Overcome inhibition of anaerobic digestion of chicken manure under ammonia-stressed condition by lowering the organic loading rate

Journal Pre-proof Overcome inhibition of anaerobic digestion of chicken manure under ammonia-stressed condition by lowering the organic loading rate

Ahmed Mahdy, Shaojie Bi, Yunlong Song, Wei Qiao, Renjie Dong PII:

S2589-014X(19)30249-X

DOI:

https://doi.org/10.1016/j.biteb.2019.100359

Reference:

BITEB 100359

To appear in:

Bioresource Technology Reports

Received date:

2 December 2019

Accepted date:

2 December 2019

Please cite this article as: A. Mahdy, S. Bi, Y. Song, et al., Overcome inhibition of anaerobic digestion of chicken manure under ammonia-stressed condition by lowering the organic loading rate, Bioresource Technology Reports(2018), https://doi.org/10.1016/ j.biteb.2019.100359

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© 2018 Published by Elsevier.

Journal Pre-proof Overcome inhibition of anaerobic digestion of chicken manure under ammoniastressed condition by lowering the organic loading rate Ahmed Mahdya,b, Shaojie Bi a,c, Yunlong Songa,c, Wei Qiao a,c*, Renjie Dong a,c College of Engineering, China Agricultural University, Beijing 100083, China

b

Department of Agricultural Microbiology, Faculty of Agriculture, Zagazig University, 44511 Zagazig, Egypt

c

State R&D Center for Efficient Production and Comprehensive Utilization of Biobased Gaseous Fuels, Energy Authority, National Development, and Reform Committee, Beijing 100083, China

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a

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Abstract

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The synergetic inhibition of fatty acids accumulation and ammonia on the metabolic

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capability of methanogens limits the efficiency of nitrogen-rich substrates anaerobic

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digestion (AD). In this study, AD of chicken manure under different experimental conditions (namely, organic loading rate (OLR) of 1.25, 2.5 and 5 g.TS L-1d-1) with

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ammonia-stressed system (⁓6000 mg-NH4+-N/L) were investigated. The results

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demonstrated an inhibited-steady state of methane production at OLR of 5.0 g.TS Ld which resulted in up to 30%-37% methane yield reduction. The highest methane

yield (356 mL g VSin-1) and organics removal efficiency (66%) were achieved at OLR of 2.5 g.TS L-1d-1 by extending the hydraulic retention time to 40 days. Methanosarcina was found more tolerant to ammonia stress but Methanobacterium and Methanoculleus dominated in the higher methane yield digester. The results indicated that the proper trade-off between high OLR and ammonia inhibition may develop a high efficiency AD for nitrogen-rich materials. Key words: anaerobic digestion; chicken manure; ammonia inhibition; organic loading rate; microbial community -1 -

Journal Pre-proof 1.

Introduction

Anaerobic digestion (AD) is a microbial-based process which converts the organic substrates into clean energy, the biogas (mainly, methane and CO2) (Chen et al., 2014). The whole anaerobic process is catalyzed through different metabolic pathways by the action of hydrolytic bacteria, acidogens, acetogens and methanogens, respectively, which should be balanced for stable and long-term process.

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Nevertheless, during the mineralization of organic nitrogen, the excessive release of mineral nitrogen (NH3, NH4+) of nitrogen-rich substrate could easily inhibit the

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microbial groups. Specifically, optimal ammonia levels (< 200 mg NH4+-N g L-1)

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provide the sufficient buffer capacity and the essential nutrient for microbial growth,

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however, the excessive concentrations (> 1500 mg NH4+-N g L-1) could inhibit the

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microbial activity, mainly methanogens, by changing intracellular pH, depleting in intracellular cations and/or influencing the enzyme system of methane production

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(Yenigun and Demirel 2013). So far, the ammonia inhibition in AD of nitrogen-rich

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materials is still a big challenge.

In an attempt to increase the quantities of substrates degradation and thus increase the treatment capacity of the plants, increasing organics load (short HRT) was used as an applicable solution (Wandera et al., 2019). However, this scenario cannot be withdrawn with the digestion of nitrogen-rich wastes such as chicken manure because of the excessive ammonia production during the degradation process (Chen et al., 2008; Yenigun and Demirel 2013). In this manner, 30% loss in methane yield of fullscale biogas digesters treating protein-rich substrates was attained due to ammonia inhibition (Fotidis et al., 2014). In such conditions, hydrogenotrophic methanogens were reported to be more tolerant to high ammonia level compared with acetoclastic -2 -

Journal Pre-proof methanogens (Wang et al., 2016). For instance, hydrogenotrophic methanogens became the dominant methanogens when the ammonia concentration exceeded 3 NH4+-N g L-1 in anaerobic digestion process (Wiegant and Zeeman,1986). Accordingly, the enrichment of Methanoculleus and Mehtanobrevibacter in AD process was used as a bio-augmentation tool to improve the process performance under high ammonia levels (Fotidis et al., 2014; Yang et al., 2019). Nevertheless, it has been reported that the doubling time of hydrogenotrophic methanogens

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(Methanoculleus) increased from 10-13 day to 23-50 day when total-ammonia

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nitrogen (TAN) increased from 0.5-2.8 to 4.8 g L-1 (Westerholm et al., 2019).

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Furthermore, some acetate oxidizing bacteria (SAO), the syntophic partner of

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hydrogenotrophic methanogens, were proposed to have relatively long doubling time, exceeding 25d (De Vrieze et al., 2012). This fact in turn highlights the important role

considering

that

SAO

pathway

coupled

with

hydrogenotrophic

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substrate,

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of the adequate HRT (low OLR) during the biodegradability of nitrogen-rich

methanogens will be the main route for methane production. The proper OLR and

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HRT are supposed to mitigate ammonia inhibition effect on methanogens and protect functional microbes to be washout due to extending their doubling time under such conditions.

Although the proper OLR has proved its efficiency for healthy AD performance of chicken manure, no comparison has been made between the microbial community profiles of healthy and overloaded systems, under inhibited and well-performed steady-state of methane yield, to understand the mechanism of inhibition. Most of the previous research were focused on the methane yield and the intermediates (Zhang et al., 2017a; Bi et al., 2019; Busato et al., 2020). The other studies focused only on the microbial community of the healthy process (Song et al., 2019). Consequently, the -3 -

Journal Pre-proof influence of accidental increase in ammonia levels, as a results of increasing the loading rate of nitrogen-rich substrates, on the methanogenic community should be further examined under well-performed and overloaded systems, to select a proper OLR and HRT for promoting desirable and less sensitive methanogens and metabolic pathways. This study therefore aimed at providing insights into the underling processes that were

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suspected to circumvent or alleviate the ammonia inhibition at adequate OLR. OLR

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impacts were assessed in a long-term study of high ammonia digesters operated at different HRT of 20, 40 and 60 days. Process performance, VFAs accumulation and

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mass balances were comprehensively investigated under well-performed and

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overloaded systems. 16S rRNA analyses were utilized to determine the microbial

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community profiles dynamics, with the intention of determining the key microorganisms related to process stability and enhanced productivity of methane

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yield at high ammonia levels.

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2. Material and methods

2.1. Substrate and inoculum

The nitrogen-rich substrate used in the current study was chicken manure and was collected from a laying hen farm in Beijing, China. The raw manure which had a total solid (TS%) concentration of 32% was diluted by tap water to required concentration (TS<10%) and stored at 4°C (not more than 2-3 weeks) until being used. The inoculum was obtained from a long-term mesophilic continuously stirred tank reactor (CSTR) experiment treating chicken manure for more than 48 months. The chemical characteristics of chicken manure and inoculum are depicted in Table 1.

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Journal Pre-proof 2.2. Experimental operation Three parallel CSTRs were operated for treating chicken manure at different HRT (20, 40 and 60 days), which were represented by using the abbreviation as 20dHRT, 40d-HRT and 60d-HRT digesters. Each digester had an active volume of 12 L and total volume of 16 L. Continuous mixing was provided by mechanical stirrer at 100 rpm. The digesters were operated at mesophilic condition (37°C) by passing the

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water inside a water jacket. The OLR was set at 5, 2.5 and 1.25 g TS L-1 d-1 in 20d-,

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40d- and 60d-HRT digester, respectively. The digesters were fed manually once per day. The same amount of slurry was withdrawn as effluent from each digester. The

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produced biogas was collected in a collection bag and the biogas volume was

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measured by a biogas meter every day. The 60d-HRT was firstly started up and

2.3. Microbial analysis

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provided the matured inoculum for the 40-HRT and 20-HRT digesters.

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DNA extraction was performed through the cetyltrimethyl ammonium bromide/sodium dodecyl sulfate (CTAB/SDS) procedure, including enzymatic cell lysis, purification and precipitation steps. The amplification of 16 rRNA gene was performed using forward primers (338F and 524F) and reverse primers (806R and 958R) for bacterial and archaeal sequences, respectively. The key characteristics of PCR thermal cycling were as following: denaturation one cycle for 1 min and then 30 cycles for 10 s at 98°C, annealing for 30s at 50°C and elongation for 30s and then for 5 min at 72°C. NEB Next® 157 Ultra™ DNA Library Prep Kit for Illumina (NEB, USA) was used to sequence the amplicons. The library quality was evaluated and sequenced by The Qubit@ 2.0 Fluorometer (Thermo Scientific) with Agilent -5 -

Journal Pre-proof Bioanalyzer159 2100 system and the Illumina HiSeq platform at Majorbio company (Shanghai, China), and then the reads were merged by FLASH. The reads were grouped into operational taxonomic units (OTUs) with the similarity of 97%. Taxonomic classification was conducted with a 16S rRNA reference (RDP) database. 2.4. Analytical measurements TS, volatile solid (VS), volatile suspended solid (VSS), total chemical oxygen

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demand (TCOD), soluble COD (SCOD) and TAN were determined based on standard

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methods (APHA 2005). Particulate COD (PCOD) was determined as the remaining

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fraction of TCOD after subtracting SCOD. The soluble fractions were obtained after centrifugation of the samples at 10,000 rpm for 10 minutes (Cence TGL-16M, China)

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and these were then passed through a 0.22 μm filter. Methane content and volatile

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fatty acids (VFAs) concentration were measured using different gas chromatographs as described by (Mahdy et al., 2020). Mettler-Toledo pH meter was used to determine

FAN 

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equation:

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pH variation. Free ammonia nitrogen (FAN) was calculated based on the following

10 pH  TAN 6334 exp( )  10 pH K

Where K refers to the process temperature. The relation between process parameters was identified by principal component analysis (PCA) using CANOCO 4.5 software package.

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Journal Pre-proof 3. Results and discussion 3.1. The long term performance of the digesters under different OLRs The process performance including methane yield, biogas production rate, VS removal, pH, TAN, FAN and VFA in the digesters at different HRTs is shown in Fig. 1 and the overall operational conditions of the digesters are listed in Table 2. Higher methane yields of 355±26 and 322±18 mL CH4. g VS-1 were detected when the OLRs

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were 2.5 and 1.25 g TS L-1d-1, respectively, and the yield was maintained at down to

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224±28 mL CH4. g VS-1 when the OLR was 5.0 g TS L-1d-1. The volumetric methane production rate showed opposite tendency. More specifically, the maximum biogas

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production rate of (1.7±0.14 L L-1d-1) was observed at the highest OLR while the

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biogas production rates attained at OLR of 1.25 and 2.5 g TS L-1d-1 were only 1.1±0.1

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and 0.78±0.15 L L-1d-1, respectively. This finding corresponded to the results of previous investigations demonstrating that higher OLR could increase methane

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production rate while fractional conversion of methane yield declined as the scope of

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hydrolysis reduced (Mahdy et al., 2019; Wandera et al., 2019). From methane yield results, it could be confirmed that highest OLR digester was operated at an “inhibited steady-state”, a constant but suboptimal performance, with up to 30%-37% losses in methane yield compared to the performance of the two lower OLR digesters (Fig. 2a). The methane yield attained in the current study in highest OLR digester, however, was higher than that achieved previously with the same substrate at similar OLR but longer HRT (30 day) (Molaey et al., 2018) which may be attributed to the different nutritional quality of chickens. While Belostotskiy et al. (2015) obtained similar methane yield of 250 mL. g VSin-1 in the digesters fed with chicken manure at OLR of 2.8 g L-1d-1 and TAN level of 3750 mg L-1. -7 -

Journal Pre-proof Organic matter removal efficiency is fundamentally dependent on the OLR. In highest OLR digester, the VS removal efficiency was 56%, which was lower by 10% and 12% compared with 40d- and 60d-HRT digesters, respectively (Fig 1b, 2b). These values were consistent with the results of methane yield. Data from previous studies revealed that higher OLR by shortening HRT declines the fractional conversion efficiency of volatile solids to methane according to the extent of hydrolysis (Mahdy et al., 2019 and Wandera 2019). More specifically, the VS removal efficiency was

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52% in digesters treating sewage sludge at OLR of 1.4 g L-1d-1 (20 d HRT) and

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decreased to one-half of what it was, when the OLR increased to 5.7 g L-1d-1 by

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shortening the HRT down to 5 days (Mahdy et al., 2019), signifying the finite

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digestion time. Other factors/inhibitors that have adversely impact of the key

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constituents of microbial structure performing the process, cannot be excluded. 3.2. The accumulation of VFA under higher OLR

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Despite VFAs concentrations were quite low and stable (<650 mg/L) in the two lower

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OLR digesters, VFAs in the 20d-HRT digester was as high as 5674±514 mg L-1. Therefore, it seems likely that, in the 20d-HRT digester, the metabolic capability of methanogens did not meet the activity of acetogens, and thus VFAs were not consumed at the same rate at which they were produced. Consequently, VFAs were accumulated

and

lower

methane

yield

were

produced.

Furthermore,

acetate/propionate ration obviously increased in the two lower digesters compared to 20d-HRT digester where the propionate represented a big proportion of VFAs composition, implying that acetogens were also suppressed at shortest HRT. It can be seen in Fig. 1c that, there were no differences among different tested OLRs in TAN levels in which TAN ranged between 5500 and 6000 mg L-1. That means the three -8 -

Journal Pre-proof anaerobic systems were exhibited to similar effects from TAN, implying that the VFA accumulation in the 20d digester cannot be attributed to TAN concentration since all digesters experienced almost the same TAN concentration but was mostly originated from the high OLR. Similar TAN concentration (5000 mg L-1) was observed formerly in digesters treating chicken manure at a moderate OLR (2.7 gVS L-1d-1) (Molaey et al., 2018; Ziganshin et al., 2016). The association between methane yields and VFAs results signified the key role of OLR to guarantee the balance between VFAs

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producers and consumers (Fig. 2). This fact was further supported by the results

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reported by Mahdy et al., (2019) who observed similar phenomenon when treating

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sewage sludge at short HRT.

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In this study, the pH value was around 8.2 in the 20d-HRT digester despite the sever

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VFAs accumulation which may be ascribed to high ammonia level. Indeed, ammonia is able to buffer VFAs and avoid pH drop (Zhang et al., 2017b). FAN level in 20d-

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HRT digester was significantly higher than that attained in other digesters. FAN

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concentration was around 2157±862 mg L-1, representing 2-fold higher compared to HRT of 40 and 60day (Fig. 2). The tolerance threshold of FAN was reported as high as 1450 mg L-1 when using acclimatized inocula (Callaghan et al., 1998). We, therefore, postulated that VFAs accumulation was a symptom of the of methanogens inhibition, the key constituents of the microbial structure carrying out VFAs conversion to methane, as a result of high FAN content. Nevertheless, the decreasing of the FAN in the first 60 days of the 20d HRT digester was observed. The FAN level in this reactor became almost at the same level with the other two digesters. From Fig. 1b and d, it seems probably that the fluctuation and discrepancy in the VS removal was correlated to the inhibited-steady state of methane yield and both were mediated by accumulation of VFAs and the lower methanogens activity because of mainly -9 -

Journal Pre-proof organics overloading and partially high ammonia level. Indeed, the process parameters during the long-term operation at different HRTs showed obvious variations. One the other hand, the performance of 40d-HRT and 60d-HRT digesters were functionally comparable. The methane yield in both digesters ranged 320-350 mL.gVSin-1, pH values were 7.6-7.8 and they had considerably lower levels of VFAs

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and FAN than 20d-HRT digester. Furthermore, the overall VS removal was similar in

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both digesters, representing 10% over 20d-HRT digester. Thus, considering the advantages of higher methane recovery, high VS removal with absence of both VFAs

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accumulation and ammonium inhibition (Fig. 2), the digestion of nitrogen-rich

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substrates at lower OLR and longer HRT could be more feasible.

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3.3. Substrate degradation kinetics under different OLRs

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The results of methane production kinetics during the first 24h after feeding revealed that methane production rate was clearly faster in 20d-HRT digester as

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shown in Fig. 3. The number of hours required for methane formation varied among the different OLRs. Specifically, 7-hours were needed for the generation of 50% of total methane potential in the 20d-HRT digester, while this time was extended to 24hours in the 40d-HRT and 60d-HRT digesters. In contrary, the methane yield exhibited opposite tendency, representing higher methane yield in later digesters. The measurement of methane yield through 24h after feeding in 20d-HRT, 40d-HRT and 60d-HRT digesters revealed similar levels to that attained in long-term performance. This variation in the methane yield of the three tested OLRs further reflected the positive impact of the extended retention time and the lowering organic loads on methane yield, as demonstrated in several studies investigating different substrates -10 -

Journal Pre-proof such as sludge (Wandera et al., 2019) and food waste (Algapani et al., 2018). This fact may be ascribed to the inhibited methanogens under high ammonia levels which may result in low conversion of VFAs and thus lower methane potential at shorter HRT was achieved. The VFAs results also supported this hypothesis (Fig. 1b), in which VFAs level in 20d-HRT digesters was considerably 9-fold higher than other tested HRTs.

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3.4. COD mass balance at different digesters

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The conversion of chicken manure into SCOD, VFAs, CH4 and particulate COD

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(PCOD), which represents un-degraded material, was compared in the three digesters at different HRTs. The COD mass balance (Fig. 4) showed that PCOD and SCOD

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fractions in the highest OLR digester were significantly higher (by 23% and 12%,

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respectively) relative to other digesters. This evidence indicated lower hydrolysis and acidogenesis efficiencies, which could be attributed to the finite digestion time. In

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addition, the fraction of COD-VFA constituted 6% of the total organics at shorter

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HRT while it was negligible in the two lower OLR digesters. This fact implied that the methanogenic substrates was available but methanogens were somehow inhibited to convert them to methane, resulting in lower methane yield in 20d-HRT digester. Similar observation was reported in previous study in which methane yield was dropped along with VFAs accumulation as a result of ammonia inhibition (Bi et al., 2019). For the 40d- and 60d-HRT digesters, the values of PCOD, SCOD and VFAs were comparable, supporting the results attained in Fig. 1. However, the COD-CH4 attained in both digesters slightly varied. Taking into consideration the results of methane yield attained in section 3.1 and the similar results of other COD fractions,

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Journal Pre-proof this variation may be attributed to the imprecision of the measurement of total organics in 40d-HRT digester as reported previously (Algapani et al., 2019). 3.5. Correlation between system parameters To identify the relationship between operating parameters and different OLRs, Principal Component Analysis (PCA) was performed as shown in Fig. 5. Different OLRs were mainly distinguished in two areas, one of which was linked to pH,

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ammonia and VFAs and the other one was associated with VS removal and methane

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yield. The results revealed that OLR considerably affected the process performance

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(VS removal and methane yield) and major intermediates (FAN and VFAs). More specifically, VFAs, pH and FAN were positively correlated and were clustered in the

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same side with 20d-HRT digester, implying a strong impact of these variable on the

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process performance at shorter HRT. In contrast, VS removal and methane yield were positioned on the opposite side and clustered at the same area with lower OLRs,

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signifying negative relation with former parameter (VFAs and FAN) and the strong

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positive correlation of methane yield and lower OLR. Under this scenario, it can be assumed that higher OLR (and not TAN level) resulted in process inhibition, which revealed an unbalanced equilibrium among VFAs producers and VFAs consumers and thus, VFAs accumulated which in turn could serve as process inhibitor as well. This evidence was consistent with the same tendency found in Fig. 1 and Table 2. Furthermore, as shown in Fig.5, the discrepancy between the direction of 20d-HRT digester and methane yield or VS removal, is most likely related to ammonia inhibition on methanogens (Lv et al., 2014), which could explain the inhibited-steady state detected at short HRT. These results evidenced that the important and the effective parameter in this study was OLR, which directly determines the presence or -12 -

Journal Pre-proof absence of VFAs accumulation and ammonia inhibition symptoms and consequently, reflecting on methane yield and organics depletion. Furthermore, the PCA results showed also that HRT 40d- and 60d-HRT digesters cluster closely, suggesting similar process performance in these digesters.

3.6. Response of microbial community to different OLRs

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To demonstrate the influence of OLR and consequently the effect of VFAs levels

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on the relative abundance of the microbial community, the sequencings of bacterial

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and archaea genes were performed in the digester with “inhibited-steady state’’ (20d-

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HRT digester) and in the digester with satisfied performance (40d-HRT digester). The results depicted in Fig. 6 clearly show significant changes in both bacterial and

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archaeal profiles. In both digesters, the phylum Firmicutes dominated with a relative

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abundance of (48%) in 20d-HRT digester and (63%) in 40d-HRT digester. In addition, other phyla were also abundant including Bacteroidetes (33% and 21%) and

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Cloacimonetes (9% and 10.5%) in 20d-HRT and 40d-HRT digesters, respectively. The decreased OLR appeared to gather more Firmicutes in the process, implying that Firmicutes members, which responsible for VFAs generation, played a pivotal role in the enhancement of methane yield in the 40d-HRT digesters. In fact, both Firmicutes and Bacteroidetes, which mediate the hydrolysis and acidification stages in AD process, were previously found in digesters with high TAN levels (Yan et al., 2019), indicating the robustness of these phyla to high ammonia concentration. At genus level as depicted in Fig. 6a, the most noteworthy observation was that the relative abundance of the facultative anaerobic bacteria (Fastidiosipila), which are able to ferment carbohydrate and protein and produce acetate and butyrate (Falsen et al., -13 -

Journal Pre-proof 2005), was significantly increased by 6-fold along with shifting HRT from 20 to 40 days. Likewise, the relative abundance of syntrophic bacteria Proteiniphilum was increased by 7.5-fold. Proteiniphilum can convert various organic matters to acetic acid and CO2 (Chen et al., 2005). Therefore, their relative abundances enhancement might help to form VFAs, increasing methane yield in 40d-HRT digester. Furthermore, the enhanced abundances of these genera implied that both Fastidiosipila and Proteiniphilum were vulnerable to high FAN level. Given the

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highly flexible metabolism of these genus, it seems probably that the suboptimal VS

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removal efficiency attained in 20d-HRT digester may be ascribed to the low relative

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abundance of such bacteria. The obligate anaerobic bacteria Syntrophomonas

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abundance increased from less than detectable level in 20d-HRT digester to 2.7% in 40d-HRT digester. This genus could oxidize acetate and thus serve as partners in a

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syntrophic association with a hydrogenotrophic methanogenic bacterium (Sousa et al.,

conversion

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2007). Therefore, it was expected that the production of methane through two-stage (syntrophic

acetate

oxidation

coupled

with

hydrogenotrophic

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methanogenesis) will be promoted in 40d-HRT digester. It seems likely that the enhancement of methane production and VS removal might be attributed to the increased abundance of aforementioned genera, that are involved in the production of the precursors for methanogens, as a response of bacterial community to the decreased OLR of ammonia-stressed system. Unlike, the relative abundance of Gallicola decreased from 9.4 % in 20d-HRT digester to 3.4% in 40d-HRT digester, signifying the robustness of such genus to high TAN and FAN level. Gallicola is capable of converting purines and uric acid to acetate, CO2 and NH3 (Ezaki et al., 2001). The presence of Gallicola at high relative abundance in 20d-HRT digester might, therefore, help to enhance the buffering ability and prevent pH drop (Fig. 1). -14 -

Journal Pre-proof These variations suggested that proper HRT and OLR enhanced the adaptive ammonia tolerant bacteria to work more efficiently and providing more methanogenic substrates (acetate and hydrogen) for methanogenesis and thus higher methane yield and VS removal were achieved in 40d-HRT digester. As shown in Fig. 6b, a total of 5 methanogens were identified in 20d-HRT digester, in which only Methanosarcina was the prevalent one, representing 81% of the total

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archaea. The dominance of Methanosarcina in 20d-HRT reactor could be attributed to

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several reasons. Firstly, due to their short doubling time and high growth rate. Methanosarcina needs less than 36 h to replicate themselves (Demirel and Scherer

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2008). Secondly, the slightly accumulation of VFAs attained in 20d-HRT digester

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may provide the metabolic requirement of the higher growth rate of Methanosarcina

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regarding acetate concentration (above 1 mM) (Westerholm et al., 2016). The third reason explaining the high abundance of Methanosarcina in 20d-HRT digester is that

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they are capable of tolerant high ammonia levels. In fact, this finding was in

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agreement with previous studies, where Methanosarcina was the prevalent methanogens at extreme ammonia concentration (Taubner et al., 2015). Recently, a study dedicated to acclimatize the methanogenic consortium to extreme ammonia level found that Methanosarcina was responsible for high methanization efficiency at extreme ammonia level (Yan et al., 2019). At longer HRT, a new steady-state of methanogenic community structure was established. More specifically, Methanobacterium became the dominant methanogens in 40d-HRT digester, accounting for 37% of all reads, followed by Methanoculleus (14.5%) and Methanocorpusculum (9%). All of them are hydrogenotrophic methanogens and thus the methane was supposed to be produced through CO2 -15 -

Journal Pre-proof reduction using H2 as electron donor in 40d-HRT digester, supporting that the increased methane yield was partially due to stabilization of hydrogenotrophic methanogens. The increase of hydrogenotrophic methanogens (Methanobacterium, Methanoculleus and Methanocorpusculum) was in tangent with the enrichment of syntrophic bacteria (Proteiniphilum and Syntrophomonas) in 40d-HRT digester. Similarly, Ziganshin et al., (2016) have reported the dominancy of hydrogenotrophic methanogens in digester fed with chicken and cattle manure and the TAN level was

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close to 6000 mg L-1. Noteworthy to mention that, the doubling time of certain

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hydrogenotrophic methanogens i.e. Methanoculleus could exceed 20 days under high

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ammonia level (Westerholm et al., 2019). In addition, the relatively slow growth of

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their partners (syntrophic acetate oxidizers, 28day) (De Vrieze et al., 2012) indicating that the retention time of 40 days might help to avoid the washout effect of such

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genera. Furthermore, the absence of ammonium transportation system into some

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hydrogenotrophs cells i.e., Methanoculleus may support them to be adapted in high TAN levels (Molaey et al., 2018) and not in high FAN level. It can be concluded that

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however, hydrogenotrophic methanogens are widely reported to be more active when experiencing ammonia inhibition (Moestedt et al., 2016), the adequate HRT should be provided to guarantee process stability under such condition. Therefore, the dissimilarities between microbial profiles in 20d-HRT and 40d-HRT despite the stability of operating condition except OLR evidenced that OLR had an important influence on the shaping of microbial composition and thus the process performance changed accordingly as depicted in Fig. 1. This conclusion was further supported by the major depletion of accumulated VFAs despite the same TAN levels in both digesters.

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Journal Pre-proof Overall, the AD of chicken manure has been frequently applied at HRT of 20 days, however, the methane yield and VS removal were considerably lower compared to a slight lower OLR. This unsatisfactory results in terms of methane recovery and organic matter stabilization were most probably as a result of ammonia inhibition in finite digestion time that did not provide the enough time for the proliferation of ammonia-tolerant methanogens Therefore, applying AD of nitrogen rich substrates at relative lower OLR by extending HRT would be preferred to alleviate ammonia

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effects and thus more methane recovery and higher organic matters removal will be

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achieved.

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Conclusion

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The AD process can proceed well under ammonia-stress condition (around 6000

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mg/L) when the suitable OLR (OLR of 2.5 g TSL-1d-1) and HRT (40d) were set. The significant VFA accumulation and the low methane production were mainly induced

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by the higher OLR instead of the high TAN level. The enhancement of SAO members

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and the shift of the dominant methanogens from Methanosarcina to hydrogenotrophic methanogens (Methanobacterium, Methanoculleus) indicated the most tolerant microorganism do not always support the high process performance. However, further studies are crucial to economically judge the feasible of process capacity reduction as a result of lowering OLR. Acknowledgements This work was partially supported by the National Key Research and Development Program of China (2016YFD0501403), the Beijing Natural Science Foundation (6182017) and the State Administration of Foreign Experts Affairs P.R.China. (Project No. WQ20180011). -17 -

Journal Pre-proof References 1. Algapani, D.E., Qiao, W., Pumpo, F., Bianchi, D., Wandera, S.M., Adani, F., Dong, R., 2018. Long-term bio-H2 and bio-CH4 production from food waste in a continuous two-stage system: Energy efficiency and conversion pathways. Bioresour. Technol. 248, 204-213. 2. Algapani, D.E., Qiao, W., Ricci, M., Bianchi, D., Wandera, S.M., Adani, F., Dong, R., 2019. Bio-hydrogen and bio-methane production from food waste in a two-stage anaerobic digestion process with digestate recirculation. Renew. Energ. 130, 1108-1115.

of

3. APHA, 2005. Standard Methods for the Examination of Environmental Reviews, 18(f Water and Wastewater). American Public Health Association, Washington DC.

-p

ro

4. Belostotskiy, D.E., Ziganshina, E.E., Siniagina, M., Boulygina, E.A., Miluykov, V.A., Ziganshin, A.M., 2015. Impact of the substrate loading regime and phosphoric acid supplementation on performance of biogas reactors and microbial community dynamics during anaerobic digestion of chicken wastes, Bioresour. Technol. 193, 42-52.

lP

re

5. Bi, S., Qiao, W., Xiong, L., Ricci, M., Adani, F., Dong, R., 2019. Effects of organic loading rate on anaerobic digestion of chicken manure under mesophilic and thermophilic conditions. Renew. Energ. 139, 242-250.

na

6. Busato, C. J., Ros, C.D., Rellay, R., Barbierato, P., Pavan, P., 2020. Anaerobic membrane reactor: Biomethane from chicken manure and high-quality effluent. Renew. Energy 145, 1647-1657.

Jo ur

7. Callaghan, F.J., Wase, D.A.J., Thayanithy, K., Forster, C.F., 1998. An examination of the continuous anaerobic co-digestion of cattle slurry and fish offal. Process Saf. Environ. 76(3),224-228. 8. Chen, J.L., Ortiz, R., Steele, T.W.J., Stuchey, D.C., 2014. Toxicants inhibiting anaerobic digestion: A review. Biotechnol. Adv. 32, 1523-1534. 9. Chen, S.Y., Dong, X.Z., 2005. Proteiniphilum acetatigenes gen. nov., sp nov., from a UASB reactor treating brewery wastewater. Int. J. Syst. Evol. Microbiol. 55, 2257-2261. 10. Chen, Y., Cheng, J.J., Creamer, K.S., 2008. Inhibition of anaerobic digestion process: a review. Bioresour. Technol. 99, 4044-4064. 11. De Vrieze, J., Hennebel, T., Boon, N., Werstraete, W., 2012. Methanosarcina: The rediscovered methanogen for heavy duty biomethanation. Bioresour. Technol. 112, 1-9. 12. Demirel, B., Scherer, P., 2008. The roles of acetotrophic and hydrogenotrophic methanogens during anaerobic conversion of biomass to methane: a review. Rev. Environ. Sci. Biotechnol. 7,173-190. 13. Ezaki, T., Kawamura, Y., Li, N., Li., Z.Y., Zhao, L., Shu, S., 2001. Proposal of the genera Anaerococcus gen. nov., Peptoniphilus gen. nov. and Gallicola gen. -18 -

Journal Pre-proof nov. for members of the genus Peptostreptococcus. Int. J. Syst. Evol. Microbiol. 51, 1521-1528. 14. Falsen, E., Collins, M.D., Welinder-Olsson, C., Song, Y., Finegold, S.M., Lawson, P.A., 2005. Fastidiosipila sanguinis gen. nov., sp. nov., a new Grampositive, coccus shaped organism from human blood. Int. J. Syst. Evol. Microbiol. 55, 853-858. 15. Fotidis, I.A., Wang, H., Fiedel, N.R., Luo, G., Karakashev, D.B., Angelidaki, I., 2014. Bioaugmentation as a solution to increase methane production from an ammonia-rich substrate. Environ. Sci. Technol. 48, 7669-7676.

of

16. Lv, Z., Hu, M., Harms, H., Richnow, H.H., Liebetrau, J., Nikolausz, M., 2014. Stable isotope composition of biogas allows early warning of complete process failure as a result of ammonia inhibition in anaerobic digesters. Bioresour. Technol. 167, 251-259.

-p

ro

17. Mahdy, A., Wandera, S.M., Akl, B., Qiao, W., Dong, R., 2020. Biostimulation of sewage sludge solubilization and methanization by hyper-thermophilic prehydrolysis stage and the shifts of microbial structure profiles. Sci. Total Environ. 699, 134373.

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re

18. Mahdy, A., Wandera, S.M., Bi, S., Song, Y., Qiao, W., Dong, R., 2019. Response of the microbial community to the methanogenic performance of biologically hydrolyzed sewage sludge with variable hydraulic retention times. Bioresour. Technol. 288,121581.

na

19. Moestedt, J., Muller, B., Westerholm, M., Schnurer, A., 2016. Ammonia threshold for inhibition of anaerobic digestion of thin stillage and the importance of organic loading rate. Microb. Biotechnol. 9 (2), 180-194.

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20. Molaey, R., Bayrakdar, A., Surmeli, R.O., Calli, B., 2018. Influence of trace element supplementation on anaerobic digestion of chicken manure: Linking process stability to methanogenic population dynamics. J. Clean. Prod. 181, 794-800. 21. Song, L., Li, D., Cao, X., Tang, Y., Liu, R., Niu, Q., Li, Y-Y., 2019. Optimizing biomethane production of mesophilic chicken manure and sheep manure digestion: Mono-digestion and co-digestion kinetic investigation, autoflourescence analysis and microbial community assessment. J. Environ. Manage. 237, 103-113. 22. Sousa, D.Z., Smidt, H., Alves, M.M., Stams, A.J.M., 2007. Syntrophomonas zehnderi sp. nov., an anaerobe that degrades long-chain fatty acids in coculture with Methanobacterium formicicum. Int. J. Syst. Evol. Microbiol. 57,609-615. 23. Taubner, R.-S., Schleper, C., Firneis, M.G., Rittmann, S.K.M.R., 2015. Assessing the Ecophysiology of Methanogens in the Context of Recent Astrobiological and Planetological Studies. Life (Basel, Switzerland) 5, 16521686.

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Journal Pre-proof 24. Wandera, S.M., Westerholm, M., Qiao, W., Yin, D., Jiang, M., Dong, R., 2019. The correlation of methanogenic communities’ dynamics and process performance of anaerobic digestion of thermal hydrolyzed sludge at short hydraulic retention times. Bioresour. Technol. 272, 180-187. 25. Wang, H., Zhang, Y.F., Angelidaki, I., 2016. Ammonia inhibition on hydrogen enriched anaerobic digestion of manure under mesophilic and thermophilic conditions. Water Res. 105, 314-319. 26. Westerholm, M., Dolfing, J., Schnürer, A., 2019. Growth characteristics and thermodynamics of syntrophic acetate Oxidizers. Environ. Sci. Technol. 53, 5512-5520.

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27. Westerholm, M., Mosestedt, J., Schnurer, A., 2016. Biogas production through syntrophic acetate oxidation and deliberate operating strategies for improved digester performance. Appl. Energy 179, 124-135.

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28. Wiegant, W.M., Zeeman, G., 1986. The mechanism of ammonia inhibition in the thermophilic digestion of livestock wastes. Agric. Wastes 16, 243-253.

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-p

29. Yan, M., Fotidis, I.A., Tian, H., Khoshnevisan, B., Treu, L., Tsapekos, P., Angelidaki, I., 2019. Acclimatization contributes to stable anaerobic digestion of organic fraction of manucipal solid waste under extreme ammonia levels: Focusing on microbial community dynamics. Bioresour. Technol.286, 121376.

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30. Yang, Z., Wang, W., Liu, C., Zhang, C., Liu, G., 2019. Mitigation of ammonia inhibition through bioaugmentation with different microorganisms during anaerobic digestion: Selection of strains and reactor performance evaluation. Water Res. 155, 214-224.

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31. Yenigun, O., Demirel, B., 2013. Ammonia inhibition in anaerobic digestion: A review. Process Biochem. 48 (5-6), 901-911.

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32. Zhang, J., Loh, K-C., Lee, J., Wang, C-H., Dai, Y., Tong, Y.W., 2017b. Three-stage anaerobic co-digestion of food waste and horse manure. Sci. Rep. 7,1269. 33. Zhang, W., Lang, Q., Pan, Z., Jiang, Y., Liebetrau, J., Nelles, M., Dong, H., Dong, R., 2017a. Performance evaluation of a novel anaerobic digestion operation process for treating high-solids content chicken manure: Effect of reduction of the hydraulic retention time at a constant organic loading rate. Waste Mange. 64, 340-347. 34. Ziganshin, A.M., Ziganshina, E.E., Kleinsteuber, S., Nikolausz, M., 2016. Comparative analysis of methanogenic communities in different laboratoryscale anaerobic digesters. Archaea https://doi.org/10.1155/2016/3401272.

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Journal Pre-proof Table 1 The chemical characteristics of substrate and inoculum

Parameters

Unit

Chicken manure

Inoculums

Mean±SD

Mean±SD

TS

%

103±2

46±3

VS

%

71±2

34±2

pH

/

7.4±0.2

7.9±0.3

TCOD

g·L-1

99±5

/

SCOD

-1

5±1

VFAs

mg·L

/

268±12

NH4+-N

mg·L-1

/

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40±2

-1

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g·L

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4680±22

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Table 2 Average operation performance of the mesophilic CSTR under different HRTs Parameters

Unit

HRT=20d

HRT=40d

HRT=60d

OLR

g-TS L-1·d-1

5

2.5

1.25

TS effluent

%

5.3±0.6

4.9±0.4

4.7±0.2

VS effluent

%

3.3±0.8

2.4±0.1

2.2±0.2

SCOD effluent

g·L-1

20±9.8

5±1.2

4±0.6

Gas production

L·L·d-1

1.7±0.2

1.1±0.1

0.8±0.2

Methane yield

mL·g-VSin-1

223.8±28

354.6±26

321.6±18

CH4

%

60±4

63±2

64±5

CO2

%

40±4

37±2

36±5

8.2±0.4

7.8±0.2

7.8±0.1

pH

o J

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/

l a

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f o

NH4+-N

g·L-1

5596±813

5634±242

5933±150

TVFA

mg·L-1

5709±513

635±180

324±81

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Fig. 1. The process performance in the long-term operation (Methane yield, TAN, FAN, pH, VFA, VS removal and biogas production) at different HRTs.

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Fig. 2. Variation of different parameters in the digesters operated at different HRTs.

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Fig. 3. Biogas production (a) and methane yield (b) for 24 hours after feeding the digesters at different HRTs.

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Fig. 4. Mass balance attained at different HRTs.

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Fig. 5. Principal component analysis (PCA) to identify the relationship between process parameters (including, methane yield, VS removal, FAN, TAN, pH and VFAs) at HRT of 20 (red), 40 (blue) and 60 days (yellow).

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Fig. 6. Bacteria (a) and archaea (b) community at genus level based on 16S rRNA gene sequencing in 20d- and 40d-HRT digesters.

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Journal Pre-proof Declaration of interests

☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

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☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

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Journal Pre-proof Highlights 

The ammonia inhibition effect was investigated under different OLRs.



The inhibited–steady state was induced by high OLR regardless TAN level.

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Microbial community was characterized by 16S rDNA cloning library.



Methanosarcina was more tolerant to ammonia but did not support high

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performance.

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Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6