Effect of bioaugmentation on the microbial community and mono-digestion performance of Pennisetum hybrid

Waste Management 78 (2018) 741–749

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Effect of bioaugmentation on the microbial community and mono-digestion performance of Pennisetum hybrid Li Lianhua a,b,c,1, Li Ying a,c,1, Sun Yongming a,c,⇑, Yuan Zhenhong a,c,d, Kang Xihui a,b,c, Zhang Yi a,b,c, Yang Gaixiu a,c a

Guangzhou Institute of Energy Conversion, CAS Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, PR China University of Chinese Academy of Sciences, Beijing 100049, PR China Guangdong Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, PR China d Collaborative Innovation Centre of Biomass Energy, Henan Province, Zhengzhou 450002, PR China b c

a r t i c l e

i n f o

Article history: Received 1 December 2017 Revised 24 May 2018 Accepted 15 June 2018

Keywords: Pennisetum hybrid Mono-digestion Bioaugmentation Stability Microorganism community

a b s t r a c t In this study, bioaugmentation with methanogenic propionate-utilizing enrichment was investigated as a method to improve the mono-digestion performance of Pennisetum hybrid in a semi-continuous mode. The effect of bioaugmentation on the microbial community was analyzed as well. The results demonstrate that the steady-state organic loading rate (OLR) of the bioaugmented reactor increased to 4.0 g VS/(L
d) with a volumetric biogas production of 1.95 ± 0.17 m3/(m3
d). In contrast, the nonbioaugmented reactor failed at an OLR of 2.0 g VS/(L
d) accompanied with the accumulation of volatile fatty acids (VFAs). The results of whole genome pyrosequencing analysis suggest that the decrease in relative abundance of syntrophic butyrate and propionate oxidizers, such as Syntrophomonas, Syntrophobacter, and Syntrophorhabdus, reduced the conversion efficiency of butyrate and propionate which leads to the accumulation of butyrate and propionate, influencing the performance of the mono-digestion reactor. Conversely, in the bioaugmented reactor, the higher density of protein- and amino acid-utilizing bacteria, such as Proteiniphilum, Thermovirga, and Lutaonella, as well as the syntrophic association of Syntrophomonas spp. coupled with the methanogens Methanosarcina and Methanocella has a positive effect on system stability and performance. Ó 2018 Published by Elsevier Ltd.

1. Introduction Pennisetum spp., a genus of Paniceae, is widely distributed and used mostly as animal forage (Farrell et al., 2002). Currently, Pennisetum spp. has been deemed as a promising C4 perennial herbaceous plant for biofuel production due to its utilization efficiencies in water and nitrogen, high levels of photosynthesis rates, huge biomass yields, and satisfactory material quality (Nizami et al., 2009; Somerville et al., 2010; Tilman et al., 2006). The feasibility and performance of anaerobic digestion of Pennisetum spp. have been covered in previous literatures (Carvalho et al., 2016; Surendra and Khanal, 2015). Additionally, the technological parameters of the harvest, storage, pretreatment, and processing conditions for Pennisetum spp. have been studied and optimized

⇑ Corresponding author at: Guangzhou Institute of Energy Conversion, CAS Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, PR China. E-mail address: [email protected] (S. Yongming). 1 The first two authors contributed equally to this paper. https://doi.org/10.1016/j.wasman.2018.06.031 0956-053X/Ó 2018 Published by Elsevier Ltd.

(Li et al., 2012, 2016b; Nizami and Murphy, 2010; Pakarinen et al., 2008). Previous studies have reported that the mono-digestion of grass failed in long-term operation due to nutrient imbalance and mechanical failure. Failure caused by crashing in an agitation system was reported for the mono-digestion of grass at an OLR of 2.5 g VS/(L
d) (Thamsiriroj et al., 2012). Similarly, failure in the recovery and stabilization of the mono-digestion system by the addition of trace elements was also reported (Janke et al., 2016). Codigestion of grass and manure has been shown to enhance the efficiency of anaerobic digestion. Studies have found that adding livestock manure can increase the methane yield and improve stability by balancing the nutrient and microbial composition (Jagadabhi et al., 2008; Lehtomaki et al., 2007; Wall et al., 2014, 2013). However, this method was limited by the availability of manure in the mixture. Bioaugmentation with specific stress-resistant or stressefficient microbes is considered to be one of the most effective methods in improving the performance of anaerobic digestion, and has been employed in the phases of hydrolysis, acidogenesis,

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acetogenesis, and methanogenesis to improve conversion efficiencies (Herrero and Stuckey, 2015; Nzila, 2017; Tale et al., 2015). For example, an improved methane yield was found by bioaugmentation with Acetobacteroides hydrogenigenes in a previous study (Zhang et al., 2015). The deterioration of an unstable digestion system was prevented and the failing digester was recovered by the enrichment of methanogenic propionate-degrading microorganisms (Li et al., 2017b). For lignocellulosic-like feedstock, the hydrolysis phase has been proven to be the rate-limiting step because of its recalcitrant structure (Rouches et al., 2016). Therefore, bioaugmentation with a hydrolytic/fermentative culture would be beneficial to reinforce the hydrolysis efficiency and improve the economic feasibility of lignocellulose-rich feedstock as biogas plants. In another study, an increase in methanogenic performance of corn waste was observed after periodic bioaugmentation with cellulolytic microorganisms (Martin-Ryals et al., 2015). The methane production of brewery spent grain increased by 17.80% with bioaugmentation with the hydrolytic bacterium P. xylanivorans Mz5T (Cater et al., 2015). A variety of inhibitory substances and intermediate products, including free ammonia (FA), volatile fatty acids (VFAs), and heavy metals are the primary cause of anaerobic digester failure (Chen et al., 2008; Yuan and Zhu, 2016). For lignocellulosic-like feedstock, system failure was always accompanied with VFAs accumulation. Propionate and acetate are the main components of VFAs and are important intermediate products which easily accumulate, resulting in system imbalance or overload. Bioaugmentation with methanogenic propionateutilizing enrichment cultures that are able to convert acetate and propionate to methane may lead to improved digestion (Tale et al., 2015). Tale et al. investigated the feasibility of bioaugmentation with a propionate-degrading enrichment culture to recover transiently overloaded digesters and found that bioaugmented digesters recovered more rapidly, requiring approximately 25 days less to recover (Tale et al., 2011). As mentioned above, bioaugmentation has been applied in the degradation of lipid-rich wastes, lignocellulosic-like feedstock, and in the reduction in the recovery time of digesters that have been exposed to toxicants. Currently, little information is available regarding bioaugmentation with a methanogenic propionate-utilizing enrichment culture for stabilizing the long-term mono-digestion of grasses. The present study aimed at: (1) comparing the stability of mono-digestion with and without bioaugmentation in a semicontinuous mode to evaluate the feasibility of methanogenic propionate-utilizing enrichment in the improvement of anaerobic digestion performance; (2) investigating the effects of bioaugmentation on the microbial community to determine the dominant bacteria and archaea in the stabile mono-digestion of grasses. 2. Material and methods 2.1. Material and inocula Grass (Pennisetum hybrid) was taken from Zengcheng, Guangdong, China (Li et al., 2016b). The collected grass was ensiled after

cutting it into particles of 1–2 cm. Ground silage grass was used for tests. Table 1 shows the physicochemical parameters of the feedstock. Mesophilic CSTR reactors provided the inocula which TS and VS content were 1.87 ± 0.03% and 1.10 ± 0.003%, respectively. The reactors were fed an artificially prepared substrate composed of peptone, yeast powder, xylose, cellulose, starch, and glucose. 2.2. Enrichment of bioaugmentation consortia Bioaugmentation culture was taken from a digester enriched in propionate-degrading bacteria according to a previous report (Li et al., 2017b). Specifically, the effluent from an anaerobic digester feeding energy crops was cultivated in a BioReactor Simulator (2 L) for the enrichment of bioaugmentation consortia. The conditions for cultivation of bioaugmentation consortia were controlled at 35 ± 1 °C with no-stirring, a hydraulic retention time (HRT) of 20 days, and organic loading rate (OLR) elevating from 0.5 g propionate (Hpr)/(L
d) to 3.0 g Hpr/(L
d). The nutrient medium for enrichment contained the following [mg/L]: FeCl3
6H2O [55]; (NH4)2HPO4 [80]; CaCl2
2H2O [120]; KCl [400]; MgSO4
6H2O [250]; NH4Cl [400]; the trace metal salts [each at 0.5 mg/L] ZnCl2, Na2SeO3, H3BO3, Na2WO4
2H2O, AlCl3
6H2O, CuCl2
2H2O, MnCl2
4H2O, NiCl2
6H2O and CoCl2
6H2O, and sodium propionate was used as a carbon source. According to whole genome pyrosequencing analysis, Proteobacteria, Chloroflexi, Synergistetes and Bacteroidetes were the primary bacteria of bioaugmentation, coupled with Methanomicrobiales, Methanomicrobiales, Methanobacteriales and Methanosarcinales as the dominant archaea. The accession number for the bioaugmentation was PRJNA391398. The effluent from the upper chamber of the digester was used as bioaugmentation consortia. 2.3. Experimental setup and procedure Two continuous stirred tank reactors (CSTR, working volume: 2 L) were run at semi-continuous and mesophilic (35 ± 1 °C) conditions (Bioprocess Control Sweden AB). A vertical mixer was used to periodically stir at a frequency of 60 sec running and 180 sec pausing. Daily biogas yields were automatically recorded by the data acquisition system. Each reactor contained 1800 mL of inocula and began at an OLR of 2.0 g VS/(L
d). The HRT of 30 d was used for each OLR. All OLRs were run for 29–93 d according to the operating performance. The non-bioaugmented reactor was used as a control (R1). In previous studies, the enriched culture of 10 mL/d was suited to enhance the anaerobic digestion of high C/N ratio feedstock (Li et al., 2018). Therefore, for the bioaugmented reactor (R2), a dosage of 10 mL/d (1–51 d) and 20 mL/d (52–91 d) of enrichment culture with 0.25–0.28% g VS was used to optimize the additional dosage at the OLR of 2.0 g VS/(L
d). Subsequently, a rise in OLR from 2.0 g VS/(L
d) to 4.0 g VS/(L
d) at an interval of 0.5 g VS/(L
d) was carried out to evaluate the effect of bioaugmentation on the long-term operation performance of mono-digestion system.

Table 1 The characteristic of Pennisetum hybrid. Material

Pennisetum hybrid

TS (%) VS (%) VS/TS pH C (%) N (%) C/N

15.87 ± 0.62 12.81 ± 0.45 82.30 ± 0.01 – 39.59 ± 0.04 0.92 ± 0.021 43.28 ± 0.96

‘‘–” meaning no data available.

20.94 ± 0.84 18.17 ± 0.81 86.74 ± 0.01 5.04 43.41 ± 0.54 0.70 ± 0.02 62.50 ± 2.68

19.05 ± 1.15 16.48 ± 1.18 86.48 ± 0.01 4.88 40.51 ± 0.33 0.66 ± 0.021 61.88 ± 2.51

16.68 ± 0.49 13.61 ± 0.96 82.37 ± 0.03 4.35 38.98 ± 0.57 0.74 ± 0.021 53.06 ± 2.31

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2.4. Analytical methods The TS, VS, carbon (C) and nitrogen (N) content were determined according to the published study (Li et al., 2012). Total ammonia-nitrogen (TAN), volatile fatty acids (VFAs) and methane content were analyzed by HACH spectrophotometer, HPLC system (Waters e2698, USA) and GC-2014 gas chromatograph (Shimadzu, Japan) (Li et al., 2017a; Zhang et al., 2017b). The titration method was used to determine the total (TA) and partial alkalinity (PA) at the pH endpoints of 4.3 and 5.7; the intermediate alkalinity (IA) was the difference of TA and PA. The metal concentrations of Fe, Co and Ni were analyzed by Inductive Coupled Plasma Emission Spectrometer (OPTIMA 8000DV, PerkinElmer, USA). The degradation degree of volatile solids (VSremoved) was calculated according to the reported method (Koch, 2015). 2.5. Whole genome pyrosequencing analysis 2.5.1. Sampling for microorganism community analysis To analyze the bacteria and archaea community during the process, samples from R1 were collected on days 21 (MD-2-21d), 55 (MD-2-55d), 75 (MD-2-75d) and 85 (MD-2-85d). Samples from R2 were collected at OLR of 2 (10 mL and 20 mL), 2.5, 3, 3.5 and 4 g VS/(L
d) which signed as SQ-Y-2-10 (SQ-Y-2–20), SQ-Y-2.510, SQ-Y-3-10, SQ-Y-3.5-10 and SQ-Y-4-10, respectively. These samples were stored at 20 °C prior to further analysis. 2.5.2. DNA extraction Samples (4 mL) were centrifuged at 10,000 rpm for 3 min, then the supernatant was decanted and the residual biomass was used for DNA extraction (E.Z.N.ATM Mag-Bind Soil DNA Kit, OMEGA). The standard protocol provided in the kit was followed. The concentration of extracted DNA was estimated by gel electrophoresis. 2.5.3. Polymerase chain reaction The programs for amplifying the V3-V4 regions of the bacterial 16S ribosomal RNA gene by polymerase chain reaction (PCR) were covered in previous work (Li et al., 2017a). The V3-V4 regions of the archaea 16S ribosomal RNA gene were amplified twice by nested PCR using primers 340F (CCCTAYGGGGYGCASCAG)/1000R (GGCCATGCACYWCYTCTC) and 349F (GYGCASCAGKCGMGAAW)/806R (GGACTACVSGGGTATCTAAT). The PCR programs for bacteria and archaea were as follows: 94 °C for 3 min, then 94 °C for 30 s, 45 °C for 20 s, 65 °C for 30 s for 5 cycles, then 94 °C for 20 s, 55 °C for 20 s, 72 °C for 30 s for 20 cycles, finally 72 °C for 5 min. The amplified PCR products were evaluated by gel electrophoresis. 2.5.4. Sequencing and phylogenetic analysis Sequencing was performed using the Illumina MiSeq system (Illumina Miseq2x300bp, USA). The effective sequences were identified to the order, phylum, class, family and genus level according to the RDP classifier via the database of PDR (http://rdp.cme.msu. edu/misc/resources.jsp), Silva (http://www.arb-silva.de/), and NCBI 16S (http://ncbi.nlm.nih.gov/). The OTUs were defined by clustering sequences at a confidence threshold of 97%. 3. Results and discussion 3.1. The anaerobic digestion performance in long-term operation 3.1.1. Mono-digestion of Pennisetum hybrid without bioaugmentation A failure in long-term operation was evident for the monodigestion of Pennisetum hybrid without bioaugmentation, coupled with a decrease in volumetric biogas yield and an increase in VFAs

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concentration (Figs. 1 and 2). The volumetric and specific biogas yield at day 60–93 decreased by 87% and 88%, respectively, in comparison with the values at day 1–59. Additionally, the methane content fell from 55.08% to 21.93% (Table 2). The highest VFAs value of 6091 mg/L was obtained with acetic acid, butyric acid, and propionic acid occupying 62%, 27%, and 11% of the total concentration, respectively (Fig. 1b), attaining inhibition thresholds of 1.5–2.5 g/L for acetic acid and 5.8–6.9 g/L for VFAs (Solli et al., 2014). The TAN level ranged between 256 and 684 mg/L, far below the reported inhibitory concentration of 1500–7000 mg N/L (Fig. S1). The pH value decreased to 5.01 (Fig. 1a). The stability indicators, VFAs/TA and IA/PA, obtained sizeable variances with the values increasing to 1.43 and 9.59, respectively (Fig. 2), well beyond the reported stability threshold of 0.8 for VFAs/TA and 0.9 for IA/PA (Callaghan et al., 2002; Ferrer et al., 2010). 3.1.2. Mono-digestion of Pennisetum hybrid with bioaugmentation A stable long-term operation was observed for the bioaugmented mono-digestion of Pennisetum hybrid in comparison with the control system. For R2, the best performance was obtained at an OLR of 2.0 g VS/(L
d) and a dosage of 10 mL, which achieved the maximum specific biogas yield of 610.16 ± 72.30 mL/g VS (Table 2). A slight decrease in volumetric and specific biogas yield of 2–3% was observed with the bioaugmentation volume of 20 mL at the same OLR. A similar methane content of 54.97–56.98% was obtained with different dosages. Therefore, the dosage of 10 mL was selected for the elevated OLR experiments. Raising the OLR from 2.0 g VS/(L
d) to 4.0 g VS/(L
d) with 10 mL bioaugmentation resulted in an increase in volumetric biogas yield, while the specific biogas yield decreased to 435.80 ± 40.49 mL/g VS (Table 2). The pH values were in the range of 6.95–7.66. The IA/PA and VFAs/TA values ranged in 0.02–0.36 and 0–0.12, respectively for the OLR of 2.0, 2.5 and 3.5 g VS/(L
d); all of these values are considered to be in a suitable range for stable operation (Li et al., 2016a; Wall et al., 2014). Fluctuations in IA/PA and VFAs/TA were observed at OLRs of 3.0 and 4.0 g VS/(L
d). The IA/PA and VFAs/ TA climbed to the maximum values of 0.99 and 0.49, respectively at the OLR of 3.0 g VS/(L
d), reaching the upper value for process stability. This increase was ascribed to the accumulation of acetic acid (Fig. 1a), which was gradually consumed to a low level, indicating that R2 have a certain resistance for higher VFAs concentration. A decreasing trend from 0.41 to 0.27 for IA/PA was evident at OLR of 4.0 g VS/(L
d), and a similar trend was observed for VFAs/TA with its value reduced from 0.30 to 0.09. In this phase, propionic acid achieved a maximum value of 1839 g/mL, which gradually decreased to 394 g/mL, suggesting that R2 has better conversion efficiency for propionate. 3.2. The microbial community structure in different conditions A total of 626,476 and 427,546 non-chimeric sequence reads of bacteria and archaea 16 S rRNA gene, respectively, were recovered from all the samples (Table S1). These sequence reads revealed a total of 11,109 OTUs for bacteria and 4050 OTUs for archaea. For the non-bioaugmented system, the OTUs for bacteria and archaea were in the range of 880–1285 and 401–530, respectively. Changes in Chao1 were also used to estimate the total number of OTUs; they showed a trend similar to that of the OTUs. Both indicators, OTUs and Chao1, demonstrated that the richness of the bacteria community is higher than that of archaea. For the bioaugmented system, the number of OTUs for bacteria decreased from 1355 to 907 with an increased OLR and from 1355 to 1086 with an elevated dosage. Meanwhile, the number of OTUs for archaea and bacteria decreased from 474 to 126 and to 440, respectively with an increased OLR and dosage. Similar trends in Chao1 were also observed except for SQ-Y-2-20. The values of OTUs and Chao1 for

L. Lianhua et al. / Waste Management 78 (2018) 741–749

3

4

2

3 1

2 1

0 9

pH value

5

(a)

0

50

100

150

200

0 5

250

8

4

7

3

6

Mono-digestion without bioaugmentation Mono-digestion with bioaugmentation Organic loading rate

5 4

Organic loading rate (g VS/L.d)

Volumetric biogas yield (m3/m3.d)

744

2 1 0

0

50

100

150

200

250

Time (d) 5

(b) Mono-digestion with bioaugmentation

4

VFAs concentration (mg/L)

2000

3

1000

2 0 5000

0

50

100

150

200

250

1 4

4000 3000 2000 1000 0

Acetic acid Propionic acid Butyric acid

3

Organic loading rate

2

Organic loading rate (g VS/L.d)

3000

Mono-digestion without bioaugmentation 1

0

50

100

150

200

250

Time (d) Fig. 1. The anaerobic digestion performance of different mono-digestion systems.

the bioaugmented system suggest that changes in OLR have an effect on the richness of microorganisms. The rarefaction curve also confirmed these findings (Fig. S2). The Shannon index, an indicator of the diversity of microbial populations, fluctuated within the range of 3.64–4.20 for R1 and decreased from 4.05 to 3.61 for R2, showing a reduction in bacterial diversity with an elevated OLR. Similar to bacteria, the Shannon index of the archaea ranged from 1.66 to 2.25 for R1 and decreased from 2.40 to 0.84 for R2. The coverage values were more than 0.99, suggesting that most bacteria and archaea community groups were detected. The dominant bacteria in all samples at the phyla level were Bacteroidetes, Firmicutes, Proteobacteria, Chloroflexi, Synergistetes and Verrucomicrobia, ranging between 92.40 and 98.02% of all bacterial reads (Fig. 3). The dominant archaea at the order level were

Methanosarcinales, Methanomicrobiales, Methanobacteriales, Desulfurococcales, and Methanomassiliicoccales, accounting for 90.43– 99.89% of all archaeal reads (Fig. 4). 3.2.1. The microbial community of the non-bioaugmented monodigestion system For the bacterial community of the non-bioaugmented monodigestion system, an evident decrease in the relative abundance of Bacteroidetes (from 32.88% to 15.65%), and Chloroflexi (from 21.93% to 2.11%) was observed, while the relative abundance of Firmicutes (from 28.85% to 65.80%) sizeably increased with anaerobic digestion processed. The relative abundance of Proteobacteria and Synergistetes fluctuated in 9.84–12.77% and 1.24–2.11%, respectively. At the genus level, an increase in the relative abundance

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2.0

5

VFAs/TAformono-digestion without bioaugmentation VFAs/TAformono-digestion with bioaugmentation

4 3

1.0 2 0.5 1 0.0 16

0

IA/PA

12

50 100 150 200 IA/PAformono-digestion without bioaugmentation IA/PAformono-digestion with bioaugmentation

0 5

250

4 3

8

Organic loading rate (g VS/L.d)

VFAs/TA

1.5

2

OLRformono-digestion without bioaugmentation OLRformono-digestion with bioaugmentation

4

0

1 0

0

50

100

150

200

250

Time(d) Fig. 2. The stability analysis of anaerobic digestion under different conditions.

Table 2 The volumetric and specific biogas yield for different mono-digestion systems. OLRa

Dose (mL)

Specific biogas yieldc

Volumetric biogas yieldb

Methane contentd

VS reduction rated

Mono-digestion

2.0 (1–59d) 2.0 (60–93d)

0 0

564.12 ± 79.25 66.73 ± 121.71

1.12 ± 0.16 0.15 ± 0.24

55.08 21.93

53.72 ± 3.22 /

Mono-digestion with bioaugmentation

2 2.5 3 3.5 4 2

10

610.16 ± 72.30 519.54 ± 67.62 474.16 ± 62.46 435.80 ± 40.49 488.10 ± 43.41 598.13 ± 89.66

1.23 ± 0.14 1.30 ± 0.11 1.42 ± 0.19 1.53 ± 0.14 1.95 ± 0.17 1.19 ± 0.16

54.97 54.32 53.39 55.54 57.38 56.98

52.26 ± 8.46 68.11 ± 5.23 58.94 ± 3.38 57.57 ± 4.29 39.10 ± 5.83 61.68 ± 7.68

20

‘‘–”meaning no data available. a OLR in g VS/(L
d). b Volumetric biogas yield in m3/(m3
d). c Specific biogas yield in mL/g VS. d Methane content and VSremoved in %.

of Clostridium IV (Firmicutes, from 0.14% to 32.04%), Ethanoligenens (Firmicutes, from 0.01% to 9.27%), Megasphaera (Firmicutes, from 0.00% to 9.24%), Intestinimonas (Firmicutes, from 0.30% to 1.74%), Fonticella (Firmicutes, from 0.00% to 2.79%), Prevotella (Bacteroidetes, from 0.01% to 3.81%), Acinetobacter (Proteobacteria, from 0.31% to 2.07%), Sutterella (Proteobacteria, from 0.00% to 1.71%), and Defluviimonas (Proteobacteria, from 0.00% to 1.71%) was observed with the anaerobic digestion proceeded. However, the relative abundance of Ruminococcus (Firmicutes, from 3.63% to 0.02%), Saccharofermentans (Firmicutes, from 3.00% to 0.65%), Syntrophomonas (Firmicutes, from 1.48% to 0.24%), Anaerovorax (Firmicutes, from 0.94% to 0.32%), Oscillibacter (Firmicutes, from 1.01% to 0.21%), Mariniphaga (Bacteroidetes, from 4.13% to 0.01%), and Meniscus (Bacteroidetes, from 1.91% to 0.02%), Syntrophobacter (Proteobacteria, from 0.92% to 0.27%) and Syntrophorhabdus (Proteobacteria, from 3.33% to 0.26%), Levilinea (Chloroflexi, from 18.75% to 1.62%), and Ornatilinea (Chloroflexi, from 2.37% to 0.42%) decreased. For the archaeal community of the non-bioaugmented monodigestion system, a dramatic decrease of 81% in Methanosarcinales was observed during anaerobic digestion, which was caused by the reduction in genus of Methanothrix. Meanwhile, a sizable increase

in the relative abundance of Methanomicrobiales (from 27.74% to 79.81%), Methanobacteriales (from 0.37% to 1.63%), and Methanomassiliicoccales (from 0.76% to 5.94%) was attributed to the enrichment of Methanolinea, Methanobacterium, Methanomassiliicoccus, and Methanosphaerula. The shift of dominant archaea from acetoclastic methanogens (Methanosarcinales) to hydrogenotrophic methanogens (Methanomicrobiales, Methanobacteriales, and Methanomassiliicoccales) was consistent with the variation in the bacterial community (Table 3). The enrichment of hydrogenproducing bacteria, such as Clostridium IV, Ethanoligenens and Megasphaera, boosted the growth of hydrogenotrophic methanogens. 3.2.2. The microbial community of the bioaugmented mono-digestion system For the bacterial community of R2 with different dosages of bioaugmentation, the dominant phyla ranged in the relative abundance of 34.75–39.77% for Bacteroidetes, 18.01–25.58% for Firmicutes, 8.98–12.98% for Proteobacteria, 12.97–13.33% for Chloroflexi, 5.33–8.60% for Synergistetes, 1.30–6.18% for Verrucomicrobia, 1.90–2.37% for Euryarchaeota, and 0.37–1.10% for Planctomycetes.

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Relative abundance

a

100% 80% 60% 40% 20% 0%

Bacteroidetes Verrucomicrobia Planctomycetes Hydrogenedentes

Firmicutes Euryarchaeota Actinobacteria Atribacteria

Proteobacteria Spirochaetes Thermotogae Fibrobacteres

Chloroflexi Cloacimonetes Acidobacteria unclassified

Synergistetes Parcubacteria Latescibacteria others

Fig. 3. Relative abundance of bacteria 16S rRNA gene at the phylum level (A) and class level (B) at different condition.

At the genus level, minor changes in the relative abundance of Clostridium IV, Intestinimonas, Exiguobacterium, Saccharofermentans, Syntrophomonas, Syntrophomonas, Clostridium III, Oscillibacter, Mariniphaga, Meniscus, Acinetobacter, Smithella, Syntrophobacter, Syntrophorhabdus, Levilinea, Ornatilinea, and Cloacibacillus were observed. The relative abundance of Ruminococcus decreased from 9.07% to 0.27%, while the relative abundance of Aminivibrio, Puniceicoccus and Anaerovorax increased from 2.28% to 6.40%, 0.00% to 4.86%, and 0.38% to 1.41%, respectively, with the increased dosage. For the archaea community of R2, at the order level, the relative abundance of Methanosarcinales, Methanomicrobiales, Methanobacteriales, and Methanomassiliicoccales showed a range of 53.46– 55.75%, 28.21–36.21%, 0.48–0.62%, and 0.82–0.95%, respectively. At the genus level, the dominant genera were Methanothrix, Methanospirillum, and Methanolinea, with relative abundance of 55.47–52.30%, 9.48–13.73%, and 7.08–10.59%, respectively. With increasing the OLR of R2 from 2 g VS/(L
d) to 4 g VS/(L
d), the bacterial and archaea community dramatically changed. For the bacterial community, Bacteroidetes was the most prevalent group at the phylum level, accounting for 34.75–50.73% of total

OTUs, followed by Firmicutes (22.92–32.08%), Proteobacteria (2.54–12.98%), Chloroflexi (0.13–13.33%), Synergistetes (3.78– 11.06%), Verrucomicrobia (1.30–6.59%), and Spirochaetes (0.30– 3.03%). At the OLR of 2.0–2.5 g VS/(L
d). The dominant bacteria at the genus level included Levilinea (Chloroflexi, 8.54–10.37%), Ruminococcus (Firmicutes, 3.02–9.07%), Saccharofermentans (Firmicutes, 1.61–1.99%), Mariniphaga (Bacteroidetes, 2.07–3.41%), Aminivibrio (Synergistetes, 2.28–2.46%), Cloacibacillus (Synergistetes, 1.11–1.75%), and Syntrophobacter (Proteobacteria, 1.01–1.48%). Some of the genera dramatically changed; for example, the relative abundance of Clostridium IV, Clostridium III, Puniceicoccus, Prevotella, Proteiniphilum, and Snaerovorax increased from 0.13% to 1.21%, 0.39% to 2.75%, 0.00% to 2.16%, 0.23% to 1.42%, 0.02% to 2.15%, and 0.38% to 1.20%, respectively. Meanwhile, the relative abundance of Syntrophorhabdus, Ornatilinea, and Sedimentibacter decreased. With the OLR increasing to 3.0–3.5 g VS/(L
d), a sizable increase in the relative abundance of Proteiniphilum, Clostridium XIVa, Aminivibrio, Puniceicoccus, Prevotella, Syntrophobacter, Cloacibacillus, and Lutaonella was observed, while the relative abundance of Levilinea, Ornatilinea, and Smithella decreased. When

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a

OLR was further elevated to 4.0 g VS/(L
d), Methanomicrobiales become the most prevalent group with a relative abundance of 80.31%, which most OTUs were assigned to Methanoculleus (80.13%).

Relative abundance

100% 80% 60% 40%

3.3. Comparison of the performance and microorganism community of bioaugmented and non-bioaugmented mono-digestion systems

20% 0%

Methanosarcinales Desulfurococcales

Methanomicrobiales Methanomassiliicoccales

Methanobacteriales other

b Relative abundance

100% 80% 60% 40% 20% 0%

Methanothrix Methanoculleus unclassified

Methanolinea Methanospirillum other

Methanobacterium Methanomassiliicoccus

Methanosarcina Methanosphaerula

Fig. 4. Relative abundance of archaea 16S rRNA gene at the order level (A) and genus level (B) at different condition.

the OLR was further elevated to 4.0 g VS/(L
d), the relative abundance of Proteiniphilum, Ruminococcus, Syntrophobacter, Lutaonella, and Sphaerochaeta obtained the maximum values of 29.79%, 9.72%, 3.47%, 9.05% and 2.74%, respectively. For the archaea community of R2 at elevated OLR levels, the dominant orders were Methanosarcinales and Methanomicrobiales at the OLR of 2.0–2.5 g VS/(L
d), with the relative abundance accounting for 83.96% and 85.85%, respectively. The dominant genera assigned to these two orders included Methanothrix, Methanolinea, and Methanospirillum, which the relative abundance was 45.90–55.47%, 7.08–11.23%, and 3.64–13.73%, respectively. With the OLR increasing to 3.0–3.5 g VS/(L
d), the dominant orders were Methanosarcinales, Methanomicrobiales, and Methanobacteriales. Among them, the relative abundance of Methanosarcinales fluctuated between 54.98 and 45.94%. The prevalent genera were Methanothrix (23.50–23.97%) and Methanosarcina (22.44–31.00%). For the order of Methanomicrobiales, the relative abundance decreased from 21.87% to 9.44%, and most OTUs were assigned to Methanospirillum. Additionally, the order of Methanobacteriales showed an increase in relative abundance from 18.50% to 42.48%, and most OTUs were assigned to Methanobacterium. When the

The non-bioaugmented mono-digestion system failed in longterm operation at a feeding rate of 2.0 g VS/(L
d). This value is under the reported OLR values for the stable operation of the mono-digestion of grass (Thamsiriroj et al., 2012; Voelklein et al., 2016), ascribing to the differences in temperature, feedstock, supplementary nutrients, and reactor type. Thermophilic operation, trace element addition, and effluent recirculation were effective in improving the stability of the mono-digestion of grass. From the aspect of the microbial community, the relative abundance of butyrate-, propiontic-, and hydrogen-producing bacteria, such as Clostridium IV, Ethanoligenens, Megasphaera, Intestinimonans, Fonticella and Prevotella increased when the non-bioaugmented reactor became unstable (Bui et al., 2016; Xing et al., 2006; Zhang et al., 2017a; Zhou et al., 2015). Meanwhile, the relative abundance of syntrophic butyrate and propiontic oxidizers, such as Syntrophomonas, Syntrophobacter and Syntrophorhabdus decreased, leading to the accumulation of propionate and butyrate (Suyun et al., 2018). For the archaea community, a shift of dominate methanogens from acetoclastic to hydrogenotrophic was observed, which was correlated with the change in the composition of the bacterial community. These results suggest that the conversion of propionate and butyrate may be the rate-limiting step at an unstable system of anaerobic digestion. In comparison with the R1 system, the stable running OLR of R2 increased 1-fold with bioaugmentation. From the aspect of the nutrient concentrations, no obvious differences were observed for elemental Fe and Ni, and the concentration of Co was lower than the detection limit (Fig. S3). For bacteria, some unique genera were observed such as Proteiniphilum, Thermovirga, and Lutaonella, which have been detected in the enriched bioaugmentation media. Proteiniphilum, affiliated to the phylum of Bacteroidetes, uses protein as a fermentative substrate and produces carbon dioxide, hydrogen, and acetic acid (Cardinali-Rezende et al., 2016). Lutaonella, an amino acid-degrading acidogen, was also affiliated to the phylum of Bacteroidetes (Arun et al., 2009). Thermovirga, amino acid-degrading bacterium, utilizes proteinous substrates, some single amino acids, and a limited number of organic acids, but not sugars, fatty acids or alcohols (Dahle and Birkeland, 2006). Meanwhile, the relative abundance of Sphaerochaeta dramatically increased. Sphaerochaeta, a hydrolytic bacterium, is associated with the breakdown of cellulosic materials (Rui et al., 2015). In addition, the relative abundance of genus Syntrophomonas increased with the elevated OLR and reached maximum abundance

Table 3 The relative abundance of acetoclastic and hydrogenotrophic methanogens. SQ-Y-2-10 SQ-Y-2-20 SQ-Y-2.5-10 SQ-Y-3-10 SQ-Y-3.5-10 SQ-Y-4-10 MD-2-21d MD-2-55d MD-2-75d MD-2-85d Acetoclastic methanogens

Methanothrix Methanosarcina Total

Hydrogenotrophic Methanolinea methanogens Methanobacterium Methanoculleus Methanospirillum Methanomassiliicoccus Methanosphaerula Total

55.47 0.08 55.55

52.3 0.62 52.92

45.9 0.12 46.02

23.97 31 54.97

23.5 22.44 45.94

1.34 0.72 2.06

56.06 0.05 56.11

62.45 0.13 62.58

24.24 0.05 24.29

10.76 0.92 11.68

7.08 0.18 0.78 13.73 0.82 0.22 22.81

10.59 0.51 2.87 9.48 0.95 3.19 27.59

11.23 3.92 0.52 3.64 4.16 0.31 23.78

2.81 18.48 0.63 10.64 1.87 0.08 34.51

0.24 42.47 0.97 6.89 1.48 0.17 52.22

0.08 11.02 80.13 0.07 6.28 0.01 97.59

8.77 0.15 1.1 4.16 1.38 0.22 15.78

5.98 0.54 1.66 1.83 2.16 0.42 12.59

38.21 0.48 0.1 1.45 0.76 0.58 41.58

38.43 1.63 1.19 3.02 5.94 0.95 51.16

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at the OLR of 4.0 VS/(L
d). These results indicate that the stable operation of R2 was ascribed to (1) the improved utilization of protein- and amino acid-components by the Proteiniphilum, Thermovirga, and Lutaonella, resulting in an enhancement in the buffer capacity for acidification; (2) the enhanced propionate degradation performed by Syntrophomonas. For the archaeal community, the methanogenesis pathway shift from acetoclastic to hydrogenotrophic with elevated OLR was ascribed to the following: (1) Methanothrix was dominant at low OLR at the conditions of low acetate concentrations; (2) Methanosarcina and other hydrogenotrophic methanogens have a higher resistance for extreme conditions and thus perform better in higher loaded conditions (Conklin et al., 2006); (3) the syntrophic association of Syntrophomonas spp. together with the methanogens Methanosarcina and Methanocella were beneficial to retain the stability of the reactor. 4. Conclusion The mono-digestion of the Pennisetum hybrid failed during long-term operation at an OLR of 2.0 g VS/(L
d), coupled with the accumulation of VFAs and the imbalance of the microbial community. Improved anaerobic digestion was obtained by bioaugmentation. The OLR at a stable reaction status increased to 4.0 g VS/(L
d) and the maximum volumetric biogas yield reached up to 1.95 ± 0. 17 m3/(m3
d). Bioaugmentation had an obvious effect on the structure of the microbial community, with the genus Proteiniphilum, Thermovirga, and Lutaonella sizably increased, and the hydrogenotrophic Methanoculleus becoming the most prevalent archaea in the group. Acknowledgments Funding: The work was supported by National Key Research and Development Program of China [2017FYD0800801]; National Natural Science Foundation of China [51708538]; Bureau of International Cooperation, Chinese Academy of Sciences [182344KYSB20170009]; Science and Technology Planning Project of Guangdong Province [2017A050501049, 2017B020 238002]; Science and Technology Program of Guangzhou [201707010201]; The Natural Science Foundation for research team of Guangdong Province [2016A030312007]. Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at https://doi.org/10.1016/j.wasman.2018.06. 031. References Arun, A.B., Chen, W.M., Lai, W.A., Chou, J.H., Shen, F.T., Rekha, P.D., Young, C.C., 2009. Lutaonella thermophila gen. nov., sp nov., a moderately thermophilic member of the family Flavobacteriaceae isolated from a coastal hot spring. Int. J. Syst. Evol. Microbiol. 59, 2069–2073. Bui, T.P.N., Shetty, S.A., Lagkouvardos, I., Ritari, J., Chamlagain, B., Douillard, F.P., Paulin, L., Piironen, V., Clavel, T., Plugge, C.M., de Vos, W.M., 2016. Comparative genomics and physiology of the butyrate-producing bacterium Intestinimonas butyriciproducens. Environ. Microbiol. Rep. 8, 1024–1037. Callaghan, F.J., Wase, D.A.J., Thayanithy, K., Forster, C.F., 2002. Continuous codigestion of cattle slurry with fruit and vegetable wastes and chicken manure. Biomass Bioenergy 22, 71–77. Carvalho, A.R., Fragoso, R., Gominho, J., Saraiva, A., Costa, R., Duarte, E., 2016. Waterenergy nexus: Anaerobic co-digestion with elephant grass hydrolyzate. J Environ Manage 181, 48–53. Cardinali-Rezende, J., Rojas-Ojeda, P., Nascimento, A.M.A., Sanz, J.L., 2016. Proteolytic bacterial dominance in a full-scale municipal solid waste anaerobic reactor assessed by 454 pyrosequencing technology. Chemosphere 146, 519–525.

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