©Haramaya University, 2020 ISSN 1993-8195 (Online), ISSN 1992-0407(Print) East African Journal of Sciences (2020) Volume 14 (2) 99-110 Licensed under a Creative Commons *Corresponding Author. E-mail: olyaderba@gmail.com Attribution-NonCommercial 4.0 International License. Evaluating Rumen Fluid-Inoculated Lignocellulosic Substrates for Biogas Production Yimeslal Atnafu Sema, Olyad Erba Urgessa*, and Ameha Kebede Ayele School of Biological Sciences and Biotechnology, Haramaya University, P.O. Box 138, Dire Dawa, Ethiopia Abstract Background: The continuous decline in reserves of fossil and petroleum-based fuel and its undesirable environmental effects is a major problem faced by humanity. Biofuel production from lignocellulosic biomass such as agricultural, forest and crop residues, food waste and energy grasses is cost effective and could provide a sustainable solution to the problem. However, since a lignocellulosic substrate is recalcitrant, the choice of more effective digestion inoculums is crucial to achieve an improvement in the efficiency of digesting the substrate. Microbial community present in rumen is efficient in hydrolysis of complex organics at mesophilic temperature. Objective: The objective of this study was to evaluate and compare biogas production potential of watercrown, potato peel, wheat and sorghum straw and sugarcane bagasse separately inoculated with rumen fluid of cow, goat and sheep. Material and Methods: Total solid, volatile solid, C/N ratio and pH of the slurry before and after anaerobic digestion were determined following standard procedures. The volume of biogas produced was measured using water displacement method for 32 days and energy was estimated from a general methane composition of biogas and 1 m3 of methane contains 34 MJ of energy. Results: The initial C/N ratio of slurries of potato peel ranged from 15.33 to 16.33. The highest cumulative biogas yield of 1318.83 mL g-1 VSadded-1 was obtained from potato peel inoculated with cattle rumen fluid. The cumulative biogas yield of watercrown inoculated with cattle and sheep rumen fluid was higher than that of all the slurries of sugarcane bagasse and wheat straw. Conclusions and implications: Slurries of potato peel and watercrown grass produced the highest amount of biogas and could be used potentially for biogas production at the pilot scale. Keywords: Anaerobic digestion; Biomass; Cumulative biogas; Watercrown 1. Introduction The increasing energy requirements along with the consequences of climate change, and depletion of unsustainable and polluting fossil fuels have driven the search for renewable energy sources (Cesaro and Belgiorno, 2015; Zhanga et al., 2019). Biomass is a renewable energy source that has potential for conversion into bioenergy via fermentation and anaerobic digestion (Cesaro and Belgiorno, 2015). Biofuel production from abundant and readily accessible resources such as lignocellulosic biomass (agricultural residues, forest residues, and crop residues) is cost effective and is a sustainable solution to the continuous decline in reserves of existing fossil and petroleum-based fuel, undesirable environmental effects, and associated unfavorable prices (Saratale and Oh, 2012). In biofuel production, a priority research area is looking for alternatives to conventional energy crops (Zhang et al., 2019). A promising alternative source is the agricultural sector which generates natural wastes such as manure and arable by-products that could be used for renewable energy generation by anaerobic digestion (Spence et al., 2019). Grasses are potential alternative source of renewable energy due to their fast growing rate even in infertile land, low cultivation costs, high accessibility, consumption of whole plants and lower environmental impacts when compared to other plants (Pantawong et al., 2015). Perennial grasses, especially C4 grasses, are excellent substrate for renewable energy production because of a high potential of dry matter yields and fast growth compared to annual crops (Karp and Shield, 2008). Watercrown (PA S PA L I D I U M G E M I N A T U M ) is aquatic, herbaceous and perennial grass. It can become locally abundant under extended warm season flooding (CABI, 2014). Growing tips and stem fragments of watercrown for lake restoration is an environmentally and economically useful way of obtaining biomass for biogas production (Mallison et al., 2006). Harvesting stem fragments of watercrown has a minimal, short term effect on offshore populations and within three weeks, emergent regrowth of the source plants can advance to the status of pre-harvest densities (Mallison and Thompson, 2010). The choice of more effective digestion inoculums is crucial to achieve the improvement in the anaerobic fermentation efficiency of lignocellulosic substrate (He et al., 2018). Ruminants can digest a wide range of cellulosic substrates, including many bioenergy-relevant substrates such as corn, stover, switchgrass and miscanthus (Patra and Saxena, 2009) because the rumen, as part of the digestive tract in herbivores, houses a mailto:olyaderba@gmail.com Yimeslal et al. East African Journal of Sciences Volume 14 (2) 99-110 100 microbial community that naturally and efficiently hydrolyzes cellulose (Christopherson and Suen, 2013). Microbial community present in rumen is efficient in hydrolysis of complex organics at mesophilic temperature. Especially, the community present in the fluid of rumen is stable (in terms of production) over time and it is resistant to contamination (Weimer, 1996). Rumen community of microbes together hydrolyzes and ferment chitin which is the second most abundant organic polymer after cellulose (Weimer et al., 2009). The major fermentation products produced by most rumen microorganisms are methane, hydrogen and a diverse set of short-chain fatty acids. These products have potential for use as bioenergy-relevant alternative fuels (Weimer, 1996; Weimer et al., 2009). Several studies were conducted on biogas production from anaerobic digestion of potato peel (Liang and McDonald, 2015; Mu et al., 2017; Achinas et al., 2019), sugarcane bagasse (Janke et al., 2015; Adarme et al., 2017; Eshore et al., 2017; Lima et al., 2018; Nostratpour et al., 2018; Hashemi et al., 2019; Vats et al., 2019), wheat straw (Ferraro et al., 2018; He et al., 2018; Kong et al., 2018; Mancini et al., 2018; Moset et al., 2018; Kumer et al., 2019; Liu et al., 2019), sorghum straw (Garcia et al., 2019) and different grass species (Mähnert, 2005; Uzodinma and Ofoefule, 2009). Sludge/digestate of anaerobic digestions was used as inoculums in most of these studies. In some, bacteria isolated from bovine rumen (Eshore et al., 2017), anaerobic digestate mixed with fresh bovine manure and/or rumen (Uzodinma and Ofoefule, 2009; Lima et al., 2018), cattle rumen fluid (He et al., 2018) and anaerobic fungi isolate (Ferraro et al., 2018) were used as inoculums. Comparative studies have not yet been done to investigate the potential of inoculums of rumen fluid of cow, goat and sheep for biogas production. Furthermore, there is no study conducted on biogas production potential of watercrown. Given the effect of feeding pattern of rumen animal on biogas production (Uzodinma and Ofoefule, 2009) and the synergy of rumen microbe in carrying out food digestion (Christopherson and Suez, 2013), the effect of agriculturally important rumen animal such as cattle, sheep and goat rumen fluid on biogas production needs to be comparatively investigated. Therefore, the overall objective of this study was to evaluate and compare biogas production potential from watercrown with that from well investigated potato peel, sugarcane bagasse, wheat straw and sorghum straw separately inoculated with rumen fluids of cow, goat and sheep. 2. Materials and Methods 2.1. Collection Lignocellulosic Substrates and Inoculum Wheat and sorghum straw were collected from Haramaya University research farm and sugarcane bagasse was collected from Wonji-Shewa Sugar Factory. Watercrown (P. geminatum) was collected from marshland around Lake Denbal (Ziway). Potato peels were collected from the student cafeteria of Haramaya University. The collected lignocellulosic substrates were dried in oven at 60 °C (Bozym et al., 2015) until constant weight was obtained. The dried substrates were ground in an electric grinder and the powders were stored in air tight plastic bags at 28 °C (Kumer et al., 2019). Fresh rumen fluid of cattle, goat, and sheep were collected from Haramaya town abattoir under aseptic conditions (Begum et al., 2013). The rumen fluids were stored at 6 °C to avoid undesirable fermentation processes (Achinas et al., 2019). 2.2. Characterization of the Slurry In many studies, it is customary to investigate total solid, volatile solid and C/N ratio of the inoculums and substrate, separately. In this study, the slurries were formulated by mixing lignocellulosic substrates with the rumen fluids at 0.1:1 (w/v) ratio that based on study set up for biogas production by Achinas et al. (2019). Then, the slurries were characterized in terms of their total solid (TS in g), volatile solid (VS in g), C/N ratio and pH. These parameters were measured before and after anaerobic digestion. The amount of TS and VS was determined by modifying methods described by Eshore et al. (2017). Briefly, the powders of each substrate (5 g) and each inoculum (50 mL) were mixed separately in flask. The slurries were transferred and wrapped up in pre-weighted aluminum foil, and their fresh matter weights (FMW) were measured separately by incubating at 105 °C in a hot air oven till constant weights (DMW) were obtained. These constant dry matter weights (DMW) were TS weight of the slurries and the percentage of the total solid (%TS) was determined as follows: % TS = TS(g) FMW(g) x 100 The obtained total solids were separately transferred to crucible, heated at 500 °C for 2 h in a muffle furnace and their weights were measured. These weights were volatile solid (VS) weight of the slurries and the percentage of the volatile solid (%VS) was determined as follows: % VS = VS(g) TS(g) x 100 C/N ratio was determined by analysis of carbon and nitrogen composition of slurries. The percentage of carbon (%C) found in the slurry samples was determined as described by (Shaw, 1959). The percentage of nitrogen (%N) found in the slurry samples was determined as described by (SD, 2003). The measurement of TS, VS and C/N ratio after anaerobic digestions was conducted after decanting overlaying liquid solution found in the anaerobic digesters. Yimeslal et al. Biogas from Lignocellulose Digestion Using Rumen Fluid 101 The pH of the slurries before and after the anaerobic digestion was measured using a pH meter (3310, Jenway). Measurement was conducted following operating procedures of the manufacturer meter manual. The pH meter was warmed up for 15 minutes and its electrode was calibrated before each pH measurements. After calibration, pH was measured by rinsing electrodes with distilled water and submerging it into the slurries. 2.3. Anaerobic Digestion Prior to the anaerobic digestion experiments, each rumen fluid was incubated anaerobically at 37 °C for 5 days (Kumer et al., 2019). Biogas production was carried out in triplicate using 500 mL plastic bottles as digester. The digestion experiments were carried out in batch mode following the method of Achinas et al. (2019). Briefly, 10 g of lignocellulosic substrate powder was separately weighed into the digesters. Anaerobic digestion was initiated by adding 100 mL rumen fluid in to the digesters. For all experiments, 100 mL of distilled water was added, and no additional nutrients/ trace elements were added to the digesters as it was assumed that they were provided by the inoculums. Thereafter, the digesters were sealed using glue and they were incubated at 37 °C for 32 days. The method used to estimate biochemical biogas potential was a volumetric test which considers the amount of displaced liquid as the measurement of biogas produced. For this purpose, 500 mL of plastic bottles were filled with acidified brine solution and were connected to anaerobic digesters. The volume of displaced brine solution was measured every day. 2.4. Biogas Conversion to Energy Energy was estimated from the produced cumulative biogas from a general %65 methane composition of biogas and 1 m3 of methane contains 34 MJ of energy (IRENA, 2016). 1 m3 of biogas = 22 MJ of energy 2.5. Statistical Data Analysis Statistics Package for Social Sciences (SPSS) version 20 was used to carry out the analysis of total solid (%), volatile solid (%), C/N ratio (%), pH (change) and cumulative biogas yield. Microsoft excels 2007 used to carry out analysis of daily biogas yield and rate. During SPSS operation, paired sample t-test was used to compare percentage of total solid, volatile solid and C/N ratio at the beginning and at the end of anaerobic digestion and single-factor analysis of variances (ANOVA) was used to compare cumulative biogas yield. The means were separated using Duncan. Statistical significance was established at p-value < 0.05 level. 3. Results and Discussion 3.1. Characterization of the Slurry For potato peel inoculated with cattle rumen fluid (PPCRF), the volatile solid (%) after anaerobic digestion was significantly less than volatile solid (%) before anaerobic digestion (Table 1). This might be one of the factors that contributed to the highest cumulative biogas production from PPCRF as compared to other slurries. In line with this, a previous study showed as volatile solid (%) removal was increased, cumulative biogas yield was also increased (Achinas et al., 2019). In fact, volatile solid content is the proportion of the solid material that can be digested by the micro-organisms and turned into biogas in the digester (IRENA, 2016). Another important factor that affects biogas production is C/N ratio. Initial C/N rations of potato peel slurries were lower than that of other slurries. For potato peel slurries, values found (15.33 ± 0.48, 16.73 ± 0.97 and 14.26 ± 0.76 for PPCRF, PPGRF and PPSRF, respectively) were lower than the optimum value recommended for biogas production (20–30: 1) (Pontoni et al., 2015; Ajeej et al., 2016; Perazzolo et al., 2016; Achinas et al., 2019). Too high or low ratios as compared to the recommended one lead to process inhibition through surplus nitrogen conversion into free ammonia (BRC, 2014). As a remedy to inhibition caused by toxic levels of ammonia, stripping off ammonia as a measure to overcome too high levels and adjusting pH by addition acid shifting the NH3/NH4+ balance in the system towards less free ammonia were suggested (BRC, 2014). For sugarcane bagasse, watercrown, and wheat straw slurries, the initial C/N ratios were beyond the optimum level. From the C/N ratios of these slurries, higher carbon content is clearly noted. However, these lignocellulosic substrates relatively produced lower biogas as compared to potato peel slurries that might occur due to bioavailability of not all of the carbon content in the substrate for anaerobic digestion. A previous study showed a C/N ratio close to 15–30 for almost all of the investigated lignocellulosic substrate (Garcia et al., 2019). Eshore et al. (2017) reported 90.8 ± 0.60% TS, 80.48 ± 0.78% VS for sugarcane bagasse. Janke et al. (2015) reported extremely higher C:N ratio (116:1) than that of sugarcane bagasse slurries found in this study (44.78 ± 1.53, 39.11 ± 0.25 and 32.55 ± 1.51 for SbCRF, SbGRF and SbSRF, respectively). The difference was that the measured C/N ratios in this study were for slurries (blend of sugarcane bagasse and rumen fluid). The added rumen fluid might have a reduced C/N value. Uzodinma and Ofoefule (2009) reported 89.80% TS, 75.85% VS and 28.29 C/N ratio for Panicum maximum blended with cow manure. The related substrate watercrown (P. geminatum) blended with rumen fluid in this study showed lower %TS and %VS, and higher C:N ratio. Yimeslal et al. East African Journal of Sciences Volume 14 (2) 99-110 102 Table 1. Characteristics of slurry used in batch anaerobic digestion. Slurry Total solid (%FM) Volatile solid (%TS) C/N Before After Before After Before After PPCRF 45.40±2.40a 43.02±1.92b 43.95±2.40a 41.57±1.92b 15.33±0.48a 11.69±1.07b PPGRF 44.27±4.16a 43.52±4.85a 42.82±4.16a 42.07±4.85a 16.73±0.97a 11.49±2.18b PPSRF 46.33±1.22a 44.47±4.79a 44.88±1.22a 43.02±4.79a 14.26±0.76a 9.807±0.71b SbCRF 94.36±1.30a 85.24±1.51b 92.86±1.30a 83.74±1.51b 44.78±1.53a 42.00±8.28a SbGRF 94.28±1.22a 87.38±1.56b 92.28±1.22a 85.38±1.56b 39.11±0.25a 30.95±1.65b SbSRF 95.70±1.86a 87.05±0.81b 93.20±1.86a 84.55±0.81a 32.55±1.51a 32.19±0.60a WcCRF 66.44±1.81a 62.38±1.04a 62.60±1.81a 58.54±1.04b 46.22±1.13a 37.59±2.19b WcGRF 65.33±0.88a 61.15±1.24b 61.49±0.88a 57.31±1.24b 38.67±0.70a 37.28±2.79a WcSRF 66.97±1.6a 63.17±1.11b 63.13±1.61a 59.33±1.11b 40.14±0.20a 30.87±0.21b WsCRF 95.25±1.32a 87.80±2.92b 92.25±1.32a 84.80±2.92b 40.14±2.72a 33.89±3.20b WsGRF 91.37±3.47a 79.04±1.12b 87.47±3.47a 75.14±1.12b 33.89±2.41a 33.15±1.30a WsSRF 85.56±2.03a 79.25±4.45a 80.76±2.03a 74.45±4.45a 44.33±1.16a 38.79±0.71b Note: PPCRF, PPGRF and PPSRF are slurries of potato peel and cow, goat and sheep rumen fluid mixture, respectively; SbCRF, SbGRF and SbSRF are slurries of sugarcane bagasse and cow, goat and sheep rumen fluid mixture, respectively; WcCRF, WcGRF and WcSRF are slurries of watercrown and cow, goat and sheep rumen fluid mixture, respectively; WsCRF, WsGRF and WsSRF are slurries of wheat straw and cow, goat and sheep rumen fluid mixture, respectively. Letters compare means across row within characteristics of the slurries. Different letters show significant difference at p < 0.05. 3.2. pH of the Slurry The initial pH was found to range from 6.53 to 8.75. The initial pH for potato peel slurries were 7.24, 6.86 and 6.53 for PPCRF, PPGRF and PPSRF, respectively (Figure 1). The maximum daily biogas yield was achieved in a shorter retention time for potato peel slurries than for the other lignocellulosic slurries (Figure 3, 4 and 5). This might be due to relatively lower pH values of potato peel slurries. The shorter retention time is needed for digestion of lignocellulosic substrate as the pH of the anaerobic digestion medium closer to the optimum pH. Consistent with this suggestion, Lan et al. (2013) showed that a pH between 5.2 and 6.2 was needed to obtain maximal hydrolysis of lignocelluloses. Uzodinma and Ofoefule (2009) recorded pH 7 for grass field (P. maximum) blended with cow dung at charging. In the present study, pH 8.69 was recorded initially for related slurry (WcCRF). The final pH ranged from 5.54 to 8.24 (Figure 1). The initial pH values of WsSRF (8.62), WcCRF (8.69) and WsCRF (8.75) were beyond the range 6.5 to 8.5 in which methane fermentation took place in anaerobic digestion systems (Achinas et al., 2019). After the anaerobic digestion, pH of all the slurries was within the range, except the pH of PPSRF. It was reported that the pH less than 6.5 inhibited the growth of methanogenic bacteria and that subsequently the volume of produced biogas was reduced (Food Technology et al., 2016; Achinas et al., 2019). In contrast to this result, the pH of PPSRF fell below the range from 6.53 to 5.54 after anaerobic digestion and it produced a higher cumulative biogas compared to PPGRF which showed higher value of pH. The lower pH of PPSRF might have reduced the toxic level of NH3. BRC (2014) reported that the amount of NH3 present in liquid anaerobic digestion medium is dependent on the total N-NH4+ levels in combination with pH and temperature, i.e. the higher the pH and/or temperature, the more NH3. Yimeslal et al. Biogas from Lignocellulose Digestion Using Rumen Fluid 103 Figure 1. pH of slurry before and after anaerobic digestion. The effect of change in pH is shown in Figure 2. PPCRF showed the lowest change (0.17) and SbGRF showed the highest change (1.02). From the change in pH, it is expected that SbGRF produced the lowest cumulative biogas yield. But, the lowest cumulative biogas yield was obtained from SbCRF (Table 2) which showed 0.70 value of change in pH. This showed that the decrease final pH of all slurries resulted in a narrow change in which the activities of natural anaerobic microbes in the slurries were maintained. Various research reports have confirmed that the methanogenic microorganisms are highly sensitive to change in pH and survive the pH range of 6.0 to 8.5 i.e. change in pH of 2 (Mösche and Jördening 1999; Wang et al. 1999). The narrow change in pH of the slurries shows strong buffer capacity of lignocellulosic substrates blended with rumen fluid. According to Mösche and Jördening (1999) and Wang et al. (1999), the pH value decreases by accumulation of VFA in the anaerobic digestion process. But, the accumulation of VFA will often not always result in a pH drop due to the buffer capacity of the substrate. For each lignocellulosic substrate, the lower values of changes in pH, the higher cumulative biogas yield. Figure 2. The effect of change in pH on cumulative biogas yield. 3.3. Biogas Production from the Slurries Experimental data for comparative biogas production of watercrown with other substrates were presented as daily biogas production, daily biogas production rate, cumulative biogas production and energy estimation. 3.3.1. Daily Biogas Production of Watercrown Compared to Potato Peel The maximum daily biogas yield achieved in shorter retention time for potato peel slurries as compared to other lignocellulosic slurries. This might be due to 5.00 5.50 6.00 6.50 7.00 7.50 8.00 8.50 9.00 p H o f sl u rr y Slurry of anaerobic digestion Before After Yimeslal et al. East African Journal of Sciences Volume 14 (2) 99-110 104 relatively lower initial pH values of potato peel slurries (Figure 1). As compared to the slurries of potato peel, all the counterpart slurries of watercrown produced lower maximum daily biogas yield and they took longer period of time to produce the maximum daily biogas yield (Figure 3A). For the slurries of potato peel, the maximum daily biogas yield reached 64.33 mL g-1 VSadded-1, 61.95 mL g-1 VSadded-1 and 70.67 mL g-1 VSadded-1 on 12 day for PPCRF, PPGRF and PPSRF, respectively. For the slurries of watercrown, the maximum daily biogas yield reached 28.85 mL g-1 VSadded-1, 28.36 mL g-1 VSadded-1 and 33.10 mL g-1 VSadded-1 on 17, 11 and 14 days for WcCRF, WcGRF and WcSRF, respectively. All the slurries of watercrown produced lower daily biogas yield rate during the retention time of anaerobic digestion (Figure 3B). For the slurries of watercrown, the peak yield rate reached 19.42 mL g-1 VSadded-1 day-1, 17.73 mL g-1 VSadded-1 day-1 and 21.47 mL g-1 VSadded-1 day-1 on 23, 15 and 19 days for WcCRF, WcGRF and WcSRF, respectively. For the slurries of potato peel, the peak yield rate reached 47.86 mL g-1 VSadded-1 day-1, 39.28 mL g-1 VSadded-1 day-1 and 49.31 mL g-1 VSadded-1 day-1 on 20, 18 and 19 days for PPCRF, PPGRF and PPSRF, respectively. Figure 3. Daily biogas production of watercrown slurry compared to potato peel slurry. Daily biogas yield (A). Daily biogas yield rate (B). PPCRF, PPGRF and PPSRF are slurries of potato peel and cow, goat and sheep rumen fluid mixture, respectively; WcCRF, WcGRF and WcSRF are slurries of watercrown and cow, goat and sheep rumen fluid mixture, respectively. 3.3.2. Daily Biogas Production of Watercrown Compared to Sugarcane Bagasse Compared to the slurries of sugarcane bagasse, slurries of watercrown maintained producing higher daily biogas yield during 1 to 26, 5 to 19 and 6 to 26 retention times for WcCRF, WcGRF and WcSRF, respectively (Figure 4A). For slurries of sugarcane bagasse, the maximum daily biogas yield reached 12.33 mL g-1 VSadded-1 , 15.82 mL g-1 VSadded-1 and 18.46 mL g-1 VSadded-1 on 25, 20 and 20 days for SbCRF, SbGRF and SbSRF, respectively. The slurries of sugarcane bagasse took longer retention time to reach the highest daily biogas yield and produced lower values of the highest yield. Yimeslal et al. Biogas from Lignocellulose Digestion Using Rumen Fluid 105 All the slurries of watercrown produced higher daily biogas yield rate starting from 7-day retention time of anaerobic digestion. WcCRF produced higher daily biogas yield rate than that of SbCRF during entire retention time. WcGRF produced higher daily biogas yield rate than that of SbGRF starting from 6-day retention time. WcSRF produced higher daily biogas yield rate than that of SbSRF starting from 7-day retention time (Figure 4B). For the slurries of sugarcane bagasse, the peak daily biogas yield rate reached 9.50 mL g-1 VSadded-1 day-1, 11.85 mL g-1 VSadded-1 day-1 and 12.33 mL g-1 VSadded-1 day-1 on 27, 25 and 23 days for SbCRF, SbGRF and SbSRF, respectively. The plot of daily biogas yield rate of the two slurries showed different pattern. The plot of slurries of sugarcane bagasse showed more constant pattern. Figure 4. Daily biogas production of watercrown slurry compared to sugarcane bagasse slurry. Daily biogas yield (A). Daily biogas yield rate (B). SbCRF, SbGRF and SbSRF are slurries of sugarcane bagasse and cow, goat and sheep rumen fluid mixture, respectively WcCRF, WcGRF and WcSRF are slurries of watercrown and cow, goat and sheep rumen fluid mixture, respectively. 3.3.3. Daily Biogas Production of Watercrown Compared to Wheat Straw Compared to the slurries of wheat straw, slurries of watercrown maintained producing higher daily biogas yield in some period of retention times. WcCRF maintained producing higher daily biogas yield than that of WsCRF starting from 5-day of retention time. WcGRF maintained producing higher daily biogas yield than that of WsGRF in the 6 to 15 retention times, then after, it maintained producing lower daily biogas yield. WcSRF maintained producing higher daily biogas yield than that of WsSRF in the 6 to 22 retention times (Figure 5A). For the slurries of wheat straw, the highest daily biogas yield reached 18.44 mL g-1 VSadded-1, 21.60 mL g-1 VSadded-1 and 21.32 mL g-1 VSadded-1 on 21, 25 and 22 days for WsCRF, WsGRF and WsSRF, respectively. The slurries of wheat straw took longer retention time to reach the highest daily biogas yield and produced lower values of highest daily biogas yield. All the slurries of watercrown produced higher daily biogas yield rate than that of counterpart slurries of wheat straw starting from 5-day retention time of anaerobic digestion. WcCRF produced higher daily biogas yield rate than that of WsCRF starting from 5-day Yimeslal et al. East African Journal of Sciences Volume 14 (2) 99-110 106 retention time. WcGRF produced higher daily biogas yield rate than that of WsGRF and WcSRF produced higher daily biogas yield rate than that of WsSRF during entire retention time (Figure 5B). For the slurries of wheat straw, the peak daily biogas yield rate reached 13.30 mL g-1 VSadded-1 day-1, 15.63 mL g-1 VSadded-1 day-1 and 13.73 mL g-1 VSadded-1 day-1 on 22, 28 and 22 days for SbCRF, SbGRF and SbSRF, respectively. The plot of daily biogas yield rate of the two slurries showed different pattern. Figure 5. Daily biogas production of watercrown slurry compared to wheat straw slurry. Daily biogas yield (A). Daily biogas yield rate (B). WsCRF, WsGRF and WsSRF are slurries of wheat straw and cow, goat and sheep rumen fluid mixture, respectively; WcCRF, WcGRF and WcSRF are slurries of watercrown and cow, goat and sheep rumen fluid mixture, respectively. 3.3.4. Cumulative Biogas Production and Energy Conversion The cumulative biogas yield (mL g-1 VSadded-1) from anaerobic digestion of potato peel, sugarcane bagasse, watercrown and wheat straw inoculated with rumen fluid of cow, goat and sheep were shown in Table 2. After 32 days, the highest cumulative biogas yield (1318.83 mL g-1 VSadded-1) was obtained from PPCRF and the lowest cumulative biogas yield (286 mL g-1 VSadded-1) was obtained from SbCRF. The highest cumulative biogas production of slurries of potato peel was might be attributed to C/N ratio close to optimum range and significant removal of percent of volatile solid (Table 1). The cumulative biogas yield of WcCRF (541.03 mL g- 1 VSadded-1) and WcSRF (547.75 mL g-1 VSadded-1) were higher than that of all the slurries of sugarcane bagasse and wheat straw. This might be due to a higher lignin percentage in sugarcane bagasse and wheat straw. A review conducted by Cesario and Bolgia (2015) showed 20% lignin content in bagasse and 8.3 to 24% lignin content in wheat straw. Uzodinma and Ofoefule (2009) Yimeslal et al. Biogas from Lignocellulose Digestion Using Rumen Fluid 107 reported the cumulative gas yield of 225.80 L per total mass of slurry from anaerobic digestion of Panicum maximum blended with cow dung for 30 days. Inoculums significantly affected cumulative biogas yield of potato peel, sugarcane bagasse, watercrown and wheat straw (p < 0.05) (Table 2). PPGRF produced significantly the lowest cumulative biogas yield. The order of cumulative biogas yield was PPCRF > PPSRF > PPGRF. For sugarcane bagasse, SbSRF produced significantly the lowest cumulative biogas yield and the cumulative yield order was SbSRF > SbGRF > SbSRF. For watercrown anaerobic digestion, WcGRF produced significantly the lowest cumulative biogas yield and the order was WcSRF > WcCRF > WcGRF. For wheat straw anaerobic digestion, WsGRF produced significantly the highest cumulative biogas yield and the order was WsGRF > WsCRF > WsSRF. The result showed variation of cumulative biogas production based on inoculums and the type of substrates. These variations were might be due to the difference in feeding habit of cow, goat and sheep. The direct influence of diet on the ruminal microbial community was previously reported indicating that particular groups of microbes are better adapted for fermentation of specific feedstocks (Patra and Saxena, 2009). According to Christopher and Suez (2013), it is practical to feed ruminant animals a preferred substrate and screen a stable microbial consortium that is well adapted to the fermentation of the feed. Biogas production is usually measured or estimated in cubic meters over a period of time, but it should be converted and reported in energy units (IRENA, 2016). Accordingly, the cumulative biogas produced was converted to energy unit and result was included in Table 2. PPCRF produced the highest cumulative CH4 (857.35 ± 20.18 mL g-1 VSadded-1) with total energy estimation of 29.15 ± 0.69 MJ. Whereas, SbCRF produced the lowest cumulative CH4 (185.90 ± 3.25 mL g-1 VSadded-1) with total energy estimation of 6.32 ± 0.11 MJ. Table 2. Cumulative biogas production of the slurries and energy estimation. Slurry Cumulative Biogas yield (mL g-1 VSadded-1) Cumulative CH4 (mL g-1 VSadded-1) Total Energy Estimation (MJ) PPCRF 1318.83±31.31g 857.35 ± 20.18g 29.15 ± 0.69g PPGRF 997.73 ± 17.84f 648.48 ± 11.41f 22.05 ± 0.39f PPSRF 1311.94 ± 7.13g 852.80 ± 4.55g 29.00 ± 0.16g SbCRF 286.00 ± 5.21a 185.90 ± 3.25a 6.32 ± 0.11a SbGRF 334.33 ± 1.31b 217.32 ± 0.75b 7.39 ± 0.02b SbSRF 339.70 ± 2.06b 221.00 ± 1.30b 7.51 ± 0.05b WcCRF 541.03 ± 10.77e 351.65 ± 7.03e 11.96 ±0.24e WcGRF 397.43 ± 6.37c 258.48 ± 4.18c 8.79 ± 0.14c WcSRF 547.75 ± 5.12e 356.20 ± 3.38e 12.11 ± 0.12e WsCRF 387.15 ± 4.26c 251.77 ± 2.63c 8.56 ± 0.09c WsGRF 483.33 ± 15.10d 314.17 ± 9.78d 10.68 ± 0.33d WsSRF 381.45 ± 5.20c 247.87 ± 3.58c 8.43 ± 0.12c Note: Letters compare means across column. Different letters show significant difference at p < 0.05. 4. Conclusions This study revealed that the use of rumen fluid resulted in fast degradation of lignocellulosic substrate. Potato peel slurries produced the highest cumulative biogas yield in a relatively short period of time. Therefore, it is economical and environmentally friendly to use potato peel waste from domestic and food processing industries for fast and potential biogas production contributing to the solid waste management. The results demonstrate that the second highest biogas yield was obtained from watercrown slurry. Since watercrown is fast-growing, high-yielding, available substrate in plenty, it can be used for sustainable biogas production. Nutrient composition of watercrown slurry should be determined for its application for a large scale biogas production. 5. Acknowledgements This paper is part of Haramaya University Research Grant coded HURG-2018-06-01-97. Therefore, Office of the Vice-President for Research Affairs, Haramaya University, deserves gratitude for funding this research project. 6. References Achinas, S., Li, Y., Achinas, V. and Euverink, G.J.W. 2019. 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