Effect of hydraulic retention time on anaerobic co-digestion of cattle manure and food waste

Renewable Energy 150 (2020) 213e220

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Effect of hydraulic retention time on anaerobic co-digestion of cattle manure and food waste Shaojie Bi a, Xiujie Hong b, Hongzhi Yang c, Xinhui Yu a, Shumei Fang a, Yan Bai a, Jinli Liu a, Yamei Gao a, Lei Yan a, Weidong Wang a, Yanjie Wang a, * a Heilongjiang Provincial Key Laboratory of Environmental Microbiology and Recycling of Agro-Waste in Cold Region, College of Life Science and Technology, Heilongjiang Bayi Agricultural University, Daqing, 163319, China b Commission of Agriculture of Daqing City, Ministry of Agriculture, Daqing, 163311, China c College of Food Science, Heilongjiang Bayi Agricultural University, Daqing, 163319, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 15 March 2019 Received in revised form 2 November 2019 Accepted 20 December 2019 Available online 28 December 2019

Anaerobic digestion of cattle manure has a low efficiency due to the high hydraulic retention time (HRT) required to degrade the abundant degradation-resistant compositions, co-digestion with food waste is effective at improving the methane production. Lowering the HRT can therefore increase the methanogenic efficiency during co-digestion. This study considered the effects of different HRTs (25, 20, 15, 10, 7, 5, and 4 days) on cattle manure and food waste co-digestion. The highest methane production was achieved at 1.48 L/L/d with an HRT of 5 days. The maximum methane yields (236e257 mL/g-VS) were attained at HRT
15 days and decreasing the HRT to 10-5 days resulted in low methane yields and complete process failure at HRT 4 days, due to volatile fatty acids accumulated and microorganisms washed out. From a high HRT of 20 days to a low HRTs of 5 days, Bacteroidetes and Firmicutes were the dominant bacteria and the percentage of syntrophic acetate oxidizing bacteria (mainly Pelotomaculum and Pseudothermotoga) clearly increased. The dominant methanogen changed from the acetotrophic Methanosaeta to the hydrogenophilic Methanobrevibacter. These results enable biogas plants to utilize surplus amounts of cow manure and food waste in a sustainable manner with high process capacity and methane recovery. © 2020 Elsevier Ltd. All rights reserved.

Keywords: Cattle manure Food waste Hydraulic retention time Microbial structure Biogas generation

1. Introduction The utilization of fossil fuels for energy production has caused serious pollution in recent decades [1]. In order to address climate change and reduce carbon emissions, the Chinese government announced its goal to decrease economic carbon intensity (CO2 per unit of gross domestic product) by 60e65% compared with 2005 and to produce 20% of the country’s energy from non-fossil fuels by 2030 [2]. This initiative includes a nationwide effort to increase the mobilization of livestock manure for energy production [3]. Annual livestock manure production in China is about 3.8 billion tons, of which cattle manure accounts for a large proportion [4].

* Corresponding author. E-mail addresses: [email protected] (S. Bi), [email protected] (X. Hong), [email protected] (H. Yang), [email protected] (X. Yu), [email protected] (S. Fang), [email protected] (Y. Bai), 961665969@qq. com (J. Liu), [email protected] (Y. Gao), [email protected] (L. Yan), [email protected] (W. Wang), [email protected] (Y. Wang). https://doi.org/10.1016/j.renene.2019.12.091 0960-1481/© 2020 Elsevier Ltd. All rights reserved.

Anaerobic digestion (AD) is one of the most popular technologies for manure treatment and energy recovery, allowing its use for heating, electricity, and transportation fuel [5,6]. However, the methane yield and methane production rate from cattle manure as a sole substrate is low, due to its high content of non-biodegradable and degradation-resistant substances [7,8]. The co-digestion of cattle manure with other easily degradable organic materials is a suitable solution for this problem. Food waste is an ideal co-substrate given its high carbohydrate content, rapid hydrolysis, and acidification of organic matter [9]. In turn, cattle manure can provide buffering capacity which might be beneficial for co-digestion with easily degradable food waste [10,11]. About 600 million tons of food waste is generated annually in China, approximately 90% of which is disposed of in landfills or open dumps; such improper treatment of food waste can cause serious environmental problems [12]. Thus, co-digesting food waste with cattle manure to produce renewable energy could improve the nutrient balance of the process, enhance methane

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production, and minimize the negative environmental impacts of these waste streams [13]. Hydraulic retention time (HRT) is one of the most important parameters affecting AD performance; reducing HRT is effective at increasing methane production and economic efficiency. Furthermore, changes in HRT can affect the structure of the microbiome community. The imbalance between fast growing microorganisms (hydrolytic bacteria and acidogens) and slow growing methanogens as a result of improper HRT results in problems such as insufficient utilization of hydrolysis-acidogenesis products and/or washout effect of methanogens [14]. Furthermore, acetoclastic methanogens are commonly more sensitive to environmental change than hydrogenotrophic methanogens [15]. For example, the methane produced from syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (SAO-HM) increases at low HRTs when sewage sludge was degraded under mesophilic conditions [16]. It has been reported that the variations in microbial structure profiles are significantly governed by many operational variables such as HRT [14]. Therefore, understanding the correlations between microbial community structure and functions with operational conditions is pivotal for an efficient and stable AD performance. However, few studies have considered the effect of HRT on the co-digestion of cattle manure and food waste, especially with regard to the microbial community over the long term. This study aimed, therefore, to investigate the effect of HRT on cattle manure and food waste co-digestion by operating AD process at sequentially reduced HRT (from 25 to 4 day) with the aim of optimizing the HRT of AD of such wastes. The process performance and biodegradation behavior of organic matters and different intermediates was empirically evaluated. In addition, the correlations between the microorganisms’ relative abundance at short HRT (5days) and long HRT (20-days) were evaluated using next generation 16S rRNA gene sequencing. AD kinetics and Monod model for constant VS were also assessed for further elucidation of how HRT affects process performance. 2. Materials and methods 2.1. Substrate and inoculum Food waste was collected after lunch time from a student canteen at Heilongjiang Bayi Agricultural University, China. Undesirable components such as bones, plastics, and wastepaper were removed manually. The residual food waste was mixed for 5 min using a blender (Joyoung JYLC012). Cattle manure was scraped off of the feed lanes of a farm in Daqing, China. Both samples were stored at 4  C. A mixture of cattle manure and food waste (2:1 wt/ weight on VS basis to reach to the ideal C/N ratio for anaerobic digestion i.e., 20 to 30 [17], was diluted with tap water to adjust VS content to the required level at each HRT prior to being used as a substrate. The inoculum was taken from a laboratory-scale digester containing a treated mixture of cattle manure and food waste operated at 37  C for more than 400 days. The characteristics of the substrate and inoculum are given in Table 1.

Table 1 Substrate and inoculum characteristics. Parameter

Unit

Substrate

Inoculum

TSa VSb VS/TS TCODc SCODd C H O N S C/N

% % % g/L g/L % DMe % DMe % DMe % DMe % DMe /

7.7 ± 0.5 6 ± 0.3 76.2 ± 1.1 74.6 ± 3.5 26.5 ± 1.4 49.4 ± 0.6 5.8 ± 1.1 41 ± 0.6 2.2 ± 0.4 1.6 ± 0.3 22.5 ± 0.6

2.7 ± 0.5 1.4 ± 0.3 49.1 ± 2.3 NA NA NA NA NA NA NA NA

COD is chemical oxygen demand. a TS is total solids. b VS is volatile solids. c TCOD is the total influent COD. d SCOD is the soluble influent COD. e DM is the dry matter.

10, 7, 5 and 4 days, respectively. The reactor was run for approximately 2 HRTs at each stage. During the process, biogas volume and composition (CH4 and CO2) and pH values were measured every day. Volatile fatty acids (VFAs), total ammonium nitrogen (TAN), total chemical oxygen demand (TCOD), soluble chemical oxygen demand (SCOD), and effluent alkalinity were measured every four days. 2.3. Analysis methods Duplicate samples for analyses of TS, volatile suspended solids (VSS), VS, TCOD, SCOD, TAN, and total alkalinity were analyzed using standard methods [18]. In brief, TS and VS were measured by the gravimetric method after the sample was dried at 105  C and 600  C for more than 24 h and 6 h, respectively. TCOD and SCOD were detected using a potassium dichromate solution. Prior to testing, the SCOD sample was centrifuged at 8000 rpm for 15 min and filtered through a 0.22 mm filter. TAN was measured by spectrophotometry at 420 nm after the sample was treated with Nessler’s reagent. Total alkalinity was measured by hydrochloric acid titration to pH 4.5. pH values were measured using a pH meter (HORIBA B-712, Japan). Biogas production was calibrated to standard conditions (0  C, 1.013 bar). Biogas composition was analyzed by a biogas analyzer (Geotech GA 5000, UK). VFA composition and elemental composition (C, H, O, and N) was analyzed using the methods defined in a previous study [19]. The fraction of organics destroyed was calculated based on gas production data (gCOD-CH4) observed for each HRT stage [19]. 2.4. Process kinetics analysis The process kinetics were calculated using modified Monod equations [19,20]:

Cs ¼ ðKs xDÞ=ðumax  DÞ

(1)

2.2. Experimental set-up and operation A 20 L continuous stirred tank reactor (CSTR) with a working volume of 16 L was used as the digester. The digestion temperature (37  C) was maintained using a heated water bath (HH-60, China). The reactor was agitated using biogas circulation with a flow rate of 6 L/min. Biogas production was measured and recorded using a wet biogas meter (LML-1, China). The digester was fed with the mixture of substrates with increasing organic loading rates (OLR) from 2.4 to 3, 4, 6, 8.6, 12 and 15 g-VS/(L$d), by reducing HRT from 25 to 20, 15,

Px ¼ DxYx=s xðCs0 

Cx ¼ Yx=s xðCs0 

Ks x D Þ umax  D

Ks x D Þ umax  D

(2)

(3)

where Cs is the volatile solid concentration, g-VS/L; D is the dilution rate, 1/HRT, 1/d; Px is the methane production (L/L/d), and Cx is the microbial concentration (g-VSS/L). Ks, umax, Yx/s and Cso are

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constants and were fixed using Origin fitting analysis. 2.5. Sampling and microbial community pyrosequencing and illumina sequencing In order to identify the microbial community at HRTs of 5 and 20 days, samples were collected and mixed at the 85th to 87th days and 190th to 192nd days and stored at 20  C. DNA was extracted from the mixtures using a genomic DNA rapid extraction kit purchased from Abigen Corporation in Beijing. 16S rDNA was amplified by polymerase chain reaction (PCR) after confirming the concentration and purity of the DNA. The primers were 520F (50 -AYTGGGYDTAAAGNG-30 ) and 802R (50 -TACNVGGGTATCTAATCC-30 ) for bacteria and U789F (50 -TAGATACCCSSGTAGTCC-30 ) and U1068R (50 CTGACGRCRGCCATGC-30 ) for archaea. PCR reactions were performed, according to previous studies [21,22]. All sequences were grouped into operational taxonomic units (OTUs) after the DNA sequencing was performed using the Illumina high-throughput

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sequencing platform [23]; the microbial diversity was then analyzed [19]. 3. Results and discussion 3.1. Potential methane yield of the co-digested substrates Based on the elemental composition in Table 1, the organic part of the mixture can be characterized as C26.51H37.69O14.11N. A theoretical methane production potential of 530 mL/g-VS was calculated using the Buswell equation (C26.5H37.7O14.1Nþ27.1H2O /14.2CH4 þ 12.1CO2 þ NH4HCO3) [24]. Based on a batch test, a biochemical methane potential (BMP) of 259 mL/g-VS was obtained [25]. This value is much higher than those obtained in previous studies of anaerobic treatment of cattle manure in batch trials [26] and in continuous reactors [7]. The C/N ratio was within the reported optimal range (20e30) for anaerobic microorganism growth [17].

Fig. 1. Performance of anaerobic co-digestion of cattle manure and food waste while decreasing HRT.

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3.2. Process performance along with HRT shorting The highest specific methane yield was 257 mL/g-VS at an HRT of 25 days, which was comparable with the results obtained from the batch assay (259 mL/g-VS) of the same mixture [25]. This value was much higher than that obtained in Co-AD of food waste and cattle manure at ratio of 3:1 [27] but was much lower than that reported by Zhang et al. (2013) [13]. These differences may be attributed to the different qualities of specific manure and/or food waste. The specific methane yield decreased to 246 and 236 mL/gVS when the AD process was operated at HRTs of 20 and 15 days, respectively. During the first three stages, the corresponding volumetric methane production rate gradually increased from 0.62 to 0.74 and 0.94 L/L/d, respectively (Fig. 1, Table 2). The volumetric methane production rate continued to grow from 1.19 to 1.46 and 1.48 L/L/d when HRT decreased from 10 to 7 and 5 days. However, an obvious decline in methane yield appeared at an HRT of 10 days. The methane yield decreased by 16%, 33%, and 65% when HRT decreased from 15 to 10, 7, and 5 days, respectively, then mostly disappeared at an HRT of 4 days. These changes in methane production were reflected in the trends of VS removal efficiency. More specifically, during the first three stages (25, 20, and 15 days HRT), 57e59% VS was removed, but this value decreased by 16% at 10 days HRT and decreased by 60% at 4 days HRT (Fig. 2). Thus, for this mixture, a stable AD process could be achieved at an HRT
15 days, while an HRT <15 days could cause a considerable decline in methane yield and organic removal efficiency. The TAN concentrations in the digester were maintained around 0.6 g/L during the entire process except at 4 days HRT (0.45 g TAN/ L), these values were far below the reported TAN inhibition threshold for anaerobic digestion [28]. The total VFA concentration was less than 0.5 g/L during HRTs of 25, 20, and 15 days. Total VFA and TAN resulted in a corresponding alkalinity of 3.33e3.56 g CaCO3/L (Fig. 1e), which provided a strong buffer maintaining the almost constant pH of 7.3e7.4 (Fig. 1d). The VFA to alkalinity ratio was within the early warning threshold of 0.3e0.4 [29], and the AD

Fig. 2. VS removal efficiency while decreasing HRT.

process was stable. The total VFAs slowly increased alongside with decreasing HRT less than 15 days and finally rose up to 2.6 and 23.1 g/L at 5 and 4 days HRT, respectively. Acetic, propionic, and butyric acids were the main acids composing VFAs. It has been reported that a VFA concentration above 4 g/L would exert inhibitory effects on an AD process [30] and 1.5 g/L was also stablished as a threshold for a healthy AD process [31]. As the VFAs accumulated, the ratio of VFAs to alkalinity generally rose to 16.5, indicating the breakdown of the AD process [32]. This indicated that at HRTs
15 days, the performance of AD process was stable. AD process inhibition due to VFA accumulation appeared at HRTs <10 days; an HRT of 4 days caused AD process failure. 3.3. COD mass balance vs. decreasing HRT The transformation of organic matter into SCOD*, VFAs, and methane was compared considering decreasing HRT (Table 2). The

Table 2 Long-term performance of anaerobic co-digestion of cattle manure and food waste. Parameter

Unit

HRT 25

20

15

10

7

5

4

Organic loading rate Duration pH Methane production Methane yield CH4 content Volatile fatty acids Acetate Propionate Isobutyrate Butyrate Isovalerate Valerate Hexanoate Total alkalinity Total ammonia nitrogen PCODe/TCODina SCOD*d/TCODin CODVFAb/TCODin CODCH₄c/TCODin

kg-VS/(m3$d) d / L/L/d ml/g-VSin % g/L g/L g/L g/L g/L g/L g/L g/L g/L mg/L % % % %

2.4 1e50 7.4 ± 0.1 0.62 ± 0.02 257 ± 9 67 ± 1 0.42 ± 0.05 0.18 ± 0.08 0.12 ± 0.03 0.03 ± 0.03 0.06 ± 0.05 0.04 ± 0.04 0 0 3.56 ± 0.18 0.6 ± 0.04 35 ± 4 8±1 1±1 57 ± 3

3 51e92 7.4 ± 0.1 0.74 ± 0.02 246 ± 7 68 ± 1 0.43 ± 0.02 0.34 ± 0.02 0.07 ± 0.01 0.02 ± 0.02 0 0 0 0 3.44 ± 0.11 0.6 ± 0.03 35 ± 4 9±1 1±1 56 ± 2

4 93e129 7.3 ± 0.1 0.94 ± 0.03 236 ± 6 66 ± 1 0.45 ± 0.09 0.27 ± 0.12 0.09 ± 0.02 0.03 ± 0.02 0.01 ± 0.03 0.02 ± 0.03 0 0.02 ± 0.01 3.33 ± 0.19 0.64 ± 0.05 35 ± 3 8±1 1±1 54 ± 1

6 130e156 7.1 ± 0.1 1.19 ± 0.04 198 ± 7 67 ± 2 0.54 ± 0.07 0.18 ± 0.03 0.08 ± 0.03 0.06 ± 0.01 0.05 ± 0.01 0.08 ± 0.01 0.04 ± 0.02 0.05 ± 0.03 3.15 ± 0.12 0.56 ± 0.02 44 ± 4 8±1 1±1 45 ± 2

8.6 157e178 6.9 ± 0.1 1.46 ± 0.06 170 ± 7 62 ± 2 0.83 ± 0.15 0.35 ± 0.18 0.23 ± 0.12 0.05 ± 0.05 0.04 ± 0.04 0.06 ± 0.05 0.04 ± 0.03 0.06 ± 0.04 2.71 ± 0.1 0.68 ± 0.04 46 ± 2 10 ± 1 1±1 41 ± 1

12 179e194 6.8 ± 0.1 1.48 ± 0.08 126 ± 22 56 ± 1 2.6 ± 0.28 1.06 ± 0.21 0.39 ± 0.09 0.18 ± 0.05 0.39 ± 0.3 0.23 ± 0.05 0.15 ± 0.08 0.21 ± 0.14 2.71 ± 0.11 0.58 ± 0.04 49 ± 2 19 ± 2 4±1 28 ± 1

15 195e207 5.4 ± 0.1 / / / 23.1 ± 0.1 8.28 ± 0.3 3.87 ± 0.08 1.19 ± 0.01 7.33 ± 0.3 1.67 ± 0.06 0.42 ± 0.02 0.34 ± 0.01 1.4 ± 0 0.44 ± 0.03 / / / /

Notes: COD is chemical oxygen demand. a SCODin is the soluble influent COD. b CODVFA is the calculated COD base of individual VFAs in the effluent. c CODCH₄ is the calculated COD based on the conversion coefficient of COD and methane production under the standard conditions (350 mL-CH4/g-COD). d SCOD* is the SCOD other than VFAs, calculated as SCOD* ¼ SCOD-CODVFA. e PCOD is particulate chemical oxygen demand, calculated as PCOD ¼ TCOD-SCOD; “/” means data not available.

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CH4-COD fraction ranged from 54 to 57% at HRTs of 25, 20, and 15 day, which was similar to the VS removal rate (57e59%), indicating most VS was converted to methane. Then CH4-COD fraction decreased to 45%, 41% and 28% at HRTs of 10, 7, and 5 day, indicating a decline in methanization. The fraction of particulate chemical oxygen demand (PCOD), reflecting the ratio of non-degraded organic matter, was stable at 35% for the 25, 20, and 15 day HRTs, then began to rise as HRT was reduced below 10 days. The higher PCOD in effluent revealed that hydrolysis was impaired with decreasing HRT. In addition, the percentage of VFAs was 1% throughout the fermentation process, apart from 4% at an HRT of 5, indicating that methanogens cannot effectively consume the transformed VFAs at 4d HRT. Furthermore, the SCOD* fraction ranged between 8 and 10% at all tested HRTs. Exception made for 5 day HRT where SCOD* increased up to 19% indicating that acidogenic process was slightly inhibited. 3.4. Microbial concentration and VS removal efficiency along with HRT shorting The choice of HRT is critical for anaerobic fermentation. If HRT is too long, the operational cost will be increased to a considerable extent. If HRT is insufficient, the biomass flowing out of the reactor per unit time increases, resulting in decreasing microbial content and a lower degradation rate of organic matter [16]. A CSTR tends to be unstable at a dilution rate of 1/HRT, which is close to the methanogens’ proliferation rate [19]. The impact of HRT on microbial concentration, methane production, and VS removal efficiency was calculated using Eqs. (1)e(3) (Fig. 3). Methane production rose with decreasing HRT, peaked around an HRT of 6 days, then decreased and ceased at an HRT of 4 days. At the same time, the VS in the digestate increased and approached that in the feeding source at an HRT of 4 days. The microbial concentration continued to decrease and almost disappeared at an HRT of 4 days. Methane production from organics proceeds mainly through hydrolysis, acidification, and methanogenesis. Methanogenesis is often recognized as a rate-limiting step at a short HRT, due to the slow growth rate of methanogens,

217

especially for acetotrophic methanogens. Furthermore, VFA accumulation (acetate first, followed by propionate) caused by limited methanogenesis, leads to the process failure at an HRT of 4 days. 3.5. Impact of HRT on microbial community structure As shown in Fig. 4a, representatives of the phyla Bacteroidetes and Firmicutes were dominant bacteria at HRTs of both 20 and 5 days. Hydrolyzing bacteria in the anaerobic reactor were mainly comprised of Firmicutes, which are involved in the degradation of complex organic matter and dominate the AD process for food waste and cattle manure [33,34]. They are recognized as cellulosedegrading and lipid-degrading bacteria as well as acetic-acidforming bacteria in anaerobic digestion [35], including several recently described syntrophic acetate oxidizing bacteria (SAOB), which can grow associated with hydrogenotrophic methanogens and promote methane production under stress conditions [36]. The Bacteroidetes are also well known as cellulose-degrading bacteria in anaerobic digestion [37]. Even a few unidentified SAOBs are considered to be members of this phylum [38]. Syntrophic acetate oxidation coupled with hydrogenotrophic methanogenesis (SAOHM) has been suggested as the dominant pathway for methane production under low HRTs [19]. Table 3 summarizes the changes in SAOBs between HRTs of 20 and 5 days. Pelotomaculum sp. and Pseudothermotoga sp. were detected in the digester; the latter were recognized as an important SAOB in the AD process, as they can degrade lipids to produce fatty acids and glycerol [39]. The total relative abundance of SAOBs increased to 7.1% at an HRT of 5 days, enhancing the proportion of SAO-HM pathways in response to the decreasing HRT. The obligate acetotrophic genera (e.g., Methanosaeta at 86.7%), dominated the archaeal community at an HRT of 20 days (Fig. 4b). According to a previous study, acetotrophic methanogens contributed 70% of methane production in the anaerobic digestion process [40]. Methanosaeta sp. has been recognized as the dominant methanogen in stable biogas processes [41]. At an HRT of 5 days, there was less diversity in the archaeal community and Methanobrevibacter sp. prevailed in the system. These are strictly

Fig. 3. Effect of HRT on methane production, volatile solid and mircobial concentration.

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Fig. 4. Composition of (a) bacterial and (b) archaeal communities at HRTs of 20 and 5 days.

Table 3 Percentage of potential syntrophic acetate oxidation bacteria (SAOB) and hydrogenotrophic partners. HRT (days)

SAOB

Hydrogenotrophic partners

Pelotomaculum sp. Pseudothermotoga sp. Total Methanocorpusculum sp. Methanobacterium sp. Methanoculleus sp. Methanobrevibacter Total

20

5

0.2 0.3 0.5 5.2 3.3 0.8 0.2 9.5

4.5 2.6 7.1 0 4.2 0 95.8 100

hydrogenotrophic methanogens that are dominant in slaughterhouse waste and leachate biogas fermentation at low HRTs (e.g., 5e6.1 d), due to a short doubling time of 3 h [42e44]. Compared with acetotrophic methanogens, hydrogenotrophic methanogens are less sensitive to environmental changes including HRT [45]. Besides, the growth rate of hydrogenotrophic methanogens is much higher than that of acetotrophic methanogens. The doubling time of the latter ranged from 1.5 to 4.0 d, while that of some of the former was <6 h. In addition, under low HRT conditions, acetotrophic methanogens were more easily washed out [46,47]. Therefore, hydrogenotrophic methanogens are more likely to become the dominant archaea under stressful conditions such as high ammonia levels, high loading, or low HRTs [48e50]. Hydrogenotrophic methanogens can thus play an important role in compensating for the washout of acetotrophic methanogens and SAO-HM is the suggested dominant methanogenic pathway under low HRTs. The calculated parameters of equations (1)e(3) was provided in Table S1. 4. Conclusions This study demonstrated that anaerobic digestion supplemented with co-substrates (cow manure and food waste) could

successfully operate with high methane yield (236e257 mL/g-VS), high VS removal (57e59%) and stable performance at an HRT range of 15e25 days. While an inhibited steady state of methane production was observed at 10, 7, 5-d HRT, the methane production totally ceased as a result of VFA accumulation over the inhibited threshold at 4-HRT. The methane was exclusively generated by H2mediated CO2 reduction pathway at short HRT, judging by the prevalence of hydrogenotrophic methanogens (mediated by Methanobrevibacter) with the absence of acetotrophic methanogens. These results indicated that correlation between microbial community and HRT could be utilized as a bio-indicator for better process performance and to foresee process failure. In addition, high AD process capacity of the biogas plant could be achieved with highest methane yield by appropriate adjustment of HRT. Author contributions Shaojie Bi, Weidong Wang and Yanjie Wang designed and performed experiments, analyzed data and co-wrote the paper. Shaojie Bi, Xiujie Hong, Hongzhi Yang, and Xinhui Yu performed experiments. Shaojie Bi, Shumei Fang and Yan Bai performed kinetics analyses and microbial analyses. Jinli Liu, Yamei Gao, and Lei Yan co-wrote the paper. Declaration of competing interest The authors declared that they have no conflicts of interest to this work. Acknowledgments This work was supported by the National Key Research and Development Program of China (2017YFD0800802-03). This work partly adopted the research logic and data presenting methodologies published by Dr. Wei Qiao who work at china agricultural university. Dr. Ahmed Mahdy help to polish the English during the revision of this paper. We would also like to thank Editage [www. editage.cn] for English language editing.

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