Bioresource Technology 196 (2015) 606–612
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Comparing activated carbon of different particle sizes on enhancing methane generation in upflow anaerobic digester Suyun Xu a,⇑, Chuanqiu He a, Liwen Luo a, Fan Lü b, Pinjing He b, Lifeng Cui a a b
Department of Environment & Low-Carbon Science, School of Environment and Architecture, University of Shanghai for Science and Technology, Shanghai 200093, China State Key Laboratory of Pollution Control and Resource Reuse, Tongji University, Shanghai 200092, China
h i g h l i g h t s Both PAC and GAC could promote the syntrophic metabolism of alcohol and VFAs. PAC is superior to GAC on the enhancement of biomethane generation process. PAC provides more abundant micropore–mesopore structure for bacteria to colonize. Microbial community colonized on PAC and GAC was characterized and compared.
a r t i c l e
i n f o
Article history: Received 17 June 2015 Received in revised form 3 August 2015 Accepted 8 August 2015 Available online 14 August 2015 Keywords: Activated carbon Archaea Methanogenesis Syntrophic acetate oxidization Volatile fatty acid
a b s t r a c t Two sizes of conductive particles, i.e. 10–20 mesh granulated activated carbon (GAC) and 80–100 mesh powdered activated carbon (PAC) were added into lab-scale upflow anaerobic sludge blanket reactors, respectively, to testify their enhancement on the syntrophic metabolism of alcohols and volatile fatty acids (VFAs) in 95 days operation. When OLR increased to more than 5.8 g COD/L/d, the differences between GAC/PAC supplemented reactors and the control reactor became more significant. The introduction of activated carbon could facilitate the enrichment of methanogens and accelerate the startup of methanogenesis, as indicated by enhanced methane yield and substrate degradation. High-throughput pyrosequencing analysis showed that syntrophic bacteria and Methanosarcina sp. with versatile metabolic capability increased in the tightly absorbed fraction on the PAC surface, leading to the promoted syntrophic associations. Thus PAC prevails over than GAC for methanogenic reactor with heavy load. Ó 2015 Elsevier Ltd. All rights reserved.
1. Introduction In China, brewery wastewater is 1.5–2.0% of the total wastewater production in the country (Feng et al., 2008). Due to its high concentration of biodegradable organic matters, biological treatment is a good choice for the treatment of brewery wastewater (Parawira et al., 2005). Generally, aerobic treatment has been successfully applied for the treatment of brewery wastewater and recently anaerobic systems have become an attractive option (Simate et al., 2011). One of the most popular anaerobic processes for wastewater treatment is the Upflow Anaerobic Sludge Blanket (UASB). Simultaneously, methane derived from anaerobic treatment of brewery wastewater in anaerobic digesters is an alternative fuel.
⇑ Corresponding author. Tel./fax: +86 21 5527 5979. E-mail address:
[email protected] (S. Xu). http://dx.doi.org/10.1016/j.biortech.2015.08.018 0960-8524/Ó 2015 Elsevier Ltd. All rights reserved.
However, as anaerobic bacteria are slow growing microorganisms, the major problem of anaerobic digester is the long start-up periods and difficulty in spontaneous development of granulation. The reduction of start-up time is one of the key parameters to increase the competitiveness of high-rate anaerobic reactors. The presence of porous media such as plastic-, ceramic- or carbonbased material, is known to enhance the performance of anaerobic reactors by offering a rough and fissured surface for microorganisms to settle and colonize easily (Fernández et al., 2007; Kindzierski et al., 1992). Among these materials, bacteria preferably adhered to the solid supports made of carbon material (Kuroda et al., 1988). For instance, anaerobic biofilm reactor packed with Granular Activated Carbon (GAC) has been successfully used to achieve effective decontamination for olive mill wastewater even at high organic loads of 15.7–55.6 g COD/L/d (Bertin et al., 2004). Due to its inherent absorption capacity, AC can help to reduce the organic shock loading impact on the process of biomethane generation (Aktasß and Çeçen, 2007).
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Furthermore, in recent studies, granular activated carbon (GAC) was reported to facilitate direct interspecies electron transfer (DIET) between defined species such as Geobacter metallireducens and Methanosarcina barkeri (Liu et al., 2012), or mixed culture within methanogenic aggregates (Luo et al., 2015). In fact, electron transfer to methanogens during the syntrophic metabolism can be realized indirectly by taking H2 or formate as the interspecies electron transfer carries, or directly via biological electrical connections or a combination of biological and abiological electron transfer components (Chen et al., 2014). Related studies suggested that syntrophic metabolism, such as the syntrophic acetate oxidization played a important role in the initiation of methanogenesis, especially in the stressed environmental conditions (Hao et al., 2010). DIET was the primary mechanism of interspecies electron transfer in UASB reactors treating brewery wastewater (Liu et al., 2012; Morita et al., 2011; Shrestha et al., 2014). Thus multiple lines of evidence suggested that the strategy of introducing conductive carbon materials into anaerobic digester may help to strengthen the syntrophic associations between bacteria and methanogens and therefore to enhance the digester’s effectiveness (Zhao et al., 2015). Nevertheless, the colonization of bacteria was affected by the characteristics of the supporting materials. Bacteria preferably adhered to the moderately rough surfaces that have pores measuring a few tenths of a micron in diameter more than the polished surfaces and rough surfaces (Kuroda et al., 1988). It found that the outer pores of AC are inaccessible for the organisms, which can therefore attach only to the external surface and enter only some large macropores and fissures (Kuroda et al., 1988; Voice et al., 1992). Meanwhile, there are intrinsic correlation between microbial community and the conductivity of anaerobic sludge aggregates (Shrestha et al., 2014). Thus it can foresee that the adhesion of microbial communities on AC with different size and macroporous structures might be different, leading to the differentiated efficiencies of DIET and methane conversion. However, to the authors’ knowledge, the relevant studies are quite few, especially about the selective colonization of functional microbes on carbon materials with different particular sizes. Therefore, two kinds of AC with different particle sizes (i.e. 0.84–2.00 mm and 75–177 lm) have been supplemented into UASB reactors and the reactors’ performance were compared by studying the stability, substrate bioconversion efficiency and CH4 productivity under a large range of high organic loading rates. Additionally, microorganism colonized on the surface of AC was characterized by scanning electron microscopy (SEM) and highthroughput 16S rRNA gene pyrosequencing analysis in an attempt to explain the effects of the AC properties on the adhesion and colonization of bacteria.
2. Methods
powered activated carbon (PAC) were added into R1 and R2, respectively, each of which received 5 g/L of AC. R0 without AC was operated as a control. Synthetic brewery wastewater was used in this work for feeding the reactors. Ethanol and glucose were used as carbon source, which concentrations were 28.15 g/L and 17.79 g/L, respectively. Additionally, 2.59 g urea, 0.20 g yeast extract, 1.91 g K2HPO4, 1.24 g KH2PO4 and 2 mL trace element solution were added (per liter) to supply necessary nutrient for microbial growth. The total COD of original solution for synthetic wastewater is 65.3 g/L. During the experiment, the original solution was diluted with deionized water into different organic loads according to the experiment operation conditions (Table 1). The pH of diluted influent solution was adjusted to 7.2 using Na2CO3 and HCl solutions. Reactors were operated in batch mode, and 2 L of diluted synthetic wastewater was fed into each reactor every 48 h. Three reactors were placed in a temperature-control incubator at 35 ± 2 °C without light. 2.2. Analytical methods 2.2.1. Physiochemical analysis Biogas produced from each reactor was trapped by gas bags, the volume of which was measured with syringe. A cumulative value of biogas generated within two-day batch operation was recorded. Biogas compositions (CH4, CO2 and H2) were determined by a gas chromatograph (GC9890B, Shanghai Linghua Co., China) equipped with a thermal conductivity detector. Before feeding fresh substrate into reactors every two days, the effluent of each reactor was sampled and analyzed to monitor the variations of pH, total organic carbon (TOC) and volatile fatty acids (VFAs). After filtration (0.45 lm), the concentrations of VFAs were analyzed using high performance liquid chromatography (Waters 2695/2489, USA). TOC was analyzed by using Total Carbon/Total Nitrogen analyzer (Multi N/C 3100, Jena Co., Germany). The microorganism attached on AC was observed by scanning electron microscope (SEM), which detailed method was described in Supporting Information. 2.2.2. Microbiological analyses 2.2.2.1. Spatial fractionation of sludge samples. To study the spatial distribution of bacteria and archaea on AC, the microorganisms in the sludge samples were divided into three parts, i.e. suspended, loosely attached and tightly adsorbed fractions according to the method developed by (Luo et al., 2015). Then the total DNA of three fractions were extracted using the PowerSoilTM DNA isolation kit (Mo-Bio Laboratories Inc., CA), and labeled with ‘‘S”, ‘‘L” and ‘‘T” respectively. The concentration of DNA samples were analyzed with UV–Vis spectrophotometer (Nanodrop 2000, Thermo, USA). Each sample has been conducted in triplicate, then the combined DNA solution was stored for the following analysis.
2.1. Reactor design and operation Three identical lab-scale upflow anaerobic digesters (internal diameter of 160 mm and height of 360 mm) were used in this study, each of which had a working volume of 5.6 L. The seed sludge was taken from the secondary sedimentation tank of a full-scale wastewater treatment plant (Quyang Sewage Treatment Plant) located in Shanghai, China. After four months acclimation, seed sludge was sieved through 0.5 mm mesh before feeding into UASB reactors. The final concentration of volatile suspended solids (VSS) in reactors was adjusted to 6.0 g/L, the ratio of VSS to total suspended solids (TSS) was 66%. Two different particle sizes of coal-based AC, i.e. 10–20 mesh (0.84–2.00 mm) of GAC and 80–100 mesh (75–177 lm) of
2.2.2.2. High-throughput 16S rRNA gene pyrosequencing. The microbial community of samples collected from suspended sludge and biofilm were analyzed by using high-throughput pyrosequencing
Table 1 Operation conditions for upflow anaerobic digesters. Stage
I II III IV
Run time (day)
1–10 11–68 69–83 84–95
OLR (g COD/L/d)
HRT (d)
R0
R1
R2
2.9 5.8 5.8 5.8
2.9 5.8 7.0 12.0
2.9 5.8 7.0 12.0
5.6 5.6 5.6 5.6
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on an Illumina platform (Illumina Miseq PE300). Amplicon libraries were constructed for pyrosequencing using bacterial primers 515F (50 -GTG CCA GCM GCC GCG GTA A-30 ) and 806R (50 -GGA CTA CHV GGG TWT CTA AT-30 ) for the V4–V5 region of the microbial 16S rRNA gene, which were selected as the sequencing primer set to simultaneously obtain bacterial and archaeal information (Bates et al., 2011; Luo et al., 2015). The raw pyrosequencing data were deposited to the NCBI Sequence Read Archive database (PRJNA285717). Then the barcodes and primers from the resulting sequences were trimmed for subsequent analysis.
acidogenesis, which proportion was influenced by various factors such as pH, temperature, hydraulic retention time etc (Xu et al., 2014). During the acetogenesis process, acetate can be generated from the oxidization of propionate and butyrate according to Eqs. (1) and (2), while the production of methane is mainly resulted from two pathways, i.e. hydrogenotrophic and acetotrophic ways (Abbasi et al., 2012). As seen in Fig. 2, butyrate was the predominant species for R0, while acetate and propionate predominated in R1 and R2, which suggested that the addition of AC might accelerate the oxidization of butyrate into acetate. Anaerobic oxidation of fatty acids
3. Results and discussion
CH3 CH2 CH2 COO þ 2H2 O ! 2CH3 COO þ Hþ þ 2H2
ð1Þ
3.1. Variations of biogas production, pH and TOC
CH3 CH2 COO þ 2H2 O ! CH3 COO þ CO2 þ 3H2
ð2Þ
Total four stages of operation (Stage I–IV) were conducted in 95 days, which OLRs were 2.9, 5.8, 7.0 and 12.0 g COD/L/d, respectively. The variations of biogas generation from three UASBs under each stage are shown in Fig. 1. The OLR of Stage I was 2.9 g COD/L/ d, and the volumes of biogas generated from three reactors were found to be at a low level, which can be regarded as the acclimation and adaptation period of inoculum to the water quality. When OLR increased to 5.8 g COD/L/d at Stage II, pH levels in R1 and R2 were relatively stable, which almost lay in the range of 7.0– 7.8. Whereas, the pH of R0 declined sharply from 7.8 to 5.5 after one week (Fig. 1c), due to the acid accumulation as shown in Fig. 2a. The acid crisis led to the inhibition of biogas production, which even ceased occasionally. The acidification was not easy to be alleviated by taking a series of pH adjustment measurements, thus the organic load was no longer increased in R0 and maintained at 5.8 g COD/L/d till to the end of experiment. However, biogas volume of R1 and R2 generally increased when OLR was increasing. Meanwhile, TOC concentration of effluent from in R0 tended to increase with the continuous operation, indicating the poor performance of reactor. Whereas TOC removal efficiencies of R1 and R2 were much higher, mostly higher than 90%. These results indicate a significant improvement on the reactor performance with the augmentation of AC in R1 and R2 compared with R0. Furthermore, it is interesting to find that, both the biogas volume and the methane content in R2 are higher than R1 (Figs. 1 and S1). The average methane content of R1 was 64.1 ± 5.7%, while it was 70.4 ± 4.4% under the stable operation. However, the TOC removal efficiency of R2 was slightly lower than R1. It indicates that the conversion rate of substrate into methane was slightly higher in R2, which might be resulted from a more efficient syntrophic cooperation. Nevertheless, this hypothesis needs to be proved by further researches. 3.2. Variations of volatile fatty acids (VFAs) Fig. 2 shows the variations of VFAs in effluent from three reactors. It is easily to find that R0 was undergoing acidification when increasing OLR to 5.8 g COD/L/d, as evidenced by the much higher level of VFAs concentrations, which even went up to 24.7 g/L by the end of experiment. This trend was in accordance with the acidic pH in R0. VFAs concentrations of R1 and R2 increased to about 4 g/L on the beginning of Stage II, which decreased in the following days. It indicates the faster acclimation of microorganism to the elevated OLR in R1 and R2 due to the addition of AC. This result is similar to the previous studies, showing that the addition of AC could accelerate the startup of methanogenesis (Kindzierski et al., 1992). Acetate, butyrate and propionate were the predominant VFAs in this study. In the digester taking glucose as the substrate, acetate, butyrate and propionate were reported to be the major products of
By the end of Stage II, the increase of total VFAs concentration was found occasionally in R1 and R2. It might be related to the failure of temperature control due to instrument trouble, which was repaired before starting Stage III. When OLR increased to 12.0 g COD/L/d in Stage IV, VFA and TOC concentrations of R1 increased suddenly, whereas they were much more stable in R2. It indicates that the syntrophic microbial community attached to PAC might be much adaptable for high density wastewater. 3.3. Microbial community revealed by high-throughput sequencing By the end of digestion, sludge samples were taken to characterize the spatial colonization of bacteria and archaea in solution and tightly or loosely bound on the GAC and PAC, which results were illustrated in Table 2 and Figs. 3 and 4. As shown in Fig. 3, the taxonomic compositions of inoculum and R0 were distinctly differentiated from R1 and R2. The detected total sequences number was 9446–15,900. The relative proportion of archaea increased significantly from less than 1% in inoculum to 5.1–31.1%. Furthermore, the archaea taxonomic number in the tightly bound fractions, i.e. the R1-T and R2-T (25.0% and 31.1%), was much higher than the L and S fractions (4.1–6.8%), which indicates that the archaea tend to reside in the core area of AC. The similar result was found by Satoh et al. (2007), a layered structure of the anaerobic sludge granule was revealed by the application of fluorescence in situ hybridization (FISH), where the outer layer of granule was dominated by Bacteria and the inner layer was dominated by Archaea. Thus, this result proved that the introduction of AC could accelerate the startup of methanogenesis, leading to the faster conversion of VFAs into methane gas. 3.3.1. Archaeal taxonomic distribution A total of 35 OTUs were detected for Archaea, and seven predominant OTUs were selected for further discussion. As shown in Fig. 4a, both the hydrogenotrophic methanogens and acetoclastic methanogens were found in R0, which was constituted by the species clustered to the genera of Methanosarcina, Methanosaeta, Methanobacterium and Methanobrevibacter. Only two predominant genera was found in R0, i.e. Methanobacterium (81.6%) and Methanosaeta (14.3%). For R1 and R2, the proportion of Methanosaeta decreased significantly, and the proportion of Methanosarcina increased accordingly compared to R0. Methanosarcina accounted for 4.6–19.2% in the ‘‘S” and ‘‘L” fractions of R1 and R2, and 50.0% and 58.6% in the ‘‘T” fractions of R1 and R2, respectively. It is generally accepted that two-thirds or more of the methane was produced in an anaerobic bioreactor is derived directly from acetate (acetoclastic methanogenesis). Only two methanogenic genera, i.e. Methanosaeta and Methanosarcina, are known to grow by producing methane from acetate (Zinder, 1993). Methanosaeta concilii is solely an acetoclastic bacterium and is the only
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I
III
II
I
IV
III
II
IV
(a)
R0 R1 R2
15,000
(c) 8
7 10,000
pH
Volume of biogas (mL)
20,000
6
5,000 5 0 4
100
(d)
12000 80
TOC of effluent (mg.L -1)
Propotion of methane(%)
(b)
60
40
20
0
8000
4000
0 0
20
40
60
80
Run time (day)
100
0
20
40
60
80
100
Run time (day)
Fig. 1. Reactor performance under various OLRs, (a) biogas volume; (b) the methane proportion of biogas; (c) pH of effluent; (d) TOC of effluent.
mesophilic species of its genus, other species being thermophiles; nevertheless M. barkeri is metabolically the most versatile of all the mesophilic methanogenic bacteria, since it can not only form methane from acetate, but also from H2 and CO2 (hydrogenotroph), and from methanol and methylamines (methylotroph) (Rocheleau et al., 1999). It has been reported that under the stressed environmental conditions, the syntrophs of acetate oxidizing bacteria and hydrogenotrophic methanogens outcompeted the acetoclastic methanogens to be the dominant acetate utilizers (Hao et al., 2010; Hattori, 2007). It is corresponded to the results of present study. In R1 and R2, the fraction of acetoclastic methanogens decreased, which was accompanied with the increase of Methanosarcina, a species with more versatile metabolic ability (Ho et al., 2013). Furthermore, Methanoculleus was only found in R1 and R2 augmented with AC, and it preferred to be suspended in solution or loosely attached to AC. The genus Methanoculleus, is composed of eight species, nearly all species can grow on H2/CO2 or formate as substrates for methanogenesis, two species (Methanoculleus chikugoensis and Methanoculleus palmolei) can also use alcohols. The increased abundance of Methanoculleus may also be associated with the alteration of predominant pathway for methanogenesis. Comparing the archaeal community distributed in the different fractions of sludge in R1 and R2, it is found that hydrogenotrophic methanogens preferred to predominate in the planktonic phase and the outer layer of biofilm attached to AC, whereas acetoclastic methanogens and hydrogenotrophic methanogens co-existed in the inner layer of biofilm attached to AC. This result is corresponding to the study of Luo et al. (2015), who also found that Methanobacterium tended to reside in the suspended solution, while Methanosarcinales resided deep within the pore channel of the coarse biochar.
3.3.2. Bacterial taxonomic distribution A total of 363 OTUs were detected for bacteria, total 23 OTUs were selected for further discussion. The predominant species include Proteiniphilum, Bacteroidales, Alcaligenes, Planococcaceae, Clostridiaceae, Corynebacterium, and Spirochaete. R0 was dominated by the members of Proteiniphilum (24.3%), Planococcaceae (13.0%), Alcaligenes (8.9%) and Clostridiaceae (7.6%). Nevertheless, in R1 and R2 the percentage of Planococcaceae and Clostridiaceae decreased and the the fractions of Bacteroidales (12.4–29.9%), Corynebacterium (3.9–19.1%) and Spirochaete (up to 12.5%) increased accordingly. The genus Proteiniphilum was found to be one of the most abundant species in all the reactors, which belongs to the family of Porphyromonadaceae. The species Proteiniphilum acetatigenes was previously found in the reactors accepting brewery wastewater, which can use pyruvate as carbon resource and convert it to acetic acid and CO2. The syntrophic degradation of fatty acids by coculturing this syntrophic strain and Methanobacterium sp. has also been proved (Chen and Dong, 2005). Comparing R1 with R2, the fractions of Bacteroidales, Desulfuromonas and Thermotogaceae were relatively higher in R2. Thermotogaceae was also found in the AD sludge (Siegert et al., 2015). Thermotogales-related phylotypes were theorized to be involved in the syntrophic acetate oxidation with hydrogenotrophic methanogens (Balk et al., 2002), or biodegradation of hydrocarbons via syntrophic or fermentation oxidation under methanogenic or sulfidogenic conditions (Cheng et al., 2013; Sun et al., 2014). Bacteroidales and Desulfuromonas have also been detected in the methanogenic consortium (Luo et al., 2015). Furthermore, Spirochaete tends to distribute in planktonic phase (S and L fractions). Whereas Corynebacterium, Enterococcus and Propionimicrobium tends to attach on the inner layer of aggregates,
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I
III
II
IV
24
(a)R0
VFAs (g/L)
20 16 12 8 4 0
Fig. 3. Similarity of taxonomic composition.
0
20
40
60
80
100
8
(b)R1 VFAs (g/L)
6 4 2 0 0
20
40
60
80
100
8
(c)R2
butyrate propionate acetate
VFAs (g/L)
6 4 2 0 20
40
60
80
100
Time (d) Fig. 2. Changes of VFAs under various OLRs, (a) R0, (b) R1, (c) R2.
showing much higher abundance for the tightly absorbed fraction (10.9–19.1%, 4.0–8.1% and 7.7–18.3%) than in the S and L fractions (1–3%). These results showed that the syntrophic bacterium was crucial for the methanogenic reactor with alcohol and VFAs as substrate. AC augmentation led to the increment of syntrophic acetate oxidizing bacteria, which could enhance the syntrophic degradation of alcohol and VFAs in brewery wastewater.
4. Discussions Biomethane generation from organic matters is fulfilled by the syntrophic relationships between acetogens and methanogens. The first mechanism described for electron exchange in methanogenic systems was interspecies hydrogen transfer and formate (Schink, 1997). The capability of exchanging electron via electrically conductive filaments or soluble medium, such as multiheme c-type cytochrome, OmcS has also been found for some specific
microbes (Reguera et al., 2006). Recent studies indicate that interspecies electron transfer could occur in co-culture of Geobacter and Methanosaeta species within aggregates or via granulated activated carbon or carbon nanotube (Morita et al., 2011; Rotaru et al., 2014). However, the effect of these inert material on the degradation of organic matters in natural communities is less clear. Above results showed that, by adding PAC and GAC, a higher methane production rate was found in the anaerobic digester with natural microorganism (using pre-cultured sewage sludge as inoculum). Enhanced performance could be correlated to the increased microbial population of methanogenic bacteria and syntrophic metabolism bacteria, such as Bacteroidales and Spirochaete. Methanosarcina, which have a high level of metabolic capability, increased in the tightly absorbed fraction on the PAC surface, while Methanoculleus was the second predominant species in the looselybound fraction of PAC. The fractions of Bacteroidales, Desulfuromonas and Thermotogaceae were also found to be higher in R2 with PAC. Thermotogaceae was one of the limited number of syntrophic acetate oxidation bacteria have been isolated and characterized until now, the fraction of which was found to be increased in the anaerobic digester with stressed environment condition, such as reduced hydraulic retention time (Ho et al., 2013). Nevertheless, the detected OTUs clustered to Geobacter was relatively low in present study, which plays important role in the DIET between syntrophic species. It indicates that there might be other microbes participant in the DIET, which needs further investigation. Furthermore, the morphological feature of microorganism colonized on the PAC and GAC also showed diversity. It found that both materials shows extensive colonization within the pore structure but the PAC appeared to be more completely and consistently colonized (Fig. S2). Li et al. (2015) has found the bacteria were densely packed with their induced extracellular polymer substance (EPS) in anaerobic sludge aggregates under the exposure to carbon nanotube. It can also be observed that the bacteria attached on PAC were closely clustered with EPS to present a larger aggregates in the pores measuring a few tenths lm. Nevertheless, millions of smaller pores were observed in GAC and PAC measuring less than 10 lm, which appeared to be inaccessible for bacteria. Similar result has been observed by Voice et al. (1992). PAC provided abundant micropore–mesopore structure for methanogens and syntrophic VFAs utilizing bacteria to colonize, meanwhile EPS surrounded microorganism clusters might also play role in enhancing the interspecies mass transfer and electron transfer. From this perspective, PAC prevails over than GAC especially when reactor was operated under heavy load.
Table 2 Total sequences number detected by high-throughput sequencing.
Total sequences number Sequences number of archaea Ratio of archaea (%)
Inoculum
R0
R1_L
R1_S
R1_T
R2_L
R2_S
R2_T
15,111 30 0.20
9466 488 5.15
11,563 475 4.11
12,965 877 6.761
13,336 3334 25
15,900 656 4.13
12,871 658 5.11
13,238 4112 31.06
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(a)
1.0
Proportion
0.8 others Thermoplasmata Methanoculleus Miscellaneous Methanobrevibacter Methanobacterium Methanosarcina Methanosaeta
0.6
0.4
0.2
0.0 Inoculum
R0
R1_L
R1_S
R1_T
R2_L
R2_S
R2_T
(b) 1.0
Proportion
0.8
0.6
0.4
0.2
0.0
Inoculum
R0
R1_L
R1_S
R1_T
R2_L
R2_S
R2_T
Others Aminobacterium Clostridiaceae Anaerolineaceae Mollicutes Thermovirga Synergistaceae Erysipelothrix Dehalobacter Christensenellaceae Planococcaceae Enterococcus Saprospiraceae Acetobacterium Pseudomonas Propionimicrobium Spirochaete Alcaligenes Petrimonas Corynebacterium Thermotogaceae Desulfuromonas Bacteroidales Proteiniphilum
Fig. 4. (a) Archaeal taxonomic distributions; (b) bacterial taxonomic distributions.
5. Conclusions
Appendix A. Supplementary data
This study shows that AC can enhance the biogas production and methane content from anaerobic digesters with synthetic brewery wastewater, meanwhile shorten the start-up time. Both GAC and PAC enhanced the ability of microbes in resisting the organic loading shock. Comparatively, the more abundant mesopore structure of PAC was favorable for the colonization of specific bacteria, such as Methanosarcina sp. and syntrophic VFAs-oxidizing bacteria, leading to the enhanced syntrophic associations between bacteria and methanogens. Thus anaerobic digester supplemented with PAC will be a cost-effective strategy for converting waste to biofuel.
Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.biortech.2015.08. 018.
Acknowledgements The authors wish to thank the financial supports from National Natural Science Foundation of China (51308337), Natural Science Foundation of Shanghai (13ZR1458400), and the supports from State Key Laboratory of Pollution Control and Resource Reuse Foundation (PCRRF13007) and Shanghai Municipal Education Commission (slg13028).
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