N ratio feedstock with methanogenic enrichment culture

Accepted Manuscript Bioaugmentation strategy for enhancing anaerobic digestion of high C/N ratio feedstock with methanogenic enrichment culture Ying Li, Lianhua Li, Yongming Sun, Zhenhong Yuan PII: DOI: Reference:

S0960-8524(18)30262-1 https://doi.org/10.1016/j.biortech.2018.02.069 BITE 19578

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Bioresource Technology

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9 January 2018 10 February 2018 14 February 2018

Please cite this article as: Li, Y., Li, L., Sun, Y., Yuan, Z., Bioaugmentation strategy for enhancing anaerobic digestion of high C/N ratio feedstock with methanogenic enrichment culture, Bioresource Technology (2018), doi: https://doi.org/10.1016/j.biortech.2018.02.069

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Bioaugmentation strategy for enhancing anaerobic digestion of high C/N ratio feedstock with methanogenic enrichment culture Ying Li, Lianhua Li, Yongming Sun∗ Zhenhong Yuan a

b

c

Laboratory of Biomass Bio-chemical Conversion, GuangZhou Institute of Energy Conversion, Chinese Academy of Sciences, Guangzhou 510640, PR China

Key Laboratory of Renewable Energy, Chinese Academy of Sciences, Guangzhou 510640, PR China

Guangdong Provincial Key Laboratory of New and Renewable Energy Research and Development, Guangzhou 510640, PR China

Abstract: To investigate whether bioaugmentation could improve the digestion performance of high C/N ratio feedstock without co-digestion with nitrogen-rich substrate, different forms of enriched methanogenic culture were introduced to the continuous feed digesters. The performance efficiency of bioaugmentation on digestion improvement was compared. The effect of bioaugmentation on microbial community composition was revealed as well. Results demonstrated that routine bioaugmentation with liquid culture (containing the microbes and the medium remains) showed the best performance, with the organic loading rate (OLR), methane percentage, volumetric methane production (VMP) and volatile solid methane production (VSMP) higher at 1.0 g L-1d-1, 24%, 0.22 L L-1 d-1 and 0.23 Lg-1VSd-1 respectively, compared to the non-bioaugmentation control. Whole genome pyrosequencing analysis suggested that consecutive microbial consortium addition could reconstruct the methanogens community by increasing the populations of acetoclastic methanogens Methanothrix, which could accelerate the degradation of acetate and methane production.

Keywords: Bioaugmentation, high C/N ratio, methanogenic propionate-degrading culture, microbial community 1. Introduction Anaerobic digestion (AD) is a promising technology that combines with the organic waste treatment and renewable energy production. Many kinds of organic waste have been used as feedstock for AD, such as manure, agricultural crops, food waste, sewage sludge, wastewater and microalgae. C/N ratio of the substrates is a crucial factor in the production of biogas because an appropriate nutrient balance is required by anaerobic microorganism

∗

Corresponding author at: No. 2 Nengyuan Rd, Tianhe District, Guangzhou 510640, PR China. E-mail: [email protected]

for their growth as well as for maintaining a stable environment (Zhang et al., 2014).Generally, a C/N ratio of 20–30 is considered optimal for anaerobic bacterial growth in an AD system (Hassan et al., 2017; Jain et al., 2015; Xu et al., 2018). Inappropriate C/N ratios could result in high volatile fatty acids (VFAs) accumulation and/or high ammonia released in the digester, both of which are potential inhibitors in the AD process (Jain et al., 2015).For the carbon-rich or nitrogen-rich substrates, it is difficult to keep an optimal C/N ratio in mono-digestion. Thus, anaerobic co-digestion with high C/N ratio and low C/N ratio substrates has been widely studied to increase the biogas yield, such as food waste with cattle slurry, farm residue with animal slurry, pig slurry with agricultural biomass, energy crops with wastes, etc (Tiwary et al., 2015). Although co-digestion offers several advantages involved regulation of nutrient balance, improvement of buffer capacity, alleviation of inhibitory effects caused by high ammonia and/or VFA concentration during the anaerobic digestion process (Mata-Alvarez et al., 2014), improper selection of co-substrates, co-substrate composition and operating conditions can lead to process instability and reduction of methane production (Xie et al., 2016). Moreover, the feedstock for co-digestion is limited by seasons or regions to some degree, which is one of the challenges for the continuous operation of a scaled AD. Therefore, it is necessary to seek an alternative solution for the stable mono-digestions in case of the shortages of the co-substrates. Bioaugmentation, a practice of adding specific microorganisms to a system, is considered as a method to enhance the performance of AD. As the previous studies reported, bioaugmentation has successfully reduced the start-up period (Lins et al., 2014), shortened hydraulic retention time (Baek et al., 2016), decreased the recovery time of anaerobic digesters stressed by oxygen (Bhattad et al., 2017; Schauer-Gimenez et al., 2010) or organic overloading (Tale et al., 2011; Tale et al., 2015),increased the methane production from cellulosic waste(Cater et al., 2015; Ecem Oner et al., 2017; Martin-Ryals et al., 2015; Nkemka et al., 2015), microalgae(Lu et al., 2013), digested sludge (mainly proteins and polysaccharides) (Lu et al., 2014), and ammonia-rich substrate (Fotidis et al., 2013; Fotidis et al., 2014). Furthermore, bioaugmentation also has been studied to accelerate VFA degradation (Town & Dumonceaux, 2016) and improve the stability of AD (Acharya et al., 2015; Li et al., 2017b). However, implementation of bioaugmentation to enhance anaerobic digestion of high C/N ratio feedstock has

been less well addressed. Further studies are needed in this area since VFAs accumulation inhibit methane production in the anaerobic digesters suffered by C/N ratio imbalance. Moreover, the addition form of bioaugmentation culture is poorly explored. It is also important to consider the nutrient, in particular trace elements, in the bioaugmentation liquid culture, which may have a significant effect on the microbial diversity and abundance within the digester. In an attempt to investigate whether bioaugmentation could improve the digestion performance of high C/N ratio feedstock without a supply of nitrogen-rich co-substrate or pH adjustment, methanogenic culture acclimated in our previous work (Li et al., 2017a) was chosen as bioaugmented inoculum to accelerate degradation of propionate and acetate, as well as enhance the density of methanogens. The bioaugmentation performance with different forms of the enrichment (liquid /solid) was compared to affirm the best strategy for bioaugmentation. A deeper understanding of correlation relationship between the adding culture and endogenous microbes from the digester was provided based on the microbial community analysis.

2. Materials and methods 2.1 Experimental set-up The inoculum was taken from an anaerobic digester treating food waste (Guangzhou Institute of Energy Conversion, Chinese Academy of Sciences, China). Before use it was sieved through a 1 mm mesh to remove grit and other solids. The experiment was carried out in the mesophilic semi-continuous CSTR-type reactors with a working volume of 2 L/ 1 L, which was initially 100% inoculated with 2 L sieved inoculum, with headspace flushed with a N2:CO2 gas mixture (mixed in 80:20 ratio v/v). An identical hydraulic retention time (HRT) of 20 days was maintained by removing appropriate volume of reactor content and replacing it with the same volume of feed once per day. To simulate a kind of high C/N ratio substrate that is prone to VFAs accumulation, the artificial feedstock was made up with rice flour, whole egg powder and milk powder. The dry weight ratio of rice flour, whole egg powder milk powder was 3:1:1. The basic characteristics of artificial feedstock as follows: total solid (TS): 93.85%, volatile solid (VS): 92.42%, C%: 46.15, N%: 1.16, H%: 6.8, S%: 0.03, C/N: 39.78. The volume of the fresh feed was made

up by H2O. The basic characteristics of inoculum as follows: TS: 1.87%, VS: 1.11%. 2.2 Bioaugmentation culture The bioaugmentation culture was taken from a propionate-degrading bioreactor (Li et al., 2017a). Sodium propionate was the sole carbon substrate .The volume of the feed was made up by nutrient medium. The nutrient medium contained the following [mg L-1]: NH4Cl [400]; MgSO4·6H2O [250]; KCl [400]; CaCl2·2H2O [120]; (NH4)2 HPO4 [80]; FeCl3·6H2O [55]; and the trace element salts (CoCl2·6H2O, NiCl2·6H2O, MnCl2·4H2O, CuCl2 ·2H2 O, AlCl3· 6H2 O, ZnCl2, Na2 WO4 ·2H2 O, H3 BO3, Na2SeO3 and Ma2MoO4 ·2H2O) [each at 0.5] According to whole genome pyrosequencing analysis, the main methanogens (relative abundance above 1%) in the bioaugmentation culture added between day 121 and 200 were Methanothrix (53~70%), Methanoculleus (10~25%), Methanobacterium (1~2%), Methanospirillum (0.1~1.1%). Other 5 genera, Methanosarcina, Methanosphaerula, Methanomassiliicoccus, Methanosphaera and Methanoregula, were with a low abundance (below 1%). Syntrophobacter (40~63%) was the dominant bacterial genus. The different forms of bioaugmentation culture were prepared as follows: 1）Whole active culture：The bioaugmentation seed (10mL) was taken from the bioreactor by sterile syringe and added into reactor RL+S immediately. Whole active culture contains the enriched solid microbes and the liquid remains of the culture medium. 2）Sterile liquid culture：Taking the bioaugmentation seed from the bioreactor by sterile syringe, then which was autoclaving for 20mins under 121℃. Sterile liquid culture contains the dead cell biomass and the remains of the liquid culture medium. 3）Solid culture：The enriched liquid culture was centrifuged at 7000 g for 5mins at room temperature and was resuspended with ddH2O, then centrifuged again under the same conditions to collect the microbial precipitate for bioaugmentation. Solid culture mainly contained the enriched microbial biomass. 2.3 Experimental procedure The whole experiment lasted for 200 days with three different experimental phases: phase I (0–110 day), phase

II (111-120 day), and phase III (121–200 day). The main strategic operational conditions of each experimental reactor are shown in Table 1. During the first phase (0–110 d), the experiment was carried out in a laboratory-scale semi-continuously reactor (R0) with a working volume of 2 L. The OLR was 0.5 g VS L-1d-1 by adding appropriate amount of the artificial food waste. For phase II (111–120 d), to make the reactors for bioaugmentation comparison operating from the same staring point, the digestate in R0 was divided into four parts homogeneously on day 111 and maintained in four 1 L reactors named RC (control), RL+S (liquid and solid), RL (liquid), and RS (solid) .The initial volume of digestate in each reactor was 0.5 L. To make the working volume up to 1L, four reactors was running under OLR of 0.5 g VS L-1 d-1 with daily feed but without discharge (day 111-120).The reactors were restarted to operate in daily fill-and-draw mode until the volume of the digestate were supplemented to 1 L. In phase III (121–200 d), RL+S was bioaugmented by active liquid methanogenic culture (10 mL /d); RL was added sterile culture (10 mL /d) to rule out the impact of microbial organisms; RS was daily introduced solid biomass collected from 10 mL liquid culture to avoid the effect of remains in the culture medium; RC was the control digester without bioaugmentation. In this phase, OLR was step-wise increased from 0.75 g VS L-1 d-1 to 2.0 g VS L-1 d-1. 2.4 Analytical methods for digestion performance Methane (CH4) and carbon dioxide (CO2) contents were analyzed by GC-2014 gas chromatograph (Shimadzu, Japan) which equipped with TCD detector (120°C) Porapak Q column（70 °C）and argon as carried gas (20 mL⋅min-1). pH was determined using a FE28-Standard meter (Mettler -Toledo, Switzerland) with glass electrode calibrated in buffers at pH 7.0 and 4.0. Alkalinity was measured by a Titroline 5000 titrator (Julabo,German) with 0.25 N H2SO4 to endpoints of pH 5.7 and 4.3, allowing calculation of total alkalinity (TA), partial alkalinity (PA) and intermediate alkalinity (IA). VFAs were quantified by HPLC system (Waters e2698, USA), equipped with Bio-RAD column at 50 °C and 0.5 mM H2SO4 as the mobile phase at the flow rate of 0.5 mL/min. 2.5 Metagenomic DNA extraction and amplification

DNA extraction was performed using the fast DNA spin kit for soil (QBIOgene Inc., Carlsbad, CA, USA), according to the manufacturer’s instructions. DNA quality was assessed using gel electrophoresis (1% agarose) and DNA concentrations were determined using a Qubit Fluorometer (Thermo, USA). The extracted DNA sample was then handled according to the protocol of genomic DNA sample preparation kit (Illumina). The DNA fragmentation was firstly performed using Covaris S2 Ultrasonicator, and the DNA fragments were then processed by end reparation, A-tailing, adapter ligation, DNA size-selection. PCR reaction and products purification were based on Illumina Miseq2x300 instructions. For Archaea, the 16S rRNA genes were amplified through three rounds of PCR. The primers for the first round were 340Fand 1000R. Then the PCR products were used as templates for a second PCR with 349F and 806R, and the third round PCR amplified with Illumina nested primers. 2.6 Whole genome pyrosequencing analysis Before sequencing, PCR products of different samples were normalized in equimolar amounts in the final mixture, which was used to construct the PCR amplicon libraries. Sequencing was carried out on an Illumina HiSeq 2000. The obtained sequences were phylogenetically allocated down to the phylum, class, and genus level with the RDP classifier(http://rdp.cme.msu.edu/misc/resources).To define the relative abundance of a given phylogenetic group, the number of sequences affiliated to that group was divided by the total number of obtained sequences. The results were used for the analysis and comparison of microbial community structure differences.

3. Results and discussion 3.1 Independent digestion performance of high C/N ratio feedstock Fig. 1 shows the digestion performance of R0 before bioaugmentation in terms of VMP, methane percentage, pH, alkalinity and VFA concentration. OLR was kept at 0.5 g L-1d-1 druing the first 110 days. It is clear that it took nearly three retention times to start up. The VMP was below 0.1 L L-1d-1 during first 50 days. From then on a fluctuated increase of VMP was observed from 0.05 L L-1 d-1 to 0.19 L L-1d-1. At the later stage (85-110 d), VMP was stable at around 0.14 L L-1d-1, and methane percentage was between 46% and 63%.

Acetic acid started to accumulate at the early stage, the concentration of which was up to approximately 2000 mgL-1after one retention time, resulting in a poor methane production. Propionic acid was gradually becoming the main accumulated VFAs since the third HRT, with the concentration fluctuating between 1200 mgL-1 and 1800 mgL-1.Except the accumulation of acetic acid and propionic acid, butyric acid accumulated temporarily during the third HRT. Accumulation of VFAs in anaerobic digestion systems are an indicator of substrate overload and/or stress caused by changes in environmental conditions such as pH, temperature, and carbon source (Gerardi, 2003). This increase in the concentration of VFAs indicated either an increase in the activity of the VFA-forming bacteria or a decrease in the activity of VFA-consuming bacteria and/or archaea (Vasquez & Nakasaki, 2016). Total VFA concentration was increasing first and then decreased slightly, leading to methane production was inhibited at the early period and then increasing at later stage. The pH decreased from 7.7 to 6.5 after 45 days running and subsequently stable around 6.7.This might be due to the accumulated VFAs. IA: PA was increasing first and then fluctuated between 0.44 and 0.51. The above results suggest that the anaerobic digestion of high C/N ratio feedstock was not performing well. The reasons could be explained in the follow aspects: 1) The accumulation of intermediate products, especially propionate and acetate, reduced the methane production from the substrate. Studies also indicated that single stage mono-digestion of easily degradable feedstock is unstable due to the VFA accumulation (Guo et al., 2014); 2) The dropping pH, mainly caused by the accumulation of VFAs, negatively affected the growth of methanogens, which led to the lower methane production (Chen et al., 2008); 3) As previous studies suggest that the inoculum which was selected influenced the methane yield (Han et al., 2016; Wilkins et al., 2015). Thus, the inoculum used in this study might be not with high methanogenic abundance and activity. 3.2 Digesters performance with each form of bioaugmentation To evaluate the best strategy of bioaugmentation, the digestate from R0 was inoculated into four digesters on day 111 and then added different form of bioaugmented culture on day 121.The digestion performance of each reactor is shown in Fig.2. It is clear that the performance of RC running without any introduced microbes was worst compared to other three reactors.

RL+S, adding the whole active methanogenic culture (10 mL /d), showed the best performance among the four digesters in terms of VMP, methane percentage, pH, alkalinity and VFA concentration (Fig.2, Fig.3 and Table 2). The OLR for stable running could increase from 0.5 g L-1 d-1 to 1.5 g L-1d-1, correspondingly the VMP rose from 0.11 L L-1 d-1to 0.43 L L-1d-1, which might ascribe to the degradation of the accumulated VFAs (Fig.3). Methane percentage of RL+S was between 50% and 63%, which was also higher than other three digesters. The pH was decreasing before bioaugmentation (day 111-120), and then increased slightly to approximately 7.0 with the addition of methanogenic culture. RL, a sterile control, was adding sterile liquid culture to investigate the impact of the remains in the culture medium and dead cell on the digestion performance. As Fig.2 (Table 2) shows, VMP was increasing from 0.12 L L-1 d-1 to 0.22 L L-1d-1with the OLR rose from 0.5 g L-1d-1 to 1.0 g L-1d-1, and the methane percentage was between 48% and 56%, while the digestion was failing when the OLR further elevated to 1.5 g L-1 d-1.pH was lower than that of RL+S, with the value of approximately 6.6. Since day 170, IA:PA was above 1.0, indicated an unstable digestion (Ripley et al., 1986). The pH dropped sharply after day 180 due to the accumulation of VFAs (Fig.3). RS was introducing active solid methanogenic culture (the biomass collected from liquid culture) to rule out the effect of the nutrient remained in the culture medium. The performance of R S was second only to RL+S during the first HRT (121-140 d) with OLR of 0.75 g VS L-1d-1, while the methane production was dropping significantly since day 147 (Fig 2, Table 2), indicating the digester bioaugmentation with solid culture could not operate stably under the OLR of 1.0 g L-1d-1 . The pH of Rs was lower than both RL and RL+S (Fig 2), implying the liquid constituents in the bioaugmented culture might enhance the buffer capacity of the digestion since perhaps some NH4+ provided in the culture medium still remained. More VFAs accumulated with the elevated OLR in the later stage in Rs, resulting in a lower pH, which inhibited the survival of the added culture. Most methanogens work optimally in a pH range from 6.5 to 7.2, with a methanogenesis step failure below pH of 5.5 (Fisgativa et al., 2016). It should be noted that the VMP of RS was higher than that of RL in the early period of bioaugmentation (121-151 d), suggesting that introducing microbial biomass could shorten the effective period for enhancing the performance of digestion compared to adding sterile liquid culture, since adding the microbial biomass could directly increase the density of functional microbes, while it took time for adding nutrient containing in the sterile

liquid culture to enhance the endogenous methanogens populations. Fig.3 shows the VFA concentration in each reactor. The total volatile fatty acids (TVFAs) of four digesters on day 110 were approximately 1800 mg L-1, mainly propionic acid and acetic acid. TVFAs, especially acetic acid in RC kept increasing from day110, and almost no degradation of the accumulated propionic acid was observed in RC. In the contrary, the concentration of propionic acid, acetic acid and TVFAs in RL+S reduced sharply after introducing special culture, which was responsible for the increasing methane production. Comparing to R L, the slight lower TVFAs concentration in RS before day 150 led to its better performance, while propionic acid and acetic acid started to accumulate in RS, resulting in the poor performance during the later experimental period. The above results demonstrated that bioaugmentation with the methanogenic propionate-degrading culture could enhance anaerobic digestion and accelerate the degrading of accumulated VFA, especially acetic acid and propionic acid. As the previous studies suggested, using propionate-, butyrate-, and acetate-degrading cultures as inocula could improve AD system stability with efficient degradation of the high concentration of VFAs in a two-stage reactor fed with simulated wastewater (Acharya et al., 2015). In addition, daily bioaugmented with both anaerobic and aerated, methanogenic propionate enrichment cultures decreased recovery time of the overload digesters fed with non-fat dry milk and basal medium (Tale et al., 2015). 3.3 Statistical comparison of reactors performance For better comparing the performance of different forms of bioaugmentation, the average performance characteristics of each HRT were calculated with statistical analysis shown in Table 2. During day 111-120, methane percentage, VMP and VSMP of four digesters were similar, which means four reactors shared the same started point before bioaugmentation. The performance of RL+S, RL, and RS were all better than RC since day 121, suggesting that introducing methanogenic propionate-degrading consortium or the remains containing in its culture medium to the digesters could both improve the digestion performance of high C/N ratio feedstock. This might because that the introducing active solid microbial biomass could enhance the density of the benefit microbes like methanogens. While adding the sterile liquid culture could also improve the digestion performance, due to some trace element or other nutrient

was remained in the sterile culture, which could promote the endogenous microbial growth and methanogensis, however, these remained chemicals could be not enough for supporting the stable operation of the reactor at relative higher OLR (above 1.0 g VS L-1 d-1). For RL+S, with bioaugmentation, the maximum OLR, the average methane percentage, VMP and VSMP during the period of day 121-160 was enhanced by 1.0 g L-1d-1(~200%), 24.04% (~77%), 0.22 L L-1 d-1(~367%) and 0.23 Lg-1VSd-1(~288%) respectively than that without bioaugmentation (Rc, day 121-160). Comparing to the digester before bioaugmentation (R0, day 0-110), after daily bioaugmentation, RL+S (day 103-123) also exhibited a pronounced increase in methane production. The average methane percentage, VMP and VSMP were higher at 1.84% (~3%), 0.22 L L-1 d-1(~200%) and 0.09 Lg-1VSd-1(~41%), respectively. RL+S showed the best digestion performance among the four digesters, indicating that special microbes, coupled with some growth factors would be the best form for bioaugmentaion. On one hand, the growth factors can contribute to the survival of exogenous bioaugmentation seed. On the other hand, they are also helpful for the growth of endogenous microbes within the digesters. If trace elements such as Fe、Co、Ni are chosen as growth factors to assist the bioaugmentation, the methanogensis would be improve as well, since trace elements positively affect some essential co-factors of enzymes involved in the methanogensis (Banks et al., 2012; Moestedt et al., 2016; Zhang et al., 2015). Comparing the AD performance with other works (Uçkun Kiran et al., 2014), it seems that specific methane production and OLR in this study are not high even with bioaugmentation. This might be mainly due to the substrate itself which was high C/N ratio and easily degradable. Just as studies suggested that substrate and selected operating parameters could be the main drivers for both process performance and microbial community (Liu et al, 2017). The more substrates are soluble, easily fermentable, and rich in VFA, the lower can be their loading rate into digesters (Schievano et al, 2010). Moreover, a higher dosage of bioaugmentation might enhance the digestion performance (Li et al, 2017b). 3.4 Effect of bioaugmentation on methanogen community shift Fig .4 shows the shift of Archaeal in five digesters on genus level. Methanospirillum (~56%) and

Methanosarcina (~25%) were the dominant methanogens before bioaugmentation in R0 on the first day. On day 120, the day before bioaugmentation, Methanospirillum(~39%), Methanobacterium (~30%) and Methanothrix (~26%) were the dominant methanogens in RC, R L+S, RL, and RS. After day 120, four digesters showed quite different shifts on methanogen community. For RL+S, the most abundant methanogen was replaced by Methanothrix (35~45%), followed by Methanospirillum (27~35%) and Methanobacterium (18~25%) in the period of day 140-200. While in RC and RL both Methanospirillum and Methanothrix lost their dominance, instead the members of Methanobacterium were becoming the predominant methanogens, with the abundance of 66% in RC on day 160 and 80% in RL on day 200. In R S, Methanoculleus (58%) and Methanomassiliicoccus (32%) dominated the methanogen on day 180. Table 3 compares the abundance of methanogenic archaea in four reactors at bioaugmentation start and end points. It is clear that acetoclastic methanogens accounted for low percentage in RC, RL, and R S, with the proportion of below 7%, which might be the main reason for the accumulation of acetic acid and poor methane production in three digesters (Fig 2, Fig 3 and Table 2).On the contrary, the enhancement of Methanothrix density in RL+S led to its relative higher population of acetoclastic methanogens, which could the main contributor for its better performance and the relative stable digestion. For the continuous reactors the major challenge for bioaugmentation is to ensure that the introduced microorganisms are able to thrive and are not washed out of the reactor (Fotidis et al., 2014; Mohan et al., 2005). Both Methanothrix and Methanoculleus are the main methanogens in the adding bioaugmentation culture (Li et al., 2017a). Routine addition of active liquid culture led to Methanothrix members growth well in RL+S, whereas, introducing the solid bioaugmentation seed were not able to make the members of Methanothrix surviving well in the later period in RS, which might due to the acidic environment in RS. The other group of methanogens in the bioaugmentation culture, Methanoculleus, hydrogenotrophic methanogens(Garcia et al ,2006), did not play important role in the improvement of methane production in RL+S or Rs, which might be ascribed to the substrate (hydrogen) competition of other hydrogenotrophic methanogens. The present study sought to determine that bioaugmentation could be an alternative way of co-digestions. Its

application in mono-digestion could avoid the selection and limitation of available co-substrates. Daily bioaugmentation was implemented in this study to favor survival and prolonged activity of the exogenous microbial population (Martin-Ryals et al., 2015). Nevertheless, in consideration of the cost and benefit, future study to reduce the bioaugmentation dose and frequency is warranted to help make bioaugmentation more practically viable at full scale.

4. Conclusions This study demonstrated that bioaugmentation with methanogenic culture is a successful solution to improve the anaerobic digestion of high C/N ratio feedstock without co-digestion with nitrogen-rich substrate. The dropping density of acetoclastic methanogen was the main reason for the poor performance of high C/N ratio feedstock. Bioaugmentation with liquid methanogenic culture could increasing the abundance of Methanothrix, a group of acetoclastic methanogen, which could contribute to the improvement of digestion performance in terms of maximum OLR,

methane percentage, VMP and VSMP. Compared to the solid microbial consortium, adding

liquid culture containing some nutrient might be the better choice for bioaugmentation. E-supplementary data for this work can be found in e-version of this paper online. Acknowledgements Thanks are due to National Nature Science Foundation of China (51708538), National key research and development program of China (2017FYD0800801), Science and Technology Planning Project of Guangdong Province, China (2013B010204053), Natural Science Foundation of Guangdong Province, China (2016A030312007, 2017A030310443) and Bureau of International Cooperation, Chinese Academy of Sciences (182344KYSB20170009) for supporting this research. References 1.

Acharya, S.M., Kundu, K., Sreekrishnan, T.R. 2015. Improved stability of anaerobic digestion through the use of selective acidogenic culture. J. Environ. Eng., 141, 8.

2.

Baek, G., Kim, J., Shin, S.G., Lee, C. 2016. Bioaugmentation of anaerobic sludge digestion with iron-reducing bacteria: process and microbial responses to variations in hydraulic retention time. Appl. Microbiol. Biotechnol.,

100, 927-937. 3.

Banks, C.J., Zhang, Y., Jiang, Y., Heaven, S. 2012. Trace element requirements for stable food waste digestion at elevated ammonia concentrations. Bioresour. Technol., 104, 127-35.

4.

Bhattad, U., Venkiteshwaran, K., Maki, J.S., Zitomer, D.H. 2017. Biochemical methane potential assays and anaerobic digester bioaugmentation using freeze dried biomass. Environmental Science-Water Res. Technol., 3, 1152-1161.

5.

Cadillo-Quiroz, H., Yavitt, J.B., Zinder, S.H. 2009. Methanosphaerula palustris gen. nov., sp nov., a hydrogenotrophic methanogen isolated from a minerotrophic fen peatland. Int. J. Syst. Evol.Microbiol., 59, 928-935.

6.

Cardinali-Rezende, J., Debarry, R.B., Colturato, L.F.D.B., Carneiro, E.V., Chartone-Souza, E., Nascimento, A.M.A. 2009. Molecular identification and dynamics of microbial communities in reactor treating organic household waste. Appl. Microbiol. Biotechnol., 84, 777-789.

7.

Cater, M., Fanedl, L., Malovrh, S., Logar, R.M. 2015. Biogas production from brewery spent grain enhanced by bioaugmentation with hydrolytic anaerobic bacteria. Bioresour. Technol., 186, 261-269.

8.

Chen, Y., Cheng, J.J., Creamer, K.S. 2008. Inhibition of anaerobic digestion process: a review. Bioresour. Technol., 99, 4044-64

9.

Ecem Oner, B., Akyol, C., Bozan, M., Ince, O., Aydin, S., Ince, B. 2017. Bioaugmentation with Clostridium thermocellum to enhance the anaerobic biodegradation of lignocellulosic agricultural residues. Bioresour. Technol., 249, 620-625.

10. Fisgativa, H., Tremier, A., Dabert, P. 2016. Characterizing the variability of food waste quality: A need for efficient valorisation through anaerobic digestion. Waste Manage., 50, 264-274. 11. Fotidis, I.A., Karakashev, D., Angelidaki, I. 2013. Bioaugmentation with an acetate-oxidising consortium as a tool to tackle ammonia inhibition of anaerobic digestion. Bioresour. Technol., 146, 57-62. 12. 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. 13. Garcia, J.-L., Ollivier, B., Whitman, W.B. 2006. The Order Methanomicrobiales. in Dworkin M. In: Falkow S, Rosenberg E, Schleifer, KH, Stackebrandt E (Eds.), The Pro-karyotes. Springer, New York,pp,208-230. 14. Gerardi, M. H. 2003. Biogas. The Microbiology of Anaerobic Digesters. John Wiley & Sons, Inc. New Jersey. 15. Guo, X., Wang, C., Sun, F., Zhu, W., Wu, W., 2014. A comparison of microbial characteristics between the thermophilic and mesophilic anaerobic digesters exposed to elevated food waste loadings. Bioresour. Technol. 152,

420-428. 16. Hassan, M., Ding, W., Umar, M., Rasool, G. 2017. Batch and semi-continuous anaerobic co-digestion of goose manure with alkali solubilized wheat straw: A case of carbon to nitrogen ratio and organic loading rate regression optimization. Bioresour. Technol., 230, 24-32. 17. Jain, S., Jain, S., Wolf, I.T., Lee, J., Tong, Y.W. 2015. A comprehensive review on operating parameters and different pretreatment methodologies for anaerobic digestion of municipal solid waste. Renew. Sust. Energ. Rev.s, 52, 142-154. 18. Kendall, M.M., Boone, D.R. 2006. The Order Methanosarcinales. in Dworkin M. In: Falkow S, Rosenberg E, Schleifer, KH, Stackebrandt E (Eds.), The Pro-karyotes. Springer, New York, pp. 244-256. 19. Kroeninger, L., Berger, S., Welte, C., Deppenmeier, U. 2016. Evidence for the involvement of two heterodisulfide reductases in the energy-conserving system of Methanomassiliicoccus luminyensis. Febs J.283, 472-483. 20. Li, Y., Sun, Y., Yang, G., Hu, K., Lv, P., Li, L. 2017a. Vertical distribution of microbial community and metabolic pathway in a methanogenic propionate degradation bioreactor. Bioresour. Technol., 245, 1022-1029. 21. Li, Y., Zhang, Y., Sun, Y., Wu, S., Kong, X., Yuan, Z., Dong, R. 2017b. The performance efficiency of bioaugmentation to prevent anaerobic digestion failure from ammonia and propionate inhibition. Bioresour. Technol., 231, 94-100. 22. Lins, P., Reitschuler, C., Illmer, P. 2014. Methanosarcina spp., the key to relieve the start-up of a thermophilic anaerobic digestion suffering from high acetic acid loads. Bioresour. Technol., 152, 347-54. 23. Liu, T., Sun, L., Mueller, B., Schnurer, A. 2017. Importance of inoculum source and initial community structure for biogas production from agricultural substrates. Bioresour. Technol., 245, 768-777. 24. Lu, F., Ji, J., Shao, L., He, P. 2013. Bacterial bioaugmentation for improving methane and hydrogen production from microalgae. Biotechnology for Biofuels, 6,92.1-11 25. Lu, F., Li, T., Wang, T., Shao, L., He, P. 2014. Improvement of sludge digestate biodegradability by thermophilic bioaugmentation. Appl. Microbiol. Biotechnol., 98, 969-977. 26. Martin-Ryals, A., Schideman, L., Li, P., Wilkinson, H., Wagner, R. 2015. Improving anaerobic digestion of a cellulosic waste via routine bioaugmentation with cellulolytic microorganisms. Bioresour. Technol., 189, 62-70. 27. Mata-Alvarez, J., Dosta, J., Romero-Gueiza, M.S., Fonoll, X., Peces, M., Astals, S. 2014. A critical review on anaerobic co-digestion achievements between 2010 and 2013. Renew. Sust. Energ. Rev., 36, 412-427. 28. Maus, I., Wibberg, D., Stantscheff, R., Cibis, K., Eikmeyer, F.-G., Koenig, H., Puehler, A., Schlueter, A. 2013. Complete genome sequence of the hydrogenotrophic Archaeon Methanobacterium sp Mb1 isolated from a

production-scale biogas plant. J. Biotechnol. 168, 734-736. 29. Moestedt, J., Nordell, E., Shakeri Yekta, S., Lundgren, J., Marti, M., Sundberg, C., Ejlertsson, J., Svensson, B.H., Bjorn, A. 2016. Effects of trace element addition on process stability during anaerobic co-digestion of OFMSW and slaughterhouse waste. Waste Manag., 47(Pt A), 11-20. 30. Mohan, S.V., Rao, N.C., Prasad, K.K., Sarma, P.N., 2005. Bioaugmentation of an anaerobic sequencing batch biofilm reactor (AnSBBR) with immobilized sulphate reducing bacteria (SRB) for the treatment of sulphate bearing i.

chemical wastewater. Process Biochem. 40, 2849-2857.

31. Nkemka, V.N., Gilroyed, B., Yanke, J., Gruninger, R., Vedres, D., McAllister, T., Hao, X.Y. 2015. Bioaugmentation with an anaerobic fungus in a two-stage process for biohydrogen and biogas production using corn silage and cattail. Bioresour. Technol., 185, 79-88. 32. Schauer-Gimenez, A.E., Zitomer, D.H., Maki, J.S., Struble, C.A. 2010. Bioaugmentation for improved recovery of anaerobic digesters after toxicant exposure. Water Res., 44, 3555-3564. 33. Schievano, A., D’Imporzano, G., Malagutti, L., Fragali, E., Ruboni, G., Adani, F., 2010.Evaluating inhibition conditions in high-solids anaerobic digestion of organic fraction of municipal solid waste. Bioresour. Technol., 101, 572-5732 34. Tale, V.P., Maki, J.S., Struble, C.A., Zitomer, D.H. 2011. Methanogen community structure-activity relationship and bioaugmentation of overloaded anaerobic digesters. Water Res., 45, 5249-56. 35. Tale, V.P., Maki, J.S., Zitomer, D.H. 2015. Bioaugmentation of overloaded anaerobic digesters restores function and archaeal community. Water Res., 70, 138-147. 36. Tiwary, A., Williams, I.D., Pant, D.C., Kishore, V.V.N. 2015. Emerging perspectives on environmental burden minimisation initiatives from anaerobic digestion technologies for community scale biomass valorisation. Renew. Sust. Energ. Rev., 42, 883-901. 37. Town, J.R., Dumonceaux, T.J. 2016. Laboratory-scale bioaugmentation relieves acetate accumulation and stimulates methane production in stalled anaerobic digesters. Appl. Microbiol. Biotechnol., 100, 1009-1017. 38. Uçkun Kiran, E., Trzcinski, A.P., Ng, W.J., Liu, Y., 2014. Bioconversion of food waste to energy: a review. Fuel, 134, 389-399. 39. Vasquez, J., Nakasaki, K. 2016. Effects of shock loading versus stepwise acclimation on microbial consortia during the anaerobic digestion of glycerol. Biomass Bioenergy, 86, 129-135. 40. Wilkins, D., Rao, S., Lu, X., Lee, P.K.H. 2015. Effects of sludge inoculum and organic feedstock on active microbial communities and methane yield during anaerobic digestion. Front. Microbiol., 6,1-11.

41. Xie, S., Hai, F.I., Zhan, X., Guo, W., Ngo, H.H., Price, W.E., Nghiem, L.D. 2016. Anaerobic co-digestion: A critical review of mathematical modelling for performance optimization. Bioresour. Technol., 222, 498-512. 42. Xu, R., Zhang, K., Liu, P., Khan, A., Xiong, J., Tian, F., Li, X. 2018. A critical review on the interaction of substrate nutrient balance and microbial community structure and function in anaerobic co-digestion. Bioresour. Technol., 247, 1119-1127. 43. Zhang, C., Su, H., Baeyens, J., Tan, T. 2014. Reviewing the anaerobic digestion of food waste for biogas production. Renew. Sust. Energ. Rev., 38, 383-392. 44. Zhang, W., Wu, S., Guo, J., Zhou, J., Dong, R. 2015. Performance and kinetic evaluation of semi-continuously fed anaerobic digesters treating food waste: Role of trace elements. Bioresour. Technol., 178, 297-305.

Fig.1 The digestion performance of high C/N ratio feedstock

Fig.2 The effect of bioaugmentation on digestion performance (RC was the control digester without bioaugmentation; RL+S was bioaugmented with active liquid methanogenic culture; RL was added sterile liquid culture; RS was introduced solid biomass collected by centrifuging the liquid culture)

Fig.3 The effect of bioaugmentation on VFA concentration (RC was the control digester without bioaugmentation; R L+S was bioaugmented with active liquid methanogenic culture; RL was added sterile liquid culture; RS was introduced solid biomass collected by centrifuging the liquid culture)

Fig. 4 The effect of bioaugmentation on Archaeal community structure on genus level (R0 was the digester before bioaugmentation ; RC was the control digester without bioaugmentation; RL+S was bioaugmented with active liquid methanogenic culture; RL was added sterile liquid culture; RS was introduced solid biomass collected by centrifuging the liquid culture. 0, 80, 120, 140, 160 ,180 and 200 represent days of running)

Table1. The main strategic operation for the experiment Phase I: Independent digestion

Phase II: Divided into four reactors

Phase III: Bioaugmentation comparison

0–110 day

111-120 day

121-140 day

141-180 day

181-200 day

0.5 g VS L-1d-1

0.5 g VS L-1d-1

0.75 g VS L-1d-1

1.0 g VS L-1d-1

1.5 g VS L-1d-1

R0 (2L)

RC

Inoculated from R0 and making

R C: non-bioaugmentation

RL+S

up volume from 0.5L to 1L by

RL+S: adding whole active culture

RL

feeding daily without discharge

RL: adding sterile liquid culture

RS

R S: adding active solid culture (centrifuged)

Table 2 Statistical analysis of the digestion performance during each stage of four bioreactors Period/ d CH 4%/ %

-1 -1

VMP/ LL d

VSMP/ Lg-1VSd-1

OLR /g L-1d-1

RC

RL+S

RL

RS

111-120

0.5

45.78（1.51）

45.88（1.65）

45.88（1.77）

45.95（2.32）

121-140

0.75

46.50（1.81）

53.36（2.08）

51.58（2.45）

52.33（2.29）

141-160

1.0

16.32（14.25） 57.59（2.28）

51.64（3.09）

47.56（9.48）

161-180

1.0

-

61.93（3.11）

55.23（1.09）

2.56（1.15）

181-200

1.5

-

56.00（0.55）

26.92（19.06）

111-120

0.5

0.13（0.01）

0.13（0.01）

0.12（0.01）

0.13（0.01）

121-140

0.75

0.11（0.03）

0.21（0.05）

0.14（0.02）

0.18（0.03）

141-160

1.0

0.01（0.01）

0.34（0.02）

0.22（0.02）

0.16（0.11）

161-180

1.0

-

0.34（0.02）

0.22（0.03）

0.00（0.00）

-

181-200

1.5

-

0.42（0.01）

0.07（0.09）

111-120

0.5

0.25（0.02）

0.25（0.02）

0.25（0.02）

0.25（0.02）

121-140

0.75

0.15（0.03）

0.28（0.06）

0.19（0.02）

0.23（0.05）

141-160

1.0

0.00（0.00）

0.34（0.02）

0.22（0.02）

0.16（0.11）

161-180

1.0

-

0.33（0.03）

0.21（0.02）

0.00（0.00）

181-200

1.5

-

0.28（0.01）

0.04（0.06）

Values are expressed as mean values with standard deviation shown in parentheses

-

-

Table 3. Relative abundance of methanogenic archaea in each bioreactor Methanogen

Methanogenesis type

Relative abundance (%) RC

Methanothrix*

Acetoclastic(Kendall & Boone, 2006)

RL+S

RL

RS

120d

160d

120d

200d

120d

200d

120d

180d

26

5

26

35

26

5

26

4

Methanosarcina

Acetoclastic (Kendall & Boone, 2006)

4

2

4

1

4

2

4

2

Methanospirillum

Hydrogenotrophic(Garcia et al., 2006)

39

10

39

30

39

3

39

6

Methanobacterium

Hydrogenotrophic(Maus et al., 2013)

30

66

30

25

30

80

30

16

Methanoculleus*

Hydrogenotrophic(Garcia et al ,2006)

1

13

1

8

1

4

1

58

Methanomassiliicoccus

Hydrogenotrophic(Kroeninger et al., 2016)

<1

3

<1

1

<1

6

<1

13

Hydrogenotrophic(Cardinali-Rezende et al.,

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

Methanosphaera

2009) Hydrogenotrophic(Cadillo-Quiroz

et

al.,

Methanosphaerula

2009)

Methanolinea

Hydrogenotrophic(Garcia et al ,2006)

<1

<1

<1

<1

<1

<1

<1

<1

Methanoregula

Hydrogenotrophic(Garcia et al ,2006)

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

<1

Total acetoclastic methanogen

30

7

30

36

30

7

30

6

Total hydrogenotrophic methanogen

70

93

70

64

70

93

70

94

Methanobrevibacter

* The dominant methanogens in the bioaugmentation inoculum.

1. VFAs accumulation resulted in poor performance of high C/N ratio feedstock. 2. Bioaugmentation can enhance the anaerobic digestion of high C/N ratio feedstock. 3. Acetoclastic methanogens Methanothrix was increased with bioaugmentation.