Rapid start-up of thermophilic anaerobic digestion with the turf fraction of MSW as inoculum

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  • dAust

    Inoculum

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    2011 Elsevier Ltd. All rights reserved.

    c digesdue toet al.,

    tion (Drobic danaerosome

    researchers have succeeded in starting up thermophilic anaero-bic digestion within a month by using anaerobic mesophilicsludge (Lepist and Rintala, 1997; Boukov et al., 2005). How-ever, during the transition of temperature from 37 to 55 C, aproper strategy is necessary, to avoid sludge washout, as a direct

    tally sustainable MSW processing and treatment.The biological conversion of the organic fraction of municipal

    solid waste (OFMSW) to methane has been described to be ableto be accomplished via thermophilic anaerobic digestion (Bolzo-nella et al., 2003; Walker et al., 2006a; Forster-Carneiro et al.,2007, 2008). In this process thermophilic methanogens are be-lieved to be introduced into the reactor at least in part by thefeedstock material (Charles et al., 2009). Based on the ready avail-ability of MSW and the presence of thermophilic methanogens inMSW, MSW could be an alternative start-up seed for thermophilicanaerobic digestion.

    Corresponding author. Present address: Department of Science and Engineering,School of Biological Science and Biotechnology, Murdoch University, South Street,Murdoch, Western Australia 6150, Australia. Tel.: +61 8 93602403; fax: +61 893107084.

    Bioresource Technology 102 (2011) 77627767

    Contents lists availab

    Bioresource T

    elsE-mail address: R.Cord-Ruwisch@murdoch.edu.au (R. Cord-Ruwisch).(De Baere, 2000), due to the difculties in control and start-up pro-cesses. Of these disadvantages, the start-up process is the most sig-nicant drawback of thermophilic anaerobic digestion. Due to thelimited numbers of thermophilic anaerobic reactors, thermophilicinocula are not readily available. Without a source of thermophilicinoculum, the start-up period of thermophilic anaerobic digestioncan be prolonged, with Ahring (1994) reporting up to one yearfor a reactor to reach steady state.

    To decrease the duration of the start-up period, some

    thermophilic anaerobic digestion. Hence, the generation of suitablethermophilic inocula, which are readily available in large quantity,is a worthwhile goal for a research project.

    The last decade has witnessed an increase in the generation ofmunicipal solid waste (MSW). In 2006, the amount of MSW pro-duced globally was 2.02 billion tones, with this amount predictedto have increased by 37% between 2007 and 2011 (Global WasteManagement Market Assessment, 2007). This rapid increase inwaste production has sparked enhanced interest in environmen-1. Introduction

    Currently, thermophilic anaerobias a method to treat organic wastesvolumetric biogas production (Grifn2000) and facilitate pathogen reducwhen compared to mesophilic anaeadvantages, full-scale thermophiliclimited applications, conned to only0960-8524/$ - see front matter 2011 Elsevier Ltd. Adoi:10.1016/j.biortech.2011.06.008tion is of wide interestits potential to increase1998; Ahn and Forster,ugba and Zhang, 1999)igestion. Despite thesebic digestion has hadregions such as Europe

    result of temperature transition, resulting in a start-up periodspanning several months (Fang and Lau, 1996). To start-upnew commercial anaerobic reactors, it is still difcult to obtaina large amount of mesophilic anaerobic seed. This issue is partic-ularly relevant in rural areas where anaerobic digestion is rarelyin operation.

    Despite the increasing interest in thermophilic anaerobic diges-tion, studies of thermophilic methanogenic sources remain limited.Inoculum source is a signicant factor for the successful start-up ofThermophilic anaerobic digestionMSW

    are specied. Stirring of the culture was not conducive for successful start-up as it resulted specicallyin propionate accumulation.Rapid start-up of thermophilic anaerobicas inoculum

    Suwat Suwannoppadol, Goen Ho, Ralf Cord-RuwischFaculty of Sustainability, Environmental and Life Science, Murdoch University, Western

    a r t i c l e i n f o

    Article history:Received 18 March 2011Received in revised form 3 June 2011Accepted 4 June 2011Available online 12 June 2011

    Keywords:TurfRapid start-up

    a b s t r a c t

    This study aims to determiobic digestion. Within dayattempt to narrow down wvacuum cleaner dust, banaphilic methanogenesis witthermophilic acetate degraanaerobically incubated genabling successful start-u

    journal homepage: www.ll rights reserved.igestion with the turf fraction of MSW

    ralia 6150, Australia

    suitable start-up conditions and inoculum sources for thermophilic anaer-f incubation MSW at 55 C, methane was produced at a high rate. In anh components of typical MSW contained the thermophilic methanogens,eel, kitchen waste, and garden waste were tested as inoculum for thermo-etate as the substrate. Results singled out grass turf as the key source ofg methanogenic consortia. Within 4 days of anaerobic incubation (55 C),turf samples produced methane accompanied by acetate degradation

    of thermophilic anaerobic digestion. Other essential start-up conditions

    le at ScienceDirect

    echnology

    evier .com/locate /bior tech

  • The main purpose of this study was to nd a source of thermo-philic methanogens suitable for the rapid and sustained startup ofthermophilic anaerobic digestion that is readily available in largequantities. We dene rapid sustained start-up by the followingparameters:

    i. A short lag time before the production of methane.

    ii. Signicant methane production rate (>0.2 L/L/d) after the lagtime.

    iii. The presence of acetate dependent methanogenesis.

    To succeed in rapid start-up of anaerobic digestion, the lag time

    (Wheaton), performed in duplicate (Sections 3.4 and 3.5) andsealed with butyl rubber stoppers and aluminum crimps. To estab-lish anaerobic conditions, the headspaces of all asks were ushedwith N2/CO2 (80%/20%) for 30 s. All samples were incubated at55 C in either a water bath (Paton, model RW 1812) with shaking(30 oscillations/min (O.P.M.)) or, in Section 3.3, a heating magneticstirrer (RCT basic IKAMAG control stirred at 800 rpm). Followinginitial set-up, all serum vials were depressurized to atmosphericpressure, after the rst hour of incubation.

    To measure methane production in the 1 L Schott bottles, CO2was scrubbed from the biogas by passing it through sodiumhydroxide solution (1 M). The gas stream (assumed to be methane)was measured by the downward displacement of oil (Dow Coming

    sed.

    ayum

    S. Suwannoppadol et al. / Bioresource Technology 102 (2011) 77627767 7763has to be shorter than 1 week. In addition, acetate, the most signif-icant methanogenic substrate must be degraded to ensure a sus-tainable operation. Other aims of this current study were to:

    Quantify the effect of aerobic pretreatment of MSW on thestart-up of thermophilic anaerobic digestion.

    Investigate the effect of stirring on thermophilic start-up. Identify the fraction of MSW responsible for the source of ther-mophilic methanogens.

    2. Methods

    2.1. Inoculum sources

    Different potential sources of methanogens such as cow man-ure, MSW, turf were tested as a seed for start-up of thermophilicanaerobic digestion. The types of inocula used in each experimentare shown in Table 1. The MSW, collected from the Perth Metropol-itan area, Western Australia, was mechanically sorted and theOFMSW (wet bulk density = 578 kg m3; C:N = 18:1; total volatilesolids = 0.56 g/g TS (total solids); moisture content = 55%) preparedas described by Walker et al. (2009).

    Cow manure produced on the day was collected from the veter-inary farm located on the Murdoch University campus, Perth, Wes-tern Australia and used immediately as inoculum. Fresh grasssamples were taken from three different locations: a grass lawnat Murdoch University, a local Perth turf supplier, and from a localhousehold in Perth.

    2.2. Experimental design

    Acetate, as a carbon source for methanogenesis, sodium bicar-bonate (NaHCO3) as buffer, and deionised water, to adjust workingvolume, were used in all experiments as shown in Table 1.Experiments were conducted in either 1 L Schott bottles (Sections3.13.3) sealed with rubber stoppers or in 100 mL serum vials

    Table 1Summary of the types of inocula and initial bicarbonate and acetate concentrations u

    Experiment Types of inocula

    3.1 100 g of Cow manure, or100 g of MSW

    3.2 100 g of MSW, or100 g of pre-aerated MSW,

    3.3 100 g of MSW3.4 3 g of Turf, mixed tree leaves, mixed tree bark, mixed dry soil (aw

    from turf), mixed vegetable wastes, or dust from a household vacucleaner

    3.5 15 g of Turf from different sampling locations, or

    200 g of Turf

    Remark N.A. = Not added. The initial pH of all experiments was between 8.0 and 8.4.200 Fluid 50 CS). In the case of serum vials, the volume of biogasover-pressure produced was measured using a 50 ml glass syringe(Popper & sons, Inc.). Aerobic pre-treatment of MSW samples (Sec-tion 3.2) was performed as described by Walker et al. (2006a).

    2.3. Analysis

    The VFA concentrations of samples were analyzed by gas chro-matography (GC) using a Varian Star 3400 equipped with a Varian8100 auto sampler and a ame ionization detector (FID) as de-scribed by Walker et al. (2009). The methane concentration in bio-gas was analyzed by Varian Star 3400 gas chromatograph (GC)equipped with a thermal conductivity detector as described byCharles et al. (2009). Total solids (TS), volatile solids (VS) andchemical oxygen demand (COD) were performed according toAmerican Public Health Association (2005).

    3. Results and discussion

    3.1. Comparison of thermophilic methanogenic activity in cow manureand MSW

    The knowledge that cow manure is a good source of methano-gens is well established in the literature (Chachkhiani et al.,2004). Moreover, composting of MSW has been reported to pro-duce methane (Beck-Friis et al., 2000; Jckel et al., 2005). This im-plies that methanogens may already be present in MSW. Alsoliterature studies on the anaerobic digestion of MSW reported thatthe development of thermophilic methanogenesis occurred, with-out supplying an external inoculum such as anaerobic digestersludge (Charles et al., 2009).

    To investigate the potential source of thermophilic methano-gens, the following experiment aims at comparing the relativethermophilic methanogenic activity in cow manure with that inMSW. Because cowmanure is deprived of readily degradable mate-rial such as acetate, 80 mM of acetate was added to the tests with

    Bicarbonateconcentrationadded (mM)

    Acetateconcentrationadded (mM)

    Finalworkingvolume (mL)

    Daily addition ofNaOH to control pHat 7.0

    100 80 1000 NO250 N.A. 1000 No100 N.A. 1000 Yes100 N.A. 1000 Yes250 N.A. 1000 No100 30 50 No

    250 30 40 No

    250 100 1000 No

  • cow manure, to ensure the availability of acetate as methanogenicsubstrate.

    As methanogenic acetate conversion (encompassing direct acet-oclastic methanogenesis and syntrophic acetate conversion) is crit-ical for anaerobic digestion, it is relevant to establish whetheracetate dependent methanogenesis is present or not. The thermo-philic incubation of cow manure and MSW produced methane gaswithin 4 days in both cases (Fig. 1A). Over 2 weeks of incubation,methane from MSW was continuously produced until it reached9.5 L/L compared to only about 1.0 L/L from cow manure. Further-more, the batch reactor seeded with cow manure did not lead toacetate degradation (Fig. 1B). This showed that the thermophilicconsortium in cow manure did not serve as a suitable inoculumfor rapid thermophilic acetate degradation, while acetate degrada-tion in the reactor seeded with MSWwas obvious from the volatilefatty acids prole (Fig. 1B).

    By using non-radioactive polymerase chain reaction-singlestrand conformation polymorphism (PCR-SSCP) methods and 16SrDNA sequences, Chachkhiani et al. (2004) revealed that therewere bothMethanosarcina thermophila andMethanoculleus thermo-philus in cow manure. Their initial biogas production rate wasapproximately 0.1 L/L/day after 3 days of cow manures incubationand the methane content in the biogas reached 88% on day 10. Theauthors concluded that the methanogens present within the cowmanure were suitable to start up thermophilic anaerobic digestion.By contrast the current study found that cow manure was not anappropriate thermophilic anaerobic inoculum due to lack of ace-tate degradation during methanogenesis. This indicates that cowmanure does not always contain acetate degrading methanogensor syntrotophs. This was also found by the study by Shin et al.(2004), analyzing Archaea in cow rumen by PCR and 16S rDNA

    sequences, nding that the predominant Archaea belonged to theMethanomicrobiaceae family, which are hydrogen utilizing metha-nogens. This would make sense for the rumen environment, asVFA are the prime feed source for the animal.

    3.2. The effect of aerobic pretreatment of MSW on methane production

    The results above suggest that the MSW collected in the metro-politan area of Perth, Western Australia can be used as an inoculumfor the rapid start-up of thermophilic anaerobic digestion. How-ever MSW digestion can lead to overload conditions resulting in or-ganic acid accumulation and acidication (Charles et al., 2009) andan unsuccessful start-up of anaerobic digestion. To avoid this, someresearchers have applied an aerobic pretreatment to lower the le-vel of easily-degradable carbon in MSW (OKeefe and Chynoweth,2000). As methanogens are strictly anaerobic microorganisms,their survival in such aerobic pretreatment remains unclear. Toinvestigate whether aerobic pretreatment of MSW is benecial tostart-up of a subsequent thermophilic anaerobic digestion, freshMSW was partially composted.

    During two weeks of anaerobic incubation, the aerated MSW(3.7 L/L) produced two times less methane than untreated MSW(8.0 L/L) (Fig. 2). Nevertheless, the aerated MSW sample could stillbe used as an anaerobic inoculum to start-up thermophilic anaer-obic digestion. The aerated pretreatment of MSW causes the oxida-

    FAs

    7764 S. Suwannoppadol et al. / Bioresource Technology 102 (2011) 776277670

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    Fig. 1. Comparison of methane production (A) and volatile fatty acid prole (B)during thermophilic anaerobic incubation of 100 g MSW or cow manure (+80 mMacetate) in a 1 L batch reactor. The initial acetate concentrations were 80 mM forcow manure and 0 mM for MSW respectively. The initial bicarbonate concentra-

    tions were 100 mM and 250 mM for cow manure and MSW respectively. Legend(A): reactor seeded with MSW(d) and Cowmanure (h). Legend (B): closed symbols:MSW, open symbols: cow manure. Acetate ds, Propionate jh, and Butyrate N4.00 2 4 6 8 10 12 14V

    Incubation Time (Days)

    Fig. 2. Effects of aerobic pretreatment of MSW on methane production (A) and VFAproles (B) of thermophilic anaerobic batch digestion of 100 g MSW. Both reactorstion of degradable organics by aerobic microbes and hence isexpected to lower the methane production potential. This observa-tion has been described for MSW during aerobic/anaerobic treat-ment and quantied by electron ow balance by Walker et al.(2006b).

    The aeration pretreatment of 5 days tested here did not affectthe viability of anaerobic acetate degraders in the MSW as theoverall acetate degradation capacity was similar to the controlwithout prior aeration treatment (Fig. 2). When using the initialrates of methane production as an indicator of the number of ac-tive methanogens present (Fig. 2A, days 58), it can be concluded

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    (B)contained 100 mM bicarbonate. Legend (A): reactor seeded with untreated MSW(s) and aerated MSW (j). Legend (B): closed symbols: aerobically pretreated, opensymbols: untreated; Acetate ds, Propionate jh, Butyrate N4.

  • in the presence of acetate and bicarbonate. Methane was not de-tected over a period of 30 days in any of the other samples consist-ing of mixed tree leaves, mixed tree bark, mixed vegetable waste(including banana peel, carrot, potato, mushroom), dust from ahousehold vacuum cleaner, dry soil from 3 separate locations ofMurdoch University campus (away from lawn).

    Within a short incubation of about 4 days the turf grass sampleinitiated methane production. This suggests that the turf grasssample was a signicant source of thermophilic methanogens inthe synthetic MSW.

    3.5. Comparison of methane production of turf grass samples fromdifferent sampling locations

    The turf grass tested in the previous experiment was taken froma grass lawn on the campus of Murdoch University, Perth, WesternAustralia. To establish whether it is a general phenomenon thatgrass lawn contains a signicant inoculum of thermophilic metha-nogens, samples from different grass lawns around Perth weretested. This included grass lawns located on campus, from a localturf supplier and from household gardens around the Perth metro-politan area.

    Results showed that all turf grass samples displayed high ther-mophilic methanogenic activity within 7 days of incubation (Fig. 4)conrming previous results. Acetate analysis after 21 days showedthat of the initial acetate concentration of 30 mM, less than 13 mMwas left in all cases. This acetate degradation suggests that the turfgrass samples contained acetate degrading methanogens or syn-trophic bacteria, which are essential for stable proper start-up ofanaerobic digestion.

    To conrm the presence of acetate driven methanogenesis, VFA

    ce Tthat the aerobic pretreatment did not have a signicant adverse ef-fect on the survival of methanogens. In order to maintain stabledigestion, the untreated reactor required approximately two timesmore (20.1 g/kg solid) sodium hydroxide addition than the aerobi-cally pretreated reactor (12 g/kg solid) for neutralizing pH.

    Although a mechanism of survival of oxygen sensitive methano-gens during aerobic pretreatment is still unclear, a similar resultwas found in the literature. Charles et al. (2009) found that notonly had acetoclastic methanogens survived in MSW during aero-bic treatment but the amount of hydrogenotrophic methanogenshad increased approximately 100-fold during aerobic pretreatmentof MSW.

    Overall results suggest that when microbes present in MSWserve as the sole source of methanogens, an aerobic pretreatmentis an acceptable method to lower the easily degradable carbon inMSW, and reduce the risk of acidication, without adversely affect-ing the methanogenic inoculum present.

    3.3. The effect of stirring during the initial start-up period

    According to the literature, stirring improves anaerobic diges-tion, by

    Providing uniform distribution of organisms and substrates(Lema et al., 1991).

    Enhancing substrate and microbes contact (Smith et al., 1996).

    However, it has also been recommended by some researchers toavoid intense stirring during the start-up phase (McMahon et al.,2001; Stroot et al., 2001; Hoffmann et al., 2008). To clarify thesearguments, this experiment compared vigorously stirred and un-stirred reactors during the start-up phase.

    During the start-up phase of anaerobic digestion, stirring insidethe reactor is an important operational process that needs to beconsidered. Fig. 3 displays that vigorous stirring inhibited propio-nate degradation resulting in its accumulation. Similar results havebeen reported under both thermophilic (Kaparaju et al., 2008) andmesophilic conditions (Stroot et al., 2001). Therefore, to ensure asuccessful start-up process, vigorous stirring should be avoidedas accumulated propionate (>20 mM) has been found to inhibitacetate degradation, especially at low pH (Barredo and Evison,1991).

    3.4. Quantication of viable thermophilic methanogenic activities indifferent fractions of MSW

    The previous results revealed the presence of thermophilicmethanogens in fresh MSW. While there is a signicant variationin MSW, depending on culture, and season, it is generally recog-nized as comprising of similar types of domestic refuse includingfood, fruit and vegetables, and gardening wastes.

    During the past decade, most studies relating to thermophilicmethanogens in compost have focused on potential methane emis-sions, factors that lead to methane emissions, and thermophilicmethanogenic communities in compost materials (Beck-Friiset al., 2000; Jckel et al., 2005; Thummes et al., 2007). However,there has been no research investigating the origin of thermophilicmethanogens in the start-up material such as MSW. The aim of thisexperiment is to test for the presence of thermophilic methanogen-ic activity in the different fractions typical of MSW. Samples offractions typically contained in MSWwere collected and separatelyused as a potential source of methanogens for thermophilic anaer-obic digestion.

    S. Suwannoppadol et al. / BioresourResults clearly indicated that, of the samples tested (all contain-ing 30 mM acetate and 200 mM bicarbonate), only the turf grasssample enabled the development of thermophilic methanogens0

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    Fig. 3. Effects of reactor stirring on start-up of thermophilic anaerobic digestion ofnon-sterile MSW (A) and VFA proles (B) of 200 g of MSW (unstirred and stirredreactor). Both reactors contained 250 mM bicarbonate. Legend (A): unstirredreactor (s) and stirred reactor (j). Legend (B): closed symbols: stirred reactor,open symbols: unstirred reactor; Acetate ds, Propionate jh, Butyrate N4.

    echnology 102 (2011) 77627767 7765degradation proles were recorded during the start-up of a larger1 L reactor inoculated with turf. The time course of methane pro-duction (Fig 5A) and acetate degradation (Fig 5B) demonstrated

  • ce T0

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    Fig. 4. Comparative methane production of 15 g of turf grass samples taken from:Murdoch University (s), local Perth turf supplier (N), and household located in East() and South Perth (h). All assays (40 mL working volume) contained 30 mMacetate and 250 mM bicarbonate.

    7766 S. Suwannoppadol et al. / Bioresourthe presence of acetoclastic methanogenesis or syntrophic acetatedegraders. After an initial increase of acetate (days 19), explainedby the fermentation of plant carbohydrates, acetate was rapidlymethanized at 15 mM/L/day (Fig. 5B). Methane formation stoppedwhen acetate was depleted.

    The spike addition of additional acetate (Fig 5B, day 15) re-sumed acetate conversion to methane showing that a properstart-up of thermophilic anaerobic digestion associated with ace-tate conversion to methane was accomplished. The high methaneproduction rate obtained (1 L/L/d) was in the order of well estab-lished full-scale anaerobic digesters.

    Although methanogens have been found in various environ-ments such as human dental plaque (Kulik et al., 2001), and ricepaddy (Watanabe et al., 2006), the current study shows the rstevidence of the systematic presence of thermophilic methanogensin grass lawn. To explain this nding, further study is requiredaddressing questions such as below:

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    Fig. 5. Methane production (A) and volatile fatty acid prole (B) during thermo-philic anaerobic incubation of 200 g of grass turf in a 1 L batch reactor. The initialacetate and bicarbonate concentration were 100 and 250 mM, respectively. Legend(B): Acetate (s), Propionate (h), Butyrate (4).s Is the observation that thermophilic methanogens are presentin grass lawn consistent globally?

    s What part/s of turf grass: leaf and/or root and/or soil is/are themain source of methanogens?

    s What are the species of methanogens in grass lawn?

    4. Conclusions

    The overall conclusions based on the results of this study can besummarized as follows: (1) MSW was a more effective thermo-philic anaerobic methanogenic seed than cow manure. (2) Withinthe fractions of MSW, it was the turf, or grass clippings, that con-tained the source of thermophilic methanogens for fast digesterstart-up including acetate conversion. (3) During the start-up per-iod, the provision of high buffer capacity and slow stirring is rec-ommended. (4) To minimize the need for buffer or alkali additionduring the start-up, aerobic pretreatment was a suitable method.

    Acknowledgements

    We thank Dr. Lee Walker from AnaeCo for helpful discussionand for providing representative samples of the organic fractionof MSW.

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    S. Suwannoppadol et al. / Bioresource Technology 102 (2011) 77627767 7767

    Rapid start-up of thermophilic anaerobic digestion with the turf fraction of MSW as inoculum1 Introduction2 Methods2.1 Inoculum sources2.2 Experimental design2.3 Analysis

    3 Results and discussion3.1 Comparison of thermophilic methanogenic activity in cow manure and MSW3.2 The effect of aerobic pretreatment of MSW on methane production3.3 The effect of stirring during the initial start-up period3.4 Quantification of viable thermophilic methanogenic activities in different fractions of MSW3.5 Comparison of methane production of turf grass samples from different sampling locations

    4 ConclusionsAcknowledgementsReferences

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