The research was carried out in microbiology laboratory of department of biology, Debre Markos University, East Gojjam, Ethiopia. The university is found in Debre Markos town. Debre Markos is located at latitude and longitude of 10°20’N 37°43’E/10.330°N 37.717°E, elevation of 2,446 meters above sea level. It is 300 km away from Addis Abeba, capital city of Ethiopia and 265 km from Bahir Dar regional state of Amhara. In Debre Markos, there are 107,684 residents, comprising 49,893 men and 57,791 women ( Aynalem et al., 2014). The average annual rainfall is 380 mm, while the lowest and maximum temperatures are 150°C and 220°C, respectively.

Test of strain Saccharomyces cerevisiae isolate MUTJ0F (OR209280.1) with accession numbers from the National Center for Biotechnology Information was acquired from previously isolated traditional fermented alcoholic beverage (Tej) in Ethiopia. These strains of Saccharomyces cerevisiae isolate MUTJ0F (OR209280.1) were obtained in our stock cultures from the previous study ( Fentahun and Andualem, 2024).

The study was aimed at evaluating the effect of ruminant fluids and Saccharomyces cerevisiae isolate MUTJ0F (OR209280.1) on biogas generation from various mixed organic wastes under co-digestion with cattle manure. Various wastes from fruits and vegetables were gathered from the town fruit houses, leftovers food from Debre Markos University student cafeteria, fresh cattle manure (CM) from Monkorer Agroindustry Enterprise and rumen fluids (RL) from nearby slaughterhouse was taken and used as activator for biogas production.

Waste from fruits and vegetables was gathered from Debre Markos town’s juice shops. Unwanted which are non-digestible materials was carefully separated from the substrate. Food scraps from leftovers were gathered from cafeteria of students found in the Main Campus of Debre Markos University daily for a week. Indigestible wastes, such bones, were carefully removed from the substrate from the gathered meal. The mixture of substrates includes peels of bread, injera, spaghetti, papaya, mango, banana, and avocado were used in this research work. To improve and maintain anaerobic digestions process, the organic wastes were manually chopped to a size of 1-4 mm ( Leta et al., 2015). The CM were separated and allowed to dry for two days in direct sunlight on a plastic tray then it was shredded to an average particle size of 2 mm and kept in a refrigerator at 4°C ( Tamrat et al., 2013). After measuring the samples of total solids (TS), the de-sized cattle manure and food waste were mixed separately with distilled water in 1:5 (solid waste: distilled water) volume ratio, in order to maintain the total solid in the digester between 8 to 15%, which is the optimum value for wet anaerobic digestion ( Ituen et al., 2007).

After filtering the rumen fluids, the filtrate was stored in a refrigerator until used. Then, different amount of the filtrate was added into each digester to start up the reaction ( Aurora, 1983; Genet et al., 2018). The S. cerevisiae isolate MUTJ0F inoculum was prepared in Yeast Extract Peptone Dextrose Broth (YEPD Broth) (Sigma-Aldrich (Oxoid Limited, USA) medium containing (g/l): yeast extract 10, peptone 20, and dextrose 20. The media was sterilized at 121°C for 15 min in an autoclave. A loop full of a chosen 48 hrs old culture was inoculated into a 250 ml flask with 100 ml of the medium, and it was then shaken at room temperature at 25°C on a rotary shaker (SHKA4450-1CE) (121rpm) for 72 hrs. The inoculums were specific to each digester.

Total solids (TS), volatile solids (VS), moisture content, organic carbon, and pH were measured in each sample of biodegradable cattle manure, cafeteria leftovers, and fruit and vegetable wastes using the standard methods ( APHA, 1999).

2.5.1 Total solids (TS)

An evaporating dish (crucible) was cleaned and dried for one hour at 105°C, cooled in desiccators and weighed immediately before use 5 gram of cattle manure, fruit and vegetable waste. Five gram of sample was weighed separately applying for an standard analytical balance (LX200ABL) and placed on a pre-dried and weighed evaporating dish. Then the dish (crucible) was placed inside an oven (Contherm 260M) maintained at 105°C. The dish (crucible) was allowed to stay in the oven (Contherm 260M) for 24 hrs and then removed and allowed to cool in desiccators and weighed.

Using the formula stated in APHA (1999), the percentage of the TS was determined as follows. % TS = mDS mFS × 100

Where,

% TS = percentage of total solid

mDS = mass of dry sample

mFS = mass of fresh sample.

2.5.2 Volatile solids (VS)

The total solid was ignited at 550°C in a muffle furnace (BiBBY, Stuart) for 3 hrs to determine the volatile and fixed solids of the sample. Then volatile solid content in the sample was determined using the formula: APHA (1999). % VS = mDS − m ( ash ) mDS × 100

Where, mDS= mass of dry samples whereas m(ash) = mass of ash

2.5.3 Organic carbon (C)

According to Haug (1993), using data from volatile solids and an empirical equation, the organic carbon was calculated, and the organic carbon content of the sample was calculated by taking into account the volatile solids content, which was expressed as a percentage: % carbon = % VS 1.8

2.5.4 Moisture content determination

Oven-dying method was used to figure out the amount of moisture as the percentage of wet (initial) weight of the material lost through heating. To achieve this, 10 g of sample was dried in an oven (Contherm 260M) at 105°C for 24 hrs and weighed. Moisture content was then calculated using the formula ( Elias et al., 2010): % MC = W − D W × 100 where W is the sample’s initial weight (g), D is the sample’s weight during a 24 hrs drying period at 105°C, and M is the moisture content.

2.5.5 pH determination

A digital pH meter was applied for measurement the pH value (Hanna ECI pH meter, Hanna scientific, USA). The pH meter was calibrated using pH 4.0 and 7.0 buffers prior to measuring the samples of pH, resulting in a measurement that was within the proper pH range ( Arogo et al., 2009).

This study was consisted of anaerobic digestion of substrate in 12 treatments. The 12 treatment types used for anaerobic co-digestion digestion was cattle manure (CM), fruit and vegetable wastes (FVW) and leftover foods (LF) in mixtures. The first factor was dosage of rumen liquid i.e. 0 ml/100 gram, 25 ml/100 gram, 50 ml/100 gram, 75 ml/100 gram, and 100 ml/100 gram, while the second factor was dosage of S. cerevisiae isolate MUTJ0F i.e. 5 ml/100 gram, and 10 ml/100 gram of the mixed organic waste. Three replicates were used for the treatments. The study was conducted at the room temperature (30°C). Each digester water content was calculated in accordance with the suggestion of ( Ituen et al., 2007). Feed stocks was mixed with distilled water to get about 8% of TS suspension. The following formula was then used to determine how much water needed to be added: 8 % = mFTS A + B where A is the mass of the freshly added sample, B is the amount of water and inoculums to be added in order to achieve an 8% TS suspension in the digester, and mFTS is the mass of the fixed total solid.

Anaerobic digesters (plastic bottle) were constructed for bench-scale experiments with which biogas was produced out of the degradation of substrates in 0.6 L digester. The three plastic bottles were set up so that the substrate was in the first bottle, the acidified brine solution was in the center, and the last bottle was collecting the brine solution that was expelled from the second container. All the three containers were interconnected with a plastic tube having a diameter of 1 cm. The lids of all digester were sealed tightly using super glue in order to control the entry of oxygen and loss of biogas.

An acidified brine solution was produced by adding NaCl to water until a solution that was supersaturated formed. The brine solution was then acidified by adding two to three drops of sulfuric acid by the method of ( Elijah et al., 2009). Finally, this formed solution was contained in the second chamber. The biogas was moved to the second chamber while it was being produced in the fermentation chamber. A pressure buildup served as the catalyst for the solution displacement because the biogas is insoluble in it. The amount of gas collected was equal to the amount of water that was pushed from the cylinder. By looking at the cylinder’s graduation, the displacement of water was measured. According to Budiyono et al. (2010), the “liquid displacement method” was used to measure the amount of biogas produced.

The measurement cylinder was filled with gas after collecting. The lit match sticks were positioned close to the cylinder mouth. The gas formed in the digester that contains methane was burnable. If no flame forms the gas produced in the digester was non-burnable.

Version 23.0 of SPSS (IBM SPSSInc., Chicago, IL, SPSS (RRID:SCR_002865), https://www.ibm.com/support/pages/downloading-ibm-spss-statistics-23) was used to analyze the data. The mean and standard deviations of the triplicates analysis were calculated using analysis of variance (ANOVA).

The physical-chemical properties of different mixed wastes, including their moisture contents, pH, organic carbon, total solids, and volatile solids, are shown in Table 1. Pre-digestion of three mixed wastes, including cattle manure, cafeteria leftovers, and vegetable and fruit wastes were varied in the amount of the composition, which was due to the variability in the composition of the samples of the different substrates. Each digester mixed wastes had between 6.8 and 7.6 in pH. High moisture content percentage of mixed wastes were ranged from 62.5 to 77.6, which makes anaerobic digestion facilitates ( Fernández et al., 2008). The mixed wastes physicochemical characteristics were showed a low percentage of volatile solids relative to total solids. The VS of cafeteria leftover food waste was greater (30.8%) than that of cattle manure and fruit and vegetable wastes, suggesting a comparatively higher energy content that is advantageous for biogas production. The VS/TS ratios for cafeteria leftover food waste and cattle manure were 78.3% and 91.2%, respectively. For effective biogas generation, the amount of biodegradable organic matter should be between 70 and 95 percent of the dry matter content ( Steffen et al., 1998; Buffiere et al., 2006).

All of the substrates were considered acceptable for anaerobic digestion because all the mixed wastes had the highest ratio of VS to TS in Table 1. Cattle dung is used to speed up the production of biogas by promoting bacterial growth in the digester. Co-digestion has a positive synergistic effect by neutralising pH, increasing buffering capacity, reducing the effects of harmful compounds, and supplying more balanced nutrients like vitamins, trace metals, and other substances required for microbial growth ( Fang, 2010; Aragaw et al., 2013; Jianzheng et al., 2011).

The amount of biogas generated from biogas production using rumen fluids and S. cerevisiae isolate MUTJ0F as a fermentation activator is presented in Table 2. The production of biogas at the mesophilic temperature (30°C) using rumen fluid and the S. cerevisiae isolate MUTJ0F was more intense than that without inoculums. Rumen fluid and S. cerevisiae isolate MUTJ0F inoculums increased production biogas more than by four times in compare to a mixed substrate without inoculums. This implies that the high anaerobic bacterial concentration in rumen fluid efficiently breaks down organic substrate from a mixed waste. The findings are consistent with the work of other researchers ( Tamirat, 2012; Sakar et al., 2008; Yitayal, 2011; Forster-Carneiro et al., 2008; Abdullahi et al., 2011).

According to the results, the maximum amount of biogas (6900.3 ml) was produced by combining 100 ml of rumen fluids with 10 ml of S. cerevisiae isolate MUTJ0F/100 gram mixed waste, followed by 100 ml of rumen fluids and 5 ml of S. cerevisiae isolate MUTJ0F/100 gram mixed waste. As the digester rumen content varies, the results also showed that the amount of biogas generated increases when the dose of S. cerevisiae isolate MUTJ0F is increased from 5 ml to 10 ml. The addition of the S. cerevisiae isolate MUTJ0F culture to the digester enhanced the number of ruminal bacteria and their activity while also improving the digestibility of dry matter, crude protein, and hemicelluloses ( Lynd et al., 2002; Wiedmeier et al., 1987; Wilson, 2011). According to the findings, 100 ml of rumen fluid mixed with 10 ml of S. cerevisiae isolate MUTJ0F was the optimum quantity to provide the best performance of biogas production.

The pH value was checked every 10 days to examine the effect of change during digestion on bacterial activity ( Figure 1). The pH value drops rapidly, reached 4.3 on day forty. After that, the pH value then increased to 6.4 over the period of the following sixty days. This low pH value was permitted very little methanogenic bacterial activity and the acid-formers might yet be able to proliferate and generate large amounts of volatile acids, which lowers the pH of the digester’s contents ( Joyce et al., 2018; Li et al., 2018; Stabnikova et al., 2005; Lin et al., 2011). On the other hand, the pH starts to rise on day forty of fermentation. This could be because proteins break down to release ammonia, which causes alkalinity ( Gerardi, 2003). This also brings the pH closer to neutral, which makes it easier for methanogenic microbes to multiply and produce methane. Methanogenic bacteria the optimum pH values between 6.8 and 7.2 ( Anunputtikul and Rodtong, 2004; Budiyono et al., 2010).

A Bunsen burner attached to the digester gas outlet was utilized to evaluate the biogas combustibility after the 60 ^th day of the digestion period in a 0.6 L digester. The flammable gas was observed at the burner’s mouth confirming presence of methane in the biogas ( Figure 2).