Effects of volatile fatty acids, ammonium and agitationon thermophilic methane production from biogas plantsludge in lab-scale experiments

Philipp Lins & Paul Illmer

Received: 9 December 2011 /Accepted: 4 January 2012 /Published online: 17 May 2012# Institute of Microbiology, Academy of Sciences of the Czech Republic, v.v.i. 2012

Abstract The effects of different volatile fatty acids (VFA,formate, acetate, propionate and butyrate), ammonium(NH4

+) and agitation on methane (CH4) production weredetermined in 120-mL serum bottles. We showed that theaddition of formate did not lead to an inhibition of methano-genesis until a concentration of 120 mmol/L. A completeinhibition of methanogenesis was detected in variants con-taining 360 mmol/L formate or propionate until day 3 butthe production started afterwards within next 2 days. Thismight indicate a kind of adaptation to the higher volatilefatty acid concentrations. Increasing NH4

+ concentrationsled to higher initial CH4 production, with an optimum at120 mmol/L. The addition of 720 mmol/L NH4

+ led to acomplete inhibition until day 3; subsequently, CH4 produc-tion started again on day 5 though it was still significantlylower compared to the other variants. Finally, also the speedof agitation showed significant effects on methanogenesis.The CH4 production from complex carbon sources was mostfavourable at a moderate agitation of 150 rpm of thelab-scale serum bottles. A lower or higher speed broughtabout a distinct reduction of CH4 production.

Introduction

Anaerobic digestion is a common treatment to reduce thevolume of organic waste and because of the production ofbiogas (methane and/or hydrogen) more than just analternative to composting. Several parameters shouldbe monitored and controlled during anaerobic digestion;

among them are the concentration of volatile fatty acids(VFAs) such as acetate and propionate, the concentration ofNH4

+ and the pH value (Ahring et al. 1995; Illmer andGstraunthaler 2009). In digesters where the substrate consistsof protein- and nitrogen-rich content, the level of ammonia(NH3) and/or ammonium (NH4

+) might be of interest becauseat high concentrations they might show toxic effects (Illmer etal. 2009). Especially during thermophilic digestion NH3mightreach higher concentrations because with a rise in temperatureand pH the level of undissociated NH3 will increase (Scherer2001). Apart from these parameters, the mode of stirring andagitation is a parameter that attracts civil and process engi-neers as well as applied microbiologists.

We investigated the effects of relevant VFAs and NH4+

added in different concentrations on methanogenesis. Inaddition, the pH optimum for CH4 production was de-termined and the influence of different agitation speedsof lab-scale serum bottles was determined.

Materials and methods

The minimal medium for the VFA, pH and NH4+ experiment

was prepared according to Wagner et al. (2010) with the onlyexception that pure N2 gas was used for establishing anaerobicconditions. The complex medium for the agitation experimentwas prepared according to Lins et al. (2010), the carbon andenergy sources being carboxymethylcellulose, yeast extractand peptone from casein. These complex substrates weretaken to stimulate the cooperation of the different trophiclevels involved for a complete anaerobic degradation(Schink 1997). After autoclaving, 50 mL of the media wereanaerobically and in sterile manner dispensed into 120-mLserum bottles which were flushed with oxygen-free cylindergas (pure N2 or N2–CO2 at 70:30). Then the stock solutions of

P. Lins (*) : P. IllmerInstitute of Microbiology, University of Innsbruck,Technikerstr. 25,6020 Innsbruck, Austriae-mail: Philipp.Lins@uibk.ac.at

Folia Microbiol (2012) 57:313–316DOI 10.1007/s12223-012-0132-7

VFAs and NH4Cl were added. The following concentrationswere used: controls (assumed to be log 0), 3.6 mmol/L (log0.556), 12 mmol/L (log 1.079), 36 mmol/L (log 1.556),120 mmol/L (log 2.079), 360 mmol/L (log 2.556) and720 mmol/L (log 2.857). The latter concentration was usedonly in the NH4

+ experiment. The pH was controlled by pHindicator paper after autoclaving and addition of the stocksolutions, and at the end of the experiments the pH wasmeasured by pH metre. A volume of 5 mL of a 1:5 dilutedsludge (with distilled water) from a 750,000-L biogas plant(Tyrol, Austria) was used as an inoculum, which is describedelsewhere (Illmer and Gstraunthaler 2009). Determination ofthe gas quality and quantity was performed according to Linset al. (2010), except for the quantity of the gas productionduring the agitation experiment, which was measured with amanometer. The serum bottles were incubated at 52±1°Cfor 5 or 77 days for the agitation experiment until the gasproduction ceased.

Results and discussion

At the beginning of the investigation different concentrationsof the important VFAs formate, acetate, propionate and buty-rate were added separately as sole carbon sources. The cumu-lative CH4 production as dependent on the different VFAconcentration is shown in Fig. 1. Due to the inoculation minorbiogas-producing plant sludge residues were introduced intothe bottles, which might explain the CH4 production withinthe control bottles. Therefore, a low amount of CH4 mighthave been produced without converting the added VFAs.

The addition of formate (Fig. 1a) led to a different effecton the CH4 production compared to the other three VFAs(Fig. 1b–d). Obviously formate did not lead to an inhibitionof the CH4 production until a concentration of 120 mmol/L.Only at the highest concentration, i.e. 360 mmol/L, no CH4

production was detectable within 3 days but after two addi-tional days the methanogenic population has recovered(Fig. 1a). The positive effect of 120 mmol/L formate mightindicate a high number of active hydrogenotrophic, formate-utilising methanogens present in the diluted biogas plantsludge, like e.g. species of the order Methanobacteriales(Malin and Illmer 2008; Lins P, Schwarzenauer T,Reitschuler C, Wagner AO, Illmer P (in press) Methanogenicpotential of formate in thermophilic anaerobic digestion.WasteManag Res). The variation of acetate, the most importantintermediate in the anaerobic digestion, led to an inhibitionbeyond 36 mmol/L. Propionate and butyrate led to a remark-able inhibition beginning at 36 mmol/L, and no CH4 produc-tion could be detected even at the highest propionateconcentration. However, at the higher concentrations of ace-tate, propionate and butyrate it seems that, like with the highestformate concentration, a kind of adaptation might have taken

place because CH4 production was detected. Finally, the 5-dayincubationmight have been too short to produce large amountsof CH4 from acetate, propionate and butyrate, since no signif-icant production could be achieved compared to the control. Itmight have been that the number of acetoclastic metha-nogens (acetate-degraders) was too low or that theywould have needed more time to adapt to the artificialmedium. The methanization from acetate, propionateand butyrate requires a decarboxylation and/or β-oxidation step, followed by subsequent methanogenesis bythe slow growing acetoclastic methanogens (Schink 1997).

During this study it was also important to determine thepH optimum for methanogenesis, which was found to be ina wide range of pH 7.2–8.2 (data not shown). The pH valueplays a central role because it affects the process bothdirectly and indirectly. Directly, because in the first step ofthe anaerobic degradation hydrolytic microorganisms favouran acidic pH but methanogens at the end of the degradationcascade prefer a neutral up to slightly alkaline pH (Layet al. 1998). In a well-working digester both reactionsrun concurrently and thus no significant accumulationsoccur. Indirectly, because the pH changes the ratio ofthe dissociated and undissociated species of VFAs andNH4

+/NH3, which might have toxic effects on themicroorganisms because the latter species are able to diffusethrough the membrane and cause irreversible damage bychanging the intracellular pH (Kadam and Boone 1996).

For the next experiment, increasing concentrations ofNH4

+, ranging from 0 to 720 mmol/L (010.08 g NH4+–N

per litre), were established in the serum bottles (Fig. 2).Based on the previous experiment, 36 mmol/L formatewas chosen as sole carbon source since this concentrationdid bring about a distinct CH4 production and no sign ofstress. An obvious increase of CH4 production with increas-ing concentration of NH4

+ up to 120 mmol/L (∼1.68 gNH4

+–N per litre) could be detected after 3 days of incuba-tion. This might rely on the fact that NH4

+ is a favourablenitrogen source readily available for microorganisms. Atfirst glance the optimum in this study seems to be relativelyhigh but the original biogas plant sludge also contains about2.8 g NH4

+–N per kilogram (Illmer and Gstraunthaler 2009)and the microorganisms might have therefore already adap-ted to high NH4

+ concentrations.Figure 3 shows the effects of different agitation speeds on

the cumulative CH4 and gas production. On day 36 (abouthalf of the incubation time), a significantly higher CH4 andgas production was determined for a moderate agitation(150 rpm) of the lab-scale serum bottles compared to thecontrol. In addition, distinct differences to the other variantswere also obvious; however, they were not significant withall variants. This points to the assumption that CH4

production from complex substrates might favour mod-erate agitation. It increases the liquid–gas transfer and

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distributes the microorganisms and their substrates andend products. Further increase of the agitation speed ledto an inhibition of the degradation and the subsequentCH4 production, which might be directed to physicaldisruption and stress of mutually associated syntrophicmicrobial communities. However, this outcome might

vary with the shape of the bottles used for cultivation. At theend of the experiment on day 77, the gas production hadceased due to a depletion of the substrates, and differencesbetween variants have equalised. During the first day ofincubation a gradual increase of hydrogen production wasmeasured with increasing agitation speed, indicating an

Fig. 2 Effect of increasing concentrations of NH4+ on methane pro-

duction from 36 mmol/L formate after 3 and 5 days of incubation;means ± SD of three replicates

Fig. 3 Changes in cumulative methane and gas production with in-creasing agitation on day 36 after incubation. Characters indicatehomogeneous groups (α00.05); means ± SD of five replicates

Fig. 1 Effects of increasing concentrations of formate (a), acetate (b), propionate (c) and butyrate (d) on methane production after 3 and 5 days ofincubation; means ± SD of three replicates

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interruption of the syntrophic H+-transfer, which again pointsto the importance of agitation.

References

Ahring BK, Sandberg M, Angelidaki I (1995) Volatile fatty acids asindicators of process imbalance in anaerobic digestors. ApplMicrobiol Biotechnol 43:559–565

Illmer P, Gstraunthaler G (2009) Seasonal changes in quantities ofbiowaste on full scale anaerobic digester performance. WasteManag 29:162–167

Illmer P, Schwarzenauer T, Malin C, Wagner AO, Miller LM (2009)Process parameters within a 750,000 litre anaerobic digester dur-ing a year of disturbed fermenter performance. Waste Manag29:1838–1843

Kadam PC, Boone DR (1996) Influence of pH on ammonia accumu-lation and toxicity in halophilic, methylotrophic methanogens.Appl Environ Microbiol 62:4486–4492

Lay JJ, Li YY, Noike T (1998) Influences of pH and moisture contenton the methane production in high-solids sludge digestion. WaterEnviron Res 70:1075–1082

Lins P, Malin C, Wagner AO, Illmer P (2010) Reduction of accumu-lated volatile fatty acids by an acetate-degrading enrichmentculture. FEMS Microbiol Ecol 71:469–478

Malin C, Illmer P (2008) Ability of DNA content and DGGE analysisto reflect the performance condition of an anaerobic biowastefermenter. Microbiol Res 163:503–511

Scherer PA (2001)Mikrobiologie der Vergärung von festen Abfallstoffen.In: Kämpfer P, Weißenfels WD (eds) Biologische Behandlung vonorganischen Abfällen. Springer, Heidelberg, pp 45–80

Schink B (1997) Energetics of syntrophic cooperation in methanogenicdegradation. Microbiol Mol Biol Rev 61:262–280

Wagner AO, Gstraunthaler G, Illmer P (2010) Utilisation of singleadded fatty acids by consortia of digester sludge in batch culture.Waste Manag 30:1822–1827

316 Folia Microbiol (2012) 57:313–316