F1000Research F1000Research 2046-1402 F1000 Research Limited London, UK 10.12688/f1000research.161561.2 Research Article Articles Enhancing biogas production from co-digestion of organic wastes mixed with cattle manure using rumen fluid and Saccharomyces cerevisiae isolate MUTJ0F

[version 2; peer review: 1 approved with reservations, 2 not approved]

 Fentahun Mulugeta Conceptualization Data Curation Formal Analysis Funding Acquisition Investigation Project Administration Resources Software Supervision Validation Visualization Writing – Original Draft Preparation Writing – Review & Editing https://orcid.org/0009-0007-3025-7156 a 1 Kahsay Birhanu Conceptualization Data Curation Investigation Methodology Resources Software Validation Writing – Review & Editing 2 College of Natural and Computational Sciences, Department of Biology, Debre Markos University, Debre Markos, Amhara, 269, Ethiopia Institute of Biotechnology, Mekelle University, Mek'ele, Tigray, Ethiopia mulugetafanatahun@dmu.edu.et

No competing interests were disclosed.

 19 12 2025 2025 14 199 16 12 2025 Copyright: © 2025 Fentahun M and Kahsay B 2025 This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Background

Biogas production from different types of biodegradable waste is an alternative to fossil fuels for energy consumption and biodegradable waste management. Biogas is a cost-effective, environmentally friendly, and sustainable energy source that provides a reliable method for handling food waste.

 Methods

This study evaluated the effects of rumen fluid and S. cerevisiae isolate MUTJ0F as fermentation activators on biogas production from the co-digestion of cattle manure, fruit and vegetable residues, and cafeteria food waste at Debre Markos University in Ethiopia. Under mesophilic conditions, 12 treatments with different rumen fluid (0–150 ml/100 g) and S. cerevisiae isolate MUTJ0F inoculum (5–10 ml/100 g) dosages were assessed. Biogas was produced from mixed waste using plastic digesters (0.6 L) at mesophilic temperature (30°C) after the 60-day retention period. Standard techniques were used to measure the physicochemical characteristics of the mixed waste, such as pH, total solids, volatile solids, organic carbon, and moisture. This study was conducted experimentally using factorial patterns and a completely randomized 6 × 2 design. The water displacement method was used to estimate the average amount of biogas generated by a 0.6 L digester.

 Result

Rumen fluid with S. cerevisiae isolate MUTJ0F increased biogas generation by more than four times compared to the uninoculated control. The combination of 100 ml rumen fluid and 10 ml S. cerevisiae isolate MUTJ0F per 100 g of mixed waste produced the highest volume of biogas (6900.3 ml). pH monitoring revealed normal acidogenesis followed by a gradual return to methanogenic conditions, while combustibility tests showed the presence of methane in the biogas.

 Conclusion

These results suggest that using rumen fluid and the S. cerevisiae isolate MUTJ0F as inoculants in a biogas digester can enhance methane production and biodegradation efficiency.

 Anaerobic digestion Biogas production Inoculum Rumen fluid Debre Markos University This research was supported by Debre Markos University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Amendments from Version 1

The manuscript has undergone extensive revisions in response to the reviewers' feedback. The abstract has been updated to clarify the study's background, methodology, and key findings. The introduction was revised to enhance scholarly quality and clarity, with the novelty and unique contributions of the study clearly highlighted. The Materials and Methods section was significantly enhanced by providing step-by-step procedures, which improved transparency and reproducibility. The Results and Discussion sections have been rearranged to incorporate up-to-date literature and provide a stronger interpretation of the results. The Conclusions section was revised to state the main findings and the recommendations concisely. Additionally, the entire manuscript was carefully edited in English to improve clarity, grammar, and readability, and new citations were added to support the study's relevance and scientific context.

 1. Introduction

The search for sustainable energy alternatives is increasing owing to growing concerns about the effects of fossil fuel consumption on the environment and the rapid depletion of the world's petroleum reserves ( Akinbami et al., 2001; Imri and Valeria, 2007). To address these challenges, long-term strategies for sustainable development place greater emphasis on switching to renewable energy sources that can lessen reliance on finite fossil fuels and mitigate environmental degradation ( Farid, 2022). Among the available options, renewable energy technologies, especially biogas have become practical, economical, and environmentally sustainable for energy generation ( Alemayehu, 2014; Abdulkareem, 2005; Arthur and Brew-Hammond, 2010; Alemayehu, 2014). Anaerobic digestion of biomass or organic waste produces biogas, which is used for various household and commercial purposes, such as cooking, heating, lighting, electricity production, and vehicle fuel ( Harris, 2008; Corral et al., 2008; Nasir et al., 2012).

Biogas production depends on the activity of various bacteria and microorganisms that break down organic matter in anaerobic digestion ( Membere et al., 2012). An innovative solution to increase anaerobic waste digestion yields is co-digestion, which is an inexpensive and simple technology that enhances the anaerobic digestion rate by creating a better nutrient balance from the materials mixed to feed the digester, provides positive synergism for bacterial growth, and increases biogas production ( Sosnowski et al., 2003; Mata-Alvarez et al., 2000; Mshandete et al., 2004; Leta et al., 2015). Several studies have used co-digestion to increase the rate at which organic matter is converted biologically in the biogas system to improve biogas performance ( Huang et al., 2016; Alemayehu, 2014; Mata-Alvarez et al., 2014; Abbas et al., 2021).

Hydrolysis, acidogenesis, acetogenesis, and methanogenesis are the four interdependent microbial stages of anaerobic digestion, which are carried out by fermentative, acidogenic, and methanogenic microbial communities ( Jia et al., 2020). Rumen fluid is a waste product from slaughterhouses that is regularly dumped into drainage systems ( Zhang et al., 2016). A diverse range of fungi, bacteria, protozoa, and archaea inhabit the rumen, an anaerobic microbial habitat ( Sylvester et al., 2004; Sonakya et al., 2003). The rumen contains a variety of microorganisms, including cellulolytic and methanogenic bacteria ( Lopes et al., 2004; Yue and Yu, 2009). The rumen could be helpful as an activator in the anaerobic fermentation process that produces biogas. This fermentation process resembles the biogas digester process ( Achmad et al., 2011). Saccharomyces cerevisiae is an anaerobic microorganism that can increase fiber degradation, stimulate cellulolytic bacterial and fungal growth, and increase the pH in digesters through organic acid production ( Lynd et al., 2002; Achmad et al., 2011).

Debre Markos University is one of Ethiopia’s federal universities, and currently, there are more than 20,000 resident students on the main campus during the regular academic year and summer. For this reason, these students have their meals at the university. We have an enormous amount of organic waste and leftover food, which can be utilized as inputs for the generation of anaerobic biogas. The primary ingredients of the meals provided to students at Debre Markos University include bread, injera, spaghetti, rice, meat, and various stews and sauces. The majority of food leftovers were disposed of near the fence at the back of the male student home, which produced an unpleasant odor and a suitable environment for the growth of harmful bacteria. However, various juice house wastes are among the municipal wastes that are becoming difficult to manage. This kind of waste is typically dumped in landfills along with other household wastes, which greatly pollutes the environment in Debre Markos town. Therefore, finding alternative ways to handle organic waste and convert it into a source of energy is necessary.

This study offers an innovative strategy for increasing biogas production by combining rumen fluid, a naturally abundant source of hydrolytic, cellulolytic, and methanogenic microorganisms, with a locally isolated strain of S. cerevisiae (MUTJ0F) as dual fermentation activators during the co-digestion of cafeteria food waste, juice house waste, and cattle manure. Although there is information on biogas production from organic waste or single inoculums ( Alemayehu, 2014; Abayneh et al., 2014; Hammad et al., 2018; Earnest and Singh, 2013) but there is limited reports on methods for increasing biogas generation, particularly those that use rumen fluid and S. cerevisiae. This study aimed to determine the effect of rumen fluid and S. cerevisiae isolate MUTJ0F as fermentation activators on the amount of biogas generated from food waste co-digestion under anaerobic conditions and cattle manure, and to optimize the volume of rumen fluid and S. cerevisiae isolate MUTJ0F for biogas production. Therefore, the results may help universities, municipalities, and small businesses implement effective waste-to-energy management, encourage renewable energy, and reduce environmental pollution.

 2. Methods 2.1 The study area

The research was carried out in the microbiology laboratory of the Department of Biology, Debre Markos University, East Gojjam, Ethiopia. The university is located in Debre Markos. 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 Ababa, the capital city of Ethiopia, and 265 km from Bahir Dar, the capital city of the Amhara region. 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.

 2.2 Yeast strain used in experiments

The strain Saccharomyces cerevisiae isolate MUTJ0F (OR209280.1) with accession number was used in this study. The Saccharomyces cerevisiae strain MUTJ0F (OR209280.1) was acquired from stock cultures from a previous isolated traditional fermented alcoholic beverage (Tej) in Ethiopia. The methods used for their isolation and identification have been described in our previous studies. Sequenced data was deposited in GenBank in the NCBI database, and accession numbers were obtained ( Fentahun and Andualem, 2024).

 2.3 Substrate collection and preparation

This study aimed to evaluate the effects of ruminant fluids and S. cerevisiae isolate MUTJ0F (OR209280.1) on biogas generation from various mixed organic wastes co-digested with cattle manure. Various wastes from fruits and vegetables were gathered from the town fruit houses, leftover food from the Debre Markos University student cafeteria, fresh cattle manure (CM) from Monkorer Agroindustry Enterprise, and rumen fluids (RL) from a nearby slaughterhouse and used as activators for biogas production.

Waste from fruits and vegetables was collected from the juice shops in Debre Markos town. Unwanted, non-digestible materials were carefully separated from the substrate. Food scraps from leftovers were gathered daily for a week from the cafeteria of students found in the Main Campus of Debre Markos University. Indigestible waste, such as bones was carefully removed from the substrate of the gathered meal. A mixture of substrates, including peels of bread, injera, spaghetti, papaya, mango, banana, and avocado was used in this study. To improve and maintain the anaerobic digestion process, the organic wastes were manually chopped to a size of 1-4 mm ( Leta et al., 2015). The CM was 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 ( Tamirat et al., 2013). After measuring the total solids (TS) of the samples, the de-sized cattle manure and food waste were mixed separately with distilled water in a 1:5 (solid waste: distilled water) volume ratio to maintain the total solid in the digester between 8 and 15%, which is the optimum value for wet anaerobic digestion ( Ituen et al., 2007).

 2.4 Preparation of inoculums

After filtering the rumen fluid, the filtrate was stored in a refrigerator until use. Then, different amounts of the filtrate were added to each digester to initiate the reaction ( Aurora, 1983; Genet et al., 2018). The S. cerevisiae isolate MUTJ0F inoculum was prepared in Yeast Extract Peptone Dextrose Broth (YEPD) (Sigma-Aldrich (Oxoid Limited, USA) medium containing (g/l): yeast extract 10, peptone 20, and dextrose 20. The medium 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 inoculum was specific to each digester.

 2.5 Analysis of the physic-chemical properties of the substrate

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 waste using standard methods ( APHA, 1999).

2.5.1 Total solids (TS)

According to APHA (1999), the gravimetric method was used to determine the total solids (TS) content of each sample. An evaporating dish (crucible) was first carefully cleaned, dried for an hour at 105°C in an oven, cooled in a desiccator, and precisely weighed. Using a standard analytical balance (LX200ABL), five grams of each sample of cattle manure, fruit, and vegetable waste was weighed independently and added to a crucible that had already been weighed. Subsequently, the crucibles containing the samples were placed in an oven (Contherm 260M) set to 105°C for 24 hrs to dry. The crucibles were dried, cooled to room temperature in a desiccator, and weighed again.

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 previously dried total solids were ignited in a muffle furnace (BiBBY, Stuart) at 550°C for 3 hrs to determine the volatile solids (VS) and fixed solids (FS). The crucibles were removed after ignition, allowed to cool in a desiccator, and then precisely weighed. The volatile solids were represented by the weight loss during combustion, and the fixed solids were represented by the remaining residue. 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

The moisture content of each sample was calculated using the oven-drying method, which measures the percentage of water lost relative to the initial wet weight of the sample. After carefully weighing 10 grams of each sample, it was placed in oven (Contherm 260M) at 105°C for 24 hrs set to 105°C for an entire day. The samples were weighed again after drying and cooling in a desiccator. 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

The pH of each sample was determined using a digital pH meter (Hanna ECI pH meter, Hanna Scientific, USA) in accordance with standard procedures. The pH meter was calibrated using standard buffer solutions of pH 4.0 and 7.0 before measurement to ensure accuracy throughout the relevant pH range. After calibration, the electrode was submerged in the substrate samples, rinsed with distilled water, and the pH values were recorded ( Arogo et al., 2009).

 2.6 Experimental set up

This study consisted of the anaerobic digestion of substrates in 12 treatments. The 12 treatment types used for anaerobic co-digestion were cattle manure (CM), fruit and vegetable waste (FVW), and leftover food (LF) in mixtures. The first factor was the dosage of rumen liquid i.e. 0 ml/100 g, 25 ml/100 g, 50 ml/100 g, 75 ml/100 g, and 100 ml/100 g), while the second factor was the dosage of S. cerevisiae isolate MUTJ0F i.e. 5 ml/100 g, and 10 ml/100 g) of the mixed organic waste. Three replicates were used for each treatment. The study was conducted at room temperature (30°C). The water content of each digester was calculated according to the suggestion of ( Ituen et al., 2007). Feed stock was mixed with distilled water to obtain approximately 8% of TS suspension. The following formula was then used to determine the amount of water 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.

 2.7 Construction of the anaerobic digesters for batch system

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

 2.8 Biogas measurements

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.

 2.9 Burning test

A burning test was performed to qualitatively determine whether the biogas contained methane. After the biogas was collected, a measurement cylinder containing the gas was carefully placed. A lit matchstick was brought close to the mouth of the cylinder while observations were made. If the gas started a fire and created a flame, it was a sign that the biogas contained flammable methane. However, if there was no flame, the gas was considered non-combustible, indicating that there was very little or no methane in the sample.

 2.10 Data analysis

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).

 3. Results and Discussion 3.1 Analysis of the physic-chemical properties and composition of the mixed wastes

The physicochemical properties of the different mixed wastes, such as their moisture content, pH, organic carbon, total solids, and volatile solids are shown in Table 1. The pre-digestion of three mixed wastes, including cattle manure, cafeteria leftovers, and vegetable and fruit wastes, varied in the amount of composition owing to the variability in the composition of the samples of the different substrates. The pH of the mixed waste ranged from 6.8 to 7.6, which is within the ideal range for anaerobic digestion. This near-neutral pH is useful for microbial activity, especially methanogens, which thrive in stable pH environments ( Budiyono et al., 2010). The high moisture content of the mixed wastes ranged from 62.5 to 77.6%, which increases microbial accessibility and substrate solubilization for anaerobic digestion ( Fernández et al., 2008). The physicochemical characteristics of the mixed waste showed a low percentage of volatile solids relative to the 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.

Physico chemical properties of mixed wastes (mean ± SD).
No Component Percent by weight (% w/w) Total solid (TS) Volatile solids (VS) Organic carbon (C) Moisture content (MC) pH VS/TS (%)
1 Cafeteria leftover food 22.5 ± 0.15 39.4 ± 0.06 30.8 ± 0.12 41.6 ± 0.23 62.5 ± 0.21 7.6 ± 0.21 78.3 ± 0.36
2 Fruit waste and Vegetable 17.3 ± 0.1 31.5 ± 0.3 25.2 ± 0.06 34.4 ± 0.15 66.3 ± 0.06 6.8 ± 0.06 80.2 ± 0.87
3 Cattle manure waste 47.8 ± 0.12 21.3 ± 0.15 19.4 ± 0.1 25.3 ± 0.1 77.6 ± 0.12 6.8 ± 0.06 91.2 ± 1
Total 100 - - - - - -

The VS/TS ratios of the cafeteria food waste and cattle manure were 78.3% and 91.2%, respectively. These values indicated that there were sufficient biodegradable volatile solids in each substrate to enable effective anaerobic digestion ( Li et al., 2013; Pagliacci et al., 2016; Kawai et al., 2014). The amount of biodegradable organic matter in the dry matter content should be between 70 and 95% for efficient biogas production ( Wu et al., 2021; Buffiere et al., 2006).

Among with significant potential for biogas production ( Panahi et al., 2022; Zeng et al., 2022). All the substrates, cafeteria leftover food had the highest organic carbon content (41.6%), which is indicative of its rich protein, fat, and carbohydrate content. This makes it a high-energy feedstock 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 accelerate biogas production by promoting bacterial growth in the digester. Co-digestion has a positive synergistic effect by neutralizing pH, increasing buffering capacity, reducing the effects of harmful compounds, and supplying more balanced nutrients, such as vitamins, trace metals, and other substances required for microbial growth ( Fang, 2010; Aragaw et al., 2013; Jianzheng et al., 2011).

 3.2 The effect of rumen liquid and <italic toggle="yes">S. cerevisiae</italic> isolate MUTJ0F on biogas production

The amount of biogas generated from digester using rumen fluids and S. cerevisiae isolate MUTJ0F as a fermentation activator is presented in Table 2. The 12 methane production treatments varied significantly, reflecting variations in the mixed-waste composition and inoculum dosage. The combination of mixed waste with rumen fluid and S. cerevisiae isolate MUTJ0F significantly improved anaerobic digestion at mesophilic temperature (30°C) compared to the control (without inoculum). This implies that the high anaerobic bacterial concentration in rumen fluid efficiently breaks down organic substrates from mixed waste. These findings are consistent with those of other researchers ( Tamrat, 2012; Sakar et al., 2008; Yitayal, 2011; Forster-Carneiro et al., 2008; Abdullahi et al., 2011).

The effect biogas production on 12 treatments used in anaerobic digestion of mixed wastes combinations with rumen fluid and <italic toggle="yes">S. cerevisiae</italic> isolate MUTJ0F doses (ml).
Treatments Mixed Wastes (MW) 100 g Rumen fluid S. cerevisiae isolate MUTJ0F Methane (mean ± SD)
T1 MW 1 0 ml RF 5 ml 1600 ± 0.49 ml
T2 MW 2 0 ml RF 10 ml 1600 ± 0.62 ml
T3 MW 3 25 ml RF 5 ml 2100.7 ± 0.67 ml
T4 MW 4 25 ml RF 10 ml 2300.3 ± 0.17 ml
T5 MW 5 50 ml RF 5 ml 3400.4 ± 0.49 ml
T6 MW 6 50 ml RF 10 ml 3800.6 ± 0.56 ml
T7 MW 7 75 ml RF 5 ml 6000.4 ± 0.17 ml
T8 MW 8 75 ml RF 10 ml 6500.5 ± 0.25 ml
T9 MW 9 100 ml RF 5 ml 6800.4 ± 0.12 ml
T10 MW 10 100 ml RF 10 ml 6900.3 ± 0.26 ml
T11 MW 11 150 ml RF 5 ml 6300.57 ± 0.47 ml
T12 MW 12 150 ml RF 10 ml 6350.13 ± 0.06 ml
T control MW 0 0 ml RF 0 ml 1500.2 ± 0.1 ml

T, Treatments: MW, Mixed Waste: RF, Rumen fluid.

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. These results demonstrate that co-inoculation with S. cerevisiae isolate MUTJ0F and rumen fluid significantly increases biogas productivity by enriching the microbial community involved in acidogenesis, hydrolysis, and methanogenesis. The digester with the lowest methane volume (1500.2 ml) was the control digester (without inoculum), which contained only mixed waste without yeast or rumen fluid. This demonstrates that external microbial stimulants significantly increase the efficiency of anaerobic digestion.

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; Wilson, 2011; Wandera et al., 2018).

 3.3 pH variations in digestor T10 during fermentation

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; Wilson, 2011). On the other hand, the pH starts to rise on day forty of fermentation. This increase is due to ammonification processes in which the breakdown of proteins releases ammonia, which buffers the system and increases alkalinity ( Gerardi, 2003; Zhang et al., 2014; Yaichurrozi et al., 2016). 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).

T10 pH variation during the digestion process. 3.4 Combustibility of biogas

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

Performance of biogas production after 60 days observation.

A. Mix of wastes which was subjected to digesters; B. 0.6 L plastic bottle digesters; and C . Combustibility of Biogas.

 4. Conclusion

This study showed that the co-digestion of mixed organic waste with S. cerevisiae isolate MUTJ0F and rumen fluid is a feasible method for significantly improving biogas production in anaerobic environments. Among all treatments, a mixture of 100 ml rumen fluid and 10 ml S. cerevisiae isolate MUTJ0F/100 g of mixed waste produced the highest volume of biogas (6900.3 ml). Biodegradation, microbial strength, and biogas generation can all be enhanced by using rumen fluid and the S. cerevisiae isolate MUTJ0F as an inoculum in biogas digesters. Using rumen fluid and S. cerevisiae as bioactivators provides a valuable, affordable, and effective way to increase the production of biogas from readily accessible organic wastes, such as fruit leftovers, cattle manure, and cafeteria leftovers. Institutions such as universities, agro-industries, and municipalities that produce large amounts of food and fruit-vegetable waste should implement co-digestion systems using these inoculants to improve waste management, lower environmental pollution, and produce renewable energy. Additionally, by offering a cleaner, sustainable alternative for household and industrial energy, expanding this strategy could improve public health, reduce reliance on firewood, and mitigate deforestation.

 Author contribution statement

Conceptualization, M.F. and B.K; Methodology, B.K.; Data analysis, M.F.; Investigation, M.F. and B.K.; Resources, M.F. and B.K.; Writing – original draft, M.F.; Writing – review and editing, M.F. and B.K.; Visualization, M.F.; Funding acquisition, M.F. All authors have read and agreed to the published the manuscript.

 Consent for publication

Not applicable.

 Ethical approval

Not applicable.

 Data availability statement Underlying data

Figshare: The effect biogas production on 12 treatments used in anaerobic digestion of mixed wastes combinations with rumen fluid and S. cerevisiae isolate MUTJ0F doses (ml) of data analysis. https://doi.org/10.6084/m9.figshare.28284806 ( Fentahun and Kashay, 2025).

This project contains the following underlying data:

Raw data. Physico chemical properties of mixed wastes of data analysis.

Data are available under the terms of the Creative Commons Attribution 4.0 International license (CC-BY 4.0).

 Acknowledgments

We want to express our gratitude to Debre Markos University for financial support.

 References Abayneh L Kebede A Muthuswamy M : Biogas production from co-digestion of coffee pulp waste and cow dung under mesophilic conditions. The Journal of Energy and Environmental Science. Photon. 2014;128:444–450. Abbas Y Yun S Wang K : Static-magnetic-field coupled with fly-ash accelerant: A powerful strategy to significantly enhance the mesophilic anaerobic-co-digestion. Bioresource Technology. 2021;327:124793. 33581377 10.1016/j.biortech.2021.124793 Abdulkareem AS : Refining biogas produced from biomass: An alternative to cooking gas. Leonardo. Journal of Science. 2005;7:1–8. Abdullahi I Ismail A Musa OA : Effect of Kinetic Parameters on Biogas Production from Local Substrate using a Batch Feeding Digester. European Journal of Scientific Research. 2011;2011(57):626–634. Achmad KT Gani SA Sunarto SI : The use of rumen liquid and Saccharomyces cerevisiae as activators in biogas production from fresh market garbage. Lucrări Ştiinţifice. 2011;56. Akinbami JFK Ilori MO Oyebisi TO : Biogas energy use in Nigeria: current status, future prospects and policy implications. Renewable and Sustainable Energy Reviews. 2001;5:97–112. 10.1016/S1364-0321(00)00005-8 Alemayehu G : Anaerobic co-digestion of biodegradable municipal solid waste with human excreta for biogas production: A review. American Journal of Applied Chemistry. 2014;2:55–62. 10.11648/j.ajac.20140204.12 Anunputtikul W Rodtong S : Laboratory scale experiments for biogas production from cassava tubers. The Joint International Conference on “Sustainable Energy and Environment”. 2004;1:238–243. APHA (American Public and Health Association): Standard method for the Examination of water and waste water. Parts 1000 and 4000, 216, 240. Hand book of water purity and quality. 1999. Aragaw T Mebeaselassie A Amare G : Co-digestion of cattle manure with organic kitchen waste to increase biogas production using rumen fluid as inoculums. International Journal of the Physical Sciences. 2013;8:443–450. Arogo J Wen O Ignosh J : Bio-methane Technology College of Agriculture and Life Sciences. Virginia Polytechnic Institute and State University publication;2009; pp.442–881. Arthur R Brew-Hammond A : Potential biogas production from sewage sludge: A case study of the sewage treatment plant at Kwame Nkrumah university of science and technology, Ghana. International Journal of Energy and Environment. 2010;1:1009–1016. Aurora SP : Microbial Digestion in Ruminants. Indian Journal of Agricultural Research. 1983;2:85–94. Aynalem A Helmut K Terefe D : Evaluation of the 2007 Ethiopian Census Data with an Emphasis on Coverage in Amhara Region. Ethiopian Journal of Development Research. 2014;36. Budiyono W Johari IN Sunarso S : The kinetic of biogas production rate from cattle manure in batch mode. International journal of chemical and biological enginnering. 2010;3:39–44. Buffiere P Loisel D Bernet N : Towards new indicators for the prediction of solid waste anaerobic digestion properties. Water Science & Technology. 2006;53:233–241. 16784208 10.2166/wst.2006.254 Corral MM Samani ZA Hanson G : Anaerobic digestion of municipal solid waste and agricultural waste and the effect of co-digestion with dairy cow manure. Bioresource Technology. 2008;99:8288–8293. 18482835 10.1016/j.biortech.2008.03.057 Earnest VP Singh LP : Biomethanation of Vegetable and Fruit Waste in Co-digestion Process. International Journal of Emerging Technology and Advanced Engineering. 2013;3:250–2459. Elias J Hameed S Araya A : Renewable biogas energy production from Cladodes of opuntiaficus- indica. Journal of Food and Agriculture Science. 2010;1:44–48. Elijah T Iyagba IA Yahaya SM : The study of cow dung as co-substrate with rice husk in biogas production. Scientific Research Essay. 2009;4:861–866. Fang C : Biogas production from food-processing industrial wastes by anaerobic digestion. Technical University of Denmark;2010. PhD Thesis. Farid G : The new global energy order: shifting players, policies, and power dynamics. Chapter 2. Public Responses to Fossil Fuel Export. 2022; pp.25–44. Fentahun M Andualem B : Characterization of wild indigenous yeasts that can withstand different stresses from traditional fermented beverages in the Amhara Region, Ethiopia. Food Science and Applied Biotechnology. 2024;7:262–277. 10.30721/fsab2024.v7.i2.405 Fentahun M Kashay B : Figshare: The effect biogas production on 12 treatments used in anaerobic digestion of mixed wastes combinations with rumen fluid and S. cerevisiae isolate MUTJ0F doses (ml) of data analysis. 2025. 10.6084/m9.figshare.28284806 Fernández J Pérez M Romero LI : Effect of substrate concentration on dry mesophilic anaerobic digestion of organic fraction of municipal solid waste (OFMSW). Bioresource Technology. 2008;99:6075–6080. 10.1016/j.biortech.2007.12.048 18255282 Forster-Carneiro T Pérez M Romero LI : Influence of total solid and inoculum contents on performance of anaerobic reactors treating food waste. Bioresource Technology. 2008;99:6994–7002. 18295478 10.1016/j.biortech.2008.01.018 Genet T Birhanu A Solomon K : Optimization of bio gas production from slaughterhouse wastes. African Journal of Environmental Science and Technology. 2018;12:283–295. 10.5897/AJEST2017.2456 Gerardi MH : The Microbiology of anaerobic digesters. Wiley Interscience;2003; pp.1–85. Hammad EI Al-Agha MR El-Nahhal Y : Enhancing Biogas Production: Influence of Mixing Cow and Chicken Manures. Energy and Power Engineering. 2018;10:383–397. 10.4236/epe.2018.108024 Harris P : Biogas Notes. AFEST, University of Adelaide and IOBB;2008. Haug RT : The practical handbook of compost engineering. Florida, USA: Lewis Publishers;1993. Huang X Yun S Zhu J : Mesophilic anaerobic co-digestion of aloe peel waste with dairy manure in the batch digester: Focusing on mixing ratios and digestate stability. Bioresource Technology. 2016;218:62–68. 27347799 10.1016/j.biortech.2016.06.070 Imri K Valeria N : Experiments on the maximum biogas production. 7 th International Multidisciplinary Conference. Baia Mare, Romania:2007; pp.309–310. Ituen EE John N Bassey M : Biogas production from organic waste in AkwaIbom. Appropriate Technologies for Environmental Protection in the Developing World. 2007; pp.93–99. Jia B Yun S Shi J : Enhanced anaerobic mono- and co-digestion under mesophilic condition: Focusing on the magnetic field and Ti-sphere core-shell structured additives. Bioresource Technology. 2020;310:123450. 32388352 10.1016/j.biortech.2020.123450 Jianzheng L Ajay K Jha J : Assessment of the effects of dry anaerobic codigestion of cow dung with waste water sludge on biogas yield and biodegradability. International Journal of the Physical Sciences. 2011;6:3679–3688. Joyce A Ijaz UZ Nzeteu C : Linking microbial community structure and function during the acidified anaerobic digestion of grass. Frontiers in Microbiology. 2018;9:540. 29619022 10.3389/fmicb.2018.00540 PMC5871674 Kawai M Nagao N Tajima N : The effect of the labile organic fraction in food waste and the substrate/inoculum ratio on anaerobic digestion for a reliable methane yield. Bioresource Technology. 2014;157:174–180. 24556370 10.1016/j.biortech.2014.01.018 Leta D Solomon L Chavan RB : Production of biogas from fruit and vegetable wastes mixed with different wastes. Environment and Ecology Research. 2015;3:65–71. Li D Ran Y Chen L : Instability diagnosis and syntrophic acetate oxidation during thermophilic digestion of vegetable waste. Water Research. 2018;139:263–271. 29656191 10.1016/j.watres.2018.04.019 Li Y Zhang R Liu X : Evaluating methane production from anaerobic mono- and co-digestion of kitchen waste, corn stover, and chicken manure. Energy Fuel. 2013;27:2085–2091. 10.1021/ef400117f Lin J Zuo J Gan L : Effects of mixture ratio on anaerobic co-digestion with fruit and vegetable waste and food waste of China. Journal of Environmental Science. 2011;23:1–15. Lopes WS Leite VD Prasad S : Influence of inoculum on performance of anaerobic reactors for treating municipal solid waste. Bioresource Technology. 2004;94:261–266. 15182832 10.1016/j.biortech.2004.01.006 Lynd LR Weimer PJ Van ZWH : Microbial Cellulose Utilization: Fundamentals and Biotechnology. Microbiology and Molecular Biology Reviews. 2002;66:506–577. 12209002 10.1128/MMBR.66.3.506-577.2002 PMC120791 Mata-Alvarez J Dosta J Romero-Güiza M : A critical review on anaerobic co-digestion achievements between 2010 and 2013. Renewable and Sustainable Energy Reviews. 2014;36:412–427. 10.1016/j.rser.2014.04.039 Mata-Alvarez JS Macé S Llabrés P : Anaerobic digestion of organic solid wastes: An overview of research achievements and perspectives. Bioresource Technology. 2000;74:3–16. 10.1016/S0960-8524(00)00023-7 Membere EA John U Akan UE : Biomechanization Potential of Organic Fraction of Municipal Solid Waste (OFMSW) from Co-Digestion of Pig and Cow Dung. International Journal of Environmental Science. 2012;2:2387–2399. Mshandete AA Kivaisi A Rubindamayugi B : Anaerobic digestion of sisal pulp and fish wastes. Bioresource Technology. 2004;95:19–24. 15207289 10.1016/j.biortech.2004.01.011 Nasir IM Mohd GTI Omar R : Anaerobic Digestion Technology in Livestock Manure Treatment for Biogas Production: A Review. Engineering in Life. 2012;12:258–269. 10.1002/elsc.201100150 Pagliaccia P Gallipoli A Gianico A : Single stage anaerobic bioconversion of food waste in mono and co-digestion with olive husks: impact of thermal pretreatment on hydrogen and methane production. International Journal of Hydrogen Energy. 2016;41:905–915. 10.1016/j.ijhydene.2015.10.061 Panahi HK Dehhaghi M Guillemin GJ : Bioethanol production from food wastes rich in carbohydrates. Current Opinion in Food Science. 2022;43:71–81. 10.1016/j.cofs.2021.11.001 Sakar S Yetilmezsoy K Kocak E : Anaerobic digestion technology in poultry and livestock treatment - a literature review. Waste Management & Research. 2008;27:3–18. Sonakya V Raizada N Dalhoff R : Elucidation mechanism of organic acids production from organic matter (grass) using digested and partially digested cattle feed. Water Science & Technology. 2003;48:255–259. 14682594 10.2166/wst.2003.0476 Sosnowski P Wieczorek A Ledakowicz S : Anaerobic codigestion of sewage sludge and organic fraction of municipal solid wastes. Advances in Environmental Research. 2003;7:609–616. 10.1016/S1093-0191(02)00049-7 Stabnikova O Ang S Liu X : The use of hybrid anaerobic solid liquid system for the treatment of lipid-containing food waste. Journal of Chemical Technology & Biotechnology. 2005;80:455–461. 10.1002/jctb.1225 Sylvester JT Karnati SKR Yu TZ : Development of an assay to quantify rumen ciliate protozoal biomass in cows using real-time PCR. The Journal of Nutrition. 2004;134:3378–3384. 15570040 10.1093/jn/134.12.3378 Tamirat A Mebeaselassie A Amare G : Co-digestion of cattle manure with organic kitchen waste to increase biogas production using rumen fluid as inoculums. International Journal of Physical Sciences. 2013;8:443–450. Tamrat A : Effects of Co-Digestion of Cattle Manure with Organic Kitchen Waste Using Rumen Fluid as Inoculums on the Rate and Amount of Biogas Yield. Haramaya university;2012. Master’s thesis. Wandera SM Qiao W Algapani DE : Searching for possibilities to improve the performance of full scale agricultural biogas plants. Renewable Energy. 2018;116:720–727. 10.1016/j.renene.2017.09.087 Wilson DB : Microbial diversity of cellulose hydrolysis. Current Opinion in Microbiology. 2011;14:259–263. 10.1016/j.mib.2011.04.004 Wu D Li L Peng Y : State indicators of anaerobic digestion: a critical review on process monitoring and diagnosis. Renewable and Sustainable Energy Reviews. 2021;148:111260. 10.1016/j.rser.2021.111260 Yaichurrozi R Rusdi T Hidayat A : Kinetics Studies Impact of Initial pH and Addition of Yeast Saccharomyces cerevisiae on Biogas Production from Tofu Wastewater in Indonesia. International Journal of Engineering. 2016;29:1037–1046. Yitayal A : Study on biogas energy production from leaves of Justicia schimperiana (hochst.ex a. Nees) t. Anders. Addis Ababa University, School of graduate studies Environmental Science Program;2011. Yue ZB Yu HQ : Anaerobic batch degradation of cattail by rumen cultures. International Journal of Environmental Pollution. 2009;38:299–308. 10.1504/IJEP.2009.027230 Zeng J Zeng H Wang Z : Review on technology of making biofuel from food waste. International Journal of Energy Research. 2022;46:10301–10319. 10.1002/er.7868 Zhang H Zhang P Ye J : Improvement of methane production from rice straw with rumen fluid pretreatment: A feasibility study. International Biodeterioration & Biodegradation. 2016;113:9–16. 10.1016/j.ibiod.2016.03.022 Zhang W Wei Q Wu S : Batch anaerobic co-digestion of pig manure with dewatered sewage sludge under mesophilic conditions. Applied Energy. 2014;128:175–183. 10.1016/j.apenergy.2004.04.071 10.5256/f1000research.193404.r449755 Reviewer response for version 2 Marousek Josef 1 Referee https://orcid.org/0000-0001-5175-1961 Technical University of Košice, Košice–Sever, Slovakia

Competing interests: No competing interests were disclosed.

 13 1 2026 Copyright: © 2026 Marousek J 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

The urgency and significance of the research hypothesis needs to be better justified and placed into [economic/industrial/environmental] context. It is appropriate to adapt he methodology to the current state of knowledge. The applicability needs to be strenghtened [cost breakdown and corresponding financial analyses are hard to find]. It is advisable to improve the conclusions in terms of synthesis and fertilization.

Is the work clearly and accurately presented and does it cite the current literature?

No

If applicable, is the statistical analysis and its interpretation appropriate?

Partly

Are all the source data underlying the results available to ensure full reproducibility?

No

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

No

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

phytotechnology, commercialization

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Fentahun Mulugeta Department of Biology, Debre Markos University, Debre Markos, Amhara, Ethiopia

Competing interests: No competing interests were disclosed.

 20 3 2026

Mulugeta Fentahun

Professor Josef Marousek thank you very much for your insightful comments and the time you dedicated to review our article.

I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:

Comment1: The urgency and significance of the research hypothesis needs to be better justified and placed into [economic/industrial/environmental] context.

Response: Thank you for this valuable comment. We have revised the manuscript to better clarify the urgency and significance of the research hypothesis within economic, industrial, and environmental contexts. Refer to the introduction part of the updated manuscript.

Comment2: It is appropriate to adapt the methodology to the current state of knowledge

Response: Thank you for this helpful comment. We have revised the methodology to align with the current state of knowledge by updating the experimental method based on recent literature. Refer to the method section of the updated manuscript.

Comment3: The applicability needs to be strengthened [cost breakdown and corresponding financial analyses are hard to find].

Response: Thank you for this important comment. We have strengthened the applicability of the study by considering the economic feasibility and practical potential of the study.

Comment4: It is advisable to improve the conclusions in terms of synthesis and fertilization.

Response: Thank you for this valuable suggestion. We have revised the conclusion section (refer to the revised manuscript).

 Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.

Best regards,

    Authors

 10.5256/f1000research.193404.r445154 Reviewer response for version 2 Yellezuome Dominic 1 Referee https://orcid.org/0000-0002-3676-2942 University of Mines and Technology, Tarkwa, Western Region, Ghana

Competing interests: No competing interests were disclosed.

 9 1 2026 Copyright: © 2026 Yellezuome D 2026 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation approve-with-reservations

This manuscript aimed at enhancing biogas production from a co-digestion of various organic wastes using rumen fluid and Saccharomyces cerevisiae isolate MUTJ0F as activators. The manuscript requires a careful major revision before it could possibly be accepted.

 

There is too much information in the methods paragraph in the Abstract Section, in which some of it can not be found in the Methods Section, such as “This study was conducted experimentally using factorial patterns and a completely randomized 6 × 2 design.”

And some of them are not important to be in the Abstract Section. Such as “Standard techniques were used to measure the physicochemical characteristics of the mixed waste, such as pH, total solids, volatile solids, organic carbon, and moisture. This study was conducted experimentally using factorial patterns and a completely randomized 6 × 2 design. The water displacement method was used to estimate the average amount of biogas generated by a 0.6 L digester.”

These sentences are similar; hence should be merged.

“Biogas production from different types of biodegradable waste is an alternative to fossil fuels for energy consumption and biodegradable waste management. Biogas is a cost-effective, environmentally friendly, and sustainable energy source that provides a reliable method for handling food waste.”

“An innovative solution to increase anaerobic waste digestion yields is codigestion, which is an inexpensive and simple technology that enhances the anaerobic digestion rate by creating a better nutrient balance from the materials mixed to feed the digester, provides positive synergism for bacterial growth, and increases biogas production (Sosnowski et al., 2003; Mata-Alvarez et al., 2000; Mshandete et al., 2004; Leta et al., 2015). Several studies have used co-digestion to increase the rate at which organic matter is converted biologically in the biogas system to improve biogas performance (Huang et al., 2016; Alemayehu, 2014; Mata-Alvarez et al., 2014; Abbas et al., 2021).”

“A diverse range of fungi, bacteria, protozoa, and archaea inhabit the rumen, an anaerobic microbial habitat (Sylvester et al., 2004; Sonakya et al., 2003). The rumen contains a variety of microorganisms, including cellulolytic and methanogenic bacteria (Lopes et al., 2004; Yue and Yu, 2009).”

“Institutions such as universities, agro-industries, and municipalities that produce large amounts of food and fruit-vegetable waste should implement co-digestion systems using these inoculants to improve waste management, lower environmental pollution, and produce renewable energy. Additionally, by offering a cleaner, sustainable alternative for household and industrial energy, expanding this strategy could improve public health, reduce reliance on firewood, and mitigate deforestation.”

Authors cited lists of 3 or more references without proper justification of the relevance of each one. Hence, avoid citing more than two references and outdated references.  

“Although there is information on biogas production from organic waste or single inoculums (Alemayehu, 2014; Abayneh et al., 2014; Hammad et al., 2018; Earnest and Singh, 2013) but there is limited reports on methods for increasing biogas generation, particularly those that use rumen fluid and S. cerevisiae.” This paper is not clear in the description of the scientific novelty in comparison to related previously published and referenced in your work below:

 Achmad KT, Gani SA, Sunarto SI, et al.: The use of rumen liquid and Saccharomyces cerevisiae as activators in biogas production from fresh market garbage. Lucrări Ştiinţifice. 2011; 56.

Aragaw T, Mebeaselassie A, Amare G: Co-digestion of cattle manure with organic kitchen waste to increase biogas production using rumen fluid as inoculums. International Journal of the Physical Sciences. 2013; 8: 443–450.

Tamirat A, Mebeaselassie A, Amare G: Co-digestion of cattle manure with organic kitchen waste to increase biogas production using rumen fluid as inoculums. International Journal of Physical Sciences. 2013; 8: 443–450.

Tamrat A: Effects of Co-Digestion of Cattle Manure with Organic Kitchen Waste Using Rumen Fluid as Inoculums on the Rate and Amount of Biogas Yield. Haramaya university; 2012. Master’s thesis

Yue ZB, Yu HQ: Anaerobic batch degradation of cattail by rumen cultures. International Journal of Environmental Pollution. 2009; 38: 299–308.

Zhang H, Zhang P, Ye J, et al.: Improvement of methane production from rice straw with rumen fluid pretreatment: A feasibility study. International Biodeterioration & Biodegradation. 2016; 113: 9–16.

“…there are 107,684 residents, comprising 49,893 men and 57,791 women (Aynalem et al., 2014).” These statistics are outdated.

“This study aimed to evaluate the effects of ruminant fluids and S. cerevisiae isolate MUTJ0F (OR209280.1) on biogas generation from various mixed organic wastes co-digested with cattle manure. Various wastes from fruits and vegetables were gathered from the town fruit houses, leftover food from the Debre Markos University student cafeteria, fresh cattle manure (CM) from Monkorer Agroindustry Enterprise, and rumen fluids (RL) from a nearby slaughterhouse and used as activators for biogas production.” These sentences contains repititions in the next paragraph, hence should be avoided.

“… the de-sized cattle manure and food waste were mixed separately with distilled water in a 1:5 (solid waste: distilled water) volume ratio …”  What is the ratio between cafeteria leftovers, cattle manure, and fruit and vegetable waste? How was the solid waste measured in volume?

What is the size of the filter used in filtering the rumen fluid?

Some of the citations are not necessary, such as

“Then, different amounts of the filtrate were added to each digester to initiate the reaction (Aurora, 1983; Genet et al., 2018).”

“After calibration, the electrode was submerged in the substrate samples, rinsed with distilled water, and the pH values were recorded (Arogo et al., 2009).”

In subsection 2.5.1, how was the measured empty crucible used in the formula?

In subsection 2.5.2, the authors indicated the measurement of fixed solids, but it was not presented in Table 1.

Authors should be consistent with the way they explain parameters in an equation, whether horizontal or vertical.

The procedure for moisture content determination is the same as that of total solids, so you can indicate the formula of MC there and delete subsection 2.5.4

“After calibration, the electrode was submerged in the substrate samples, rinsed with distilled water, and the pH values were recorded (Arogo et al., 2009).” Are the authors sure this was the sequence used in measuring the pH value?

“The first factor was the dosage of rumen liquid i.e. 0 ml/100 g, 25 ml/100 g, 50 ml/100 g, 75 ml/100 g, and 100 ml/100 g), …” Why is the 150 ml rumen fluid not stated here but in Table 2?

“The study was conducted at room temperature (30°C).” How did the authors maintain this temperature in the room?

“Feed stock was mixed with distilled water to obtain approximately 8% of TS suspension.” Is this information similar to the “1:5 (solid waste: distilled water) volume ratio” indicated in Section 2.3?

In section 2.6, the retention time or duration of the anaerobic digestion was not mentioned.

Section 2.7 should come before Section 2.6. Also, the authors need to provide an image of the three plastic bottles set up for anaerobic digestion, since it was constructed for this study. What is the working volume of the digester?

Sections 2.8 and 2.7 can be merged. Was the biogas measured daily or at what interval?

In Section 3.1, what does the use of “mixed wastes” mean? For example: “The pH of the mixed waste ranged from 6.8 to 7.6, …” Does it mean the mixture of the various feedstock or the individual feedstock?

“The VS/TS ratios of the cafeteria food waste and cattle manure were 78.3% and 91.2%, respectively.” What about fruit and vegetable waste?

“Cattle dung is used to accelerate biogas production by promoting bacterial growth in the digester.” Provide a reference.

“Co-digestion has a positive synergistic effect by neutralizing pH, increasing buffering capacity, reducing the effects of harmful compounds, and supplying more balanced nutrients, such as vitamins, trace metals, and other substances required for microbial growth (Fang, 2010; Aragaw et al., 2013; Jianzheng et al., 2011).” The relevance of this information in the Results and Discussion Section is not clear since it has already been stated in the Introduction Section with different references cited.

“The 12 methane production treatments varied significantly, reflecting variations in the mixed-waste composition and inoculum dosage.” Are the authors sure that the variations in the mixed-waste composition may have caused the significant difference in biogas production?

“This implies that the high anaerobic bacterial concentration in rumen fluid efficiently breaks down organic substrates from mixed waste.” Is it only the rumen fluid that increased biogas production?

“According to the results, the maximum amount of biogas (6900.3 ml) …” Normally, biogas production is evaluated using methane yield (mL/gVS or mL/gTS) to enable comparison with other published studies.

In Table 2, why was only rumen fluid not investigated? The heading “Methane (mean ±  SD)”, did the authors measure methane or biogas?

In Figure 1, please plot pH on the y-axis and days on the x-axis. Why plot only T10? It is better to plot all the treatments. Why was the pH measured up to 80 days, but the anaerobic digestion was observed for 60 days, as indicated in Figure 2, the Abstract and Section 3.4?

Figure 2A and B should come before Figure 1.

These sentences could have been verified by plotting the daily biogas production.

“This low pH value was permitted very little methanogenic bacterial activity …”

“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).”

 The authors should clarify which equipment was used for the combustion test, either “a lit matchstick” as indicated in Section 2.9 or “a Bunsen burner” as indicated in Section 3.4. Also, Figure 2C was not well captured to help identify which equipment was used.  In section 3.4, it is better use “Figure 2C” rather than “Figure 2”.

“Biodegradation, microbial strength, and biogas generation can all be enhanced by using …” How were biodegradation and microbial strength measured in this study?

Future perspectives or studies could be added to the Conclusion section.

The language must be checked thoroughly.

Some statements are not clear or too long and should be revised. For examples:

 “However, various juice house wastes are among the municipal wastes that are becoming difficult to manage.” Also, indicate why they are difficult to manage.

“… food waste co-digestion under anaerobic conditions and cattle manure, …”

“The strain Saccharomyces cerevisiae isolate MUTJ0F (OR209280.1) with accession number was used in this study.”

“A mixture of substrates, including peels of bread, injera, spaghetti, papaya, mango, banana, and avocado was used in this study.” Which substrates?

“The CM was separated …” Should it be “separated” or “spread”?

“The inoculum was specific to each digester.”

“The crucibles were dried, cooled to room temperature in a desiccator, and weighed again.” Should it be “dried” or “removed”?

“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:”

“The first factor was the dosage of rumen liquid i.e. 0 ml/100 g, 25 ml/100 g, 50 ml/100 g, 75 ml/100 g, and 100 ml/100 g), while the second factor was the dosage of S. cerevisiae isolate MUTJ0F i.e. 5 ml/100 g, and 10 ml/100 g) of the mixed organic waste.” What does the “factor” stands for?

“… until a solution that was supersaturated formed.”

“After the biogas was collected, a measurement cylinder containing the gas was carefully placed.”

“… varied in the amount of composition owing to the variability in the

composition of the samples of the different substrates.”

“Among with significant potential for biogas production (Panahi et al., 2022; Zeng et al., 2022). All the substrates, cafeteria leftover food had the highest organic carbon content (41.6%), …”

“This makes it a high-energy feedstock substrates were considered acceptable for anaerobic digestion because all the mixed wastes had the highest ratio of VS to TS in Table 1.”

“This low pH value was permitted very little methanogenic bacterial activity …”

 

Avoid stating the mesophilic condition twice in the abstract section.

"physic-chemical properties" = "physicochemical properties"

Use SI units. For example “hrs” = “h”

These sentences should be merged to improve clarity.

 “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.”

“These values indicated that there were sufficient biodegradable volatile solids in each substrate to enable effective anaerobic digestion (Li et al., 2013; Pagliacci et al., 2016; Kawai et al., 2014). The amount of biodegradable organic matter in the dry matter content should be between 70 and 95% for efficient biogas production (Wu et al., 2021; Buffiere et al., 2006).”

“This increase is due to ammonification processes …” Since this sentence was not verified by measurement, it would be better to express it as “This increase may be due to ammonification processes …”

Is the work clearly and accurately presented and does it cite the current literature?

Partly

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Partly

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Anaerobic digestion, pyrolysis and gasification

I confirm that I have read this submission and believe that I have an appropriate level of expertise to confirm that it is of an acceptable scientific standard, however I have significant reservations, as outlined above.

Fentahun Mulugeta Department of Biology, Debre Markos University, Debre Markos, Amhara, Ethiopia

Competing interests: No competing interests were disclosed.

 20 3 2026

Mulugeta Fentahun

Thanks to Dr. Dominic Yellezuome for your constructive comments. We conducted a number of changes.

I greatly appreciate your feedback and suggestions. Please find my responses to your comments below:

Comment 1: There is too much information in the methods paragraph in the Abstract Section, in which some of it can not be found in the Methods Section. Some of them are not important to be in the Abstract Section.

Response: Thank you for this helpful comment. We have revised the abstract according to your feedback. Refer to the abstract part in the updated manuscript

Comment 2: These sentences are similar; hence should be merged.

Response: Thank you for this observation. We have revised the manuscript by merging similar sentences to improve readability and eliminate redundancy (Refer to the revised manuscript).

Comment 3: Authors cited lists of 3 or more references without proper justification of the relevance of each one. Hence, avoid citing more than two references and outdated references (Refer to the revised manuscript).

Response: Thank you for this constructive comment. We have revised the manuscript by limiting citations and avoiding outdated references (Refer to the revised manuscript).

Comment 4: Although there is information on biogas production from organic waste or single inoculums (Alemayehu, 2014; Abayneh et al., 2014; Hammad et al., 2018; Earnest and Singh, 2013) but there is limited reports on methods for increasing biogas generation, particularly those that use rumen fluid and S. cerevisiae.” This paper is not clear in the description of the scientific novelty in comparison to related previously published and referenced in your work below:

Response: Thank you for this important comment. We have revised the manuscript based on your comments (Refer to the revised manuscript).

Comment 5: There are 107,684 residents, comprising 49,893 men and 57,791 women (Aynalem et al., 2014).” These statistics are outdated.

Response: Thank you for pointing this out. We have updated the demographic information using the most recent and reliable data sources to ensure accuracy and relevance in the manuscript.

Comment 6: This study aimed to evaluate the effects of ruminant fluids and S. cerevisiae isolate MUTJ0F (OR209280.1) on biogas generation from various mixed organic wastes co-digested with cattle manure. Various wastes from fruits and vegetables were gathered from the town fruit houses, leftover food from the Debre Markos University student cafeteria, fresh cattle manure (CM) from Monkorer Agroindustry Enterprise, and rumen fluids (RL) from a nearby slaughterhouse and used as activators for biogas production.” These sentences contains repititions in the next paragraph, hence should be avoided.

Response: Thank you for this comment. We have revised the manuscript to remove the repetitive sentences .

Comment 7: The de-sized cattle manure and food waste were mixed separately with distilled water in a 1:5 (solid waste: distilled water) volume ratio …”  What is the ratio between cafeteria leftovers, cattle manure, and fruit and vegetable waste? How was the solid waste measured in volume?.

Response: Thank you for this insightful comment. We have revised the manuscript to clarify the mixing ratios and measurement procedures (Refer to the revised manuscript).

Comment 8: What is the size of the filter used in filtering the rumen fluid?

Response: Thank you for this comment. We have revised the manuscript based on your comments (Refer to the revised manuscript).

Comment 9: Some of the citations are not necessary, such as ○ “Then, different amounts of the filtrate were added to each digester to initiate the reaction (Aurora, 1983; Genet et al., 2018).” “After calibration, the electrode was submerged in the substrate samples, rinsed with distilled water, and the pH values were recorded (Arogo et al., 2009).

Response: Thank you for this comment. We have revised the manuscript by removing unnecessary citations.

Comment 10: In subsection 2.5.1, how was the measured empty crucible used in the formula?

Response: Thank you for this comment. We have revised and clarified the method in subsection 2.5.1 based on your feedback (Refer to the updated manuscript).

Comment 11: In subsection 2.5.2, the authors indicated the measurement of fixed solids, but it was not presented in Table 1.

Response: Thank you for this comment. We have revised the manuscript by include the measured fixed solids in Table 1 or clarified in the manuscript.

Comment 12: Authors should be consistent with the way they explain parameters in an equation, whether horizontal or vertical.

Response: Thank you for this comment. We have revised the manuscript to present all equation parameters consistently in a horizontal format (Refer to the updated manuscript).

Comment 13: The procedure for moisture content determination is the same as that of total solids, so you can indicate the formula of MC there and delete subsection 2.5.4.

Response: Thank you for this suggestion. We have removed subsection 2.5.4 and included the formula for moisture content within the total solids section, streamlining the methods and avoiding redundancy.

Comment 14: After calibration, the electrode was submerged in the substrate samples, rinsed with distilled water, and the pH values were recorded (Arogo et al., 2009).” Are the authors sure this was the sequence used in measuring the pH value?

Response: Thank you for this comment. We have revised the correct sequence and refer to the updated the manuscript.

Comment 15: The first factor was the dosage of rumen liquid i.e. 0 ml/100 g, 25 ml/100 g, 50 ml/100 g, 75 ml/100 g, and 100 ml/100 g), …” Why is the 150 ml rumen fluid not stated here but in Table 2?

Response: Thank you for this comment. We have revised the manuscript to include the 150 ml/100 g rumen fluid level in the text for consistency with Table 2.

Comment 16: The study was conducted at room temperature (30°C).” How did the authors maintain this temperature in the room?

Response: Thank you for this comment. We have clarified in the manuscript that the study was conducted under mesophilic conditions at 38 °C using an oven, and the text has been corrected to fix the typographical error.

Comment 17: Feed stock was mixed with distilled water to obtain approximately 8% of TS suspension.” Is this information similar to the “1:5 (solid waste: distilled water) volume ratio” indicated in section 2.3?

Response: Thank you for this comment. We have clarified in the manuscript that the 8% TS suspension is distinct from the 1:5 ratio described in section 2.3.

Comment 18: In section 2.6, the retention time or duration of the anaerobic digestion was not mentioned.

Response: Thank you for this comment. We have revised section 2.7 based on your comment.

Comment 19: Section 2.7 should come before Section 2.6. Also, the authors need to provide an image of the three plastic bottles set up for anaerobic digestion, since it was constructed for this study. What is the working volume of the digester?

Response: Thank you for this comment. We have revised the manuscript and we have added an image of the three plastic bottle digesters used in this study and the working volume of digester.

Comment 20: Sections 2.8 and 2.7 can be merged. Was the biogas measured daily or at what interval?

Response: Thank you for this comment. We have merged sections 2.7 and 2.8 for clarity and streamlined the methods. We also clarified that biogas production was measured at regular daily intervals throughout the experiment.

Comment 21: In section 3.1, what does the use of “mixed wastes” mean? For example: “The pH of the mixed waste ranged from 6.8 to 7.6, …” Does it mean the mixture of the various feedstock or the individual feedstock?

Response: Thank you for this comment. We have clarified in section 3.1 based on your comments. Refer to the results and discussion parts in the updated manuscript .

Comment 22: The VS/TS ratios of the cafeteria food waste and cattle manure were 78.3% and 91.2%, respectively.” What about fruit and vegetable waste?

Response: Thank you for this comment. We have revised the manuscript based on your comments.

Comment 23: Cattle dung is used to accelerate biogas production by promoting bacterial growth in the digester.” Provide a reference.

Response: Thank you for this comment. We have added a relevant reference. Refer to the updated manuscript.

Comment 24: Co-digestion has a positive synergistic effect by neutralizing pH, increasing buffering capacity, reducing the effects of harmful compounds, and supplying more balanced nutrients, such as vitamins, trace metals, and other substances required for microbial growth (Fang, 2010; Aragaw et al., 2013; Jianzheng et al., 2011).” The relevance of this information in the Results and Discussion Section is not clear since it has already been stated in the Introduction Section with different references cited.

Response: Thank you for this comment. We have revised the results and discussion section and remove the redundant statement.

Comment 25: The 12 methane production treatments varied significantly, reflecting variations in the mixed waste composition and inoculum dosage.” Are the authors sure that the variations in the mixed-waste composition may have caused the significant difference in biogas production?

Response: Thank you for this comment. We have revised the manuscript based on your comments.

Comment 26: This implies that the high anaerobic bacterial concentration in rumen fluid efficiently breaks down organic substrates from mixed waste.” Is it only the rumen fluid that increased biogas production?

Response: Thank you for this comment. We have revised the manuscript based on your comments.

Comment 27: According to the results, the maximum amount of biogas (6900.3 ml) …” Normally, biogas production is evaluated using methane yield (mL/gVS or mL/gTS) to enable comparison with other published studies.

Response: Thank you for this comment. We clarify that our results were reported based on the total biogas volume (mL), and we did not measure total solids or volatile solids after digestion (methane yield (mL/gVS or mL/gTS)  in this experiment.

Comment 28: In Table 2, why was only rumen fluid not investigated? The heading “Methane (mean ± SD)”, did the authors measure methane or biogas?

Response: Thank you for this comment. We have clarified that Table 2 presents biogas production and not methane specifically; the heading has been corrected accordingly. Additionally, rumen fluid alone was not included as a treatment because the study focused on co-digestion with mixed wastes to evaluate the synergistic effect of dual inoculants.

Comment 29: In Figure 1, please plot pH on the y-axis and days on the x-axis. Why plot only T10? It is better to plot all the treatments. Why was the pH measured up to 80 days, but the anaerobic digestion was observed for 60 days, as indicated in Figure 2, the Abstract and Section 3.4?

Response: Thank you for this comment. We have revised Figure 1 to plot pH on the y-axis against days on the x-axis and included all treatments for clarity. Additionally, we clarified that pH measurements were continued up to 80 days to monitor post-digestion stabilization, even though the main anaerobic digestion period lasted 60 days.

Comment 30: Figure 2A and B should come before Figure 1.

Response: Thank you for this comment. We have revised the manuscript so that Figures 2A and 2B now appear before Figure 1.

Comment 31: These sentences could have been verified by plotting the daily biogas production.

Response: Thank you for this comment. We have revised the sentences in the updated manuscript.

Comment 32: The authors should clarify which equipment was used for the combustion test, either “a lit matchstick” as indicated in Section 2.9 or “a Bunsen burner” as indicated in Section 3.4. Also, Figure 2C was not well captured to help identify which equipment was used.  In section 3.4, it is better use “Figure 2C” rather than “Figure 2”

Response: Thank you for this comment. We have revised the manuscript in the method section and the results and discussion sections.

Comment 33: Biodegradation, microbial strength, and biogas generation can all be enhanced by using” How were biodegradation and microbial strength measured in this study

Response: Thank you for this comment. We have revised the manuscript based on your comments.

Comment 34: Future perspectives or studies could be added to the Conclusion section.

Response: Thank you for this comment. We have revised the conclusion section to include future perspectives.

Comment 35: The language must be checked thoroughly.

Response: Thank you for this comment. We have carefully reviewed and revised the manuscript to improve language, grammar, and clarity throughout the text.

  Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.

Best regards,

Authors

 10.5256/f1000research.177603.r401840 Reviewer response for version 1 Emmanuel Jovine Kamuhabwa 1 Referee https://orcid.org/0000-0002-2580-1539 Chemistry, University of Dar es Salaam, Dar es Salaam, Dar es Salaam, Tanzania

Competing interests: No competing interests were disclosed.

 19 8 2025 Copyright: © 2025 Emmanuel JK 2025 This is an open access peer review report distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. recommendation reject

Title

The title captures the content of the current work.

Abstract

The abstract has no information on the study, methodology used and key findings and recommendation based on findings. Authors must re-write this to reflect the current work. Other comments are found in the main document.

Introduction

The section has not been written properly and not easy to follow and understand. Authors must write this in a scholarly manner to reflect an article for publication.  The novelty is not well feature thus, authors must reveal the novelty of the work. Other comments are found in the main document.

Materials and methods

Proper details and step after step must be provided for readers to follow. Authors need to take time and re-do it properly. Other comments are found in the main document.

Results and discussion

Results are not well organized and discussed with previous work. Authors need to re-think on how they can organize these data for publication. Other comments are found in the main document.

Conclusions and recommendations

This section is not well written thus, must be re-written for clarity. Only key findings and recommendation must be presented and not otherwise. Other comments are found in the main document.

General comment

The manuscript must benefit from a serious English Language editing before being indexed.

The manuscript must be enriched with up-to-date citations which are available in the literature. Other comments are found in the main document.

Is the work clearly and accurately presented and does it cite the current literature?

No

If applicable, is the statistical analysis and its interpretation appropriate?

Not applicable

Are all the source data underlying the results available to ensure full reproducibility?

Partly

Is the study design appropriate and is the work technically sound?

Yes

Are the conclusions drawn adequately supported by the results?

Partly

Are sufficient details of methods and analysis provided to allow replication by others?

Partly

Reviewer Expertise:

Catalysis, materials science and bioenergy

I confirm that I have read this submission and believe that I have an appropriate level of expertise to state that I do not consider it to be of an acceptable scientific standard, for reasons outlined above.

Fentahun Mulugeta Department of Biology, Debre Markos University, Debre Markos, Amhara, Ethiopia

Competing interests: No competing interests were disclosed.

 11 12 2025

Mulugeta Fentahun

Dr. Jovine Kamuhabwa Emmanuel thank you very much for your insightful comments and the time you dedicated to review.

We greatly appreciate your feedback and suggestions. We provide a detailed response to each comment, along with the corresponding changes made in the revised manuscript below:

Title:

Comment: The title captures the content of the current work.

Response: Thank you for your positive feedback.

Abstract Revision:

Comment: The abstract has no information on the study, methodology used, and key findings and recommendations based on the findings. Authors must re-write this to reflect the current work.

Response: Thank you for your insightful comment. We have thoroughly revised the abstract part in response to your suggestions (see the revised manuscript).

Introduction Revision:

Comment: The section has not been written properly and is not easy to follow and understand. Authors must write this in a scholarly manner to reflect an article for publication. The novelty is not well featured; thus, authors must reveal the novelty of the work.

Response: We sincerely thank the reviewer for your constructive feedback. We have thoroughly revised the introduction section to improve clarity, coherence, and scholarly manner. Additionally, we have clearly highlighted the novelty of the study by emphasizing its unique contributions in the revised manuscript (refer to the revised manuscript).

Methodology Revision:

Comment: Proper details and step after step must be provided for readers to follow. Authors need to take time and re-do it properly.

Response: Thank you for this valuable observation. We have substantially revised the materials and methods section to improve clarity, completeness, and reproducibility. The updated version now provides detailed, step-by-step procedures for the experimental work, enabling readers to follow the methodology accurately and enhancing the scientific rigor of the manuscript (see the revised manuscript).

Results and Discussion Revision:

Comment: Results are not well organized and discussed with previous work. Authors need to re-think on how they can organize these data for publication.

Response: We sincerely thank the reviewer for your constructive feedback. We have carefully revised the results and discussion section to enhance clarity and coherence. The revised version enriched with up-to-date citations (recent literature) and emphasizes the key findings. Please refer to the updated section in the revised manuscript.

Conclusions and recommendations Revision:

Comment: This section is not well written thus, must be re-written for clarity. Only key findings and recommendation must be presented and not otherwise.

Response: Thank you for your insightful comment. We have revised the conclusions section to clearly highlight the study’s key findings and recommendation. Please see the updated section in the revised manuscript.

General comment:

Comment: The manuscript must benefit from a serious English Language editing before being indexed.

Response: We sincerely thank the reviewer for the valuable suggestion regarding the language quality of our manuscript. We have thoroughly revised the manuscript to improve English clarity, grammar, and readability.

Comment: The manuscript must be enriched with up-to-date citations which are available in the literature.

Response: Thank you for your comments. We have updated the manuscript by incorporating recent literature, as suggested (see the revised manuscript).

 Thank you once again for your valuable feedback. Your comments have significantly contributed to improving the quality of this paper.

Best regards,

Authors