Anaerobic Digestion for Simultaneous Sewage Sludge Treatment and

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Anaerobic Digestion for Simultaneous Sewage Sludge Treatment and CO Biomethanation: Process Performance and Microbial Ecology Gang 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 of both nonrenewable and renewable sources including biomass and coal, and it consists mainly of CO, CO2, and H2. In this paper we aim to bioconvert CO in the syngas to CH4. A novel technology for simultaneous sewage sludge treatment and CO biomethanation in an anaerobic reactor was presented. Batch experiments showed that CO was inhibitory to methanogens, but not to bacteria, at CO partial pressure between 0.25 and 1 atm under thermophilic conditions. During anaerobic digestion of sewage sludge supplemented with CO added through a hollow fiber membrane (HFM) module in continuous thermophilic reactors, CO did not inhibit the process 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 time of 0.2 d. Results from high-throughput sequencing analysis showed clear differences of the microbial community structures between the samples from liquid and biofilm on the HFM in the reactor with CO addition. Species close to Methanosarcina barkeri 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 carboxidotrophic activities test showed that CO was converted by both archaea and bacteria, the bacterial species responsible for CO conversion are unknown.

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INTRODUCTION Anaerobic digestion is a technologically simple and effective biological process for treatment of organic wastes, because it can reduce the environmental impact of wastes and at the same time produce energy in the form of biogas. There are many full scale biogas plants treating sewage sludge from wastewater treatment plants, cattle manure, and others.1−3 However, there are still considerable amounts of organics remaining after digestion (e.g., 50−70% of the organic matter left for sewage sludge and cattle manure4−6). These organics, after dewatering and drying,7 could be further converted to synthetic gas (syngas) by thermal gasification, achieving complete removal of the organics in the wastes.8 The thermal conversion of relatively dry and nonreadily biodegradable organic residues to syngas is attracting much attention for the production of renewable energy. Nowadays, syngas is mostly produced from nonrenewable sources, such as coal.9 Syngas mainly consists of CO, CO2, and H2 when the thermal gasification is conducted in the absence of oxygen or partially combusted in the presence of a limited oxygen supply.10 Although syngas can be used as fuel directly, the volumetric energy density of syngas is only about 50% of natural gas (mainly CH4). The conversion of syngas to methane is an important step to meet the increasing demand for natural gas. Additionally, natural gas infrastructure is well developed and natural gas grids are present in many countries. © 2013 American Chemical Society

There are already several projects in China to produce natural gas from syngas derived from abundant coal resources.11,12 The conversion of CO and H2 to CH4 by microorganisms has been studied previously. Methanosarcina acetivorans, Methanosarcina barkeri, and Methanothermobacter thermautotrophicus are able to convert CO to CH4.13 CO has the potential to be biologically converted to CH4 in a biogas reactor treating sewage sludge or other organic wastes considering the high microbial diversity inside the reactor.8,14 The conversion of H2 to CH4 is a well-known biological reaction occurring in biogas reactors.15 By integrating digestion of wastes/wastewaters with syngas biomethanation in biogas reactors, several advantages can be achieved such as decrease of costs for syngas biomethanation (chemical catalyst additions are not be required) and savings in process energy consumption (mild operation conditions compared to alternative chemical processes). Hydrogen can be either separated from syngas for further usage as fuel and chemical16 or combined with CO for biomethanation to increase CH4 production. In this study, only Received: Revised: Accepted: Published: 10685

March 11, 2013 August 16, 2013 August 16, 2013 August 16, 2013 dx.doi.org/10.1021/es401018d | Environ. Sci. Technol. 2013, 47, 10685−10693

Environmental Science & Technology

Article

then CO was added to the headspace of the closed bottles to achieve corresponding CO partial pressure. For bottles at CO partial pressure of 1 atm, the bottles were purged with pure CO for 10 min. Finally, the initial total pressure in all the above bottles was adjusted to 1.5 atm by injecting N2 in the headspace of the bottles. All the bottles were then placed in a thermostatic shaker at 55 °C. The shaker was controlled at 300 rpm to overcome the gas−liquid mass transfer limitation. The gas composition in each bottle was measured every two days. After each gas composition measurement, the CO in the headspace was refreshed by repeating the above steps to make sure that the CO partial pressure in the headspace of each bottle was close to the set initial value. In addition, the bottles with only inoculum and water were used as negative controls. The bottles with cellulose (1:1 mixture of Avicel and cellulose powder) and inoculum were used as positive controls.19 The cellulose concentration was the same as the VS concentration in the bottles with sewage sludge. All the tests were prepared in triplicate. The production of methane from sewage sludge undergoes hydrolysis, acidification and methanation.20 Therefore, the effect of CO on individual steps was also studied. Batch experiment 2 was conducted to study the effect of CO on hydrolysis and acidification of sewage sludge. Similar procedure was adopted as batch experiment 1. The only difference was that 2-bromoethanesulfonic acid (BES) (25 mM) was added to all the bottles to inhibit the activity of methanogens. The SCOD concentration was monitored as a measure of the extent of hydrolysis (solubilization) of sewage sludge. The acidification was estimated by measuring VFA concentration in the liquid. As aceticlastic methanogenesis is the main pathway during sewage sludge digestion,21 batch experiment 3 was conducted to study the effect of CO on aceticlastic methanogenesis. Similar procedure was adopted as batch experiment 1 except that sodium acetate (30 mM) was used instead of sewage sludge as substrate. Both negative controls and positive controls were included in batch experiments 2 and 3 as batch experiments 1. The only difference was that the COD concentration of added cellulose was the same as the COD concentration of the added sodium acetate in batch experiment 3. Batch experiment 2 was run for 2 days, while batch experiment 3 was run for 12 days. Reactor Operation. Two identical 600 mL continuously stirred tank reactors (CSTR) (A and B) with working volumes of 400 mL were used. A hollow fiber membrane (HFM) module for bubbleless CO distribution was installed in reactor A. The HFM module contained a bundle of 600 microporous polypropylene HFMs with 40% porosity and 0.04 um pore size (Membrana, Germeny). The outside diameter, internal diameter, and length of the fiber were 300 μm, 220 μm, and 20 cm, respectively, providing a total surface area of 1130 cm2 for the HFM module. CO was pumped into the HFM module from a gas bag using gastight tube. The daily CO flow rate was calculated by measuring the initial and residual CO inside the gas bag using a 100 mL gastight syringe. Different CO flow rates were obtained by adjusting the speed of the peristaltic pump. The pressure inside the HFM module was monitored by a gas pressure meter. Initially, both reactors were inoculated with digested sewage sludge and fed with sewage sludge under thermophilic conditions (55 °C) with 10 d hydraulic retention time. The reactors were mixed with a magnetic stirrer at a stirring speed of 150 rpm, and sewage sludge was fed to both reactors once per day. Similar performance was observed in the

biomethanation of CO in the syngas was investigated to test the technical possibilities of the new concept of integrating digestion of wastes/wastewaters with syngas biomethanation in biogas reactors. There are some challenges for bioconversion of CO in biogas reactors, such as potential toxicity of CO to active microorganisms in the biogas reactors. CO has been reported to be toxic for many microorganisms due to its high affinity to metal-containing enzymes.13 The anaerobic process is very complex, involving various microorganisms active in different steps.17 It is crucial to understand the effect of CO on the activity and ecology of the microorganisms in the anaerobic reactor, which determines the degradation of organic wastes in the biogas reactor. The rapid development of next-generation sequencing technologies makes it possible to reveal the diversity and structure of the microbial community, with high sequencing depth. The Ion Torrent PGM (Life Technologies) was launched in early 2011, and it has the highest throughput compared with 454 GS Junior (Roche) and Miseq (Illumina), which makes the sequencing cost-effective and time-saving.18 It can provide significant insight into the microbial community in the anaerobic reactors. Until now, there are no reports describing the microbial ecology in a mixed culture that could convert CO to methane. The microbial community study based on Ion Torrent PGM sequencing would give insight into the microbial ecology involved in the CO conversion. Based on the above considerations, this work aimed to elucidate the possibility to integrate CO biomethanation and sewage sludge treatment in the anaerobic reactor. The effect of CO addition to the anaerobic reactor on the sewage sludge degradation was studied. The changes of microbial community diversity and structure upon CO addition using Ion Torrent PGM sequencing based on the 16S rRNA genes were examined.

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MATERIALS AND METHODS Inoculum and Sewage Sludge. The inoculum used in this study was digested sewage sludge obtained from a thermophilic anaerobic reactor in a wastewater treatment plant (Hillerod, Denmark). The sewage sludge (mixture of primary and secondary sludge) was also obtained from the same 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. The characteristics 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 ± 1.3 g/L, carbohydrates 4.1 ± 0.4 g/L, lipids 2.1 ± 0.3 g/L. For the continuous experiments, the characteristics of the inoculum were 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 sludge were as follows: pH 6.8 ± 0.1, TS 44 ± 1.6 g/L, VS 33.5 ± 1.3 g/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 1 atm were tested in the batch experiments. Three main batch experiments were conducted. Batch experiment 1 was conducted to study the effect of CO on the biomethanation process of sewage sludge. Thirty milliliters of inoculum and 10 mL of sewage sludge were added to 118 mL serum bottles. The bottles were then sealed with butyl stoppers and aluminum crimps. For bottles at CO partial pressures of 0, 0.1, 0.25, and 0.5 atm, they were purged with pure nitrogen for 10 min and 10686

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


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Table 1. Summary of Performance in Reactors A and B during Steady Statesa I (1−35)

a

II (36−67)

A

B

A

gas pressure (bar) CO flow rate (mL/d) CO flow rate (mol CO/(m2·d)) gas RT (d) biogas production rate (mL/(L·d))

0.16 ± 0.02 448 ± 50 0.18 0.9 2232 ± 115

− 0 − − 1101 ± 33

0.38 ± 0.02 985 ± 92 0.39 0.4 3463 ± 298

CH4 CO2 CO CH4 production rate (mL/(L·d)) measured CH4 from CO/theoretical CH4 from CO (%) pH acetate (mM) propionate (mM) VS removal efficiency (%) dissolved CO (μM)

42.2 ± 1.3 56.5 ± 1.2 0 943 ± 66 92.8

62 ± 1.5 36.9 ± 1.6 − 683 ± 25 −

7.10 ± 0.04 0.34 ± 0.02 0.03 ± 0.01 39.4 ± 2.5 1.4 ± 1.2

7.25 ± 0.05 0.38 ± 0.06 0.02 ± 0.02 41.4 ± 3.2 −

III (68−99) B

A

IV (100−131) B

A

B

− 0 − − 1080 ± 38

1.58 ± 0.05 3790 ± 240 1.5 0.1 10375 ± 300

− 0 − − 1036 ± 39

35.5 ± 1.3 63.4 ± 1.2 0 1230 ± 114 97.2

− 0.93 ± 0.03 0 1946 ± 125 − 0.77 − 0.2 1027 ± 22 6075 ± 258 Biogas Composition (%) 61.4 ± 1.1 29.8 ± 1 38.6 ± 1.5 68.9 ± 0.8 − 0 631 ± 18 1811 ± 74 − 93.5

62.1 ± 0.7 36.6 ± 0.9 − 674 ± 29 −

19.2 ± 1.5 44.5 ± 4.4 35.2 ± 4 1992 ± 190 93.7

61 ± 0.9 38.3 ± 0.6 − 628 ± 29 −

7.03 ± 0.02 0.44 ± 0.1 0.06 ± 0.05 37.4 ± 2.8 2.5 ± 1.3

7.28 ± 0.03 0.56 ± 0.09 0.08 ± 0.05 39.5 ± 2.3 −

7.24 ± 0.05 0.31 ± 0.1 0.03 ± 0.02 42.4 ± 3.3 −

7.17 ± 0.02 0.41 ± 0.06 0.02 ± 0.02 37.4 ± 3.5 5.7 ± 2.5

7.29 ± 0.06 0.48 ± 0.09 0.03 ± 0.02 39.4 ± 3.8 −

7.03 ± 0.04 0.30 ± 0.05 0.01 ± 0.01 40.2 ± 3.1 3.3 ± 2.3

“±” means standard deviation. All the calculations were based on five measurements during steady states.

machine with 316 chip using the Ion Sequencing 200 kit (all Life Technologies) according to the standard protocol (Ion Xpress Plus gDNA and Amplicon Library Preparation, Life Technologies). The results were deposited into the DDBJ sequence-read archive database (DRA000940). The low-quality sequences without exact matches to the forward and reverse primers, with length shorter than 100 bp, and containing any ambiguous base calls, were removed from the raw sequencing data by RDP tools23 (http://pyro.cme.msu.edu/). Chimeras were removed from the data by using the Find Chimeras web tool (http://decipher.cee.wisc.edu/FindChimeras.html). The numbers of high quality sequences were 30638 (AL), 34682 (AM), and 10893 (control) for archaea with average length of 144 bp, 153164 (AL), 52293 (AM), and 85696 (control) for bacteria with average length 175 bp. To facilitate the comparison between different samples, the numbers of sequences were normalized to the same sequencing depths (archaeal 10 000 sequences, bacteria 50 000 sequences) by MOTHUR program24 (http://www.mothur.org/wiki/Sub. sample). The sequences were phylogenetically assigned to taxonomic classifications by RDP Classifier with a confidence threshold of 50% (http://rdp.cme.msu.edu/classifier/classifier. jsp). RDP has been widely used in 16s rRNA gene-based microbial community analysis for taxonomic assignment because it is fast and easy to use,22,25,26 and it has generally consistent results with MEGAN, which is also a software for taxonomic assignment.26 The sequences were clustered into operational taxonomic units (OTU) by setting a 0.03 distance limit by MOTHUR program (http://www.mothur.org/wiki/ Mothur_manual). Rarefaction curves, Shannon diversity index, species richness estimator of Chao1, diversity coverage, venn diagrams comparing the number of overlapping OTUs among the samples, and dendrograms based on Bray−Curtis similarity matrix were also generated by MOTHUR program. Analytical Methods. TS, VS, and COD were analyzed according to APHA.27 Protein and carbohydrate were measured according to ref20. The concentrations of acetate, butyrate, and propionate were determined by gas chromatograph (GC) (Hewlett-Packard, HP5890 series II) equipped with a flame ionization 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

two reactors for approximately 1 month; thereafter, continuous CO supply was initiated to reactor A. The operation parameters are shown in Table 1. Specific Methanogenic Activity (SMA) Assays. Specific methanogenic activity assays on specific substrates during steady-state conditions were carried out for both reactors. Five milliliter samples were immediately transferred from the reactors to 20 mL serum bottles. The samples were supplemented with different substrates: acetate (20 mM) or H2/CO2 (80/20, 1 atm). Bottles with reactor samples only, but without substrates, were used as controls. In phase III, carboxydotrophic (CO bioconversion) activity of the sludge from reactor A was tested by using CO/N2 (10/90, 1 atm) as substrate. To determine the CO conversion route, we performed batch experiments, in which 25 mM BES (to inhibit methanogens) was added to the bottles besides CO/N2 (10/ 90, 1 atm).14 All the bottles were incubated in a shaker at 55 °C with shaking speed 300 rpm. All the tests were prepared in triplicate. The SMA and carboxydotrophic activity was calculated as the initial, linear CH4 accumulation, or CO consumption rate divided by the biomass VS content in each series. High-Throughput 16S rRNA Gene Sequencing and Analysis. Samples from liquid (AL) and biofilm (AM) on the HFM in reactor A, and from liquid (control) in the control reactor B, were collected after one month running in phase III, because the solids retention time (SRT) was 10 days and changes in the microbial community composition should be established in three SRTs from a change in conditions.22 In addition, stable performances were also obtained in both reactor A and B after one month running in phase III. Total genomic DNA was extracted from each sample using QIAamp DNA Stool Mini Kit (QIAGEN, 51504) according to the manufacturer’s instructions. PCR was conducted using the extracted DNA (Detailed information about PCR can be found in Supporting Information). The PCR products were purified using the QIAquick spin columns (QIAGEN) to remove the excess primer dimers and dNTPs, and the concentration of PCR amplicons was measured by NanoDrop spectrophotometer.22 Then the samples were sent out for the barcoded libraries preparation and sequencing on an Ion Torrent PGM 10687



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

mm stainless steel column packed with Molsieve SA (10/80). CH4 and CO were analyzed with GC-TCD fitted with paralled columns of 1.1 m × 3/16 in. Molsieve 137 and 0.7 m × 1/4 in. chromosorb 108. Dissolved CO in the liquid was measured according to the method previously reported for dissolved H2 measurement.28 Detailed information about the operation conditions of the above GC was described previously.29 An analysis of variance (ANOVA) was used to test the significance of results, and p < 0.05 was considered to be statistically significant. The gas volume reported in this study was calibrated to standard temperature (273 K) and pressure (1 atm).

CO + H 2O → H 2 + CO2

(ΔGo ′ = −20 kJ/mol CO) (1)

4CO + 2H 2O → CH4 + 3CO2

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(ΔGo ′ = −53 J/mol CO)

RESULTS AND DISCUSSION Effect of CO Partial Pressure on the Anaerobic Digestion of Sewage Sludge. The cumulative CH 4 production during the anaerobic digestion of sewage sludge at different CO partial pressures is shown in Figure 1. The CH4

(2)

To elucidate the effect of CO partial pressure on the individual steps of anaerobic digestion, batch experiments were set up. In batch experiment 2 (BES addition), CO was not consumed during the experimental period (2 days), which was probably due to the lack of acclimatization of the inoculum to CO. It was consistent with batch experiment 1, where the CO was consumed only after around 2 days. The SCOD in the bottles with sewage sludge increased from the initial SCOD around 120 mg/L to around 1500 mg/L in all bottles (Figure S3, Supporting Information). There was no significant difference among the bottles, and it indicated that the addition of CO up to a partial pressure of 1 atm did not affect hydrolysis/solubilization of sewage sludge. The VFA concentration and distribution are shown in Figure S4 (Supporting Information), and the CO partial pressures up to 1 atm did not have influence on the VFA distribution. In batch experiment 3 where acetate was used as the substrate instead of sewage sludge, the CH4 production in the bottle with CO partial pressure 0.1 atm (35.7 ± 1.5 mL) was higher than that in the bottles without CO (28.5 ± 2.4 mL) (Figure S5, Supporting Information). The higher CH4 production was due to the conversion of CO to CH4. By subtracting the CH4 production from CO, the CH4 production in the bottles with 0.1 atm CO was 27.1 ± 2.3 mL, which was close to the values of the bottle without CO addition. Consistently with batch experiment 1, where sewage sludge was used as substrate, further increase of the CO partial pressure resulted in decreased CH4 production. The above data suggest that the CO inhibition of the anaerobic digestion of sewage sludge was mainly due to the inhibition of methanogens. On the contrary, CO did not obviously inhibit the hydrolysis and acidification of sewage sludge in the studied range of CO partial pressure (from 0 to 1 atm). Reactor Performance. The simultaneous anaerobic digestion of sewage sludge and CO biomethanation was then tested in a continuous thermophilic anaerobic reactor. The CO conversion is strongly limited by the gas−liquid mass transfer because of its low solubility.30 Therefore, a hollow fiber membrane module which could transfer the gas to the liquid without bubble31 was used. Different operation conditions were applied, and the reactor performance at steady state at each operation condition is summarized in Table 1. During phase I, the CO pressure inside the HFM was 0.16 atm, which corresponded to a CO flow rate of 448 mL/d and gas retention time of 0.9 d. CO was not detected in the biogas of reactor A, indicating complete consumption of the added CO. An increased 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 CO pressure inside the HFM during phases II and III was followed by a further increase of the CH4 production rate, and complete conversion of CO was still obtained. However, when the CO

Figure 1. Cumulative CH4 production from sewage sludge at different CO partial pressures. NC means negative control; PC means positive control.

production from negative and positive controls were 9.1 ± 1.3 mL and 124 ± 7.6 mL, respectively. Based on this, the CH4 yield from cellulose was calculated as 385 mLCH4/g cellulose, and it was close to the theoretical value 413 mL CH4/g cellulose, which showed that the batch experiments in our study were correctly carried out. When the CO partial pressure was 0.1 atm, the cumulative CH4 production was 18.6% higher than that from control without CO addition. The higher methane production could be attributed to that part of CO that was converted to methane. The consumption of CO is shown in Figure S1, Supporting Information. Previous study showed that CO was converted first to H2 by eq 1 and then to CH4 by anaerobic sludge under thermophilic conditions.14 However, H2 was not detected during the digestion process with CO addition (0.1 atm) (Figure S2, Supporting Information). Therefore, we could assume that the consumed CO was fully converted to CH4 according to eq 2. When the methane production from CO was subtracted from the total methane production, 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 at CO partial pressure 0.1 atm. However, when the CO initial partial pressure was increased to 0.25 atm, serious inhibition to methane production was observed. The total methane production after 16 days digestion was only 71% (CO 0.25 atm) and 50% (CO 0.5 atm) of that from the control. There 10688



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Figure 2. Taxonomic classification of the archaea communities. Relative abundance was defined as the number of sequences affiliated with that taxon divided 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 the reactor 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 were classified as “others”.

The SMA results were shown in Figure S6, Supporting Information. It was obvious that the activities of hydrogenotrophic methanogens were increased upon the addition of CO, while the activities of aceticlastic methanogens were not affected. These results imply that H2 was an intermediate during CO biomethanation. The results were consistent with previous studies where CO was reported to be converted to CH4 by anaerobic sludge under thermophilic conditions and H2 was found to be an intermediate.8,14 The carboxidotrophic activities tested by BES addition which would specifically inhibit methanogens (Table S1, Supporting Information) further demonstrated that H2 was the dominant intermediate. The carboxydotrophic activities of the whole mixed culture and bacteria (with BES addition) were 5.2 ± 1.1 and 2.3 ± 0.7 mMCO/gVS.d (Table S1, Supporting Information), respectively, which showed that CO was converted by bacteria. The lower carboxydotrophic activities with BES addition indicated that archaea also played an important role in the CO conversion. The carboxydotrophic activity of 5.2 mM-CO/gVS.d was relatively lower than that (20.9 mM CO/gVSS.d) reported previously,8 where granular sludge was used under thermophilic condition. It could be due to the fact that in our study the VS contained both microorganisms and undigested sewage sludge, while in that study VSS only contained microorganisms. On the basis of the above results, we conclude that the two processes, i.e., CO and sewage sludge biomethanation, could be achieved simultaneously in the anaerobic reactor without negative impact on each other. Diversity and Structure of the Microbial Communities Revealed by High-Throughput Sequencing. The parameters related to microbial community diversity are shown in Table S2, Supporting Information. The species richness of the liquid phase (AL) and biofilm on the HFM (AM) in reactor A for both archaea and bacteria were higher than those of the liquid phase in reactor B (control), which was reflected by the large numbers of OTUs and Chao 1. The rarefaction curves (Figure S7, Supporting Information) of the three samples at 0.03 distance suggested that the sequencing depths for both archaea (10 000) and bacteria (50000) were still not enough to cover the whole diversity. However, the coverage values for archaea (>98%) and bacteria (>97%) indicated that most common OTUs were detected in our study. The Shannon

pressure was increased to 1.58 atm, CO appeared in the biogas at a concentration of 35.2%. The dissolved CO was measured to be 5.7 μM, which would only correspond to 0.97% CO in the gas phase at thermodynamic equilibrium calculated based on 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 could be due to the CO bubble formation on the surface of the HFM. When the gas pressure inside the HFM was higher than the bubble point, bubbles were formed and accumulated on the membrane surface and were finally released to the gas phase.32 To demonstrate this, an additional experiment was conducted to find the bubble point of the HFM. Bubble formation was observed when the CO pressure was around 1.2 atm. During the operation of reactor A, the CO flow rate was increased with the increase of CO pressure and it was kept relatively stable under each operational condition, which indicated that membrane 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 be easily explained by eq 2. It could be expected that if H2 in the syngas was also added to the reactor, the CH4 content would be higher because H2 could be converted to CH4 with CO2.13 The pH of reactor A (7.0−7.17) was slightly lower than that of reactor B (7.24−7.29) but still in the optimal range for anaerobic digestion. The acetate and propionate concentration in the liquid of both reactors were very low. There was no obvious difference of VS removal efficiency between reactors A and B. The above observations show that CO addition in the present study did not affect the anaerobic digestion of sewage sludge. It could be due to the fast consumption of CO by the microorganisms in reactor A which kept the dissolved CO at a low level (1.4−5.7 uμM). It was consistent with the batch experiment where CO partial pressure even at 0.1 atm (corresponding to 60 μM dissolved CO) did not lead to inhibition on anaerobic digestion of sewage sludge. Assuming that the CH4 produced from sewage sludge was the same from both reactors A and B, we could calculate the CH4 coming from CO by subtracting the CH4 originating from sewage sludge. As shown in Table 1, the conversion efficiencies of CO were between 92% and 97%, which showed that the consumed CO was almost fully converted to CH4. 10689



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Figure 3. Taxonomic classification of the bacteria communities. AM, sample from the biofilm on the HFM in reactor A; AL, sample from the liquid in 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 three samples were classified as “others”.

The archaea mediating hydrogenotrophic and aceticlastic methanogenesis were found mainly within four orders (Methanobacteriales, Methanococcales, Methanomirobiales, and Methanosarcinales).21 Therefore, the sequences of archaea from all the three samples were assigned to the order and genus level using RDP classifier. The order level identification of the archaea communities is illustrated in Figure 2. Although Methanosarcinales dominated (>50%) in all the three samples, clear differences among the samples were found. The hydrogenotrophic methanogens-Methanomicrobiales also represented a dominant portion (18%) of AL, which was consistent with the increased activities of hydrogenotrophic methanogens (Figure S6, Supporting Information). Another order (Methanobacteriales) that mediates hydrogenotrophic methanogenesis was dominant in AM which made up around 39% of the total number of sequences. The results indicate that different microbial community structures were formed depending on the different external factors and conditions applied on the reactors where the three samples were retrieved. The genus level identification showed that Methanosaeta, a strict aceticlastic methanogen genus, was dominant in both AL and control. Dominance of Methanosaeta was also found in the anaerobic reactors treating sewage sludge previously which was attributed to the low levels of acetate.21 However, Methanosaeta comprised only 1.8% of the total number of sequences in AM, while Methanosarcina and Methanothermobacter made up 56.8% and 37.8% of the total number of sequences, respectively. Several known species (Methanosarcina acetivorans, Methanosarcina barkeri, Methanothermobacter thermautotrophi-

diversity index provides not only species richness but also the evenness of the species among all the species in the community. For both archaea and bacteria, AM and AL had higher diversity compared to the control reactor. The above results showed that CO addition to the anaerobic reactor treating sewage sludge increased the diversity of microbial communities in the liquid phase and biofilm on the HFM. It could be attributed to the fact that CO conversion to methane is performed by many different species 33 which would be enriched during the operation of the reactor and thereby increase the microbial diversity. The shared OTUs among the three samples for archaea were only 8, which accounted for 0.7% of the total 926 OTUs (Figure S8, Supporting Information). For archaea, members of AM seem to have little shared OTUs with both AL and control, indicating that very different archaeal community structure was formed in AM compared with AL and control. For bacteria, the shared OTUs among the three samples were higher (18.1% of the total 5974 OTUs). The OTUs belonging to AM and AL, but not to control, might be related to the CO conversion. Differences among the three samples were also assessed by generating dendrograms from Bray−Curtis similarity matrices, which took into account the abundance of sequences in each OTU.34 As shown in Figure S9, Supporting Information, AL and control were clustered together for both archaea and bacteria, while AM was separated from AL and control. It demonstrates that a clearly different microbial community structure was formed on the biofilm of the HFM compared to that for the liquid samples (both AL and control). 10690



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cus) mediating CO biomethanation were found belonging to these two genera.13 We further assigned the sequences to species level by choosing representative sequences from each dominant OTU (sequences >1%). One dominant OTU had a 94% similarity to M. barkeri, and three dominant OTUs had 89−92% similarities to M. thermautotrophicus for sample AM (Table S, Supporting Information3). These four OTUs accounted for 76.5% of the total number of sequences. We could not exclude that the left OTUs also had similarity to these two species. Therefore, at least 76.5% of the sequences could mediate CO biomethanation in AM. M. barkeri (5%) was also found in sample AL. Nevertheless, M. thermautotrophicus was not found in the AL sample, and it could be due to the longer doubling time of M. thermautotrophicus (>200h) compared with M. barkeri (65 h) utilizing CO as sole energy and carbon source,13 which would result in wash out of M. thermautotrophicus from the reactor. Although M. thermautotrophicus has shorter doubling time (around 5 h) utilizing H2 as carbon source (65−70 °C), they were outcompeted by other hydrogenotrophic methanogens due to the suboptimal conditions in the liquid.35 It has been reported that H2 is formed as an intermediate during the conversion of CO to CH4 by both M. barkeri and M. thermautotrophicus.13 H2 can be used either by M. barkeri and M. thermautotrophicus directly or by other hydrogenotrophic methanogens.36 The enrichment of genus Methanolinea in AL, mediating hydrogenotrophic methanogenesis, might be related to the indirect utilization of H2. The phylogenetic classification of bacteria sequences from the three samples is summarized in Figure 3. All samples showed a high diversity reflected by the numbers (16 AL, 15 a.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) which indicated that unknown bacteria existed in the reactors. Firmicutes was dominant in all the samples, and it mainly consisted of the genus Coprothermobacter. The dominance of Coprothermobacter was also found in a previous study treating sewage sludge under thermophilic conditions.37 Coprothermobacter are thermophilic proteolytic bacteria, and its presence was related to the high protein content in the sewage sludge. The difference of phylum distribution was not so obvious between AL and control, but AM showed significant difference compared with that of AL and control. A higher percentage of Proteobacteria in AM (24.4%) was found compared with that in AL (7.3%) and control (9.3%). The class level identification of the bacterial communities further showed that Gammaproteobacteria belonging to Phylum Proteobacteria was enriched in AM. However, most of the sequences belonging to class Gammaproteobacteria in AM had unclassified standing on the genus level. The identified dominant genera (relative abundance higher than 1%) in all the three samples were Fervidobacterium, Anaerobaculum, Stenotrophomonas, Coprothermobacter, and Soehngenia, which were not reported to have the ability to metabolize CO (Table S4, Supporting Information). The known thermophilic bacterial species mediating CO in Table S4 (Supporting Information) were also absent in AM and AL in the other identified minor genera (relative abundance lower than 1%). The carboxidotrophic activities test had demonstrated that there were bacteria mediating CO conversion in the liquid of reactor A. It could be due to the existence of some unknown species mediating CO biomethanation, 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 be deduced that unknown CO-related bacteria might belong to some unclassified phylum because the percentage of unclassified phylum was higher in both AL and AM than in control. It was also possible that the unknown CO-related bacteria might belong to the class Gammaproteobacteria in AM because they were enriched in AM compared with AL and control. However, we could not exclude the possibility that the enrichment of the class Gammaproteobacteria in AM was related to the biofilm formation on the HFM, because a previous study showed that the suspended growth microbial communities structure was different from attached growth (Biofilm) microbial communities structure.38 On the basis of the above results and analysis, we conclude that unknown bacteria species played an important role in the CO conversion. More studies should be carried out to identify the unknown carboxidotrophic bacteria species. Outlook. This study demonstrated a novel technology for simultaneous sewage sludge treatment and CO biomethanation in the anaerobic reactor. Further efforts should be made to improve 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 HFM that we used to supply dissolved CO to the liquid phase due to its relatively low bubble point. It has been reported that dense HFM or composite HFM had a bubble point higher than that of microporous HFM,39 and it could be expected that even lower gas retention time could be applied with full conversion of CO by using another type of HFM. Second, although only the biomethanation of CO was investigated in the present study, the results could be extrapolated to bioconversion of syngas in a biogas reactor. However, syngas has a more complex composition. It also contains H2 which could contribute to additional methane production and other trace compounds such as tar, ethane, ethylene, and acetylene,40 which may affect the bioprocess. Real syngas instead of CO alone should be tested in a future study. For the first time, we applied the newly developed Ion Torrent PGM to sequence the 16s RNA genes of the environmental samples and revealed the diversity and structure of microbial communities that could achieve simultaneous sewage sludge treatment and CO biomethanation. However, the sequencing results can only provide a semiquantitative analysis of the microbial community structures due to PCR bias.41 Direct metagenomic sequencing of the total genomic DNA extracted from the samples by Ion Torrent PGM should be used in the future study to eliminate PCR bias. Furthermore, metagenomic sequencing will provide not only the information of taxonomic diversity but also the functional gene diversity of the microbial communities.42 It will reveal how the CO addition affected the distribution of functional genes (e.g., the genes of Ni-containing CO dehydrogenases, acetyl-CoA synthases, and energy-converting hydrogenases33) in the mixed culture, although we did not identify any known bacteria species related to CO conversion in the present study.

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ASSOCIATED CONTENT

S Supporting Information *

PCR conditions, Tables S1−S4, and Figures S1−S9. This material is available free of charge via the Internet at http:// pubs.acs.org. 10691



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(18) Loman, N. J.; Misra, R. V.; Dallman, T. J.; Constantinidou, C.; Gharbia, S. E.; Wain, J.; Pallen, M. J. Performance comparison of benchtop high-throughput sequencing platforms. Nat. Biotechnol. 2012, 30 (5), 434−+. (19) Hansen, T. L.; Schmidt, J. E.; Angelidaki, I.; Marca, E.; Jansen, J. L.; Mosbaek, H.; Christensen, T. H. Method for determination of methane potentials of solid organic waste. Waste Manage. 2004, 24 (4), 393−400. (20) Yuan, H. Y.; Chen, Y. G.; Zhang, H. X.; Jiang, S.; Zhou, Q.; Gu, G. W. Improved bioproduction of short-chain fatty acids (SCFAs) from excess sludge under alkaline conditions. Environ. Sci. Technol. 2006, 40 (6), 2025−2029. (21) Karakashev, D.; Batstone, D. J.; Angelidaki, I. Influence of environmental conditions on methanogenic compositions in anaerobic biogas reactors. Appl. Environ. Microbiol. 2005, 71 (1), 331−338. (22) Zhang, H.; Banaszak, J. E.; Parameswaran, P.; Alder, J.; Krajmalnik-Brown, R.; Rittmann, B. E. Focused-pulsed sludge pretreatment increases the bacterial diversity and relative abundance of acetoclastic methanogens in a full-scale anaerobic digester. Water Res. 2009, 43 (18), 4517−4526. (23) Cole, J. R.; Wang, Q.; Cardenas, E.; Fish, J.; Chai, B.; Farris, R. J.; Kulam-Syed-Mohideen, A. S.; McGarrell, D. M.; Marsh, T.; Garrity, G. M.; Tiedje, J. M. The Ribosomal Database Project: Improved alignments and new tools for rRNA analysis. Nucleic Acids Res. 2009, 37, D141−D145. (24) Schloss, P. D.; Westcott, S. L.; Ryabin, T.; Hall, J. R.; Hartmann, M.; Hollister, E. B.; Lesniewski, R. A.; Oakley, B. B.; Parks, D. H.; Robinson, C. J.; Sahl, J. W.; Stres, B.; Thallinger, G. G.; Van Horn, D. J.; Weber, C. F. Introducing Mothur: Open-source, platformindependent, community-supported software for describing and comparing microbial communities. Appl. Environ. Microbiol. 2009, 75 (23), 7537−7541. (25) Zheng, X.; Su, Y. L.; Li, X.; Xiao, N. D.; Wang, D. B.; Chen, Y. G. Pyrosequencing reveals the key microorganisms involved in sludge alkaline fermentation for efficient short-chain fatty acids production. Environ. Sci. Technol. 2013, 47 (9), 4262−4268. (26) Ye, L.; Zhang, T. Pathogenic bacteria in sewage treatment plants as revealed by 454 pyrosequencing. Environ. Sci. Technol. 2011, 45 (17), 7173−7179. (27) APHA. Standard methods for the examination of water and wastewater, 19th ed.; American Public Health Association: New York, 1995. (28) Luo, G.; Angelidaki, I. Co-digestion of manure and whey for in situ biogas upgrading by the addition of H2: process performance and microbial insights. Appl. Microbiol. Biotechnol. 2013, 97 (3), 1373− 1381. (29) Luo, G.; Talebnia, F.; Karakashev, D.; Xie, L.; Zhou, Q.; Angelidaki, I. Enhanced bioenergy recovery from rapeseed plant in a biorefinery concept. Bioresour. Technol. 2010, 102 (7), 1310−1313. (30) Munasinghe, P. C.; Khanal, S. K. Syngas fermentation to biofuel: Evaluation of carbon monoxide mass transfer coefficient (k(L)a) in different reactor configurations. Biotechnol. Prog. 2010, 26 (6), 1616− 1621. (31) Sahinkaya, E.; Hasar, H.; Kaksonen, A. H.; Rittmann, B. E. Performance of a sulfide-oxidizing, sulfur-producing membrane biofilm reactor treating sulfide-containing bioreactor effluent. Environ. Sci. Technol. 2011, 45 (9), 4080−4087. (32) Dong, W. Y.; Wang, H. J.; Li, W. G.; Ying, W. C.; Gan, G. H.; Yang, Y. Effect of DO on simultaneous removal of carbon and nitrogen by a membrane aeration/filtration combined bioreactor. J. Membr. Sci. 2009, 344 (1−2), 219−224. (33) Sokolova, T. G.; Henstra, A. M.; Sipma, J.; Parshina, S. N.; Stams, A. J. M.; Lebedinsky, A. V. Diversity and ecophysiological features of thermophilic carboxydotrophic anaerobes. FEMS Microbiol. Ecol. 2009, 68 (2), 131−141. (34) Burke, C.; Thomas, T.; Lewis, M.; Steinberg, P.; Kjelleberg, S. Composition, uniqueness and variability of the epiphytic bacterial community of the green alga Ulva australis. ISME J. 2011, 5 (4), 590− 600.

AUTHOR INFORMATION

Corresponding Author

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

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS

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REFERENCES

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

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Anaerobic Digestion for Simultaneous Sewage Sludge Treatment and

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