Ammonia inhibition in anaerobic digestion: A review

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Contents lists available at SciVerse ScienceDirect

Process Biochemistry

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mmonia inhibition in anaerobic digestion: A review

rhan Yenigün, Burak Demirel ∗

nstitute of Environmental Sciences, Bogazic i University, Bebek, Istanbul 34342, Turkey

a r t i c l e i n f o

rticle history:eceived 27 February 2013eceived in revised form 11 April 2013ccepted 17 April 2013

eywords:mmonianaerobic digestion

a b s t r a c t

Even though ammonia is an essential nutrient for bacterial growth, it may inhibit methanogenesis duringanaerobic digestion process if it is available at high concentrations. Therefore, ammonia is regarded as apotential inhibitor during anaerobic digestion, particularly when dealing with complex type of substratessuch as manure or the organic fraction of municipal solid waste (OFMSW). Ammonia is produced throughbiological degradation of nitrogenous matter. Ammonium ion (NH4

+) and free ammonia (NH3) are the twoprincipal forms of inorganic ammonia nitrogen. Both forms can directly and indirectly cause inhibition inan anaerobic digestion system. Particularly, free ammonia (FAN) is a powerful inhibitor in an anaerobic

nhibitionecovery

digester above threshold concentrations. Process inhibition is related to the particular characteristics ofthe substrate to be anaerobically digested, pH, process temperature (mesophilic or thermophilic), typeof the seed sludge (inoculum), the reactor configuration and to the concentrations of ammonium andammonia. In this paper, ammonia inhibition in anaerobic digestion systems and the recovery efforts afterinhibition are discussed. Furthermore, the impacts of ammonia inhibition on the microbial populationavailable in anaerobic digesters, namely bacteria and Archaea, are also evaluated in detail.

© 2013 Elsevier Ltd. All rights reserved.

ontents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 002. Early studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 003. Mesophilic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 004. Thermophilic digestion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 005. Mesophilic vs thermophilic . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 006. Microbiology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 007. Recovery after inhibition. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 008. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

Acknowledgment . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

. Introduction

Anaerobic digestion processes have gained extreme importanceithin the last three decades, since biogas, a form of renewable

nergy, can be produced through biological treatment of wastes andastewaters with different characteristics. Research activities on

modeling, and process microbiology. Furthermore, the activity andfate of two distinct groups of microorganisms, namely acidogensand methanogens, also make the study of anaerobic digestion morechallenging [1]. However, anaerobic digestion processes are vul-nerable to inhibition by certain accumulating chemicals, amongwhich ammonia (NH3) and ammonium ion (NH4

+) are the most

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arious aspects of anaerobic digestion have recently taken a moreccelerated pace, particularly focusing on the effects of changesn the operational and environmental parameters on the processerformance and stability, inhibition and toxicity, optimization,

∗ Corresponding author. Tel.: +90 212 359 46 00; fax: +90 212 257 50 33.E-mail addresses: [email protected] (O. Yenigün),

[email protected], [email protected] (B. Demirel).

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significant inhibitors. Although some ammonium is beneficial forbacterial growth, undesirably high concentrations may be reachedduring the breakdown of proteins available in the substrate.

2. Early studies

ibition in anaerobic digestion: A review. Process Biochem (2013),

Anaerobic digestion has been used to treat municipal sludge,animal wastes and industrial wastes of high organic contentsince the beginning of the twentieth century. The conventional

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ubstrates were primary sludge or slurried organic wastes. Afterid 50s, thickened sludges were introduced to, so called, high-

ate digesters, however frequent reactor failures were observed.igh alkalinity concentrations of up to 6500 mg/l and pH valuess high as 7.4, first led to the suggestion that the bacterial activ-ty was hindered due to some toxic effect of the alkalinity [2].nother common observation was the rapid increase of volatile

atty acid (VFA) concentrations observed simultaneously duringeactor failures. McCarty and McKinney showed that the decreasen bacterial activity was actually due to salt toxicity [3]. Use ofarious salts in the digesters for pH control was a common exer-ise. Among those used, sodium, potassium and ammonium saltsere found to cause toxic effects [2–5]. It was generally agreed

hat for high rate digesters, total ammonia nitrogen (TAN), i.e. freemmonia nitrogen + ammonium nitrogen concentrations of around700–1800 mg/l caused reactor failure [2,6]. McCarty and McKin-ey further proposed that the inhibition/toxicity was due to freemmonia (FA) in solution rather than the ammonium ions, equilib-ium concentrations being dependent on pH and temperature [3].hey also found out that 150 mg/l free ammonia nitrogen (FAN)oncentration was completely inhibitory to anaerobic digestion.elbinger and Donnellon studied the relationship between organic

oading rate (OLR) and ammonia inhibition [6]. The authors foundut that the bacteria could be acclimated to ammonium, if it was fedt slowly increasing concentrations, thus in such high rate digestersAN concentrations of 2700 mg/l could exist without signs of slowown or failure. In another study with piggery wastes as the sub-trate, partial inhibition was observed at 3000 mg/l total ammoniaTA), i.e. free ammonia + ammonium ion concentration [7]. Furtheresearch on the acclimation of Archaea to higher TAN concentra-ions revealed that levels up to 5000 mg/l could be tolerated inigested sewage sludge and 3075 mg/l in piggery manure [8]. Theseesults were confirmed by Braun et al. [9]. All early studies men-ioned above were concentrated on the causes of inhibition andelationships with pH, loading rates, VFA and salt concentrations,ynergistic and antagonistic effects and what could be done toncrease bacterial toleration.

During the 70s, the importance of energy recovery and renew-ble energy became increasingly apparent. In time, as somedvantages of anaerobic treatment over aerobic treatment wereecognized, the necessity of more research focusing on otherspects of the anaerobic digestion process became evident. Amonghese were studies related to process stability with respect torocess parameters, biogas recovery, microbiological aspects androcess modeling. Many different types of wastes and co-digestionractices with various mixtures were considered. Digestion underesophilic and thermophilic conditions was also compared. Nearly

n all these studies, stretching the limits of certain experimentalonditions led to process slow down or failure, mainly due to inhibi-ion/toxicity caused by the accumulation of various inhibitor/toxichemicals [10]. In time, a great amount of work has been particu-arly carried out about inhibition and toxicity.

Ammonia (NH3) and ammonium (NH4+), apart from being

resent in the sludge/slurry to be digested, also accumulate dur-ng breakdown of proteins and are the foremost inhibitors to thenaerobic digestion process. The important factors here are initialoncentrations, process temperature, pH, organic loading rate andcclimation of inoculum, all of which have direct or indirect effectn inhibitory concentrations. A summary of selected works aboutmmonia inhibition in anaerobic digestion from literature coveringoth mesophilic and thermophilic processes is given in Table 1.


. Mesophilic digestion

Mesophilic anaerobic digestion generally refers to the pro-ess temperatures between 30 and 40 ◦C. Most of the full scale

PRESShemistry xxx (2013) xxx– xxx

anaerobic digesters are operated under mid-mesophilic condi-tions, around 35 ◦C. As mentioned above, TAN concentrations ofaround 1700–1800 mg/l were completely inhibitory with unaccli-mated inoculum, although with acclimation, inhibitory TAN levelscould increase up to 5000 mg/l. Another concern was the processstability [11]. It was possible to maintain a stable digestion of asynthetic acetic acid substrate at inhibitory TAN levels in excessof 5000 mg/l, adjusting pH to 8.0, with corresponding 256 mgFAN/l. Stable digester operation was therefore possible with lowermethane yield. Slight increases of TAN caused progressively lowermethane production and at a TAN level of 6700 mg/l, completeinhibition was observed. In another study, a different syntheticsubstrate with a chemical oxygen demand (COD) concentration of10,000 mg/l was digested with slow additions of NH4Cl [12]. Thedigesters were flow-through and the pH was kept around 7.0–7.2.When TAN concentration reached 7850 mg/l, slight inhibition wasobserved (corresponding FAN concentration was 80–100 mg/l). A50% inhibition at 11,780 mg TAN/l and pH 6.8 was the outcome.Near complete inhibition was encountered at 18,300 mg/l TAN anda pH of 6.6. In a similar study, during continuous NH4Cl additionto slurried poultry manure in a 5-liter laboratory digester, a TANconcentration of 5980 mg/l depressed biogas yields by 27% at apH of 7.43 and 145 mg FAN/l [13]. Using potato juice as substratein batch reactors, ammonia inhibition of acclimated (to 2315 mgTAN/l) granular sludge [14] was investigated to higher ammo-nium concentrations by ammonium ion addition [15]. It was foundthat at 11,831 mg TAN/l, with pH of 7.57, methanogenic activity of0.04 g COD/g VS/day was maintained. Another noteworthy conclu-sion here was the operation of a full scale upflow anaerobic sludgeblanket (UASB) reactor with TAN concentration between 5000 and7500 mg/l, which had a maximum specific methanogenic activ-ity of 0.1–0.15 kg COD/kg VS/day and an average sludge content of40 kg VS/m3.

Ammonia inhibition versus sludge retention time (SRT) wasstudied with slug and continuous additions of NH4Cl to anaerobicacetate and propionate enrichment cultures [16]. High SRT systems(40 days) were more tolerant to TAN concentration of 5000 mg/l incomparison with low SRT systems (25 days). 55 mg FAN/l was themaximum tolerable concentration in these experiments. Heinrichset al. employed acclimated sludge (to >1500 mg TAN/l) and a syn-thetic substrate comprising acetic, propionic acids and mixtures aswell as lactic acid [17]. FAN levels causing 50% inhibition variedbetween 106 and 183 mg/l. Digestion of diluted poultry manure(10–14% total solids) in 5 l reactors gave satisfactory results in termsof biogas yield at TAN concentrations of up to 7700 mg/l within apH range of 8.1–8.5 [18].

The substrates with high initial TAN and protein concentra-tions such as sea-food industry wastes and wastewaters couldbe treated anaerobically using acclimated sludges containing upto 3000 mg/l TAN concentrations (corresponding to 150 mg FAN/lat pH of 7.6) [19]. Working with macerated fish offal and cat-tle slurry mixtures in continuous anaerobic digesters resulted inreduced methane yields under 100 mg/l FAN concentration. How-ever, the cause of inhibition was attributed to the presence oflong chain fatty acids (LCFAs) associated with the lipids in thefish offal [20]. The same research group also studied batch anaero-bic digestion with a variety of organic mixtures including slurriesof cattle manure, chicken manure, fish offal, fruit and vegetablewastes and brewery sludge [21]. Successful reactor operationand satisfactory methane yields were obtained in this study. Thedigesters that contained chicken manure slurry exhibited depres-sion in methane yields (up to 65%) at FAN concentrations of about


1000 mg/l, although a total reactor failure was not observed. In allexperiments, unacclimated bacterial cultures were used and thedigesters containing chicken manure had lag periods of up to 9days.


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Table 1A summary of selected works focusing on ammonia inhibition in anaerobic digestion.

Substrate Reactor type Loading rate Temperature pH TAN (critical conc. or asspecified)

FAN (critical conc. or asspecified)

Acclimation Reference

Sludge Laboratory-scalebatch reactors

– 30 ◦C 7.2–7.4 >5000 mg/l – Yes [8]

Piggery manure Laboratory-scalebatch reactors

– 30 ◦C 7.2–7.4 >3075 mg/l Yes [8]

MSW/Sludge Semi continuous – 39 ◦C 8.0 2800 mg/l – No [24]Synthetic wastewater UASB 1.2 kg COD/m3/d 35 ◦C 7.7–8.1 6000 mg/l 0.8 g/l Yes [27]Slaughterhouse waste +

organic fraction ofMSW

CSTR 3.7 kg VS/m3/d 34 ◦C 7.5 4100 mg/l 337 mg/l Yes [31]

Sewage sludge Semi continuous 2.0 kg VS/m3/d 35 ◦C 8.0 3000 mg/l 400 mg/l Yes [41]Cattle manure CSTR – 45 ◦C 7.4–7.9 6000 mg/l 0.7 g/l Yes [54]Organic fraction of MSW High solids reactor 6.5 g VS/kg/d 55 ◦C 7.0 2500 mg/l (100% inhibition) – Yes [56]Non-fat dry milk CSTR 4 g COD/l/d 55 ◦C 6.5–8.0 5.77 g/l (64% inhibition) – Yes [57]Pig manure CSTR 9.4 g VS/l/d 51 ◦C 8.0 11 g/l (50% inhibition) 1450 mg/l (50% inhibition) Yes [59]Organic fraction of MSW Batch – Mesophilic–

thermophilic215–468 mg/l (50% inhibition) – [67]

Critical concentration is defined as the concentration at which inhibition starts.TAN, total ammonia nitrogen; FAN, free ammonia nitrogen.


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The effect of pH at various TAN levels was investigated dur-ng high-solids sludge digestion [22]. The reactors were 120 mllass bottles. The pH range was chosen to vary between 6.5 and.0, while TAN concentration range varied from 0 to 6000 mg/l.t all pH levels, 5000 mg TAN/l reduced the methanogenic activ-

ty by 50%. At pH of 9.0 and FAN concentration of 900 mg/l, theigester could be operated satisfactorily and the authors con-luded that the TA, rather than FA, was a more significant factorn inhibiting methanogenic activity in high-solids digestion. Whenag (acclimation) periods were investigated with respect to TANnd FAN concentrations, it was found that within a pH range of.5–9.0, acclimation periods and ammonium ion concentrationsere not correlated, whereas acclimation periods increased with

ncreasing FAN levels. This finding suggested that the FA was aore sensitive factor for the inhibition of an unacclimated bac-

erial system. Another high-solids digestion study was reported,n which the substrate was chicken manure, with total solidsanging from 5.0 to 21.7% [23]. In reactors with unacclimatedewage sludge, the methanogenesis was severely inhibited after50 mg/l FAN. Municipal solid waste and sludge were digested at

slightly higher mesophilic temperature of 39 ◦C with unadaptedeed sludge [24]. The TAN concentration causing 50% inhibition wasround 2400 mg/kg (141 mM), and the total failure took place at800 mg/kg (165 mM).

Anaerobic glucose degradation in batch reactors was inhibitedy about 70% at 3500 mg/l TAN concentration and at pH of 8.0 [25].lthough a seed sludge, which was acclimated to 3100 mg TAN/las used, glucose consuming acidogenic bacteria could not stand

hese TAN concentrations. In another study with a pH value of 8.1,table reactor operation was possible with 587 mg FAN/l (4718 mgAN/l), using fishery effluents as substrate [26]. Here ammoniadapted anaerobic filter reactors were used and NH4Cl was addedo test the extent of inhibition. Using a synthetic substrate com-osed of acetic, propionic, butyric acids and yeast extract, ammonia

nhibition was investigated in five laboratory digesters started-upith different seed sludges [27]. Ammonium hydroxide was grad-ally added to elevate TAN concentrations from 1000 mg/l up to000 mg/l, and similarly, the initial pH of 7.7 was gradually raisedo 8.1, with FAN concentration reaching to 800 mg/l. At the end of50 days of experiment, all five reactors were stably in operationlthough decreasing COD removal efficiencies were obtained.

Sludge from juvenile salmon hatcheries was treated in a lab-ratory size anaerobic digester using inoculum from a digesterperated using saline fish farming sludge [28]. A severe inhibi-ion was reported at TAN of 6390–7460 mg/l at pH being around.4–7.5, corresponding to 197–230 mg FAN/l at pH 7.5. How-ver, the concentrations of LCFA and VFAs were also high and ahared responsibility of inhibition was the conclusion. Alfalfa silageminced and mixed with water) was used as the substrate in a study,here three 10 l anaerobic digesters were operated under different

oading conditions [29]. The pH varied between 7.2 and 7.8 and ini-ial TAN concentration was 1600 mg/l, which went up to 5000 mg/l,ausing severe inhibition in one of the digesters in which effluentecirculation was employed.

Anaerobic digestion of the solid fraction of pig slurry was inves-igated using the seed from a sewage sludge digester [30]. FANoncentrations in the batch digesters reached as high as 388 mg/l.

combination of diluted solid slaughterhouse wastes and organicraction of municipal solid waste was treated in laboratory digestersith gradual increase of organic loading rates and with ammonium

cclimated seed sludge [31]. At pH values varying between 7.5 and.0, stable reactor operation could be achieved with TAN levels of


100 mg/l.Waste sludge from a high load activated sludge plant treating

laughterhouse wastes was co-digested with various meat indus-ry wastes (cow manure, ruminal wastes and pig-cow waste slurry)


at different mixing ratios in 1 l batch reactors [32]. The seed wasa digested material from a conventional anaerobic digester, andit was not particularly adapted to high ammonia concentrations.It was found out by the authors that 50% inhibition of methaneproduction occurred at a co-digestion ratio of 90:10 waste sludgeto pig-cattle slurry with a TAN concentration of 1130 mg/l (corre-sponding to 70 mg FAN/l) and 80% inhibition at 65:35 waste sludgeto pig-cattle slurry ratio with a TAN concentration of 2770 mg/l(230 mg FAN/l). Swine waste was used as the substrate in anotherstudy, in which anaerobic digestion was carried out in laboratoryscale anaerobic squenching batch reactors [33]. The seed was asludge from a digester treating pre-acidified brewery wastewa-ter. At a sub-mesophilic temperature of 25 ◦C, the reactors with4000 mg/l TAN produced 45% lower methane yields compared withthe reactors containing 1600 mg TAN/l (pH varying between 7.5 and7.6). Raising the operating temperature to 35 ◦C, the methane yieldsof reactors increased but with only 13% lower methane yields forthe reactors containing 4000 mg/l TAN concentrations. During thisstage of operation, the methane yields were reported to changebetween 0.31 and 0.36 l CH4/g VSfed. Efficient digester performancewith a TAN concentration of 5200 mg/l could be obtained near theend of the operational period.

Anaerobic digestion of meat and bone meal (animal food) wascarried out at different solid contents of up to 10% in laboratoryscale batch reactors [34]. Methane production was inhibited inthe digesters with 5 and 10% solid substrate. FAN concentrationsreached up to 1000 mg/l, and the authors concluded that at suchhigh FAN concentrations, Archaea could still sustain activity andinhibition was reversible. Leather fleshing wastes were co-digestedwith organic fraction of municipal solid waste using various carbonto nitrogen (C:N) ratios and controlled pH values ranging between4.5 and 8.5 [35]. Biogas yields were satisfactory in general exceptfor the reactor with low carbon to nitrogen ratio of 5 (TAN concen-tration here was 3600 mg/l) and the reactor with leather fleshingwaste only (TAN concentration here was 4289 mg/l).

Organic municipal wastes were digested in laboratory-scalereactors with gradual increases of TAN concentrations from 800to 6900 mg/l [36]. At pH of 7.9 and 3300 mg TAN/l concentration,the methane yields were optimum. However, as TAN went up to5500 mg/l, 50% reduction in methane production was observed.Wastewaters obtained from settling and filtering of piggery slurrywere treated anaerobically in serum bottles with additions of VFA,nutrient solutions and inoculum sludge obtained from full-scaleanaerobic digesters [37]. FAN concentrations of around 40 mg/lcaused 50% inhibition in methanogenic activity. In a parallel study,the liquid fraction of settled pig slurry was fed to an upflow anaero-bic sludge blanket (UASB) reactor containing ammonia unadaptedseed sludge [38]. The OLR was gradually increased to preventammonia inhibition and thus, FAN levels up to 375 mg/l did notaffect the COD removal efficiency.

Ground chicken feathers (380–440 g) were fed to 42 l anaerobicdigesters and co-digested with ammonia adapted swine manureand slaughterhouse sludge (35 l each) at a sub-mesophilic tem-perature of 25 ◦C and a pH between 7.6 and 7.9 [39]. The initialTAN concentrations of swine manure and slaughterhouse sludgewere 4800 mg/l and 3200 mg/l, respectively. Methane productionin digesters with feathers was substantially higher in comparisonto those in the control digesters without feathers. Besides, TAN con-centrations increased to 6900 mg/l and 3500 mg/l in feather feddigesters with swine manure and slaughterhouse sludge, respec-tively. No inhibition could be observed. Glucose was used as thesubstrate in another batch study, where inocula from various full-


scale anaerobic reactors were operated [40]. At pH values rangingbetween 7.6 and 8.4, 50% inhibition was observed at a TAN con-centration of about 5000 mg/l. Dewatered sludge was digested inlaboratory size reactors using inoculum from an anaerobic digester


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f a conventional wastewater treatment plant [41]. Biogas produc-ion was moderately inhibited at TAN levels of 3000 mg/l (400 mgAN/l at pH 8.0) and severe inhibition was observed when TAN con-entration was 4500 mg/l with corresponding FAN concentration of00 mg/l.

The impacts of transient overloads on the performance of laboratory-scale mesophilic anaerobic ammonium oxidizingnaerobic baffled reactor was investigated through increasingubstrate concentration or influent flow rate using syntheticastewater [42]. The authors reported that when the concentra-

ions of ammonium and nitrite exceeded threshold, they becamenhibitors. During shock loading, the concentration of free ammoniaanged between 8.2 and 97.1 mg/l. The effect of ammonia inhibitionas studied during the enhanced anaerobic treatment of digested

ffluent from a full-scale CSTR using a laboratory-scale mesophilicnaerobic reactor [43]. The reactor was operated within an OLRange of 1.5–3.5 kg COD/m3/d at a HRT of 1.5 d. It was reportedy the authors that the SCOD/TAN (soluble COD/total ammoniaitrogen) ratio was a key parameter and the threshold value of theCOD/TAN was determined to be 2.4 (TAN concentration of about250 mg/l) at an influent pH of 8.5–9.0.

The impact of ammonia removal from piggery wastewater bymmonia stripping was investigated as a pre-treatment method inrder to increase methane yield from anaerobic digestion of piggeryastewater [44]. When ammonia was air-stripped at pH values of

.5 and 10, significant improvements in methane yields from anaer-bic digestion were achieved, also preventing the process failure.race element demand of anaerobic digesters at elevated ammoniaoncentrations fed with food waste was studied [45]. It was con-luded that both selenium (Se) and cobalt (Co) were required fornterspecies electron transfer at high ammonia levels to preventccumulation of propionic acid.

The effects of Fe, HCl and trace element addition on processtability of mesophilic anaerobic digestion of pig slaughterhouseaste was investigated using laboratory-scale CSTRs in order to

chieve higher OLRs [46]. The concentrations of TAN in all reac-ors generally ranged from 1.5 to 2.5 g/l, while it increased to 3.5 g/ln one of the reactors. The concentrations of FAN were calculatedo be between 0.05 and 0.22 g/l in all reactors. These values did notause inhibition in any of the reactors in this work. The authors alsoeported that addition of Fe, HCl and trace element allowed oper-tion of mesophilic CSTRs at higher OLRs around 2.25 kg VS/m3/d.esophilic anaerobic batch co-digestion of dairy manure, chickenanure and wheat straw were investigated in a laboratory-scale

tudy, particularly focusing on the C:N ratio [47]. The authorseported that the C:N ratios of 25, 30, and 35 provided low andtable TAN and FAN concentrations of 712, 604, and 444 mg/l, and.1, 7.5, and 2.2 mg/l, respectively. On the contrary, the C:N ratiof 15 resulted in higher TAN and FAN concentrations of 2614 and23 mg/respectively. The optimization of the C:N ratio resulted in

stable co-digestion process.

. Thermophilic digestion

Thermophilic anaerobic digestion processes are carried outithin a temperature range of 45–65 ◦C. Thermophilic process has

dvantages over mesophilic digestion for achieving higher rates ofigestion, greater conversion of waste organics to gas, faster solid-

iquid separation, and minimization of bacterial and viral pathogenccumulation [48]. Due to the high energy density of the sub-trates and high loading rates, self-heating effects have brought


bout an increase in operating temperatures from mesophilic toub-thermophilic and thermophilic temperatures more recently49]. The disadvantages of thermophilic operation are poor super-atant quality and poor process stability [50]. One of the earlier

PRESShemistry xxx (2013) xxx– xxx 5

studies investigating the effect of various operating parameters onthe thermophilic anaerobic digestion of cattle wastes concludedthat at high loading rates and pH levels above 7.8, the process couldbe inhibited by TAN concentrations of above 1700 mg/l [51]. Thiswas in agreement with inhibitory ammonia concentrations foundin earlier mesophilic studies. Diluted cow manure was digested ina semi-continuous 120 l reactor, where the inoculum was freshcow manure [52]. Inhibition started at 1700 mg TAN/l concen-tration. Cattle manure was used as the substrate in continuouslyfed laboratory scale reactors in another work [53]. NH4Cl wasgradually administered for adaptation and the pH was kept con-stant. The first signs of inhibition occurred at a TAN concentrationof 4000 mg/l, corresponding to 900 mg FAN/l. The process insta-bility due to ammonia led to VFA accumulation, which loweredthe pH. Therefore, the decreased FAN concentration eventuallyresulted in a stable, but lowered methane yield, which was calledby the authors as the “inhibited steady state”. A parallel studyby the same researchers investigating the effect of temperaturechanges within the thermophilic range revealed that with adaptedinoculum, 6000 mg TAN/l could be tolerated at 45 ◦C [54]. The keyconclusion here was that the decreasing the process temperatureovercame ammonia inhibition. In a follow-up research, digestionof swine manure was investigated in laboratory scale batch andcontinuously stirred tank reactors (CSTRs) [55]. A severe inhibitionand a very low methane yield were observed at a TAN concentra-tion of 6000 mg/l and the methane yield decreased with increasingtemperature (within the thermophilic range). It was concludedthat a threshold of 1100 mg/l FAN concentration was required forintroducing inhibition.

Biodegradable organic fraction of municipal solid waste wasdigested in a pilot scale high-rate anaerobic digester at differentmass retention times and gradually increasing ammonia concen-trations [56]. The start of ammonia inhibition, 50% inhibition andtotal reactor failure occurred at 1000, 1500 and 2500 mg/l TANconcentrations, respectively. Impacts of stepwise increases in TANconcentrations were studied on anaerobic digestion of soluble non-fat dry milk [57]. A TAN concentration of 4920 mg/l caused 39%inhibition in specific methanogenic activity.

Diluted cattle manure containing 1300–1400 mg TAN/l wasdigested in laboratory-scale CSTRs [58]. TAN concentrations weregradually elevated with pulse additions of NH4Cl and tryptone. A38% reduction in methane yield was associated with 6200 mg/lTAN concentration. Pig manure was digested in laboratory scaleCSTRs with inoculum adapted to high concentrations of ammonia[59]. With periodic pulse additions of NH4Cl, ammonia inhibitionlevels were investigated at pH ranges between 7.91 and 8.03. A50% decrease in methane yield was observed at 11,000 mg TAN/l,corresponding to 1450 mg FAN/l. At these inhibitory levels, VFAconcentrations did not vary in comparison to those in the controlreactor (without the pulse additions), a result in contradiction tothose in previous studies [53,60].

5. Mesophilic vs thermophilic

Some investigators have exclusively studied the effect of tem-perature on anaerobic digestion in relation to ammonia inhibition.Lower biogas production at thermophilic temperatures in compar-ison to mesophilic temperatures was attributed to FA inhibition[61]. Hashimoto studied the anaerobic digestion of beef cattlemanure by gradual additions of NH4Cl [60]. In unadapted digestion,ammonia inhibition started at 2500 mg TAN/l both for mesophilic


and thermophilic operations. However, as FA increased with tem-perature, the calculated FAN concentrations were 30 mg/l for themesophilic and 200 mg/l for the thermophilic digesters. In adaptedoperations, 4000 mg TAN/l could be tolerated.


IN PRESSG Model

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6 ss Biochemistry xxx (2013) xxx– xxx

tatFwdiawct

5[nccpihct7mo

rima

6

tmcbmdlfaqDthmc[atpTstabcA

iph

Table 2The classification of methanogens.

Class I. Methanobacteria (known to grow on H2/CO2 and formate as C source)Order I. Methanobacteriales

Family I. MethanobacteriaceaeGenus I. Methanobacterium

Genus II. MethanobrevibacterFamily II. Methanothermaceae

Class II. Methanococci (known to grow on H2/CO2 and formate as C source)Order I. Methanococcales

Family I. MethanococcaceaeFamily II. Methanocaldococcaceae

Class III. Methanomicrobia (known to grow on H2/CO2 and formate as C source)Order I. Methanomicrobiales

Family I. MethanomicrobiaceaeGenus IV. Methanogenium

Family II. MethanocorpusculaceaeFamily III. Methanospirillaceae

Genus I. MethanospirillumOrder II. Methanosarcinales (known to be acetato- and methylotrophic)

Family I. MethansarcinaceaeGenus I. Methanosarcina

Family II. Methanosaetaceae



The organic fraction of household waste was digested in con-inuous CSTRs at mesophilic and thermophilic temperatures, withmmonia unadapted inoculum [62,63]. Ammonium ion was addedo the assays at various concentrations. At pH of 7.5, 220 mgAN/l caused a 50% inhibition of mesophilic methane production,hereas 690 mg FAN/l caused a 50% inhibition at thermophilic con-itions. As for peptone deamination and methane generation, 50%

nhibition was observed with 88–92 mg FAN/l in the mesophilicnd 251–297 mg FAN/l in the thermophilic assays. Poultry manureas digested in serum vials at gradually increasing ammonium con-

entrations [64]. At about 4000 mg TAN/l, independent of operatingemperatures, 50% inhibition took place.

Effect of stepwise and pulse temperature increase from 35 to5 ◦C was investigated in pilot-scale and laboratory-scale reactors65]. The substrates were maize silage and pig manure, and ammo-ia adapted bioslurries were used as seed. At 45 ◦C, the critical TANoncentration was found to be 5500 mg/l. Temperature increasesaused process disturbances such as drops in methane yields andropionic acid accumulation. Two laboratory scale CSTRs operat-

ng at 35 and 55 ◦C digested secondary residuals obtained fromigh rate anaerobic digestion of brewery wastewaters [66]. FANoncentrations climbed up to 640 mg/l in the thermophilic reac-or, but could be lowered to 358 mg/l by decreasing pH from 7.6 to.3, before any sign of inhibition. Both reactors exhibited similarethane yields rendering the mesophilic reactor superior in terms

f energy consumption.The organic fraction of municipal solid waste was digested in

eactors with supernatant recycling [67]. The methane yield wasnhibited by 50% at FAN concentrations of 215 and 468 mg/l under

esophilic and thermophilic conditions, corresponding to 3860nd 5600 mg TAN/l, respectively.

. Microbiology

Anaerobic microbial communities can be classified intowo domains, namely bacteria and Archaea [68]. Four major

etabolic groups are involved in a stable ongoing anaerobiconversion process. These groups are hydrolytic-fermentativeacteria, proton-reducing acetogenic bacteria, hydrogenotrophicethanogens and acetoclastic methanogens [69]. During anaerobic

egradation of a particulate substrate, carbohydrates, proteins andipids are firstly hydrolyzed to organic monomers by hydrolytic-ermentative bacteria. The carbonic products of these reactionsre either acetate or propionate and butyrate, which are subse-uently converted to acetate and hydrogen by acetogenic bacteria.uring methanogenesis, methane is produced from acetate by ace-

oclastic methanogens or from hydrogen and carbon dioxide byydrogenotrophic methanogens. Methyl groups (methylamines,ethanol) are also converted into methane in this stage. A classifi-

ation of the methanogens reported in this paper is given in Table 268]. The performance of an anaerobic reactor is directly associ-ted with the structure of the microbial community present withinhe reactor. The operational and environmental parameters of therocess eventually influence the fate of the microbial community.herefore, the concentrations of ammonia, coming directly with theubstrate or produced during anaerobic degradation, is a parame-er that has to be monitored carefully in order to achieve a stablend efficient process, since excess concentrations of ammonia haveeen often reported to inhibit the activity of the anaerobic microbialommunities in anaerobic reactors treating a variety of substrates.

summary of selected works from literature is also given in Table 3.


Methanobacterium formicicum was reported to be partlynhibited at a TAN concentration of 3000 mg/l at pH 7.1, while com-lete inhibition took place at 4000 mg TAN/l [7]. Methanospirillumungatei exposed to ammonia lost up to 98% of the cytoplasmic K+

Genus I. Methanosaeta

via an ammonia/K+ exchange reaction [70]. NH4OH or methylamineadditions were most effective resulting in depletion of K+ at alka-line pH, indicating that ammonia was the chemical species crossingthe membrane. The authors concluded that methanogenesis wassensitive to both pH of the cytoplasm and the medium.

The impact of TAN concentration between 680 and 2601 mg/lon methanogenic sludge was investigated in batch experiments[71]. The accumulation of acetate at a TAN concentration at2601 mg/l showed that, above the threshold concentration of1700 mg/l, ammonia-N had relatively more negative effect onacetate-consuming methanogens than on hydrogen consumingmethanogens; although the opposite was true for TAN levels belowthis threshold.

Transport of K+ in M. hungatei pretreated with ammonia wasstudied and it was observed that the methanogenic activity waslost, but it could be recovered through addition of Ca+2 or Mg+2

[72]. Ca+2 or Mg+2 addition seemed to activate cells so that theycould make ATP and transport K+. The toxicity of ammonia to thegrowth of several methanogenic bacteria was evaluated in terms ofan ammonia/potassium exchange reaction and in terms of inhibi-tion of methanogenesis [73]. Growth of Methanobrevibacter smithii,Methanobrevibacter arboriphilus and Methanobacterium strain G2Rwas normal in media containing up to 400 mM NH4Cl (5.55 g/l TAN),with no observation of inhibition of methane production. How-ever methane synthesis from Methanothrix concilii was completelyinhibited at TAN levels of 560 mg/l, while methane formationfrom Methanosarcina barkeri was not inhibited at 2800 mg TAN/l.Another observation was that certain cations countered the toxiceffects which ammonia had on methane synthesis, notably Ca2+ inM. concilii and Na+ in M. barkeri.

During thermophilic anaerobic digestion of livestock wastes,inhibition at high ammonia concentrations was reported to influ-ence the formation of methane from H2 and CO2 [74]. Ammoniahad only a minor effect on methane formation from acetate, whichcould be shown by the independence of the specific growth rate ofacetate-consuming methanogens from a TAN concentration up to4500 mg/l. The effect of NH4Cl on methanogenesis by pure culturesof M. hungatei, M. barkeri, Methanobacterium thermoautotrophicum,and M. formicicum at a pH of 6.5 was investigated [75]. M. barkeri, M.thermoautotrophicum, and M. formicicum were found to be resistant


to ammonia concentrations over 10,000 mg TAN/l, while M. hun-gatei was more sensitive with 50% of inhibition of methanogenesisat 4200 mg TAN/l.


ARTICLE IN PRESSG Model

PRBI-9822; No. of Pages 11

O. Yenigün, B. Demirel / Process Biochemistry xxx (2013) xxx– xxx 7

Table 3A summary of research findings for impact of ammonia on Archaea in anaerobic digestion.

Substrate Temperature(◦C)

pH Critical TAN conc.or as specified(mg/l)

Critical FAN conc.or as specified(mg/l)

Organismsaffected/present

Moleculartechniqueapplied

Reference

– 38 ◦C – 4000 (100% inhibition) – Methanobacterium formicicum – [7]– – 6.5 4200 (50% inhibition) – Methanospirillum hungatei – [75]– 60 ◦C 6.9 4000 – Methanobacterium [79]

7.0 6000 (50% inhibition) Thermoformicucum (present)Swine waste 25 ◦C – ≥3500 – Methanomicrobiales 16S rRNA gene

analysis[82]

MethanosarcinaSynthetic wastewater 35 ◦C 8.0 6000 (100% inhibition) >700 (100% inhibition) Methanosarcina FISH/DGGE [85]Synthetic wastewater 35 ◦C 7.7 – >100 (100% inhibition) Methanosaeta-related species FISH/DGGE [86]Sodium acetate – – 7000 (acclimated) – Methanosarcinaceae spp. FISH [93]

5000 (non-acclimated) Methanococcales spp.

hao0iimt[wT7tm

hia[aTtcapaibetdsim

rm1Tmdtlptta

Cattle excreta + olivemill waste

37/55 ◦C – 1300 –

The effect of ammonia on the maximum growth rate (�m) ofydrogen-consuming methanogens was studied at different pHnd temperature values [76]. The maximum inhibited growth ratef hydrogenotrophic methanogens present in sludge appeared to be.126 h−1 at pH and temperature of 7.0 and 37 ◦C, respectively. The

ncrease in pH from 7.0 to 7.8 at 37 ◦C seemed to enhance ammonianhibition. During anaerobic digestion of dairy cattle manure, the

ethanogenic community was inhibited at high TAN concentra-ions as indicated by changes in relative rates of acetate utilization77]. The maximum growth rate (�m) of acetoclastic methanogensas investigated during anaerobic digestion of poultry manure at

AN concentrations from 7700 to 10,400 mg/l and a pH of between.80 and 7.93 [78]. It was reported that pH and the TAN concentra-ion were the predominant inhibition factors for the acetoclastic

ethanogens.The effects of ammonia on pure cultures of thermophilic

ydrogen-utilizing methanogens including M. thermoautotroph-cum, Methanobacterium thermoformicicum, Methanogenium sp. and

putative M. thermoautotrophicum were investigated in batch tests79]. For all strains, initial inhibition started at 3000–4000 mg TAN/lnd 50% decrease in growth rates were observed at around 6000 mgAN/l. In addition, slow growth and aggregate formation of M.hermoformicicum could be observed at 9000 TAN/l. A further con-lusion of this study was that the thermophilic hydrogen utilizersre less sensitive towards ammonia then their mesophilic counter-arts. During thermophilic anaerobic digestion of livestock waste,mmonia concentrations of 4000 mg N/l or more was found tonhibit digestion of cattle manure. In another previous study, M.arkeri was found to be more sensitive to excess ammonia lev-ls than Methanobacterium bryantii [80]. TAN concentration higherhan 5000 mg/l was reported to inhibit thermophilic anaerobicigestion of cattle manure in UASB reactors [81]. The authors alsotated that the acetoclastic methanogens showed a higher sensitiv-ty to the presence of ammonia in comparison to hydrogenotrophic

ethanogens.During operation of a full-scale anaerobic sequencing batch

eactor (ASBR) of 600 m3 volume for treating swine waste,ethanogenic population changes were monitored at a HRT of

5 days and at a TAN concentration of about 3600 mg/l [82].he 16S ribosomal RNA (rRNA) levels of the acetate-utilizingethanogens of the genus Methanosarcina was observed to

ecrease from 3.8 to 1.2% (expressed as a percentage of theotal 16S rRNA levels) during this period, while the 16S rRNAevels of Methanosaeta concilii remained below 2.2%. Methane


roduction and the reactor performance were not affected ashe 16S rRNA levels of the hydrogen-utilizing methanogens ofhe order Methanomicrobiales increased from 2.3 to 7.0%. Theuthors concluded that the anaerobic digestion of swine waste

Methanosarcina PCR/16S rRNAgene analysis

[94]

could be sustainable at TAN levels exceeding 3500 mg/l, dueto the ability of the hydrogen-utilizing methanogens of theorder Methanomicrobiales to be active at these high TAN lev-els.

Microbial diversity of two different laboratory-scale anaero-bic reactors (namely an UASB and a hybrid bed) treating a younglandfill leachate with TAN concentrations up to 2700 mg/l, wasinvestigated using FISH (fluorescent in situ hybridization) andDGGE (denaturing gel gradient electrophoresis) techniques [83].During low acetate levels in the reactors, the stability of operationwas supported by the abundance of Methanosaeta, and in both reac-tors the presence of Methanobacteriaceae was also detected, whileother methanogenic species were not encountered. The effect ofammonia on methanogenic activity of anaerobic films enriched bymethylaminotrophic methane producing Archaea was studied inbatch tests operated at a pH of 7.5 and a temperature of 37 ◦C [84].The concentrations of ammonia used in the experiments were 48.8,73.8, 98.8, 148.8, 248.8, 448.8, and 848.8 mg FAN/l, respectively.The findings indicated that then highest methanogenic activitytook place at 48.8 mg FAN/l, and above 148.8 mg FAN/l inhibitioncould easily be observed. The lowest methanogenic activity was at848.8 mg FAN/l.

During investigation of high free ammonia (FAN) concentra-tions on the performance of UASB reactors operated with syntheticwastewater and within a TAN concentration ranging from 1000to 6000 mg/l, the results of FISH analysis showed that propionatedegrading acetogenic bacteria was more sensitive to free ammoniathan Archaea and they were significantly inhibited at a FAN of above200 mg/l [27]. The impact of FAN concentrations up to 750 mg/lwas investigated in laboratory-scale UASB reactors and themethanogenic population was monitored by using FISH and DGGE[85]. The FISH results indicated the abundance of Methanosarcina-like acetoclastic methanogens in reactors. Increase in FAN levelabove 700 mg/l resulted in disintegration of large Methanosarcinaclusters. It was also reported by the same authors that, after astart-up period of 140 days in mesophilic laboratory-scale UASBreactors, when FAN concentration was gradually increased from50 to 130 mg/l, Methanosaeta-related species coming from theseed sludge have noticeably lost their activities and their filamen-tous forms deteriorated when FAN level exceeded 100 mg/l [86].Although some Methanobacterium and Methanospirillium-relatedhydrogenotrophic methanogens could then be detected, at theend of start-up period mainly Methanosarcina-like acetoclasticmethanogens were abundant in reactors. The authors concluded


that the coccus shaped Methanosarcina species were more resis-tant to elevated FAN levels than rod shaped Methanosaeta cells asan advantage of their high volume to surface ratio and formationof big clusters.


ING Model

P

8 ss Bioc

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7

bd



The effects of ammonia concentration and zeolite addition onhe specific methanogenic activity (SMA) of anaerobic sludges werenvestigated in batch tests conducted at a pH of 6.8–7.2 and at5 ◦C [87]. Piggery, malting production and urban sludges obtainedrom full-scale anaerobic reactors were employed in this work. Itas reported that piggery sludge was mostly affected by ammo-ia concentrations and addition of zeolite at doses from 0.01 to.1 g/g VSS reduced the inhibitory effect of ammonium. The micro-ial population detected by 16S rRNA technique was composed ofethanococcaceae, Methanosarcina, and Methanosaeta.

Mesophilic anaerobic digesters of 3.6 l were continuously runt 35 ◦C to treat poultry waste [88]. Increasing ammonia levelsnd organic loading rate did not result in a microbial communityhift, however, Crenarchaeota archaeal populations were detectedn reactors by PCR instead of Euryarchaeota methanogens. Thempact of carbon textile fibers (CTF) addition to thermophilic

ethanogenic bioreactors was investigated under increasingmmonia concentrations [89]. The reactors were operated at a pHf 7.8 and synthetic garbage slurry was used as substrate. Under000 mg TAN/l, the methanogenic archaea was dominated byethanobacterium sp. and Methanosarcina sp. as detected by 16S

RNA gene analysis. In the control reactor without CFT addition,ethanosarcina sp. was inhibited and washed out at a TAN con-

entration of 1500 mg/l, while Methanobacterium sp. continuedo proliferate. According to the results obtained, the authorsoncluded that CFT enables proliferation of the methanogens byreventing ammonia inhibition. In a recent work, Methanosarcinap. was reported to be quite robust to ammonia toxicity whenompared with other methanogens [90]. Methanosarcina sp. couldolerate a TAN of up to 7000 mg/l. Bioaugmentation of syntrophiccetate-oxidizing culture in biogas reactors fed with whole stillagend cattle manure [91]. Mesophilic laboratory-scale anaerobiceactors were operated under gradually increasing ammoniaoncentrations from 1.5 to 11 g TAN/l, and a fixed volume of syn-rophic acetate-oxidizing culture was daily added to the reactors,owever, addition of the culture did not seem to affect the stabilitynd performance of the biogas reactors, and the reactors eventu-lly deteriorated at high ammonia concentrations. In a previoustudy, it was reported that during a gradual increase in ammoniaoncentration up to 7 g TAN/l in a mesophilic semi-continuousaboratory-scale anaerobic digester fed with source-separatedrganic fraction of municipal solid waste at a HRT of 30 days,

shift from the acetoclastic methanogenesis to syntrophiccetate oxidation took place when the TAN concentration rosebove 3 g/l [92].

In a recent study, the effect of ammonium and acetate onethanogenic pathway and methanogenic community was inves-

igated using batch tests and FISH technique [93]. When theulture wax acclimatized to acetate and ammonia, thermophilicultures were reported to change their acetate bioconversionathway from syntrophic acetate oxidation with subsequentydrogenotrophic methanogenesis to acetoclastic methanogene-is mediated by Methanosarcinaceae spp. The adapted mesophiliculture showed no pathway shift. At high ammonia levels of 7 gAN/l, the acetoclastic Methanosarcinaceae spp. was determinedo be the dominant methanogen in nonacclimatized thermophiliculture. Mesophilic and thermophilic Methanococcales spp. fromonacclimatized cultures were found to be tolerant to ammoniaoncentrations up to 5 g TAN/l.

. Recovery after inhibition


Previous experimental studies indicated that the ammonia inhi-ition could be removed through lowering of pH and subsequentecrease of free ammonia concentration, suggesting that, although


digester toxicity might indirectly be related to free ammoniaconcentration, it was directly related to unionized volatile acidconcentration [11].

Serum bottle experiments were conducted at 35 ◦C to evalu-ate the reversibility characteristics of methane formation systemsexposed to ammonium concentrations of 4000, 8000, and24,000 mg/l TAN [95]. The authors observed that the ammo-nium toxicity was very reversible and if the ammonium in thesupernatant were removed, the system could rapidly recover tofull biogas production. The threshold dose for ammonium wasreported to be less than 2500 mg/l. In another early study, itwas reported that the TAN concentrations at 1900–2000 mg/lcaused failure of methanogenesis. However, after an adaptationperiod, methanogenesis seemed to be possible even at higher TANconcentrations [14]. After the adaptation period, the maximumspecific methanogenic activity at a TAN concentration of 2315 mg/lwas higher than the maximum specific methanogenic activity at1900 mg TAN/l.

Anaerobic digestion of potato juice at extreme concentrationsof ammonia was investigated in a laboratory-scale study usingreactors with working volumes of 4.5 l operated at 30 ◦C [15].The highest TAN concentration, at which methanogenesis wasfound to be possible, was 11.8 g/l. The authors stated that afterbeing adapted, which meant having gained the ability to gener-ate methane at ammonia concentrations exceeding the thresholdlevel, the microbial community could produce methane again at aTAN concentration of 11.8 g/l, showing that toxicity was reversibleeven at extremely high ammonia concentrations. Furthermore, theacidogenic population seemed to be hardly affected within a TANconcentration range of 4051–5734 mg/l, while the methanogenicpopulation lost 56.5% of its activity under these conditions.

Anaerobic treatment of sea food processing wastewater wasinvestigated using an industrial pilot plant operated at 37 ◦C[19]. During the operation, TAN and FAN concentrations variedbetween 1 and 3 g/l, and 25 and 150 mg/l, respectively. Higherprotein content in influent wastewater increased the forma-tion of FA, and when the free ammonia concentrations reached300 mg/l, inhibition took place. When the protein content inthe influent wastewater was decreased, inhibition could then bereversed and normal treatment efficiencies could be achieved after10 days.

The influence of ammonium on anaerobic treatment of poultrymanure was investigated in a laboratory-scale work [96]. It wasobserved that within a NH4Cl concentration range of 2 and 10 g/l,production of methane was not affected, but at higher concentra-tions from 10 to 30 g/l, methane production significantly decreased.The authors tried to recover the system through addition of 10%(w/v) powdered phosphorite ore and this external supplemen-tation increased production of biogas and methane up to NH4Clconcentration of 30 g/l. However, at concentrations above 50 g/l,addition of phosphorite even did not help to recover inhibition ofmethanogenesis.

During thermophilic anaerobic digestion of swine manure, a lowmethane yield of 67 ml CH4/g VS was achieved at a TAN level of 6 g/land several methods were investigated to increase the methaneyield [97]. Through addition of 1.5% (w/w) activated carbon, 10%(w/w) glauconite or 1.5% (w/w) activated carbon and 10% (w/w)glauconite, the methane yield increased to 126, 90 and 195 mlCH4/g VS, respectively.

Thermophilic pilot-scale biogas reactors were operated usingthe organic fraction of MSW as substrate in order to investigateammonia inhibition [98]. The authors reported that the inhibition


took place at a TAN concentration of 1200 mg/l. Dilution of digestercontent with water or adjustment of substrate C:N ratio of 30–40were suggested to mitigate ammonia inhibition. In another work[35], adjustment of C:N ratio and pH to maximize biogas production


ING Model

P

ss Bioc

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opreoico

dcrccp

8

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nd keep TAN and FAN concentrations under control gave bestesults when C:N ratio was 15 at a pH of 6.5.

During thermophilic batch and CSTR anaerobic treatment of cat-le manure, dilution of the biomass with water, reactor effluentnd manure seemed to improve the recovery speed and stability ofn ammonia-inhibited biogas digester fed with cattle manure [99].mong these recovery options, dilution with manure provided aigh production of methane, while dilution with reactor effluenteemed to provide the most stable recovery process.

In a more recent work, 500 mg/l FA was reported to result innhibition during mesophilic batch anaerobic digestion of meatnd bone meal at TS concentrations of 10% [34]. According to theuthors, with a decrease of pH from 8.0 to 7.5, inhibition could beeversed and the methanogens could adapt themselves to higherA concentrations up to 998 mg/l.

Thermophilic anaerobic batch experiments were conductedsing digested piggery effluent in order to mitigate FA inhibition100]. Based on the experimental findings, the authors reportedhat the reduction of pH from initial value of 8.3–6.5, and zeo-ite treatment ranging from 10 to 20 g/l seemed to be effectiveptions to mitigate ammonia inhibition during thermophilic anaer-bic treatment of piggery wastewater.

During thermophilic pilot-scale anaerobic digestion of vari-us substrates such as food waste, fruit and vegetable waste,aper waste, and green waste up to OLRs of 7–10 kg VS/m3/d andetention time up to 19 days, FA accumulation/inhibition wasncountered in the system [101]. A carbon to nitrogen (C:N) ratiof 32 in feedstock seemed to result in 30% less ammonia formationn the digester than a C:N ratio of 27. The authors recommendedhanging the C:N ratio, and a higher OLR in order to reduce andvercome ammonia inhibition.

The feasibility of continuous ammonia removal in an anaerobicigestion process was evaluated using a hollow fiber membraneontactor module [102]. Mesophilic laboratory-scale anaerobiceactors were run using slaughterhouse waste and within TANoncentrations from 6 to 7.4 g/l. Use of hollow fiber membraneontactor helped in reduction of FAN concentration by about 70%,roviding a more stable operation.

. Conclusions

Anaerobic digestion process is not only an efficient tool for wasteanagement, but also an important technology for recovery of bio-

as from organic substrates as a source of renewable energy as longs the process is carefully controlled and monitored. On the otherand, inhibitory compounds such as FAN can be formed duringiological degradation of various substrates. Above threshold con-entrations, FAN is a powerful inhibitor in an anaerobic digester,nd can easily cause process instability indicated by a decrease inoth biogas and methane yields, which can eventually lead to fail-re of the reactor. Therefore, control and monitoring of ammoniaoncentrations in substrate and digester, pH and process tempera-ure enable a safe and stable process, particularly during anaerobicigestion of substrates such as manure (especially poultry manure,ow manure and piggery waste). As long as the microbial commu-ity in the anaerobic digester is gradually acclimated to increasing

evels of ammonia, it has been shown that the digester can operateven at very high concentrations of ammonia without jeopardiz-ng its safety. Pre-treatment of substrate before anaerobic digestionrocess will also decrease the possible adverse impacts of ammo-ia beforehand. If the process is inhibited by high concentrations of


AN, recovery options such as dilution of the substrate, dilution ofhe reactor contents, adjustment of process pH, adjustment of C:Natio of the substrate, external addition of compounds like zeolite,lauconite, and activated carbon can be employed to increase


biogas and methane yields and to achieve a stable and efficientprocess.

Acknowledgment

Orhan Yenigün acknowledges the support provided by Dr. Pra-tim Biswas, Head of the Department of Energy, Environmental andChemical Engineering, at the Washington University in St. Louis,USA, in the preparation of this manuscript during his sabbaticalleave from Bogazic i University.

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ING Model

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accumulation in a pilot-scale thermophilic dry anaerobic digester. Bioresour



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Documents

Ammonia inhibition in anaerobic digestion: A review