Enhancing anaerobic degradation of corn stover residues and biogas production via rumen microorganisms

Enhancing anaerobic degradation of corn stover residues and biogas production via rumen microorganisms | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Enhancing anaerobic degradation of corn stover residues and biogas production via rumen microorganisms Anastasia Makri, Spyridon Ntougias, Paraschos Melidis This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4174541/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 24 Oct, 2024 Read the published version in Environmental Processes → Version 1 posted 8 You are reading this latest preprint version Abstract Corn is one of the most common cultivations in Greece and worldwide. Raw materials like corn stover contain a remarkable amount of organic content, which could be transformed anaerobically into biogas, using as an alternative energy source. The development of efficient methods to overcome the limitations arisen from the nature of lignocellulosic biomass is a challenge since pretreatment is required to break down its complex structure. An economically-feasible biological approach to disrupt the structure of lignocellulosic materials, like corn stover, is through the valorization of hydrolytic potential of microbial communities present in rumen. Rumen microbiota has demonstrated the ability to break down lignocellulosic biomass. Thus, this work aims to enhance biogas production from corn stover residues using rumen fluid microbiota. The anaerobic digestibility of corn stover in BMP (Biochemical Methane Potential) tests and CSTR (continuous stirring tank reactors) was examined using rumen fluid as inoculum, in presence of control. Three organic loading rates (OLR), i.e. 1, 2, and 3 g VS/L.d, were tested, to define the optimum OLR for corn stover digestion. Moreover, experiments to define the optimum corn stover to rumen fluid ratio to optimize biogas production were carried out. Addition of rumen inoculum into the anaerobic digester at daily basis was found to be essential to enhance biogas production from corn stover. The optimum corn stover residues concentration in rumen fluid for optimum biogas production was 4% w/v. Addition of rumen fluid microbiota in the CSTR operating under various OLRs enhanced biogas production by 2–6.3 times. agricultural residues anaerobic digestion biogas enhancement lignocellulosic biomass hydrolysis rumen microbiota Figures Figure 1 Figure 2 Highlights High corn stover residues availability for biogas production. Effective breakdown of lignocellulose can be achieved by biological pretreatment. Pretreatment by rumen microbiota improved biogas production by 2-6.3 times. Optimum corn stover concentration for high biogas production was 4% in rumen fluid. 1 Introduction In recent years, the depletion of fossil fuels has raised the need to reduce dependence on fossil fuels. The daily energy demand is high, because of the increasing energy consumption, due to the overpopulation (Monga et al. 2023 ). The shift toward renewable energy production is necessary and crucial. The valorization of agricultural residues for generating energy and recovering nutrients has recently gained ground as an effective sustainable solution (Rahil Hasan et al. 2023 ). Looking for sustainable, abundant, and easy-to-be-found raw materials, agricultural wastes and residues could be a very attractive feedstock to contribute to energy recovery. Moreover, the daily amount of biowaste is increasing, indicating difficulties in terms of waste management (Srivastava et al. 2023 ). On the other hand, biowaste has the potential to be converted into an environmental-friendly fuel through various bioengineering processes, within the biorefinery concept (Srivastava et al. 2021 ). Anaerobic digestion is a biological process for energy recovery. It is a process where the organic content is anaerobically decomposed by a microbial consortium to produce biogas (Roubaud and Favrat 2005). It does not only provide an alternative energy source, but also consists of a way to decompose organic waste and reduce greenhouse gas emissions (Frigon and Guiot 2010 ). It includes four biological steps, i.e. biomass hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Li et al. 2010 ). Hydrolysis is the rate-limiting step in the breakdown of lignocellulosic biomass (Cirne et al. 2007 ). To improve the efficiency of biomass hydrolysis and consequently the biogas yield, it is essential to carry out a pretreatment step of lignocellulosic materials prior to anaerobic degradation. The pretreatment aims to break down the structural barriers of lignocellulosic biomass, making the cellulosic and hemicellulosic chains more accessible to microbial breakdown (Zheng et al. 2014 ). This can be achieved with various pretreatment approaches, such as physical, chemical and biological methods, or combinations of them (FitzPatrick 2010). Lignocellulosic biomass has an advantage over other feedstock choices, as it does not compete directly with food or feed production. Such residues contain a remarkable amount of organic content, which could be considered as an alternative energy source, and achievement of higher biogas yields can make these crop residues ideal for biogas production (McKendry 2002 ). Due to the economic concerns of feedstock collection and transportation, the use of lignocellulosic biomass as a feedstock for anaerobic digestion is greatly influenced by feedstock accessibility and availability (Li et al. 2011). Several lignocellulosic materials derived from agro-industrial and forestry activities have been used as feedstock, as they are abundant and available all over the year (Montoneri et al. 2009 ). Such residues are estimated to be over 10 million tons annually (Nguyen et al. 2019 ). Corn stover is common agricultural byproduct, which is widely available in corn production areas. It is one of the most popular grains in Greek fields and around the world, resulting in a significant amount of corn stover residues. Since now, the most common technique to manage these residues is burning. However, it is not advised to burn corn stover residues, due to the negative ecological impact of the process, and therefore alternatives must be found. According to Gu et al. ( 2014 ), such residues can be converted into renewable energy through anaerobic fermentation, so this process may solve numerous environmental and energy-related issues. Although lignocellulosic biomass has high organic content and energy value, its structure highly resists to hydrolysis. Most microbes cannot breakdown its structure without pretreatment, so it is considered difficult to ferment such biomass. Some anaerobic bacteria can successfully degrade the lignocellulosic biomass, but in slow rates (Zhou et al. 2014 ). Therefore, their use as a renewable energy source has been reported to require the application of pretreatment methods to enhance the digestibility of lignocellulosic biomass (Mankar et al. 2021 ). An economically-feasible and environmental-friendly method to break down the lignocellulose structure is its hydrolysis with the specialized microbial community found in the rumen fluid of ruminant animals. The rumen fluid microbiota enhance lignocellulose hydrolysis and biomass fermentation through the secretion of hydrolytic enzymes targeting β-glucosidic bonds (Yu et al. 2013 ). This symbiotic interaction of rumen microorganisms with their animal host evolved for millions of years, thus considering rumen microbiota as specialized lignocellulose degraders. Recently, the exploitation of rumen microbiome for enhancing lignocellulosic biomass degradation has led to promising results (Takizawa et al. 2018 ; Wang et al. 2018). This research work examines the effectiveness of applying rumen fluid inoculum as a pretreatment approach for the disruption of lignocellulosic corn stover residues and its valorization through anaerobic digestion. A scale-up approach was employed through the initial performance of Biochemical methane potential (BMP) tests to assess the anaerobic digestibility of corn stover in the presence of rumen fluid inoculum, followed by the investigation of the beneficial effect of rumen microbiota in the hydrolysis of its lignocellulosic biomass in Continuous Stirring Tank Reactors (CSTRs) to define optimum operating conditions regarding organic loading rate, biogas yield, methane content and amount of rumen fluid inoculum. 2 Materials and Methods 2.1 Lignocellulosic biomass and inoculum source Corn stover residues were collected from corn fields after harvest. They were oven-dried at 60 o C for 24 h, milled through a grinder and passed through a sieve of less than 2 mm size. Both total solids (TS) and volatile solids (VS) were determined in the sieved product and stored at room temperature for further use in downstream experiments. Corn stover residue dry content was 92.8 ± 0.92%, whereas corn stover organic matter was determined to be 71.6 ± 3.6%. Anaerobic sludge was obtained from a full-scale anaerobic digester nearby the city of Xanthi, Greece (40.99, 24.89), with the TS and VS of the anaerobic sludge being estimated 7.06 ± 0.20% w/v and 2.28 ± 0.02% w/v, respectively. The inoculum was left for 2 weeks to be inactivated before the inoculation with the rumen microbiota. Rumen fluid, which served as the hydrolytic inoculum, was collected from the first compartment of cow's stomach and drained through a filter prior its use. The pH of the rumen fluid was 6.15 ± 0.10, with their TS and VS being estimated as 3.62 ± 0.20% w/v and 0.99 ± 0.10% w/v, respectively. Table 1 shows the main characteristics of the lignocellulosic feedstock, rumen fluid and anaerobic sludge inocula. Table 1 Basic characteristics of hydrolytic inoculum and anaerobic sludge. Sample pH Total Solids (TS) (% w/v) Volatile Solids (VS) (% w/v) NH 4 + -N (g/L) COD (g/L) Rumen fluid 6.15 ± 0.77 3.62 ± 0.73 0.99 ± 0.14 0.15 ± 0.01 19.20 ± 1.18 Anaerobic sludge 7.15 ± 0.02 7.06 ± 0.20 2.28 ± 0.02 0.49 ± 0.01 13.20 ± 0.80 2.2 Biochemical methane potential (BMP) tests Biochemical methane potential (BMP) tests were carried out in batch reactors of 250 mL volume, with a working volume of 150 mL, which run in triplicate for a period of 28 days. The substrate-to-anaerobic inoculum (S/I) ratio in all tests was kept at 3:1, wt/wt. Each experimental setup included anaerobic sludge (blank), anaerobic sludge with corn stover residues (control), and anaerobic sludge with corn stover residues and addition of rumen fluid (sample for evaluation). In BMP tests, 4%, 6% and 12% w/v corn stover residues in rumen fluid was examined. The pH in batch reactors was adjusted to 7, and their content was flushed with N 2 /CO 2 (4/1, v/v) to create anaerobic conditions. Then, batch reactors were placed in a water bath at 37.5 o C and the produced biogas was measured through the manometric method (Angelidaki et al. 2009 ). The cumulative methane production for each BMP test was determined by subtracting the inoculum contribution (blank). 2.3 Assessment of anaerobic digestibility of corn stover residues in continuous stirred tank reactors (CSTRs): optimum operating conditions and biogas yield Two continuous stirred tank reactors (CSTRs) with a working volume of 2 L, were used to assess the anaerobic digestibility of corn stover residues. The first reactor was filled with anaerobic sludge and corn stover residues and served as the control (R ref ), while the second reactor contained anaerobic sludge and corn stover residues, in the presence of rumen fluid (Rc/r) applied at various concentrations. CSTRs were tested under various organic loading rates to identify optimum corn stover residue concentration (corn stover concentrations of 1, 2 and 3 g VS/L were tested). The hydraulic retention time (HRT) in both bioreactors was set at 38 days during the whole experimental period. The pH in the reactors was adjusted to 7.2 and mesophilic conditions were achieved in a water bath set at 37.5 o C. Rc/r was initially inoculated with rumen fluid containing corn stover and anaerobic sludge at 1:1, vol/vol, and biogas yield was monitored. A gradual decline in biogas production in Rc/r led to the addition rumen fluid in various amounts at both Rc/r and R ref . 2.4 Analytical methods TS, VS, COD, TKN and NH 4 + -N were analyzed according to APHA standard methods (APHA 2005 ). The pH was measured by a pH meter (Multi 3510 IDS, WTW) and the electrical conductivity (EC) by an EC meter (HANNA HI9033). In the CSTRs, the volumetric method based on acidic water displacement was employed to calculate biogas production (Angelidaki et al. 2009 ). The methane content in the produced biogas was measured using a 4 N NaOH solution, in which CO 2 was entrapped and separated from CH 4 (Pertiwiningrum et al. 2019 ). All the analyses were performed on centrifuged samples (4000 rpm for 5 min). Table 2 shows the characteristics of Rc/r and Rref during startup. Table 2 CSTRs’ feeding characteristics during startup. TS (% w/v) VS (% w/v) pH EC (mS/cm) COD (g/L) Rref 3.40 ± 1.90 2.20 ± 1.30 7.15 8.50 6.60 ± 3.10 Rc/r 2.20 ± 0.20 1.30 ± 1.60 7.20 10.50 5.70 ± 1.20 3 Results and discussion 3.1 Biochemical methane potential (BMP) tests of corn stover Corn stover residues are characterized by a tough lignocellulose structure, which for the common hydrolytic microorganisms are not easy to hydrolyze (Liew et al. 2012 ). The complexity and low bioavailability of lignocellulose lead to longer digestion time and lower biogas yield (He et al. 2008 ). Use of ruminal microbial consortia can be considered as an economically-feasible and technologically-effective treatment approach to facilitate anaerobic digestion of lignocellulose and enhance biogas production (Yıldırım et al. 2017 ; Meyer et al. 2021). BMP tests were implemented in order to explore the ability of rumen microbiota to disrupt the complex lignocellulose structure, in the presence of controls (absence of rumen microbiota). The results of the BMP tests are presented in Fig. 1. The cumulative biogas production during an experimental period of twenty-eight (28) days was comparatively evaluated in BMP tests in the presence and absence of rumen fluid (control) (Fig. 1a). A low biogas yield was determined during evaluation of BMP in trials with anaerobic sludge and corn stover residues, without the addition of rumen fluid. The maximum biogas production was 41.6 mL/g VS fed , with an average of 25.04 mL/g VS fed . The addition of 4%, 6% and 12% w/v corn stover residues in rumen fluid was tested in BMP tests resulted in cumulative biogas yields of 282.57, 119.08 and 87.95 mL/g VS fed , respectively. Addition of rumen microbiota enhanced the breakdown of lignocellulose content of corn stover residues into simpler compounds, resulting in a two- to seven-fold increase in biogas production (biogas production was at least 153% greater than that in the reference bioreactor, in the absence of rumen fluid). Approximately 70% of the total biogas was produced within the first 12 days of anaerobic digestion. Especially, in BMP tests applying the lowest concentration of corn stover in rumen fluid resulted in the highest biogas production. Biogas yield was affected by the addition of rumen microbial consortium, as 72% of the total cumulative biogas production was reached at day 12, while in non-rumen fluid corn stover residue control was 55% at the same time period. The daily biogas production in BMP tests containing rumen fluid showed a sharp increase (Fig. 1b). Rumen microbiota resulted in an increase in hydrolysis rate since rumen fluid addition in these trials resulted in a faster and higher biogas production. During the first 10 days of operation, the average biogas production in BMP tests in the presence of 4%, 6% and 12% w/v corn stover in rumen fluid was equal to 11.7, 6.3 and 4.3 mL/g VS respectively, while, without the addition of rumen fluid, the biogas production was only 1.6 mL/g VS. After the day 11, biogas production in the tests with 6% and 12% w/v corn stover in rumen fluid gradually decreased, estimating an average biogas release of 1.8 and 1.5 mL/g VS fed respectively, which was similar to biogas production without rumen fluid addition (1.8 mL/g VS), while, in the experiment with 4% w/v corn stover in rumen fluid, a high biogas production of 10.3 mL/g VS was maintained. From day 21 until the end of the experimental period (day 28), biogas production in the presence of 6% and 12% w/v corn stover in rumen fluid and without rumen microbiota was below 1 mL/g VS. At the same period, the biogas production in the presence of 4% w/v corn stover in rumen fluid also reduced, but in a lesser extent, recording an average of biogas yield of 2.7 mL/g VS. 3.2 Anaerobic digestibility of corn stover residues in CSTRs using rumen fluid as hydrolytic inoculum Based on the promising results of the BMP tests, where corn stover residues were treated with rumen microbiota, the next step was to evaluate the effectiveness of rumen microorganisms as hydrolytic inoculum in bench-scale CSTRs. The Rc/r treating corn stover residues was operated under a ratio of 1:1 v/v anaerobic sludge: rumen fluid, while in the Rref reactor, corn stover residues were digested using anaerobic sludge in the absence of rumen fluid. To determine the biogas production derived from the rumen fluid, a third bioreactor was initially setup and operated. Corn stover is rich in lignin, hemicellulose and cellulose (Ruan et al. 2019 ; Sues 2013 ) and, due to their slow rate of hydrolysis, is considered as difficult to decompose by the anaerobic sludge microbiota. The average daily biogas production in the reference bioreactor (Rref) was 0.14 ± 0.03 L/g VS feeding . Meanwhile, anaerobic digestion of rumen fluid in the absence of corn stover residues resulted in the biogas production of 0.15 ± 0.02 L/gVS feeding , indicating a limited contribution of rumen fluid alone in the overall biogas production. Thus, biogas production from either corn stover residues or rumen fluid alone is restricted in the presence of anaerobic sludge. In the CSTR treating corn stover residues in the presence of anaerobic sludge and rumen fluid at ratio of 1:1 v/v (Rc/r), 3.73 times more biogas was produced compared to the reference reactor (R ref ). During the first 15 days of operation, the average daily biogas production in the Rc/r was 0.64 ± 0.13 L/g VS feeding , whereas, thereafter, the average daily biogas production was reduced to 0.50 ± 0.10 L/g VS feeding and remained stable for the rest of the experimental period. Thus, biogas production in the Rc/r was much higher compared to the reference bioreactor (R ref ). Rumen fluid microbiota clearly enhanced lignocellulose degradation through the secretion of hydrolytic enzymes targeting the beta-glycosidic bonds (Fang et al. 2016 ), so assisting the hydrolysis of corn stover residues into monosaccharides, which can be easily fermented to VFAs and low MW alcohols, biotransformed to acetate through acetogenesis and successively bioconverted to biogas via methanogenesis. Although rumen microorganisms are capable of degrading lignocellulosic residues, they possess limited ability to sustain their hydrolytic activity for extended period of time. In an anaerobic digester operating under neutral pH, it appears more difficult for this specialized microbiota to reproduce and sustain their hydrolytic activity since the optimum pH in ruminant animals’ stomachs is usually below 7, with the pH value being influenced from animals’ diet (Grünberg and Constable 2009). Due to the above fact, rumen fluid was needed to be periodically added in the Rc/r reactor to maintain hydrolytic activity at high level. 3.3 Improvement of biogas production by rumen microbiota Figure 2 demonstrates the biogas production in each bioreactor during the three experimental periods examined. At OLR of 1 g VS/L.d (Fig. 2a), Rc/r, which operated with 4% w/v corn stover residues in rumen fluid, resulted in a biogas production of 1.01 ± 0.15 L/g VS added , corresponding to 6.6-fold increase in biogas yield compared to R ref (biogas production in the R ref was equal to 0.15 ± 0.05 L/g VS added ), while Rc/r operation under 10% and 20% w/v corn stover in rumen fluid produced 0.69 ± 0.07 and 0.51 ± 0.05 L biogas/g VS added , thus increasing biogas production compared to the reference reactor by 4.6- and 3.6-fold respectively. This result is in accordance with the findings of previous studies, which reported that corn stover hydrolysis by the cellulases of hydrolytic microbiota is more effective at the beginning of the hydrolysis stage, and at low lignocellulosic biomass load (Li et al. 2017). Indeed, to achieve the highest biogas production, an optimal balance should exist among the hydrolysis of lignocellulosic biomass from rumen microbiota to reduced sugars, their fermentation to VFAs and low MW alcohols by acidogenic bacteria and yeasts, and subsequently their bioconversion to biogas by acetogenic bacteria and methanogenic archaea. During the second experimental period (Fig. 2b), OLR was increased to 2 g VS/L.d in the CSTRs. Rc/r performance was tested under 8%, 20% and 40% w/v corn stover in rumen fluid. Among the three corn stover residues to rumen fluid ratios tested, better performance regarding biogas yield was observed at 8% w/v corn stover in rumen fluid, producing 0.76 ± 0.10 L biogas/g VS added , whereas biogas production at 20% and 40% corn stover in rumen fluid were 0.73 ± 0.04 L/g VS added and 0.70 ± 0.08 L/g VS added , respectively. In contrast, biogas yield in the reference reactor was equal to 0.32 ± 0.06 L/g VS added . This increase in biogas production in the Rc/r compared to R ref is indicative of the beneficial effect of rumen fluid microbiota on the hydrolysis of lignocellulosic biomass. These findings are in accordance with the results obtained from the BMP tests, where the lowest lignocellulosic biomass to rumen fluid ratio examined resulted in the highest biogas production. However, biogas production in the Rc/r at OLR 2 g VS/L.d was much lower than the biogas produced at OLR of 1 g VS/L.d. The highest biogas production at OLR 2 g VS/L.d compared to OLR 1 g VS/L.d, which was observed in the presence of 20% w/v corn stover in rumen fluid, may be attributed to the higher biomass of easily biodegradable part of corn stover biomass, which was more digestible. Li et al. (2017) reported that the easily to hydrolysis part of lignocellulosic biomass is firstly broken down by the hydrolytic enzymes of rumen microbiota. At OLR of 3 g VS/L.d (Fig. 2c), Rc/r was operated under 12%, 30% and 60% w/v corn stover residues in rumen fluid. At 12% w/v corn stover residues in rumen fluid, biogas production was high and equal to 0.84 ± 0.05 L/g VS added . At corn stover residues of 30% and 60% w/v in rumen fluid, biogas production was determined to be 0.70 ± 0.08 L/g VS added and 0.66 ± 0.06 L/g VS added , respectively. The higher biogas production in Rc/r compared to R ref (biogas yield of 0.3 ± 0.04 L/g VS added ) demonstrates the positive impact of rumen fluid addition on biogas production from lignocellulosic substrates. Under various OLR and corn stover residues to rumen fluid ratios, the Rc/r produced more biogas than the reference reactor, resulting in biogas production enhancement by 2–6.3 fold since rumen microbiota facilitate the degradation of corn stover residues. In the reference reactor (Rref), biogas production was restricted at all OLRs, estimating to range between 0.15 and 0.34 L/g VS fed , since anaerobic sludge microbiota are not particularly capable of breaking down the lignocellulose structure of corn stover residues. Similar to our study, Wang et al. (2017) reported limited biogas production, i.e. 0.19 L/g VS fed during anaerobic digestion of corn stover alone. The activity of the rumen microbiota not only improved biogas yield during anaerobic digestion of corn stover residues, but also the methane content in the biogas. In particular, during the anaerobic digestion of corn stover, biogas composition in methane in the reference reactor (R ref ) was 50%, while the respective percentage in the Rc/r was 68% on average. Thus, the use of rumen microbiota to enhance biogas production and methane content during anaerobic digestion of lignocellulosic biomass is a promising, economically-feasible, sustainable bioengineering approach. 4 Conclusions As a biological treatment method, rumen fluid microorganisms proved to be effective in hydrolyzing corn stover residues and breaking down its complex structure, indicating the feasibility of this treatment approach in valorizing lignocellulosic biomass. According to the BMP tests, 4% w/v corn stover residues in rumen fluid resulted in the highest biogas production (biogas yield 181.7 mL/g VS added ). In the CSTR, corn stover digestibility significantly increased by 2 to 6.3 fold in the addition of rumen fluid, with 4% w/v corn stover residues in rumen fluid at OLR of 1 g VS/L.d resulted in the highest biogas yield. Indeed, corn stover, an abundant agricultural residue, with considerable energy potential, can be effectively exploited with the use of rumen fluid inocula to enhance the hydrolysis of lignocellulosic biomass and substantially the biogas yield. Declarations Author contribution Conceptualization: A.M. and P.M.; methodology: A.M., S.N. and P.M.; formal analysis and investigation: A.M.; data curation: A.M.; writing—original draft preparation: A.M.; writing—review and editing: A.M., S.N. and P.M.; resources: A.M. and P.M. Funding This research received no external funding. Data Availability The authors confirm that the data supporting the findings of this study are available within the article. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication Not applicable. Competing interests The authors declare no competing interests. References Angelidaki I, Alves M, Bolzonella D, Borzacconi L, Campos JL, Guwy AJ, Kalyuzhnyi S, Jenicek P, van Lier JB (2009) Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Sci Technol 59(5): 927–934. https://doi.org/10.2166/wst.2009.040 APHA (2005) Standard Methods for the Examination of Water and Wastewater, 21 th ed. American Public Health Association, Washington DC, USA. Baek DJ, Kwon HC, Mun AL, Lim JR, Park SW, Han JS (2022) A comparative analysis of rumen pH, milk production characteristics, and blood metabolites of Holstein cattle fed different forage levels for the establishment of objective indicators of the animal welfare certification standard. Anim Biosci 35(1):147. https://doi.org/10.5713/ab.21.0079 Cirne DG, Lehtomäki A, Björnsson L, Blackall LL (2007) Hydrolysis and microbial community analyses in two-stage anaerobic digestion of energy crops. J Appl Microbiol 103:516e27. https://doi.org/doi:10.1111/j.1365-2672.2006.03270.x Fang W, Zhang P, Gou X, Zhang H, Wu Y, Ye J, Zeng G (2016) Volatile fatty acid production from spent mushroom compost: Effect of total solid content. Int Biodeter Biodegr 113:217–221. https://doi.org/10.1016/j.ibiod.2016.03.025 FitzPatrick MF, Champagne P, Cunningham M, Whitney RA (2010) A biorefinery processing perspective: Treatment of lignocellulosic materials for the production of value-added products. Bioresour Technol 101(23):8915–8922. https://doi.org/10.1016/j.biortech.2010.06.125 Frigon JC, Guiot SR (2010) Biomethane production from starch and lignocellulosic crops: a comparative review. Biofuels, Bioprod Biorefin 4(4):447–458. https://doi.org/10.1002/bbb.229 Gu Y, Chen XH, Liu ZG, Zhou XF, Zhang YL (2014) Effect of inoculum sources on the anaerobic digestion of rice straw. Bioresour Technol 158:149e155. https://doi.org/doi:10.1016/j.biortech.2014.02.01 Hasan MR, Anzar N, Sharma P, Malode S J, Shetti NP, Narang J, Kakarla RR (2023) Converting biowaste into sustainable bioenergy through various processes. Bioresour Technol Rep 101542. https://doi.org/10.1016/j.biteb.2023.101542 He Y, Pang Y, Liu Y, Li X, Wang K (2008) Physicochemical characterization of rice straw pretreated with sodium hydroxide in the solid state for enhancing biogas yield. Energy Fuels 22:2775–2781. https://doi.org/10.1021/ef8000967 Li F, Zhang P, Zhang G, Tang X, Wang S, Jin S (2016) Enhancement of corn stover hydrolysis with rumen fluid pretreatment at different solid contents: Effect, structural changes and enzymes participation. Int Biodeter Biodegr 119:405–412. https://doi.org/10.1016/j.ibiod.2016.10.038 Li Y, Park SY, Zhu J (2010) Solid- State anaerobic digestion for methane production from organic waste. Renew Sust Energy Rev 15(1):821–826. http://dx.doi.org/10.1016/j.rser.2010.07.042 Liew, L, Shi J, Li Y (2012) Methane production from solid-state anaerobic digestion of lignocellulosic biomass. Biomass Bioenergy 46:125–132. http://dx.doi.org/10.1016/j.biombioe.2012.09.014 Mankar AR, Pandey A, Modak A, Pant KK (2021) Pretreatment of lignocellulosic biomass: A review on recent advances. Bioresour Technol 334:125235. https://doi.org/10.1016/j.biortech.2021.125235 McKendry P (2002) Energy production from biomass (part 2): conversion technologies. Bioresour Technol 83(1): 47–54. https://doi.org/10.1016/S0960-8524(01)00119-5 Meyer G, Okudoh V, van Rensburg E (2022) A rumen based anaerobic digestion approach for lignocellulosic biomass using barley straw as feedstock. S Afr J Chem Eng 41:98–104. https://doi.org/10.1016/j.sajce.2022.05.005 Monga D, Shetti NP, Basu S, Kakarla RR (2023) Recent advances in various processes for clean and sustainable hydrogen production. Nano-Struct Nano-Objects 33:100948. https://doi.org/10.1016/j.nanoso.2023.100948 Montoneri E, Savarino P, Bottigliengo S, Boffa V, Prevot AB, Fabbri D, Pramauro E (2009) Biomass wastes as renewable source of energy and chemicals for the industry with friendly environmental impact. Fresenius Environ Bull 18(2):219–223. Nguyen LN, Nguyen AQ, Johir MAH, Guo W, Ngo HH, Chaves AV, Nghiem LD (2019) Application of rumen and anaerobic sludge microbes for bio harvesting from lignocellulosic biomass. Chemosphere 228:702–708. https://doi.org/10.1016/j.chemosphere.2019.04.159 Pertiwiningrum A, La’aliya I, Yusiati LM, Harto AW (2019) The performance of biogas combustion after carbon dioxide absorption using sodium hydroxide (NaOH). In: IOP Conference Series: Earth and Environmental Science, vol. 387, no. 1, p. 012051. IOP Publishing. https://doi.org/10.1088/1755-1315/387/1/012051 Ruan Z, Wang X, Liu Y, Liao W (2019) Chapter 3: Corn. Integrated Processing Technologies for Food and Agricultural By-Products. Cham, Switzerland: Springer International Publishing. Srivastava RK, Shetti NP, Reddy KR, Kwon EE, Nadagouda MN, Aminabhavi TM (2021) Biomass utilization and production of biofuels from carbon neutral materials. Environ Pollut 276:11673. https://doi.org/10.1016/j.envpol.2021.116731 Srivastava RK, Shetti NP, Reddy KR, Nadagouda MN, Badawi M, Bonilla Petriciolet A, Aminabhavi TM (2023) Valorization of biowastes for clean energy production, environmental depollution and soil fertility. J Environ Manage 332:117410. https://doi.org/10.1016/j.jenvman.2023.117410 Sues T (2013) Analysis of corn stover structural properties for mechanical processing applications. J Undergrad Res 11:article 9. Takizawa S, Baba Y, Tada C, Fukuda Y, Nakai Y (2018) Pretreatment with rumen fluid improves methane production in the anaerobic digestion of paper sludge. Waste Manage 78:379e384. https://doi.org/10.1016/j.wasman.2018.05.046 Wang S, Li F, Wu D, Zhang P, Wang H, Tao X, Ye J, Nabi M (2018a) Enzyme pretreatment enhancing biogas yield from corn stover: feasibility, optimization, and mechanism analysis. J. Agric Food Chem 66(38): 10026-10032. https://doi.org/10.1021/acs.jafc.8b03086 Wang S, Zhang G, Zhang P, Ma X, Li F, Zhang H, Tao X, Ye J, Nabi M (2018b) Rumen fluid fermentation for enhancement of hydrolysis and acidification of grass clipping. J Environ Manage 220:142e148. https://doi.org/10.1016/j.jenvman.2018.05.027 Yıldırım E, Ince O, Aydin S, Ince B (2017) Improvement of biogas potential of anaerobic digesters using rumen fungi. Renew. Energy 109:346–353. https://doi.org/10.1016/j.renene.2017.03.021 Yu S, Zhang G, Li J, Zhao Z, Kang X. (2013) Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge. Bioresour Technol 146:758–761. http://dx.doi.org/10.1016/j.biortech.2013.07.087 Zheng Y, Zhao J, Xu F, Li Y (2014) Pretreatment of lignocellulosic biomass for enhanced biogas production. Prog Energy Combust Sci 42:35–53. http://dx.doi.org/10.1016/j.pecs.2014.01.001 Zhou M, Yang J, Wang H, Jin T, Hassett DJ, Gu T (2014) Chapter 9 - Bioelectrochemistry of microbial fuel cells and their potential applications in bioenergy. Bioenergy Research: Advances and Applications, pp. 131–152. https://doi.org/10.1016/B978-0-444-59561-4.00009-7 Zhu J, Wan C, Li Y (2010) Enhanced solid-state anaerobic digestion of corn stover by alkaline pretreatment. Bioresour Technol 101:7523–7528. https://doi.org/10.1016/j.biortech.2010.04.060 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 24 Oct, 2024 Read the published version in Environmental Processes → Version 1 posted Editorial decision: Revision requested 07 Aug, 2024 Reviews received at journal 09 Jun, 2024 Reviewers agreed at journal 03 Jun, 2024 Reviewers agreed at journal 10 Apr, 2024 Reviewers invited by journal 05 Apr, 2024 Editor assigned by journal 29 Mar, 2024 Submission checks completed at journal 28 Mar, 2024 First submitted to journal 27 Mar, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4174541","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":285287039,"identity":"b920d9be-f199-4d5e-bea2-8b13ac1aa141","order_by":0,"name":"Anastasia Makri","email":"","orcid":"","institution":"Democritus University of Thrace","correspondingAuthor":false,"prefix":"","firstName":"Anastasia","middleName":"","lastName":"Makri","suffix":""},{"id":285287040,"identity":"392f3052-9f97-444b-917e-04c885dbfa94","order_by":1,"name":"Spyridon Ntougias","email":"","orcid":"","institution":"Democritus University of Thrace","correspondingAuthor":false,"prefix":"","firstName":"Spyridon","middleName":"","lastName":"Ntougias","suffix":""},{"id":285287041,"identity":"def58d9b-5c66-4d81-abbc-767fd4ad1301","order_by":2,"name":"Paraschos Melidis","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA4ElEQVRIiWNgGAWjYHACxgMJbBIM/GA2GwODATF6wFokGyBaJIjTAjb8ALFazMUOHzjwoMzC3vh47zEJhrLDdeYM7Nc+4NNiOTst4UDCOYnEbWfOpUkwnDssYdnAUzwDnxaD2zkGBxLbJBLMbuQYGzC2HZYwOMCTjNdhBrfzP4C02BvPf0O0lhwGkBbGDRI8hg8gWtgP49UC9IsB2C8zzuQYPkg4ly654TAPM14t5tLJDx/+KKuz528/Y3DgQ5k1v8Hx9sf4HYbCSwARzDz4owabLPsDvFpGwSgYBaNgxAEAUsNLJsoSRMUAAAAASUVORK5CYII=","orcid":"","institution":"Democritus University of Thrace","correspondingAuthor":true,"prefix":"","firstName":"Paraschos","middleName":"","lastName":"Melidis","suffix":""}],"badges":[],"createdAt":"2024-03-27 08:27:22","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4174541/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4174541/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s40710-024-00738-y","type":"published","date":"2024-10-24T15:57:37+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":53921542,"identity":"802a45f3-7415-4d58-99a0-982b4236ac3b","added_by":"auto","created_at":"2024-04-02 09:00:28","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":51824,"visible":true,"origin":"","legend":"\u003cp\u003eCumulative (a) and daily (b) biogas yield of corn stover residues using rumen fluid as inoculum.\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4174541/v1/c0e4ed3690534062be93a9c9.png"},{"id":53921543,"identity":"814cc3ab-4c5e-4a69-a308-6b1502211a05","added_by":"auto","created_at":"2024-04-02 09:00:28","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":46948,"visible":true,"origin":"","legend":"\u003cp\u003eImpact of rumen fluid addition on biogas production during anaerobic digestion of corn stover residues under various organic loading rates tested; a) OLR of 1 g VS/L.d, b) OLR of 2 g VS/L.d, and c) OLR of 3 g VS/L.d.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4174541/v1/e020c86337c7e7906062ce12.png"},{"id":67681876,"identity":"cce3d605-2b79-4a74-bdbd-cccb00a713f1","added_by":"auto","created_at":"2024-10-28 16:10:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":646699,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4174541/v1/80abe6d5-1e33-413c-8504-7d4c212eb213.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Enhancing anaerobic degradation of corn stover residues and biogas production via rumen microorganisms","fulltext":[{"header":"Highlights","content":"\u003cul type=\"disc\"\u003e\n \u003cli\u003eHigh corn stover residues availability for biogas production.\u003c/li\u003e\n \u003cli\u003eEffective breakdown of lignocellulose can be achieved by biological pretreatment.\u003c/li\u003e\n \u003cli\u003ePretreatment by rumen microbiota improved biogas production by 2-6.3 times.\u003c/li\u003e\n \u003cli\u003eOptimum corn stover concentration for high biogas production was 4% in rumen fluid.\u003c/li\u003e\n\u003c/ul\u003e"},{"header":"1 Introduction","content":"\u003cp\u003eIn recent years, the depletion of fossil fuels has raised the need to reduce dependence on fossil fuels. The daily energy demand is high, because of the increasing energy consumption, due to the overpopulation (Monga et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). The shift toward renewable energy production is necessary and crucial. The valorization of agricultural residues for generating energy and recovering nutrients has recently gained ground as an effective sustainable solution (Rahil Hasan et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2023\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eLooking for sustainable, abundant, and easy-to-be-found raw materials, agricultural wastes and residues could be a very attractive feedstock to contribute to energy recovery. Moreover, the daily amount of biowaste is increasing, indicating difficulties in terms of waste management (Srivastava et al. \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). On the other hand, biowaste has the potential to be converted into an environmental-friendly fuel through various bioengineering processes, within the biorefinery concept (Srivastava et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAnaerobic digestion is a biological process for energy recovery. It is a process where the organic content is anaerobically decomposed by a microbial consortium to produce biogas (Roubaud and Favrat 2005). It does not only provide an alternative energy source, but also consists of a way to decompose organic waste and reduce greenhouse gas emissions (Frigon and Guiot \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). It includes four biological steps, i.e. biomass hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Li et al. \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Hydrolysis is the rate-limiting step in the breakdown of lignocellulosic biomass (Cirne et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2007\u003c/span\u003e). To improve the efficiency of biomass hydrolysis and consequently the biogas yield, it is essential to carry out a pretreatment step of lignocellulosic materials prior to anaerobic degradation. The pretreatment aims to break down the structural barriers of lignocellulosic biomass, making the cellulosic and hemicellulosic chains more accessible to microbial breakdown (Zheng et al. \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This can be achieved with various pretreatment approaches, such as physical, chemical and biological methods, or combinations of them (FitzPatrick 2010).\u003c/p\u003e \u003cp\u003eLignocellulosic biomass has an advantage over other feedstock choices, as it does not compete directly with food or feed production. Such residues contain a remarkable amount of organic content, which could be considered as an alternative energy source, and achievement of higher biogas yields can make these crop residues ideal for biogas production (McKendry \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Due to the economic concerns of feedstock collection and transportation, the use of lignocellulosic biomass as a feedstock for anaerobic digestion is greatly influenced by feedstock accessibility and availability (Li et al. 2011).\u003c/p\u003e \u003cp\u003eSeveral lignocellulosic materials derived from agro-industrial and forestry activities have been used as feedstock, as they are abundant and available all over the year (Montoneri et al. \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). Such residues are estimated to be over 10\u0026nbsp;million tons annually (Nguyen et al. \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Corn stover is common agricultural byproduct, which is widely available in corn production areas. It is one of the most popular grains in Greek fields and around the world, resulting in a significant amount of corn stover residues. Since now, the most common technique to manage these residues is burning. However, it is not advised to burn corn stover residues, due to the negative ecological impact of the process, and therefore alternatives must be found. According to Gu et al. (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), such residues can be converted into renewable energy through anaerobic fermentation, so this process may solve numerous environmental and energy-related issues.\u003c/p\u003e \u003cp\u003eAlthough lignocellulosic biomass has high organic content and energy value, its structure highly resists to hydrolysis. Most microbes cannot breakdown its structure without pretreatment, so it is considered difficult to ferment such biomass. Some anaerobic bacteria can successfully degrade the lignocellulosic biomass, but in slow rates (Zhou et al. \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Therefore, their use as a renewable energy source has been reported to require the application of pretreatment methods to enhance the digestibility of lignocellulosic biomass (Mankar et al. \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAn economically-feasible and environmental-friendly method to break down the lignocellulose structure is its hydrolysis with the specialized microbial community found in the rumen fluid of ruminant animals. The rumen fluid microbiota enhance lignocellulose hydrolysis and biomass fermentation through the secretion of hydrolytic enzymes targeting β-glucosidic bonds (Yu et al. \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). This symbiotic interaction of rumen microorganisms with their animal host evolved for millions of years, thus considering rumen microbiota as specialized lignocellulose degraders. Recently, the exploitation of rumen microbiome for enhancing lignocellulosic biomass degradation has led to promising results (Takizawa et al. \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang et al. 2018).\u003c/p\u003e \u003cp\u003eThis research work examines the effectiveness of applying rumen fluid inoculum as a pretreatment approach for the disruption of lignocellulosic corn stover residues and its valorization through anaerobic digestion. A scale-up approach was employed through the initial performance of Biochemical methane potential (BMP) tests to assess the anaerobic digestibility of corn stover in the presence of rumen fluid inoculum, followed by the investigation of the beneficial effect of rumen microbiota in the hydrolysis of its lignocellulosic biomass in Continuous Stirring Tank Reactors (CSTRs) to define optimum operating conditions regarding organic loading rate, biogas yield, methane content and amount of rumen fluid inoculum.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Lignocellulosic biomass and inoculum source\u003c/h2\u003e \u003cp\u003eCorn stover residues were collected from corn fields after harvest. They were oven-dried at 60\u003csup\u003eo\u003c/sup\u003eC for 24 h, milled through a grinder and passed through a sieve of less than 2 mm size. Both total solids (TS) and volatile solids (VS) were determined in the sieved product and stored at room temperature for further use in downstream experiments. Corn stover residue dry content was 92.8\u0026thinsp;\u0026plusmn;\u0026thinsp;0.92%, whereas corn stover organic matter was determined to be 71.6\u0026thinsp;\u0026plusmn;\u0026thinsp;3.6%. Anaerobic sludge was obtained from a full-scale anaerobic digester nearby the city of Xanthi, Greece (40.99, 24.89), with the TS and VS of the anaerobic sludge being estimated 7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% w/v and 2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02% w/v, respectively. The inoculum was left for 2 weeks to be inactivated before the inoculation with the rumen microbiota. Rumen fluid, which served as the hydrolytic inoculum, was collected from the first compartment of cow's stomach and drained through a filter prior its use. The pH of the rumen fluid was 6.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10, with their TS and VS being estimated as 3.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20% w/v and 0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10% w/v, respectively. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e shows the main characteristics of the lignocellulosic feedstock, rumen fluid and anaerobic sludge inocula.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBasic characteristics of hydrolytic inoculum and anaerobic sludge.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eSample\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTotal Solids (TS)\u003c/p\u003e \u003cp\u003e(% w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eVolatile Solids (VS)\u003c/p\u003e \u003cp\u003e(% w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eNH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N\u003c/p\u003e \u003cp\u003e(g/L)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCOD\u003c/p\u003e \u003cp\u003e(g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eRumen fluid\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e6.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.77\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.73\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.99\u0026thinsp;\u0026plusmn;\u0026thinsp;0.14\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e19.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.18\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAnaerobic sludge\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e7.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.06\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.28\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e0.49\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e13.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.80\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Biochemical methane potential (BMP) tests\u003c/h2\u003e \u003cp\u003eBiochemical methane potential (BMP) tests were carried out in batch reactors of 250 mL volume, with a working volume of 150 mL, which run in triplicate for a period of 28 days. The substrate-to-anaerobic inoculum (S/I) ratio in all tests was kept at 3:1, wt/wt. Each experimental setup included anaerobic sludge (blank), anaerobic sludge with corn stover residues (control), and anaerobic sludge with corn stover residues and addition of rumen fluid (sample for evaluation). In BMP tests, 4%, 6% and 12% w/v corn stover residues in rumen fluid was examined. The pH in batch reactors was adjusted to 7, and their content was flushed with N\u003csub\u003e2\u003c/sub\u003e/CO\u003csub\u003e2\u003c/sub\u003e (4/1, v/v) to create anaerobic conditions. Then, batch reactors were placed in a water bath at 37.5\u003csup\u003eo\u003c/sup\u003eC and the produced biogas was measured through the manometric method (Angelidaki et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The cumulative methane production for each BMP test was determined by subtracting the inoculum contribution (blank).\u003c/p\u003e \u003cp\u003e \u003cb\u003e2.3 Assessment of anaerobic digestibility of corn stover residues in continuous stirred tank reactors (CSTRs): optimum operating conditions and biogas yield\u003c/b\u003e \u003c/p\u003e \u003cp\u003eTwo continuous stirred tank reactors (CSTRs) with a working volume of 2 L, were used to assess the anaerobic digestibility of corn stover residues. The first reactor was filled with anaerobic sludge and corn stover residues and served as the control (R\u003csub\u003eref\u003c/sub\u003e), while the second reactor contained anaerobic sludge and corn stover residues, in the presence of rumen fluid (Rc/r) applied at various concentrations. CSTRs were tested under various organic loading rates to identify optimum corn stover residue concentration (corn stover concentrations of 1, 2 and 3 g VS/L were tested). The hydraulic retention time (HRT) in both bioreactors was set at 38 days during the whole experimental period. The pH in the reactors was adjusted to 7.2 and mesophilic conditions were achieved in a water bath set at 37.5\u003csup\u003eo\u003c/sup\u003eC. Rc/r was initially inoculated with rumen fluid containing corn stover and anaerobic sludge at 1:1, vol/vol, and biogas yield was monitored. A gradual decline in biogas production in Rc/r led to the addition rumen fluid in various amounts at both Rc/r and R\u003csub\u003eref\u003c/sub\u003e.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Analytical methods\u003c/h2\u003e \u003cp\u003eTS, VS, COD, TKN and NH\u003csub\u003e4\u003c/sub\u003e\u003csup\u003e+\u003c/sup\u003e-N were analyzed according to APHA standard methods (APHA \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2005\u003c/span\u003e). The pH was measured by a pH meter (Multi 3510 IDS, WTW) and the electrical conductivity (EC) by an EC meter (HANNA HI9033). In the CSTRs, the volumetric method based on acidic water displacement was employed to calculate biogas production (Angelidaki et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). The methane content in the produced biogas was measured using a 4 N NaOH solution, in which CO\u003csub\u003e2\u003c/sub\u003e was entrapped and separated from CH\u003csub\u003e4\u003c/sub\u003e (Pertiwiningrum et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). All the analyses were performed on centrifuged samples (4000 rpm for 5 min). Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e shows the characteristics of Rc/r and Rref during startup.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eCSTRs\u0026rsquo; feeding characteristics during startup.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"6\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTS\u003c/p\u003e \u003cp\u003e(% w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eVS\u003c/p\u003e \u003cp\u003e(% w/v)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003epH\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eEC (mS/cm)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eCOD (g/L)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRref\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.90\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.30\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.15\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e8.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e6.60\u0026thinsp;\u0026plusmn;\u0026thinsp;3.10\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eRc/r\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e2.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.60\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e7.20\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e10.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c6\"\u003e \u003cp\u003e5.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.20\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3 Results and discussion","content":"\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e3.1 Biochemical methane potential (BMP) tests of corn stover\u003c/h2\u003e \u003cp\u003eCorn stover residues are characterized by a tough lignocellulose structure, which for the common hydrolytic microorganisms are not easy to hydrolyze (Liew et al. \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). The complexity and low bioavailability of lignocellulose lead to longer digestion time and lower biogas yield (He et al. \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Use of ruminal microbial consortia can be considered as an economically-feasible and technologically-effective treatment approach to facilitate anaerobic digestion of lignocellulose and enhance biogas production (Yıldırım et al. \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Meyer et al. 2021). BMP tests were implemented in order to explore the ability of rumen microbiota to disrupt the complex lignocellulose structure, in the presence of controls (absence of rumen microbiota). The results of the BMP tests are presented in Fig.\u0026nbsp;1. The cumulative biogas production during an experimental period of twenty-eight (28) days was comparatively evaluated in BMP tests in the presence and absence of rumen fluid (control) (Fig.\u0026nbsp;1a). A low biogas yield was determined during evaluation of BMP in trials with anaerobic sludge and corn stover residues, without the addition of rumen fluid. The maximum biogas production was 41.6 mL/g VS\u003csub\u003efed\u003c/sub\u003e, with an average of 25.04 mL/g VS\u003csub\u003efed\u003c/sub\u003e. The addition of 4%, 6% and 12% w/v corn stover residues in rumen fluid was tested in BMP tests resulted in cumulative biogas yields of 282.57, 119.08 and 87.95 mL/g VS\u003csub\u003efed\u003c/sub\u003e, respectively. Addition of rumen microbiota enhanced the breakdown of lignocellulose content of corn stover residues into simpler compounds, resulting in a two- to seven-fold increase in biogas production (biogas production was at least 153% greater than that in the reference bioreactor, in the absence of rumen fluid). Approximately 70% of the total biogas was produced within the first 12 days of anaerobic digestion. Especially, in BMP tests applying the lowest concentration of corn stover in rumen fluid resulted in the highest biogas production. Biogas yield was affected by the addition of rumen microbial consortium, as 72% of the total cumulative biogas production was reached at day 12, while in non-rumen fluid corn stover residue control was 55% at the same time period.\u003c/p\u003e \u003cp\u003eThe daily biogas production in BMP tests containing rumen fluid showed a sharp increase (Fig.\u0026nbsp;1b). Rumen microbiota resulted in an increase in hydrolysis rate since rumen fluid addition in these trials resulted in a faster and higher biogas production. During the first 10 days of operation, the average biogas production in BMP tests in the presence of 4%, 6% and 12% w/v corn stover in rumen fluid was equal to 11.7, 6.3 and 4.3 mL/g VS respectively, while, without the addition of rumen fluid, the biogas production was only 1.6 mL/g VS. After the day 11, biogas production in the tests with 6% and 12% w/v corn stover in rumen fluid gradually decreased, estimating an average biogas release of 1.8 and 1.5 mL/g VS\u003csub\u003efed\u003c/sub\u003e respectively, which was similar to biogas production without rumen fluid addition (1.8 mL/g VS), while, in the experiment with 4% w/v corn stover in rumen fluid, a high biogas production of 10.3 mL/g VS was maintained. From day 21 until the end of the experimental period (day 28), biogas production in the presence of 6% and 12% w/v corn stover in rumen fluid and without rumen microbiota was below 1 mL/g VS. At the same period, the biogas production in the presence of 4% w/v corn stover in rumen fluid also reduced, but in a lesser extent, recording an average of biogas yield of 2.7 mL/g VS.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Anaerobic digestibility of corn stover residues in CSTRs using rumen fluid as hydrolytic inoculum\u003c/h2\u003e \u003cp\u003eBased on the promising results of the BMP tests, where corn stover residues were treated with rumen microbiota, the next step was to evaluate the effectiveness of rumen microorganisms as hydrolytic inoculum in bench-scale CSTRs. The Rc/r treating corn stover residues was operated under a ratio of 1:1 v/v anaerobic sludge: rumen fluid, while in the Rref reactor, corn stover residues were digested using anaerobic sludge in the absence of rumen fluid. To determine the biogas production derived from the rumen fluid, a third bioreactor was initially setup and operated. Corn stover is rich in lignin, hemicellulose and cellulose (Ruan et al. \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Sues \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2013\u003c/span\u003e) and, due to their slow rate of hydrolysis, is considered as difficult to decompose by the anaerobic sludge microbiota. The average daily biogas production in the reference bioreactor (Rref) was 0.14\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03 L/g VS\u003csub\u003efeeding\u003c/sub\u003e. Meanwhile, anaerobic digestion of rumen fluid in the absence of corn stover residues resulted in the biogas production of 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.02 L/gVS\u003csub\u003efeeding\u003c/sub\u003e, indicating a limited contribution of rumen fluid alone in the overall biogas production. Thus, biogas production from either corn stover residues or rumen fluid alone is restricted in the presence of anaerobic sludge. In the CSTR treating corn stover residues in the presence of anaerobic sludge and rumen fluid at ratio of 1:1 v/v (Rc/r), 3.73 times more biogas was produced compared to the reference reactor (R\u003csub\u003eref\u003c/sub\u003e). During the first 15 days of operation, the average daily biogas production in the Rc/r was 0.64\u0026thinsp;\u0026plusmn;\u0026thinsp;0.13 L/g VS\u003csub\u003efeeding\u003c/sub\u003e, whereas, thereafter, the average daily biogas production was reduced to 0.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 L/g VS\u003csub\u003efeeding\u003c/sub\u003e and remained stable for the rest of the experimental period. Thus, biogas production in the Rc/r was much higher compared to the reference bioreactor (R\u003csub\u003eref\u003c/sub\u003e). Rumen fluid microbiota clearly enhanced lignocellulose degradation through the secretion of hydrolytic enzymes targeting the beta-glycosidic bonds (Fang et al. \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), so assisting the hydrolysis of corn stover residues into monosaccharides, which can be easily fermented to VFAs and low MW alcohols, biotransformed to acetate through acetogenesis and successively bioconverted to biogas via methanogenesis.\u003c/p\u003e \u003cp\u003eAlthough rumen microorganisms are capable of degrading lignocellulosic residues, they possess limited ability to sustain their hydrolytic activity for extended period of time. In an anaerobic digester operating under neutral pH, it appears more difficult for this specialized microbiota to reproduce and sustain their hydrolytic activity since the optimum pH in ruminant animals\u0026rsquo; stomachs is usually below 7, with the pH value being influenced from animals\u0026rsquo; diet (Gr\u0026uuml;nberg and Constable 2009). Due to the above fact, rumen fluid was needed to be periodically added in the Rc/r reactor to maintain hydrolytic activity at high level.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e3.3 Improvement of biogas production by rumen microbiota\u003c/h2\u003e \u003cp\u003eFigure\u0026nbsp;2 demonstrates the biogas production in each bioreactor during the three experimental periods examined. At OLR of 1 g VS/L.d (Fig.\u0026nbsp;2a), Rc/r, which operated with 4% w/v corn stover residues in rumen fluid, resulted in a biogas production of 1.01\u0026thinsp;\u0026plusmn;\u0026thinsp;0.15 L/g VS\u003csub\u003eadded\u003c/sub\u003e, corresponding to 6.6-fold increase in biogas yield compared to R\u003csub\u003eref\u003c/sub\u003e (biogas production in the R\u003csub\u003eref\u003c/sub\u003e was equal to 0.15\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 L/g VS\u003csub\u003eadded\u003c/sub\u003e), while Rc/r operation under 10% and 20% w/v corn stover in rumen fluid produced 0.69\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07 and 0.51\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 L biogas/g VS\u003csub\u003eadded\u003c/sub\u003e, thus increasing biogas production compared to the reference reactor by 4.6- and 3.6-fold respectively. This result is in accordance with the findings of previous studies, which reported that corn stover hydrolysis by the cellulases of hydrolytic microbiota is more effective at the beginning of the hydrolysis stage, and at low lignocellulosic biomass load (Li et al. 2017). Indeed, to achieve the highest biogas production, an optimal balance should exist among the hydrolysis of lignocellulosic biomass from rumen microbiota to reduced sugars, their fermentation to VFAs and low MW alcohols by acidogenic bacteria and yeasts, and subsequently their bioconversion to biogas by acetogenic bacteria and methanogenic archaea.\u003c/p\u003e \u003cp\u003eDuring the second experimental period (Fig.\u0026nbsp;2b), OLR was increased to 2 g VS/L.d in the CSTRs. Rc/r performance was tested under 8%, 20% and 40% w/v corn stover in rumen fluid. Among the three corn stover residues to rumen fluid ratios tested, better performance regarding biogas yield was observed at 8% w/v corn stover in rumen fluid, producing 0.76\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10 L biogas/g VS\u003csub\u003eadded\u003c/sub\u003e, whereas biogas production at 20% and 40% corn stover in rumen fluid were 0.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 L/g VS\u003csub\u003eadded\u003c/sub\u003e and 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 L/g VS\u003csub\u003eadded\u003c/sub\u003e, respectively. In contrast, biogas yield in the reference reactor was equal to 0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 L/g VS\u003csub\u003eadded\u003c/sub\u003e. This increase in biogas production in the Rc/r compared to R\u003csub\u003eref\u003c/sub\u003e is indicative of the beneficial effect of rumen fluid microbiota on the hydrolysis of lignocellulosic biomass. These findings are in accordance with the results obtained from the BMP tests, where the lowest lignocellulosic biomass to rumen fluid ratio examined resulted in the highest biogas production. However, biogas production in the Rc/r at OLR 2 g VS/L.d was much lower than the biogas produced at OLR of 1 g VS/L.d. The highest biogas production at OLR 2 g VS/L.d compared to OLR 1 g VS/L.d, which was observed in the presence of 20% w/v corn stover in rumen fluid, may be attributed to the higher biomass of easily biodegradable part of corn stover biomass, which was more digestible. Li et al. (2017) reported that the easily to hydrolysis part of lignocellulosic biomass is firstly broken down by the hydrolytic enzymes of rumen microbiota.\u003c/p\u003e \u003cp\u003eAt OLR of 3 g VS/L.d (Fig.\u0026nbsp;2c), Rc/r was operated under 12%, 30% and 60% w/v corn stover residues in rumen fluid. At 12% w/v corn stover residues in rumen fluid, biogas production was high and equal to 0.84\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05 L/g VS\u003csub\u003eadded\u003c/sub\u003e. At corn stover residues of 30% and 60% w/v in rumen fluid, biogas production was determined to be 0.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08 L/g VS\u003csub\u003eadded\u003c/sub\u003e and 0.66\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06 L/g VS\u003csub\u003eadded\u003c/sub\u003e, respectively. The higher biogas production in Rc/r compared to R\u003csub\u003eref\u003c/sub\u003e (biogas yield of 0.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04 L/g VS\u003csub\u003eadded\u003c/sub\u003e) demonstrates the positive impact of rumen fluid addition on biogas production from lignocellulosic substrates.\u003c/p\u003e \u003cp\u003eUnder various OLR and corn stover residues to rumen fluid ratios, the Rc/r produced more biogas than the reference reactor, resulting in biogas production enhancement by 2\u0026ndash;6.3 fold since rumen microbiota facilitate the degradation of corn stover residues. In the reference reactor (Rref), biogas production was restricted at all OLRs, estimating to range between 0.15 and 0.34 L/g VS\u003csub\u003efed\u003c/sub\u003e, since anaerobic sludge microbiota are not particularly capable of breaking down the lignocellulose structure of corn stover residues. Similar to our study, Wang et al. (2017) reported limited biogas production, i.e. 0.19 L/g VS\u003csub\u003efed\u003c/sub\u003e during anaerobic digestion of corn stover alone.\u003c/p\u003e \u003cp\u003eThe activity of the rumen microbiota not only improved biogas yield during anaerobic digestion of corn stover residues, but also the methane content in the biogas. In particular, during the anaerobic digestion of corn stover, biogas composition in methane in the reference reactor (R\u003csub\u003eref\u003c/sub\u003e) was 50%, while the respective percentage in the Rc/r was 68% on average. Thus, the use of rumen microbiota to enhance biogas production and methane content during anaerobic digestion of lignocellulosic biomass is a promising, economically-feasible, sustainable bioengineering approach.\u003c/p\u003e \u003c/div\u003e"},{"header":"4 Conclusions","content":"\u003cp\u003eAs a biological treatment method, rumen fluid microorganisms proved to be effective in hydrolyzing corn stover residues and breaking down its complex structure, indicating the feasibility of this treatment approach in valorizing lignocellulosic biomass. According to the BMP tests, 4% w/v corn stover residues in rumen fluid resulted in the highest biogas production (biogas yield 181.7 mL/g VS\u003csub\u003eadded\u003c/sub\u003e). In the CSTR, corn stover digestibility significantly increased by 2 to 6.3 fold in the addition of rumen fluid, with 4% w/v corn stover residues in rumen fluid at OLR of 1 g VS/L.d resulted in the highest biogas yield. Indeed, corn stover, an abundant agricultural residue, with considerable energy potential, can be effectively exploited with the use of rumen fluid inocula to enhance the hydrolysis of lignocellulosic biomass and substantially the biogas yield.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eConceptualization: A.M. and P.M.; methodology: A.M., S.N. and P.M.; formal analysis and investigation: A.M.; data curation: A.M.; writing\u0026mdash;original draft preparation: A.M.; writing\u0026mdash;review and editing: A.M., S.N. and P.M.; resources: A.M. and P.M.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis research received no external funding.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability\u003c/strong\u003e The authors confirm that the data supporting the findings of this study are available within the article.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einterests\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAngelidaki I, Alves M, Bolzonella D, Borzacconi L, Campos JL, Guwy AJ, Kalyuzhnyi S, Jenicek P, van Lier JB (2009) Defining the biomethane potential (BMP) of solid organic wastes and energy crops: a proposed protocol for batch assays. Water Sci Technol 59(5): 927\u0026ndash;934. https://doi.org/10.2166/wst.2009.040\u003c/li\u003e\n\u003cli\u003eAPHA (2005) Standard Methods for the Examination of Water and Wastewater, 21\u003csup\u003eth\u003c/sup\u003e ed. American Public Health Association, Washington DC, USA.\u003c/li\u003e\n\u003cli\u003eBaek DJ, Kwon HC, Mun AL, Lim JR, Park SW, Han JS (2022) A comparative analysis of rumen pH, milk production characteristics, and blood metabolites of Holstein cattle fed different forage levels for the establishment of objective indicators of the animal welfare certification standard. Anim Biosci 35(1):147. https://doi.org/10.5713/ab.21.0079\u003c/li\u003e\n\u003cli\u003eCirne DG, Lehtom\u0026auml;ki A, Bj\u0026ouml;rnsson L, Blackall LL (2007) Hydrolysis and microbial community analyses in two-stage anaerobic digestion of energy crops. J Appl Microbiol 103:516e27. https://doi.org/doi:10.1111/j.1365-2672.2006.03270.x \u003c/li\u003e\n\u003cli\u003eFang W, Zhang P, Gou X, Zhang H, Wu Y, Ye J, Zeng G (2016) Volatile fatty acid production from spent mushroom compost: Effect of total solid content. Int Biodeter Biodegr 113:217\u0026ndash;221. https://doi.org/10.1016/j.ibiod.2016.03.025\u003c/li\u003e\n\u003cli\u003eFitzPatrick MF, Champagne P, Cunningham M, Whitney RA (2010) A biorefinery processing perspective: Treatment of lignocellulosic materials for the production of value-added products. Bioresour Technol 101(23):8915\u0026ndash;8922. https://doi.org/10.1016/j.biortech.2010.06.125 \u003c/li\u003e\n\u003cli\u003eFrigon JC, Guiot SR (2010) Biomethane production from starch and lignocellulosic crops: a comparative review. Biofuels, Bioprod Biorefin 4(4):447\u0026ndash;458. https://doi.org/10.1002/bbb.229\u003c/li\u003e\n\u003cli\u003eGu Y, Chen XH, Liu ZG, Zhou XF, Zhang YL (2014) Effect of inoculum sources on the anaerobic digestion of rice straw. Bioresour Technol 158:149e155. https://doi.org/doi:10.1016/j.biortech.2014.02.01\u003c/li\u003e\n\u003cli\u003eHasan MR, Anzar N, Sharma P, Malode S J, Shetti NP, Narang J, Kakarla RR (2023) Converting biowaste into sustainable bioenergy through various processes. Bioresour Technol Rep 101542. https://doi.org/10.1016/j.biteb.2023.101542\u003c/li\u003e\n\u003cli\u003eHe Y, Pang Y, Liu Y, Li X, Wang K (2008) Physicochemical characterization of rice straw pretreated with sodium hydroxide in the solid state for enhancing biogas yield. Energy Fuels 22:2775\u0026ndash;2781. https://doi.org/10.1021/ef8000967\u003c/li\u003e\n\u003cli\u003eLi F, Zhang P, Zhang G, Tang X, Wang S, Jin S (2016) Enhancement of corn stover hydrolysis with rumen fluid pretreatment at different solid contents: Effect, structural changes and enzymes participation. Int Biodeter Biodegr 119:405\u0026ndash;412. https://doi.org/10.1016/j.ibiod.2016.10.038\u003c/li\u003e\n\u003cli\u003eLi Y, Park SY, Zhu J (2010) Solid- State anaerobic digestion for methane production from organic waste. Renew Sust Energy Rev\u003cem\u003e \u003c/em\u003e15(1):821\u0026ndash;826. http://dx.doi.org/10.1016/j.rser.2010.07.042 \u003c/li\u003e\n\u003cli\u003eLiew, L, Shi J, Li Y (2012) Methane production from solid-state anaerobic digestion of lignocellulosic biomass. Biomass Bioenergy 46:125\u0026ndash;132. http://dx.doi.org/10.1016/j.biombioe.2012.09.014\u003c/li\u003e\n\u003cli\u003eMankar AR, Pandey A, Modak A, Pant KK (2021) Pretreatment of lignocellulosic biomass: A review on recent advances. Bioresour Technol 334:125235. https://doi.org/10.1016/j.biortech.2021.125235 \u003c/li\u003e\n\u003cli\u003eMcKendry P (2002) Energy production from biomass (part 2): conversion technologies. Bioresour Technol 83(1): 47\u0026ndash;54. https://doi.org/10.1016/S0960-8524(01)00119-5\u003c/li\u003e\n\u003cli\u003eMeyer G, Okudoh V, van Rensburg E (2022) A rumen based anaerobic digestion approach for lignocellulosic biomass using barley straw as feedstock. S Afr J Chem Eng 41:98\u0026ndash;104. https://doi.org/10.1016/j.sajce.2022.05.005 \u003c/li\u003e\n\u003cli\u003eMonga D, Shetti NP, Basu S, Kakarla RR (2023) Recent advances in various processes for clean and sustainable hydrogen production. Nano-Struct Nano-Objects 33:100948. https://doi.org/10.1016/j.nanoso.2023.100948\u003c/li\u003e\n\u003cli\u003eMontoneri E, Savarino P, Bottigliengo S, Boffa V, Prevot AB, Fabbri D, Pramauro E (2009) Biomass wastes as renewable source of energy and chemicals for the industry with friendly environmental impact. Fresenius Environ Bull 18(2):219\u0026ndash;223.\u003c/li\u003e\n\u003cli\u003eNguyen LN, Nguyen AQ, Johir MAH, Guo W, Ngo HH, Chaves AV, Nghiem LD (2019) Application of rumen and anaerobic sludge microbes for bio harvesting from lignocellulosic biomass. Chemosphere 228:702\u0026ndash;708. https://doi.org/10.1016/j.chemosphere.2019.04.159\u003c/li\u003e\n\u003cli\u003ePertiwiningrum A, La\u0026rsquo;aliya I, Yusiati LM, Harto AW (2019) The performance of biogas combustion after carbon dioxide absorption using sodium hydroxide (NaOH). In: IOP Conference Series: Earth and Environmental Science, vol. 387, no. 1, p. 012051. IOP Publishing. https://doi.org/10.1088/1755-1315/387/1/012051\u003c/li\u003e\n\u003cli\u003eRuan Z, Wang X, Liu Y, Liao W (2019) Chapter 3: Corn. Integrated Processing Technologies for Food and Agricultural By-Products. Cham, Switzerland: Springer International Publishing.\u003c/li\u003e\n\u003cli\u003eSrivastava RK, Shetti NP, Reddy KR, Kwon EE, Nadagouda MN, Aminabhavi TM (2021) Biomass utilization and production of biofuels from carbon neutral materials. Environ Pollut 276:11673. https://doi.org/10.1016/j.envpol.2021.116731\u003c/li\u003e\n\u003cli\u003eSrivastava RK, Shetti NP, Reddy KR, Nadagouda MN, Badawi M, Bonilla Petriciolet A, Aminabhavi TM (2023) Valorization of biowastes for clean energy production, environmental depollution and soil fertility. J Environ Manage\u003cem\u003e \u003c/em\u003e332:117410. https://doi.org/10.1016/j.jenvman.2023.117410\u003c/li\u003e\n\u003cli\u003eSues T (2013) Analysis of corn stover structural properties for mechanical processing applications. J Undergrad Res 11:article 9.\u003c/li\u003e\n\u003cli\u003eTakizawa S, Baba Y, Tada C, Fukuda Y, Nakai Y (2018) Pretreatment with rumen fluid improves methane production in the anaerobic digestion of paper sludge. Waste Manage 78:379e384. https://doi.org/10.1016/j.wasman.2018.05.046 \u003c/li\u003e\n\u003cli\u003eWang S, Li F, Wu D, Zhang P, Wang H, Tao X, Ye J, Nabi M (2018a) Enzyme pretreatment enhancing biogas yield from corn stover: feasibility, optimization, and mechanism analysis. J. Agric Food Chem 66(38): 10026-10032. https://doi.org/10.1021/acs.jafc.8b03086 \u003c/li\u003e\n\u003cli\u003eWang S, Zhang G, Zhang P, Ma X, Li F, Zhang H, Tao X, Ye J, Nabi M (2018b) Rumen fluid fermentation for enhancement of hydrolysis and acidification of grass clipping. J Environ Manage 220:142e148.\u003cem\u003e \u003c/em\u003ehttps://doi.org/10.1016/j.jenvman.2018.05.027 \u003c/li\u003e\n\u003cli\u003eYıldırım E, Ince O, Aydin S, Ince B (2017) Improvement of biogas potential of anaerobic digesters using rumen fungi. Renew. Energy 109:346\u0026ndash;353. https://doi.org/10.1016/j.renene.2017.03.021\u003c/li\u003e\n\u003cli\u003eYu S, Zhang G, Li J, Zhao Z, Kang X. (2013) Effect of endogenous hydrolytic enzymes pretreatment on the anaerobic digestion of sludge. Bioresour Technol 146:758\u0026ndash;761. http://dx.doi.org/10.1016/j.biortech.2013.07.087\u003c/li\u003e\n\u003cli\u003eZheng Y, Zhao J, Xu F, Li Y (2014) Pretreatment of lignocellulosic biomass for enhanced biogas production. Prog Energy Combust Sci 42:35\u0026ndash;53. http://dx.doi.org/10.1016/j.pecs.2014.01.001\u003c/li\u003e\n\u003cli\u003eZhou M, Yang J, Wang H, Jin T, Hassett DJ, Gu T (2014) Chapter 9 - Bioelectrochemistry of microbial fuel cells and their potential applications in bioenergy. Bioenergy Research: Advances and Applications, pp. 131\u0026ndash;152. https://doi.org/10.1016/B978-0-444-59561-4.00009-7\u003c/li\u003e\n\u003cli\u003eZhu J, Wan C, Li Y (2010) Enhanced solid-state anaerobic digestion of corn stover by alkaline pretreatment. Bioresour Technol 101:7523\u0026ndash;7528. https://doi.org/10.1016/j.biortech.2010.04.060\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"environmental-processes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"enpr","sideBox":"Learn more about [Environmental Processes](https://www.springer.com/journal/40710)","snPcode":"40710","submissionUrl":"https://submission.nature.com/new-submission/40710/3","title":"Environmental Processes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"agricultural residues, anaerobic digestion, biogas enhancement, lignocellulosic biomass hydrolysis, rumen microbiota","lastPublishedDoi":"10.21203/rs.3.rs-4174541/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4174541/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eCorn is one of the most common cultivations in Greece and worldwide. Raw materials like corn stover contain a remarkable amount of organic content, which could be transformed anaerobically into biogas, using as an alternative energy source. The development of efficient methods to overcome the limitations arisen from the nature of lignocellulosic biomass is a challenge since pretreatment is required to break down its complex structure. An economically-feasible biological approach to disrupt the structure of lignocellulosic materials, like corn stover, is through the valorization of hydrolytic potential of microbial communities present in rumen. Rumen microbiota has demonstrated the ability to break down lignocellulosic biomass. Thus, this work aims to enhance biogas production from corn stover residues using rumen fluid microbiota. The anaerobic digestibility of corn stover in BMP (Biochemical Methane Potential) tests and CSTR (continuous stirring tank reactors) was examined using rumen fluid as inoculum, in presence of control. Three organic loading rates (OLR), i.e. 1, 2, and 3 g VS/L.d, were tested, to define the optimum OLR for corn stover digestion. Moreover, experiments to define the optimum corn stover to rumen fluid ratio to optimize biogas production were carried out. Addition of rumen inoculum into the anaerobic digester at daily basis was found to be essential to enhance biogas production from corn stover. The optimum corn stover residues concentration in rumen fluid for optimum biogas production was 4% w/v. Addition of rumen fluid microbiota in the CSTR operating under various OLRs enhanced biogas production by 2\u0026ndash;6.3 times.\u003c/p\u003e","manuscriptTitle":"Enhancing anaerobic degradation of corn stover residues and biogas production via rumen microorganisms","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-02 09:00:19","doi":"10.21203/rs.3.rs-4174541/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-08-07T08:46:11+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-06-09T19:22:18+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"246379443265609351604703831602921954516","date":"2024-06-03T06:43:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"7819599b-3ad3-41d4-812e-69cc617b02ad","date":"2024-04-10T15:45:39+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-05T15:35:39+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-03-29T07:52:30+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-03-28T12:36:56+00:00","index":"","fulltext":""},{"type":"submitted","content":"Environmental Processes","date":"2024-03-27T08:24:40+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"environmental-processes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"enpr","sideBox":"Learn more about [Environmental Processes](https://www.springer.com/journal/40710)","snPcode":"40710","submissionUrl":"https://submission.nature.com/new-submission/40710/3","title":"Environmental Processes","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"fe47abc5-33f3-47f6-a8dd-845f6be8bf6c","owner":[],"postedDate":"April 2nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-10-28T16:02:05+00:00","versionOfRecord":{"articleIdentity":"rs-4174541","link":"https://doi.org/10.1007/s40710-024-00738-y","journal":{"identity":"environmental-processes","isVorOnly":false,"title":"Environmental Processes"},"publishedOn":"2024-10-24 15:57:37","publishedOnDateReadable":"October 24th, 2024"},"versionCreatedAt":"2024-04-02 09:00:19","video":"","vorDoi":"10.1007/s40710-024-00738-y","vorDoiUrl":"https://doi.org/10.1007/s40710-024-00738-y","workflowStages":[]},"version":"v1","identity":"rs-4174541","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4174541","identity":"rs-4174541","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}