Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: What to expect from anaerobic fungus Orpinomyces sp.

Accepted Manuscript Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: what to expect from anaerobic fungus Orpinomyces sp Çağrı Akyol, Orhan Ince, Mahir Bozan, E. Gozde Ozbayram, Bahar Ince PII: DOI: Reference:

S0960-8524(19)30035-5 https://doi.org/10.1016/j.biortech.2019.01.024 BITE 20901

To appear in:

Bioresource Technology

Received Date: Revised Date: Accepted Date:

6 December 2018 6 January 2019 7 January 2019

Please cite this article as: Akyol, C., Ince, O., Bozan, M., Ozbayram, E.G., Ince, B., Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: what to expect from anaerobic fungus Orpinomyces sp, Bioresource Technology (2019), doi: https://doi.org/10.1016/j.biortech.2019.01.024

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Fungal bioaugmentation of anaerobic digesters fed with lignocellulosic biomass: what to expect from anaerobic fungus Orpinomyces sp.

Çağrı Akyol a, Orhan Ince b*, Mahir Bozan a, E. Gozde Ozbayram b, Bahar Ince a

a

Institute of Environmental Sciences, Boğaziçi University, Bebek 34342, Istanbul, Turkey

b

Department of Environmental Engineering, Istanbul Technical University, Maslak 34469, Istanbul, Turkey

*Corresponding author (O. Ince) Address: Department of Environmental Engineering, Istanbul Technical University Maslak 34469 Istanbul Turkey E-mail: [email protected] Phone: +902122856570 Fax: +902122856570

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ABSTRACT Energy-efficient biogas reactors are often designed and operated mimicking natural microbial ecosystems such as the digestive tracts of ruminants. Anaerobic fungi play a crucial role in the degradation of lignocellulose-rich fiber thanks to their high cellulolytic activity. Fungal bioaugmentation is therefore at the heart of our understanding of enhancing anaerobic digestion (AD). The efficieny of bioaugmentation with anaerobic fungus Orpinomyces sp. was evaluated in lignocellulose-based AD configurations. Fungal bioaugmentation increased the methane yield by 15-33% during anaerobic co-digestion of cow manure and selected cereal crops/straws. Harvesting stage of the crops was a decisive parameter to influence methane production together with fungal bioaugmentation. A more efficient fermentation process in the bioaugmented digesters was distinguished by relatively-higher abundance of Synergistetes, which was mainly represented by the genus Anaerobaculum. On the contrary, the composition of the methanogenic archaea did not change, and the majority of methanogens was assigned to Methanosarcina.

Keywords: anaerobic digestion; anaerobic fungus; bioaugmentation; cereal crops; lignocellulosic biomass; metagenomics

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1. Introduction Plant biomass is a promising alternative for biofuel refineries as it is the most abundant and sustainable feedstock in context of renewable energy sources (Guo et al., 2018). Plant materials with high lignin content (complex chains of phenol molecules, 10-20%) of lignified plant cell walls are defined as lignocellulosic biomass, which also contains hemicellulose (polymer of xylose, mannose and galactose, 15-35%), cellulose (polymer of glucose, 30-50%) and small amounts of ash, proteins and pectins (Bhutto et al., 2017; Nzila, 2017; van Kuijk et al., 2015). Further, lignocellulose content and composition varies between species and changes during plant maturation stages (Grabber, 2005; van Kuijk et al., 2015). Recalcitrant structure of lignocellulosic biomass is one of the major limitations in biogas production thus necessitating efficient and novel strategies (Shrestha et al., 2017). To date, remarkable number of strategies have been proposed for enhancing the efficiency of lignocellulose-based anaerobic digestion (AD) (Paudel et al., 2017). Among these alternatives, microbial strategies are eagerly referred to enhance the hydrolysis rate during AD of lignocellulosic biomass (Nzila, 2017). Microorganisms in herbivorous’ digestive systems, known as rumen microorganisms, are capable of effectively-digesting lignocellulosic compounds (Ozbayram et al., 2018b). The microbiota of the fermentation chamber of the ruminant gut is comprised of prokaryotic (bacteria and archaea), and eukaryotic (protozoa and fungi) organisms (Bayané and Guiot, 2011) which rely on symbiotic associations with each other (Yue et al., 2013). Among this diverse microbial consortium, anaerobic fungi are known to be key players in the degradation of lignocellulosic materials (Gruninger et al., 2014). Members of the anaerobic fungi (phylum Neocallimastigomycota) are highly fibrolytic organisms and represent a promising source of lignocellulolytic enzymes (Struchtemeyer et al., 2014). For instance, Orpinomyces sp. strain C1A exhibits great diversity of these carbohydrate active enzymes (Morrison et al., 2016). This

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defined lignocellulolytic enzyme cocktail indicates rumen anaerobic fungi (i.e. Orpinomyces sp.) as a remarkable biomass degrader and, renders them as promising agents for biofuels production (Couger et al., 2015; Youssef et al., 2013). Close association between anaerobic fungi and methanogens is well stated. Along with crossfeeding, hydrogen transfer also influences fungal catabolic pathways and specific enzyme profiles which in return shifts end products (Cheng et al., 2009). Syntrophic interactions between acetogenic bacteria and methanogens are also well known to occur in the biogas microbiome (Demirel and Scherer, 2008). Thus, the idea of augmenting biogas reactors with anaerobic fungi therefore seems a coherent alternative to overcome the limitation arising from the biodegradation of recalcitrant feedstock since anaerobic fungi show improved growth in the presence of methanogens (Dollhofer et al., 2015). Bioaugmentation of anaerobic digesters has been successfully applied using a wide range of cellulolytic microorganisms, leading significant increases on methane yield (Nzila, 2017). Although most of these studies focused on bacterial bioaugmentation which considered adding either single species (Öner et al., 2018; Tsapekos et al., 2017) or mixed culture of bacteria (Martin-Ryals et al., 2015; Ozbayram et al., 2018a), a few studies also applied methanogenic bioaugmentation (Fotidis et al., 2014) and fungal bioaugmentation (Ferraro et al., 2018; Kazda et al., 2014). Earlier attempts have been made using anaerobic fungi to enhance methane production from energy crops (Procházka et al., 2012) and further combine hydrogen and methane production in a two-stage system fed with corn silage and cattail (Nkemka et al., 2015). In a recent study, methane production from microalgae was significantly increased by 41% using mix culture of rumen anaerobic fungi in batch assays (Aydin et al., 2017). Furthermore, enzyme activities of different strains belonging to anaerobic fungi were reported and recommended to be harnessed for industrial applications ((Dagar et al., 2018). Shrestha and colleagues also highlighted the need for future research to

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use of anaerobic fungi in AD and evaluate its use by analysing the microbial community structure and the transcriptome in their comprehensive review paper (Shrestha et al., 2017). Orpinomyces sp., together with other anaerobic fungi species, was already detected in fullscale biogas plants fed with lignocellulosic biomass (Dollhofer et al., 2017). However, further investigation was recommended for efficient fungal bioaugmentation strategies. Afore-mentioned reasons created a big motivation for us to take advantage of highlycellulolytic anaerobic fungus Orpinomyces sp. to enhance the anaerobic biodegradability of lignocellulosic biomass with respect to a bioaugmentation approach. Up to now, few studies characterized biogas microbiome in anaerobic digesters upon fungal bioaugmentation (Aydin et al., 2017; Yıldırım et al., 2017). Findings from these recent researches indicated that indigenous biogas microbiome was greatly influenced by fungal bioaugmentation using a mix culture of anaerobic fungi. However, it is well known that the efficiency of bioaugmentation and biomethanation is strongly dependent on the bioaugmentation culture as well as the type of feedstock. More specifically, to the best of our knowledge, no research was conducted to investigate fungal bioaugmentation of cereal crops in AD and evaluate its effect on biogas microbiome. The present study therefore aimed to demonstrate a comprehensive research on the effect of fungal bioaugmentation with Orpinomyces sp. on methane production from cereal crops and straws (i.e. barley, triticale, wheat, rye). These selected crop materials were harvested at different maturation stages and subjected to anaerobic digestion (AD). Cow manure was used as the co-substrate in mesophilic AD tests to balance C/N ratio and nutrient content. Bacterial and methanogenic archaeal populations were characterized using high throughput 16s rRNA gene amplicon sequencing. Since cereal crops and straws hold great potential as candidate for biofuel production, we wanted to determine the key players of lignocellulose degradation and reveal the impacts of fungal bioaugmentation approach on biogas microbiome. As the presence and activity of anaerobic fungi in agricultural biogas

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plants are recently revealed, this study may provide useful information for implementing biological strategies to enhance anaerobic biodegradability of lignocellulosic biomass. 2. Materials and methods 2.1. Substrates and inoculum Cereal crops (i.e. wheat, rye, barley and triticale) were cultivated in the fields of Faculty of Agriculture, Uludağ University, Bursa, Turkey, and harvested between May-July 2016. Whole crop materials of barley and triticale were considered with respect to different harvesting stages, namely early harvest (grain in the milk stage), late harvest (maturity complete stage) (Amon et al., 2007) and harvesting residues (straw parts); meanwhile, only harvesting residues of wheat and rye were utilized in AD tests. Total solids (TS) and volatile solids (VS) of the cereal crops were in the range of 40.6-48.8% and 35.2-40.0% in earlyharvested samples, 90.5-90.7% and 69.6-73.1% in late-harvested samples and 88.2-89.6% in harvesting residues, respectively. Carbon to nitrogen (C:N) ratio ranged between 21:1-33:1, 22:1-28:1 and 74:1-87:1 in early-, late-harvested samples and residues, respectively. Cellulose contents (% TS) of the early-harvested, late-harvested and harvesting residues ranged between 12.03-19.28%, 11.25-12.85% and 50.39-51.39%, respectively. Hemicellulose contents (% TS) of the early-harvested, late-harvested and harvesting residues ranged between 14.15-18.39%, 12.13-27-79% and 20.64-24.5%, respectively. Finally, lignin contents (% TS) of the earlyharvested, late-harvested and harvesting residues ranged between 1.15-2.78%, 0.93-2.09% and 7.61-7.75%, respectively. Cow manure was selected and used as the co-substrate to each crop material in AD tests and obtained from a healthy, non-medicated cow that was kept at the barn of Veterinary Faculty of Istanbul University, Istanbul, Turkey. Physicochemical characteristics of the cow manure were: pH: 7.5, TS: 14.3%, VS: 11.5%, C:N: 25:1, cellulose (% TS): 35.9%, hemicellulose (% TS): 17.19% and lignin (% TS): 14.52%. Anaerobic seed sludge used as inoculum was taken from a full-scale biogas reactor fed with cattle manure and

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agro-industrial process leftovers in Bursa, Turkey. The main characteristics of the anaerobic seed sludge were as follows: pH: 8.35, TS: 7.2%, VS: 4.5%, VS/TS: 62%. 2.2. Isolation and cultivation of Orpinomyces sp. Anaerobic basal media was prepared according to (Orpin, 1976) with few modifications. Basal media contained following ingredients: 150 mL/L clarified rumen fluid -supernatant of centrifuged rumen fluid, 150 mL/L Mineral Solution I (0.3% K2HPO4), 150 mL/L Mineral Solution II (0.3% KH2PO4, 0.6% NaCl, 0.6% (NH4)2SO4, 0.06% CaCl2, and 0.06% MgSO4) 6 g/L NaHCO3, 2.5 g/L yeast extract, 10 g/L peptone, and 1 mg/L resazurin. Mixture was stirred and heated until boiling for 1 h in order to replace O 2 with CO2. 5 g/L cellobiose was added to basal media as carbon source and 1 g/L L-cystein.HCl was added to mixture to give final reduction, Hungate tubes and serum bottles were filled with final media and tightly sealed with rubber stopper prior to sterilization by autoclave at 121 oC for 15 min. Anaerobic rumen fungus Orpnimoyces sp. was obtained from the culture collection of Department of Zootechnics, Kahramanmaraş Sütçü İmam University, Kahramanmaraş, Turkey (Çömlekçioğlu et al., 2008). Isolation of anaerobic rumen fungi was conducted by applying dilution methodology carried out by (Theodorou et al., 1993). Briefly, 10 g animal manure was taken as microbial source sample and added to 100 mL basal media. 1 mL of mixture was transferred to Hungate tubes containing 9 mL basal media and wheat straw; furthermore, serial dilution was applied to samples up to 10 3. Antibiotics (Cloramphenicol: 100 μg/mL, Ampicillin: 100 μg/mL, Streptomycin: 140 μg/mL, Erythromycin: 200 μg/mL) were supplemented to media to prevent bacterial contamination. Whole procedure was carried out under anaerobic conditions by supplying CO2 to the samples. The diluted samples were left for incubation at 39 oC for 3-10 days. Roll Tube method, which was developed by (Joblin, 1981), was then applied to obtain single colonies from diluted samples. 0.5 mL of the diluted sample was injected to melted basal

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anaerobic agar media and rolling method was applied during solidification of media. After incubation at 39 oC for 3-4 days, single colonies were selected and transferred to basal broth media for further morphological analyzing under Olympus BX51 light microscopy. Isolated Orpinomyces sp. was maintained and subcultured by utilizing basal media containing cellobiose. Before implementing AD tests, 80 mL of media in 120 mL serum bottles was inoculated with Orpinomyces sp. culture and incubated for 5 days at 39 oC. Following the final incubation, the fungus and spent medium were directly utilized for the bioaugmentation of anaerobic digesters. 2.3. Anaerobic digestion Anaerobic co-digestion trials were conducted subjecting cow manure and early-harvested barley (EB) and triticale (ET), late-harvested barley (LB) and triticale (LT) as well as harvest residues of barley (RB), triticale (RT), rye (RR) and wheat (RW) for bioaugmentation (_B) and non-bioaugmentation (_NB) set-ups. Methane production from each crop material-cow manure mixture with and without bioaugmentation (10% v/v of Orpinomyces sp. and spent medium) (Nkemka et al., 2015) were performed in batch assays using 1 L glass reactors with a working volume of 750 mL. Optimum crop material to cow manure ratio (1:1, VS basis) was determined in a previous study (Akyol et al., 2016). Each substrate mixture was inoculated with the anaerobic seed sludge at the inoculum to substrate ratio of 1:1 (VS basis). Another run was performed as the blank subjecting the anaerobic seed sludge to digestion and background biogas production was subtracted from the experimental digesters. The theoretical background biogas production from the spent medium was calculated and subtracted from the recorded biogas production in the bioaugmented digesters. The anaerobic digesters were operated in an incubating shaker at 37 ± 1 oC at a mixing speed of 100 rpm. No pH adjustment was made, and the digesters were flushed with N2 to maintain anaerobic

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conditions. The experiments were monitored until there was no significant change in biogas production (p < 0.05). 2.4. Analytical methods and chemicals Total solids (TS), volatile solids (VS), sCOD, TKN and alkalinity were measured according to Standard Methods (APHA/AWWA/WEF, 2012). pH was measured by benchtop pH meter (FEP20, Mettler Toledo). Carbon to nitrogen (C:N) ratio was analyzed using Elemental Combustion System (Costech, CHNSO, USA) with dried samples. Cellulose, hemicellulose and lignin contents of crop materials and cow manure were determined by Standard Forage Analysis (Goering and Van Soest, 1970). Reduction in cellulose content in anaerobic digesters was determined according to a method previously reported by (Siegert and Banks, 2005). Biogas generation in anaerobic digesters was cumulatively-recorded by Milligas counters (MGC-1, Ritter Bochum, Germany). Representative samples were taken from the digesters once on every 10 days of AD for analytical analyses. Gas composition were determined as described in a previous study (Akyol et al., 2016). All chemicals were purchased from Sigma-Aldrich (St.Louis, USA). 2.5. Metagenomic analysis Triplicate subsamples were collected from each anaerobic digester and total genomic DNA was extracted from 500 μL of slurry samples using MN NucleoSpin Soil DNA isolation kit (Macherey-Nagel, Germany) and a ribolyser (Fast PrepTM FP120 Bio 101 Thermo Electron Corporation, Belgium). Total DNA was quantified using absorbance measurement on the NanoPhotometer P-Class (Implen, Germany). Extracted DNA samples for each AD trial were then pooled to have a representative sample and stored at -20 °C for further analyses. Bacterial and methanogenic community compositions were processed and analyzed with the ZymoBIOMICSTM Service - Targeted Metagenomic Sequencing (Zymo Research, Irvine, CA). 16S ribosomal RNA gene targeted sequencing was performed using the Quick-16 ™

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NGS Library Preparation Kit (Zymo Research, Irvine, CA). The 16S primers used amplified the V3-V4 region of the 16S rRNA gene. The final PCR products are quantified with qPCR fluorescence readings and pooled together based on equal molarity. The final pooled library was cleaned up with Select-a-Size DNA Clean & ConcentratorTM (Zymo Research, Irvine, CA), then quantified with TapeStation® and Qubit®. The final library was sequenced on Illumina® MiSeqTM with a V3 reagent kit (600) cycles. The sequencing was performed with >10% PhiX spike-in. The sequences obtained from this study were deposited under the EMBL-EBI accession number PRJEB29114. 2.6. Bioinformatics Amplicon sequences were inferred from raw reads using the Dada2 pipeline (Callahan et al., 2016). Chimeric sequences were also removed with the Dada2 pipeline. Taxonomy assignment, alpha diversity indices (Shannon, Simpson, Chao1 and Pielou), beta-diversity analyses and heatmaps were performed and prepared with Qiime v.1.9.1 to identify and compare significant features of the samples (Caporaso et al., 2010). Taxa that have an abundance significantly different among groups were identified by LefSe (linear discriminant effect size) (Segata et al., 2011) with default settings if applicable. In-house scripts were used for data visualization. 2.7. Statistical analysis One-way-analysis of variance (ANOVA) was conducted using SPSS 21 software, and statistical significance was assumed at a level of (p < 0.05). The differences in the microbial communities between bioaugmented and non-bioaugmented AD trials were evaluated by principal coordinates analysis (PCoA) in Fast UniFrac (http://bmf.colorado. Edu/fastunifrac/). 3. Results and discussion 3.1. Overall AD performance

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Bioaugmentation of the anaerobic digesters with rumen anaerobic fungus Orpinomyces sp. significantly improved (p < 0.05) the methane production in all biochemical methane potential (BMP) trials (Fig. 1). Highest methane yield was achieved during the anaerobic codigestion of cow manure and early-harvested barley (EB_B) as 430 mL CH4/g VS, an increment by 14% comparing to its control (EB_NB) (Fig. 1a). Comparatively elevated impact of fungal bioaugmentation was observed in late-harvested barley (LB) trials. There was 22% increase in methane yield in LB_B compared to LB_NB. Similarly, fungal bioaugmentation of barley residues (RB_B) digesters enhanced the methane yield by 23%. The effect of addition of Orpinomyces sp. to AD was seen at the highest when the cosubstrate of cow manure was selected as triticale (Fig. 1b). Fungal bioaugmentation of the digesters fed with early-harvested (ET_B) and late-harvested triticale (LT_B) boosted the methane yield up to 254 mL CH4/g VS and 244 mL CH4/g VS, performing 33% and 26% higher than their control digesters ET_NB and LT_NB, respectively. Meanwhile, methane production in the anaerobic digesters fed with cow manure triticale residues (RT) were 161 mL CH4/g VS and 136 mL CH4/g VS in bioaugmented (RT_B) and non-bioaugmented (RT_NB) set-ups, respectively. Methane yields of the anaerobic digesters during the anaerobic digestion of cow manure and wheat straw (RW) and rye straw (RR) are also shown in Fig. 1c. The methane yields of these AD trials differed insignificantly among each other (p > 0.05) as 118 mL CH4/g VS and 115 mL CH4/g VS in RW_NB and RR_NB, respectively. On the other hand, fungal bioaugmentation contributed to an increase in methane yields by 15% and 17% in RW_B and RR_B comparing to their controls, respectively. In addition, pH was in the range of 7.0-7.5 during the operation thanks to excess alkalinity of the seed sludge. Reduction in the cellulose content of anaerobic digesters are shown in Fig. 2. Cellulose degradation in the non-bioaugmented digesters fed with early-harvested crops, namely EB_NB and ET_NB, were calculated as 44% and 47%, respectively. Meanwhile, the digesters

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fed with late-harvested crops (LB_NB and LT_NB) performed better in terms of cellulose reduction as 56% and 57%, respectively. Fungal bioaugmentation resulted in significantly higher cellulose degradation (p < 0.05) of 72%, 69%, 66% and 76% in EB_B, ET_B, LB_B and LT_B, respectively. Meanwhile, cellulose reduction in the non-bioaugmented digesters were calculated as 43% in RB_NB, 43% in RT_NB, 44% in RW_NB and 36% in RR_NB. Cellulose degradation in bioaugmented digesters reached up to 80%, 79%, 82% and 86% in RB_B, RT_B, RW_B and RR_B, respectively. According to BMP results, fungal bioaugmentation with Orpinomyces sp. boosted the methane yield by 15-33% in all examined trials in this particular study. Selection of cosubstrate to cow manure attracts great attention here since it influenced both methane yield and enhancement degree of bioaugmentation. Hence, early-harvested barley was found to be the highest-yielding cereal crop material, which highlighted that harvesting cut of barley also greatly affected the methane production. Comparatively lower lignin and/or higher organic content in early-harvested barley can be contributed to this differentiation among other crop materials. Meanwhile, there was no significant effect of harvesting stage of triticale on methane yields in both non-bioaugmented and bioaugmented AD. On the other hand, the effect of fungal bioaugmentation on methane yield was most apparent in AD fed with triticale. Straw parts of all cereal crops produced lower methane production as expected since they exhibited higher amounts of lignocellulose comparing to the main crops. Nevertheless, a remarkable increase in methane yields were still observed in straw-added digesters upon bioaugmentation. Besides the enhancement of methane yields, bioaugmentation with Orpinomyces sp. increased the initial methane production rate in all AD trials. In other words, fungal bioaugmentation contributed to shorten the microbial lag-phase of anaerobic biodegradation, which can be noteful for reducing the retention time of biogas reactors. A similar outcome was also mentioned by (Nkemka et al., 2015) during anaerobic digestion of

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corn silage and cattail. Although the authors did not initially receive any affirmative effects of fungal bioaugmentation with anaerobic fungus P. rhizinflata YM600 in BMP batch tests, they further increased the methane yield by 3-10% in a two-stage system (leach bed-UASB reactor) and highlighted the potential of fungal bioaugmentation of AD. An innovative perspective was followed by (Ferraro et al., 2018) to enhance biogas production from wheat straw and mushroom spent straw. The authors combined bioaugmentation with anaerobic ruminal fungi (i.e. Neocallimastix sp. and Orpynomyces sp.) and fermentative bacteria (a fermenting-acidogenic component, consisting of a hydrogen-producing bacterial pool (F210)) and achieved a major increase in methane yield. In another study, the addition of anaerobic fungal cultures increased the methane yield by 4-22% from energy crops. 3.2. Microbial community dynamics An average number of 51159 raw reads per sample were produced from the sequencing analysis with an average length of 250 bp. Details of the sequencing data of the samples can be found in E-supplement file. Rarefraction curves were depicted to evaluate the alpha diversity representing the microbial diversity of each sample (E-supplement file). Alpha diversity indices of all samples taken from the digesters are illustrated in Table 1. The highest values of indices were determined in the control digesters. Bioaugmentation application caused a decrease in the diversity of microbial communities in general. Furthermore, a moderate decrease was also calculated in the evenness of the bioaugmented AD trials. Since, the highest estimated richness based on Chao1 was calculated for the non-bioaugmented AD trials with harvest residues of wheat (RW_NB), bioaugmented AD trials with early-harvested barley (EB_B) had the least Chao1 value. Only in the RB trials, the richness increased after bioaugmentation (RB_B) application compared to its control (RB_NB). The Shannon diversity indices were in the range of 3.99-4.40 and 3.44-3.95 in the non-bioaugmented and bioaugmented digesters, respectively. Besides, there was a remarkable difference in the

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Simpson indices in the EB, RT and RW trials. Moreover, EB_NB had the most even microbial community. Fig.3 represents the beta diversity displayed as PCA plot that shows microbial diversity differences between the samples. A clear distance in microbial diversity between the bioaugmentation and non-bioaugmentation set-ups were revealed. In each set-up, the samples were divided into 2 different groups with respect to the use of the whole crop or crop residues (straw). In detail, whole crop-fed (i.e. EB, ET, LB, LT) AD configurations were clustered together in groups in the bioaugmented and non-bioaugmented digesters. Similarly, strawadded AD (i.e. RB, RT, RW, RR) showed comparatively higher similarity and were clustered together. 3.3. Microbial diversity The relative abundances of the bacterial phyla and families in AD are depicted in Fig.4a and Fig.4b, respectively. The most abundant bacterial phyla belonged to Firmicutes (52%-75%) and Bacteroidetes (19-40) in all AD systems. Synergistetes (8-9%) was the following predominant phylum but only in the bioaugmented digesters, followed by Proteobacteria (1%-9%), OP9 (2%-4%) and Thermotogae (1%-4%). In the non-bioaugmented digesters, Proteobacteria and Tenericutes were also detected in minor abundances. Although most abundant bacterial families were similar in AD samples, their proportions differed between bioaugmented and non-bioaugmented digesters. Accordingly, an unclassified family of the order MBA08 (25%-37%) and Porphyromonadaceae (13%27%) dominated the bioaugmented digesters, followed by Anaerobaculaceae (8%-9%), Caldicoprobacteraceae (5%-8%), and unclassified family of the order Bacteroidales (3%8%), Clostridiaceae (5%-6%), Ruminococcaceae (2%-4%), TIBD11 (2%-4%), Thermotogaceae (1%-4%), Tissierellaceae (1%-4%), Bacteroidaceae (1%-3%) and Syntrophomonadaceae (1%-2%). Meanwhile, Porphyromonadaceae (13%-27%), an

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unclassified family of the order MBA08 (13%-24%) and Clostridiaceae (8%-14%) were the most abundant bacterial families in the non-bioaugmented digesters, followed by Caldicoprobacteraceae (5%-14%), Tissierellaceae (6%-8%), Bacteroidaceae (1%-5%), Erysipelotrichaceae (1%-3%), Ruminococcaceae (1%-3%), an unclassified family of the order Bacteroidales (1%-3%), Lachnospiraceae (1%-2%) and Syntrophomonadaceae (1%2%). Figs. 4c and 4d display the methanogenic archaeal diversity at the family and genus level, respectively. Methanogenic archaea accounted on average for 1.3% of the whole AD microbiome. By far, Methanosarcinaceae dominated all digesters with and without fungal bioaugmentation. Methanomicrobiaceae, Methanomassiliicoccaceae and Methanobacteriaceae were also other methanogens responsible for methane production. At the genus level, Methanosarcina (family Methanosarcinaceae) was the most abundant methanogen; whereas, Methanosphera (family Methanobacteriaceae), Methanomassiliicoccus (family Methanomassiliicoccaceae), Methanoculleus (family Methanomicrobiaceae) and Methanobrevibacter (family Methanobacteriaceae) were also detected in the digesters. The relative abundances of major bacterial groups were influenced by both bioaugmentation and usage of the whole crop or crop residues (Fig. 5). As it is clear from the clustering of OTUs, two main groups and four subgroups were determined. The results revealed that the bacterial community structures varied in the samples due to the bioaugmentation application and formed two main groups. Whereas the first subgroup was composed of EB_B, ET_B and LB_B digesters, the second subgroup was formed by the bioaugmented AD trials with harvest residues namely RB_B, RT_B, RR_B and RW_B and as well as LT_B. Surprisingly, the third subgroup only included the non-bioaugmented digester with late-harvested barley (LB_NB) and differed from the other non-bioaugmented digesters which together formed the forth

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subgroup. Whereas the most dominant OTU was MBA08 in bioaugmented AD trials, Porphyromonadaceae confronted as the most dominant OTU in the non-bioaugmented digesters. Fig. 6 displays the LEfSe cladogram of all detected bacterial compositions from phylum to specie levels both in bioaugmented and non-bioaugmented AD trials. The samples were clustered into 2 groups of leaf, where the non-bioaugmented ADs (controls) indicated in green colour and the bioaugmented ADs trials leaves were indicated in red colour. The bioaugmented trials had four phylum level markers, Fibrobacteres, Armatimonadetes, Acidobacteria, and OP9, showed the highest abundance in these digesters comparing to the controls. Furthermore, Synergistales, Bifidobacteriales, Lachnospiraceae and Enterobacteraceae species were significantly enriched in the bioaugmented digesters. On the other hand, Alphaproteobacteria species became forward in the control digesters. Fungal bioaugmentation, together with the selection of crop material to be fed to AD, showed some remarkable outcomes in terms of microbial diversity. Major lesson learned from the microbial ecology of this particular AD systems is that fungal bioaugmentation was the driving factor to cluster microbial groups, followed by the use of whole crop or straw parts. Despite the core microbiome of AD were represented by the typically-detected bacteria (i.e. Firmicutes and Bacteroidetes); Synergistetes, which was mainly assigned to the genus Anaerobaculum, only enriched in the bioaugmented digesters. The members of the phylum are known to ferment sugars to acetate and H2 (Maune and Tanner, 2012). Higher abundance of Synergistetes marked that efficient syntrophic oxidations were achieved since syntrophic oxidizers relieves the accumulation of propionate and butyrate (Deng et al., 2018). Relatively higher abundance of these species can be attributed to a more efficient fermentation in the anaerobic digesters upon fungal bioaugmentation. Similarly, Thermotogae, which was only represented by the family Thermotogaceae, were only abundant in the bioaugmented digesters

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fed with the whole crops (i.e. EB_B, ET_B, LB_B, LT_B). Although bacterial diversity differed with respect to the application of fungal bioaugmentation, methanogenic archaeal diversity was similar and dominated by Methanosarcina. Methanosarcina sp. are able to use both acetoclastic and hydrogenotrophic methanogenesis pathways and reported to be more tolerant to stress conditions than other methanogens (De Vrieze et al., 2012). It is known that the addition of pure and/or mix culture of microorganisms into AD via bioaugmentation can stress out the existing microbiome (Nzila, 2017). Hence, higher tolerance of Methanosarcina sp. can be one of the reasons for the absence of distinction between bioaugmented and nonbioaugmented digesters. These findings are in agreement with other studies focusing on the microbial communities of biogas reactors fed with lignocellulosic substrates (Grohmann et al., 2018; Li et al., 2018; Tsapekos et al., 2017). Samples taken from lab-scale biogas reactors fed with sugarcane filter cake alone or together with bagasse showed the predominance of the bacterial families Bacteroidaceae, Prevotellaceae and Porphyromonadaceae (phylum Bacteroidetes) and Synergistaceae (phylum Synergistetes), and the methanogenic genera Methanosarcina and Methanobacterium (Leite et al., 2016). Firmicutes and Bacteroidetes are stated as the most commonly found bacterial phyla in biogas plants treating lignocellulosic biomass (Bozan et al., 2017). In another study, Clostridium (phylum Firmicutes) dominated the samples together with Methanobrevibacter and Methanosarcina as the most abundant methanogens in agricultural AD systems (Liu et al., 2017). A few recent studies also focused on the microbial characterization of fungal-bioaugmented AD few with different substrates (Aydin et al., 2017; Yıldırım et al., 2017). Similar bacterial communities were reported as the most abundant phyla, namely Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes as well as Synergistetes. On the other hand, Methanosaeta and Methanolinea were reported as the most abundant methanogens when the substrates were microalgae and animal manure, respectively.

17

The variations can be easily attributed to the initial microbial composition of the anaerobic seed sludge, different bioaugmentation cultures and other environmental and operating conditions. 3.4. Assessment of fungal bioaugmentation of lignocellulose-based AD Bioaugmentation of anaerobic digesters is often limited to lab-scale, yet it has a large potential to be extended to pilot/large-scale. Although pre-treatment methods are frequently used in full-scale processing, the cost and partial hydrolysis are among the main limitations (Nzila, 2017). An increase in the time length of AD is also another major drawback since it eventually increases the process cost (Sindhu et al., 2016). The cost of bacterial bioaugmentation in batch fermentation was roughly calculated and concluded to be not practically feasible; however, further continuous operation was proposed to be economically feasible (Peng et al., 2014). Economic evaluation of routine bacterial bioaugmentation was also conducted to stimulate how it could work in practical application. When the amount of methane production, reduction of waste disposal and maintenance of bioaugmentation culture were all considered, the results indicated that routine bioaugmentation with mixed hydrolytic bioculture of AD treating corn waste could improve the economics by $27-$34/dry tonne (Martin-Ryals et al., 2015). Since the authors obtained the bioaugmentation culture commercially, on-site cultivation of such microorganisms would be more cost-effective. Feasibility of similar bioaugmentation approaches in practical applications highly depend on the methane potential of utilized substrates, reactor configuration, type of bioaugmentation culture and its expenses (chemicals, cultivation equipment etc.). The results obtained in this study indicated that fungal bioaugmentation of biogas reactors can be a promising approach; however, further research on continuous operation with repetitive bioaugmentation seems to be necessary to investigate the survival and activity of anaerobic fungi during AD. The increment in methane yield can still be further elevated by combining

18

biological pretreatment with bioaugmentation and/or simultaneous addition of different microbial groups in AD. Thus, the substrate utilization rate and energy recovery can be improved. Further studies are needed both in lab-scale and pilot/full scale to improve the process efficiency and investigate the feasibility of various bioaugmentation approaches. 4. Conclusions Bioaugmentation with Orpinomyces sp. significantly increased the methane yield, in which early-harvested barley was the highest-yielding co-substrate to cow manure with 430 mL CH4/g VS upon fungal bioaugmentation. Although no significant effect of harvesting stage was observed for triticale, Orpinomyces sp. further boosted the methane yield by 33%. In terms of straws, the enhancement in methane yield varied between 15-23%, and triticale straw performed slightly higher than that of barley, wheat and rye. Correspondingly, the application of fungal bioaugmentation and the use of whole crop or straw were the most critical factors to cluster microbial groups with respect to similarity. Acknowledgements The authors kindly acknowledge Prof. Emin Özköse and his research group for culturing Orpinomyces sp. at the Department of Zootechnics, Kahramanmaraş Sütçü İmam University, Kahramanmaraş, Turkey. Büşra Ecem Öner and Ömer Uzun are also acknowledged for their support in the AD experiments. The authors thank Pakize Ozlem Kurt Polat and Koksal Yagdi for the cultivation and harvesting of the cereal crops. Illumina Sequencing was performed with ZymoBIOMICSTM Service – Targeted Metagenomic Sequencing (Zymo Research, Irvine, CA). This study was supported by The Scientific and Technological Research Council of Turkey (TUBITAK) (Project No: 115Y597). Appendix A. Supplementary data Supplementary data of this work can be found in online version of the paper. References

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Figure captions Fig. 1. Cumulative methane yields in non-bioaugmented and bioaugmented trials during anaerobic co-digestion of cow manure and (a) barley at different harvesting stages (b) triticale at different harvesting stages (c) wheat straw and rye straw. Fig. 2. Cellulose loss in non-bioaugmented and bioaugmented anaerobic digesters. Fig. 3. Two dimensional plot of a 3D principal component analysis based on the beta diversity of microbial communities. Fig. 4. Bubble diagram displaying the relative abundances of (a) bacterial communities at the phylum level (b) bacterial communities at the family level (c) methanogenic archaeal communities at the family level (d) methanogenic archaeal communities at the genus level. Fig. 5. Heatmap displaying the unique sequence abundance of microbial communities. Fig. 6. Cladogram displaying the taxonomy of 16S rRNA gene sequences.

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29

30

31

Table 1. Summary of the estimated richness and evenness of the microbial communities. Sample

OTUs

Chao1

Shannon

Simpson

Pielou's evenness

EB_NB

231

231

4.34

0.98

0.80

ET_NB

235

235

4.21

0.97

0.77

LB_NB

221

221

4.12

0.96

0.76

LT_NB

291

291

4.30

0.97

0.76

RB_NB

204

204

3.99

0.96

0.75

RT_NB

307

308

4.30

0.97

0.75

RW_NB

409

412

4.40

0.97

0.73

RR_NB

289

290

4.23

0.97

0.75

32

EB_B

133

133

3.44

0.94

0.70

ET_B

201

201

3.68

0.95

0.69

LB_B

171

171

3.69

0.95

0.72

LT_B

233

234

3.70

0.94

0.68

RB_B

340

344

3.95

0.95

0.68

RT_B

277

278

3.81

0.93

0.68

RW_B

212

212

3.67

0.93

0.69

RR_B

239

240

3.87

0.95

0.70

Highlights 

Fungal bioaugmentation with Orpinomyces sp. increased the methane yield by 1533%.



Harvesting stage of the crops greatly influenced the process performance.



Microbial groups were clustered based on bioaugmentation and crop material.



Anaerobaculum was only enriched in the bioaugmented digesters.



Methanogenic archaeal diversity was not affected by fungal bioaugmentation.

33

Batch Anaerobic Digesters

Anaerobic Microbiome Whole crops - Bioaugmented 0.1

PC2

Crop residues - Non.bioaugmented

0.0

Whole crops - Non.bioaugmented -0.1

Crop residues - Bioaugmented

-0.2 -0.3

-0.2

-0.1 Bioaugmented

0.1

0.2

0.3

Non.Bioaugmented

ET_NB

EB_NB

LT_NB

RB_NB

RR_NB

RT_NB

RW_NB

RW_B

LB_NB

RT_B

RB_B

LT_B

RR_B

LB_B

ET_B

Bioaugmentation

EB_B

Cereal crops

0.0 PC1