acclimation of an anaerobic consortium capable of effective biomethanization of mechanically-sorted...

$: Acclimation of an anaerobic consortium capable of effective biomethanization of mechanically-sorted organic fraction of municipal solid waste through a semi-continuous enrichment procedure$
Research ArticleReceived: 17 December 2011 Revised: 3 March 2012 Accepted: 4 March 2012 Published online in Wiley Online Library:

(wileyonlinelibrary.com) DOI 10.1002/jctb.3809

Acclimation of an anaerobic consortiumcapable of effective biomethanizationof mechanically-sorted organic fractionof municipal solid waste througha semi-continuous enrichment procedureLorenzo Bertin,∗ Cristina Bettini, Giulio Zanaroli, Serena Fraraccio,Andrea Negroni and Fabio Fava

Abstract

BACKGROUND: Municipal solid waste is generally disposed of by incineration or landfilling. Higher combustion efficiencies orlower waste amounts to be dumped, respectively, can be obtained through recovery of the waste wet fraction, which couldbe valorized by means of anaerobic digestion processes with the production of a CH4-rich biogas. However, such a fraction istypically poorly digestible. The aim of this work was to obtain a methanogenic consortium capable of effective biomethanizationof the wet fraction collected from a municipal solid waste.

RESULTS: An up-flow recirculated column bioreactor (0.36 L) was initially filled with cattle manure and then, after a short batchworking period, fed with the above mentioned target substrate according to a semicontinuous scheme. More than 90% ofthe overall COD fed during the whole 5-months experiment was depleted. The methane production yield gradually increasedthroughout the process to 0.35 L g−1 of depleted COD. Molecular analysis of the microbial consortium indicated that thearchaeal population, which consisted of two acetoclastic Methanosarcina sp. strains, persisted during the whole experiment,whereas several hydrolytic and acidogenic Firmicutes and Bacteroidetes were enriched throughout the process, concomitantlyto the increase of the biomethanization potential of the consortium.

CONCLUSIONS: An anaerobic consortium capable of efficiently converting the wet fraction of municipal solid waste intomethane was obtained through a dedicated enrichment procedure in an up-flow anaerobic recirculated reactor. This result wasmainly ascribed to the acclimation and enrichment of bacterial species.c© 2012 Society of Chemical Industry

Keywords: anaerobic digestion; mechanically sorted municipal solid waste; denaturing gradient gel electrophoresis; bacterialcommunity; archaea community

INTRODUCTIONAnaerobic digestion (AD) has been extensively proposed duringrecent years as a promising option for the disposal and valorizationof the source sorted organic fraction of municipal solid waste(OFMSW).1 – 3 Biomethanization processes of the wet fractionobtained from mechanical separation processes of municipalsolid waste (MSW) (i.e. mechanically sorted OFMSW, normallydesignated as MS-OFMSW) were also proposed. The developmentof effective AD processes able to biomethanize wet MS-OFMSWwould contribute to promote the separation of wet and dryfractions of MSW: this approach can significantly increase the wastetreatment effectiveness, since the disposal of dried matter wouldallow higher combustion efficiencies or lower waste amountsto be dumped if incineration or landfilling, respectively, areapplied as the treatment procedure. However, most studieswere related to co-digestion processes, in which MS-OFMSWwas fed to the digester together with remarkable amounts

of highly digestible co-substrates,4 – 6 whereas few successfulexamples of direct AD of MS-OFMSW have been reported inthe literature.7 – 14 Furthermore, all these studies were carried outunder batch conditions, often in the presence of recirculatedleachate, with, to the very best of our knowledge, a lackof data related to effective AD (semi)continuous processesdedicated to the direct digestion of MS-OFMSW occurs. Thisevidence can be mostly ascribed to the poor efficiency of ADtowards MS-OFMSW organic matter, which is mainly due tothe occurrence of heavy metals and organics able to adversely

∗ Correspondence to: Lorenzo Bertin, Department of Civil, Environmental andMaterial Engineering (DICAM), Faculty of Engineering, University of Bologna,via Terracini 28, 40131 Bologna, Italy. E-mail: [email protected]

Department of Civil, Environmental and Material Engineering (DICAM),University of Bologna, 40131 Bologna, Italy

J Chem Technol Biotechnol (2012) www.soci.org c© 2012 Society of Chemical Industry

www.soci.org L Bertin et al.

affect key constituents of the microbial community carrying outthe process.15 Recent studies have shown that the inhibitionof methane production due to MS-OFMSW pollutants can bemitigated via tailored acclimation of the AD microbial communityto the MS-OFMSW.5,16 The selection of robust pollutant-resistantAD microbial community can be achieved through a dedicatedenrichment procedure applied to a MS-OFMSW native microfloragrown in the presence of an organic matter-rich biowaste, suchas cattle manure (CM). The last matrix can provide additionalmicroorganisms, promptly fermentable carbon sources andessential nutrients.5

The main purpose of this study was to develop and characterizean anaerobic consortium capable of effective biomethanizationof MS-OFMSW in the absence of co-substrates. To this aim, aculture enrichment strategy based on semicontinuous cultivationof an AD microbial consortium obtained from a preliminary batchco-digestion of CM and MS-OFMSW, in the presence of increasingamounts of MS-OFMSW, was developed. An integrated chemical,microbiological and molecular monitoring strategy was adoptedto assess the biomethanization potential and the structure ofthe microbial community throughout the sequential cultureenrichments.

EXPERIMENTALSubstratesThe experimental MS-OFMSW used in the experiments was ob-tained from the municipal solid waste treatment plant of Ostellato(Ferrara, Italy). It was collected after the following mechanicalpre-treatments of unsorted MSW: chopping, screening, mag-netic deferrization, pressing (via Doppstaadt extruder DSP 20-5,Werner Doppstadt Umwelttechnik GmbH & Co.KG, Germany)and separation of the wet fraction (from the dry one); then,it was diluted with the leachate of a not pressed fraction ofthe same unsorted MSW, biostabilized through a compostingprocess. Before its use, the waste stream resulting from theprevious pretreatment was milled and sieved (cut off: 3 mm).The main chemical-physical features of the resulting experi-mental MS-OFMSW are reported in Table 1. Volatile suspendedsolids (VSS) represented about half of the total suspended solids(TSS), while a large part of the waste soluble COD was dueto volatile fatty acids (VFAs), mainly responsible for its low pH.The experimental MS-OFMSW was found to be contaminated byheavy metals (about 0.5 mg g−1

TSS), in particular Mn (25 µg g−1TSS), Fe

(13 µg g−1TSS) and Al (12 µg g−1

TSS). B, Sr, Cu, Zn and Pb were alsodetected at relevant concentrations (about 7, 7, 4, 3 and 1 µg g−1

TSS,respectively).

The matrix with which the reactor was initially filled was obtainedby milling, sieving (cut off: 3 mm) and diluting a cattle manure (CM)with tap water. As a result of the pretreatment, it contained 3.07%of TSS and 2.24% of VSS, 6.20 g L−1 of soluble COD, 3.81 g L−1 ofVFA and its pH was 6.8 (Table 1).

Reactor features and setupA recirculated up-flow anaerobic reactor was developed consistingof a thermostated glass column (inner diameter 35 mm; height350 mm) whose reaction volume, including the external recycleline, was 0.363 L. The reactor was equipped with an external recycleline, whose flow rate was about 360 mL h−1. The inlet and outletlines intersected the recycle line in the proximity of the columnbottom. The reactor head space (0.050 L) was connected to a‘Mariotte’ bottle by which the volume of the biogas produced wasdetermined. A pH probe (81-04 model, ATI Orion, Boston, MA) wasplaced at the top of the bioreactor. The operational temperaturewas 35 ◦C. The reactor was completely filled with pre-treated CM asdescribed above (‘Substrate’ section) and it was forced to operateunder batch conditions until methane was detected in the biogasproduced (2 weeks).

Experimental approachThe reactor was fed with the MS-OFMSW and was operatedaccording to a semicontinuous scheme in order to achieve theprogressive acclimation of the anaerobic microflora to increasingconcentrations of the waste constituents. To this aim, 62 successiveshort-time batch processes were carried out in an overallexperimental period of 149 days, so that each batch process lastedon average about 58 h. Since the recycling flow ratio allowedcomplete recirculation of the reactor medium in about 1 h, i.e.in a considerably shorter period than that of the short batchprocesses reported above, the reactor broth was assumed tobe homogeneous. At the beginning of each short batch period,about 11 mL of digestate were collected from the outlet line;then, an equal volume of the experimental MS-OFMSW was fedin the system via the inlet line with a high inlet flow, so thatthe feeding step lasted on average about 1 min. The hydraulicretention time (HRT) (which was calculated by consideringhow much time was necessary to feed the rector with anexperimental MS-OFMSW volume corresponding to the reactionvolume) and the organic load rate (OLR) were about 82 daysand 0.7 g L−1 day−1, respectively, (Table 2). Such slow loadingconditions were specifically aimed at obtaining an acclimatedmethanogenic consortium capable of effective biomethanizationof MS-OFMSW. As a consequence of the experimental approach,the organic matter in the bioreactor was a homogenous mixtureof the experimental MS-OFMSW and CM. According to theperfectly mixed reactor hypothesis, and without considering thesubstrate biological consumption, the theoretical amount (g) ofthe experimental MS-OFMSW in the reactor within each shortbatch period was calculated as follows:

(MS − OMMSW)j+1 = (MS − OFMSW)j − (MS − OFMSW)j · Q

V+ (MS − OFMSW)in (1)

where (MS-OFMSW)j represents the waste amount in the reactorcorresponding to the jth experimental period, Q and V represent

Table 1. Main chemical–physical parameters of the organic wastes employed in the study

pHTSS%

VSS%

CODg L−1

Total VFAsgCOD L−1

Acetic acidgCOD L−1

Propionic acidgCOD L−1

Butyric acidgCOD L−1

CM 6.8 3.07 ± 0.12 2.24 ± 0.05 6.20 ± 0.18 3.81 ± 0.19 0.70 ± 0.07 0.48 ± 0.03 0.40 ± 0.02

MS-OFMSW 4.8 4.77 ± 0.31 2.46 ± 0.15 57.51 ± 0.97 29.85 ± 1.41 4.20 ± 0.01 2.39 ± 0.12 6.09 ± 0.46

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Table 2. Main process parameters

Process lengthday

Number of shortbatch cycles

Average feedper cycle mL

HRTday

Inlet CODg L−1

OLRg L−1 day−1

Average CODremoval %

Overall CODremoval %

149 62 10.63 ± 0.16 82.04 57.51 ± 0.97 0.70 26.46 ± 1.16 91.46

the volume of the outlet stream and of the reactor, respectively,and (MS-OFMSW)in represents the amounts of the experimentalMS-OFMSW fed at the beginning of each period by the inlet stream.

The COD removal related to each jth period was calculatedby taking into account the COD occurring in the reactor at thebeginning and at the end of each short batch phase; on the basisof such data, an average COD removal was calculated. High levelsof biodegradation of the MS-OFMSW fed at the beginning ofeach batch period do not necessary result in high COD removals,since the amount of such added matrix represented about 3%of the whole reaction volume. Thus, the persistence of a non-biodegradable fraction in the reactor throughout the experimentwould induce low COD removals. On the other hand, if the latterfraction was very low with respect to the total MS-OFMSW fedduring the whole experiment, high overall COD removal could beachieved anyway, the overall COD removal being calculated byconsidering the overall COD which entered the reactor, includingthat due to the CM that was initially introduced in the column,and the whole amount of COD persisting in the overall effluentcollected at the end of all batch periods.

Analytical methodsTSS and VSS parameters were analyzed according to standardmethods.17 COD values were obtained via catalytic oxidationand spectroscopic analysis following the Hach Mn(III) method.18

The volume of the biogas produced was determined via waterdisplacement in a Mariotte flask, while the biogas compositionwas analyzed daily through a microGC 3000◦ Agilent Technologycoupled with a TCD detector under the following conditions: injec-tor temperature 90 ◦C; column temperature 60 ◦C; sampling time20 s; injection time = 50 ms; column pressure 25 psi; run time 44 s.Nitrogen was employed as the carrier gas phase. VFAs were moni-tored as reported elsewhere.19 Heavy metals were determined byinductively coupled plasma optical emission spectroscopy (ICP-OES). Analytical samples were previously digested with 65% nitricacid until the disappearance of suspended particles; then, the ex-hausted acid was diluted with 1 mol L−1 nitric acid and the liquidsample analyzed by ICP-OES.

Molecular biology analysesA sample of processed waste was collected for molecular analysisafter 7, 28, 51 and 116 days from the start up of the process,i.e. after approximately 0, 1, 2 and 4 months of AD consortiumenrichment. Genomic DNA was extracted from approximately60 mg of pellet obtained after centrifugation of the waste at13 000 rpm for 10 min. DNA was extracted with the UltraCleanSoil DNA kit (Mo Bio Laboratories, Carlsbad, CA, USA) accordingto the manufacturer’s instructions20 as described elsewhere.21

Archeal 16 S rRNA gene DGGE analysis was performed with primersGC-344f and 915r22 and PCR conditions described previously.21

PCR products were resolved with a D-Code apparatus (Bio-Rad,Milan, Italy) on a 7% (w/v) polyacrylamide gel (acrylamide-N,N′-methylenebisacrylamide, 37 : 1) in 1 × TAE with a denaturing

gradient from 40% to 60% denaturant, where 100% denaturantis 7 mol L−1 urea and 40% (v/v) formamide. The electrophoresiswas run at 55 V for 16 h at 60 ◦C. The gel was stained in asolution of 1 × SYBR-Green (Sigma Aldrich, Milwaukee, WI) in1 × TAE for 30 min and its image captured in UV transilluminationwith a digital camera supported by a Gel Doc apparatus (Bio-Rad, Milan, Italy). Bands with the highest intensities, togetherwith those characteristic of a specific DGGE profile, were cutfrom the gel with a sterile scalpel and DNA was eluted in50 µL of sterile deionized water at 4 ◦C for 16 h. 4 µL of thesolution were then used as template to re-amplify the bandfragment using the same primers without the GC-clamp and thesame PCR conditions described above. The bacterial and archaealamplicons obtained were then sequenced with primer 357f and344f, respectively, after amplicon purification with EXOSAP (USBCorporation, Cleveland, Ohio, US) according to the manufacturer’sinstructions. Sequencing reactions and runs were performed byBMR Genomics (Padova, Italy). For each 16 S rRNA gene sequence,the most closely related sequence and the sequence of the mostclosely related cultured bacterial strain were retrieved from theRibosomal Database Project-II website with the SEQMATCH tool.The phylogenetic affiliation of each sequence was retrieved fromthe same website with the CLASSIFIER tool.

RESULTSPerformances of the anaerobic digestion processAccording to Equation (1), which describes the theoretical increasein the experimental MS-OFMSW concentration in the hypotheticalabsence of biological activity, the same target waste would haverepresented almost all of the organic matter occurring in thereactor at the end of the experiment (99.48% of the whole reactorvolume). Thus, the theoretical overall COD concentration, in thecase of no biodegradation processes, would have been higherthan 57 g l−1 at the end of the experiment (Fig. 1). In contrast,the COD of the processed medium slowly decreased during thefirst 46 days of the experiment, from 7.1 to about 4.4 g L−1; then,it remained constant to the end of the study (Fig. 1). The CODremoval observed within each short batch period generally variedin the range 20–35% (average value: 26.46 ± 1.16%, Fig. 1 andTable 2); as a result of such biodegradation activity, the overallCOD removal was 91.46% (Table 2).

The methane volumetric production increased to about0.150 L day−1 during the first month, after which it remainedalmost stable until the end of the experiment (Fig. 2). By combiningthe latter data with COD removals, the profile of methaneproduction yields related to each short time batch periodswere observed to increase throughout the experiment, up toits maximum theoretical value (i.e. 0.35 L g−1, Fig. 2).

The concentration of total VFAs generally followed a similarprofile to that of COD. However, an almost complete acidification ofthe reactor broth was observed during the first week of operations,when VFAs represented up to 90% of the total COD. Such aratio constantly decreased within the first experimental month,

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0

10

20

30

40

50

60

0 30 60 90 120 150

time (day)

CO

D (

mg

L-1

)

0

10

20

30

40

50

60

70

80

90

100

110

120

CO

D r

emov

ed (

%)

Figure 1. Theoretical COD in the hypothetical absence of COD removals(�); contribution to the theoretical COD of MS-OFMSW (�) and CM (♦);actual COD (•); COD removal percentage related to each single short-timebatch period (∗).

0

50

100

150

200

250

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 30 60 90 120 150

CH

4 (m

L L

-1)

CH

4/C

OD

rem

(L

g-1

)

time (day)

Figure 2. Methane production rate (�, L day−1) and yield (♦, L g −1)observed throughout the experiment.

concomitantly with the increase of biomethane production. Afterthe first month of operation, the overall VFAs concentration (about3.1 gCOD L−1) stably represented about 70% of the effluent COD.Also the concentrations of single VFAs were observed to be almoststable after the first experimental month. Acetic acid was alwaysthe main acid among VFA mixture compounds: during the firstmonth, it represented up to 80% of the whole VFA fraction, andtherefore more than 70% of the total COD (day 18); then, itsconcentration was significantly lowered (to 0.75 gCOD L−1), thusrepresenting about 25% of the VFA mixture. After the first month,the amount of acetic acid became comparable with those ofpropionic and butyric acids (0.50 and 0.35 gCOD L−1, respectively),i.e. the other two main VFAs produced.

Main features of the microbial consortium enrichedin the reactorThe samples analyzed were obtained at the start up of thebioreactor and after 7, 28, 51 and 116 days of treatment(m0, m1, m2 and m4, respectively). At the beginning of theenrichment process (m0, Fig. 3(a)), the bacterial communityconsisted of a Corinebacterium phylotype (band 23), 4 phylotypesbelonging to Bacteroidetes (bands 2, 5, 9 and 22), 5 Firmicutes(bands 7, 13, 14, 15 and 16), an Alphaproteobacterium (band20) and a Synergistetes phylotype (band 18) (Fig. 3(a), Table 3).Phylotypes having high sequence similarity to the BacteroidetesRuminobacillus xylanolyticum G1 (band 5) and the FirmicutesSedimentibacter saalensis ZF2 (band 7), Clostridium maritimum G12

(band 15) and uncultured bacterium MBA02 (band 16) were themost abundant, based on relative band intensities. The archaealcommunity (m0, Fig. 3(b)) was represented by 2 DGGE bands (1Aand 2A) having 98% sequence identity to each other and 93%to 94% sequence identity with several aceticlastic Methanosarcinamazei, Methanosarcina barkeri and Methanosarcina lacustris strains.

No differences were observed in the DGGE profile of thearchaeal community throughout the whole enrichment process(Fig. 3(b)). In contrast, a remarkable change in the structure andcomposition of the bacterial community occurred during the firstmonth of enrichment (m1, Fig. 3(a)), as evidenced by the lowSorensen’s similarity index among the DGGE banding patterndetected at month 0 (m0) and month 1 (m1) (0.65). In particular,two Bacteroidetes (band 2 and 22), the Corynebacterium and theSynergistetes phylotypes (bands 23 and 18, respectively) initiallyoccurring in the microbial consortium became undetectable atmonth 1, whereas an unclassified bacterium (band 4), threeBacteroidetes (bands 1, 8 and 10), and five Firmicutes phylotypes(bands 3, 11, 12, 17 and 21) were found to enrich during thesame time (Fig. 3(a), Table 3). Further additions of MS-OFMSWinduced only minor changes in the structure and compositionof the microbial community, since m1 and m2 showed identicalDGGE banding patterns and high Sorensen’s similarity scores wereobserved among the DGGE banding patterns of m1 and m2 withrespect to that of m4 (0.89). In particular, a Bacteroidetes distantlyrelated to the Mucilaginibacter genus (band 6) and a Firmicutes,namely a Synthophomonas sp. (band 19), were detected at month4 (m4) (Fig. 3(a); Table 3), while the relative intensity of bands 4 and8 increased (m4). Finally, based on the relative intensity of bands,the most prominent phylotypes at the end of the enrichment werethe uncultured bacterium 42B BS1 4 (band 4), a Proteiniphilum sp.(band 5), a Sedimentibacter saalensis strain (band 7), two Alistipessp. (bands 8 and 10), a Clostridium sp. (band 11), a Moorella sp. (band16) and a Syntrophomonas zehnderi strain (band 19) (Fig. 3(a)).

DISCUSSIONThe present work was addressed to develop an anaerobicconsortium capable of effective biomethanization of the organicmatter occurring in an experimental MS-OFMSW, which wasdemonstrated to be a very low digestible substrate in preliminarybatch tests (data not shown). The occurrence of heavy metals couldhave significantly contributed to the latter evidence, since theytypically inhibit anaerobic consortia, particularly methanogenicpopulations, at concentrations comparable with those observedin the feed adopted in this study.23 – 25 However, we cannot excludethat other chemicals or/and factors could have adversely affectedbiomethanization of the MS-OFMSW during preliminary tests.

To the above mentioned research aim, a tailored protocol foracclimating a native AD microbial consortium to the experimentalMS-OFMSW was developed. A semicontinuous mode of operation,consisting of a stepwise replacement of digested material withfresh MS-OFMSW, was chosen to expose the microbial communityto an increasing amount of waste by trying, at the same time,to favour the persistence of those members of the microbialcommunity that were capable of growing in the presence ofthe low digestible experimental matrix. All this considered theexperimental approach proposed in this study can be considereda solution for the start-up of effective (semi)continuous anaerobicdigestion processes entirely fed with a low digestible substrate,such as MS-OFMSW. Since effective AD processes fed with MS-OFMSW in the absence of co-substrates were reported only under



1

2

3

4

567891011

121314

1516

19 18

202122

23

17

A

m0 m1 m2 m4

1A

2A

m0 m1 m2 m4

B

Figure 3. PCR-DGGE analysis of the bacterial (A) and archaeal (B) communities throughout the enrichment process. m0, m1, m2 and m4 indicate samplesobtained approximately 0, 1, 2 and 4 months after start up of the process (7, 28, 51 and 116 days, respectively). Arrows indicate bands that were excisedfrom the gel and sequenced. Band numbers and positions are indicated on the right hand side of the picture.

batch conditions,7 – 14 the present work represents an encouragingcontribution to increasing the processing of the target waste inanaerobic digesters. As a consequence of the proposed approach,the microflora occurring in the bioreactor acquired a remarkableability to convert MS-OFMSW COD into biomethane throughoutthe 149 days of semicontinuous treatment. In particular, methaneproduction yield achieved at the end of the study correspondedto the maximum theoretical value (0.35 L g−1) (Fig. 2) and wasaccompanied by quite high overall COD removals (Table 2).Recently, a study dedicated to the selection and acclimation ofan effective methanogenic consortium in the biomethanization ofMS-OFMSW was carried out by continuously feeding an identicallydeveloped reactor with the same experimental matrix employedin the present research.26 Even though encouraging results havebeen obtained, lower biomethane yields and COD removals wereachieved both as a result of a first 3-months run (0.15 L g−1 and84%, respectively), carried out by initially filling the reactor withcattle manure (such as in this study), and of a second run, carriedout under steady state conditions after a further batch period(0.25 L g−1 and 89%, respectively).26 This evidence suggests thatthe semicontinuous approach proposed in this research allowedbetter acclimation of the selected anaerobic consortium. However,a comparison of the performances of the two different approachesshould take into consideration that the process developed undercontinuous conditions was carried out with a lower HRT and ahigher OLR.

Remarkable production of some VFAs was also observedthroughout the experiment; acetic acid, which was the main VFAaccumulated during the first part of the study, decreased duringthe second part of it, probably as a result of increased activity ofacetoclastic methanogens of the enriched AD community.

Interestingly, all main monitored process parameters becamestable after the first month of treatment (Figs 1 and 2). This canbe ascribed to the fact that the organic matter inside the reactorat that time was completely due to MS-OFMSW. This assumptionis apparently not in line with the estimated COD data reported inFig. 1, according to which CM contribution to the whole COD wasstill significant in the second month of operations. However, suchestimation does not include COD depletion due to the biologicalactivity occurring in the reactor.

PCR-DGGE analysis of microbial community prevailing in thereactor throughout its semicontinuous feeding evidenced thatthe archaeal community consisted of acetoclastic methanogensand that it did not change in the presence of increasing amounts ofthe experimental MS-OFMSW (Fig. 3(b)). In contrast, a remarkablechange of the bacterial community structure was observedunder the same conditions (Fig. 3(a)). This strongly suggeststhat the increased experimental MS-OFMSW biomethanizationpotential of the AD microbial community observed throughoutthe study was mainly due to the adaptation of the bacterialpopulation rather than of the archaeal one. This conclusionis supported by the evidence that the main changes in the



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composition of the bacterial community occurred during the firstmonth of operations (Fig. 3(a)), i.e. when major changes in VFAsconcentrations, together with remarkable conversion yields ofCOD into methane, were observed (Figs 1 and 2). Thus, the mainchemical parameters became stable when a stable compositionof the microbial consortium was also achieved. Obtaining aneffective and microbiologically stable methanogenic consortiumrepresented the main target of this study.

Major changes in the bacterial community consisted in thedisappearance of the Actinobacteria and Synergistetes members ofthe population, along with the enrichment of several Firmicutes,which accounted for about 65% of the overall phylotypesdetected at the end of the enrichment process, and fewBacteroidetes. Several members of these phyla are known to havehydrolytic and acidogenic activity.27 In particular, the prominentbands in the DGGE profile of the culture at the end of theenrichment (5, 7, 11, 16, 17, 19) revealed the presence ofstrains related to Proteiniphilum acetatigenes, a proteolytic andamino acids fermenting bacterium that produces mainly acetateand propionate,28 Sedimentibacter saalensis, an amino acidsto acetic and butyric acid fermenting bacterium,29 Clostridiumstraminisolvens and Clostridium clariflavum, two cellulolytic andcellobiose to acetate and lactate fermenting bacteria,30,31 Moorellaperchloratireducens, a sugar to acetate fermenting bacterium32

and Syntrophomonas zehnderi, a syntrophic fatty-acid-oxidizingbacterium.33 In addition, the main phylotype that enrichedthroughout the process (band 4) was distantly related toSyntrophothermus lipocalidus, a syntrophic fatty-acid-oxidizingbacterium isolated from the granular sludge of a thermophilicupflow anaerobic sludge blanket reactor fed with an artificialwastewater containing sucrose, acetate and propionate as themajor carbon sources;34 however, the low similarity among their16 S rRNA gene sequence (77%) and the different temperature ofthe two AD processes in which these phylotypes were detectedsuggest that the contribution of band 4 phylotype to the oxidationof organic matter in our reactor might be different from thatobserved for Syntrophothermus lipocalidus. Finally, other bacterialphylotypes that enriched at the end of the process were relatedto the Alistipes genus (bands 8, 10), whose members are typicallyfound in the human intestinal microbiota35 – 37 and that havenot previously been reported to enrich in AD processes; thus,their possible contribution to the methanization of MS-OFMSWremains unclear. On the basis of the composition of the bacterialcommunity enriched, the overall main fermentation productexpected would be acetate, which is in accordance with thedetection of acetate as the major VFA, among those detected inthe bioreactor, and the detection of aceticlastic methanogens asthe sole Archaea members of the community.

CONCLUSIONSIn conclusion, an anaerobic consortium capable of effectivebiomethanization of a polluted experimental MS-OFMSW recalci-trant to AD was obtained. It is composed of a large variety of ADtypical bacteria along with few acetoclastic archea species. Thesemicontinuous cultivation protocol adopted in this study mightrepresent a novel approach to force the adaptation of AD micro-bial consortia to contaminated organic fractions collected fromunsorted MSW. The results of this research are of special interest interms of applying the acclimated consortium in biotechnologicalprocesses tailored to the valorization of MS-OFMSW obtained bymechanical separation processes.

ACKNOWLEDGEMENTSThe authors thank Dr Andrea Simoni and Professor Claudio Ciavatta(Department of Agroenvironmental Science and Technologies,University of Bologna) for performing heavy metal analyses.Lorenzo Bertin particularly thanks Dr. Filippo Mingozzi (RecuperaS.r.l., Ferrara, Italy) for his help in the collection of the experimentalMS-OFMSW.

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