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Bioenergy Research Institute, Nanjing Tech University, Nanjing, Jiangsu ... Keywords: Anaerobic digestion, Biogas, Microbial community, Temperature, R...
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Biogas Production and Microbial Community Dynamics during the Anaerobic Digestion of Rice Straw at 39−50 °C: A Pilot Study Qing Yu,†,‡,§ Zhenzhen Tian,†,‡,§ Jingyuan Liu,†,‡ Jun Zhou,*,†,‡,§ Zhiying Yan,∥ Xiaoyu Yong,†,‡ Honghua Jia,†,‡ Xiayuan Wu,†,‡ and Ping Wei† †

College of Biotechnology and Pharmaceutical Engineering and ‡Bioenergy Research Institute, Nanjing Tech University, Nanjing, Jiangsu 211816, China § Jiangsu National Synergetic Innovation Center for Advanced Materials (SICAM), Nanjing, Jiangsu 211816, China ∥ Key Laboratory of Environmental and Applied Microbiology, CAS, Environmental Microbiology Key Laboratory of Sichuan Province, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China ABSTRACT: The purpose of this study was to investigate the anaerobic digestion (AD) of rice straw at different temperatures in a 300 m3 bioreactor. The results showed that the biogas yield was 401.9 m3/ton (dry straw weight) in this AD system. The contents of total solids, volatile solids, chemical oxygen demand, pH, NH4+-N, and volatile fatty acids were all in the optimal range, indicating that the entire AD system was stable and efficient. In addition, the phylum Bacteroidetes was the main type of bacteria found during the AD process. When the temperature increased, the relative abundance ratio of Methanoculleus, Methanosarcina, and Firmicutes increased. The phyla Bacteroidetes and Fibrobacteres predominated in straw samples, and the relative abundance ratio of Firmicutes and Bacteroidetes in thermophilic AD was much greater than that in mesophilic AD. This study provides new evidence regarding the effects of temperature on the community changes of specific microbiota in the AD of rice straw. to mesophilic conditions. Lin et al.9 investigated the relationship between methane production and microbial gene expression at different temperatures (25 °C−55 °C) in a 2 L anaerobic flask with swine manure as the raw material, and it was found that temperature affected methane production through centralization of the microbial community in the AD system. Watanabe et al.13 found that the methane production rate and microbial community were also influenced by temperature in the Brazilian waterweed Egeria densa AD system. However, there are only a few reports in the literature regarding the operating temperature when only straw is used to produce biogas in commercial reactors. In particular, an understanding of both process performance and the microbial community during AD is necessary to determine the optimum operating conditions in commercial reactors. The Chinese government has strongly supported the building of biogas plants for waste treatment for a long time. To date, many biogas plants have been built to treat the agricultural waste, and about 30% of these use straw as the feedstock. Biogas plants designed for agricultural waste are classified as small-, medium-, large-, and super-large-scale based on the daily biogas production. However, the annual biogas production is low.14,15 Biogas plants have the pervasive problems of low material conversion rates and low volumetric biogas production rates.16 Furthermore, as anaerobic bacteria grow slowly, the major problem in an anaerobic digester is the long start-up period and difficulty in achieving spontaneous development of granulation.17 Therefore, in this study, experiments were conducted

1. INTRODUCTION Biomass resources are the fourth largest energy resources after coal, fossil oil, and natural gas, accounting for 14% of the total energy consumption worldwide. As a large agricultural country, approximately 700 million tons of straw are produced in China every year, which is almost 1/3 of the amount generated worldwide.1,2 Approximately 400 million tons can be used as biomass energy. However, most of this straw is burned in the field, which causes serious environmental pollution and is a waste of a natural resource. AD is considered a promising means of crop straw disposal because it prevents environmental pollution and, at the same time, generates a renewable energy source.2−4 AD is a complex biochemical process, and several major factors can influence AD such as the digestion temperature, carbon-to-nitrogen ratio (C/ N), initial pH and substrate concentration, and pretreatment of the substrate.5−7 Digestion temperature is one of the most important factors and affects the stability and performance of straw AD in both batch and continuous mode.8 In an AD system, the thermodynamic equilibrium of biochemical reactions and microbial community structure, activities, and diversity can be greatly influenced by the digestion temperature.9 Researchers have researched the effect of temperature on AD and have obtained different results. Li et al.10 found that total methane production increased with increased temperature, while acidification occurred easily in a thermophilic environment, leading to the control of biogas production. Mashad et al.11 investigated the start-up performance of AD under different temperatures, and confirmed that the AD process can be accelerated when AD systems are operated under thermophilic conditions (55.8 °C). Kim et al.12 found a similar phenomenon when more biogas was produced during AD under thermophilic conditions compared © XXXX American Chemical Society

Received: December 22, 2017 Revised: March 2, 2018 Published: March 16, 2018 A

DOI: 10.1021/acs.energyfuels.7b04042 Energy Fuels XXXX, XXX, XXX−XXX

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Energy & Fuels under thermophilic conditions in a 300 m3 reactor. The objectives of this study were to assess the effects of temperature on biogas yield and methane yield from straw. The pH, ammonia nitrogen (NH4+-N), chemical oxygen demand (COD), volatile fatty acid (VFA) concentration, and straw components were measured to investigate the AD process performance in detail. In addition, the relationship between biogas production and the microbial community structure at different temperatures was determined.

clustered into operational taxonomic units (OTUs), and an OTU table was created for statistical analysis.21 Taxonomy was assigned using the Ribosomal Database Project classifier. We mainly calculated the αdiversity and β-diversity analyses of the samples.22 2.6. Statistical Analysis. To analyze the differences in the experimental data, Origin 8.5 software was used to draw the figures. Representational difference analysis (RDA) and principal coordinates analysis (PCoA) in Fast UniFrac were used to evaluate the differences in the structure of the microbial communities.

3. RESULTS AND DISCUSSION 3.1. Effects of Different Temperatures on Biogas and Methane Production. In this study, temperature was the key parameter influencing biogas production during the AD process. Daily biogas production volume in this fermentation system at different temperatures is shown in Figure 1a. When the reactor was fed with 800 kg of rice straw every day, the daily biogas production improved with increasing temperature. These results indicated that the daily biogas production peak in the fermentation system was 335 m3 (day 50). The methane and CO2 concentrations during the AD process at different temperatures are shown in Figure 1b. The initial methane and carbon dioxide concentrations were both approximately 50%. The daily methane content improved as the temperature increased, and the peak content in this fermentation system was 67% (day 40). After that, the methane concentration was stable at about 60%, and the CO2 concentration was stable at about 40%. The daily methane production peak in this fermentation system was 204 m3 (day 50). The trends in daily biogas production, daily methane content, and daily methane production were consistent, and increased slowly from day 0 to day 24, increased quickly to a peak (day 50), and then remained stable. In conclusion, this fermentation system ran steadily, and biogas production improved with increased temperature. At all high-temperature fermentation stages, the TS biogas production was 401.9 m3/ton (dry weight). The chemical properties of rice straw are presented in Table 1. The water was added to promise that the TS was suitable for fermentation. The results of fermented liquid’s TS and VS are shown in Figure 1c. In this study, the TS value and VS value were maintained within a suitable range. 3.2. Effects of Different Temperatures on COD, NH4+-N, pH, and VFA. The COD, NH4+-N, pH, and VFA were important parameters for evaluating the microenvironment in the AD system (Figure 2). In this high-temperature AD system, the pH value ranged from 7.01 to 7.27, and the VFA concentration ranged from 13.27 to 19.90 mM/L (Figure 2a). As previously reported, the pH value can directly affect the growth of microorganisms; pH 7.0 is optimal for the growth of methanogens, and pH 5.5−6.5 is beneficial for the growth of acidogens.23,24 In the present study, the pH values were maintained within the optimal range throughout the entire experimental process. It is known that VFAs are generated by hydrolysis and acidogenesis. In the AD system, VFAs are subsequently consumed by methanogenesis.25 In the present study, the VFAs decreased at different temperatures. This indicated that the VFAs were digested via methanogenesis when the temperature increased. However, other VFAs, with the exception of acetic acid, were infrequently found; thus, Figure 2a only shows the change in acetic acid with temperature. The COD and NH4+-N concentrations are shown in Figure 2b. The results indicated that the COD ranged from 4934.2 to 6568.3 mg/L, and the NH4+-N concentration ranged from 750.4 to 1008.5 mg/L. According to previous reports, when the NH4+-N concentration

2. MATERIALS AND METHODS 2.1. Substrates and Inoculum. Rice straw was collected from Siyang (Jiangsu Province, China), and the stalk samples were compressed into bulk for transportation. Before use, the rice straw was shattered into sections 10 mm long and added in the fermentation tank, mixed with the fermented liquid and water. The inoculum was the original fermented liquid from the fermentation tank. 2.2. Experimental Design and Setup. A 300 m3 anaerobic bioreactor was used in this study. The experiments were conducted in the 300 m3 anaerobic bioreactor at 39 to 50 °C for 50 days. The initial content of total solid (TS) in the substrate in the digester was 8%. The digester was fed with rice straw (800 kg) every day. The biogas production and methane concentration were measured automatically. The TS, volatile solid (VS), pH value, VFA, COD, and NH4+-N were measured regularly. 2.3. Measurement of Chemical Properties. The biogas production and temperature of the fermentation system were evaluated with an intelligent monitoring system. The contents of biogas, including methane and carbon dioxide, were also analyzed with intelligent monitoring. The biogas yield and methane content were used to calculate methane production.18 In addition, samples were regularly withdrawn to measure related chemical properties. The contents of TS and total VS were measured using standard methods.19 The total C and total N in the rice straw were measured using a Vario EL element analyzer (Elementar Analysensysteme GmbH, Germany). Samples were dried and triturated for the measurement of the total C and total N.1 The cellulose, hemicellulose, and lignin of the rice straw were measured using a fiber analyzer (ANKOM A200i, America). The gas chromatography (Model 6890A, Agilent Technologies) was used to measure the VFA concentration. Before the VFA was measured, the samples were drawn from the supernatant after the centrifuging of the fermented liquid and then acidized by methanoic acid (1 mL of supernatant added to 30 μL of methanoic acid). The conditions of gas chromatography (GC) were: capillary column (HP-INNOWAX, 60 m × 0.32 mm), carrier gas of He (40 cm/sec), the temperature profile maintained at 100 °C for 5 min and then increased to 250 °C by 10 °C/min and maintained 250 °C for 12 min; the split ratio was 1:50; and the type of detector was a flame ionization detector at 300 °C. The COD and NH4+-N were measured using a water-quality detector (LianHua 5B-3C). The pH value was measured using a pH meter (model PHSJ-5, INESA). 2.4. DNA Extraction and Sampling. The biogas slurry samples were centrifuged at 1684g for 10 min to separate the liquid and solids before extracting the DNA. The total microbial DNA extraction (DNA isolation and purification) was performed using PowerSoil DNA Isolation Kit Components (Mo-Bio, Carlsbad, CA). A NanoDrop spectrophotometer was used to measure DNA concentration. In addition, DNA was diluted and stored at −40 °C for downstream use.18 2.5. 16S rRNA Gene Sequences. The samples were sequenced with the Illumina Miseq platform at Chengdu Institute of Biology, Chinese Academy of Sciences (Sichuan, China). The 16S rRNA gene was amplified with the primers 515F-909R for pyrosequencing.20 All PCR products were pooled together with an equal molar amount from each sample and quantified using a NanoDrop spectrophotometer. QIIME Pipeline version 1.7.0 (http://qiime.org/) was used to process the sequence data. Based on the barcodes, all sequence reads were assigned and trimmed. The sequences with length >300 bp, without ambiguous base “N”, and an average base quality score of >30 were used for downstream analysis. Sequences at a 97% identity threshold were B

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Energy & Fuels Table 1. Characteristics of the Substrates Used in the Anaerobic Digestion Experiments parameter

rice straw

TSb (%) VSc (%) C/N lignin (%) cellulose (%) hemicellulose (%)

93.49% ± 0.12a 85.83% ± 0.09a 70.88 ± 0.48a 30.04 ± 0.74a 39.06 ± 0.02a 23.87 ± 0.58a

Values are expressed as the mean ± deviation (n = 3). bTS: Total solid. cVS: Volatile solid.

a

3.3. Differences in Prokaryote Diversity at Different Temperatures. In total, 27 duplicate samples were collected at nine time points on day 0 (39 °C), 8 (40 °C), 12 (41 °C), 23 (43 °C), 31 (45 °C), 39 (48 °C), 40 (50 °C), 42 (50 °C), and 47 (50 °C). The samples were marked ZJ-6, ZJ-7, ZJ-8, ZJ-9, ZJ-10, ZJ11, ZJ-12, ZJ-13, and ZJ-14 to analyze the prokaryotic community diversity at different temperatures. All high-quality sequences were acquired and clustered to calculate the OTUs, and 1352 to 1605 OTUs were generated at a sequencing depth of 14270 reads (Table 1). As the temperature increased, the observed species, Shannon’s diversity indices, and Chao1 estimator of richness changed a little (Table 1). Diversity of the prokaryotic communities in the different temperature samples was also evaluated by PCoA (Figure 3a). There were three distinct community structures at different temperatures. The samples from similar temperatures were clustered together. The structure of the microbial communities at ZJ-6, ZJ-7, and ZJ8 were similar; the structure of the microbial communities at ZJ-9 and ZJ-10 were similar; and the structure of the microbial communities at ZJ-11, ZJ-12, ZJ-13, and ZJ-14 were similar at different times under similar temperatures. These results indicated that similar microbial communities occurred at similar temperatures. As the temperature increased, the microbial communities changed considerably during the AD process. RDA was mainly used to reflect the relationship between flora and environmental factors and was applied on the basis of the OTUs. It was found that 88% and 4.9% of the variations could be explained at the OTUs level by the x and y-axis, respectively (Figure 3b). These results showed that the influence of COD, VFAs, NH4+-N, and temperature on the microorganisms was relatively large. The attributed values of ZJ-6 and ZJ-7 were similar under the different environmental factors; the attributed values of ZJ-11, ZJ-12, ZJ-13, and ZJ-14 were similar. Samples ZJ6 and ZJ-7 were taken at similar temperatures; samples ZJ-11, ZJ12, ZJ-13, and ZJ-14 were taken at similar temperatures. These findings indicated that the community compositions of different samples were different and were similar when the samples were taken at a similar temperature. The relationships between environmental factors were different, as temperature was positively correlated with NH4+-N and pH and was negatively correlated with COD and VFAs. 3.4. Dynamic Changes in the Prokaryotic Community. Figure 4 shows the composition of major bacterial phyla (relative abundances more than 0.1%). These results indicated that the prokaryotic microflora was composed of 24 phyla in the samples, and there were 8 types of high relative abundance ratio microbial colonies at the phylum level in the thermophilic AD of rice straw (phyla with relative abundances more than 1%), which were Firmicutes, Bacteroidetes, Crenarchaeota, Chloroflexi, Tenericutes, Proteobacteria, Thermotogae, and Fibrobacteres. What is

Figure 1. (a) Dynamics of daily volumetric biogas production, (b) methane and carbon dioxide concentration, and (c) total solid and volatile solid value during the anaerobic digestion of rice straw at different temperatures.

exceeds 3000 mg/L, the production of methanogens is inhibited.26,27 During this AD system at different temperatures, the NH4+-N concentration was maintained in the optimal range. These results indicated that the entire AD system was stable and efficient. Furthermore, the continuous 300 m3 anaerobic bioreactor performed well at different temperatures during biogas production. C

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Figure 2. (a) Dynamic change in volatile fatty acids (VFAs) and pH value and chemical oxygen demand (COD) and (b) ammonia nitrogen (NH4+-N) concentration during the anaerobic digestion of rice straw at different temperatures.

Figure 3. (a) Principal coordinates analysis (PCoA) based on the overall prokaryotic community at genus level at different temperatures in the anaerobic digestion of rice straw; (b) distance-based redundancy analysis (dbRDA) of environmental variables (VFAs, pH, NH4+-N, COD, and temperature), and prokaryotic community compositions at the genus level, according to the species fit range of 60−100%; values on the axes indicated the percentages of total variation explained by each axis. Samples were collected at different temperature: ZJ-6 (39 °C), ZJ-7 (40 °C), ZJ-8 (41 °C), ZJ-9 (43 °C), ZJ-10 (45 °C), ZJ-11 (48 °C), ZJ-12 (50 °C), ZJ-13 (50 °C), and ZJ-14 (50 °C).

relative abundance ratio of Tenericutes increased from 0.15% to 2.45% and the relative abundance ratio of Thermotogae increased from 0.24% to 4.99%, while the relative abundance ratio of Crenarchaeota decreased from 16.23% to 5.64% and the relative abundance ratio of Chloroflexi decreased from 9.27% to 1.97%. Chloroflexi can decompose glucose and soluble microbial products to acetic acid and hydrogen at the hydrolysis phase during the AD process.32 The relative abundance of Chloroflexi in this study was higher than that in other literature reports, indicating the high biogas production of the bioreactor in this study.18 The relative abundance ratio of Proteobacteria decreased from 3.45% to 1.51%, which was correlated with lignocellulose degradation at the hydrolysis phase during the AD process. In addition, the relative abundance ratio of Fibrobacteres decreased from 2.92% to 0.88%, which confirmed that it is involved in cellulose hydrolysis. Overall, Firmicutes and Bacteroidetes were the main cellulolytic bacteria in the thermophilic AD. The relative abundance of Firmicutes increased slightly with increasing temperature and explained why the bioreactor in this study had a high methane production rate. In addition, Chloroflexi, Proteobacteria, and Fibrobacteres were beneficial in producing

more, the methanogens all belonged to Euryarchaeota at the phylum level. The relative abundance ratio of the Euryarchaeota increased from 1.44% to 2.96% with increased temperature. Of these, Firmicutes and Bacteroidetes were the main cellulolytic bacteria in thermophilic AD, and their relative abundance ratios were higher than those of other microbial colonies. During the entire fermentation period, the relative abundance ratio of Bacteroidetes was almost unchanged and was maintained at 35.94%. The phylum Bacteroidetes usually degrades lignocellulose at the hydrolysis and acidogenesis stages during the AD process.28 Cellulose, hemicellulose, and lignin are main components of rice straw, which could limit the hydrolysis rate in the AD system.29,30 Therefore, Bacteroidetes was the major phylum, and the content of Bacteroidetes also remained stable at different temperatures. Accordingly, Firmicutes was the next major bacterial phylum in these samples. With increased temperature, the relative abundance of Firmicutes increased from 9.27% to 50.19%. It is known that the phylum Firmicutes contain acetogenic bacteria, which degrade VFAs to produce acetic acid.31 The VFA concentration remained low (below 20 mM/L) with increased temperature, indicating many methanogenic archaea in the AD system. With increased temperature, the D

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Figure 4. Relative abundance of prokaryotes taxonomic groups in digester samples collected at different temperatures and operating times. The taxonomic classification of prokaryotes reads at the phylum level. Prokaryotes groups accounting for less than 0.1% of all classified sequences are summarized in the group “others”. Samples were collected at different temperature: ZJ-6 (39 °C), ZJ-7 (40 °C), ZJ-8 (41 °C), ZJ-9 (43 °C), ZJ-10 (45 °C), ZJ-11 (48 °C), ZJ-12 (50 °C), ZJ-13 (50 °C), and ZJ-14 (50 °C).

Figure 6. (a) Relative abundance of prokaryote taxonomic groups in slurry (ZJ-6) and straw (ZJ-15) samples collected at the same temperature (39 ± 1 °C); (b) relative abundance of prokaryote taxonomic groups in slurry (ZJ-12) and straw (ZJ-16) samples collected at the same temperature (50 ± 1 °C). The taxonomic classification of prokaryotes reads at the phylum level. Prokaryote groups accounting for less than 0.1% of all classified sequences are summarized in the group “others”.

Figure 5. Composition of methanogens at the genus level in the anaerobic digestion system at different temperatures. Samples were collected at different temperature: ZJ-6, (39 °C); ZJ-7, (40 °C); ZJ-8, (41 °C); ZJ-9, (43 °C); ZJ-10, (45 °C); ZJ-11, (48 °C); ZJ-12, (50 °C); ZJ-13, (50 °C); ZJ-14, (50 °C).

increased temperature, the relative abundance ratio of Methanolinea decreased from 35.86% to 3.40%, and the relative abundance ratio of Methanosaeta decreased from 55.53% to 4.11%, which indicated that these two types of Archaea are mesophilic methanogenic Archaea. Methanoculleus is a hydrogen nutrition methanogenic archaeon that can produce methane using H2 and CO2.33 With increased temperature, the relative abundance ratio of Methanoculleus increased from 0% to 48.18%, and the relative abundance ratio of Methanosarcina (which can use multiple substrates to synthesize methane) also increased from 1.11% to 22.2%. It is known that methanogenesis has a low growth rate; thus, these organisms may be much more

methane, but their relative abundance ratios decreased. This was because they were unable to adapt to the high-temperature environment. 3.5. Dynamic Changes in the Methanogen Community. There were five main types of methanogens in the thermophilic AD of rice straw, which were Methanobacterium, Methanoculleus, Methanolinea, Methanosaeta, and Methanosarcina, and all are Archaea (Figure 5). Of these, Methanolinea and Methanosaeta are acid nutrition methanogenic Archaea.18 With E

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many prokaryotes were not adapted for a high-temperature environment. However, the relative abundance ratio of Firmicutes and Bacteroidetes in thermophilic AD was much greater than in mesophilic AD. Thus, rice straw was easily digested in thermophilic AD, which increased methane production.

Table 2. Prokaryotic Diversity Indices Based on the 97% Identity of 16S rRNA Gene Sequences Chao1c

sample b

ZJ-6 ZJ-7b ZJ-8b ZJ-9b ZJ-10b ZJ-11b ZJ-12b ZJ-13b ZJ-14b

2525.78 ± 105 2180.37 ± 117a 2261.42 ± 205a 2363.84 ± 164a 2481.52 ± 201a 2768.79 ± 119a 2769.54 ± 213a 3060.37 ± 156a 2763.35 ± 146a a

observed OTUd

Shannon indexe

1496.40 ± 88 1352.40 ± 96a 1383.50 ± 105a 1393.73 ± 78a 1402.60 ± 66a 1477.60 ± 82a 1510.13 ± 74a 1605.80 ± 36a 1464.10 ± 68a

8.08 ± 0.08a 7.94 ± 0.02a 7.86 ± 0.03a 7.73 ± 0.15a 7.64 ± 0.03a 7.61 ± 0.12a 7.64 ± 0.09a 7.87 ± 0.05a 7.59 ± 0.18a

a

4. CONCLUSIONS With increased temperature, the daily biogas production and methane content increased, and the biogas yield was 401.9 m3/ ton dry rice straw. The microbial community compositions were markedly changed when the temperature increased; the relative abundance ratio of methanogens, which can produce methane using H2 and CO2 or multiple substrates, increased in the thermophilic AD. Furthermore, the relative abundance ratios of Firmicutes and Bacteroidetes in the thermophilic AD system were higher than in mesophilic AD system. This pilot scale study provided a better understanding of the AD process resulting from specific microbiota and their different spatial distributions in a pure rice-straw system.

Values are expressed as the mean ± deviation (n = 3). bSamples were collected at different temperature: ZJ-6 (39 °C), ZJ-7 (40 °C), ZJ-8 (41 °C), ZJ-9 (43 °C), ZJ-10 (45 °C), ZJ-11 (48 °C), ZJ-12 (50 °C), ZJ-13 (50 °C), and ZJ-14 (50 °C). cChao1: the estimator of community richness. dObserved operational taxonomic units (OUT). e Shannon index is the index of community diversity. a

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susceptible to environmental changes than other microorganisms.34,35 Based on our analysis, there are three main methanogenesis pathways: acid nutrient, methylotrophic, and hydrogenotrophic methanogens in the AD process. This was also shown by the aforementioned changes in the methanogens in thermophilic AD of rice straw. Furthermore, a suitable microenvironment was created at different temperatures, which led to a high methane production rate in the thermophilic AD. 3.6. Differences in Prokaryotes Associated with Straw and Slurry. Figure 6 shows the differences in microflora between straw and slurry at 39 and 50 °C. In general, Firmicutes, Bacteroidetes, Crenarchaeota, and Fibrobacteres were abundant in samples. The relative abundances of these phyla were different in the rice straw and slurry samples. The specific relative abundance ratios of these prokaryotic communities are shown in Table 2. These results indicated that Firmicutes, Spirochaetes, and Fibrobacteres were mainly found in straw samples, and Bacteroidetes, Crenarchaeota, and Chloroflexi were the major phyla in the slurry at 39 °C. However, the phyla Bacteroidetes and Fibrobacteres were mainly found in straw samples, and Firmicutes, Crenarchaeota, Euryarchaeota, and Proteobacteria were the major phyla in the slurry at 50 °C. Straw-associated microbiota mainly degraded straw to soluble substrates for fermentation and methanogenesis in the slurry (see Table 3). In addition, when the results in Figure 6a,b were compared, it was found that the number of prokaryotic phyla in mesophilic AD was greater than in thermophilic AD. This indicated that

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Phone/fax: +86-25-58139929. ORCID

Jun Zhou: 0000-0002-3980-487X Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by the National Key Research and Development Program of China (grant no. 2016YFE0112800), the Key Science and Technology Project of Jiangsu Province (grant no. BE2016389), and the National Natural Science Foundation of China (grant nos. 21777069 and 31700092) as well as The Jiangsu Synergetic Innovation Center for Advanced Bio-Manufacture.

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REFERENCES

(1) Yan, Z. Y.; Song, Z. L.; Li, D.; Yuan, Y. X.; Liu, X. F.; Zheng, T. The effects of initial substrate concentration, C/N ratio, and temperature on solid-state anaerobic digestion from composting rice straw. Bioresour. Technol. 2015, 177, 266−273. (2) Zhou, J.; Yan, B. H.; Wang, Y.; Yong, X. Y.; Yang, Z. H.; Jia, H. H.; Jiang, M.; Wei, P. Effect of steam explosion pretreatment on the anaerobic digestion of rice straw. RSC Adv. 2016, 6, 88417−88425.

Table 3. Relative Abundance of Prokaryote Taxonomic Groups in Slurry and Straw Samples Collected at the Same Temperature phylum

ZJ-6b (%)

ZJ-15b (%)

ZJ-12c (%)

ZJ-16c (%)

Firmicutes Bacteroidetes Crenarchaeota Euryarchaeota Proteobacteria Chloroflexi Spirochaetes Fibrobacteres

8.86 ± 0.96a 36.60 ± 0.09a 13.77 ± 0.13a − − 8.78 ± 0.13a 2.76 ± 0.03a 2.52 ± 0.06a

17.46 ± 0.12a 34.69 ± 0.93a 0.612 ± 0.02a − − 4.49 ± 0.09a 14.13 ± 0.15a 16.56 ± 0.66a

52.15 ± 1.25a 21.03 ± 0.96a 7.49 ± 0.12a 3.55 ± 0.06a 2.31 ± 0.03a − − 0.74 ± 0.01a

33.24 ± 0.82a 39.64 ± 0.65a 0.58 ± 0.01a 0.93 ± 0.01a 0.60 ± 0.02a − − 12.37 ± 0.16a

a Values are expressed as the mean ± deviation (n = 3). bRelative abundance of prokaryote taxonomic groups in slurry (ZJ-6) and straw (ZJ-15) samples collected at the same temperature (39 ± 1 °C). cRelative abundance of prokaryotes taxonomic groups in slurry (ZJ-12) and straw (ZJ-16) samples collected at the same temperature (50 ± 1 °C).

F

DOI: 10.1021/acs.energyfuels.7b04042 Energy Fuels XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.energyfuels.7b04042 Energy Fuels XXXX, XXX, XXX−XXX