Role of Microbial and Organic Amendments for the Enrichment of Methane Production in Bioreactor

Abstract

Studies were carried out on lab-scale levels for biogas production using two different wastewaters, that is, herbal pharmaceutical wastewater and food processing wastewater. A total of eight methane bacteria were isolated from cattle dung and mass culturing was carried out to study their feasibility in biogas escalation. Optimization of methane bacteria that could increase biogas production was identified. Among the methane bacteria, two species Bacillus sk1 and Bacillus sk2 were found to enhance the biogas production to a maximum level. Gas analysis showed CH4 content of 63% in the case of food processing wastewater and around 67% with herbal pharmaceutical wastewater. Bacillus sk1 was found to be more suitable for both wastewater and biogas production and was found to be 46.4% in food processing wastewater and 43.3% in herbal pharmaceutical wastewater. Amendment of Bacillus sk2 in food processing wastewater produces 39.7% and 30.3% of biogas in herbal pharmaceutical wastewater was observed. Enzyme Bacillidine™ (P-COG-concentrate aqueous base) was also tried but results were not very encouraging. Comparative studies on both the wastewater have been discussed in detail in this article.

1. Introduction

Due to industrialization and excessive exploitation of natural resources, as well as the population explosion at the global level affects the environment at large [1, 2]. Textile industries, municipal sewage, dairy waste, pharmaceutical industries, swine, and aquaculture sectors release wastewater on a regular basis [3, 4]. Wastewater contains a variety of unfavorable chemical components and microbes that show short- and long-term environmental and human health implications [5, 6]. Untreated wastewater if utilized directly for irrigation may cause undesirable implications in the environment and groundwater [7]. The challenges with wastewater treatment include high energy consumption and laborious work [8]. In recent decades, a new goal has gained attraction on resource recovery from wastewater paired with its treatment technologies.

Regarding the crisis of energy use, the widespread usage of fossils fuels may deplete in the next 50 years [9]. So, to cope with the future energy demand, it is critical to seek innovative renewable energy sources [10, 11]. Other technologies for harnessing renewable energy sources, such as solar, wind, hydraulic, and geothermal energy, have been created [12]. All of these technologies have been developed very well and are commercially accessible to meet rising energy demand to some extent. Using wastewater as a source of energy can also help to relieve pressure on other technologies. Fresh microbial biomass or residual biomass after lipid extraction can be used directly for bioenergy generation using dark fermentation (biohydrogen production), fermentation (bio alcohols), and anaerobic digestion (methane) [13, 14, 15].

Presently, the global pharmaceutical sector has been quickly expanding and contributing to great economic development. But on the other hand, it is generating significant environmental degradation by releasing effluents [16]. Pharmaceutical manufacturing based on chemicals employs a number of chemical processes that result in complicated effluent with high salt content and poor nutritional value that is difficult to biodegrade. The wastewater generated by various pharmaceutical businesses is not uniform, and the composition of each type of wastewater is impacted by the techniques used. Antibiotics, steroids, reproductive hormones, analgesics, beta-lactamides, antidepressants, detergents, as well as unspent solvent and heavy metals make up the majority of the substance [17]. The treatment of wastewater includes several processes that are usually expensive. Anaerobic digestion (AD) is a low-cost technique for treating organic wastes while simultaneously recovering energy in the form of methane [18, 19]. The efficiency of anaerobic digestion is determined by the cooperation of numerous microorganisms that conduct hydrolysis, fermentation, and methanogenesis [20, 21]. Anaerobic digestion (AD) is a waste-to-biomethane conversion technology that has been utilized to transform sewage sludge, agricultural/livestock residues, food wastes, and other organic waste streams into biomethane [22]. Many research studies have been undertaken for the past 10 years to optimize the digestion benefits in terms of biogas production, environmental effect, and reduction of waste [23].

Therefore, looking at the present scenario of energy demand in a developing country like India, it is very much necessary to switch over to bio-methanation, as it is the ultimate environment-friendly and sustainable way of progressive development. Our study aimed to produce biogas from two types of wastewaters namely herbal medicinal wastewater and food processing wastewater using cow dung and isolated microbial species.

2. Materials and methods

Samples of influent cattle dung slurry were collected and analyzed for various physicochemical characteristics. Similarly, effluent samples were also collected from an anaerobic conventional digester for their characterization after 38 days. The physical and chemical parameters were determined according to the Standard Procedures [24]. Two different wastewaters viz., herbal pharmaceutical and food processing were also collected and its treatability studies were carried out. Herbal pharmaceutical wastewater was tried to treat with Vermi filters that produce a nutrient biosolid and vermiwash that promotes the growth of plants [25, 26].

3. Isolation of anaerobic bacteria

The potential bacterial species involved in biogas production were isolated from a fresh sample of cattle dung. During isolation, the sample was diluted as per requirement. The anaerobic agar medium was used for the selective isolation of the anaerobes. The spread-plate method was employed for bacterial-cell isolation. A total of 0.5 ml of diluted bacterial samples was spread onto an anaerobic agar plate. The plates were then incubated in an anaerobic jar provided with alkaline pyrogallol to make the environment free of molecular oxygen. Incubation was carried out for 72–96 h, at 37°C for bacteria proliferation.

Microscopic examination was carried out to study the presence of methane bacteria and their motility. A total of eight methanogenic cultures were isolated and detailed biochemical tests were carried out, such as the sugar fermentation test and IMViC test. Apart from these two tests Indole test, methyl red test, Voges Proskauer test, citrate utilization test, etc., were also evaluated. Enzyme like catalase test, oxidase test was performed to authenticate the presence of these enzymes. To confirm the methanogenic nature of the bacteria fluorescence test was carried out. Identification of the eight cultures of the methane bacteria was carried out by subjecting the isolates to scanning electron microscopy.

A total of eight isolates of bacterial species were obtained from the predigested cow dung slurry, which was inoculated for the comparative assessment of methane production individually by each species. The studies were conducted for two months, based on which the isolates 2 and 4 were contributing maximum. So, by taking these two isolates the experiment was designed by taking wastewaters from two different industries, namely the food processing industry and herbal pharmaceutical industry. Along with that, the enzyme Bacillidine was also taken for the experiment, which was isolated from the same material (pre-digested cow dung slurry). The details of different combinations taken for the experiment have given in Table 1.

Digester	Concentration
First set
FPW	200 ml seed + 800 ml food processing wastewater (FPW)
Bacillus sk2	200 ml seed + 800 ml FPW + isolate no. 2 (2 ml)
Bacillus sk1	200 ml seed + 800 ml FPW + isolate no. 4 (2 ml)
Enzyme bacillidine	200 ml seed + 800 ml FPW + enzyme Bacillidine (2 ml)
Second set
HPW	200 ml seed + 800 ml herbal pharmaceutical wastewater (HPW)
Bacillus sk2	200 ml seed + 800 ml HPW + isolate no. 2 (2 ml)
Bacillus sk1	200 ml seed + 800 ml HPW + isolate no. 4 (2 ml)
Enzyme bacillidine	200 ml seed + 800 ml HPW + enzyme Bacillidine (2 ml)

Table 1.

Different digester mixture with seed.

Sterilized seeds were used.

4. Experimental set up

4.1 Part 1

Fresh cow dung slurry was prepared, for that, a total solid content was diluted in water to make a slurry of 1:1 ratio to get a total solid content of approximately 8.0% solids. Every digester was filled with 950 ml of diluted cattle dung slurry plus 50 ml of seed slurry from a working biogas plant. So, the working volume of digesters was kept to be 1 liter. A total of eight species of methane bacteria were isolated and they were subjected to mass culturing. Out of the nine digesters, one was kept as control with only cattle dung, while others were used for experimental purposes and named 1, 2, 3, 4, 5, 6, 7, and 8, respectively. These eight cultures individually were added (2 ml) to each of the above eight cattle dung digesters.

The digesters with maximum gas production were identified and further experimental work was initiated. Two bacterial species, that is, isolate 2 and isolate 4 resulted in maximum biogas production. The biogas production by individual species is given in Figure 1. DNA sequencings for 16s RNA studies of isolates 2 and 4 show that isolate 2 is Bacillus sk2 and isolate 4 is Bacillus sk1 (Figures 2 and 3).

Figure 1.

Daily biogas production by eight types of isolates. *C: control and 1, 2, 3, 4, 5, 6, 7, 8 are the isolates no 1, 2, 3, 4, 5, 6, 7, 8.

Figure 2.

Daily biogas production in food processing wastewater with Bacillus sk1, Bacillus sk2, and enzyme Bacillidine. *FPW: food processing wastewater.

Figure 3.

Daily biogas production by herbal pharmaceutical wastewater with Bacillus sk1, Bacillus sk2, and enzyme Bacillidine. *HPW: herbal pharmaceutical wastewater.

4.2 Part 2

Eight digesters of 2-liter capacities were taken for two different wastewaters for different combinations with Bc (isolate 2) and Bt (isolate 4) and enzyme Baccillidine along with control as H and B. The biogas was collected in the inverted measuring cylinder of 2-liter capacity filled with 20% sodium sulfate [27]. Every day the digesters were manually shacked for 3–4 times a day and daily gas production was monitored. This situation was maintained for a period of 2 months till the gas production ceased completely. The digester where maximum gas production was obtained was selected for further experimental work using wastewaters as an organic amendment.

5. Results and discussion

There were many industrial wastewaters studied by anaerobic treatment by various authors. These wastewaters may have properties that accelerate the process of digestion. Some of the properties, such as temperature, pH, alkalinity, total ammonia nitrogen, volatile acids, total solids, total volatile solids, and phosphate, may also influence the enzymatic reaction of anaerobic treatment. In the present study, both the wastewaters were containing plant materials as organic matter. Therefore, it was easily degrading when processed anaerobically.

Karray et al. [28] utilized anaerobic digestion of green algae Ulva rigida with sugar industry influent and tried to increase biogas generation. The results showed that this combination helps to compost algae with anaerobic sludge and water yielded the optimal inoculum for producing biogas and feeding an anaerobic reactor, providing 408 mL of biogas. When sugar co-substrate was employed, a maximum methane generation yield of 114 mL g⁻¹ was received with 75% methane. Biogas was produced anaerobically from wastewater of the Colombian palm oil mill industry by Nabarlatz et al. [29]. Using two distinct inoculums, anaerobic digestion tests were carried out in batch mode to assess the effects of pH and inoculum to substrate ratio on anaerobic digestion. The best-suited inoculum was determined to be a 1:1 v/v mixture of urban WWTP (wastewater treatment plant) anaerobic sludge/pig manure at a ratio of 2 g volatile solids (VS) inoculum/g VS substrate, which produced the largest amount of accumulated methane, attaining 2740 mL methane without neutralizing pH. Anaerobic digestion of brewery wastewater to enhance biogas production using UASB (upflow anaerobic sludge blanket) reactor was carried out by Enitan et al., [30]. Using a modified methane generation model, 1.46 L CH₄/g COD was generated. Similarly, Debowski et al. [31] investigated the anaerobic treatment of dairy wastewater in a multi-section horizontal flow reactor (HFAR) using microwave and ultrasonic generators. The study's findings in terms of wastewater treatment efficiency, biogas output, and economic analysis results demonstrated that the HFAR can compete with existing industrial technologies for food wastewater treatment [31]. Ounsaneha et al. [32], evaluated biogas generation during the digestion of municipal wastewater and food waste in semi-continuous and continuous operation with varying hydraulic retention times (HRTs). At 30 days of HRTs with a 10:90 ratio of municipal wastewater to food waste, methane outputs of 167.41 66.52 ml/g-Vs were observed in semi-continuous mode.

Initially, physicochemical parameters of seed slurry, herbal, and food processing wastewater were determined by the standard methods. The parameters that were determined included—pH, alkalinity (as CaCO₃ mg/l), total ammonia nitrogen (mg/l), volatile acids (mg/l as CH₃COOH), total solids (%), total volatile solids (%), and phosphate (mg/l). The pH of the seed slurry before the experiment was 6.91 indicating it was very less acidic and very close to the neutral pH. The alkalinity was found to be 1280 mg/l (as CaCO₃). Total ammonia nitrogen was 105.28 mg/l. Total acids were determined as CH₃COOH that was 312 mg/l. The percentages of total solids and total volatile solids were 5.50% and 3.60%, respectively. The data obtained are given in the tabular form in Table 2.

Sr. no.	Parameter	Seed slurry	HPW	FPW
1.	pH	6.91	6.07	6.78
2.	Alkalinity as CaCO3 mg/l	1280	20.00	28.40
3.	Total ammonia nitrogen mg/l	105.28	24.64	140.00
4.	Volatile acids mg/l as CH3COOH	312.00	60.00	384.00
5.	Total solids %	5.50	14.00	10.20
6.	Total volatile solids %	3.60	7.10	3.40
7.	Phosphate mg/l	—	2328.00	2067.00

Table 2.

Physicochemical characteristics of herbal and food processing wastewater.

The physicochemical characterization of herbal processing wastewater (HPW) and food processing wastewater (FPW) was done where the same parameters were determined as in the seed slurry. The pH of FPW was 6.07, slightly higher than that of HPW in which the pH was found to be 6.78. The total ammonia nitrogen in the HPW was 20 mg/l, while that in FPW was 28.40 mg/l. Volatile acid in HPW was 60 mg/l, whereas in FPW the amount was comparatively very higher at about 384 mg/l. Total solids in HPW and FPW were 14% and 10.20%, respectively. Total volatile solids in HPW were found to be 7.10% and in FPW it was 3.40%. The amount of phosphate in HPW and FPW was found to be 2328 mg/l and 2067 mg/l, respectively. All the data obtained by characterization of HPW and FPW is shown in tabular form in Table 2.

Two sets of different digester mixtures were prepared for the experiment. In the first set food processing waste (FPW) water was used, whereas in the second set herbal processing wastewater (HPW) was used to make the digester mixtures with seed slurry. Each set contained four different digester mixtures. One mixture was prepared by mixing 200 ml of seed slurry and 800 ml of wastewater only. Another mixture contained 200 ml seed slurry, 800 ml wastewater, and isolate no. 2, that is, Bacillus cereus (Bc). The third kind of mixture contained 200 ml seed slurry, 800 ml wastewater, and isolate no. 4, that is, Bacillus thuringiensis (Bt). In the fourth kind of mixture enzyme, Bacillidine was added to 200 ml seed slurry and 800 ml wastewater. In this mixture, isolates 2, 4, and enzyme (2 ml) were mixed in each reactor. Hence four kinds of mixtures were there and the total numbers of eight digester mixtures were prepared using FPW and HPW. The above-mentioned information is summarized in Table 1.

The sets prepared were used in the experiment and kept for 45 days for biogas production. After 45 days the physicochemical parameters of all eight mixtures were determined. The same parameters were determined that were found initially. The pH of the four FPW effluents was slightly acidic to almost neutral. The alkalinity was between the range of 350–580 mg/l. The total ammonia was found in between 142 and 168 mg/l. Volatile acids were in the range of 168–276 mg/l. Total solids in the four mixtures were found in the range of 8–9%. Total volatile solids were between 6 and 7.2%. The pH of the four HPW effluents was slightly acidic compared to those of FPW. All the mixtures were ranged from 6.38 to 6.72. The alkalinity was between the range of 236–450 mg/l. The total ammonia was found in between 32 and 70 mg/l. Volatile acids were in the range of 120–218 mg/l. Total solids in the four mixtures were found in the range of 8.4–8.9%. Total volatile solids were between 5.7 and 8%.

6. Selection of isolates

The second part of this experiment showed that isolate no. 2, 8, and control were continued up to the 45th day, while isolate no. 4 and 7 has stopped on the 42nd day. Isolate no. 1 and 6 were stopped on the 40th day and isolate no 3 and 5 were stopped on the 36th day. On the 45th-day total biogas production was found to be 6080 ml in control, whereas in isolate 1, 3, 5, 6, 7, and 8 it was 2115 ml, 3595 ml, 1515 ml, 5430 ml, 5555 ml, and 5445 ml, respectively. The reactor containing isolate no. 2 was able to produce 6470 ml and the reactor with isolate no. 4 was able to produce 6900 ml of biogas, which was subsequently higher than isolate no. 2. So, it proves that isolate no 2 produces 6.4% and isolate no. 4 produces 13.5% more biogas as compared to control. Figure 1 shows that isolate no. 4 was having the highest peak on the 13th day with production of 900 ml of biogas. Therefore, isolate no. 2 and 4 were selected for further studies.

A fluorescence test was conducted for the identification of methanogenic bacteria having the F₄₂₀ coenzyme, which depicts blue-green fluorescence by methanogenic bacteria and was easily differentiated from the white-yellow fluorescence observed in non-methanogenic bacteria. The isolate no. 2, 6, 7, and 8 indicated the blue-green fluorescence in ultraviolet light depicting the presence of methanogenic bacteria, whereas isolate no. 1, 3, 4, and 5 indicated the negative fluorescence activity.

7. Efficient isolate along with organic additive

The experiment conducted to prove the efficiency of isolats no. 2 and 4 along with the organic additives like food processing wastewater and herbal pharmaceutical wastewater were continued till the complete anaerobic digestion took place. The digester mixtures were prepared as given in Table 1. After completing anaerobic digestion, the physicochemical analysis shows that they are well within the limits as per standards. The physicochemical characteristic of seed slurry and wastewater is given in Table 2. Initial characteristics of seed slurry, herbal, and food processing wastewater were determined by the standard methods. The parameters included were pH, alkalinity (as CaCO₃ mg/l), total ammonia nitrogen (mg/l), volatile acids (mg/l as CH₃COOH), total solids (%), total volatile solids (%), and phosphate (mg/l). The pH of the seed slurry before the experiment was 6.91 indicating it was very less acidic and very near to the neutral pH. The characterization of herbal processing wastewater (HPW) and food processing wastewater (FPW) showed the pH of FPW was 6.07, which was slightly higher than the HPW in which the pH was found to be 6.78.

Two sets of different digester mixtures were prepared for the experiment. In the first set food processing waste (FPW) water was used, whereas in the second set herbal processing waste (HPW) water was used to make the digester mixtures with seed slurry. Each set contained four different digester mixtures. The mixture was prepared by mixing 200 ml of seed slurry and 800 ml of wastewater with an inoculum of isolate, which was given in Table 1.

The digesters containing food industrial wastewaters were continued for 43 days, while digesters of herbal pharmaceutical wastewaters were continued for 58 days. The physicochemical characteristics of completely digested effluents were given in Table 3. Results indicated pH in the range of 6.58–7.08 and volatile acid to alkalinity ratio well below 0.8 indicated good buffering. Total ammonia nitrogen was well within the limits indicating the efficient working of reactions takes place. In no instances, there was any alarming increase in either volatile acid or total ammonia nitrogen shows that the system was well-balanced methane activity. This was due to the presence of higher organic content in the wastewaters.

Sr. no.	Parameter	HPW effluents				FPW effluents
		HPW	Bacillus sk2	Bacillus sk1	Enzyme bacillidine	FPW	Bacillus sk2	Bacillus sk1	Enzyme bacillidine
1.	pH	6.72	6.58	6.66	6.38	7.07	6.93	6.84	7.08
2.	Alkalinity as CaCO3 mg/l	450.80	290.80	320.80	236.00	350.20	580.80	386.40	520.00
3.	Total ammonia nitrogen mg/l	46.20	32.00	70.56	56.28	159.60	168.00	147.84	142.80
4.	Volatile acids mg/l as CH3COOH	218.40	156.00	168.00	120.00	180.00	276.00	168.00	240.00
5.	Total solids %	8.80	8.50	8.40	8.90	8.0	8.50	9.00	8.80
6.	Total volatile solids %	8.00	6.00	7.10	5.70	7.2	6.04	6.40	6.80

Table 3.

Characteristics of effluents with herbal pharmaceutical wastewater (HPW) and food processing wastewater (FPW).

The characteristics of effluents after 45 days of food processing wastewater and herbal pharmaceutical wastewaters were given in Table 3. The pH of the four FPW effluents was slightly acidic to almost neutral. The alkalinity was between the range of 350–580 mg/l, with total ammonia as 142–168 mg/l. Whereas, volatile acids were in the range of 168–276 mg/l. Total solids in the four mixtures were found in the range of 8–9%. With total volatile solids in between 6 and 7.2%.

The pH of the four HPW effluents was slightly acidic compared to those of FPW. All the mixtures were ranged from 6.38 to 6.72. The alkalinity was between the ranges of 236–450 mg/l. The total ammonia was found in between 32 and 70 mg/l. Volatile acids were in the range of 120–218 mg/l. Total solids in the four mixtures were found in the range of 8.4–8.9%. Total volatile solids were between 5.7 and 8%. The effluent of FPW and HPW showed minimized total solids after exhausting the bioreactors.

8. Methane production

The microbial activity may get affected by some of the factors in an anaerobic digester. The design of the reactor, temperature, pH, C:N ratio, and wastewater characteristics along with the composition of complete seed material. Some of the methanogens belonging to the order viz., methanosarcinales, methanosarcinaceae, and methanosaetaceae may often be detected in accelerating methanogenicity.

Ho and Sung [33] investigated methanogenic activity in anaerobic membrane bioreactors (AnMBRs) used to treat synthetic municipal wastewater. The methanogenic activity profiles of suspended and attached sludge in AnMBRs treating synthetic municipal wastewater at 25 and 15°C were investigated using the specific methanogenic activity (SMA) assay. On day 1, AnMBR 1's methanogenic activity was 51.8 ml CH4/g VSS d, but by day 75, it had grown by 27% to 65.7 ml CH4/g VSS d. The methanogenic activity of AnMBR 2 sludge, on the other hand, was lower than that of AnMBR 1. Silva et al. [34] looked at the effects of pharmaceuticals like Ciprofloxacin (CIP), Diclofenac (DCF), Ibuprofen (IBP), and 17α-ethinylestradiol (EE2) on the activity of acetogens and methanogens in anaerobic communities. The majority of these compounds end up in wastewater treatment plants. The specific methanogenic activity was unaffected at doses of 0.01–0.1 mg/L. Acetogenic bacteria were sensitive to CIP concentrations more than 1 mg/L, whereas DCF and EE2 toxicity was only identified at concentrations greater than 10 mg/L, and IBP had no effect at any concentration. Acetoclastic methanogens were more sensitive to these micropollutants, being affected by all of the pharmaceutical chemicals tested, but to varying degrees. When compared to acetoclasts and acetogens, hydrogenotrophic methanogens were unaffected by any concentration, showing that they are less sensitive to these chemicals. CIP had the greatest impact on microbial communities, followed by EE2, DCF, and IBP, but the responses of the various microbial species differed [34]. The co-digestion of mixed sludge from wastewater treatment plants and the organic fraction of municipal solid trash were explored by Keucken et al. [35]. When co-digesting mixed sludge with organic fraction at a 1:1 ratio, based on the volatile solids (VS) concentration, the results reveal rapid adaptability of the process and an increase in biomethane output of 20–40%. The microbial community is also affected by the introduction of organic fractions. The methanogenic activity grows and adapts to acetate decomposition under 50% co-substrate and constant loading circumstances (1 kg VS/m³/d), while the community in the reference reactor, which does not have a co-substrate, remains unaffected. The methanogenic activity in both reactors increases when the load is increased (2 kg VS/m³/d), while the composition of the methanogenic population in the reference reactor remains unchanged [35].

Isolate no. 4 was found to be more suitable for herbal wastewater than food processing wastewater because biogas production was almost double in the case of herbal pharmaceutical wastewater. Enzyme Bacillidine™ (P-COG-concentrate aqueous base) was also tried but results were not very encouraging. In the case of herbal pharmaceutical wastewater also the increase in biogas production was very significant with isolate 2 with total gas production of 3085 ml as compared to 2068 ml in the case of food processing wastewater.

The bioreactor with food processing wastewater and only sterilized seed was able to produce the biogas up to the 18th day, which was 2090 ml. However, the bioreactor with Bc was able to produce the biogas for 38 days with 2920 ml, which was almost 39.7% higher than the control, while the bioreactor with Bt was able to produce 3895 ml of biogas in 43 days, which was 86.4% higher than the control. In the case of enzyme Baccilidine, the biogas production was observed to be 2320 ml in 39 days, which was only 11.0% higher than the control. Figure 2 shows that food processing wastewater produces 2090 ml of biogas in 43 days, but bioreactor amended with the culture of Bc produces 2920 ml of biogas that was 39.7% more and Bt amended bioreactor producing 3895 ml of biogas, which was 46.4% more than control. In the case of enzyme Baccillidine, only 2320 ml of biogas was produced, which was only 10.2% higher than the control.

Similar results were observed in the case of herbal pharmaceutical wastewaters also. Figure 3 shows that the bioreactor containing only wastewater and seed was able to produce 3205 ml of biogas in 45 days, whereas the bioreactor containing isolate 2 was able to produce 4600 ml of biogas in 44 days that was 43.5% higher than the control and the bioreactor containing isolate 4 was able to produce 5650 ml of biogas in 58 days, which was 76.3% higher than the control.

Enzyme baccilidine was able to produce only 2930 ml of biogas in 27 days, which was 8.6% lesser than the control. Hence it has been proved that the Bt contributes more than Bc for the biogas production, while enzyme baccilidine attenuates the biogas production in overall processes. In the case of enzyme Baccillidine, it produced only 2750 ml (14.0% less than control) of biogas.

9. Conclusion

Looking at the present scenario of the energy crisis and the environmental damage that occurs due to the use of nonrenewable sources of fossil fuel, it is the need of an hour to switch over to the use of renewable sources of energy. In the present studies, the herbal pharmaceutical wastewater and food industry wastewater were rich in organic content, which promotes the anaerobic biodegradability with a maximum production of methane by inoculating the specific bacteria isolated from the cow dung. Only cow dung seed is not able to produce more biogas as compared to isolated microbes. Baccilus sk1 (Bt, isolate no. 4) was highly capable of methane production rather than Bacillus sk2I (Bc, isolate 2). Therefore, the culture of Bacillus sk1 became the best enhancer of biogas production. Such inoculums can be cultured on large scale and may be utilized for future energy generation.

Acknowledgments

The author is grateful to University Grant Commission (UGC) for providing the fellowship.