Evaluation of sludge quality from cow feces and vegetable waste from biodigester

Corresponding author: I Gusti Nyoman Gde Bidura Copyright © 2025 Author(s) retain the copyright of this article. This article is published under the terms of the Creative Commons Attribution License 4.0. Evaluation of sludge quality from cow feces and vegetable waste from biodigester Tjokorda Sari Nindhia 1, I Gusti Nyoman GdeBidura 2, *, I Putu Sampurna 3 and Tjokorda Gde Tirta Nindhia 4 1 Study Program Doctor of Animal Husbandry, Faculty of Animal Husbandry, Udayana University, Denpasar 80232, Bali, Indonesia. 2 Faculty of Veterinary Medicine, Udayana University, Jimbaran 80361, Bali, Indonesia. 3 Faculty of Animal Husbandry, Udayana University, Jimbaran 80361, Bali, Indonesia. 4 Study Program of Mechanical Engineering, Engineering Faculty, Udayana University, Jimbaran 80361, Bali, Indonesia. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 Publication history: Received on 27September 2025; revised on 08 November 2025; accepted on 11 November 2025 Article DOI: https://doi.org/10.30574/wjbphs.2025.24.2.1005 Abstract This study aims to evaluate the quality of biodigester fermentation sludge produced by continuously filling the biodigester with cow feces and vegetable waste. The study used a 200-liter capacity horizontal portable biogas digester, equipped with an agitator filled with cow feces and vegetable waste of kale, cabbage and Chinese cabbage. The research method is experimental research (true experimental research). This study was designed with a nested pattern, consisting of two factors, the first factor is a digester filled with cow feces (experiment I), cow feces and “Kol” vegetable waste (experiment II), cow feces and “Kangkung” vegetable waste (experiment III) and cow feces and “Sawi” vegetable waste (experiment IV). The results showed that the addition of vegetable waste consistently reduced (P<0.05) the Biological Chemical Demand (BOD) value of the sludge. Statistically, the Chemical Oxygen Demand (COD) values from the four experiments were not significantly different (P>0.05). The mixture of "Sawi putih" is very efficient in decomposing solids, or "Sawi putih" itself has a lower solid content and is more easily decomposed. The addition of vegetables succeeded in creating a more balanced C/N ratio, thereby preventing excessive ammonia release. The addition of vegetable waste can suppress the pathogen population, on the contrary, the CO2 content produced is higher, especially with the addition of "Kol" vegetable waste. The addition of vegetable waste to the cow dung-based biodigester can suppress the number of pathogenic bacteria. It was concluded that the addition of vegetable waste to the cow dungbased biodigester, consistently reduced the BOD value of the sludge and lower solid content, on the contrary, it can prevent excessive ammonia release and suppress the number of pathogenic bacteria. Keywords: Sludge; Cow Feces; Vegetable Waste; Biological chemical demand; Biodigester 1. Introduction Organic waste management is a key focus for sustainable technological advancement. Naturally composting organic materials takes a significant amount of time, ranging from three months to a year. One efficient way to process waste is through the use of a biodigester. A biodigester is a system that uses anaerobic processes to decompose organic waste into biogas and organic fertilizer [1]. The biodigester process not only reduces the amount of waste produced by accelerating decomposition but also produces renewable energy in the form of biogas, which can be used for cooking and other purposes. Biogas digesters are divided into two types based on their raw material feeding system: batch and continuous. The main difference between the two types lies in the feeding method and the continuity of biogas production. Batch systems have several disadvantages, including varying biogas quality from day to day. Initially, the biogas cannot be used as fuel World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 309 due to its low or minimal methane content. This system does not allow for daily waste processing. To address these shortcomings, continuous systems were developed [2]. A continuous system allows for daily organic waste processing, producing biogas with a high methane content, which can be used as fuel daily. It can also process livestock waste, including cow dung, daily. Unlike batch systems, a continuous system requires careful attention to the volume of feed and retention time to achieve optimal results, as all four processes in an anaerobic digester (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) occur simultaneously [3]. Anaerobic digester technology can use various types of waste as a substrate to produce biogas, including large amounts of lignocellulosic waste generated from agricultural, urban and other activities, livestock waste, household and industrial waste, solid waste from activities in urban areas and food waste [4]. Ruminant feces, particularly cattle feces, have long been used as a substrate in anaerobic digesters due to their abundant availability and nutritional content. However, using a single substrate can sometimes be challenging. Cattle feces have a suboptimal carbon-nitrogen (C/N) ratio or limited micronutrient content, which can hinder the efficiency of the anaerobic digestion process and biogas production [3]. To overcome these limitations, an anaerobic co-digestion strategy, which involves mixing two or more substrates together, is proposed. Adding vegetable waste to a digester using cow feces as the primary substrate can increase the available carbon supply, help balance the C/N ratio, and provide other essential nutrients that may be lacking in cow feces alone [3]. Anaerobic digestion relies on several different parameters to achieve optimal performance, including: hydraulic retention time (HRT), temperature, pH, mixing, C/N ratio, and substrate [5,6]. Several studies have shown that the addition of vegetable waste can increase biogas production rates, methane content, and reduce hydraulic retention time in digesters [3]. This occurs because vegetable waste has very promising characteristics as a co-substrate in anaerobic digesters. The high fiber content (cellulose, hemicellulose), water, and various macroand micronutrients make vegetable waste a good source of carbon and nutrients for methanogenic bacteria [7]. Alternating substrate application reflects field conditions, where waste is abundant, and understanding how a continuous digester system responds to variations in organic load is essential for field implementation. This study aimed to evaluate the quality of biodigester fermentation sludge produced by continuously feeding the biodigester with cow feces and vegetable waste. 2. Material and methods 2.1. Research Location The research was conducted at the Simantri 369 Animal Sustainable Livestock Farmers Group (“Gapoktan Merta Sari”), Sukawati District, Gianyar Regency, Bali Province. Biogas sludge quality testing was conducted at the Analytical Laboratory of Udayana University, Bukit Jimbaran Campus, Denpasar. Microbial contamination testing (TPC and fecal coliform) was conducted at the Veterinary Health Laboratory, Denpasar Veterinary Center (BBVet), Bali, Indonesia. 2.2. Material and Research Instruments The materials used were Balinese cattle feces and elephant grass feed obtained from the “Satwa Lestari” Livestock Farmers Group (“Gapoktan Merta Sari”) Simantri 369, Sukawati District, Gianyar Regency, Bali Province. Vegetable waste was obtained from public markets around Batubulan, Gianyar, Bali, Indonesia. Horizontal Portable Biogas Digester: The method of feeding is interspersed with cow feces to ensure the availability of methanogenic bacteria in the anaerobic digester. The vegetable waste used in this study is cabbage, kale and Chinese cabbage waste from the local Public Market. A 200-liter capacity drum is operated for the study, as presented in Figure 1. The anaerobic digester is filled with slurry-like filling at the inlet (1); The digester tank (2) is completely filled with substrate; A stirrer (3) is provided to rotate the agitator to make the substrate well mixed and flow from the inlet (1) to the outlet (6) during the digester feeding. Biogas is produced and collected in the floating drum (9) by opening the inlet valve (7) and closing the biogas outlet valve (8). If the biogas in the floating drum is to be used as fuel, the inlet valve (7) must be closed and the outlet valve (8) must be closed. The digester is operated in a batch system for about 1 month initially. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 310 Figure 1 Schematic of a 200-liter continuous anaerobic digester; 1= Slurry inlet; 2= Anaerobic digester tank; 3= Scaffolding; 4= Agitator; 5= Biogas outlet; 6= Digestion outlet; 7= Biogas inlet valve; 8= Biogas outlet valve; 9= Floating drum for biogas storage 2.3. Research Design This study was designed with a nested pattern, consisting of two factors, the first factor is a digester filled with cow feces (experiment I), cow feces and “Kol” vegetable waste (experiment II), cow feces and “Kangkung” vegetable waste (experiment III) and cow feces and “Sawi” vegetable waste (experiment IV). 2.4. Variables and Data Analysis The independent variable is the provision of vegetable waste in cow feces, which consists of experiment 1 (continuous Balinese cow feces), Experiment II (continuous Balinese cow feces and vegetable waste "Kol"), Experiment III (continuous Balinese cow feces and vegetable waste "Kangkung"), and experiment IV (continuous Balinese cow feces and vegetable waste "Sawi putih") and the biogas production time for 30 days in the digester of the 4 experiments in the continuous process system. The dependent variable is a variable whose size depends on the independent variable. In this study, the dependent variables are sludge pH, sludge temperature, environmental temperature, humidity, daily gas volume, biogas formation rate and biogas composition from the 4 experiments, total bacteria and number of coliform bacteria, Biological Chemical Demand (BOD), Chemical Oxygen Demand (COD), Total Solid Suspension (TSS), ammonia NH3 from the sludge produced in the digester from the 4 experiments in the continuous process system. The data generated from the four experiments were analyzed using Analysis of Variance (ANOVA) and presented in the form of error bar graphs to determine the difference in the mean and margin of error with a 95% confidence level. The analysis procedure used IBM SPSS (Statistical Product and Service Solutions) statistics version 28 [8]. The digester sludge quality data obtained in this study were tabulated, then compared with the quality standards based on the Minister of Environment Regulation No. 05 of 2014, and the Bali Governor Regulation No. 16 of 2016 and discussed descriptively qualitatively. 3. Results 3.1. Biochemical Oxygen Demand (BOD) The study measured the Biochemical Oxygen Demand (BOD) value of sludge produced from four biodigester experiments fed with cow feces and various vegetable wastes alternately shown in Figure 2. The results showed a significant difference (P<0.05) in BOD values between experiments, which indicates the level of residual organic pollution after fermentation. Experiment I had the highest BOD value, which was 618.747 mg/L. This high value indicates that the fecal sludge contained significant organic pollutants (P<0.05) after the fermentation process. Experiment II had a BOD value of 499.130 mg/L. The addition of vegetable waste "Kol" reduced the BOD compared to experiment I. More details are presented in Figure 2. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 311 Figure 2 BOD observation graph for each experiment every 10 days for 30 days Experiment III had the lowest BOD value, which was 321.808 mg/L. The addition of “Kangkung” vegetable waste significantly reduced the BOD value. Experiment IV had a BOD value of 480.058 mg/L. The addition of “Sawi putih” vegetable waste also reduced BOD, but not as effectively as “Kangkung” vegetable waste. Statistically, experiment I was significantly different (P<0.05) from experiments II, III, and IV. While experiment II was not significantly different from experiment IV (P>0.05) but significantly different (P<0.05) from experiment III. Experiment III was not different from IV (P>0.05). The addition of vegetable waste (“Kol, Kangkung and Sawi putih”) consistently reduced the BOD value of the sludge. Kale is the most effective type of vegetable waste in reducing the organic pollutant content in the sludge after fermentation. The low BOD values in the “Kangkung” and “Sawi putih” cabbage experiments at the end of the observation indicate that the sludge from these two experiments has better quality. 3.2. ChemicalOxygenDemand (COD) Figure 3 Graph of COD observations in each experiment every 10 days for 30 days The most striking finding was that the sludge from experiment III had the highest average COD value, at 8000 mg/L. Conversely, the sludge from experiment IV showed the lowest average COD value, at 5600 mg/L. This could mean that the addition of "Sawi putih" vegetable waste did not contribute as much dissolved organic matter as other vegetables, or that the organic matter was consumed more quickly and converted into other forms (including biogas), leaving less remaining. Statistically, the COD values from the four experiments were not significantly different (P>0.05). More details can be seen in Figure 3. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 312 The graph in Figure 3 depicts changes in dissolved organic matter content (measured as COD) in the fermentation sludge over 10-day intervals. This dynamic reflects how quickly the raw materials (feces and vegetables) are broken down into simpler compounds by microorganisms in the biodigester. The most significant finding is that each type of vegetable waste creates a unique sludge decomposition profile over time. This suggests that the type of substrate significantly determines the speed and stages of the fermentation process. 3.3. Total Solid Suspension (TSS) Figure 4 presents a comparison of the total suspended solids (TSS) content in the final sludge from the four experimental treatments. The TSS value represents the number of solid particles remaining in the digester fluid after the fermentation process. In the context of a biodigester, a low TSS value at the end of the process is generally desirable, as it indicates that most of the solid organic matter has been successfully decomposed into soluble compounds and biogas. Experiment IV produced the lowest average TSS value (6577.777 mg/L). This is a strong indicator that the combination of feces and “Sawi putih” is the most effective in degrading or breaking down solid matter. In contrast, experiment I showed the highest average TSS value (23333.333 mg/L). This means that without the addition of vegetable waste, the process of decomposing solid materials in feces is not as effective as when mixed with vegetables, especially "Sawi putih". The TSS value obtained in this study is above the waste quality standard for cattle farms, Regulation of the Minister of Environment No. 05 of 2014, where the maximum TSS quality standard for cattle farm waste is 100 mg/L. Even the TSS value in fresh cattle feces samples is above the standard, namely 72,000 mg/L, as well as in the batch system at 291,000 mg/L. Figure 4 TSS observation graph for each experiment every 10 days for 30 days Experiment IV consistently showed very low TSS values throughout the observation period. This indicates that the “Sawi putih” vegetable waste mixture is highly efficient at decomposing solids, or that “Sawi putih” it self contains lower solids and is more easily decomposed. 3.4. Ammonia Figure 5 shows a comparison of ammonia (NH3) concentrations in fermentation sludge from four different experiments. Ammonia is a product of the decomposition of nitrogen-containing materials (such as proteins). Its concentration is an important indicator of nutrient balance (C/N ratio) and potential toxicity in the biodigester. Experiment I produced a significantly higher average ammonia concentration (321,283 mg/L) than the other treatments. This can be explained by the high nitrogen content of feces, which releases large amounts of ammonia during decomposition. All treatments involving the addition of vegetable waste (“Kol, Kangkung, and Sawi putih”) showed lower ammonia concentrations. This suggests that the addition of carbon-rich vegetables helps balance the carbon/nitrogen (C/N) ratio. With a more balanced C/N ratio, nitrogen is more efficiently utilized by microorganisms for cell growth and is not released as excess ammonia. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 313 Based on the research results, the ammonia levels in the sludge digesters of the four experiments were high and did not meet the requirements of the [9] regulation No. 05 of 2014, namely the ammonia quality standard in cattle farm waste is 25 mg/L. Even in fresh cow feces 105,500 mg/L and in batch 138,700 mg/L are also higher than the quality standard. Figure 5 Graph of NH3 observations in each experiment every 10 days for 30 days Ammonia is a byproduct of the decomposition of nitrogen-rich materials. Monitoring its concentration over time is crucial for understanding nutrient balance and potential inhibitors in the biogas production process. In Experiment I, ammonia concentrations showed a consistent and significant increase over 30 days. This indicates a persistent accumulation of ammonia, stemming from the high nitrogen content of feces. In contrast, the three experiments using vegetable waste (“Kol, Kangkung and Sawi putih”) showed significantly lower and relatively stable ammonia concentrations after day 10. This is strong evidence that the addition of vegetables successfully created a more balanced C/N ratio, thus preventing excessive ammonia release. 3.5. Total Plate Count (TPC) Figure 6 presents a comparison of the total microbial population (Total Plate Count - TPC) that can be grown from fermentation sludge in four experimental treatments. TPC provides an overview of the bacterial population density in the biodigester. The microbial population explosion occurred in experiment IV, which showed an unusually high average TPC value (9.07x10⁸ CFU/mL) compared to the other three treatments. Figure 6 TPC observation graph for each experiment every 10 days for 30 days This indicates that the addition of “Sawi putih” vegetable waste creates very fertile conditions that trigger massive microbial population growth. A high microbial count in the “Sawi putih” treatment does not automatically mean better World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 314 biodigester performance. A very high but unstable population could be dominated by acidogenic (acid-producing) bacteria rather than methanogenic (methane-producing) bacteria, which could actually inhibit biogas production. 3.6. Coliform Figure 7 shows a comparison of the number of coliform bacteria remaining in the fermentation sludge from the four experimental treatments. Coliform bacteria are often used as an indicator of sanitation; low counts indicate that the fermentation process has successfully reduced potential pathogens, making the final product safer for use as fertilizer. All treatments involving the addition of vegetable waste (“Kol, Kangkung and Sawi putih”) successfully reduced the number of Coliform bacteria compared to the treatment with feces alone. This is strong evidence that the co-digestion process creates an unfriendly environment for Coliform bacteria. Experiment I showed a very high average Coliform count (3.04x10⁶CFU/mL). This indicates that without the presence of vegetable co-substrate, the anaerobic digestion process alone is not effective enough to sanitize and reduce pathogens from feces. ANOVA test proved that the difference between these treatments was not significant (P>0.05). Figure 7 Coliform observation graph for each experiment every 10 days for 30 days Figure 7 shows the dynamics of changes in the number of coliform bacteria, an indicator of sanitation, during the 30day fermentation period. The goal of this process is not only to produce energy but also to reduce pathogens, so this monitoring is crucial for assessing the safety of the final product. In all three experiments, coliform counts were successfully suppressed to very low levels starting on day 10 and remained consistently low throughout the 30 days. This is strong evidence that the addition of vegetables as a co-substrate is essential for achieving sanitation goals. 4. Discussion The addition of “Kol”vegetable waste to the digester contents resulted in higher gas production compared to “Kangkung” and “Sawi putih” vegetable waste caused by several factors, such as cellulose degradation efficiency, microbial activity and the biochemical composition of the substrate. “Kol”vegetable waste produced a higher volume of gas, due to its composition and fermentation characteristics.Factors such as organic matter content and microbial activity during digestion contribute to its superior performance compared to “Kangkung” and “Sawi putih” in biogas production [5]. The addition of“Kol” can enrich certain cellulose-degrading bacteria, especially those from the phylum Bacteroidetes, to break down fibrous materials in the digester [1]. The fibrous nature of “Kol” allows for better degradation compared to “Kangkung” and “Sawi putih”, which may have fewer complex structures that do not support the same level of microbial diversity and activity [5]. The lower methane biogas composition when using vegetable waste compared to cow manure alone may be due to several factors, such as substrate characteristics and microbial dynamics during anaerobic digestion. Vegetable waste has a different chemical composition that may not be as conducive to methane production as using cow manure alone. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 315 Cow manure is rich in organic matter and has a favorable microbial community for methane production. Although “Kol” waste increases cellulase activity, the shift in the dominant microbial community may not support methane production as effectively as cow manure alone [1]. The addition of “Kol” waste may create competition among microbial species, potentially inhibiting the growth of methane-producing archaea that thrive in cow manure environments [5]. The addition of “Kol” waste to the biogas fermentation process can increase CO2 concentration compared to using feces alone, due to the specific biochemical properties of “Kubis”that affect fermentation dynamics. Cabbage (“Kol”) waste contains high levels of cellulose, which when added to the fermentation process will increase cellulase activity. This increased activity encourages the breakdown of cellulose into simple sugars, which are then fermented by bacteria, resulting in increased CO2 production as a byproduct of the metabolic process. The presence of cellulose-degrading bacteria such as those from the phylum Bacteroidetes, enriched in the system by the addition of “Kol”, will further facilitate cellulose breakdown and alter the dynamics of gas production [1]. Anaerobic digestion is influenced by the hydrogen ion concentration (pH) of the digested material. Because excessive acidity inhibits digestion, the hydrogen ion concentration of the culture medium directly affects microbial growth [6]. The pH of the system fed with cow dung was found to fluctuate up to 8.0 and down to 6.0. This condition produces biogas with a high CH4 content. Methanogens thrive in neutral to slightly alkaline conditions and die in acidic environments. The optimum pH of the system is in the range of 7–8.5 [6], where nitrogen is released and accumulates as ammonia, which in turn can increase the substrate pH [10]. Fluctuations in pH indicate that all anaerobic digestion processes (hydrolysis, acidogenesis, acetogenesis, and methanogenesis) are occurring. Meanwhile, the addition of vegetable waste “Kol”and “Kangkung” kept the pH within the neutral range for 30 days, indicating that acidogenesis did not occur and hydrolysis did not occur. Methanogens thrive in neutral to slightly alkaline conditions and die in acidic environments. Microorganism concentration plays a crucial role in anaerobic digestion. Methanogenic microorganisms generally have a long regeneration time. To avoid washout from the reactor, the hydraulic retention time should be at least 10–15 days in reactor systems that lack facilities for biomass retention and recovery [11]. In this regard, observations under an inverted phase-contrast optical microscope revealed that the concentration of microorganisms in the system fed with cow manure was higher than in the system fed with cow manure and vegetable waste (“Kol, Kangkung and Sawi putih”) alternately. Therefore, for optimal results, if vegetable waste “Kol”is introduced into the system, the retention time (residence time) should be increased [5]. Microbial diversity in anaerobic digesters depends on various factors, such as the type of pretreatment, granulation, seed inoculum, type of feedstock, temperature, stirring speed, aeration, digester design, organic loading rate, hydraulic residence time, and solids residence time. Microbial granules resemble a filamentous consortium, through which liquids and gases can flow slowly [12]. Biogas production can benefit from the use of specialized microbial consortia (inoculum) to increase biogas yield in anaerobic digesters. Inoculum can be provided from raw pig slurry using innovative procedures [13]. Unique and effective inoculums can be developed from wastewater treatment, thin stillage, and agricultural waste with varying retention times [13,14]. Another source of inoculum is sewage sludge [15]. The inoculum-to-substrate ratio must also be considered [16]. In this study, vegetable waste was simply added to the existing cow manure system without considering the effectiveness of additional inoculants from previous studies. Therefore, further research is aimed at finding suitable inoculants to improve the processing performance of cabbage waste combined with cow manure in continuous anaerobic digestion. Besides inoculum addition, another important process is acclimatization. This was not implemented in this study due to limited time and experience in this field. An example of this process is the long-term acclimatization of anaerobic sludge, which was carried out by operating a continuously stirred mesophilic anaerobic reactor with continuous feeding of food waste and cow dung. During long-term acclimatization, a continuous increase in enzyme activity was revealed, while the microbial structure tended to stabilize and had a methane yield approximately 13 times higher than the initial anaerobic sludge [15]. The acclimatization process can introduce other types of microorganisms unrelated to the established methanogenic bacteria in the system [17]. It is recommended that the methanogenic bacteria in this study undergo acclimatization to the addition of vegetable waste to improve the performance of the anaerobic digester in producing higher CH4 content in the biogas. World Journal of Biology Pharmacy and Health Sciences, 2025, 24(02), 308-317 316 5. Conclusion It was concluded that the addition of vegetable waste to the cow dung-based biodigester, consistently reduced the BOD value of the sludge and lower solid content, on the contrary, it can prevent excessive ammonia release and suppress the number of pathogenic bacteria. Compliance with ethical standards Acknowledgments The authors would like to thank the Dean of the Faculty of Animal Husbandry, Udayana University, Denpasar, the Satwa Lestari Livestock Farmers Group (“Gapoktan Merta Sari”) Simantri 369, Sukawati District, Gianyar Regency, Bali Province, and the Head of the Veterinary Health Laboratory, Denpasar Veterinary Center (BBVet), Bali, Indonesia, for the permission and facilities provided for this research. Disclosure of conflict of interest Noconflict of interestto be disclosed. References [1] Zaki MBM., Shamsudin R. And Yusoff MZM. 2021. Portable Bio-digester System for Household Use-A Review. Adv. Agric. Food Res. J., 2: a000014. [2] Nindhia T.G.T., I.W. Surata, T.S. Nindhia, D.N.K.P. Negara, and M. Diantoro. 2017. Waste of Copper Alloy Chips as Biogas Desulfurizer. International Journal of Environmental Science and Development, 8 (1): 15-18. [3] Nindhia T.G., McDonald M. And Styles D. 2021. 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