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Methane production and microbial community structure for alkaline pretreated waste activated sludge
Bioresource Technology 169 (2014) 496–501
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Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Methane production and microbial community structure for alkaline pretreated waste activated sludge Rui Sun, Defeng Xing, Jianna Jia, Aijuan Zhou, Lu Zhang, Nanqi Ren ⇑ State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
h i g h l i g h t s Alkali-pretreatment enhanced CH4 production in the anaerobic digestion of sludge. Alkali-pretreatment improved the sludge reduction in the anaerobic digestion. The shift of microbial community resulted in CH4 increase after alkali-pretreatment.
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Article history: Received 16 May 2014 Received in revised form 28 June 2014 Accepted 4 July 2014 Available online 16 July 2014 Keywords: Waste activated sludge (WAS) Alkaline pretreatment Methane production Microbial community 454 pyrosequencing
a b s t r a c t Alkaline pretreatment was studied to analyze the influence on waste activated sludge (WAS) reduction, methane production and microbial community structure during anaerobic digestion. Methane production from alkaline pretreated sludge (A-WAS) (pH = 12) increased from 251.2 mL/L d to 362.2 mL/L d with the methane content of 68.7% compared to raw sludge (R-WAS). Sludge reduction had been improved, and volatile suspended solids (VSS) removal rate and protein reduction had increased by 10% and 35%, respectively. The bacterial and methanogenic communities were analyzed using 454 pyrosequencing and clone libraries of 16S rRNA gene. Remarkable shifts were observed in microbial community structures after alkaline pretreatment, especially for Archaea. The dominant methanogenic population changed from Methanosaeta for R-WAS to Methanosarcina for A-WAS. In addition to the enhancement of solubilization and hydrolysis of anaerobic digestion of WAS, alkaline pretreatment showed significant impacts on the enrichment and syntrophic interactions between microbial communities. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Large amounts of waste activated sludge (WAS) were produced in China every year. The management and disposal of sewage sludge was estimated to account for up to 50% of the total operation and maintenance costs of waste water treatment plants. The current disposal costs were reported to be 200–650 €/t of dry weight (Foladori et al., 2010). Therefore, excess sludge, as the main by-product of the wastewater treatment plants, has become one of the most challenging issues. The conventional methods such as landfilling, incineration and beneficial uses are less preferable due to the secondary pollution, capital intensive costs and stricter regulatory pressure (Mahmood and Elliott, 2006). Anaerobic digestion of sludge which converted sludge to water, methane and carbon dioxide has gained widely attention for being a disposal ⇑ Corresponding author. Address: School of Municipal and Environmental Engineering, Harbin Institute of Technology, P.O. Box 2614, 73 Huanghe Road, Nangang District, Harbin 150090, China. Tel./fax: +86 451 86282008. E-mail addresses: [email protected] (D. Xing), [email protected] (N. Ren). http://dx.doi.org/10.1016/j.biortech.2014.07.032 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
route and energy recovery route simultaneously. With about 80% of biodegradable organic energy being converted to methane, anaerobic digestion was considered as a promising sludge reduction method for pulp-mill WAS and a highly competitive alternative for the treatment of organic solid poultry slaughter house waste (Salminen and Rintala, 2002). Due to the extensive (20–30 days) retention times and relatively low degradation efficiency (30–50%) (Lin et al., 2009), various pretreatment methods such as physical, chemical or biological pretreatment methods (or their combination) were researched to improve the digestion efficiency and sludge reduction (Vigueras-Carmona et al., 2011). While thermal, ultrasonic and enzyme pretreatment of sludge are considered to be very expensive due to the high energy demand, alkaline pretreatment was a well-known method with benefits such as simple devices, easy to operate and high efficiency (Li et al., 2012). The impact of alkaline pretreatment of sludge on the improvement of volatile suspended solids (VSS) reduction and methane production had been reported in a previous research (Li et al., 2012). 38.3% organic degradation rate and 0.65 L/gVSS biogas yield was obtained by 0.1 mol/L NaOH pretreated sludge
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(Li et al., 2012). It was also concluded that by appropriate pH pretreatment, sludge reduction and biogas production were increased by 10% and sludge dewater ability was also improved. By alkaline pretreatment, the organic compounds of sludge were destroyed or became more susceptible to enzymatic and chemical attack, which led to the enhancement of solubilization and hydrolysis (Xiao et al., 2013). However, information on the influence of alkaline pretreatment on microbial community shift in WAS was rather limited in addition to the well documented physicochemical characteristics changes (Jie et al., 2014). As a biochemical process, the performance of anaerobic digestion is highly related to the concerted activity of the interacting microorganisms including bacteria groups and archaeal groups. During anaerobic digestion, the first hydrolysis and acidogenesis process were carried out by bacteria groups and the following methanogenesis was performed by Archaea groups (Shin et al., 2010). Using denaturing gradient gel electrophoresis (DGGE) analysis, Proteocatella, Tepidibacter, and Clostridium were found to disappear when paper mill anaerobic granular sludge were pretreated at pH 9 or 10 (Huang et al., 2014), while uncultured bacterium was reported to be capable of adapting well at high pH (Jie et al., 2014). However, molecular biology methods such as DGGE often powerless to capture the comprehensive information in microbial communities due to lack of sufficient throughput (Lu et al., 2012a). 454 pyrosequencing is now increasingly chosen to provide potentially detailed insight into the diverse microbial ecosystem. In this study, the effect of alkaline pretreatment on sludge hydrolysis and methane production was investigated. The performance of the anaerobic digestion process was estimated by gas production, volatile fatty acids (VFAs) and volatile organics reduction efficiency. The microbial community structure and distribution of micro-organisms in alkaline pretreated sludge were assessed using 454 pyrosequencing.
2. Methods 2.1. Sludge samples, pretreatment and fermentation WAS was collected from the secondary sedimentation tank in Wenchang Wastewater Treatment Plant in Harbin. After 24 h sedimentation, the upper sewage was abandoned (R-WAS). The alkaline pretreated WAS (A-WAS) was adjusted to pH 12 by adding 4 M NaOH. The specific alkaline dosage was 0.92 ± 0.01 g NaOH per gram of volatile suspended solids (VSS). The characteristics of R-WAS and A-WAS were shown in Table 1. The eventual pH of the alkaline pretreated sludge was around 9–10 after another 24 h sedimentation 400 mL untreated and alkaline pretreated sludge were put into narrow-mouth digesters with effective volume of 1 L and a retention time of 16 days. To create anaerobic environment, N2 was purged for 5 min into the sludge and the top part of the bottle for removing the remained air. Then the
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sludge samples were cultured in incubator shaker at the rate of 90 r/min and temperature of 35 ± 1 °C. The sludge was sampled for further analysis on a daily basis during the anaerobic digestion process. All the experiments were performed in triplicate and mean values were applied. 2.2. Analysis methods TCOD, SCOD, TS, VS, TSS, VSS were analyzed according to the standard methods (American Public Health Association (APHA, 1998)). For the measurement of SCOD, soluble carbohydrate and soluble protein, the sludge samples were centrifuged, and the obtained supernatant were then filtered by 0.45 lm filter membrane. The filtrate was analyzed for SCOD by potassium dichromate method. As for most carbohydrates in the samples were polysaccharide, phenol – sulfuric acid colorimetric method was chosen to analyze carbohydrates concentration (Herbert et al., 1971). The protein concentration was determined using Bicinchoninic Acid Protein Assay Kit (Sigma–Aldrich) (Smith et al., 1985). The pH was measured by Shang Hai Lei Ci PHS-2F type pH meter. To analyze VFAs, gas chromatograph from U.S. Agilent Company (GC 4890) was applied. And methane content in produced biogas was determined by FULI 9790 II gas chromatograph. The gas yield and production rate were all calculated based on the gas chromatograph results. 2.3. DNA extraction, PCR amplification and 454 Pyrosequencing Alkaline pretreated and raw sludge samples were collected from the anaerobic digesters after the anaerobic digestion process for microbial community analysis. PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) was used to extract the genomic DNA of the sludge samples according to the manufacturer’s instructions. the V1–V3 region (length of 455 bp) of the bacterial 16S rRNA gene was amplified using a set of primers: 8F (50 -AGA GTTTGATCCTGGCTCAG-30 ) and 533R (50 -TTACCGCGGCTGCTGG CAC-30 ), as well as the 454 adapter A and B, barcode and linker sequence were added as previous descriptions (Lu et al., 2012a). 454 GS-FLX sequencer was then applied for pyrosequencing according to standard protocols (Lu et al., 2012a). With similarity set at 97% and a confidence threshold of 95%, the obtained sequences were phylogenetically allocated down to the phylum, class and genus level with the MOTHUR program (http://www.mothur.org/wiki/Main_Page). To define the relative abundance of a given phylogenetic group, the number of sequences affiliated to that group was divided by the total number of obtained sequences. The obtained results were used for the analysis and comparison of microbial community structure and bacterial diversity of the sludge samples before and after alkaline pretreatment. 3. Results
Table 1 Characteristics of buffered R-WAS and A-WAS.
3.1. SCOD concentration and SCOD/TCOD rate
Parameter
R-WAS
A-WAS
pH Moisture content (%) Total solids (TS, mg/L) Volatile solids (VS, mg/L) Total suspended solids (TSS, mg/L) Volatile suspended solids (VSS, mg/L) Total chemical oxygen demand (TCOD, mg/L) Soluble chemical oxygen demand (SCOD, mg/L) Soluble carbohydrate (mg COD/L) Soluble protein (mg COD/L)
6.80 ± 0.1 95.19 ± 3.1 25610 ± 71 17490 ± 269 19720 ± 15 11440 ± 13 30378 ± 929 292 ± 5 74 ± 2 337 ± 13
9.14 ± 0.1 95.30 ± 2.8 24500 ± 707 17890 ± 184 19220 ± 14 10200 ± 7 27900 ± 907 2590 ± 79 213 ± 25 6487 ± 427
Two stages were noticed during sludge solubilization (Fig. 1). During Phase I (the first two days), SCOD concentration of both A-WAS and R-WAS increased quickly, which indicated the rapid solubilization of waste sludge during anaerobic digestion. From Day 3 to Day 16 (Phase II), the SCOD concentration began to decrease slowly with an average value of 5000 mg/L for A-WAS and 3500 mg/L for R-WAS. Previous study also reported 3 phases during sludge solubilization process (Su et al., 2013). The differences could be caused by different operation condition or different sludge origins. SCOD of A-WAS was higher compared to untreated
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enzymes and weaken the biodegradability of protein (Fang and Yu, 2000). 3.3. VFAs production and composition
Fig. 1. Changes of SCOD concentration and SCOD/TCOD rate during WAS digestion.
sludge during the 16 days (Fig. 1) which meant more extracellular polymer and intracellular substances dissolved out of the cell by alkaline pretreatment. Digestibility of waste activated sludge was hence enhanced due to the additional easily degradable substrate released by alkaline pretreatment for microbial growth. On Day 2, SCOD of both A-WAS and R-WAS reached the maximum value which was 8567.9 mg/L and 5219.6 mg/L, respectively. 2 days were considered to be the most efficient treatment duration for sludge solubilization in our study. Yet 4 or 5 days were also reported in previous literatures due to different pH or operation condition (Su et al., 2013; Wu et al., 2009). The solubilization efficiencies of wasted activated sludge which was demonstrated by the value of SCOD/TCOD nearly doubled after alkaline pretreatment, especially for the first 10 days. The maximum solubilization efficiencies of A-WAS and R-WAS were 31% and 15% at Day 2.
Acetic, butyric, propionic, iso-butyric, iso-valeric acids, valeric acids and ethanol were the main type of VFAs produced in the WAS anaerobic digestion process in our study (Fig. 3). VFAs concentration of A-WAS was higher than that of R-WAS. The maximum VFAs concentration of A-WAS and R-WAS was 5178.5 mg COD/L and 4116.6 mg COD/L on Day 2. It was reported that a much lower amount of VFAs were produced by wasted activated sludge pretreated by Ca(OH)2 rather than NaOH, due to the inhabitation caused by Ca2+ to the hydrolysis of protein (Zhang et al., 2009). For both A-WAS and R-WAS, acetic acid and butyric acid were almost utilized completely. Propionic acid accumulation appeared in both A-WAS and R-WAS. While acetic acid was commonly reported to be degraded directly by methanogens (Shin et al., 2010), the accumulation of propionic acid would end in lower efficiency of the methanogenic phase and decreasing wastewater treatment efficiency (Wang et al., 2006). 3.4. Methane yield During the first 7 days, methane yield of the untreated sludge was higher than that of A-WAS (Fig. 4). Specifically, the A-WAS produced little methane from Day 1 to Day 4. The start of methane production for A-WAS was delayed by 4 days and the methane yield was reduced during the first 7 days compared to untreated sludge. Similar result had been reported by previous study (Li et al., 2012). This phenomenon might be explained by that the
3.2. VSS and protein removal As shown in Fig. 2, the removal rate of VSS increased from 61.5 ± 2.0% to 76.4 ± 0.5% after alkaline pretreatment, which indicated the better reduction amount of excess sludge. The disintegration of suspended solids by alkaline pretreatment had led to the enhancement of solubilization and higher VSS removal rate. Protein is one of the main constituents of waste sludge and protein reduction is considered as the key factor of excess sludge reduction (Feng et al., 2009). The reduction amount of the protein for A-WAS and R-WAS were 6530 ± 430 mg/L and 4830 ± 480 mg/L, respectively (Fig. 2). The improvement of protein reduction could be caused by the increased water solubility of proteins. An alkaline pH would result in the dissociation of extracellular polymeric substances and repulsions between negatively charged ones, which attributed to the increase of water solubility of proteins (Tan et al., 2010). Yet, high concentration of carbohydrate would inhibit the synthesis of protein hydrolysis related extracellular proteolytic
Fig. 2. Effect of alkaline pretreatment on VSS reduction and protein degradation.
Fig. 3. Change of VFAs concentration during WAS digestion (a) R-WAS and (b) A-WAS.
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Fig. 4. Effect of alkaline pretreatment on methane production from WAS.
activity of mesophilic methanogens would be moderately inhibited by the residual NaOH. Acid conditioning was suggested to adjust the initial pH of the pretreated sludge below 8 (Li et al., 2012). Methane yield and methane percentage of A-WAS and R-WAS reached the highest value on Day 13. The maximum methane yield was 362.2 mL/L d (methane content: 68.7%) for A-WAS and 251.2 mL/L d (methane content: 62.2%) for R-WAS, respectively. Generally, the typical value of methane content in biogas of anaerobic digestion was considered to be 48–65% (Ward et al., 2008).
3.5. Bacterial taxonomic identification From the results of 454 pyrosequencing, 5747 and 5736 reads were obtained for R-WAS and A-WAS. The maximum theoretical OTUs based on the Chao index were 1808 and 1663, which meant the community species richness of untreated sludge was higher. New bacteria species still appeared after the detected sequences reached 5000. To further reveal the diversity of microbial community, phylogenetic analysis of the 16S rRNA gene sequences was performed in phyla, class and genus level. Apparent changes were observed in microbial community structure after alkaline pretreatment (Fig. 5). Twenty phyla were detected in total with 18 for R-WAS and 14 for A-WAS. Though Chloroflexi, Firmicutes, Actinobacteria and Proteobacteria were the most dominant phyla for R-WAS and A-WAS, their distribution was quite different (Fig. 4a). The percentage of Chloroflexi, Firmicutes and Actinobacteria increased significantly after alkaline pretreatment, which was 27.6% (13.2% for R), 23.9% (17.1% for R) and 19.9% (12.8% for R), respectively. The accumulation of Chloroflexi after alkaline pretreatment was related to their resistance to extreme conditions such as high temperature and pH (Tan et al., 2010). Other than the well-known function of sludge reduction, Firmicutes and Proteobacteria were also reported to be highly associated with current production in biological electrochemical systems (BES) (Wrighton et al., 2008). The high fraction of Firmicutes and Proteobacteria might indicate the feasibility of applying alkaline pretreated sludge into BES. Interestingly, 20.6% Nitrospirae which are involved in nitrification were detected in R-WAS, while only 0.16% were observed in A-WAS. On the class level, 27 classes for R and 23 classes for A were obtained, among which Anaerolineae, Clostirida, Acidimicrobidae, Alphaproteobacteria and Planctomycetacia were the dominant ones (Fig. 5b). The percentage of Anaerolineae, Clostirida and Acidimicrobidae had shown some increase in A-WAS, while that of Alphaproteobacteria and Planctomycetacia declined. Clostirida were capable of producing organic acid by utilizing soluble organic materials and cellulolytic enzyme (Kato et al., 2004). The proportion of Bacteroidia increased to 5.5% in A-WAS, yet almost none was detected in R-WAS. Bacteroidia was identified
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as one of the few types of bacteria resistant to the extreme pH conditions, and was reported to play a critical role in sludge reduction (Tan et al., 2010). Further investigation on the genus level, more detailed information of microbial community was received (Fig. 5c). Iamia took up the largest proportion for both R-WAS and A-WAS, which was 5.0% and 10.6% respectively. Iamia was discovered to belong to the phyla Actinobacteria (Kurahashi et al., 2009) which was frequently detected in anaerobic digestion process. Previous studies had identified Actinobacteria as a producer hydrolytic enzyme or organic acid in methanogenic reactors and help to degrade organics in the supernatant (Jang et al., 2014). The proportion of Petrimonas, Syntrophomonas and Nordella were higher in A-WAS, while that of Longilinea, Novosphingobium and Leptolinea were lower. Among them, Petrimonas was known to be fermentative acidogenic bacteria.
3.6. Archaeal community Significant archaeal community shift was observed after alkaline pretreatment (Fig. 6). Methanosarcina, as the dominant Archaeon in A-WAS, took up to 63.3%, while only 6.4% was detected in R-WAS. However, the majority of archaeal populations belonged to Methanosaeta in R-WAS, with 42.6% in R-WAS and 2.0% in A-WAS. Moreover, the composition of other Archaea communities also demonstrated remarkable difference. The proportion of Methanospirillum and Methanomethylovorans was a lot higher in R-WAS compared to A-WAS, which was 27.7% (4.1% for R-WAS) and 4.3% (none for A-WAS). In contrast, Methanobacterium and Methanoculleus showed higher amount in A-WAS than R-WAS, which was 16.3% (8.5% for A-WAS) and 4.1% (none for R-WAS). As the major precursor of methane, the concentration acetate would significantly influence the composition of methanogen communities (Kim et al., 2013b). Methanosaeta and Methanosarcina are both assigned to aceticlastic methanogen families, which were Methanosaetaceae and Methanosarcinaceae. The half-maximum rate concentration (KS < 5 mg/L) of Methanosaeta was lower than that of Methanosarcina for acetate (Jetten et al., 1992). Therefore, with a higher acetate affinity Methanosaeta would dominate at low concentration of acetate (<1 mM), yet due to the higher growth rate, Methanosarcina often survives when exposed in acetate-rich conditions. This might provide a possible explanation to the priority of Methanosarcina over Methanosaeta in A-WAS. Methanospirillum and Methanoculleus both belongs to hydrogenotrophic methanogen families, which were frequently reported in mesophilic anaerobic treatment systems (Kim et al., 2013a). The higher proportion of syntrophic VFA oxidizing bacteria Clostridia along with hydrogenotrophic methanogen Methanobacterium in A-WAS explained the better performance of hydrogenotrophic methanogenesis (Jang et al., 2014).
4. Discussion Generally, organic carbon of sludge is difficult to be degraded directly by microorganisms. As the results of VFA suggested, most of the organic materials were hydrolyzed into low-molecularweight organic compounds by alkaline pretreatment which can be used immediately in the anaerobic digestion process. The methane yield was hence increased. Also, low pH resulting from the accumulation of volatile organic acids during digestion was detrimental for the maintenance and growth of methanogens. Alkaline pretreatment thus created a more favorable environment for methanogens, which resulted in the increase of methane yield. In addition, alkaline pretreatment also increased surface area
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Fig. 5. Taxonomic classification at the phylum (a), class (b) and genus (c) levels based on bacterial 16S rRNA pyrosequencing.
Fig. 6. Taxonomic classification of Archaea based on 16S rRNA clone libraries.
available for enzymatic action which contributed to the improving anaerobic digestion performance (Lin et al., 2009). The methane production in our study was comparable to previous researches. 320 mL CH4/kg VSremoval was achieved using mixed bioreactors with 700 mL worked volume by pretreating pulp and paper sludge with 8 g NaOH/100 g TSsludge during 42 days anaerobic digestion (Lin et al., 2009). The highest methane yield was 150 mL CH4/kg VS when sludge was pretreated by Ca(OH)2 which might indicate the inhabitation of Ca2+ on the hydrolysis of protein (Zhang et al., 2009). When combined alkaline pretreatment with other pretreatment, methane yield was further improved (Kim et al., 2013b). Yet these pretreatments were commonly believed to have high energy demand. Without considerable monitoring and optimization of conditions, such as oxygen, pH and organic loading rate, propionic acid often accumulated in anaerobic digestion process (Wang et al., 2006). Propionogenesis organisms are known to be facultative, and it was concluded that propionic acid accumulation typically occurred at the pH from 5.2 to 5.6, especially in micro-aerobic or shock-loading circumstance (Ren et al., 2007). Although many authors suggested that the high hydrogen partial pressure or higher biohydrogen production rate was the main reason of propionic acid accumulation (Mosey and Fernandes, 1989), other studies indicated that propionic acid accumulation was caused by high yield of NADH, independent of hydrogen partial pressure (Inanc et al., 1996). To maintain a sustainable anaerobic digestion process, the propionic acid concentrations should be kept below 1.5 g/L
(2.3 g COD/L) (Ma et al., 2009). Many solutions had been studied to avoid propionic acid accumulation. By separating acidogenic and methanogenic phases in two-stage anaerobic process, the stability and efficiency of the process had been successfully enhanced (Bolzonella et al., 2007). More specifically, an enhanced propionic acid degradation system had been proven to be effective to improve propionic acid removal rates (Ma et al., 2009). In addition, adding different hydrogen-oxidizing bacteria to enhance propionic acids degradation was also reported to be an alternative solution (Bagi et al., 2007). To date, no serious propionic acids accumulation had been reported using the newly emerging Microbial Electrolysis Cells (MECs). This might indicate the possibility of MECs being another feasible solution to propionic acids accumulation, which needs further investigation (Lu et al., 2012a,b). Apparently, high pH after alkaline pretreatment inhibited the diversity of anaerobic bacteria and the richness of the microbial community decreased. Bacteria damage and lysis was assumed to be an expected result with sludge reduction technologies (Foladori et al., 2010). Visible changes of microbial communities, especially for Archaea communities, had been detected when comparing raw sludge with alkaline pretreated sludge. Previous researches had also reported similar phenomenon (Jang et al., 2014). On the physicochemical aspects, alkaline pretreatment had accelerated the sludge hydrolysis and enhanced soluble organics concentration by swelling cells. Standing on the biological level, pretreatment might also create significant impacts on the enrichment and syntrophic interactions between microbial communities. Detailed comparison on the formation and evolution of microbial communities between various pretreatment methods would provide valuable insight into the selection of the most efficient sludge anaerobic digestion process. Further research would be needed.
5. Conclusions Methane yield was obviously increased from A-WAS digestion. The maximum methane yield of 362.2 mL/L d (methane content: 68.7%) was obtained in our study. Sludge reduction was enhanced with the VSS removal of 76.4 ± 0.5%. The improvement on SCOD
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and VFAs also indicated better solubilization and hydrolysis process. Using 454 pyrosequencing, apparent microbial community shifts especially for archaeal communities were observed after alkaline pretreatment. The dominant methanogenic population changed from Methanosaeta for R-WAS to Methanosarcina for A-WAS. This indicated the importance of further investigation into the influence of pretreatment methods on the formation and evolution of microbial communities. Acknowledgements This research was supported by National Natural Science Foundation of China (No. 31270004), the State Key Laboratory of Urban Water Resource and Environment (No. 2013DX13), the Fundamental Research Funds for the Central Universities (No, HIT.BRETIII.201232), and the Science Fund for Creative Research Groups of the National Natural Science Foundation of China (No. 51121062). References American Public Health Association (APHA), 1998. Standard Methods for the Examination of Water and Wastewater, Washington, DC. Bagi, Z., Ács, N., Bálint, B., Horváth, L., Dobó, K., Perei, K.R., Rákhely, G., Kovács, K.L., 2007. Biotechnological intensification of biogas production. Appl. Microbiol. Biotechnol. 76 (2), 473–482. Bolzonella, D., Pavan, P., Zanette, M., Cecchi, F., 2007. Two-phase anaerobic digestion of waste activated sludge: effect of an extreme thermophilic prefermentation. Ind. Eng. Chem. Res. 46 (21), 6650–6655. Fang, H.H., Yu, H., 2000. Effect of HRT on mesophilic acidogenesis of dairy wastewater. J. Environ. Eng. 126 (12), 1145–1148. Feng, L., Chen, Y., Zheng, X., 2009. Enhancement of waste activated sludge protein conversion and volatile fatty acids accumulation during waste activated sludge anaerobic fermentation by carbohydrate substrate addition: the effect of pH. Environ. Sci. Technol. 43 (12), 4373–4380. Foladori, P., Tamburini, S., Bruni, L., 2010. Bacteria permeabilization and disruption caused by sludge reduction technologies evaluated by flow cytometry. Water Res. 44 (17), 4888–4899. Herbert D., Philipps P.J.M Strange R.E., 1971. Chemical analysis of microbial cells. In: Norris, J.R. Ribbons, D.W. (eds.) Methods in Microbiology. Vol. VB. Academic Press, New York, pp. 209–344. Huang, L., Chen, B., Pistolozzi, M., Wu, Z., Wang, J., 2014. Inoculation and alkali coeffect in volatile fatty acids production and microbial community shift in the anaerobic fermentation of waste activated sludge. Bioresour. Technol. 153, 87– 94. Inanc, B., Matsui, S., Ide, S., 1996. Propionic acid accumulation and controlling factors in anaerobic treatment of carbohydrate: effects of H2 and pH. Water Sci. Technol. 34 (5), 317–325. Jang, H.M., Cho, H.U., Park, S.K., Ha, J.H., Park, J.M., 2014. Influence of thermophilic aerobic digestion as a sludge pre-treatment and solids retention time of mesophilic anaerobic digestion on the methane production, sludge digestion and microbial communities in a sequential digestion process. Water Res. 48, 1– 14. Jetten, M.S., Stams, A.J., Zehnder, A.J., 1992. Methanogenesis from acetate: a comparison of the acetate metabolism in Methanothrix soehngenii and Methanosarcina spp.. FEMS Microbiol. Lett. 88 (3), 181–197. Jie, W., Peng, Y., Ren, N., Li, B., 2014. Volatile fatty acids (VFAs) accumulation and microbial community structure of excess sludge (ES) at different pHs. Bioresour. Technol. 152, 124–129. Kato, S., Haruta, S., Cui, Z.J., Ishii, M., Yokota, A., Igarashi, Y., 2004. Clostridium straminisolvens sp. nov., a moderately thermophilic, aerotolerant and cellulolytic bacterium isolated from a cellulose-degrading bacterial community. Int. J. Syst. Evol. Microbiol. 54 (6), 2043–2047.
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Kim, J., Lee, S., Lee, C., 2013a. Comparative study of changes in reaction profile and microbial community structure in two anaerobic repeated-batch reactors started up with different seed sludges. Bioresour. Technol. 129, 495–505. Kim, J., Yu, Y., Lee, C., 2013b. Thermo-alkaline pretreatment of waste activated sludge at low-temperatures: effects on sludge disintegration, methane production, and methanogen community structure. Bioresour. Technol. 144, 194–201. Kurahashi, M., Fukunaga, Y., Sakiyama, Y., Harayama, S., Yokota, A., 2009. Iamia majanohamensis gen. nov., sp. nov., an actinobacterium isolated from sea cucumber Holothuria edulis, and proposal of Iamiaceae fam. nov.. Int. J. Syst. Evol. Microbiol. 59 (4), 869–873. Li, H., Li, C., Liu, W., Zou, S., 2012. Optimized alkaline pretreatment of sludge before anaerobic digestion. Bioresour. Technol. 123, 189–194. Lin, Y., Wang, D., Wu, S., Wang, C., 2009. Alkali pretreatment enhances biogas production in the anaerobic digestion of pulp and paper sludge. J. Hazard. Mater. 170 (1), 366–373. Lu, L., Xing, D., Ren, N., 2012a. Pyrosequencing reveals highly diverse microbial communities in microbial electrolysis cells involved in enhanced H2 production from waste activated sludge. Water Res. 46 (7), 2425–2434. Lu, L., Xing, D., Ren, N., Logan, B.E., 2012b. Syntrophic interactions drive the hydrogen production from glucose at low temperature in microbial electrolysis cells. Bioresour. Technol. 124, 68–76. Ma, J., Carballa, M., Van De Caveye, P., Verstraete, W., 2009. Enhanced propionic acid degradation (EPAD) system: proof of principle and feasibility. Water Res. 43 (13), 3239–3248. Mahmood, T., Elliott, A., 2006. A review of secondary sludge reduction technologies for the pulp and paper industry. Water Res. 40 (11), 2093–2112. Mosey, F., Fernandes, X., 1989. Patterns of hydrogen in biogas from the anaerobic digestion of milk-sugars. Water Sci. Technol. 21 (4–5), 187–196. Ren, N., Chua, H., Chan, S., Tsang, Y., Wang, Y., Sin, N., 2007. Assessing optimal fermentation type for bio-hydrogen production in continuous-flow acidogenic reactors. Bioresour. Technol. 98 (9), 1774–1780. Salminen, E., Rintala, J., 2002. Anaerobic digestion of organic solid poultry slaughterhouse waste – a review. Bioresour. Technol. 83 (1), 13–26. Shin, S.G., Lee, S., Lee, C., Hwang, K., Hwang, S., 2010. Qualitative and quantitative assessment of microbial community in batch anaerobic digestion of secondary sludge. Bioresour. Technol. 101 (24), 9461–9470. Smith, P.K., Krohn, R.I., Hermanson, G.T., Mallia, A.K., Gartner, F.H., Provenzano, M.D., Fujimoto, E.K., Goeke, N.M., Olson, B.J., Klenk, D.C., 1985. Measurement of protein using bicinchoninic acid. Anal. Biochem. 150 (1), 76–85. Su, G., Huo, M., Yuan, Z., Wang, S., Peng, Y., 2013. Hydrolysis, acidification and dewaterability of waste activated sludge under alkaline conditions: combined effects of NaOH and Ca(OH)2. Bioresour. Technol. 136, 237–243. Tan, R., Miyanaga, K., Toyama, K., Uy, D., Tanji, Y., 2010. Changes in composition and microbial communities in excess sludge after heat-alkaline treatment and acclimation. Biochem. Eng. J. 52 (2–3), 151–159. Vigueras-Carmona, S.E., Ramírez, F., Noyola, A., Monroy, O., 2011. Effect of thermal alkaline pretreatment on the anaerobic digestion of wasted activated sludge. Water Sci. Technol. 64 (4), 953–959. Wang, L., Zhou, Q., Li, F., 2006. Avoiding propionic acid accumulation in the anaerobic process for biohydrogen production. Biomass Bioenergy 30 (2), 177– 182. Ward, A.J., Hobbs, P.J., Holliman, P.J., Jones, D.L., 2008. Optimisation of the anaerobic digestion of agricultural resources. Bioresour. Technol. 99 (17), 7928–7940. Wrighton, K.C., Agbo, P., Warnecke, F., Weber, K.A., Brodie, E.L., DeSantis, T.Z., Hugenholtz, P., Andersen, G.L., Coates, J.D., 2008. A novel ecological role of the Firmicutes identified in thermophilic microbial fuel cells. ISME J. 2 (11), 1146– 1156. Wu, H., Yang, D., Zhou, Q., Song, Z., 2009. The effect of pH on anaerobic fermentation of primary sludge at room temperature. J. Hazard. Mater. 172 (1), 196–201. Xiao, B., Yang, F., Liu, J., 2013. Evaluation of electricity production from alkaline pretreated sludge using two-chamber microbial fuel cell. J. Hazard. Mater. 254– 255, 57–63. Zhang, P., Chen, Y., Zhou, Q., 2009. Waste activated sludge hydrolysis and shortchain fatty acids accumulation under mesophilic and thermophilic conditions: effect of pH. Water Res. 43 (15), 3735–3742.
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Methane production and microbial community structure for alkaline pretreated waste activated sludge Rui Sun, Defeng Xing, Jianna Jia, Aijuan Zhou, Lu Zhang, Nanqi Ren ⇑ State Key Laboratory of Urban Water Resource and Environment, Harbin Institute of Technology, Harbin 150090, China
h i g h l i g h t s Alkali-pretreatment enhanced CH4 production in the anaerobic digestion of sludge. Alkali-pretreatment improved the sludge reduction in the anaerobic digestion. The shift of microbial community resulted in CH4 increase after alkali-pretreatment.
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Article history: Received 16 May 2014 Received in revised form 28 June 2014 Accepted 4 July 2014 Available online 16 July 2014 Keywords: Waste activated sludge (WAS) Alkaline pretreatment Methane production Microbial community 454 pyrosequencing
a b s t r a c t Alkaline pretreatment was studied to analyze the influence on waste activated sludge (WAS) reduction, methane production and microbial community structure during anaerobic digestion. Methane production from alkaline pretreated sludge (A-WAS) (pH = 12) increased from 251.2 mL/L d to 362.2 mL/L d with the methane content of 68.7% compared to raw sludge (R-WAS). Sludge reduction had been improved, and volatile suspended solids (VSS) removal rate and protein reduction had increased by 10% and 35%, respectively. The bacterial and methanogenic communities were analyzed using 454 pyrosequencing and clone libraries of 16S rRNA gene. Remarkable shifts were observed in microbial community structures after alkaline pretreatment, especially for Archaea. The dominant methanogenic population changed from Methanosaeta for R-WAS to Methanosarcina for A-WAS. In addition to the enhancement of solubilization and hydrolysis of anaerobic digestion of WAS, alkaline pretreatment showed significant impacts on the enrichment and syntrophic interactions between microbial communities. Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction Large amounts of waste activated sludge (WAS) were produced in China every year. The management and disposal of sewage sludge was estimated to account for up to 50% of the total operation and maintenance costs of waste water treatment plants. The current disposal costs were reported to be 200–650 €/t of dry weight (Foladori et al., 2010). Therefore, excess sludge, as the main by-product of the wastewater treatment plants, has become one of the most challenging issues. The conventional methods such as landfilling, incineration and beneficial uses are less preferable due to the secondary pollution, capital intensive costs and stricter regulatory pressure (Mahmood and Elliott, 2006). Anaerobic digestion of sludge which converted sludge to water, methane and carbon dioxide has gained widely attention for being a disposal ⇑ Corresponding author. Address: School of Municipal and Environmental Engineering, Harbin Institute of Technology, P.O. Box 2614, 73 Huanghe Road, Nangang District, Harbin 150090, China. Tel./fax: +86 451 86282008. E-mail addresses: [email protected] (D. Xing), [email protected] (N. Ren). http://dx.doi.org/10.1016/j.biortech.2014.07.032 0960-8524/Ó 2014 Elsevier Ltd. All rights reserved.
route and energy recovery route simultaneously. With about 80% of biodegradable organic energy being converted to methane, anaerobic digestion was considered as a promising sludge reduction method for pulp-mill WAS and a highly competitive alternative for the treatment of organic solid poultry slaughter house waste (Salminen and Rintala, 2002). Due to the extensive (20–30 days) retention times and relatively low degradation efficiency (30–50%) (Lin et al., 2009), various pretreatment methods such as physical, chemical or biological pretreatment methods (or their combination) were researched to improve the digestion efficiency and sludge reduction (Vigueras-Carmona et al., 2011). While thermal, ultrasonic and enzyme pretreatment of sludge are considered to be very expensive due to the high energy demand, alkaline pretreatment was a well-known method with benefits such as simple devices, easy to operate and high efficiency (Li et al., 2012). The impact of alkaline pretreatment of sludge on the improvement of volatile suspended solids (VSS) reduction and methane production had been reported in a previous research (Li et al., 2012). 38.3% organic degradation rate and 0.65 L/gVSS biogas yield was obtained by 0.1 mol/L NaOH pretreated sludge
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(Li et al., 2012). It was also concluded that by appropriate pH pretreatment, sludge reduction and biogas production were increased by 10% and sludge dewater ability was also improved. By alkaline pretreatment, the organic compounds of sludge were destroyed or became more susceptible to enzymatic and chemical attack, which led to the enhancement of solubilization and hydrolysis (Xiao et al., 2013). However, information on the influence of alkaline pretreatment on microbial community shift in WAS was rather limited in addition to the well documented physicochemical characteristics changes (Jie et al., 2014). As a biochemical process, the performance of anaerobic digestion is highly related to the concerted activity of the interacting microorganisms including bacteria groups and archaeal groups. During anaerobic digestion, the first hydrolysis and acidogenesis process were carried out by bacteria groups and the following methanogenesis was performed by Archaea groups (Shin et al., 2010). Using denaturing gradient gel electrophoresis (DGGE) analysis, Proteocatella, Tepidibacter, and Clostridium were found to disappear when paper mill anaerobic granular sludge were pretreated at pH 9 or 10 (Huang et al., 2014), while uncultured bacterium was reported to be capable of adapting well at high pH (Jie et al., 2014). However, molecular biology methods such as DGGE often powerless to capture the comprehensive information in microbial communities due to lack of sufficient throughput (Lu et al., 2012a). 454 pyrosequencing is now increasingly chosen to provide potentially detailed insight into the diverse microbial ecosystem. In this study, the effect of alkaline pretreatment on sludge hydrolysis and methane production was investigated. The performance of the anaerobic digestion process was estimated by gas production, volatile fatty acids (VFAs) and volatile organics reduction efficiency. The microbial community structure and distribution of micro-organisms in alkaline pretreated sludge were assessed using 454 pyrosequencing.
2. Methods 2.1. Sludge samples, pretreatment and fermentation WAS was collected from the secondary sedimentation tank in Wenchang Wastewater Treatment Plant in Harbin. After 24 h sedimentation, the upper sewage was abandoned (R-WAS). The alkaline pretreated WAS (A-WAS) was adjusted to pH 12 by adding 4 M NaOH. The specific alkaline dosage was 0.92 ± 0.01 g NaOH per gram of volatile suspended solids (VSS). The characteristics of R-WAS and A-WAS were shown in Table 1. The eventual pH of the alkaline pretreated sludge was around 9–10 after another 24 h sedimentation 400 mL untreated and alkaline pretreated sludge were put into narrow-mouth digesters with effective volume of 1 L and a retention time of 16 days. To create anaerobic environment, N2 was purged for 5 min into the sludge and the top part of the bottle for removing the remained air. Then the
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sludge samples were cultured in incubator shaker at the rate of 90 r/min and temperature of 35 ± 1 °C. The sludge was sampled for further analysis on a daily basis during the anaerobic digestion process. All the experiments were performed in triplicate and mean values were applied. 2.2. Analysis methods TCOD, SCOD, TS, VS, TSS, VSS were analyzed according to the standard methods (American Public Health Association (APHA, 1998)). For the measurement of SCOD, soluble carbohydrate and soluble protein, the sludge samples were centrifuged, and the obtained supernatant were then filtered by 0.45 lm filter membrane. The filtrate was analyzed for SCOD by potassium dichromate method. As for most carbohydrates in the samples were polysaccharide, phenol – sulfuric acid colorimetric method was chosen to analyze carbohydrates concentration (Herbert et al., 1971). The protein concentration was determined using Bicinchoninic Acid Protein Assay Kit (Sigma–Aldrich) (Smith et al., 1985). The pH was measured by Shang Hai Lei Ci PHS-2F type pH meter. To analyze VFAs, gas chromatograph from U.S. Agilent Company (GC 4890) was applied. And methane content in produced biogas was determined by FULI 9790 II gas chromatograph. The gas yield and production rate were all calculated based on the gas chromatograph results. 2.3. DNA extraction, PCR amplification and 454 Pyrosequencing Alkaline pretreated and raw sludge samples were collected from the anaerobic digesters after the anaerobic digestion process for microbial community analysis. PowerSoil DNA Isolation Kit (Mo Bio Laboratories, Carlsbad, CA) was used to extract the genomic DNA of the sludge samples according to the manufacturer’s instructions. the V1–V3 region (length of 455 bp) of the bacterial 16S rRNA gene was amplified using a set of primers: 8F (50 -AGA GTTTGATCCTGGCTCAG-30 ) and 533R (50 -TTACCGCGGCTGCTGG CAC-30 ), as well as the 454 adapter A and B, barcode and linker sequence were added as previous descriptions (Lu et al., 2012a). 454 GS-FLX sequencer was then applied for pyrosequencing according to standard protocols (Lu et al., 2012a). With similarity set at 97% and a confidence threshold of 95%, the obtained sequences were phylogenetically allocated down to the phylum, class and genus level with the MOTHUR program (http://www.mothur.org/wiki/Main_Page). To define the relative abundance of a given phylogenetic group, the number of sequences affiliated to that group was divided by the total number of obtained sequences. The obtained results were used for the analysis and comparison of microbial community structure and bacterial diversity of the sludge samples before and after alkaline pretreatment. 3. Results
Table 1 Characteristics of buffered R-WAS and A-WAS.
3.1. SCOD concentration and SCOD/TCOD rate
Parameter
R-WAS
A-WAS
pH Moisture content (%) Total solids (TS, mg/L) Volatile solids (VS, mg/L) Total suspended solids (TSS, mg/L) Volatile suspended solids (VSS, mg/L) Total chemical oxygen demand (TCOD, mg/L) Soluble chemical oxygen demand (SCOD, mg/L) Soluble carbohydrate (mg COD/L) Soluble protein (mg COD/L)
6.80 ± 0.1 95.19 ± 3.1 25610 ± 71 17490 ± 269 19720 ± 15 11440 ± 13 30378 ± 929 292 ± 5 74 ± 2 337 ± 13
9.14 ± 0.1 95.30 ± 2.8 24500 ± 707 17890 ± 184 19220 ± 14 10200 ± 7 27900 ± 907 2590 ± 79 213 ± 25 6487 ± 427
Two stages were noticed during sludge solubilization (Fig. 1). During Phase I (the first two days), SCOD concentration of both A-WAS and R-WAS increased quickly, which indicated the rapid solubilization of waste sludge during anaerobic digestion. From Day 3 to Day 16 (Phase II), the SCOD concentration began to decrease slowly with an average value of 5000 mg/L for A-WAS and 3500 mg/L for R-WAS. Previous study also reported 3 phases during sludge solubilization process (Su et al., 2013). The differences could be caused by different operation condition or different sludge origins. SCOD of A-WAS was higher compared to untreated
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enzymes and weaken the biodegradability of protein (Fang and Yu, 2000). 3.3. VFAs production and composition
Fig. 1. Changes of SCOD concentration and SCOD/TCOD rate during WAS digestion.
sludge during the 16 days (Fig. 1) which meant more extracellular polymer and intracellular substances dissolved out of the cell by alkaline pretreatment. Digestibility of waste activated sludge was hence enhanced due to the additional easily degradable substrate released by alkaline pretreatment for microbial growth. On Day 2, SCOD of both A-WAS and R-WAS reached the maximum value which was 8567.9 mg/L and 5219.6 mg/L, respectively. 2 days were considered to be the most efficient treatment duration for sludge solubilization in our study. Yet 4 or 5 days were also reported in previous literatures due to different pH or operation condition (Su et al., 2013; Wu et al., 2009). The solubilization efficiencies of wasted activated sludge which was demonstrated by the value of SCOD/TCOD nearly doubled after alkaline pretreatment, especially for the first 10 days. The maximum solubilization efficiencies of A-WAS and R-WAS were 31% and 15% at Day 2.
Acetic, butyric, propionic, iso-butyric, iso-valeric acids, valeric acids and ethanol were the main type of VFAs produced in the WAS anaerobic digestion process in our study (Fig. 3). VFAs concentration of A-WAS was higher than that of R-WAS. The maximum VFAs concentration of A-WAS and R-WAS was 5178.5 mg COD/L and 4116.6 mg COD/L on Day 2. It was reported that a much lower amount of VFAs were produced by wasted activated sludge pretreated by Ca(OH)2 rather than NaOH, due to the inhabitation caused by Ca2+ to the hydrolysis of protein (Zhang et al., 2009). For both A-WAS and R-WAS, acetic acid and butyric acid were almost utilized completely. Propionic acid accumulation appeared in both A-WAS and R-WAS. While acetic acid was commonly reported to be degraded directly by methanogens (Shin et al., 2010), the accumulation of propionic acid would end in lower efficiency of the methanogenic phase and decreasing wastewater treatment efficiency (Wang et al., 2006). 3.4. Methane yield During the first 7 days, methane yield of the untreated sludge was higher than that of A-WAS (Fig. 4). Specifically, the A-WAS produced little methane from Day 1 to Day 4. The start of methane production for A-WAS was delayed by 4 days and the methane yield was reduced during the first 7 days compared to untreated sludge. Similar result had been reported by previous study (Li et al., 2012). This phenomenon might be explained by that the
3.2. VSS and protein removal As shown in Fig. 2, the removal rate of VSS increased from 61.5 ± 2.0% to 76.4 ± 0.5% after alkaline pretreatment, which indicated the better reduction amount of excess sludge. The disintegration of suspended solids by alkaline pretreatment had led to the enhancement of solubilization and higher VSS removal rate. Protein is one of the main constituents of waste sludge and protein reduction is considered as the key factor of excess sludge reduction (Feng et al., 2009). The reduction amount of the protein for A-WAS and R-WAS were 6530 ± 430 mg/L and 4830 ± 480 mg/L, respectively (Fig. 2). The improvement of protein reduction could be caused by the increased water solubility of proteins. An alkaline pH would result in the dissociation of extracellular polymeric substances and repulsions between negatively charged ones, which attributed to the increase of water solubility of proteins (Tan et al., 2010). Yet, high concentration of carbohydrate would inhibit the synthesis of protein hydrolysis related extracellular proteolytic
Fig. 2. Effect of alkaline pretreatment on VSS reduction and protein degradation.
Fig. 3. Change of VFAs concentration during WAS digestion (a) R-WAS and (b) A-WAS.
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Fig. 4. Effect of alkaline pretreatment on methane production from WAS.
activity of mesophilic methanogens would be moderately inhibited by the residual NaOH. Acid conditioning was suggested to adjust the initial pH of the pretreated sludge below 8 (Li et al., 2012). Methane yield and methane percentage of A-WAS and R-WAS reached the highest value on Day 13. The maximum methane yield was 362.2 mL/L d (methane content: 68.7%) for A-WAS and 251.2 mL/L d (methane content: 62.2%) for R-WAS, respectively. Generally, the typical value of methane content in biogas of anaerobic digestion was considered to be 48–65% (Ward et al., 2008).
3.5. Bacterial taxonomic identification From the results of 454 pyrosequencing, 5747 and 5736 reads were obtained for R-WAS and A-WAS. The maximum theoretical OTUs based on the Chao index were 1808 and 1663, which meant the community species richness of untreated sludge was higher. New bacteria species still appeared after the detected sequences reached 5000. To further reveal the diversity of microbial community, phylogenetic analysis of the 16S rRNA gene sequences was performed in phyla, class and genus level. Apparent changes were observed in microbial community structure after alkaline pretreatment (Fig. 5). Twenty phyla were detected in total with 18 for R-WAS and 14 for A-WAS. Though Chloroflexi, Firmicutes, Actinobacteria and Proteobacteria were the most dominant phyla for R-WAS and A-WAS, their distribution was quite different (Fig. 4a). The percentage of Chloroflexi, Firmicutes and Actinobacteria increased significantly after alkaline pretreatment, which was 27.6% (13.2% for R), 23.9% (17.1% for R) and 19.9% (12.8% for R), respectively. The accumulation of Chloroflexi after alkaline pretreatment was related to their resistance to extreme conditions such as high temperature and pH (Tan et al., 2010). Other than the well-known function of sludge reduction, Firmicutes and Proteobacteria were also reported to be highly associated with current production in biological electrochemical systems (BES) (Wrighton et al., 2008). The high fraction of Firmicutes and Proteobacteria might indicate the feasibility of applying alkaline pretreated sludge into BES. Interestingly, 20.6% Nitrospirae which are involved in nitrification were detected in R-WAS, while only 0.16% were observed in A-WAS. On the class level, 27 classes for R and 23 classes for A were obtained, among which Anaerolineae, Clostirida, Acidimicrobidae, Alphaproteobacteria and Planctomycetacia were the dominant ones (Fig. 5b). The percentage of Anaerolineae, Clostirida and Acidimicrobidae had shown some increase in A-WAS, while that of Alphaproteobacteria and Planctomycetacia declined. Clostirida were capable of producing organic acid by utilizing soluble organic materials and cellulolytic enzyme (Kato et al., 2004). The proportion of Bacteroidia increased to 5.5% in A-WAS, yet almost none was detected in R-WAS. Bacteroidia was identified
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as one of the few types of bacteria resistant to the extreme pH conditions, and was reported to play a critical role in sludge reduction (Tan et al., 2010). Further investigation on the genus level, more detailed information of microbial community was received (Fig. 5c). Iamia took up the largest proportion for both R-WAS and A-WAS, which was 5.0% and 10.6% respectively. Iamia was discovered to belong to the phyla Actinobacteria (Kurahashi et al., 2009) which was frequently detected in anaerobic digestion process. Previous studies had identified Actinobacteria as a producer hydrolytic enzyme or organic acid in methanogenic reactors and help to degrade organics in the supernatant (Jang et al., 2014). The proportion of Petrimonas, Syntrophomonas and Nordella were higher in A-WAS, while that of Longilinea, Novosphingobium and Leptolinea were lower. Among them, Petrimonas was known to be fermentative acidogenic bacteria.
3.6. Archaeal community Significant archaeal community shift was observed after alkaline pretreatment (Fig. 6). Methanosarcina, as the dominant Archaeon in A-WAS, took up to 63.3%, while only 6.4% was detected in R-WAS. However, the majority of archaeal populations belonged to Methanosaeta in R-WAS, with 42.6% in R-WAS and 2.0% in A-WAS. Moreover, the composition of other Archaea communities also demonstrated remarkable difference. The proportion of Methanospirillum and Methanomethylovorans was a lot higher in R-WAS compared to A-WAS, which was 27.7% (4.1% for R-WAS) and 4.3% (none for A-WAS). In contrast, Methanobacterium and Methanoculleus showed higher amount in A-WAS than R-WAS, which was 16.3% (8.5% for A-WAS) and 4.1% (none for R-WAS). As the major precursor of methane, the concentration acetate would significantly influence the composition of methanogen communities (Kim et al., 2013b). Methanosaeta and Methanosarcina are both assigned to aceticlastic methanogen families, which were Methanosaetaceae and Methanosarcinaceae. The half-maximum rate concentration (KS < 5 mg/L) of Methanosaeta was lower than that of Methanosarcina for acetate (Jetten et al., 1992). Therefore, with a higher acetate affinity Methanosaeta would dominate at low concentration of acetate (<1 mM), yet due to the higher growth rate, Methanosarcina often survives when exposed in acetate-rich conditions. This might provide a possible explanation to the priority of Methanosarcina over Methanosaeta in A-WAS. Methanospirillum and Methanoculleus both belongs to hydrogenotrophic methanogen families, which were frequently reported in mesophilic anaerobic treatment systems (Kim et al., 2013a). The higher proportion of syntrophic VFA oxidizing bacteria Clostridia along with hydrogenotrophic methanogen Methanobacterium in A-WAS explained the better performance of hydrogenotrophic methanogenesis (Jang et al., 2014).
4. Discussion Generally, organic carbon of sludge is difficult to be degraded directly by microorganisms. As the results of VFA suggested, most of the organic materials were hydrolyzed into low-molecularweight organic compounds by alkaline pretreatment which can be used immediately in the anaerobic digestion process. The methane yield was hence increased. Also, low pH resulting from the accumulation of volatile organic acids during digestion was detrimental for the maintenance and growth of methanogens. Alkaline pretreatment thus created a more favorable environment for methanogens, which resulted in the increase of methane yield. In addition, alkaline pretreatment also increased surface area
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Fig. 5. Taxonomic classification at the phylum (a), class (b) and genus (c) levels based on bacterial 16S rRNA pyrosequencing.
Fig. 6. Taxonomic classification of Archaea based on 16S rRNA clone libraries.
available for enzymatic action which contributed to the improving anaerobic digestion performance (Lin et al., 2009). The methane production in our study was comparable to previous researches. 320 mL CH4/kg VSremoval was achieved using mixed bioreactors with 700 mL worked volume by pretreating pulp and paper sludge with 8 g NaOH/100 g TSsludge during 42 days anaerobic digestion (Lin et al., 2009). The highest methane yield was 150 mL CH4/kg VS when sludge was pretreated by Ca(OH)2 which might indicate the inhabitation of Ca2+ on the hydrolysis of protein (Zhang et al., 2009). When combined alkaline pretreatment with other pretreatment, methane yield was further improved (Kim et al., 2013b). Yet these pretreatments were commonly believed to have high energy demand. Without considerable monitoring and optimization of conditions, such as oxygen, pH and organic loading rate, propionic acid often accumulated in anaerobic digestion process (Wang et al., 2006). Propionogenesis organisms are known to be facultative, and it was concluded that propionic acid accumulation typically occurred at the pH from 5.2 to 5.6, especially in micro-aerobic or shock-loading circumstance (Ren et al., 2007). Although many authors suggested that the high hydrogen partial pressure or higher biohydrogen production rate was the main reason of propionic acid accumulation (Mosey and Fernandes, 1989), other studies indicated that propionic acid accumulation was caused by high yield of NADH, independent of hydrogen partial pressure (Inanc et al., 1996). To maintain a sustainable anaerobic digestion process, the propionic acid concentrations should be kept below 1.5 g/L
(2.3 g COD/L) (Ma et al., 2009). Many solutions had been studied to avoid propionic acid accumulation. By separating acidogenic and methanogenic phases in two-stage anaerobic process, the stability and efficiency of the process had been successfully enhanced (Bolzonella et al., 2007). More specifically, an enhanced propionic acid degradation system had been proven to be effective to improve propionic acid removal rates (Ma et al., 2009). In addition, adding different hydrogen-oxidizing bacteria to enhance propionic acids degradation was also reported to be an alternative solution (Bagi et al., 2007). To date, no serious propionic acids accumulation had been reported using the newly emerging Microbial Electrolysis Cells (MECs). This might indicate the possibility of MECs being another feasible solution to propionic acids accumulation, which needs further investigation (Lu et al., 2012a,b). Apparently, high pH after alkaline pretreatment inhibited the diversity of anaerobic bacteria and the richness of the microbial community decreased. Bacteria damage and lysis was assumed to be an expected result with sludge reduction technologies (Foladori et al., 2010). Visible changes of microbial communities, especially for Archaea communities, had been detected when comparing raw sludge with alkaline pretreated sludge. Previous researches had also reported similar phenomenon (Jang et al., 2014). On the physicochemical aspects, alkaline pretreatment had accelerated the sludge hydrolysis and enhanced soluble organics concentration by swelling cells. Standing on the biological level, pretreatment might also create significant impacts on the enrichment and syntrophic interactions between microbial communities. Detailed comparison on the formation and evolution of microbial communities between various pretreatment methods would provide valuable insight into the selection of the most efficient sludge anaerobic digestion process. Further research would be needed.
5. Conclusions Methane yield was obviously increased from A-WAS digestion. The maximum methane yield of 362.2 mL/L d (methane content: 68.7%) was obtained in our study. Sludge reduction was enhanced with the VSS removal of 76.4 ± 0.5%. The improvement on SCOD
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