Anaerobic co-digestion of Pennisetum hybrid and pig manure: A comparative study of performance and microbial community at different mixture ratio and organic loading rate

Chemosphere 247 (2020) 125871

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Anaerobic co-digestion of Pennisetum hybrid and pig manure: A comparative study of performance and microbial community at different mixture ratio and organic loading rate Li Lianhua a, e, f, He Shuibin a, d, Sun Yongming a, e, f, *, Kang Xihui a, d, Jiang Junfeng a, c, Yuan Zhenhong a, e, f, Liu Dingfa b a

Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, 510640, China Guangdong Foodstuffs Imp. & Exp. (Group) Corp, Guangzhou, 510100, China Key Laboratory of Ministry of Education for Water Quality Security and Protection in Pearl River Delta, Guangdong Provincial Key Laboratory of Radionuclides Pollution Control and Resources, School of Environmental Science and Engineering, Guangzhou University, Guangzhou, 510006, China d University of Chinese Academy of Sciences, Beijing, 100049, China e Guangzhou Institute of Energy Conversion, CAS Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou, 510640, PR China f Guangdong Key Laboratory of New and Renewable Energy Research and Development, Guangzhou, 510640, PR China b c

h i g h l i g h t s  Co-digestion system with 50:50 mixture ratio obtained better stable performance.  Different issues triggered the failure co-digestion process.  Enriched protein-utilizing Proteiniphilum related to the higher ammonia level.  Shift in methanogen could enhance the stability of GP11.

a r t i c l e i n f o

a b s t r a c t

Article history: Received 1 October 2019 Received in revised form 27 December 2019 Accepted 6 January 2020 Available online 7 January 2020

To investigate how the changes in performance and the microbial community of the co-digestion system of Pennisetum hybrid and pig manure, two co-digestion systems in a semi-continuous mode were established at different grass:manure mixture ratios (50:50 and 75:25), and at variable organic loading rates (OLRs). The two reactors were in a steady-state at the OLRs of 2.0e5.0 g VS/(L$d), with the specific and volumetric biogas yields of 383.86 ± 65.13 to 574.28 ± 72.04 mL/g VS and 0.87 ± 0.07 to 2.36 ± 0.13 m3/(m3$d), respectively. The co-digestion system with a mixture ratio of 75:25 failed at an OLR of 5.5 g VS/(L,d). This failure could be attributed to the accumulation of volatile fatty acids (VFAs) owing to the imbalance between acid-production and -oxidation bacteria. By contrast, the co-digestion system with mixture ratio of 50:50 failed at an OLR of 7.0 g VS/(L,d), which was likely due to mechanical issues or improper reactor configuration. The genus Proteiniphilum contributed to the increase in total ammonia nitrogen. These findings provide useful guidance for optimizing co-digestion system, enhancing reactor performance and improving the wastes treatment. © 2020 Elsevier Ltd. All rights reserved.

Handling Editor: A Adalberto Noyola Keywords: Co-digestion Pennisetum hybrid Pig manure Performance Microbial community

1. Introduction Co-digestion has recently emerged as the optimal approach for anaerobic digestion (Hagos et al., 2017; Mehariya et al., 2018).

* Corresponding author. Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou, 510640, China. E-mail address: [email protected] (S. Yongming). https://doi.org/10.1016/j.chemosphere.2020.125871 0045-6535/© 2020 Elsevier Ltd. All rights reserved.

Numerous materials, such as wastes derived from livestock and poultry, kitchen or food processing, and agriculture, have been used as mixtures for co-digestion. Lignocellulosic materials and livestock wastes are commonly used. Livestock waste is produced in vast amounts globally, representing an environmental hazard and potential contaminant. For example, the annual output of solid waste from pig farms in China is estimated at approximately 121.7 megatons (dry weight) (Jia et al., 2018; Poirier et al., 2016). Moreover, the high ammonia levels in these nitrogen-rich wastes inhibit

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the process of anaerobic digestion (Li et al., 2018a). Pennisetum hybrid, a C4 perennial grass, shows great potential as a lignocellulosic material for co-digestion owing to its high annual biomass yield and specific methane yields reaching up to 88 MT/ha and 104e328 mL/g volatile solids (VS), respectively (Somerville et al., 2010; Yang et al., 2017). However, issues with instability in monodigestion of grass caused by nutrient deficiency and mechanical were previously reported (Janke et al., 2016; Thamsiriroj et al., 2012).Co-digestion of manure and carbon-rich lignocellulosic materials can be served as a useful substrate to enhance the performance of anaerobic digestion. With respect to co-digestion of manure and perennial grasses, Zheng et al. (2015) achieved the highest methane yield using a 2:2 switchgrass to manure ratio, and Moset et al. (2017) found that the volumetric methane yield of codigestion of cattle manure and grass increased by 20% compared to the mono-digestion of cattle manure. The performance and stability of a co-digestion system are enhanced by modulating the C/N ratio, supplying suitable trace metals, and balancing the microbial community. Zhang et al. (2016) reported that adjustment of the C/N ratio and trace element supplementation improved the performance of a co-digestion system of sorghum stem and cow manure. Similarly, supplementation of trace elements by the addition of fresh leachate, including Ni, Mo, Co, and Fe, strongly enhanced the performance of anaerobic digestion of food waste (Zhang et al., 2015). Schwede et al. (2013) attributed the positive effects of co-digestion of marine microalga and corn silage to the balanced nutrient composition, improved C/N ratio, and enhanced trace elements and alkalinity. With the development of technology, the microbial community involved into the anaerobic digestion achieved further understanding. Amha et al. (2017) observed that methane production in a co-digestion system was positively correlated with the relative activity of Syntrophomonas. Xing et al. reported that an enhancement in the hydrogenotrophic methanogens was caused by sufficient H2 and CO2 which originated from the biodegradation of lignocelluloses of cow manure co-digested with food waste(Xing et al., 2020). Xu et al. reviewed the current achievement in the relationship between microbial community and nutrient in co-digestion system, and proposed several challenges in co-digestion system, such as operation optimization, the factors affecting co-digestion performance and microbial community need further study (Xu et al., 2018). As above-mentioned, the performance and microbial community of co-digestion system could be influenced by material and operation conditions. Therefore, the aim of the present study was to examine the influence of different organic loading rates (OLRs) and mixture ratios on the performance of a co-digestion system of Pennisetum hybrid and pig manure, and to compare the microbial communities to elucidate the underlying mechanisms contributing to performance variation. These results can lay the foundation for optimizing co-digestion systems for handling waste and providing renewable energy in an environmentally friendly manner. Meanwhile, it also provided useful guidance for enhancing the performance of anaerobic digestion from the point of microbial community.

2. Materials and methods 2.1. Substrates and inoculum Pennisetum hybrid was collected from an experimental plot in Zengcheng District, Guangzhou, China (Li et al., 2016). The fresh raw materials were firstly crushed to the piece of 1e2 cm. Then the smashed samples were ensilaged by compacting it into plastic buckets, sealed, and stored at room temperature. The ensilaged samples were further pulverized, and then placed in a refrigerator until use. During the experiment, materials were collected five times. The physicochemical properties of the materials are provided in Table 1. Pig manure was collected from a pig farm in Huizhou, China. The contents of total solid (TS), volatile solid (VS), elemental C and N were 22.53 ± 0.09%, 18.65 ± 0.09%, 39.99 ± 0.14% and 2.53 ± 0.06%, respectively. Correspondingly, the C/N ratio was 15.81 ± 0.41. The inocula were taken from a continuously stirred tank reactor operated at mesophilic condition. The contents of TS and VS of inocula were 1.87 ± 0.03% and 1.10 ± 0.01%, respectively. 2.2. Equipment and experimental setup Experiments were conducted in a Bioprocess system (Bioprocess Control AB, Sweden) (Li et al., 2018b, 2018c). The system contains a continuously stirred tank reactor (CSTR) and a data acquisition system (Fig. S1). The reactor has three ports for feedstock inlet, outlet and stirrer. The stirrer is vertically mounted and automatically works at a setting frequency. The reactor with 2 L working volume was operated in semi-continuous mode and at a controlled temperature of 35 ± 1  C. Two co-digestion systems were established with different ratios of Pennisetum hybrid to pig manure of 50:50 (GP11) and 75:25 (GP31), respectively, with the C/ N ratio of 23.10e36.88(Fig. S2). A total of 1800 mL of the inoculum was added to each reactor. For both co-digestion systems, the OLR was initially set at 2.0 g VS/(L,d), and was gradually increased to 7.0 g VS/(L,d) for GP11 and to 5.5 g VS/(L,d) for GP31 at an interval of 0.5 g VS/(L,d). When the OLRs were 2.0e5.0 g VS/(L,d), the hydraulic retention time was 30 days, whereas the hydraulic retention times were decreased to 29, 26, and 22 days for OLRs of 5.5, 6.0, and 7.0 g VS/(L,d), respectively. The experiments lasted for 249 days for GP31 and 292 days for GP11. 2.3. Analytical methods The samples for determining the values of process parameters were collected at an interval of 6 day during the entire period. TS, VS, pH, concentration of total ammonia nitrogen (TAN) and contents of C and N were measured followed the previous methods (Li et al., 2012). The value of alkalinity was determined by titration method with the endpoints of pH 5.7 for partial alkalinity (PA) and 4.3 for total alkalinity (TA), respectively (Li et al., 2018c). Methane (CH4) content was measured by gas chromatograph (GC-2014, Shimadzu, Japan). The instrument has a TCD detector and Porapak Q column which operated at the temperature of 120  C

Table 1 Properties of the Pennisetum hybrid used in this study. Material

TS/%

Pennisetum hybrid

17.64 15.87 20.94 19.05 16.68

VS/% ± ± ± ± ±

0.68 0.62 0.84 1.15 0.49

16.55 12.81 18.17 16.48 13.61

C/% ± ± ± ± ±

0.66 0.45 0.81 1.18 0.96

41.76 39.59 43.41 40.51 38.98

N/% ± ± ± ± ±

0.25 0.04 0.54 0.33 0.57

0.98 0.92 0.70 0.66 0.74

C/N ± ± ± ± ±

0.04 0.02 0.02 0.02 0.02

42.86 43.28 62.50 61.88 53.06

± ± ± ± ±

1.81 0.96 2.68 2.51 2.31

L. Lianhua et al. / Chemosphere 247 (2020) 125871

and 70  C, respectively. Argon was used as carrier gas at a flow rate of 20 mL/min. The sample was firstly centrifuged at 12000 rpm for 5 min, and the supernatant was used for analysis of volatile fatty acids (VFAs). The concentration of VFAs, included acetic acid, propionic acid and butyric acid, was measured by High Performance Liquid Chromatography (Waters, e2698, USA). The instrument equipped with a Bio-RAD column which was operated at the temperature of 50  C. The mobile phase was 0.005 mM H2SO4 with a flow rate of 0.5 mL/ min (Li et al., 2018b, 2018c).

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version 2.12) (http://rdp.cme.msu.edu/misc/resources.jsp). 3. Results and discussion 3.1. Performance of anaerobic digestion systems under different conditions

2.4.3. Amplification of polymerase chain reaction (PCR) The primer pairs 341F (CCTACGGGNGGCWGCAG)/805G (ACTACHVGG GTATCTAATCC) were used to amplify the V3eV4 regions of the bacteria (Li et al., 2017). The primer pairs 340F (CCCTAYGGGGYGCASCAG)/1000R (GGCCATGCACYWCYTCTC) and 349F (GYGCASCAGKCGMGAAW)/806R (GGACTACVSGGGTATCTAAT) were used to perform the amplification of V3eV4 regions of archaea. PCR reaction was performed in a 30 mL solution which was composed of 15 mL of 2  Taq master Mix, 1 ml of each primer (10 mM), 10e20 ng genomic DNA, and distilled water. PCR programs were performed as follows: 94  C for 3 min, 5 cycles of 30 s at 94  C, 20 s at 45  C, and 30 s at 65  C, 20 cycles of 20 s at 94  C, 20 s at 55  C and 30 s at 72  C, finally extension 5 min at 72  C.

3.1.1. GP11 co-digestion system For the system operated at the OLRs of 2.0e6.0 g VS/(L,d), the pH remained relatively stable with a change in OLR, ranging from 7.12 ± 0.08 to 7.76 ± 0.53 (Fig. S3 and Table 2). The concentrations of acetic acid, propionic acid, and butyric acid were less than 1273.26, 388.33, and 382.05 mg/L, respectively. With respect to the stability of the system, the IA/PA ratio and VFAs/TA ratio of 0.120e0.368 and 0.013e0.141, respectively, were below the recommended threshold for instability (Fig. S4 and Table 2). Increasing the OLR of GP11 from 2.0 to 6.0 g VS/(L,d) resulted in an increase in the volumetric biogas yield from 0.87 ± 0.07 to 2.68 ± 0.31 m3/(m3$d) (Fig. S5 and Table 2). The maximum specific biogas yield of 553.24 ± 52.96 mL/g VS was obtained at an OLR of 2.5 g VS/(L,d) (Table 3), While the minimum value of 383.86 ± 65.13 mL/g VS appeared at the OLR of 5.0 g VS/(L,d). Compared process parameters at the OLRs of 5.0 g VS/(L,d) and 5.5 g VS/(L,d), higher VFAs concentration was reached at the OLRs of 5.0 g VS/(L,d), while no difference in TAN concentration (2958.33 ± 639.22e3022.50 ± 146.37 mg/L) was observed for the two OLRs (Fig. S3 and Table 2). Therefore, the relatively poor performance at an OLR of 5.0 g VS/(L,d) was might be related to the high VFAs concentration, resulting into a minimal biogas yield. The system failed at an OLR of 7.0 g VS/(L,d) due to sludge bulking. At this OLR, the obtained volumetric biogas yield was 2.96 ± 0.34 m3/(m3$d), with a specific biogas yield of 422.97 ± 48.23 mL/g VS and methane content of 48.23%. Bulking can be caused by system overload, overgrowth of filamentous bacteria such as Gordonia sp. and Nocardia sp. or an unsuitable stirring method or reactor type for high OLRs (Li et al., 2016). Since there was no obvious variation in operational parameters, it is more likely that system failure was due to inappropriate mechanical stirring or an unsuitable reactor configuration at this high OLR. Therefore, enhanced stirring or an optimal reactor design at high OLRs might prevent sludge bulking, which requires further investigation.

2.4.4. Sequencing and bioinformatics analysis Sequencing was performed by the Illumina HiSeq XTen plotform. The QIIME platform (version 1.8.0) was used to perform the bioinformatics analysis. The taxonomic classification at different level was performed by the Ribosomal Database Project (RDP,

3.1.2. GP31 co-digestion system Similar to GP11, there was an increasing trend in volumetric biogas yield for GP31 as the OLRs increased from 2.0 to 5.0 g VS/ (L,d) (Fig. S6). The volumetric biogas yield reached a peak of 2.36 ± 0.13 m3/(m3$d) at an OLR of 5.0 g VS/(L,d), representing a

2.4. Assessment of microbial community composition 2.4.1. Sampling procedure Samples for the analysis of microbial community were collected at OLRs of 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, and 6.0 g VS/(L,d), corresponding to day 48 (GP11e2.0 and GP31e2.0), 84 (GP11e2.5 and GP31e2.5), 114 (GP11e3.0 and GP31e3.0), 140 (GP11e3.5 and GP31e3.5), 168 (GP11e4.0 and GP31e4.0), 197 (GP11e4.5 and GP31e4.5), 229 (GP11e5.0 and GP31e5.0), 258/249 (GP11e5.5 and GP31e5.5), and 279 (GP11e6.0), respectively. 2.4.2. DNA extraction Before analysis, the samples were centrifuged at 10000 rpm for 3 min, and the solid part was used for DNA extraction. The DNA was extracted by E.Z.N.A™ Mag-Bind Soil DNA Kit (OMEGA) following the protocol provided by manufacturers.

Table 2 Process parameter of co-digestion of GP11and GP31 in semi-continuous mode. OLR g VS/(L$d)

pH

TAN (mg/L)

GP11 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0

7.12 7.34 7.30 7.32 7.29 7.36 7.28 7.76 7.54

± ± ± ± ± ± ± ± ±

0.08 0.09 0.07 0.06 0.04 0.06 0.06 0.53 0.09

VFAs (mg/L)

Stable indicator

Acetic acid

Propionic acid

Butyric acid

IA/PA

VFAs/TA

GP31

GP11

GP31

GP11

GP31

GP11

GP31

GP11

GP31

GP11

GP31

GP11

GP31

7.16 ± 0.03 7.19 ± 0.03 7.22 ± 0.03 7.2 ± 0.08 7.19 ± 0.01 7.29 ± 0.06 7.27 ± 0.1 6.70 ± 0.68 /

/ 575.00 ± 190.95 801.00 ± 261.19 1454.50 ± 519.63 1652.50 ± 498.51 2038.33 ± 87.36 2958.33 ± 639.22 3022.50 ± 146.37 1775.00 ± 615.18

382.75 ± 141.8 537.50 ± 134.13 504.50 ± 104.78 626.50 ± 34.84 782.00 ± 16.97 793.20 ± 59.23 1100.66 ± 253.03 2070.00 ± 408.07 /

104.39 99.59 85.29 130.79 151.43 155.47 1273.26 301.74 e

37.89 47.56 97.31 97.15 86.85 116.80 263.90 3690.98 /

e e e e e e 388.33 302.52 e

e e e e e e e 896.40 /

e e e e e e 304.03 382.05 e

e e e e e e e 507.23 /

0.151 0.120 0.121 0.130 0.136 0.145 0.368 0.207 /

0.140 0.103 0.159 0.163 0.160 0.140 0.160 2.591 /

0.031 0.015 0.013 0.019 0.014 0.018 0.141 0.047 /

0.013 0.008 0.017 0.014 0.008 0.015 0.025 0.537 /

“/” means no analysis. “-” lower the detection limitation.

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Table 3 Anaerobic digestion performance of GP11and GP31 in semi-continuous mode. OLR g VS/(L$d)

2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 7.0

Volumetric biogas yield m3/ (m3$d)

Specific biogas yield mL/g VS

Methane content %

Specific methane yield mL/g VS

GP11

GP11

GP11

GP31

GP11

56.66 54.29 56.70 57.41 56.95 58.27 60.29 54.91 56.75 48.23

50.24 50.72 54.98 55.48 56.06 56.30 59.87 47.11 / /

290.15 298.97 245.80 261.29 244.81 257.15 230.81 239.41 281.37 267.36

0.87 1.38 1.30 1.59 1.72 1.98 1.92 2.38 2.68 2.96

± ± ± ± ± ± ± ± ± ±

GP31 0.07 0.13 0.10 0.20 0.16 0.10 0.33 0.37 0.31 0.34

1.14 1.41 1.40 1.70 1.72 1.98 2.36 1.59 / /

± ± ± ± ± ± ± ±

0.15 0.18 0.11 0.14 0.21 0.14 0.13 0.50

511.84 553.24 433.50 454.63 430.89 439.98 383.86 432.80 445.13 422.97

GP31 ± ± ± ± ± ± ± ± ± ±

44.57 52.96 32.98 55.73 39.65 21.86 65.13 66.36 52.10 48.23

574.28 572.09 464.91 486.82 430.87 440.87 470.10 300.40 / /

± ± ± ± ± ± ± ±

72.04 64.50 36.98 41.06 52.65 32.05 29.21 73.85

GP31 ± ± ± ± ± ± ± ± ± ±

29.60 24.47 20.43 32.33 25.21 15.67 44.48 41.83 32.93 30.49

312.37 289.28 255.53 270.19 241.96 248.25 281.71 140.60 / /

± ± ± ± ± ± ± ±

43.82 27.30 20.35 26.61 31.42 18.76 17.26 70.19

“/” means no analysis.

107% increase compared to the yield obtained at an OLR of 2.0 g VS/ (L,d) (Fig. S6 and Table 3). The specific biogas yield ranged from 430.87 ± 52.65 mL/g VS to 574.28 ± 72.04 mL/g VS, and the methane content was 54.98e59.87%. There was no obvious variation in pH, VFAs/TA, and IA/PA with changes in OLR, which were 7.16 ± 0.03 to 7.29 ± 0.06, 0.008e0.025, and 0.103e0.163, respectively (Fig. S4, Fig. S7, and Table 2). The concentrations of VFAs and TAN were less than 263.90 mg/L and 1100.66 ± 253.03 mg/L, respectively, indicating that the system performed well at OLRs of 2.0e5.0 g VS/(L,d) (Fig. S7). However, at an OLR of 5.5 g VS/(L,d), the volumetric and specific biogas yields reduced by 48% and 56%, respectively, compared to the values at an OLR of 5.0 g VS/(L,d). Meanwhile, the methane content decreased to 47.11%, suggesting that the performance of GP31 reactor decreased at this OLR. Accordingly, the pH decreased to 6.70 ± 0.68 (Fig. S7 and Table 2). The TAN concentration increased to 2070.00 ± 408.07 mg/L (Fig. S7 and Table 2). The concentration of acetic acid was 3690.98 mg/L on average, and the maximum value of VFAs was 10,227.87 mg/L (Fig. S7 and Table 2), which is above the reported inhibitory concentration (Shi et al., 2017). The VFAs/TA and IA/PA ratios reached 0.537 and 2.591 (Fig. S4 and Table 2), respectively, indicating that the system had become unstable (Martin-Gonzalez et al., 2013). Therefore, severe inhibition occurred at an OLR of 5.5 g VS/(L,d), resulting in a decrease in biogas yield. This inhibition mode via VFAs production in GP31 is in contrast to the failure of the GP11 system, indicating that different factors triggered the failure of the two co-digestion systems. 3.2. Microbial communities of the co-digestion systems under different conditions The total non-chimeric sequence reads for GP11 and GP31 were 461,103e527,110 for bacteria and 196,374e225,197 for archaea, with a total of 9502e10,080 and 2383e2466 operational taxonomic units (OTU) identified for bacteria and archaea, respectively (Table S1 and Table S2). The Chao1 index was 986.24e2527.62 for bacteria and 219.38e811.00 for archaea, and the Shannon index was 2.98e4.00 for bacteria and 1.14e2.23 for archaea. The coverage values were more than 0.99, suggesting that the majority of the microbial community in the reactors was detected. 3.2.1. Bacterial communities in the co-digestion systems For both the GP11 and GP31 systems, the dominant bacteria identified at the phylum level included Bacteroidetes, Firmicutes, Chloroflexi, and Proteobacteria, which accounted for 73.98e94.82% of the total reads (Fig. S8A). With increasing OLRs, the relative abundances of Chloroflexi and Proteobacteria decreased by 87e98%

and 72e91%, whereas those of Bacteroidetes and Firmicutes increased to 32.21e67.30% and 15.91e41.34%, respectively. In the genus level, the main detected microbial included Ruminococcus, Sphaerochaeta, Turicibacter, Anaerophaga, Meniscus, Saccharofermentans, Mariniphaga, Petrimonas, Puniceicoccus, Syntrophorhabdus, Syntrophomonas, Smithella, Syntrophobacter, Geobacter, Proteiniphilum, Prevotella, Pseudomonas, Sedimentibacter, Ornatilinea, Intestinimonas, Levilinea, Leptolinea, Acinetobacter, Dysgonomonas, Intestinimonas, Clostridium, Psychrobacter, and Lutaonella, etc (Fig. S8B). Among these microbial in the genus level, Dysgonomonas, Intestinimonas, Clostridium XlVa, Clostridium III, Clostridium IV, Clostridium sensu stricto, and Prevotella were the main contributor for propionic acid and butyric acid production (Fig. 1A). The genus Dysgonomonas, in the family Porphyromonadaceae, ferments glucose to propionate and acetate (Xiong et al., 2019). The genus Prevotella grows optimally at pH 4.6e5.0, and mainly produces propionate (Liu et al., 2017; Maspolim et al., 2015). The genus Clostridium is considered a main contributor of VFAs accumulation, especially Clostridium clusters XIVa and IV that belong to the group of butyrate-producing bacteria (Jehlee et al., 2017; Zhou et al., 2015). In both reactors, the total relative abundances of these genera were 1.35e8.55% at OLRs of 2.0e5.5 g VS/(L,d), and increased by 123e1131% at an OLR of 5.5 g VS/(L,d) for GP31 and 27e340% at an OLR of 6.0 g VS/(L,d) for GP11, compared with the maximum and minimum values at lower OLRs (Fig. 1A and C). These results might explain the accumulation patterns of propionic acid or butyric acid in the reactors. Small molecules such as propionate, butyrate, and phenols are converted to biogas via syntrophic processes between bacteria and methanogens. The major bacteria involved in the process include the genus Smithella, Geobacter, Syntrophomonas, Syntrophorhabdus, and Syntrophobacter. In general, the total relative abundances of these genera decreased with an increase in the OLR from 2.0 to 5.0 g VS/(L,d). For GP31, the total relative abundance of these genera decreased from 5.91e6.73% at OLRs of 2.0e3.5 g VS/(L,d) to 0.21% at an OLR of 5.5 g VS/(L,d) (Fig. 1D). However, for GP11, the total relative abundance of these genera increased from 5.84e10.15% at OLRs of 2.0e4.0 g VS/(L,d) to 13.53e14.26% at OLRs of 5.5e6.0 g VS/ (L,d) (Fig. 1B). The genus Syntrophorhabdus has a positive syntrophic correlation with both aceticlastic and hydrogenotrophic methanogens (Ma et al., 2017). Syntrophobacter uses propionate as the only carbon source to grow in the presence of sulfate and fumarate (Centurion et al., 2018; Deng et al., 2018). Smithella is a unique syntrophic propionate or butyrate oxidizer (Kim et al., 2018; Nobu et al., 2017; Suyun et al., 2018). The genus Syntrophomonas became the dominant butyrate-oxidizing bacteria with an increase in the OLR (Xia et al., 2019). Combining the performance, process

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Fig. 1. Relative abundance of bacteria at the genus level for GP11 and GP31. (A) and (B) Dominant bacteria contributed to the accumulation and conversion of VFAs in GP11 system. (C) and (D) Dominant bacteria contributed to the accumulation and conversion of VFAs in GP31 system.

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parameter, and bacterial community, the accumulation of propionic acid in the GP31 reactor at an OLR of 5.5 g VS/(L,d) is likely due to inhibition of propionate-oxidizing bacteria, which relative abundance decreased by 97% at this OLR. By contrast, in the GP11 reactor, there was no notable accumulation of propionic acid or butyric acid because the increase in the relative abundance of Syntrophomonas ensured the conversion of propionic acid or butyric acid at OLRs of 5.5e6.0 g VS/(L,d). The total relative abundances of 4.05e18.62% in GP11 and 2.14e13.71% in GP31 were obtained for the genera Ornatilinea, Proteiniphilum, Pseudomonas, and Sedimentibacter which contributed to the degradation of protein (Fig. 2A and Fig. 2C). Among them, the relative abundance of the genus Proteiniphilum increased from 0.01e1.76% to 3.11e17.80% with an increase in the OLR from 2.5e3.5 g VS/(L,d) to 4.0e6.0 g VS/(L,d) for both reactors, becoming the predominant protein-degrading bacteria. These results indicated that the bacteria responsible for protein degradation changed with increasing OLR. Proteiniphilum, a protein-hydrolyzing bacteria, can hydrolyze peptone, yeast extract, glycine, and arginine with acetate and NH3 as the main products (Cardinali-Rezende et al., 2016; Maspolim et al., 2015), and thus might contribute to

the accumulation of NH3 during the co-digestion process. The amino acid-converting genera Cloacibacillus, Anaerovorax, Aminivibrio, Intestinimonas, Levilinea, Leptolinea, and Lutaonella accounted for a total relative abundance of 3.06e20.80% in GP11 and 3.50e17.31% in GP31 (Fig. 2B and D). Among them, the relative abundance of Levilinea decreased from the highest abundance of 18.29% to 0.03% with the OLR increased to 5.5 g VS/(L,d) for GP31. A similar decrease was observed in GP11. Levilinea metabolizes amino acids at pH 6.0e7.2, with H2, acetate, and lactate as the main products (Maspolim et al., 2015). 3.2.2. Archaeal communities in the co-digestion systems The dominant methanogens identified in the reactors at the order level included Methanosarcinales, Methanomicrobiales, Methanomassiliicoccales, and Methanobacteriales, which accounted for 37.19e98.55% of the total archaeal sequences (Fig. S9). With the OLRs increased from 2.0e5.0 g VS/(L,d) to 5.5 g VS/(L,d) of GP11 reactor, the relative abundance of Methanosarcinales and Methanomicrobiales decreased from 36.24e64.46% to 9.68% and from 20.57e42.92% to 2.95%, respectively. While an increase of 239% and 348% in the relative abundances of Methanobacteriales and

Fig. 2. Relative abundances of bacteria at the genus level for GP11 and GP31. (A) and (B) Dominant bacteria involved into the nitrogen conversion in GP11 system. (C) and (D) Dominant bacteria involved into the nitrogen conversion in GP31 system.

L. Lianhua et al. / Chemosphere 247 (2020) 125871

7

Fig. 2. (continued).

Methanomassiliicoccales was observed. Further raising the OLR to 6.0 g VS/(L,d) of GP11 reactor, an increase in relative abundance of Methanosarcinales and Methanomicrobiales appeared, while the relative abundance of Methanobacteriales and Methanomassiliicoccales decreased to 3.40% and 1.09%, respectively. For GP31 reactor, similar trend in archaea community was obtained. Generally, the relative abundance of Methanosarcinales and Methanomicrobiales decreased from the 46.68e72.98% to 4.23% and from 16.67e42.48% to 9.25% with the OLRs increased from 2.0e5.0 g VS/ (L,d) to 5.5 g VS/(L,d), while the relative abundances of Methanomassiliicoccales increased from 0.43e2.48% to 51.83%. Meanwhile, the relative abundance of Methanobacteriales ranged in 0.32e3.56%. Compared the two systems, higher relative abundance in Methanosarcinales was observed at the OLRs of 2.0e5.0 g VS/ (L,d), which related to the difference in the relative abundance of genus Methanothrix. Methanothrix was the dominant methanogen at the genus level, with a relative abundance of 35.98e64.41% for GP11 and 46.48e72.97% for GP31 at OLRs of 2.0e5.0 g VS/(L,d) Table 4. With an increase in the OLR to 5.5 g VS/(L,d), the relative abundance of Methanothrix decreased to 2.93% in GP31 and to 8.30% in GP11, with a further decrease to 0.76% at an OLR of 6.0 g VS/(L,d). Methanothrix is a typical aceticlastic methanogens and generates 70% of the methane (Ali et al., 2019; Gao et al., 2019). Comparing the two

systems, higher relative abundance of Methanothrix was observed for GP31 at the OLRs of 2.0e5.0 g VS/(L,d). This might be associated with the lower acetic acid concentration in GP31 in comparison with the GP11 system, because Methanothrix could be enriched in a condition of lower acetate concentration(Qiao et al., 2014). A decrease in the relative abundance of Methanothrix at OLRs of 5.5e6.0 g VS/(L,d) suggesting that a high ammonia and VFAs concentration inhibited the activity of Methanothrix. Methanosarcina showed different variation with a change in OLR in the two reactors. For GP11, the relative abundance of Methanosarcina ranged in 0.01e0.04% at the OLRs of 2.0e4.5 g VS/(L,d), whereas an increase in Methanosarcina (with the relative abundance of 1.38e22.28%) was obtained with the OLRs increased to 5.5e6.0 g VS/(L,d). For GP31, the relative abundance of Methanosarcina ranged in 0.01e1.31% during the whole experiments. The genus Methanosarcina comprises a group of multifunctional methanogens, which can form CH4 from H2/CO2, acetate, and methylated one-carbon compounds (Zhang et al., 2019). Compared the two systems, the genus Methanosarcina gained a great enrichment in GP11 reactor, which might be related to the VFAs and ammonia concentration. Factors, such as high levels in the VFAs (especially acetic acid) and ammonia concentration, could contribute to the increase in the relative abundance of Methanosarcina (Tian et al., 2018).

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Table 4 Relative abundance of methanogens at the genus level for GP11 and GP31. GP11

OLRs(g VS/(L,d)) 2.0

2.5

3.0

3.5

4.0

4.5

5.0

5.5

6.0

Acetoclastic methanogens

Methanothrix

35.98

54.9

51.48

55.24

60.15

64.41

36.42

8.30

0.76

Hydrogenotrophic methanogens

Methanolinea Methanobacterium Methanospirillum Methanosphaerula Methanobrevibacter Methanomethylovorans Methanoregula Methanosphaera Total Methanosarcina Methanomassiliicoccus

6.18 1.04 4.96 0.04 4.97 0.25 0.19 0.02 17.65 0.02 0.90

1.34 2.20 0.26 0.20 0.64 0.18 0.04 0.00 4.86 0.02 0.64

1.00 0.95 0.35 0.64 0.10 0.12 0.07 0.00 3.23 0.04 0.69

1.79 2.81 0.58 0.33 0.97 0.04 0.08 0.03 6.63 0.01 1.02

0.54 1.11 0.41 9.35 0.33 0.03 0.10 0.02 11.89 0.02 0.86

0.56 2.10 1.03 2.23 1.03 0.03 0.02 0.00 7.00 0.02 0.86

0.21 5.65 1.13 1.65 0.70 0.03 0.16 0.00 9.53 4.27 2.02

0.03 15.01 0.25 0.01 5.35 0.00 0.00 0.18 20.83 1.38 4.03

0.04 2.86 2.24 0.68 0.52 0.00 0.38 0.01 6.73 22.28 1.09

Both New type GP31

OLR(g VS/(L,d)) 2.5

3.0

3.5

4.0

4.5

5.0

5.5

Acetoclastic methanogens

Methanothrix

46.48

70.31

63.07

62.61

62.97

72.97

70.84

2.93

Hydrogenotrophic methanogens

Methanolinea Methanobacterium Methanospirillum Methanosphaerula Methanoculleus Methanobrevibacter Methanomethylovorans Methanoregula Total Methanosarcina Methanomassiliicoccus

20.82 0.22 4.75 0.30 0.04 0.09 0.20 0.13 26.55 0.00 0.51

4.49 0.31 1.02 0.22 0.19 0.47 0.19 0.08 6.97 0.04 1.01

8.22 0.73 1.51 0.22 0.20 0.28 0.19 0.09 11.44 0.02 0.43

6.61 0.68 1.36 0.67 0.49 0.57 0.12 0.08 10.58 0.01 1.19

2.2 2.13 3.01 4.38 0.49 0.39 0.03 0.10 12.73 0.01 2.48

0.55 3.18 2.45 0.22 0.47 0.38 0.01 0.06 7.32 0.01 1.24

0.15 2.79 3.92 0.58 0.20 0.19 0.00 0.10 7.93 0.01 1.31

0.01 2.62 0.19 0.00 8.68 0.45 0.00 0.12 12.07 1.31 51.83

Both New type

2.0

Methanomassiliicoccus showed an increase in the relative abundance with the OLR increased. For GP11, the relative abundance of Methanomassiliicoccus increased from 0.64e1.02% to 1.09e4.03% with the OLRs increased from 2.0e4.5 to 5.0e6.0 g VS/ (L,d). For GP31, the relative abundance of Methanomassiliicoccus increased from 0.43e1.19% to 1.24e51.83% with the OLRs increased from 2.0 e 3.5 to 4.0e5.5 g VS/(L,d), with a maximum value of 51.83% obtained at an OLR of 5.5 g VS/(L,d). Members of the genus Methanomassiliicoccus, assigned to the seventh order of methanogens, produce CH4 from methylamines via an H2-dependent methylotrophic pathway, but appear to be less effective in pro€ninger et al., 2019; Wang et al., 2018). An inducing methane (Kro crease in the relative abundance of this genus was accompanied with a reduction in biogas yield, indicating that the conversion efficiency of anaerobic digestion decreased. Li et al. reported that sludge co-treated by alkali and potassium ferrate did not produce methane, while an enrichment in Methanomassiliicoccus (with the relative abundance of 52.0%) occurred (Li et al., 2019). The hydrogenotrophic methanogens includes the genus Methanolinea, Methanobacterium, Methanospirillum, Methanosphaerula, and Methanobrevibacter. The total relative abundances of these methanogens were 3.04e20.65% in GP11 and 3.27e26.18% in GP31, respectively. A clear shift in dominant methanogen appeared at the OLRs of 5.5e6.0 g VS/(L,d). The genus Methanobacterium became the dominant methanogens in GP11 reactor with the relative abundance of 15.01% at the OLRs of 5.5 g VS/(L,d). Methanobacterium, a typical hydrogenotrophoic genus, use H2/CO2 and formate to produce methane. An increase in the relative abundance of Methanobacterium might be related to the TAN concentration and was beneficial to the performance of co-digestion. Hydrogenotrophic methanogens have better tolerance to the high ammonium levels and could improve the biogas yields via effective CO2 bioconversion (Pokorna et al., 2019).

3.2.3. Comparison of the conversion pathways of the co-digestion systems As shown in Fig. 3, the main microorganisms that are responsible for conversion of carbohydrates identified in the reactors were the genera Glostridium and Prevotella as the main contributor for propionate and the genera Syntrophorhabdus and Smithella as acidoxidization bacteria at OLRs of 2.0e5.0 g VS/(L,d). When the OLR was further increased, the imbalance between acid-producers and acid-oxidizers contributed to the failure of the GP31 system. In the GP11 reactor, the increase in the relative abundance of Syntrophomonas ensured the conversion of propionic acid. There was also a notable shift in microorganisms involved in the conversion of proteins and amino acids. At OLRs of 2.0e3.5 g VS/ (L,d), the primary bacteria of this group were the genera Ornatilinea, Sedimentibacter, and Levilinea. However, with increasing OLRs, the dominant bacteria were the genera Proteiniphilum, Sedimentibacter, Aminivibrio, and Levilinea. With respect to methane-forming bacteria, the genus Methanothrix was the main methanogen identified at OLRs of 2.0e5.0 g VS/(L,d). With increasing OLR, the co-existence of the genera Methanosarcina and hydrogenotrophic methanogens was responsible for methane formation in the GP11 reactor. In the GP31 reactor, the activity of methanogens decreased with increasing OLR owing to an increase in the relative abundance of Methanomassiliicoccus. 4. Conclusions Long-term operational experiments showed that a steady state was achieved in co-digestion systems of Pennisetum hybrid and pig manure at OLRs of 2.0e5.0 g VS/(L,d). The reactor with a mixture of 25% pig manure and 75% Pennisetum hybrid failed at an OLR of 5.5 g VS/(L,d) because of high concentration of TAN and VFAs, owing to an increased in the relative abundance of Proteiniphilum and an

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Fig. 3. Predicted microbial community for the conversion of carbohydrates and proteins in GP11 and GP31 system.

imbalance in acid-producer and acid-oxidation bacteria. Failure of the reactor digesting 50% pig manure and 50% Pennisetum hybrid occurred at an OLR of 7.0 g VS/(L,d), likely due to mechanical issues or an improper reactor configuration. CRediT authorship contribution statement Li Lianhua: Investigation, Writing - original draft. He Shuibin: Investigation. Sun Yongming: Supervision. Kang Xihui: Investigation, Resources. Jiang Junfeng: Investigation. Yuan Zhenhong: Conceptualization, Writing - review & editing. Liu Dingfa: Writing - review & editing. Acknowledgments This work was supported financially by National Natural Science Foundation of China [Grant number 51776208]; the Strategic Priority Research Program of Chinese Academy of Sciences [Grand number XDA21050400]; the Natural Science Foundation of Guangdong Province [Grant number 2018A0303130335]; the Science and Technology Planning Project of Guangdong Province [Grant number 2017A050501049, 2017B020238002]; Science and Technology Program of Guangzhou [Grant number 201707010201]. Appendix A. Supplementary data Supplementary data to this article can be found online at https://doi.org/10.1016/j.chemosphere.2020.125871. References Ali, S., Hua, B.B., Huang, J.J., et al., 2019. Effect of different initial low pH conditions on biogas production, composition, and shift in the aceticlastic methanogenic population. Bioresour. Technol. 289. Amha, Y.M., Sinha, P., Lagman, J., et al., 2017. Elucidating microbial community adaptation to anaerobic co-digestion of fats, oils, and grease and food waste. Water Res. 123, 277e289.

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