Bioenergy may be a significant replacement for non-renewable fossil fuels, making the path easier for sustainable development and decreasing the dependency on conventional energy sources. The world population is increasing rapidly; industrial activities are flourishing due to it. Global urbanization and industrialization lead to enhanced energy demands and more waste generation (Zhang et al., 2021). Moreover, as energy is the critical source of comfort for the modern world, most developing and underdeveloped countries have no proper access to energy services like petrol, diesel, natural gas, and electricity. Fossil fuels are the primary energy source for many countries, but their reservoirs will be scarce to meet requirements in the near future. Scientists are looking for renewable sources to overcome fuels limitations (Forgács, 2012; Gooding and Meeker, 2016).

On the other hand, globally, 17 billion tons of waste per annum is being generated due to increased industrialization and urbanization; it will reach 27 billion tons by 2050 (Patinvoh et al., 2016; Rahaman et al., 2021). Biogas is an emerging renewable energy source obtained through the degradation of organic matter by microorganisms under anaerobic conditions. The biogas production and applications provide a comprehensive and systematic guide to developing and deploying biogas supply chains and technology (Ogut et al., 2013; Kanafin et al., 2021).

According to the world’s food demands, the poultry industry is major growing strength. The necessity to cope with waste disposal needs some severe alternatives; otherwise, it will lead to global and environmental pollution (Morinval and Averous, 2021). Millions of tons of organic waste are produced annually from the poultry industry (Karuppannan et al., 2021). These wastes have high capabilities to generate methane. The poultry industry generates organic waste like feathers in a vast quantity. Feathers are composed of keratin protein, having a recalcitrant structure. The cross-linking and covalent bonds added more strength to the feather’s structure (Sypka et al., 2021). Different pretreatment methods are used like superheated water, strong acids, or alkalis puffing, and pressurized breakdown of feathers (Chao et al., 2021), and many other physiochemical methods could be applied (Wang et al., 2012, 2019).

Because of high cost, sustainability, and ecological problems, these pretreatment methods are seldom used. Recent research trends in cost-effective, ecological, and sustainable pretreatment methods with the help of microbial consortia (González et al., 2021). Different microorganisms like bacteria and fungi were isolated for pretreatment of chicken feathers (CFs). CFs were biologically pretreated, and the pretreated feathers hydrolysate was used for biogas production. Production of biogas via an anaerobic digestion (AD) process effectively reduced the waste generated in large volumes. It is a sustainable and well-established process to utilize waste for energy production. The digestate that is leftover could be still used as a source of fertilizers (Awasthi et al., 2022).

AD is a multistep, sensitive, and complex process with different microorganisms (Hagos et al., 2017). This process occurs in an oxygen-deficient environment. Methane and CO₂ are produced from a chain of different biochemical processes during AD. AD has four different stages: hydrolysis, acidogenesis, acetogenesis, and methanogenesis (Peng et al., 2021), in which the organic substances are broken down in the absence of oxygen. The organic matter that is not digested in the AD process is considered digestate. The vital operating parameters that must be maintained and optimized for an AD process are proper carbon-to-nitrogen (C/N) ratio, pH, temperature, total solid (TS) contents, and volatile solid (VS) contents (Ellacuriaga et al., 2021). Pretreatment is essential to improve the biogas production and process stability. Therefore, it will be helpful to investigate the optimal mix of substrates and the conditions of fermentation. Biological pretreatment can be carried out either by fungi or bacteria to degrade keratin waste. Bacteria use keratin as a source of carbon and energy (Chaturvedi et al., 2021). However, high nitrogen content in CFs with a low C/N ratio will not suit AD. Therefore, the feathers, a protein-rich substrate, must be co-digested with another substrate with high carbon content to balance the C/N ratio (Guo et al., 2012; Patinvoh et al., 2017; Mohanty et al., 2021). The rate of AD is affected significantly by the C/N ratio. A balance C/N ratio is required for microorganisms’ growth and proper metabolism. The reported C/N ratio for proper digestion is 20–30:1 (Zheng et al., 2021). The biomethane potential of many wastes in batch fermentation experiments has been tested, and few studies compare batch fermentation to continuous fermentation. Batch fermentations are suitable for estimating a substrate’s methane yield and biodegradability, but they do not provide information about the long-term effect of the substrate on the fermentation (Szilágyi et al., 2021).

CF contributes a significant portion to poultry waste. High content of protein in CF waste is associated with the problem of ammonia inhibition. Therefore, to address this problem, the present study was designed to increase the biogas production from CFs by biological pretreatment by Pseudomonas aeruginosa and co-digestion with carbon-rich rice husk (RH) and green grocery waste (GW) to avoid ammonia inhibition. It will help to improve the feather waste to energy conversion through the AD process.

The expanding interest toward biogas generation from bio-waste through complex AD opened new routes in improving biogas production processes and their upgradation. Waste generation and waste handling are the most critical issues to overcome these days. CF is one of the major waste, generated in a considerable amount. Feathers can be utilized for energy production by eliminating harmful environmental and catastrophic issues. In this way, on one hand, waste is properly managed, and, on the other hand, bioenergy in the form of biogas is produced (Provin et al., 2021) from the waste. Therefore, the current study was designed to evaluate the effect of different pretreatments and co-digestion and mono-digestion by AD using batch and continuous fermentation and its effect on biogas yield. The VSs of CFs, feathers hydrolysate, and RH were 70, 1.05, and 75.98%, respectively. VS depends upon the type, source, and composition of the substrate.

The results of accumulative biogas production demonstrated that, in the case of PC, most of the gas was produced in the initial 15 days of the experiment, whereas, in the case of RH pretreated co-digestion, it took 30 days. In the case of non-pretreated co-digestion, RH, and pretreated rice, there was a gradual increase in production until the end. On the other hand, in the case of feathers, the production was low until 25 days, and, after that, it was increased to reach the maximum level. The time taken by the process depends upon the substrate composition, whereas the PC contains glucose that was rapidly utilized by acidogens. Different research results reported that hydrolysis depends on substrate composition and carbohydrate hydrolyzed in hours, whereas complex compounds like cellulose and lignin took weeks to hydrolyze completely. Some other studies reported that some substrate has hardly accessible for the action of enzymes that ultimately took more time, and the hydrolysis became a rate-limiting step. The present study demonstrated (Figures 1A,B) that the microbial pretreatment was completed in 72 h of incubation. According to the research work reported by Forgács et al. (2011), with different incubation times of 1, 2, and 8 days, the biogas yield was 55, 94, and 83%, respectively. The possible reason was the VFA accumulation in the hydrolysate at the end of the experiment as the VFA measured in the hydrolysate was 875 mg/L and, in untreated feathers, was 275 mg/L. The VFA accumulation in microbial pretreated CFs’ hydrolysate was expected due to the accumulation of ammonia, which negatively affected the methanogenic population. Ammonia is linked to the presence of protein sources in the feed. Because CFs are mainly composed of protein, hydrolysis results in ammonia accumulation (Sypka et al., 2021). During AD of the nitrogenous feedstock, ammonia inhibition is one of the main challenges, resulting in lower biogas yield (Chen et al., 2008). At lower concentrations, ammonia acts as a buffering agent, but, in a low C/N ratio, the accumulation of ammonia negatively affects the methanogenic population; hence, the nitrogenous feedstock must be co-digested with the carbon-rich substrate (Christou et al., 2021). Figure 1A represents the accumulative biogas yield; the microbial pretreated feathers slightly increased the biogas yield by 1.1% compared to non-pretreated feathers. The results followed the study reported by Forgács et al. (2011) that determined the effect of microbial pretreatment using different wild and recombinant bacterial strains from CFs. Another study also reported that there was no significant increase in methane yield of CFs when biological treated with Bacillus megaterium. However, the analysis of hydrolysate showed that the total dissolved solids were increased by 1%. At the end of the incubation, VFA accumulation in the pretreated feathers was 875 mg/L, whereas, in non-pretreated, it was 275 mg/L. This clearly indicates that the microbial pretreatment increases the feathers’ hydrolysis but results in low biogas yield due to ammonia inhibition. In the case of the reactor fed with non–RH-P, accumulative biogas yield was lower than the RH-P and CFs (Figure 2). The chemical pretreatment of RH increases the biogas yield compared to non–RH-P. Different research results reported increased biogas yield due to acidic pretreatment at different concentrations and conditions. According to the result reported by He et al. (2017), the pretreatment of RH with 6% NaOH resulted in the biogas yield of 0.53N L/g VS, whereas non-pretreatment can yield 0.36 N L/g VS. The chemical pretreatment of RH showed a 32% increase in biogas yield compared to untreated RH in the case of mono-digestion. Mu et al. (2021) also checked the effect of acid mixtures like acetic and propionic acid but do not measure any variance in the treated and untreated RH. In another study by Dahunsi (2019), the pretreatment with 2% sulfuric acid may reduce the hemicellulose content by 69%. The possible reason was that the chemical pretreatment improves biodegradability, increases the biogas yield, and enhances the efficiency of the overall process. The biomass structure could be changed through chemical pretreatment methods to have more accessibility to anaerobic microorganisms, therefore enhancing the biogas production and digestion efficiency. At the end of the experiment, the VFA accumulation was 250 and 350 mg/L in RH and RH-P, respectively, which indicates the increase in hydrolysis due to pretreatment.

During the current study, the effect of co-digestion on biogas yield with pretreated and non–RH-P was determined and compared with the calculated methane yield for RH pretreated co-digestion and RH non-pretreated co-digestion. In the case of non-pretreated co-digestion, the calculated yield was determined from the yield of mono-digestion of non–RH-P and yield of hydrolysate. In the case of non-pretreated co-digestion, the accumulative biogas yield and methane yield were lower than the calculated methane yield. In the co-digestion in the case of non–RH-P, the methane yield was 22.2% decreased. The decrease in the case of non–RH-P with cattle manure is supported by Lange et al. (2016). This decrease in yield may be due to ammonia accumulation, as the feathers have high protein contents (Mohanty et al., 2021). Furthermore, RH has a complex lignocellulosic structure that is resistant to degradation. At the same time, CFs have a high concentration of proteins, whereas, during degradation of feathers, ammonia is produced due to microbial pretreatment. Ammonia is very toxic in higher concentrations (Christou et al., 2021), and at the end of the incubation, VFA accumulation in the hydrolysate was 875 mg/L, whereas, in RH, it was 250 mg/L. In the case of co-digestion, VFAs were lower than hydrolysate, which confirms that the ammonia in co-digestion affects the hydrolysis of RH, which was the reason for a lower yield in co-digestion as compared to the calculated yield (Figure 3). Magrí et al. (2017) reported that the yield of biogas depends upon the composition and characterization of the substrate. Nakamura and Asada (2021) reported that RH contains high cellulose and lignin, resistant to enzymatic degradation, leading to low biogas yield. However, the increase in biogas yield due to co-digestion depends upon the co-substrate and pretreatment. Hamed et al. (2015) reported a significant increase in biogas yield resulting from mixing cattle manure with food waste in different ratios.

According to the current research work, the methane yield from co-digestion of hydrolysate with pretreated RH was compared to calculated methane yield compared to the yield of mono-digestion of hydrolysate and yield of mono-digestion of RH-P. The co-digestion of hydrolysate with pretreated RH increases the biogas yield by 34% compared to the calculated yield as represented in Figure 4. Different studies have reported an increase in biogas yield in the case of co-digestion. Sayara and Sánchez (2019) reported a 758% increase in biogas yield in the case of co-digestion of cattle manure with pretreated RH. Dahunsi (2019) reported a 52% increase in biogas yield in co-digestion of cattle manure with cocoa husk. The increase in biogas yield was due to the pretreated RH, which has more sugar than non-pretreated. It was reported previously that chemical pretreatment improves biodegradability and increases the biogas yield and enhance efficiency of the overall process (Khan and Ahring, 2021). The biomass structure could be changed through chemical pretreatment methods to have more accessibility to anaerobic microorganisms, therefore enhancing the biogas production and digestion efficiency. The other reason for an increase in biogas production may be the effect of a more balance C/N ratio in the case of co-digestion of hydrolysate and RH-P. The nutrients and organic content present in the feedstock affect the activity and growth of the microorganisms. The essential nutrients for microorganisms are carbon and nitrogen (Mothe and Polisetty, 2021). The deficiency of any nutrient ceases the growth and activity of the microorganism. A low C/N ratio results in the formation of ammonia, whereas a high ratio results in the failure of the biogas plant. Hence, in both cases, AD failed (Nkuna et al., 2021). Appropriate C/N ratio/nutrient balance is required for the proper growth of microorganisms under stable environmental conditions. Furthermore, this is why the C/N ratio greatly influences AD (Xiao et al., 2021). Generally, the most appropriate (C/N) ratio of 25–30 was required for the suitable development of biological processes (Ramírez-Sáenz et al., 2009; Liu et al., 2016; Sillero et al., 2021). Methane production decreases because of the imbalance in the C/N ratio. Moreover, the most common problem was the inhibition by nitrogen in degrading organic wastes. Hence, the problem of the C/N ratio could be overcome by co-digestion with a waste having a high C/N ratio. At the end of the experiment, the VFA accumulation in co-digestion pretreated RH and hydrolysate was lower than both mono-digestion of hydrolysate and pretreated RHs in Figure 3. The lower VFA accumulation in the co-digestion of hydrolysate and pretreated RH means that most of the VFAs are converted to biogas, leading to high yield.

During continuous AD, biogas and methane yield for mono-digestion of microbial pretreated CFs’ hydrolysate (HL), GW, and their co-digestion (G+HL) was measured for three retention times. The biogas yield fluctuated daily with time initially, but, at steady-state, the biogas yield for microbial pretreated CFs’ hydrolysate (HL) becomes stable, as shown in Figures 5A,B. In the case of mono-digestion of hydrolysate, the biogas yield showed a continuous decline with time, leading to process failure in the third retention time. The decline in biogas yield and process failure was due to ammonia accumulation as a result of the high protein content of feathers (Mohanty et al., 2021), as, during degradation of feathers, ammonia is produced due to microbial pretreatment. Ammonia was toxic in higher concentrations (Christou et al., 2021). On the other hand, the biogas yield on green waste and co-digestion was more stable during steady-state. As a consequence of co-digestion, the biogas and biomethane yield increased compared to the average of GW and hydrolysate mono-digestions, as shown in Figures 5, 6. Co-digestion with substrates with high buffering capacity (alkalinity) such as manure can be good alternatives for the effective treatment of highly biodegradable materials. During the co-digestion of microbial pretreated CF hydrolysate with GW and animal manure, the manure provides buffering capacity and various nutrients. In contrast, the plant material provides high carbon content (Ambaye et al., 2021), resulting in a high biogas yield. The result is a more balanced C/N ratio, and the co-digestion of manure and green-grocery waste also decreases the risk of ammonia inhibition and acidification. Thus, it was better to consume microbial pretreated CF hydrolysate in co-digestion than mono-digestion for biogas production. GW has a high content of carbon and feather hydrolysate has a high amount of nitrogen, so when they both were used in co-digester to balance the C/N ratio and produce more biogas and biomethane as compared to the average of their mono-digesters, thus enhancing the biogas yield and the efficiency of overall process (Kiran et al., 2016).

Furthermore, at the start of the experiment, the pH of all the reactors was set to be 7.2, whereas, at the end of the experiment, the pH of hydrolysate increased. In the case of GW, the pH was dropped down to a critical level, whereas, in the case of co-digestion, there was no significant change in pH. The pH range varies for different groups of microorganisms for their growth, like for the growth of fermentative bacteria, pH was required about 4.0–8.5, and, for methanogenic bacteria, the pH must be in the range of 6.8–7.2; otherwise, failure of the process may occur (Amin et al., 2021). On higher pH levels, the free ammonia concentration increased, causing more toxicity for methanogens (Wang et al., 2020). The VFA accumulation was high in the case of hydrolysate and finally led to process failure. Whereas, in the case of co-digestion, the VFA accumulation was lower than mono-digestion of hydrolysate and green waste. The co-digestion enhances the process stability and reduces the toxic effect of ammonia, resulting in an increase in biogas production (Christou et al., 2021).

Non-pretreated CFs were challenging to hydrolyze by anaerobic microorganisms because of their recalcitrant nature. Microbial pretreatment of CFs by Pseudomonas aeruginosa increased its hydrolysis, but the mono-digestion of hydrolysate results in low yield of biogas due to high concentration of ammonia. On the other hand, RH’s pretreatment significantly increased biogas yield. Pretreated RH co-digestion with hydrolysate significantly increased biogas production. However, co-digestion with non–RH-P does not enhance the biogas yield. During continuous AD, the hydrolysate’s co-digestion with GW significantly increases the biogas yield. These results suggest the importance of running the bioreactor for a long term in continuous fermentation mode to test the suitability of a novel biomass substrate for industrial biogas production.

The original contributions presented in the study are included in the article/supplementary material, further inquiries can be directed to the corresponding author/s.

MS, AnK, HA, AB, ZG, AlK, and MMR contributed to design of the study, performed the experiments, and wrote the first draft of the manuscript. MB, FH, and AS wrote the comments, review, and involved in supervision of the students. SK was the main supervisor and secured funding. All authors contributed to manuscript revision, read, and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article, or claim that may be made by its manufacturer, is not guaranteed or endorsed by the publisher.