Anaerobic Digestion for Simultaneous Sewage Sludge Treatmentand CO Biomethanation: Process Performance and Microbial EcologyGang Luo, Wen Wang, and Irini Angelidaki*

Department of Environmental Engineering, Technical University of Denmark, DK-2800, Kgs Lyngby, Denmark

*S Supporting Information

ABSTRACT: Syngas is produced by thermal gasification ofboth nonrenewable and renewable sources including biomassand coal, and it consists mainly of CO, CO2, and H2. In thispaper we aim to bioconvert CO in the syngas to CH4. A noveltechnology for simultaneous sewage sludge treatment and CObiomethanation in an anaerobic reactor was presented. Batchexperiments showed that CO was inhibitory to methanogens,but not to bacteria, at CO partial pressure between 0.25 and 1atm under thermophilic conditions. During anaerobicdigestion of sewage sludge supplemented with CO addedthrough a hollow fiber membrane (HFM) module incontinuous thermophilic reactors, CO did not inhibit theprocess even at a pressure as high as 1.58 atm inside the HFM,due to the low dissolved CO concentration in the liquid. Complete consumption of CO was achieved with CO gas retention timeof 0.2 d. Results from high-throughput sequencing analysis showed clear differences of the microbial community structuresbetween the samples from liquid and biofilm on the HFM in the reactor with CO addition. Species close to Methanosarcinabarkeri and Methanothermobacter thermautotrophicus were the two main archaeal species involved in CO biomethanation.However, the two species were distributed differently in the liquid phase and in the biofilm. Although the carboxidotrophicactivities test showed that CO was converted by both archaea and bacteria, the bacterial species responsible for CO conversionare unknown.

■ INTRODUCTION

Anaerobic digestion is a technologically simple and effectivebiological process for treatment of organic wastes, because itcan reduce the environmental impact of wastes and at the sametime produce energy in the form of biogas. There are many fullscale biogas plants treating sewage sludge from wastewatertreatment plants, cattle manure, and others.1−3 However, thereare still considerable amounts of organics remaining afterdigestion (e.g., 50−70% of the organic matter left for sewagesludge and cattle manure4−6). These organics, after dewateringand drying,7 could be further converted to synthetic gas(syngas) by thermal gasification, achieving complete removal ofthe organics in the wastes.8 The thermal conversion of relativelydry and nonreadily biodegradable organic residues to syngas isattracting much attention for the production of renewableenergy. Nowadays, syngas is mostly produced from non-renewable sources, such as coal.9 Syngas mainly consists of CO,CO2, and H2 when the thermal gasification is conducted in theabsence of oxygen or partially combusted in the presence of alimited oxygen supply.10 Although syngas can be used as fueldirectly, the volumetric energy density of syngas is only about50% of natural gas (mainly CH4). The conversion of syngas tomethane is an important step to meet the increasing demandfor natural gas. Additionally, natural gas infrastructure is welldeveloped and natural gas grids are present in many countries.

There are already several projects in China to produce naturalgas from syngas derived from abundant coal resources.11,12

The conversion of CO and H2 to CH4 by microorganismshas been studied previously. Methanosarcina acetivorans,Methanosarcina barkeri, and Methanothermobacter thermauto-trophicus are able to convert CO to CH4.

13 CO has thepotential to be biologically converted to CH4 in a biogas reactortreating sewage sludge or other organic wastes considering thehigh microbial diversity inside the reactor.8,14 The conversionof H2 to CH4 is a well-known biological reaction occurring inbiogas reactors.15 By integrating digestion of wastes/waste-waters with syngas biomethanation in biogas reactors, severaladvantages can be achieved such as decrease of costs for syngasbiomethanation (chemical catalyst additions are not berequired) and savings in process energy consumption (mildoperation conditions compared to alternative chemicalprocesses).Hydrogen can be either separated from syngas for further

usage as fuel and chemical16 or combined with CO forbiomethanation to increase CH4 production. In this study, only

Received: March 11, 2013Revised: August 16, 2013Accepted: August 16, 2013Published: August 16, 2013

Article

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© 2013 American Chemical Society 10685 dx.doi.org/10.1021/es401018d | Environ. Sci. Technol. 2013, 47, 10685−10693

biomethanation of CO in the syngas was investigated to test thetechnical possibilities of the new concept of integratingdigestion of wastes/wastewaters with syngas biomethanationin biogas reactors. There are some challenges for bioconversionof CO in biogas reactors, such as potential toxicity of CO toactive microorganisms in the biogas reactors. CO has beenreported to be toxic for many microorganisms due to its highaffinity to metal-containing enzymes.13 The anaerobic processis very complex, involving various microorganisms active indifferent steps.17 It is crucial to understand the effect of CO onthe activity and ecology of the microorganisms in the anaerobicreactor, which determines the degradation of organic wastes inthe biogas reactor.The rapid development of next-generation sequencing

technologies makes it possible to reveal the diversity andstructure of the microbial community, with high sequencingdepth. The Ion Torrent PGM (Life Technologies) waslaunched in early 2011, and it has the highest throughputcompared with 454 GS Junior (Roche) and Miseq (Illumina),which makes the sequencing cost-effective and time-saving.18 Itcan provide significant insight into the microbial community inthe anaerobic reactors. Until now, there are no reportsdescribing the microbial ecology in a mixed culture thatcould convert CO to methane. The microbial community studybased on Ion Torrent PGM sequencing would give insight intothe microbial ecology involved in the CO conversion.Based on the above considerations, this work aimed to

elucidate the possibility to integrate CO biomethanation andsewage sludge treatment in the anaerobic reactor. The effect ofCO addition to the anaerobic reactor on the sewage sludgedegradation was studied. The changes of microbial communitydiversity and structure upon CO addition using Ion TorrentPGM sequencing based on the 16S rRNA genes wereexamined.

■ MATERIALS AND METHODSInoculum and Sewage Sludge. The inoculum used in

this study was digested sewage sludge obtained from athermophilic anaerobic reactor in a wastewater treatmentplant (Hillerod, Denmark). The sewage sludge (mixture ofprimary and secondary sludge) was also obtained from thesame wastewater treatment plant. For the batch experiments,the characteristics of the inoculum were as follows: pH 7.2 ±0.1, TS 23 ± 1.2 g/L, and VS 12.4 ± 1.3 g/L. Thecharacteristics of the sewage sludge were as follows: pH 6.9± 0.1, TS 38 ± 1.2 g/L, VS 28.5 ± 1.7 g/L, proteins 14.2 ± 1.3g/L, carbohydrates 4.1 ± 0.4 g/L, lipids 2.1 ± 0.3 g/L. For thecontinuous experiments, the characteristics of the inoculumwere as follows: pH 7.2 ± 0.1, TS 20 ± 1.3 g/L, and VS 11 ±1.1 g/L, respectively. The characteristics of the sewage sludgewere as follows: pH 6.8 ± 0.1, TS 44 ± 1.6 g/L, VS 33.5 ± 1.3g/L, proteins 16.2 ± 1.4 g/L, carbohydrates 4.5 ± 0.2 g/L,lipids 2.5 ± 0.1 g/L.Effect of CO on the Anaerobic Digestion of Sewage

Sludge. CO initial partial pressures of 0, 0.1, 0.25, 0.5, and 1atm were tested in the batch experiments. Three main batchexperiments were conducted. Batch experiment 1 wasconducted to study the effect of CO on the biomethanationprocess of sewage sludge. Thirty milliliters of inoculum and 10mL of sewage sludge were added to 118 mL serum bottles. Thebottles were then sealed with butyl stoppers and aluminumcrimps. For bottles at CO partial pressures of 0, 0.1, 0.25, and0.5 atm, they were purged with pure nitrogen for 10 min and

then CO was added to the headspace of the closed bottles toachieve corresponding CO partial pressure. For bottles at COpartial pressure of 1 atm, the bottles were purged with pure COfor 10 min. Finally, the initial total pressure in all the abovebottles was adjusted to 1.5 atm by injecting N2 in the headspaceof the bottles. All the bottles were then placed in a thermostaticshaker at 55 °C. The shaker was controlled at 300 rpm toovercome the gas−liquid mass transfer limitation. The gascomposition in each bottle was measured every two days. Aftereach gas composition measurement, the CO in the headspacewas refreshed by repeating the above steps to make sure thatthe CO partial pressure in the headspace of each bottle wasclose to the set initial value. In addition, the bottles with onlyinoculum and water were used as negative controls. The bottleswith cellulose (1:1 mixture of Avicel and cellulose powder) andinoculum were used as positive controls.19 The celluloseconcentration was the same as the VS concentration in thebottles with sewage sludge. All the tests were prepared intriplicate.The production of methane from sewage sludge undergoes

hydrolysis, acidification and methanation.20 Therefore, theeffect of CO on individual steps was also studied. Batchexperiment 2 was conducted to study the effect of CO onhydrolysis and acidification of sewage sludge. Similar procedurewas adopted as batch experiment 1. The only difference wasthat 2-bromoethanesulfonic acid (BES) (25 mM) was added toall the bottles to inhibit the activity of methanogens. TheSCOD concentration was monitored as a measure of the extentof hydrolysis (solubilization) of sewage sludge. The acid-ification was estimated by measuring VFA concentration in theliquid. As aceticlastic methanogenesis is the main pathwayduring sewage sludge digestion,21 batch experiment 3 wasconducted to study the effect of CO on aceticlastic methano-genesis. Similar procedure was adopted as batch experiment 1except that sodium acetate (30 mM) was used instead ofsewage sludge as substrate. Both negative controls and positivecontrols were included in batch experiments 2 and 3 as batchexperiments 1. The only difference was that the CODconcentration of added cellulose was the same as the CODconcentration of the added sodium acetate in batch experiment3. Batch experiment 2 was run for 2 days, while batchexperiment 3 was run for 12 days.

Reactor Operation. Two identical 600 mL continuouslystirred tank reactors (CSTR) (A and B) with working volumesof 400 mL were used. A hollow fiber membrane (HFM)module for bubbleless CO distribution was installed in reactorA. The HFM module contained a bundle of 600 microporouspolypropylene HFMs with 40% porosity and 0.04 um pore size(Membrana, Germeny). The outside diameter, internaldiameter, and length of the fiber were 300 μm, 220 μm, and20 cm, respectively, providing a total surface area of 1130 cm2

for the HFM module. CO was pumped into the HFM modulefrom a gas bag using gastight tube. The daily CO flow rate wascalculated by measuring the initial and residual CO inside thegas bag using a 100 mL gastight syringe. Different CO flowrates were obtained by adjusting the speed of the peristalticpump. The pressure inside the HFM module was monitored bya gas pressure meter. Initially, both reactors were inoculatedwith digested sewage sludge and fed with sewage sludge underthermophilic conditions (55 °C) with 10 d hydraulic retentiontime. The reactors were mixed with a magnetic stirrer at astirring speed of 150 rpm, and sewage sludge was fed to bothreactors once per day. Similar performance was observed in the

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two reactors for approximately 1 month; thereafter, continuousCO supply was initiated to reactor A. The operation parametersare shown in Table 1.Specific Methanogenic Activity (SMA) Assays. Specific

methanogenic activity assays on specific substrates duringsteady-state conditions were carried out for both reactors. Fivemilliliter samples were immediately transferred from thereactors to 20 mL serum bottles. The samples weresupplemented with different substrates: acetate (20 mM) orH2/CO2 (80/20, 1 atm). Bottles with reactor samples only, butwithout substrates, were used as controls. In phase III,carboxydotrophic (CO bioconversion) activity of the sludgefrom reactor A was tested by using CO/N2 (10/90, 1 atm) assubstrate. To determine the CO conversion route, weperformed batch experiments, in which 25 mM BES (to inhibitmethanogens) was added to the bottles besides CO/N2 (10/90, 1 atm).14 All the bottles were incubated in a shaker at 55 °Cwith shaking speed 300 rpm. All the tests were prepared intriplicate. The SMA and carboxydotrophic activity wascalculated as the initial, linear CH4 accumulation, or COconsumption rate divided by the biomass VS content in eachseries.High-Throughput 16S rRNA Gene Sequencing and

Analysis. Samples from liquid (AL) and biofilm (AM) on theHFM in reactor A, and from liquid (control) in the controlreactor B, were collected after one month running in phase III,because the solids retention time (SRT) was 10 days andchanges in the microbial community composition should beestablished in three SRTs from a change in conditions.22 Inaddition, stable performances were also obtained in bothreactor A and B after one month running in phase III. Totalgenomic DNA was extracted from each sample using QIAampDNA Stool Mini Kit (QIAGEN, 51504) according to themanufacturer’s instructions. PCR was conducted using theextracted DNA (Detailed information about PCR can be foundin Supporting Information). The PCR products were purifiedusing the QIAquick spin columns (QIAGEN) to remove theexcess primer dimers and dNTPs, and the concentration ofPCR amplicons was measured by NanoDrop spectrophotom-eter.22 Then the samples were sent out for the barcodedlibraries preparation and sequencing on an Ion Torrent PGM

machine with 316 chip using the Ion Sequencing 200 kit (allLife Technologies) according to the standard protocol (IonXpress Plus gDNA and Amplicon Library Preparation, LifeTechnologies). The results were deposited into the DDBJsequence-read archive database (DRA000940). The low-qualitysequences without exact matches to the forward and reverseprimers, with length shorter than 100 bp, and containing anyambiguous base calls, were removed from the raw sequencingdata by RDP tools23 (http://pyro.cme.msu.edu/). Chimeraswere removed from the data by using the Find Chimeras webtool (http://decipher.cee.wisc.edu/FindChimeras.html). Thenumbers of high quality sequences were 30638 (AL), 34682(AM), and 10893 (control) for archaea with average length of144 bp, 153164 (AL), 52293 (AM), and 85696 (control) forbacteria with average length 175 bp. To facilitate thecomparison between different samples, the numbers ofsequences were normalized to the same sequencing depths(archaeal 10 000 sequences, bacteria 50 000 sequences) byMOTHUR program24 (http://www.mothur.org/wiki/Sub.sample). The sequences were phylogenetically assigned totaxonomic classifications by RDP Classifier with a confidencethreshold of 50% (http://rdp.cme.msu.edu/classifier/classifier.jsp). RDP has been widely used in 16s rRNA gene-basedmicrobial community analysis for taxonomic assignmentbecause it is fast and easy to use,22,25,26 and it has generallyconsistent results with MEGAN, which is also a software fortaxonomic assignment.26 The sequences were clustered intooperational taxonomic units (OTU) by setting a 0.03 distancelimit by MOTHUR program (http://www.mothur.org/wiki/Mothur_manual). Rarefaction curves, Shannon diversity index,species richness estimator of Chao1, diversity coverage, venndiagrams comparing the number of overlapping OTUs amongthe samples, and dendrograms based on Bray−Curtis similaritymatrix were also generated by MOTHUR program.

Analytical Methods. TS, VS, and COD were analyzedaccording to APHA.27 Protein and carbohydrate were measuredaccording to ref20. The concentrations of acetate, butyrate, andpropionate were determined by gas chromatograph (GC)(Hewlett-Packard, HP5890 series II) equipped with a flameionization detector and HP FFAP column (30 m × 0.53 mm×1.0 μm). H2 was analyzed by GC-TCD fitted with a 4.5 m × 3

Table 1. Summary of Performance in Reactors A and B during Steady Statesa

I (1−35) II (36−67) III (68−99) IV (100−131)

A B A B A B A B

gas pressure (bar) 0.16 ± 0.02 − 0.38 ± 0.02 − 0.93 ± 0.03 − 1.58 ± 0.05 −CO flow rate (mL/d) 448 ± 50 0 985 ± 92 0 1946 ± 125 0 3790 ± 240 0

CO flow rate (mol CO/(m2·d)) 0.18 − 0.39 − 0.77 − 1.5 −gas RT (d) 0.9 − 0.4 − 0.2 − 0.1 −

biogas production rate (mL/(L·d)) 2232 ± 115 1101 ± 33 3463 ± 298 1027 ± 22 6075 ± 258 1080 ± 38 10375 ± 300 1036 ± 39

Biogas Composition (%)

CH4 42.2 ± 1.3 62 ± 1.5 35.5 ± 1.3 61.4 ± 1.1 29.8 ± 1 62.1 ± 0.7 19.2 ± 1.5 61 ± 0.9

CO2 56.5 ± 1.2 36.9 ± 1.6 63.4 ± 1.2 38.6 ± 1.5 68.9 ± 0.8 36.6 ± 0.9 44.5 ± 4.4 38.3 ± 0.6

CO 0 − 0 − 0 − 35.2 ± 4 −CH4 production rate (mL/(L·d)) 943 ± 66 683 ± 25 1230 ± 114 631 ± 18 1811 ± 74 674 ± 29 1992 ± 190 628 ± 29

measured CH4 from CO/theoreticalCH4 from CO (%)

92.8 − 97.2 − 93.5 − 93.7 −

pH 7.10 ± 0.04 7.25 ± 0.05 7.03 ± 0.02 7.28 ± 0.03 7.03 ± 0.04 7.24 ± 0.05 7.17 ± 0.02 7.29 ± 0.06

acetate (mM) 0.34 ± 0.02 0.38 ± 0.06 0.44 ± 0.1 0.56 ± 0.09 0.30 ± 0.05 0.31 ± 0.1 0.41 ± 0.06 0.48 ± 0.09

propionate (mM) 0.03 ± 0.01 0.02 ± 0.02 0.06 ± 0.05 0.08 ± 0.05 0.01 ± 0.01 0.03 ± 0.02 0.02 ± 0.02 0.03 ± 0.02

VS removal efficiency (%) 39.4 ± 2.5 41.4 ± 3.2 37.4 ± 2.8 39.5 ± 2.3 40.2 ± 3.1 42.4 ± 3.3 37.4 ± 3.5 39.4 ± 3.8

dissolved CO (μM) 1.4 ± 1.2 − 2.5 ± 1.3 − 3.3 ± 2.3 − 5.7 ± 2.5 −a“±” means standard deviation. All the calculations were based on five measurements during steady states.

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mm stainless steel column packed with Molsieve SA (10/80).CH4 and CO were analyzed with GC-TCD fitted with paralledcolumns of 1.1 m × 3/16 in. Molsieve 137 and 0.7 m × 1/4 in.chromosorb 108. Dissolved CO in the liquid was measuredaccording to the method previously reported for dissolved H2measurement.28 Detailed information about the operationconditions of the above GC was described previously.29 Ananalysis of variance (ANOVA) was used to test the significanceof results, and p < 0.05 was considered to be statisticallysignificant. The gas volume reported in this study was calibratedto standard temperature (273 K) and pressure (1 atm).

■ RESULTS AND DISCUSSIONEffect of CO Partial Pressure on the Anaerobic

Digestion of Sewage Sludge. The cumulative CH4production during the anaerobic digestion of sewage sludgeat different CO partial pressures is shown in Figure 1. The CH4

production from negative and positive controls were 9.1 ± 1.3mL and 124 ± 7.6 mL, respectively. Based on this, the CH4yield from cellulose was calculated as 385 mLCH4/g cellulose,and it was close to the theoretical value 413 mL CH4/gcellulose, which showed that the batch experiments in our studywere correctly carried out. When the CO partial pressure was0.1 atm, the cumulative CH4 production was 18.6% higher thanthat from control without CO addition. The higher methaneproduction could be attributed to that part of CO that wasconverted to methane. The consumption of CO is shown inFigure S1, Supporting Information. Previous study showed thatCO was converted first to H2 by eq 1 and then to CH4 byanaerobic sludge under thermophilic conditions.14 However,H2 was not detected during the digestion process with COaddition (0.1 atm) (Figure S2, Supporting Information).Therefore, we could assume that the consumed CO was fullyconverted to CH4 according to eq 2. When the methaneproduction from CO was subtracted from the total methaneproduction, there was no significant difference (p > 0.05)between the CH4 production with 0.1 atm CO addition (73.8± 6.2 mL CH4) and without CO addition (71 ± 5.2 mL CH4),which indicated that the CH4 production was not inhibited atCO partial pressure 0.1 atm. However, when the CO initialpartial pressure was increased to 0.25 atm, serious inhibition tomethane production was observed. The total methaneproduction after 16 days digestion was only 71% (CO 0.25atm) and 50% (CO 0.5 atm) of that from the control. There

was no obvious CH4 production when the CO partial pressurewas further increased to 1 atm. The above data clearly showedthat CO can inhibit anaerobic digestion of sewage sludge. Onthe basis of the above results, it is important to keep COconcentration in the liquid at a low level to avoid inhibition.

+ → + Δ = −′GCO H O H CO ( 20 kJ/mol CO)2 2 2o

(1)

+ → +

Δ = −′G

4CO 2H O CH 3CO

( 53 J/mol CO)2 4 2

o(2)

To elucidate the effect of CO partial pressure on theindividual steps of anaerobic digestion, batch experiments wereset up. In batch experiment 2 (BES addition), CO was notconsumed during the experimental period (2 days), which wasprobably due to the lack of acclimatization of the inoculum toCO. It was consistent with batch experiment 1, where the COwas consumed only after around 2 days. The SCOD in thebottles with sewage sludge increased from the initial SCODaround 120 mg/L to around 1500 mg/L in all bottles (FigureS3, Supporting Information). There was no significantdifference among the bottles, and it indicated that the additionof CO up to a partial pressure of 1 atm did not affecthydrolysis/solubilization of sewage sludge. The VFA concen-tration and distribution are shown in Figure S4 (SupportingInformation), and the CO partial pressures up to 1 atm did nothave influence on the VFA distribution. In batch experiment 3where acetate was used as the substrate instead of sewagesludge, the CH4 production in the bottle with CO partialpressure 0.1 atm (35.7 ± 1.5 mL) was higher than that in thebottles without CO (28.5 ± 2.4 mL) (Figure S5, SupportingInformation). The higher CH4 production was due to theconversion of CO to CH4. By subtracting the CH4 productionfrom CO, the CH4 production in the bottles with 0.1 atm COwas 27.1 ± 2.3 mL, which was close to the values of the bottlewithout CO addition. Consistently with batch experiment 1,where sewage sludge was used as substrate, further increase ofthe CO partial pressure resulted in decreased CH4 production.The above data suggest that the CO inhibition of the anaerobicdigestion of sewage sludge was mainly due to the inhibition ofmethanogens. On the contrary, CO did not obviously inhibitthe hydrolysis and acidification of sewage sludge in the studiedrange of CO partial pressure (from 0 to 1 atm).

Reactor Performance. The simultaneous anaerobicdigestion of sewage sludge and CO biomethanation was thentested in a continuous thermophilic anaerobic reactor. The COconversion is strongly limited by the gas−liquid mass transferbecause of its low solubility.30 Therefore, a hollow fibermembrane module which could transfer the gas to the liquidwithout bubble31 was used. Different operation conditions wereapplied, and the reactor performance at steady state at eachoperation condition is summarized in Table 1. During phase I,the CO pressure inside the HFM was 0.16 atm, whichcorresponded to a CO flow rate of 448 mL/d and gas retentiontime of 0.9 d. CO was not detected in the biogas of reactor A,indicating complete consumption of the added CO. Anincreased CH4 production rate was observed in reactor A(943 mL/(L·d)) compared with that in reactor B (683 mL/(L·d)). An increase of the CO flow rate by applying higher COpressure inside the HFM during phases II and III was followedby a further increase of the CH4 production rate, and completeconversion of CO was still obtained. However, when the CO

Figure 1. Cumulative CH4 production from sewage sludge at differentCO partial pressures. NC means negative control; PC means positivecontrol.

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pressure was increased to 1.58 atm, CO appeared in the biogasat a concentration of 35.2%. The dissolved CO was measuredto be 5.7 μM, which would only correspond to 0.97% CO inthe gas phase at thermodynamic equilibrium calculated basedon Henry’s law (Henry’s constant of CO 5.87 × 10−4 mol/(L·atm) at 55 °C). The high CO concentration in the biogas couldbe due to the CO bubble formation on the surface of the HFM.When the gas pressure inside the HFM was higher than thebubble point, bubbles were formed and accumulated on themembrane surface and were finally released to the gas phase.32

To demonstrate this, an additional experiment was conductedto find the bubble point of the HFM. Bubble formation wasobserved when the CO pressure was around 1.2 atm. Duringthe operation of reactor A, the CO flow rate was increased withthe increase of CO pressure and it was kept relatively stableunder each operational condition, which indicated thatmembrane fouling was not an issue in this study.The CH4 content in the biogas from reactor A in each phase

was lower than that from reactor B without CO which could beeasily explained by eq 2. It could be expected that if H2 in thesyngas was also added to the reactor, the CH4 content would behigher because H2 could be converted to CH4 with CO2.

13 ThepH of reactor A (7.0−7.17) was slightly lower than that ofreactor B (7.24−7.29) but still in the optimal range foranaerobic digestion. The acetate and propionate concentrationin the liquid of both reactors were very low. There was noobvious difference of VS removal efficiency between reactors Aand B. The above observations show that CO addition in thepresent study did not affect the anaerobic digestion of sewagesludge. It could be due to the fast consumption of CO by themicroorganisms in reactor A which kept the dissolved CO at alow level (1.4−5.7 uμM). It was consistent with the batchexperiment where CO partial pressure even at 0.1 atm(corresponding to 60 μM dissolved CO) did not lead toinhibition on anaerobic digestion of sewage sludge. Assumingthat the CH4 produced from sewage sludge was the same fromboth reactors A and B, we could calculate the CH4 coming fromCO by subtracting the CH4 originating from sewage sludge. Asshown in Table 1, the conversion efficiencies of CO werebetween 92% and 97%, which showed that the consumed COwas almost fully converted to CH4.

The SMA results were shown in Figure S6, SupportingInformation. It was obvious that the activities of hydro-genotrophic methanogens were increased upon the addition ofCO, while the activities of aceticlastic methanogens were notaffected. These results imply that H2 was an intermediateduring CO biomethanation. The results were consistent withprevious studies where CO was reported to be converted toCH4 by anaerobic sludge under thermophilic conditions and H2was found to be an intermediate.8,14 The carboxidotrophicactivities tested by BES addition which would specificallyinhibit methanogens (Table S1, Supporting Information)further demonstrated that H2 was the dominant intermediate.The carboxydotrophic activities of the whole mixed culture andbacteria (with BES addition) were 5.2 ± 1.1 and 2.3 ± 0.7 mM-CO/gVS.d (Table S1, Supporting Information), respectively,which showed that CO was converted by bacteria. The lowercarboxydotrophic activities with BES addition indicated thatarchaea also played an important role in the CO conversion.The carboxydotrophic activity of 5.2 mM-CO/gVS.d wasrelatively lower than that (20.9 mM CO/gVSS.d) reportedpreviously,8 where granular sludge was used under thermophiliccondition. It could be due to the fact that in our study the VScontained both microorganisms and undigested sewage sludge,while in that study VSS only contained microorganisms. On thebasis of the above results, we conclude that the two processes,i.e., CO and sewage sludge biomethanation, could be achievedsimultaneously in the anaerobic reactor without negative impacton each other.

Diversity and Structure of the Microbial CommunitiesRevealed by High-Throughput Sequencing. The param-eters related to microbial community diversity are shown inTable S2, Supporting Information. The species richness of theliquid phase (AL) and biofilm on the HFM (AM) in reactor Afor both archaea and bacteria were higher than those of theliquid phase in reactor B (control), which was reflected by thelarge numbers of OTUs and Chao 1. The rarefaction curves(Figure S7, Supporting Information) of the three samples at0.03 distance suggested that the sequencing depths for botharchaea (10 000) and bacteria (50000) were still not enough tocover the whole diversity. However, the coverage values forarchaea (>98%) and bacteria (>97%) indicated that mostcommon OTUs were detected in our study. The Shannon

Figure 2. Taxonomic classification of the archaea communities. Relative abundance was defined as the number of sequences affiliated with that taxondivided by the total number of sequences per sample. AM, sample from the biofilm on the HFM in reactor A; AL, sample from the liquid in thereactor A; control, sample from the liquid in the reactor B. Order and genus making up less than 1% of total composition in all three samples wereclassified as “others”.

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diversity index provides not only species richness but also theevenness of the species among all the species in the community.For both archaea and bacteria, AM and AL had higher diversitycompared to the control reactor. The above results showed thatCO addition to the anaerobic reactor treating sewage sludgeincreased the diversity of microbial communities in the liquidphase and biofilm on the HFM. It could be attributed to thefact that CO conversion to methane is performed by manydifferent species 33 which would be enriched during theoperation of the reactor and thereby increase the microbialdiversity. The shared OTUs among the three samples forarchaea were only 8, which accounted for 0.7% of the total 926OTUs (Figure S8, Supporting Information). For archaea,members of AM seem to have little shared OTUs with both ALand control, indicating that very different archaeal communitystructure was formed in AM compared with AL and control.For bacteria, the shared OTUs among the three samples werehigher (18.1% of the total 5974 OTUs). The OTUs belongingto AM and AL, but not to control, might be related to the COconversion. Differences among the three samples were alsoassessed by generating dendrograms from Bray−Curtissimilarity matrices, which took into account the abundance ofsequences in each OTU.34 As shown in Figure S9, SupportingInformation, AL and control were clustered together for botharchaea and bacteria, while AM was separated from AL andcontrol. It demonstrates that a clearly different microbialcommunity structure was formed on the biofilm of the HFMcompared to that for the liquid samples (both AL and control).

The archaea mediating hydrogenotrophic and aceticlasticmethanogenesis were found mainly within four orders(Methanobacteriales, Methanococcales, Methanomirobiales, andMethanosarcinales).21 Therefore, the sequences of archaea fromall the three samples were assigned to the order and genus levelusing RDP classifier. The order level identification of thearchaea communities is illustrated in Figure 2. AlthoughMethanosarcinales dominated (>50%) in all the three samples,clear differences among the samples were found. Thehydrogenotrophic methanogens-Methanomicrobiales also repre-sented a dominant portion (18%) of AL, which was consistentwith the increased activities of hydrogenotrophic methanogens(Figure S6, Supporting Information). Another order (Meth-anobacteriales) that mediates hydrogenotrophic methanogenesiswas dominant in AM which made up around 39% of the totalnumber of sequences. The results indicate that differentmicrobial community structures were formed depending onthe different external factors and conditions applied on thereactors where the three samples were retrieved. The genuslevel identification showed that Methanosaeta, a strict acetic-lastic methanogen genus, was dominant in both AL and control.Dominance of Methanosaeta was also found in the anaerobicreactors treating sewage sludge previously which was attributedto the low levels of acetate.21 However, Methanosaetacomprised only 1.8% of the total number of sequences inAM, while Methanosarcina and Methanothermobacter made up56.8% and 37.8% of the total number of sequences,respectively. Several known species (Methanosarcina acetivorans,Methanosarcina barkeri, Methanothermobacter thermautotrophi-

Figure 3. Taxonomic classification of the bacteria communities. AM, sample from the biofilm on the HFM in reactor A; AL, sample from the liquidin the reactor A; control, sample from the liquid in the reactor B. Phylum, class, and genus making up less than 1% of total composition in all threesamples were classified as “others”.

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cus) mediating CO biomethanation were found belonging tothese two genera.13 We further assigned the sequences tospecies level by choosing representative sequences from eachdominant OTU (sequences >1%). One dominant OTU had a94% similarity to M. barkeri, and three dominant OTUs had89−92% similarities to M. thermautotrophicus for sample AM(Table S, Supporting Information3). These four OTUsaccounted for 76.5% of the total number of sequences. Wecould not exclude that the left OTUs also had similarity tothese two species. Therefore, at least 76.5% of the sequencescould mediate CO biomethanation in AM. M. barkeri (5%) wasalso found in sample AL. Nevertheless, M. thermautotrophicuswas not found in the AL sample, and it could be due to thelonger doubling time of M. thermautotrophicus (>200h)compared with M. barkeri (65 h) utilizing CO as sole energyand carbon source,13 which would result in wash out of M.thermautotrophicus from the reactor. Although M. thermauto-trophicus has shorter doubling time (around 5 h) utilizing H2 ascarbon source (65−70 °C), they were outcompeted by otherhydrogenotrophic methanogens due to the suboptimalconditions in the liquid.35 It has been reported that H2 isformed as an intermediate during the conversion of CO to CH4by both M. barkeri and M. thermautotrophicus.13 H2 can be usedeither by M. barkeri and M. thermautotrophicus directly or byother hydrogenotrophic methanogens.36 The enrichment ofgenus Methanolinea in AL, mediating hydrogenotrophicmethanogenesis, might be related to the indirect utilization ofH2.The phylogenetic classification of bacteria sequences from

the three samples is summarized in Figure 3. All samplesshowed a high diversity reflected by the numbers (16 AL, 15a.m., and 17 control) of bacterial phyla detected. However,many sequences could not be assigned to any known phyla(percentage: 14.3% AL, 15.4% AM, and 9.9% control) whichindicated that unknown bacteria existed in the reactors.Firmicutes was dominant in all the samples, and it mainlyconsisted of the genus Coprothermobacter. The dominance ofCoprothermobacter was also found in a previous study treatingsewage sludge under thermophilic conditions.37 Coprothermo-bacter are thermophilic proteolytic bacteria, and its presencewas related to the high protein content in the sewage sludge.The difference of phylum distribution was not so obviousbetween AL and control, but AM showed significant differencecompared with that of AL and control. A higher percentage ofProteobacteria in AM (24.4%) was found compared with that inAL (7.3%) and control (9.3%). The class level identification ofthe bacterial communities further showed that Gammaproteo-bacteria belonging to Phylum Proteobacteria was enriched inAM. However, most of the sequences belonging to classGammaproteobacteria in AM had unclassified standing on thegenus level. The identified dominant genera (relativeabundance higher than 1%) in all the three samples wereFervidobacterium, Anaerobaculum, Stenotrophomonas, Coprother-mobacter, and Soehngenia, which were not reported to have theability to metabolize CO (Table S4, Supporting Information).The known thermophilic bacterial species mediating CO inTable S4 (Supporting Information) were also absent in AM andAL in the other identified minor genera (relative abundancelower than 1%). The carboxidotrophic activities test haddemonstrated that there were bacteria mediating COconversion in the liquid of reactor A. It could be due to theexistence of some unknown species mediating CO biometha-nation, and it was also reflected by the high percentage of

unclassified sequences at the genus level (48.4% in AM, 25.5%in AL). By comparison of AL, AM, and control, it could bededuced that unknown CO-related bacteria might belong tosome unclassified phylum because the percentage ofunclassified phylum was higher in both AL and AM than incontrol. It was also possible that the unknown CO-relatedbacteria might belong to the class Gammaproteobacteria in AMbecause they were enriched in AM compared with AL andcontrol. However, we could not exclude the possibility that theenrichment of the class Gammaproteobacteria in AM was relatedto the biofilm formation on the HFM, because a previous studyshowed that the suspended growth microbial communitiesstructure was different from attached growth (Biofilm)microbial communities structure.38 On the basis of the aboveresults and analysis, we conclude that unknown bacteria speciesplayed an important role in the CO conversion. More studiesshould be carried out to identify the unknown carboxidotrophicbacteria species.

Outlook. This study demonstrated a novel technology forsimultaneous sewage sludge treatment and CO biomethanationin the anaerobic reactor. Further efforts should be made toimprove the technology to accelerate its industrial application.First of all, the CO conversion efficiency needs to be increased.The limiting step in our study was the efficiency of the HFMthat we used to supply dissolved CO to the liquid phase due toits relatively low bubble point. It has been reported that denseHFM or composite HFM had a bubble point higher than thatof microporous HFM,39 and it could be expected that evenlower gas retention time could be applied with full conversionof CO by using another type of HFM. Second, although onlythe biomethanation of CO was investigated in the presentstudy, the results could be extrapolated to bioconversion ofsyngas in a biogas reactor. However, syngas has a more complexcomposition. It also contains H2 which could contribute toadditional methane production and other trace compoundssuch as tar, ethane, ethylene, and acetylene,40 which may affectthe bioprocess. Real syngas instead of CO alone should betested in a future study.For the first time, we applied the newly developed Ion

Torrent PGM to sequence the 16s RNA genes of theenvironmental samples and revealed the diversity and structureof microbial communities that could achieve simultaneoussewage sludge treatment and CO biomethanation. However,the sequencing results can only provide a semiquantitativeanalysis of the microbial community structures due to PCRbias.41 Direct metagenomic sequencing of the total genomicDNA extracted from the samples by Ion Torrent PGM shouldbe used in the future study to eliminate PCR bias. Furthermore,metagenomic sequencing will provide not only the informationof taxonomic diversity but also the functional gene diversity ofthe microbial communities.42 It will reveal how the COaddition affected the distribution of functional genes (e.g., thegenes of Ni-containing CO dehydrogenases, acetyl-CoAsynthases, and energy-converting hydrogenases33) in themixed culture, although we did not identify any known bacteriaspecies related to CO conversion in the present study.

■ ASSOCIATED CONTENT

*S Supporting InformationPCR conditions, Tables S1−S4, and Figures S1−S9. Thismaterial is available free of charge via the Internet at http://pubs.acs.org.

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■ AUTHOR INFORMATION

Corresponding Author*Phone: +45 4525 1429; fax: +45 4593 2850; e-mail: iria@env.dtu.dk.

NotesThe authors declare no competing financial interest.

■ ACKNOWLEDGMENTS

This study was funded by an individual postdoctoral grant fromThe Danish Council for Independent Research (12-126632),and the Interreg IVA programme Bioref-Øresund financed byEU.

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