is limited. Therefore the planning andbuilding of new centralized biogas plants is at a standstill atpresent.

Due to the low concentration of total solids in manure(typically 5–7% total solids in pig manure and 7–9% in ma-nure from cattle and dairy cows (Angelidaki and Ellegaard,2003)), the transport of large quantities of manure from farmsto biogas plants represents a very significant proportion ofthe operating costs for codigestion plants and is one of thereasons for the poor economic performance of biogas plantsin Denmark. In the development of more efficient treatmentprocesses, a number of different implementation techniquesfor solid–liquid separation need to be considered. In Den-mark, the most obvious application would be to replace someof the non-pretreated manure with the solid manure fractionobtained from a separation process. Utilization of the highlyconcentrated solid fraction produced by manure separationwould significantly improve the economic feasibility of codi-gestion plants because of the resulting lower transport costsper m3 of methane production and higher daily methane pro-duction per m3 of digester volume compared with untreatedmanure (Møller et al., 2004). In contrast with normal ammo-nia concentration of centralized biogas plants which is be-low 4 gNH4–N/l, however, codigestion of the solid fractions

*Corresponding author:Institute of Agricultural Engineering,Aarhus University Faculty of Agricultural Sciences, Research Cen-ter Bygholm, Schuttesvej 17, 8700 Horsens, Denmark. Phone: �4589991900; Fax: �45 89993100; E-mail: ryoh.nakakubo@agrsci.dk

produced by manure separation has been found to result ina high ammonia concentration of 5.4 gNH4–N/l (Møller etal., 2007) in digesting slurry because of the high-ammonium-N content of the solid fractions (Møller et al., 2004). Further-more, actual centralized biogas plants are designed to digestvarious kinds of proteinaceous cosubstrates together withthe solid fractions. Therefore, it is important to consider theinhibitory action of ammonia on methanogenesis, becausedigestion with proteinaceous cosubstrates increases the am-monia concentration of digesting slurry (Braun et al., 2003).However, the inhibitory ammonia concentration resultingfrom the codigestion of pig manure with solid fractions hasnot yet been investigated.

Ammonia is a necessary nutrient for the growth of bacte-ria involved in the anaerobic digestion process (McCarty,1964); several studies have shown that high ammonia con-centration may inhibit methanogenesis. McCarty (1964) in-dicated that anaerobic digestion processes are inhibited atany pH when ammonia concentration exceeds 3 gNH4–N/l.Another study reported that an ammonia concentration of2.5 gNH4–N/l resulted in some inhibition of methane pro-duction, while a concentration of 3.3 gNH4–N/l inhibitedmethanogenesis completely (Hobson and Shaw, 1976). Theadaptation of methanogenic bacteria to high ammonia con-centration improves their tolerance to inhibitory level of am-monia. Acclimated thermophilic digestion could enablemethanogens to tolerate up to concentrations of 8–13gNH4–N/l, depending on acclimation conditions and sys-tem pH (Sung and Liu, 2003). Also it was found that a con-centration of 5.77 gNH4–N/l caused a drop in methane pro-duction by as much as 64% compared to a control (Sung andLiu, 2003). However, it should be kept in mind that any po-tential inhibition by ammonia should not only be related di-rectly to the total ammonium concentration but to the con-centration of free ammonia, which has been suggested to bethe active component during inhibition (Hashimoto, 1986;Koster and Lettinga, 1984). The free ammonia concentrationdepends on the total ammonia concentration, temperature,and pH of digesting slurry (The equation is shown in Mate-rials and Methods.). It has been reported that a free ammo-nia concentration of 0.69 gNH3–N/l caused 50% inhibitionof methanogenesis under thermophilic conditions (Gallertand Winter, 1997). Another study on cattle manure at ther-mophilic temperature indicated that a free ammonia con-centration above 0.7 gNH3–N/l resulted in a poor treatmentperformance at pH values of 7.4–7.9 (Angelidaki and Ahring,1994).

Many research reports have suggested that concentrationsof individual volatile fatty acids (VFAs) are useful indicatorswhich can be correlated with process stability in the reactor.Ahring et al. (1995) suggested that a combined parameter re-flecting the concentrations of both butyrate and isobutyratecould be a reliable tool for indicating process instability. Var-ious associations between the actual concentrations of indi-vidual VFAs and process imbalance have also been sug-gested. Hill et al. (1987) reported that an acetate concentrationhigher than 13 mM would indicate process imbalance, andHill and Holmberg (1988) concluded that concentrations ofisobutyrate and isovalerate higher than 0.06 mM could in-dicate process instability. Propionate has also been suggestedto be a good indicator of process instability (Kasper andWuhrmann, 1978; Varel et al., 1977). However, some reports

have showed stable reactor performance occurring at VFAconcentrations well above these limits (Angelidaki andAhring, 1994; Ahring et al., 2001; Nielsen et al., 2004). Thusit can be concluded that it is not feasible to define an ab-solute VFA concentration, which indicates process inhibi-tion.

The purpose of this paper was to investigate the codiges-tion of pig manure with solid fractions separated from ma-nure, in order to discover both the acute inhibitory ammo-nia concentration capable of inhibiting methanogensadapted to high ammonia concentration and also to identifyuseful parameters that indicate process imbalances. In termsof biogas plant economic efficiency, this research was nec-essary to prevent ammonia inhibition from causing insuffi-cient amounts of methane gas. We induced the acute am-monia inhibition by pulsing with NH4Cl to laboratory-scalecompletely stirred tank reactor systems to simulate the sharpincrease in ammonia concentration that occurs in actual cen-tralized biogas plants when proteinaceous cosubstrates arefed to the reactors.

Materials and Methods

Completely stirred tank reactor systems

We used six anaerobic reactors (R1, R2, R3, R4, R5, andR6), each having a working volume of 2 liters. The reactorswere maintained at a thermophilic temperature of 51°C,which approximates the temperature of centralized biogasplants in Denmark of 51–55°C. Hydraulic retention time(HRT) was 13.3 days. Biogas was collected using aluminum-coated gas packs.

Substrates

The properties of the substrates and inoculum are shownin Table 1. The pig manure, approximately 14 days old, wascollected in a reception pit on a farm that specializes in pro-ducing fattening pigs. The solid fraction was collected on afarm where pig manure was separated by flocculation witha polymer (acrylamide) and dewatered using a screw press.The manure separator was produced by the Danish companyKemira Miljø. The pig manure was codigested with the solidfractions in a mass mixture ratio of 40:60 (40% pig manure,60% solid fractions). Organic loading rates of the reactorswere 9.4 gVS/ldigester/day. NH4Cl was chosen as the am-monia source in order to minimize the pH effect of ammo-nia addition. The inoculum was obtained from the reactorwhich codigested the pig manure with the solid fractions un-der stable conditions for 6 months in a mass mixture ratio of40:60 (Møller et al., 2007). The inoculum was already adaptedto high ammonia concentrations up to 5.4 gNH4–N/l.

NAKAKUBO ET AL.1488

TABLE 1. PROPERTIES OF SUBSTRATES AND INOCULUM

TS VS T-N NH4-N(%) (%) (mg/l) (g/l)

Pig manure 5.9 4.6 5.2 4.0Solid fractions 22.1 17.9 6.7 4.4Mixed material* 15.6 12.6 6.1 4.2Inoculum 12.7 9.5 6.1 4.8

*Mixture of 40% pig manure and 60% solid fractions.

Experimental methods

All reactors were fed raw material (pig manure and solidfractions) once a day after removing the same amount of di-gested slurry from the digester. We added various amountsof NH4Cl through the reactors to study the acute inhibitionprocess caused by high ammonia concentration in the di-gestate. Table 2 shows the NH4Cl pulsing rates for each re-actor. R1, which was the control reactor, was fed only rawmaterial without NH4Cl pulsing. Reactors R2–R6 werepulsed with NH4Cl together with the addition of raw mate-rial. At each pulse, the NH4Cl pulsing rate of each reactorwas increased (see Table 2). The study was conducted overa series of five pulses, with a stepwise increase of the NH4Clpulsing rate. Since we conducted this experiment to investi-gate the acute ammonia inhibition in actual centralized bio-gas plants, larger amounts of NH4Cl were re-pulsed soon af-ter observing methanogenic tolerance at each phase. Afterthe fifth pulse of NH4Cl, we continued the experiment with-out feeding in any more raw material for 24 days to observevariations in VFA concentrations.

Analytical methods

Standard procedures (DEV, 1979) were used to determinetotal solids (TS), volatile solids (VS), total Kjeldahl nitrogen(T–N), and ammonia (NH4–N). Slurry pH was determinedwith a pH meter (Radiometer A/s, Copenhagen, Denmark).Biogas production was measured by using a large syringe,as described by Steed and Hashimoto (Steed and Hashimoto,1994). The gas samples were analyzed for CO2 and CH4 con-tent using gas chromatography. Both CO2 and CH4 weremeasured on a Perkin Elmer Clarus 500 Gas Chromatographequipped with an electron capture detector (ECD) and a spe-cial Alltech CTR column. The carrier gas was He, and thetemperatures of injection port, oven, and detector were110°C, 40°C, and 150°C, respectively. Volatile fatty acid(VFA) C2–C5 concentrations were determined by means of agas chromatograph (Hewlett Packard 6850A) with a flameionization detector (FID). The column was an HP-INOWax,30 m � 0.25 mm � 0.25 �m. The carrier gas was He. The tem-perature of the column was gradually increased from 110°Cto 220°C at a rate of 10°C min�1.

Methane generation ratio (Bratio(day)) is defined as the dailymethane gas production of each reactor inhibited by NH4Clpulses, as a proportion of the daily methane gas productionof reactor R1 (no NH4Cl pulsing):

Bradio(day) � (1)BInhibitedReactor(day)���

BControl(day)

where BControl(day) is the daily methane gas production of R1.BInhibitedReactor(day) is the daily methane gas production of re-actors R2–R6 (pulsed with NH4Cl). The organic loading ratesof the reactors were equal (9.4 gVS/ldigester/day). Thereforelow methane generation ratio indicates methane gas de-creases of reactors inhibited by ammonia.

VFA inhibition ratio (VFAInhibitionRatio(day)) is defined asthe daily individual VFA concentration inhibited by NH4Clpulses, as a proportion of the daily individual VFA concen-tration of reactor R1 (no NH4Cl pulsing):

VFAInhibitionRatio(day) � (2)

where VFAControl(day) is the daily individual VFA concentra-tion of reactor R1. VFAInhibitedReactor(day) is the daily individ-ual VFA concentration of reactors R2–R6.

VFA fluctuation ratio (VFAFluctuationRatio(day)) is defined asa daily fluctuation ratio of each individual VFA concentra-tion in reactor R1, which is the control reactor, against theaverage of each individual VFA concentration of reactor R1:

VFAFluctuationRatio(day)

� (3)

where VFAControl(day) is the daily individual VFA concentra-tion of reactor R1. VFAControlAverage(day) is the average indi-vidual VFA concentration of reactor R1 from the start of theexperiment to the day of measurement. The VFA fluctuationratio indicates fluctuation sensitivity of individual VFA un-der the stable digestion process without the ammonia inhi-bition.

Concentration of free ammonia (NH3–N) was calculatedaccording to Anthonisen et al. (1976):

NH3 � N � ; � exp� � T�We used the methane generation ratio and the VFA inhi-

bition ratio to cancel the fluctuations of the methane gas pro-ductions and the VFA concentrations, which were caused bytransition to new digesting conditions of the reactors afterthis experiment started, to analyze the ammonia inhibitionon these parameters plainly.

Results and Discussion

Codigestion with solid fractions

The methane gas yield of R1 gradually increased until day10, which was probably due to the transition to new digest-ing conditions. Average methane gas yield of control reac-tor R1, which was fed only raw material with no pulsing,was 0.19 l/gVS at steady state (day 11–18). Møller et al. (2004)reported that the ultimate methane yield of fattening pig ma-nure was 0.36 l/gVS on average and that of solid fractionsseparated from pig manure was 0.25 l/gVS on average,which were obtained from a mesophilic batch experimentrunning for 100 days. Using these data, the ultimate methaneyield of R1 is estimated to be about 0.29 l/gVS. Thus, in thisexperiment, about 66% of the ultimate methane gas yield wasobtained in the control reactor at an ammonia concentrationof 4.6 g/l. Møller et al. (2007) reported that the methane yieldof thermophilic methane digestion of fattening pig manure

6344�273

Kb�Kw

(NH4 � N) � 10pH

���

�KK

w

b� � 10pH

VFAControl(day) � VFAControlAverage(day)�����

VFAControlAverage(day)

VFAInhibitedReactor(day)���

VFAControl(day)

AMMONIA INHIBITION OF METHANOGENESIS 1489

TABLE 2. NH4CL PULSING RATES

NH4Cl pulsing rates (g/l)Day 1 7 13 15 18

R1 0.0 0.0 0.0 0.0 0.0R2 1.2 2.3 6.9 7.0 24.0R3 1.9 3.3 7.5 9.8 24.5R4 3.8 4.3 7.7 9.1 28.8R5 5.8 5.4 7.4 14.1 28.4R6 7.7 5.0 9.3 19.1 27.7

with an average HRT of 23 days was 0.32 l/gVS, which was88% of the ultimate methane yield. Thus the yield of 66% ofthe ultimate methane yield is in the lower end of what is nor-mally achieved in a CSTR. The codigestion with the solidfractions seemed to result in a reduced yield due to the shortretention time, the ammonia inhibition, or the combined ef-fect, though the digestion process was stable and themethanogens were adapted to the high ammonia concen-tration.

However, R1 produced 4.6 l/l of methane gas productionper digester volume during 5 days of no feeding (no-feed-ing period) on and after day 20. This value is equal to 0.054l/gVS. Thus codigestion with solid fractions prolonged un-til a hydraulic retention time (HRT) of 18.3 days (13.3 daysof HRT � 5 days) can be estimated to produce 0.24 l/gVS(0.19 l/gVS � 0.054 l/gVS), which is about 83% of the ulti-mate methane gas yield. In addition, methane gas produc-tion of the codigestion per digester volume was 1.8 l/l/dayat steady state, which was about 150% of the methane gasproduction of only pig manure reported as 1.2 l/l in the samedigestion conditions (Møller et al., 2007). Therefore the codi-gestion of the solid fractions seemed to improve the eco-nomic feasibility of biogas plants significantly.

Inhibitory ammonia concentration

The overall performance of the reactors at each pulse ofNH4Cl is summarized in Table 3. After five NH4Cl pulses,the ammonia concentration in each reactor gradually in-

creased from 4.8 gNH4–N/l to a maximum value of 15.9gNH4–N/l, which was found in R6 (see Fig. 1). By increas-ing the amounts of the NH4Cl pulses, methane gas yields interms of loaded VS were found to decrease compared withthe control reactor R1 (see Fig. 1). Nevertheless, methane con-centration in the biogas was above 60% even after the fifthpulses of NH4Cl, which led to an apparent decrease in themethane gas yield. Generally ammonia inhibitions result inaccumulation of total VFA. But in this study, total VFA con-centrations in each reactor pulsed with NH4Cl did not varyclearly compared with R1 (see Table 3). These results dif-fered from those from previous research. Several articleshave reported that accumulation of ammonia resulted in alow methane gas concentration in the biogas and a high to-tal VFA concentration. Hashimoto (1986) reported that 7.74kgNH4–N /m3 caused by the NH4Cl pulse resulted in alower methane concentration of 27.7% and a higher totalVFA concentration of 5.96 kg/m3, compared with the con-trol reactor’s values of 56.6% and 0.92 kg/m3, respectively.Angelidaki and Ahring (1992) reported that total VFA con-centration increased from 1 to 3 g/l with an ammonia con-centration of 6 gNH4-N/l, compared to controls with a con-centration of 2.5 gNH4–N/l. Fermentative bacteria are lesssensitive than methanogens to ammonia inhibition. There-fore VFAs converted by fermentative bacteria accumulatewhen methanogens suffer from ammonia inhibition and nolonger convert VFAs to methane gas.

In this study, we used inoculum adapted to high ammo-nia concentration. Thus the methanogens had probably ac-

NAKAKUBO ET AL.1490

TABLE 3. OVERALL PERFORMANCE OF THE REACTORS AT EACH PULSE OF NH4CL

1st pulse 2nd pulse 3rd pulse 4th pulse 5th pulse No feeding*1 (day) 7 (day) 13 (day) 15 (day) 18 (day) 42 (day)

CH4 R1 45.4 62.5 63.9 64.6 65.0 71.8conc. R2 40.9 62.3 62.9 63.4 64.3 72.3(%) R3 46.7 61.7 62.5 63.0 64.1 71.1

R4 37.2 61.3 62.0 62.8 64.2 69.5R5 38.4 60.9 61.3 60.9 62.9 71.1R6 41.5 61.5 61.1 60.0 61.2 63.5

Total R1 7460 8090 6300 6620 6470 6070VFA R2 7390 7730 6180 6750 6360 5670(mg/l) R3 7140 7930 6480 6940 6700 5900

R4 7590 8030 6880 6950 6910 6610R5 7600 7780 7680 6600 6780 6630R6 7600 7390 6550 6660 6890 6990

VS R1 9.5 9.1 9.1 8.7 8.4(%) R2 9.0 9.5 9.2 9.6 10.3

R3 9.6 8.9 9.5 10.1 10.7R4 9.7 9.6 9.8 10.3 11.2R5 9.7 9.8 9.9 10.6 11.3R6 9.8 10.1 10.2 11.1 11.7

pH R1 8.07 7.69 7.78 7.85 7.98 7.94R2 8.00 7.63 7.61 7.73 7.69 7.91R3 7.99 7.69 7.60 7.72 7.68 7.96R4 8.02 7.63 7.62 7.68 7.64 8.08R5 8.01 7.60 7.62 7.68 7.63 8.05R6 7.96 7.66 7.65 7.68 7.63 8.03

*The last day of the experiment after feeding stop.

quired tolerance as strong as that of fermentative bacteria.Therefore the methanogens could convert VFAs intomethane gas without the accumulation of VFA as long as thefermentative bacteria produced VFAs without ammonia in-

hibition, which also resulted in keeping a high methane gasconcentration even after methane gas yield decreased. Thisagrees with the research of Nielsen and Ahring (2007), whoreported that in thermophilic reactors, pulses of ammonia

AMMONIA INHIBITION OF METHANOGENESIS 1491

FIG. 1. Ammonia concentration and methane gas yield of reactors pulsed NH4Cl. Dashed lines mark the points of NH4Clpulses. (No marker) R1, (�) R2, (-) R3, (�) R4, (�) R5, and (�) R6.

FIG. 2. Methane generation ratio corresponding to the total ammonia concentration and the free ammonia concentration.

(0.79 g l-1 as N) resulted in a decrease in methane produc-tion of both reactors but no immediate increases in VFA con-centrations were observed, illustrating that the ammonia in-hibition during this experiment was an overall inhibition ofthe biogas process and not only an inhibition of themethanogens.

Figure 2 shows methane generation ratio correspondingto the total ammonia concentration and the free ammoniaconcentration. A strong negative correlation (R2 � 0.91) oftotal ammonia concentration with methane generation ratio(Equation (1)) was obtained. However, R2 of a regression lineof the methane generation ratio corresponding to the freeammonia concentration was only 0.48. Thus, in this study,the total ammonia concentration seemed to reflect the anaer-obic digestion process more than the free ammonia concen-tration. This agrees with the research of Lay et al. (1998, 1997),who reported that methanogenic activity was dependent onthe level of total ammonia, but not free ammonia, indicatingthat the total ammonia was the more significant factor ratherthan the free ammonia in affecting the methanogenic activ-ity of a well-acclimatized bacterial system.

Figure 2 may not illustrate the inhibitory ammonia con-centration completely since we pulsed NH4Cl with time be-

tween the pulses of 2 to 6 days. However Figure 2 shows theacute inhibitory ammonia concentration in actual centralizedbiogas plants when proteinaceous cosubstrates are fed to thereactors. Fifty percent of the methane generation ratio wasobserved at an ammonia concentration of 11.0 g NH4–N/l.Also, the free ammonia concentration was 1.45 gNH3–N/lwhen 50% of the methane generation ratio was obtained.Gallert and Winter (1997) reported that a free ammonia con-centration of 0.69 gNH3–N/l caused a 50% inhibition ofmethanogenesis under thermophilic conditions. Angelidakiand Ahring (1994) also reported that free ammonia concen-tration above 0.7 gNH3-N/l resulted in a poor treatment per-formance at a pH of 7.4–7.9 on thermophilic cattle manuredigestion. In this study, the inhibitory free ammonia con-centration was higher due to the acclimated inoculum to thehigh ammonia concentration and the short-term exposure tothe NH4Cl pulses.

Process indicators

VFA dynamics following NH4Cl pulsing is shown in Fig.3. The VFAs of reactor R1, which was the control reactor, de-creased during the experiment. This was due to the transi-

NAKAKUBO ET AL.1492

FIG. 3. VFA concentration of (a) acetic acid; (b) propionic acid; (c) isobutyric acid; (d) butyric acid; (e) isovaleric acid; (f)valeric acid in feeding term. (No marker) R1, (�) R2, (�) R3, (�) R4, (�) R5, and (�) R6.

tion to new digesting conditions from the incubation condi-tions of the inoculum, whose total VFA concentration was7600 mg/l.

Acetic acid and propionic acid, which are the two mostabundant VFAs produced during the digestion of animalmanure, did not accumulate by increasing ammonia con-centration. Furthermore, after the third pulse of NH4Cl (day13), the propionic acid concentrations of the reactors werelower in order of the higher ammonia pulsed reactors. Thebacteria, which produced propionic acid, seemed to be moresensitive to ammonia than the other bacteria were.

Meanwhile, isobutyric acid, butyric acid, isovaleric acid,and valeric acid clearly indicated accumulation by ammoniainhibition. Table 4 shows the sensitivity of those four indi-vidual VFAs toward ammonia concentration on the day af-ter each of the NH4Cl pulses. R2, which was pulsed the low-est amount of NH4Cl, hardly accumulated any of these four

individual VFAs compared with the other reactors, thoughthe methane generation ratio was decreasing. Methanogensof R2 seemed to have a stronger tolerance against the highammonia concentration, which could have been obtained bya more gradual increase of NH4Cl pulses than in the otherreactors. However, methane gas production did not differbetween the reactors (see Fig. 2), since the amounts of thosefour individual VFAs were proportionally lower than theacetic and propionic acids.

The most rapid increase in VFA inhibition ratio (Equation(2)) through which the process imbalance can be indicated,was obtained from isobutyric acid (see Table 4). The isobu-tyric acid had already accumulated significantly by the sec-ond pulse of NH4Cl, at which time the methane generationratio (Equation (1)) was not yet affected. However, the isobu-tyric acid also showed a high VFA fluctuation ratio. The highVFA fluctuation ratio increases difficulty to monitor the VFA

AMMONIA INHIBITION OF METHANOGENESIS 1493

TABLE 4. SENSITIVITY OF ISOBUTYRIC, BUTYRIC, ISOVALERIC, AND VALERIC ACID TOWARD AMMONIA CONCENTRATION ON A DAY

AFTER EACH NH4CL PULSES. METHANE GENERATION RATIO AND AMMONIA CONCENTRATIONS ARE ALSO SHOWN

1st pulse 2nd pulse 3rd pulse 4th pulse 5th pulseVFA VFA VFA VFA VFA VFA VFA VFA VFA VFA

inhibition fluctuation inhibition fluctuation inhibition fluctuation inhibition fluctuation inhibition fluctuation(%) (%) (%) (%) (%) (%) (%) (%) (%) (%)

Isobutyric R2 101 96 107 120 141R3 97 127 140 161 198R4 101 138 149 160 207R5 101 0 157 39 197 74 170 67 226 57R6 101 134 202 213 278average 100 131 159 165 210

Butyric R2 66 100 102 112 338R3 80 103 133 176 471R4 80 0 110 2 147 23 176 29 520 28R5 92 115 163 199 609R6 75 108 146 345 907average 79 107 138 202 509

Isovaleric R2 101 99 98 105 104R3 98 101 102 112 115R4 102 0 104 1 110 12 113 09 118 9R5 104 103 127 111 124R6 104 99 112 116 129average 102 101 110 111 118

Valeric R2 89 103 113 124 196R3 97 106 147 188 225R4 104 0 123 8 160 20 191 20 237 21R5 114 115 179 200 284R6 104 107 156 230 313average 101 111 151 186 251

1st pulse 2nd pulse 3rd pulse 4th pulse 5th pulseBratio NH4-N Bratio NH4-N Bratio NH4-N Bratio NH4-N Bratio NH4-N(%) (g/l) (%) (g/l) (%) (g/l) (%) (g/l) (%) (g/l)

R2 84 5.2 105 5.6 89 6.6 69 7.7 53 11.6R3 97 5.4 102 5.8 89 7.0 72 8.7 47 12.7R4 83 5.9 100 6.4 81 7.5 62 8.9 34 13.8R5 90 6.4 99 6.9 77 7.7 59 10.1 30 14.6R6 89 6.6 90 7.1 70 8.2 48 11.5 24 15.9average 89 5.9 99 6.4 81 7.4 62 9.4 38 13.7

VFA inhibition: VFA inhibition ratioVFA fluctuation: VFA fluctuation ratioBratio: Methane generation ratio

FIG. 4. Methane gas production after stop feeding. Average gas production is shown during day 22 to 23, 23 to 24, 25 to26, and 27 to 42. The gas production of R1 was not measured after day 26 by accident. (No marker) R1, (�) R2, (�) R3, (�)R4, (�) R5, and (�) R6.

FIG. 5. VFA concentration of (a) acetic acid; (b) propionic acid; (c) isobutyric acid; (d) butyric acid; (e) isovaleric acid; (f)valeric acid after feeding stop. (No marker) R1, (�) R2, (�) R3, (�) R4, (�) R5, and (�) R6.

accumulation. Thus observation of a significant increase inisobutyric acid may possibly be difficult during operation ofactual centralized biogas plant. The second most rapid in-crease was from valeric acid and the third was from butyricacid, which had accumulated significantly by the third pulseof NH4Cl with a low VFA fluctuation ratio. The butyric acidshowed a considerably high inhibitory VFA ratio of 569% onan average value of R2 to R6 by the fifth pulse of NH4Cl,when a strong inhibition indicated by a more than 50% de-crease in methane gas production was observed. Isovalericacid did not show a significant VFA inhibition ratio, al-though the concentration increased as the ammonia concen-tration increased. In our investigation, we found that indi-vidually using isobutyric, butyric, and isovaleric acids asprocess indicators was useful. This is similar to the reportthat butyrate and isobutyrate together were particularlygood indicators (Ahring et al., 1995).

Figure 4 shows methane gas production per digester volumeduring the no-feeding period after the fifth pulse of NH4Cl. In-hibited reactors had not recovered methane gas productionduring 24 days of no feeding, although they had the potentialto produce more methane gas than R1 because they containedhigher amounts of volatile solids (see VS data in Table 3).

Figure 5 shows VFA concentrations during the no-feedingperiod after the fifth pulse of NH4Cl. During the no-feedingperiod, propionic acid concentrations were higher in order oflower ammonia concentrations (ammonia concentrations areshown in Table 3). This result supports the hypothesis that thebacteria, which produce propionic acid, are more sensitive toammonia than other bacteria. In contrast, other individualVFA concentrations during the no-feeding period were lowerin order of lower ammonia concentration. Considering thevariation of these individual VFAs and of methane gas pro-duction after feeding ceased, methanogens could not recovercompletely following inhibition. However, there is evidenceof degradation of acetic, butyric, isovaleric, and valeric acidafter feeding had ceased. Butyric acid concentration in R2, R3,and R4 decreased clearly on days 22, 26, and 42, respectively,in order of ammonia concentration as well as R1 (which wasthe control reactor). However, R5 and R6 had accumulatedbutyric acid strongly during the no-feeding period. In R2, R3,and R4, the microorganisms which convert butyric acidseemed to have degraded the butyric acid. On the other hand,those microorganisms seem to have been inactive in R5 andR6. Equally, microbial activity was also observed in R2 andR3 for acetic acid, isovaleric acid, and valeric acid. Thereforethe microorganisms that degrade acetic, butyric, isovaleric,and valeric acids were able to degrade each VFA up to am-monia concentrations of 12.1, 13.2, 12.1, and 12.1 gNH4–N/l,respectively. But microbial activities seem not to have resultedin methane gas production recovery because of low amountsof these VFAs. Isobutyric acis did not degrade during the no-feeding period at all. The microorganisms that degrade isobu-tyric acid seem to be completely inhibited at ammonia con-centrations below 11.6 gNH4–N/l. This characteristic mayexplain the reason for the fastest accumulation of isobutyricacid during ammonia inhibition.

Conclusion

Utilization of the highly concentrated solid fraction pro-duced by manure separation would significantly improve

the economic feasibility of codigestion plants. In a series ofcontinuously stirred tank reactors codigesting pig manure(40%) with the addition of solid fractions (60%), the increas-ing concentrations of ammonia caused by NH4Cl pulses re-vealed the performance of the codigestion and acute ammo-nia inhibition of methanogenesis.

The methane gas yield of the codigestion was 0.19 l/gVSat an ammonia concentration of 4.6 gNH4–N/l. The codi-gestion with the solid fractions seemed to result in a slightammonia inhibition effect though the digestion process wasstable and the methanogens were adapted to a high ammo-nia concentration. However, the methane gas production ofthe codigestion per digester volume was 1.8 l/l/day, whichwas about 150% of the methane gas production of only pigmanure.

A strong negative correlation (R2 � 0.91) between totalammonia concentration and methane gas yield was obtained.However, free ammonia concentration did not reflect theacute ammonia inhibition significantly. Compared to a con-trol reactor at an ammonia concentration of 4.6 gNH4–N/l,a 50% decrease of methane gas yield was observed at 11.0gNH4–N/l (1.45 gNH3–N/l).

Acetic and propionic acids did not indicate process im-balances. However isobutyric, butyric, and isovaleric acidswere found to be useful indicators of process imbalances.The inhibited reactors did not produce methane gas afterfeeding ceased; however, degradation of acetic, butyric, iso-valeric, and valeric acids were observed up to ammonia con-centrations of 12.1, 13.2, 12.1, and 12.1 gNH4–N/l, respec-tively.

Acknowledgement

This paper is supported by grants from The Danish En-ergy Council.

Author Disclosure Statement

The authors declare no competing financial interests exist.

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