Enhancing biogas production from co-digestion of organic wastes mixed with cattle manure using rumen fluid and Saccharomyces cerevisiae isolate MUTJ0F

Mulugeta Fentahun; Birhanu Kahsay

doi:10.12688/f1000research.161561.2

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Research Article

Revised

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 not approved]

Mulugeta Fentahun ¹, Birhanu Kahsay²

PUBLISHED 19 Dec 2025

Author details Author details

¹ College of Natural and Computational Sciences, Department of Biology, Debre Markos University, Debre Markos, Amhara, 269, Ethiopia
² Institute of Biotechnology, Mekelle University, Mek'ele, Tigray, Ethiopia

Mulugeta Fentahun
Roles: Conceptualization, Data Curation, Formal Analysis, Funding Acquisition, Investigation, Project Administration, Resources, Software, Supervision, Validation, Visualization, Writing – Original Draft Preparation, Writing – Review & Editing

Birhanu Kahsay
Roles: Conceptualization, Data Curation, Investigation, Methodology, Resources, Software, Validation, Writing – Review & Editing

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This article is included in the Energy gateway.

Abstract

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.

Keywords

Anaerobic digestion, Biogas production, Inoculum, Rumen fluid

Corresponding author: Mulugeta Fentahun

Competing interests: No competing interests were disclosed.

Grant information: 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.

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

How to cite: Fentahun M and Kahsay B. 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 not approved]. F1000Research 2025, 14:199 (https://doi.org/10.12688/f1000research.161561.2) First published: 13 Feb 2025, 14:199 (https://doi.org/10.12688/f1000research.161561.1) Latest published: 19 Dec 2025, 14:199 (https://doi.org/10.12688/f1000research.161561.2)

Revised 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.

See the authors' detailed response to the review by Jovine Kamuhabwa Emmanuel

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 = \frac{mDS}{mFS} \times 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 = \frac{mDS - m (ash)}{mDS} \times 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 = \frac{% 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 = \frac{W - D}{W} \times 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 % = \frac{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.

Table 1. 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 S. cerevisiae 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).

Table 2. 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).

Treatments	Mixed Wastes (MW) 100 g	Rumen fluid	S. cerevisiae isolate MUTJ0F	Methane (mean ± SD)

T1	MW₁	0 ml RF	5 ml	1600 ± 0.49 ml
T2	MW₂	0 ml RF	10 ml	1600 ± 0.62 ml
T3	MW₃	25 ml RF	5 ml	2100.7 ± 0.67 ml
T4	MW₄	25 ml RF	10 ml	2300.3 ± 0.17 ml
T5	MW₅	50 ml RF	5 ml	3400.4 ± 0.49 ml
T6	MW₆	50 ml RF	10 ml	3800.6 ± 0.56 ml
T7	MW₇	75 ml RF	5 ml	6000.4 ± 0.17 ml
T8	MW₈	75 ml RF	10 ml	6500.5 ± 0.25 ml
T9	MW₉	100 ml RF	5 ml	6800.4 ± 0.12 ml
T10	MW₁₀	100 ml RF	10 ml	6900.3 ± 0.26 ml
T11	MW₁₁	150 ml RF	5 ml	6300.57 ± 0.47 ml
T12	MW₁₂	150 ml RF	10 ml	6350.13 ± 0.06 ml
T control	MW₀	0 ml RF	0 ml	1500.2 ± 0.1 ml

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).

Figure 1. 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).

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.

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Version 2

VERSION 2 PUBLISHED 13 Feb 2025

Author details Author details

Birhanu Kahsay
Roles: Conceptualization, Data Curation, Investigation, Methodology, Resources, Software, Validation, Writing – Review & Editing

Competing interests

No competing interests were disclosed.

Grant information

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.

Article Versions (2)

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Published: 19 Dec 2025, 14:199

https://doi.org/10.12688/f1000research.161561.2

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Published: 13 Feb 2025, 14:199

https://doi.org/10.12688/f1000research.161561.1

© 2025 Fentahun M and Kahsay B. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Fentahun M and Kahsay B. 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 not approved]. F1000Research 2025, 14:199 (https://doi.org/10.12688/f1000research.161561.2)

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Reviewer Report 19 Aug 2025

Jovine Kamuhabwa Emmanuel, Chemistry, University of Dar es Salaam, Dar es Salaam, Dar es Salaam, Tanzania

Not Approved

https://doi.org/10.5256/f1000research.177603.r401840

Title

The title captures the content of the current work.

Abstract

The abstract has no information on the study, methodology used and key findings ... Continue reading

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

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

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
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests: No competing interests were disclosed.

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.

CITE

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Author Response 29 Dec 2025

Mulugeta Fentahun, College of Natural and Computational Sciences, Department of Biology, Debre Markos University, Debre Markos, 269, Ethiopia

29 Dec 2025

Author Response
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 ... Continue reading
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
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
Competing Interests: No competing interests were disclosed. Close
Report a concern
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COMMENTS ON THIS REPORT

Author Response 29 Dec 2025

Mulugeta Fentahun, College of Natural and Computational Sciences, Department of Biology, Debre Markos University, Debre Markos, 269, Ethiopia

29 Dec 2025

Author Response
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 ... Continue reading
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
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
Competing Interests: No competing interests were disclosed. Close
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Jovine Kamuhabwa Emmanuel, University of Dar es Salaam, Dar es Salaam, Tanzania

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19 Aug 2025 | for Version 1

Jovine Kamuhabwa Emmanuel, Chemistry, University of Dar es Salaam, Dar es Salaam, Dar es Salaam, Tanzania

12 Views Cite this report Responses(1)

Not Approved

Title

The title captures the content of the current work.

Abstract

Introduction

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

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

No
Is the study design appropriate and is the work technically sound?

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

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
Are the conclusions drawn adequately supported by the results?

Partly

Competing Interests

No competing interests were disclosed.

Reviewer Expertise

Catalysis, materials science and bioenergy

Respond to this report

Responses (1)

Author Response

29 Dec 2025

Mulugeta Fentahun, College of Natural and Computational Sciences, Department of Biology, Debre Markos University, Debre Markos, 269, Ethiopia

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

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Competing Interests

No competing interests were disclosed.

Alongside their report, reviewers assign a status to the article:

Approved - the paper is scientifically sound in its current form and only minor, if any, improvements are suggested

Approved with reservations - A number of small changes, sometimes more significant revisions are required to address specific details and improve the papers academic merit.

Not approved - fundamental flaws in the paper seriously undermine the findings and conclusions

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Enhancing biogas production from co-digestion of organic wastes mixed with cattle manure using rumen fluid and Saccharomyces cerevisiae isolate MUTJ0F

Abstract

Background

Methods

Result

Conclusion

Keywords

Revised Amendments from Version 1

1. Introduction

2. Methods

2.1 The study area

2.2 Yeast strain used in experiments

2.3 Substrate collection and preparation

2.4 Preparation of inoculums

2.5 Analysis of the physic-chemical properties of the substrate

2.6 Experimental set up

2.7 Construction of the anaerobic digesters for batch system

2.8 Biogas measurements

2.9 Burning test

2.10 Data analysis

3. Results and Discussion

3.1 Analysis of the physic-chemical properties and composition of the mixed wastes

Table 1. Physico chemical properties of mixed wastes (mean ± SD).

3.2 The effect of rumen liquid and S. cerevisiae isolate MUTJ0F on biogas production

Table 2. 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).

3.3 pH variations in digestor T10 during fermentation

Figure 1. T10 pH variation during the digestion process.

3.4 Combustibility of biogas

Figure 2. Performance of biogas production after 60 days observation.

4. Conclusion

Author contribution statement

Consent for publication

Ethical approval

Data availability statement

Underlying data

Acknowledgments

References

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