Biogeneration and physico-chemical properties of panchagavya fortied chicken feather vermicompost

An enormous amount of chicken feather waste materials released by the poultry industry creates severe environmental pollution. Vermicomposting is an eco-friendly way to degrade chicken feather waste along with microbial mixture (Panchagavya). Chicken feather waste was pre-decomposed by mixing it with fresh cow dung (T1), dry cow dung (T2), and Panchagavya (T3). Among these, T3 exhibits rapid deterioration of chicken feather waste and seven combination T3 substrates (E0-E6), taken for the vermicomposting process by Eudrilus eugeniae in 60 days. Scanning Electron Microscopy/Energy Dispersive Spectroscopy (SEM-EDS) and Fourier Transforms Infrared (FT-IR) Spectroscopy are used to assess compost maturity. The result shows that E1 (0.050:1 ratio) shows various functional groups, rich nutrients, and necessary acids than other combinations. For large-scale commercial vermistabilization of chicken feather waste, the E1 combination is suitable for manure production and thereby enhances soil fertility, agricultural production. on the 90th day. According to the previous bench-scale reports, various microorganisms have been used to degrade poultry feather waste but those methods contain several disadvantages like pH altering the microbial activities, odor production, and slow decomposition process. minimal work has been done concerning vermicomposting of poultry feather wastes. The main objectives of the present study were: (i) to collect the feather wastes from the poultry farms of Namakkal District, Tamil (ii) to prepare the Panchagavya (microbial mixture) for pre-composting/fortication of the feather waste materials; (iii) to achieve vermicomposting of the feather wastes (pre-composted) employing Eudrilus eugeniae and to evaluate the physicochemical parameters.


Introduction
In the twenty-rst century, the utilization of poultry-based products (e.g., eggs and meat) has drastically increased in Asian and European countries. Meat (chicken) is a common source of protein for children and adults. Consequently, the poultry industry is a rapidly growing industry worldwide and economically pro table (Oakley et al. 2014). About 24,000 million tons (MT) of broiler chickens are killed per annum, producing 8,500 MT of chicken feathers globally. In addition, around 350 MT of chicken feathers is eliminated during the meat process by the Indian poultry industries (Agrahari and Wadhwa 2010). The poultry feathers contain 90% of keratin proteins, 1% of lipids, as well as 8% of water (Tesfaye et al. 2017). The keratin is divided into two groups -(i) alpha-keratin, and (ii) beta-keratin (Bodner 1982), and cross-linked polypeptide chains (Gupta and Ramnani 2006). Apart from these, poultry feathers consist of several nutritive elements and pathogenic microorganisms. Improper disposal of poultry feathers creates severe environmental pollution, particularly water and soil pollution. Therefore, there is an urgent requirement for disposal technologies to minimize poultry feather wastes. Recently, vermicomposting employing different earthworm species has grasped the attention of environmental researchers.
The soil bio-indicators, particularly earthworms (Wang et al. 2020), can effectively break down several organic waste materials (Yuvaraj et al. 2020). In general, the vermicomposting operation involves the earthworm species and various microbial communities (Domínguez et al. 2019). The earthworms utilize the different polluted organic waste materials. The earthworms signi cantly alter the wastes through rapid rotation and aeration and stimulate the degradation of substrate waste materials through various digestive enzymes (Maboeta and Van Rensburg 2003). For example, the gut of the earthworms and gut-related symbionts secrete numerous enzymes, including cellulose, protease, phosphatase, amylase, urease, manase, and lipase, which e ciently stabilize the different waste materials (Ravindran et al. 2015;Gusain and Suthar 2020). The epigeic earthworm species can mainly stabilize the complex waste materials than other anecic and endogeic species, as documented by (Ravindran et al. 2021). For instance, several researchers successfully employed epigeic earthworms, such as Perionyx excavatus, Eisenia fetida, Eisenia andrei, and Eudrilus eugeniae (Yuvaraj et al. 2018(Yuvaraj et al. , 2020. Undoubtedly, vermicomposting can degrade poultry feather waste materials, which provide the essential nutritive elements that trigger crop production. Experiments of Mazotto et al. (2011) concluded that the biodegradation of poultry feathers waste employing various microbes Bacillus licheniformis 1269, Bacillus subtilis 1271, and Bacillus cereus 1268 resulted in the production of nutrient-rich substrate. Likewise, Vasileva-Tonkova et al. (2009) reported that several thermo-actinomycete strains effectively degraded the complex poultry feathers, and a considerable amount of soluble protein was increased in the nal substrate. The study of Mohan and Senthilkumar (2019) indicated that the composting of poultry feathers and physicochemical parameters signi cantly increased on the 90th day. According to the previous bench-scale reports, various microorganisms have been used to degrade poultry feather waste bags were sprinkled with water once every two days and turned up every ten days. In general, cow dung and Panchagavya contain various microbial communities which effectively degrade the fresh chicken feather waste.
Furthermore, the microbes of Panchagavya (T3) effectively stabilize the chicken feather waste material at the end of the 60th day, presented in Figure 1. Other treatments (e.g., T1 and T2) obtained a moderate level of decomposition rate. Hence, T3 substrate materials have been used for further vermicomposting operations.

Experimental con guration of vermicompost
The pre-decomposed substrate T3 was combined with cow dung in various ratios, viz. E0, E1, E2, E3, E4, E5, and E6, as described in Table 1, and was subjected to vermicomposting using Eudrilus eugeniae, commonly known as the African nightcrawler (Kinberg 1867). An experimental ratio was used for ve kg of substrate mixture in plastic containers (25 cm in diameter and 40 cm in height). Furthermore, adult E. eugeniae of 25 numbers was released into the plastic containers. Experimental containers were kept in a cool dark place and protected by a mosquito net. Humidity was maintained at 60 to 80 percent (%) during the vermicomposting process.

Analysis of physicochemical properties
Standard methods have been used for the study of physicochemical parameters of pre-decomposed and vermicompost samples. Temperature, pH, and humidity were analyzed at intervals every ten days. At the end of the 60 th day, the pH, temperature, organic carbon, organic matter, and ash content of the vermicompost (E0-E6) samples were recorded. The determined pH was 1:10 (w/v) ratio of using a double-distilled water suspension of compost, and after 30 minutes of mechanical stirring, ltered through Whatman No.1 paper and analyzed by digital pH meter (Vasanthi et al. 2014). The moisture content of the vermicompost samples was oven-dried at 105ºC until it reached a constant weight (Kato and Miura 2008; Mohee et al. 2008;Unmar and Mohee 2008), and the moisture percentage was calculated using the procedure adopted by Schwab et al. (1994). The dry ash method calculated the ash content Unmar and Mohee, 2008). Total Organic Carbon (TOC) was examined by mixed samples (5g) into the crucibles and then burned for two hours in a furnace at 550ºC or until no stains of black carbon particles were formed and tested in compliance with Mohee et al. (2008).

SEM-EDS and Fourier-Transform Infrared Spectroscopy
To analyze the surface and structural morphology of the experimental chicken feather waste (or) vermicompost, a Field Emission Scanning Electron Microscope (FESEM) was used. In the 4000-400 cm-1 frequency range on a Brucher FT-27 FT-IR spectrometer tted with software from OriginPro, an infrared Fourier (FT-IR) spectrum of the feather waste vermicompost samples was analyzed. A selection of 1 mg was combined with 100 mg KBr of spectroscopic grade and 1 MPa in pellets.

Statistical analysis
Statistical analysis was conducted using SPSS software version 26. One-Way ANOVA and Duncan Multiple Range Experiments (DMRT) were analyzed for a homogeneous data set with various parameters in pre-decomposed and vermicomposted samples of the chicken feather waste. All values at p<0.05 are considered statistically signi cant.

Results And Discussion
Decomposing of chicken feathers waste The Panchagavya consists of various materials such as cow dung, cow urine, curd, cow milk, tender coconut water, jaggery, and bananas, which strongly stimulate the microbial populations in treatments. For example, the study of Amalraj et al. (2013) demonstrated that Panchagavya contains an a uent population of actinomycetes, various bacteria, and P-solubilizers. These microbial communities effectively stabilize the complex chicken feathers waste materials during the pre-degradation process, as depicted in Table 2. The present study demonstrates that Panchagavya contains different essential nutrients that can trigger microbial populations by forti cation with substrate. Recently, Behera and Ray (2021) con rmed that various acids (e.g., malic, lactic, citric, acetic, and succinic acids) had been found in Panchagavya, which plays a pivotal role in the elimination of various soil contaminations and enhances the degradation process. The temperature of the substrate Analysis of temperature in composting units is essential to determining the maturity of the nal substrate materials. The highest level of temperature was recorded in T3 (39.63˚C) followed by T2 (30.23˚C) and T1 (29.47˚C), respectively. The substrate temperature signi cantly increased during the middle stage of composting the process, and the declining temperature level was recorded at the end of the experiment. During the decomposition process, heat is eliminated through oxidative action of various microbial communities on waste materials resulting in a decline in temperature in treatments (Peigne and Girardin 2004).

Moisture
Higher moisture content was recorded in pre-decomposed chicken feather waste materials, indicating the deterioration process progression. The highest percentage of moisture recorded in T1, T2, and T3 was 85.65%, 83.50%, and 75.78%, respectively. The subsequent ten days of moisture were improved in T1 (88.43%) and T2 (86.36%). From the 60 th day onwards, T3 (16.81%) of moisture signi cantly (at p<0.05) decreased. During the feather composting process, moisture loss was detected, which might be due to the metabolic behavior of the species that encourage heat energy or environmental factors such as air ow over ventilation (Hogan et al. 1989). A drop in moisture content <30% triggers the treatment's biological processes, and 50-60% of moisture content is optimum for the composting process (Castillo 2004).

pH
The pH of the substrate is also an essential factor in enhancing the composting process. In the present study, there was a signi cant increase in the pH of the substrate. A maximum pH was recorded in T2. In the present study, a pH of 6.0-7.0 triggered bacterial growth. Experiments by Pan and Sen (2013) demonstrated that a pH range of 6.5 to 8.0 was satisfactory for microbial growth in food wheat straw composting. This pH is one of the critical factors for the degradation of chicken feather waste. On the other hand, the substrate of the T3 had low pH (6.16) because T3 contained Panchagavya, which also effectively degraded the feather waste materials.
Vermicomposting of chicken feather waste materials and earthworm's mortality Earthworms signi cantly stabilize the pre-decomposed chicken feather waste materials. Notably, the gut wall of the earthworms and several symbionts secrete different digestives enzymes which effectually break down the chicken feather waste materials.
The earthworm mortality in experimental vermicomposting bins is presented in Table 3. Earthworm mortality was observed during the initial stages of the vermicomposting operation, and a maximum mortality rate was recorded in E5 and E6. Furthermore, zero percent of the mortality rate was observed in E0 and E1. According to our results, E1 (cow dung and 50gm pre-decomposed chicken feather waste) is suitable for nutrient-rich vermicompost production. Generally, the dried chicken feathers contain 91% of protein (Salminen and Rintala 2002). Protein-rich organic waste materials can release acidic compounds during the breakdown process (Steve 2017), which may affect the earthworm survival rate, as reported by Francesca (2017).

Physicochemical features of vermicomposting
With the progression of the vermicompost, the moisture content of the different combinations (CD with CFW) was improved, as described in Table 1.
During the vermicomposting process, the percentage of vermicompost moisture (CD and CFW combinations) ranged from 59.89% to 75.77% (Table 4). The rapid development of various microbial communities may regulate the moisture content of substrate materials. Earthworm species quickly utilize the moistened waste materials. A reduction of pH in E5 (5.74) and a signi cantly at (p<0.05) improved level of pH were recorded in E1 (7.42) and E2 (7.49). The pH of the substrate decreases by the release of organic acids at the initial stage of decomposition (Liu et al., 2011), and bioconversion of organic nitrogen into free ammonia raises the pH of the compost (Altieri et al. 2011).
The organic carbon (OC) and organic matter (OM) from earthworm worked treatments are gradually reduced during the vermicomposting process. A maximum OC and OM reduction was recorded in E6. The repaid decrease of OC might be due to the release of CO 2 from the substrate by the joint action of earthworms and various microbial communities (Prakash and Karmegam 2010). Suthar (2006) stated that reducing OC in the vermicomposting process is also responsible for microbial respiration.

Maturity analysis of substrate through Scanning Electron Microscopy (SEM)
For the surface morphological characterization of substrate materials, SEM is one of the appropriate methods. The nal vermicompost micrographs ( Figure 2) indicate the smooth surface area and substantially reduce the size of the particles. In the SEM images of E0 and E1, smaller aggregates than E2, E3, E4, E5, and E6 found larger structural surface particles. Similar results were reported by Ravindran et al. (2008) and Senthil Kumar et al. (2014).

Nutrient Analysis (SEM-EDS)
SEM-EDS suitable instruments to detect the various chemical elements (e.g., Ca, C, K, Cl, Mo, Fe, P, Na, Mg, N, S, Mn, Cr, and Co) from the substrate materials are presented in Table 5. The lowest carbon content was recorded in E0 (9.58%) and E1 (10.74%). Loss of TOC was achieved by the respiration of microorganisms and the consumption of organic compounds by microorganisms and earthworms (Sharma and Garg 2017). The nal vermicompost contained a rich amount of essential macronutrients, such as N (E1-1.24%), P (E0-4.33%), K (E0-7.43%), Ca (E3-34.48%), and Mg (E5-1.20%). Nayak et al. (2013) reported that many essential plant-based micronutrients increase during the vermicomposting process. Apart from this, the nal substrate materials contain a low level of toxic heavy metals. Lee (1985) indicated that an enormous amount of heavy metal ions could bio-accumulate in the earthworm species' internal tissue (e.g. chlorogogen tissue). The body of the earthworm contains metal-binding protein like metallothionein that effectively binds with several heavy metal ions, resulting in a minimum amount of metals found in the earthworm-treated substrate (Cherian and Nordberg 1983; Hait and Tare 2011).

FT-IR
Maturity/stability assessment was done through the FT-IR spectroscopy technique. Based on FT-IR spectroscopic techniques, the normal distribution of absorption bands in the vermicomposting samples was observed. Similar results were documented by Fleming and Williams (2019). The FT-IR spectrum of vermicomposting samples is depicted in Figure 3.
The notable peaks were recorded in E5 (3774 cm -1 ) and E6 (3848 cm -1 ), which strongly contain the N-H variety of amide compounds, and it was also con rmed by Purandaradas et al. (2018). A medium sharp peak indicated alcohol and phenol groups, as Kumar et al. (2013) documented. The amount of lower peak strength for CH 3 , CH 2 , and CH at 2920-2991cm-1 in vermicompost samples suggested a decline in the level of alkane compounds (Mothé et al., 2018). The suggestion of a diminishing pattern of aliphatic C-H stretching at 2936-2958 cm -1 supported the degradation of lipids and carbohydrates from substrate materials (Bhat et al., 2017). C=O and S-H stretching vibration of carbonyl and Mercaptans suggested the presence of two distinct weak stretches at 2425-2426 cm -1 and 2519 cm -1 . The peak at 1517 cm -1 correlated to the characteristics of the amine groups (Amide-II) from waste materials (Mothé et al. 2018). Various heights at 1417-1422 cm -1 might be due to the presence of carboxylic acid. The peak of 1460-1420 cm-1 corresponded to the vibration of lignin in the area, and 1100 cm-1 re ected the cellulose and hemicellulose C-O stretch (Mothé and De Miranda 2009). The broad peaks of 1041-1093 cm -1 were associated with anhydride, primary alcohol, and secondary alcohol stretching of CO-O-CO, S = O C-O. Furthermore, peaks of 1047-1091 cm -1 relatively humic acid content in compost and vermicompost (Kumar et al. 2015). The peak at 874 cm -1 is known to be the esters of the S-OR stretching group. The S-S bonds were absorbed adequately at the height of around 537-599 cm -1 , as con rmed by Wojciechowska et al. (1999) and Vasconcelos et al. (2008). Nitrogen-containing compounds in their infrared spectrum improved very distinctive absorption lines. C-N-C deformation was caused by mild to heavy bands at 580-418 cm-1 (Rastogi 1991).
During the biodegradation process, a substantial amount of cysteine in the chicken feathers produced a considerable NH 3 . The feather bi-sul de cysteine was broken into a stable secondary keratin structure . Earthworms' combined microbial and digestive enzymes e ciently broke down the complex feather waste materials and produced the nutrient-rich vermicompost (Zhang et al. 2000;Aira et al. 2007).

Conclusion
Our results strongly suggest that Eudrilus eugeniae and forti cation with bene cial microbes of Panchagavya convert the chicken feather waste materials into nutrient-rich vermicompost. Among the various amendment combinations, 0.050:1 ratio (E1) of chicken feather waste to cow dung provides a signi cant result in compost maturity and nutrient status and reduces environmental contamination from chicken feather waste materials. Therefore, this combination (E1) can be used for large-scale vermistabilization of chicken feather waste materials to enhance soil fertility and agricultural plant production.    Pre-decomposition of chicken feather waste materials in different treatments. T1-chicken feather waste with fresh cow dung; T2 -Chicken feather waste with dry cow dung; T3 -Chicken feather waste with Panchagavya. Figure 2 SEM micrographs indicate the morphological structures of panchagavya forti ed chicken feather during vermicomposting (E0-E6) process.

Figure 3
FT-IR analysis of panchagavya forti ed chicken feather vermicompost at different ratios.