The chemicals for the synthesis of media were purchased for authentic sources viz; tryptone (Merck Germany), yeast extract (Merck Germany), agar (Biolab Scientific UK) and NaCl Analytical grade (Merck Germany).

An anaerobic digester was designed to start the digestive process and verify the outcomes for certain bacterial strains. The digester was constructed using SS 316 stainless steel, with a thickness of 1.5 mm. The top had built-in openings for adding substrate, discharging effluent, and collecting biogas. An overhead mixing device was incorporated to ensure proper mixing and ideal interaction between the microbial population and the substrate. Temperature control for anaerobic bacteria viability and activity was ensured by using a heating element. The digester had a capacity of 8 L, ideal for experimental batch operations. This batch operation mode facilitated a controlled examination of anaerobic digestion processes, enabling the evaluation of bacterial efficiency and biogas production in specific conditions.

The experimental design was carefully planned to separate bacteria by using four parallel-operated digesters. The scheme and composition of digesters is given Table S1.

All digesters were activated simultaneously. After a five-day incubation period, 100 mL samples were carefully collected from each digester. Each sample was diluted 1:100 in distilled water for preparation for further examination. The diluted samples were incubated for two hours in a DaiHan ThermoStable™ IR-700 Low Temperature Incubator set at a precise temperature of 28±2°C. After the incubation period, the samples underwent ten consecutive dilutions to decrease the bacterial concentration to a level suitable for isolation. The isolation technique proceeded by streaking the materials onto LB agar plates, followed by incubation for 16 hours at a constant temperature of 28±2°C. Individual colonies were carefully chosen and streaked onto new LB agar plates to establish pure cultures. The streaking and incubation process was repeated until pure cultures were successfully separated. The cultures were morphologically characterized using Gram staining, which is crucial for identifying the form, kind, and general classification of the isolated bacteria. Every isolate was methodically assigned a code to facilitate tracking and analysis during the study. A figurative representation of the experimental study is given in Fig. 1.

For isolation and purification of bacteria Luria Bertani (LB) nutrient medium was used [31]. Anaerobic bacteria were isolated by purifying colonies detected on LB agar plates through subculture on fresh LB agar plates [32, 33].

One prominent way for detecting enzyme systems is by analyzing chemical reactions to nutrients and substrates, particularly colorimetric analysis. This method provides a straightforward and visually comprehensible way to demonstrate enzyme activity, making it easier to investigate bacterial physiology and diversity [34]. The catalase test is used to check the presence of catalase enzyme, an enzyme responsible for breaking down hydrogen peroxide in oxygen and water [35]. This test was not performed on anaerobic bacteria due to the oil content in the cultures. Christensen’s urea agar was carefully prepared for the urease test [36]. Skim milk agar media is used in the casein hydrolysis test to evaluate bacterial strains’ proteolytic activity on casein, the main protein in milk. The Methyl Red (MR) Test is an important instrument in microbiology used to assess bacteria’s capacity to create acids including lactic acid, acetic acid, and formic acid by fermenting glucose. Bacteria are cultivated in a glucose-enriched medium in this experiment. The carbohydrate fermentation and gas production test assess bacteria’s capacity to convert carbohydrates into organic compounds to produce energy, in addition to acid generation. This test includes a basic solution with glucose and pH indicators to track fermentation. Durham tubes are strategically positioned in the medium to collect any gas generated during the metabolic process.

An experimental setup was meticulously developed to assess the effectiveness of a bacterial consortia, using two digesters. The first digester acted as a control, receiving only effluent from baker’s yeast, while the second digester was enhanced with a specifically chosen group of isolated bacteria. 100 mL samples were collected from each digester at five-day intervals throughout a period of 30 days. The samples were thoroughly analyzed using qualitative and quantitative methods to evaluate the efficiency of the bacterial consortia in treating wastewater and producing biogas. The evaluation approach involved doing all relevant biochemical assays on the bacterial community every five days. This methodical methodology guaranteed a comprehensive comprehension of the consortium’s influence on effluent treatment effectiveness, facilitating a precise comparison of the biodegradation and biogas production capacities of the control and treated digesters. The study aims to determine the advantages of using a bacterial consortium for improved wastewater treatment and sustainable energy production through a systematic investigation.

Various morphological features such as forms, geometrical patterns, margin kinds, and elevations were noted in the bacterial isolates. Isolated bacterial colonies can be seen in Fig. S1. These colonies were picked up and re-streaked on fresh LB agar plates until purification is achieved.

A total of 23 bacterial isolates were thoroughly purified in this extensive study, including 19 fermentative and 4 anaerobic bacteria. Gram staining, an essential differential staining method illustrated in Fig. S2., was used to categorize these isolates. The gram staining results for fermentative bacteria were unequivocally conclusive. The anaerobic bacteria yielded no findings mainly because a thin layer of paraffin oil employed during the isolation step interfered with the experiment. This layer unintentionally caused the slides to crack during the heat fixation process of preparing smears for gram staining, making it difficult to acquire accurate observations for these isolates. Out of the bacteria that were identified, 18 showed gram-positive traits and were classified into three unique categories: Cocci, Bacillus, and Coccobacillus. Bacteria D1B3 was recognized as a gram-negative microorganism and categorized under the genus Streptococcus.

The Cocci group exhibited anaerobic abilities and a proficiency in generating short-chain volatile fatty acids (VFAs), specifically acetic acid. This production of acetic acids is important because VFAs play a crucial role in anaerobic digestion process, serving as substrates in microbial metabolism and in the manufacture of biomethane [37]. Bacillus bacteria also increases the production of VFAs. VFAs, such as acetic, propionic, and butyric acids, play a crucial role as intermediates in anaerobic digestion [38]. This study’s results reveal the varied metabolic abilities of the isolated bacterial strains and their individual roles in VFA generation. Anaerobic digester contains various types of strict and facultative anaerobes. These anaerobes are responsible for performing 4 different tasks. These tasks are named hydrolysis, acidogenesis, acetogenesis and methanogenesis [39]. Cocci and Bacillus are particular in performing acidogenesis while Streptococcus are specific for acetogenesis process. Production of VFAs is a very important step in anaerobic digestion. Prolonged production of VFAs causes toxication and limits the digestion process [40]. Although it is considered that methanogenesis is the rate limiting step in digestion process, acidogenesis is responsible for smooth digestion process [41]. Fig. 2. Is a graphical representation of the gram staining results compiled during the study while Fig. S2. represents the actual microscopic images of the slides prepared for gram staining.

For qualitative analysis of the bacteria, the isolated strains are subjected to a series of qualitative tests. These tests include catalase test, urease test, casein test, methyl red test, carbohydrate fermentation test and gas production test. Out of 23 strains only 3 strains are catalase positive, and the rest are catalase negative. Out of 23 strains 19 strains had the capability to produce urease enzyme and 4 strains were urease negative. Out of 23 strains 4 of the strains are casein positive and rest are negative. Out of 23 strains 20 strains can convert glucose into VFAs. Out of 23 strains only 2 strains cannot perform carbohydrate fermentation while rest can perform it. Out of 23 strains 17 strains can produce gas. CH₄, CO₂ and H₂ is produced during this test however it is difficult to differentiate the gas produced. These results are tabulated in Table 1.

Enzymes like proteases, lipases, and amylases can perform carbohydrate fermentation and can be detected using carbohydrate fermentation test [42]. These enzymes facilitate the decomposition of intricate carbohydrates into simpler molecules like sugars [39]. This conversion is necessary because the macroscopic molecules are too massive to be directly used by the bacteria in the following phases of anaerobic digestion [43]. Proteases enzymes can degrade proteins into peptides and amino acids and can be detected by using Urease Test. Amylases degrade carbohydrates, such as starches and sugars, into simpler sugars like glucose and maltose and can be detected by using both carbohydrate fermentation test and methyl red test [15, 44–47]. Acetobacterium species are important bacteria that create acetic acid from ethanol or sugars, which is crucial to produce volatile fatty acids needed for methanogenesis, methyl red test can be performed for the verification of Acetobacterium activity [48]. Clostridium is a flexible group that participates in both hydrolysis and acidogenesis [49, 50]. Eubacterium species aid in fermenting carbohydrates and proteins, resulting in the production of acetic and butyric acids. They are essential for maintaining balance in the acidogenic digesting process. Activity of Eubacterium species can be verified by using carbohydrate fermentation test and methyl red test [51]. Lactobacillus species are recognized for their ability to convert sugars into lactic acid, playing a crucial role in acidogenesis, particularly in settings rich in sugar [52]. Propionibacterium species are bacteria that have a specific role in converting sugars and lactic acid into propionic acid, acetic acid, and carbon dioxide, which adds to the variety of fermentation products [53]. Hydrogenases are involved in hydrogen metabolism, a byproduct of fermentation, affecting the redox balance and the efficiency of the digesting process. The activity of hydrogenases can be detected using gas production test [54–56]. Acidogenesis efficiency is affected by pH, to confirm the activity of a digester in acidogenic stage methyl red test can be used [57].

As acidogenesis can be evaluated using methyl red test, acetogenesis can also be checked and verified using methyl red and gas production tests [12, 58]. Hydrogenase enzymes are crucial in the usage and generation of hydrogen gas and gas production test give a hint if hydrogen is being produced or not [59] [60]. Dehydrogenase enzyme Facilitates the transformation of formate into carbon dioxide and hydrogen, providing acetogens with an alternative method to generate substances for methanogenesis, this step can also be verified using gas production test [61]. Carbon monoxide dehydrogenase (CODH) plays a crucial role in the Wood-Ljungdahl route, which is an important acetogenic mechanism [62, 63]. The gas production test also shows the possibility CODH present.

Methanogenesis is the last phase of anaerobic digestion. Crucial enzymes in this process include methyl-coenzyme M reductase (MCR), which facilitates the conversion of methyl-coenzyme M to methane, a pivotal stage in methanogenesis [64]. Formylmethanofuran dehydrogenase is one of the enzymes involved in the conversion of carbon dioxide to methane, demonstrating the intricate role of enzymes in this process. Enzymes and methanogenic archaea have a symbiotic interaction that is crucial for the efficiency of methanogenesis, which directly affects biogas production and the overall effectiveness of anaerobic digestion [65].

The combined enzyme activity of bacterial consortium is tabulated in Table 4., while in Table 3., enzyme activity in a control digester with no additives is given. It is clearly evident that the enzyme activity in digester with consortium is much better than in digester with no additives. Digester with no additives shows no catalase, urease and casein activity and intermediate acid production, carbohydrate fermentation and gas production activity. In the digester with consortium there is no catalase activity, urease activity in the first 15 days, casein activity in first 10 days, acid generation and carbohydrate fermentation in last 20 days and gas production for 30 days. Less urease activity means that there would be less ammonia production, so our system will be less prone to ammonia toxicity. Greater acid production activity and carbohydrate fermentation mean more VFAs and acetic acids production [54–57]. Positive gas production at all times means that there some gas producing at all times either it is CO₂, H₂ or CH₄ [65].

It is evident from Table 3. that isolated consortium has better performance statistics than without isolated consortium. Activity of enzyme in isolated consortium is substantially increased.

Five different parameters namely, biological oxygen demand (BOD), Chemical Oxygen Demand (COD), Total Dissolved Solids (TDS), Total Suspended Solids (TSS) and Total solids (TS) for effluent and effluent after normal AD treatment (control) and AD treatment with consortium were studied. Fig. 5. shows a comparison of five different parameters among effluent, control and effluent with consortium after 30 days batch operation of AD. For control, there is a 6.8% reduction in BOD Levels, 10.96% reduction in COD level. A drastic change in the quality of effluent after anaerobic treatment with consortium. The reduction efficiency of COD is about 45%. 51.35% reduction in BOD. The final pH of the effluent with consortium is 7.1 and about 9% reduction in total solids. The 51.35% reduction in BOD levels means that there is further potential for biological treatment. As COD is not reduced completely, further treatment either biological or chemical may be integrated with existing anaerobic digestion to completely reduce the COD level. The process of anaerobic digestion is feasible for waste waters having high COD values. Conventional wastewater treatment means can reduce or completely remove the total solids, but high COD cannot be removed by conventional methods. The consortium helped to enhance anaerobic digestion process. In Fig. 4, a comparison is shown between the quality of methane gas produced during the AD of control and effluent with consortium. In control there is no methane production till day 10 while in effluent with consortium there is methane production on day five. In control maximum methane production is at 66% which decreases afterwards. In effluent with consortium maximum methane production is at 72%. It clearly states that consortium of bacteria has improved the quality of methane produced and have the reduced incubation period of 5 days rather than 10 days for control. The four steps in AD are a complete package for the removal of COD toxicity from industrial wastewaters [66]. From hydrolysis to methanogenesis each step works in a unique way to reduce or decompose macro molecules/polymers into smaller molecules/monomers. Each molecule decomposed is a precursor for another decomposition in each step and goal is production of methane [67, 68]. Higher the methane production means the process is at good efficiency. The higher the efficiency higher will be COD removal, which leads to the solution of many environmental problems [69]. As it is evident from Fig. 3. and Fig. 4. that the values of COD and BOD have reduced in effluent with consortium than in control. Which is the clear evidence better methane production quality and enhanced AD process.