Feather Degradation Research Articles

Microbial bioconversion of feathers into antioxidant peptides and pigments and their liposome encapsulation.

The co-encapsulation of bioactive peptides obtained from degradation of chicken feathers and flexirubin-type pigment produced by Chryseobacterium sp. kr6 into phosphatidylcholine liposomes was investigated. Control empty liposomes showed mean diameter of 168.5 nm, varying to 185.4, 102.0 and 98.5 nm after the encapsulation of peptides, pigment and their co-encapsulation, respectively. Control liposomes presented zeta potential of - 20.9 mV, while the formulations containing the bioactive compounds showed values of - 30 mV or higher in magnitude. Infrared analysis revealed typical spectra for phosphatidylcholine, suggesting that no new chemical bonds were formed after encapsulation. ABTS radical scavenging assay showed that the antioxidant activity of the compounds was maintained after encapsulation. Feather waste can be a valuable substrate for simultaneous production of antioxidant peptides and pigment by Chryseobacterium sp. kr6, and their encapsulation into liposomes may be a suitable alternative for delivery of these natural antioxidants.

Optimization of Keratinase Production and Utilization of Bacillus pumilus for Feather Degradation

Soil samples were collected from the feather dumped area where Bacillus pumilus was isolated and used for keratinase production and keratinolytic activity. In the optimization study, optimal condition for enzyme production was observed at 144 h, pH 7, temperature 37°C. The organism was utilized for feather degradation study. The maximum degradation of 57% was obtained at 37°C, pH 7 and 6 days incubation. The size of keratinase was determined by SDS- PAGE and was observed as 52 KDa.

Isolation, screening, characterization of proteolytic bacteria and production of protease with its potential applications

The purpose of this study was production of protease enzyme by isolated bacteria and evaluation of their potential for various industrial applications. Two strains AN16 and AN13 exhibiting maximum proteolytic activity were used for further studies. The activity of crude enzymes was completely inhibited by phenyl-methyl-sulphobnyl fluoride (PMSF) and ethylene diamine tetra-acetic acid (EDTA) had no such negative effect on activity. Enzymes were relatively stable in presence of surface active agents such as Triton X-100, Tween-80, Tween-20 and oxidizing agent H₂O₂. The enzymes exhibited maximum activity within pH range of 7-9. The optimum temperature was recorded to be 37⁰C. The relative activity of isolate AN13 protease in presence of metal ions Ca2+, Fe2+, Mn2+, Mg2+, Zn2+, Co2+, Hg2+, Na+ was as follow: 107%, 99%, 156%, 100%, 105%, 97%, 90%, 104% and102%, 101%, 145%, 80%, 98%, 90%, 98%, 96% for AN16 protease, respectively. The degradation percentage of chicken feathers by crude protease was recorded to be 55.50% for AN16 followed by AN13 (39.79%). Study of dehairing activity of protease resulted in complete removal of hairs from hide. De-staining studies showed excellent proteinaceous stain removal efficiency. Antibacterial and antifungal activity of synthesized Silver nanoparticles (AgNPs) used against E. coli (25mm), Salmonella typhus (18mm) and fungal strain Malassezia (17mm) were also recorded. Removal of gelatin from waste x-ray films by enzyme extract showed a weight loss of about 6% (AN16) and 5% (AN13). Different low cost media were also tested to support the growth of these proteolytic bacteria exhibiting positive results. Keywords: AgNP; Dehairing; Detergents additives; Feather degradation; Protease; Silver Recovery http://dx.doi.org/10.19045/bspab.2020.90240

Isolation of Keratinolytic Microorganism Streptomyces chartreusis strain ISP 5085 from the Soil Sample: Characterization, Degradation and Microbial Identification using 16S rDNA Region

Keratinolytic microorganisms have a broad significance in feather waste degradation and it can be used for advancement of livestock feed and production of protein hydrolysates. Production of keratin into the environment in different forms is very high which leads to setback for its degradation. An approach for degradation of keratin based wastes through potential microorganism with higher efficiency can be attained. The study shows that maximum DFD% i.e. Degree of Feather (keratin) Degradation, through the isolates is achieved, when nutrient medium is kept at temperature 30 °C and pH-8.5 supplemented with dehydrated powdered form of chicken feathers as keratin source for 72 h – 120 h. Chicken feathers acts as a better substrate and source of keratin content. A molecular approach was necessary therefore; phylogenetic trees of the isolate were constructed with neighbour-joining method through the Microbial Identification using 16S rDNA Region, which showed that sequence exhibited a high level of homology with Streptomyces chartreusis strain ISP 5085. The present study deals with identification of isolates that play a significant role in the degradation of chicken feather waste as well as keratin degradation ability of the isolates and optimum conditions required for it.

Bio-degradation of Feathers Waste by the Staphylococcus epidermidis P3A

Development of industrialization is directly proportional to the development of nations. Rising industrializations also increase the pollutions. One of them is poultry industry which discharges a huge amount of keratinous waste. The present study focused on the selection of feather degrading bacteria from Barshi region of Maharashtra. Bacterial strains were grown in whole feather broth medium. Out of 16 proteolytic bacterial isolates, 9 feather degrading isolates were selected from the Parande road side feather waste dumping area. Among these isolates, P3A was selected and classified on the basis of morphological and biochemical analysis. 16S rRNA studies confirmed that the isolated strain was Staphylococcus epidermidis. Staphylococcus epidermidis P3A shows 84% feather degradation and 72 U/ml maximum keratinase activity after 96 hrs. The isolate shows potential use in feather waste disposal methods which are safe and environment friendly.

Chicken feather waste degradation by Alternaria tenuissima and its application on plant growth

The use of chicken as food is rising day today and as byproduct 8.5 billion tons, poultry feathers are produced worldwide, whereas India contributes about 350 million tons per annum. It is a waste product of poultry industries, are considered a potential high-quality protein supplement owing to their crude protein content of more than 85%. In the present study Alternaria tenuissima a keratinophilic fungus was used for feather degradation in submerged state fermentation and soil. Total Protein releases were studied in submerged state fermentation by A. tenuissima are 122µg/ml and 238µg/ml in 15 and 25 days respectively. Lysine, Methionine, Cysteine and Valine were found 15.8, 6.8, 20.2, 7.5 µg/ml in 25 days, respectively. Chicken feathers were mixed with soil and inoculated with spore suspension for degradation of complex keratin protein into simpler organic forms. A. tenuissima degraded feathers in soil and enhances nutritional value. Five-gram feathers in 250 g soil mixtures were found better growth enhancers and increased height. This work will reduce the solid waste generated in the form of feathers from the poultry industry, and convert it into a simpler organic form that can be used by plants.

Feather degradation efficiency and hide dehairing ability of a new keratinolytic bacillus halotolerans strain, isolated from a tannery wastewater

Here, we report a new keratinolytic-producing bacterium identified as Bacillus halotolerans 4BC based on 16S rRNA gene sequencing. 4BC strain isolated from a tannery wastewater, showed proteolytic activity when grown on keratin azure, bovine hair and feather meal agar plates. B. halotolerans 4BC degraded almost 88% of chicken feathers after 10 days of cultivation in feather powder broth at pH 8 and 37°C. It was also efficiently able to degrade bovine hair keratin, despite the complexity of this substrate in comparison to feather keratin. The effects of different liquid to substrate ratios, inoculum sizes and incubation times on keratinase production were studied using response surface methodology to find the optimum conditions required for maximum B. halotolerans keratinase yield. The maximum keratinase yield (1059±53 mU/ml) was found under the following conditions: incubation period of 10 days, liquid/solid ratio of 5 and inoculum size of 2.3 % v/v). The crude enzyme exhibited a remarkable activity and stability under high temperature and alkaline conditions (pH 10 at 80°C). Additionally, the touch-visual and histological results demonstrated that the enzyme treated dehaired hides exhibit similar or improved characteristics without damaging the collagen layer, which makes the crude keratinase a potential effective and eco-friendly candidate for application in leather industry to avoid pollution problems associated with the use of chemicals.

A study on optimization of biomass of Bacillus pumilus for feather degradation

Soil samples were collected from the feather dumped area, and they were screened for the presence of keratinolytic bacteria Bacillus pumilus. Based on its growth on Bacillus isolation agar, Skim milk agar, and Starch agar, it was conformed as Bacillus pumilus. The growth of bacteria was estimated by biomass estimation. In the optimization study, the optimum incubation period observed for feather degradation was 48hrs, pH 7, and temperature 40°C. Purified Keratinase enzyme was used for the feather degradation study. The maximum degradation observed was 29% at the temperature of 400C. The size of kerinase produced was estimated as 52KDa.

Optimization for Production of a Plant Growth Promoting Agent from the Degradation of Chicken Feather Using Keratinase Producing Novel Isolate Bacillus pumilus JYL

Feather is a good source of nitrogen but the highly rigid structure limits its utilization and leads to environmental pollution and resource wastage. Traditional feather degradation processes such as alkali hydrolysis and steam pressure cooking not only destroy the amino acids but also consume large amounts of energy. Therefore, searching for feather degrading bacteria to transform feather waste to nitrogen fertilizer to improve soil and plant growth is needed. And this also will solve the soil nitrogen deficiency problem in China. In this study, a bacteria that could degrade feather and promote plant growth was isolated from soil sample using dilution plate method. And the isolated bacteria was identified as Bacillus pumilus JYL according to its morphology, biochemical characteristics and 16S rRNA sequence. And the maximum keratinase activity (57.14 U/mL) was obtained under optimal conditions of 0.036% FeSO4, 0.025% MgSO4, 0.073% K2HPO4, 0.5% feather with 2% inoculant at pH 6.5 fermented for 36 h at 35 ℃ after optimization using (response surface methodology) RSM. The effect of hydrolyzate on wheat seed germination and growth was studied. The results showed that germination engergy and germination index with 3% feather hydrolyzate addition group was 94.22% and 87.52%, significantly higher than that of the control group (p < 0.05), respectively. The plant height, root length, fresh weight and dry weight in the T2 treatment (added 5 mL feather hydrolysate in 100 g soil) at 60th day were 1.10, 1.16, 1.77 and 1.73 times than that in the CK treatment (without addition of hydrolysate), respectively. And the soil enzyme activities in all treatment groups (T1, T2, T3) significantly increased compared with control group (CK) (p < 0.05). This result will provide a potential utilization method for feather valoration.

Keratinases from Coriolopsis byrsina as an alternative for feather degradation: applications for cloth cleaning based on commercial detergent compatibility and for the production of collagen hydrolysate.

Keratinases are proteolytic enzymes that emerge as an alternative for dealing with the disposal of chicken feathers. In this study, we aimed to investigate the keratin-degrading enzymes secreted by the fungus Coriolopsis byrsina and their partial biochemical characterization to adapt their use for keratin decomposition, detergent additive applications, and collagen degradation. We observed the secretion of different proteolytic enzymes that possessed caseinolytic activity that peaked at pH 7.0-9.0 and 60-70 °C and at pH 10.5 and 55-60°C, and keratinolytic activity that reached a maximum at pH 7.0-7.5 and 40-55ºC and at pH 9.0 and 55 °C. Keratinolytic activity was maintained at approximately 63% of residual activity for 1h at 50 °C. The caseinolytic activity at pH 10.5 remains stable until 1h at 50 °C, and this is in contrast to the activity at pH 8.5, where the residual activity was 50%. Caseinolytic activity was inhibited only by PMSF, while keratinolytic activity was inhibited by PMSF and EDTA. When investigating the application of C. byrsina peptidases as an additive to commercial detergent, we observed an egg stain removal performance that was similar to that demonstrated by the commercial detergent. Based on their activity and stability at alkaline pH, these enzymes appear to be attractive candidates for use in the detergent industry. Additionally, the collagenolytic activity of these enzymes potentially allows for their use in a wide array of industrial sectors that require collagenolytic enzymes, such as for the production of collagen hydrolysates from residues derived from the meat industry.

Optimization of Biomass of Keratinase Producing Bacillus sp CBNRBT2 to Utilize in Whole-Cell Immobilization for Feather Degradation

Bacillus sp CBNRBT2, obtained from Centre for Bioscience and Nanoscience Research, was used in this study. Initially, biomass was optimized for the incubation period, pH, and temperature, following biomass optimization with RSM. The obtained organism was able to produce keratinase, which was found to be a 64 KDa protein. Now the biomass produced using the optimized condition was immobilized and checked for its ability to degrade keratinase. Efficiency was compared with the whole organism. Immobilized organisms were degrading keratin and feather better. It was found to reduce featherweight by 41 %. Thus, this immobilized bacterium can be used for poultry waste treatment.

Sustainable production, biochemical and molecular characterization of thermo-and-solvent stable alkaline serine keratinase from novel Bacillus pumilus AR57 for promising poultry solid waste management

The increasing amount of recalcitrant keratinous wastes generated from the poultry industry poses a serious threat to the environment. Keratinase have gained much attention to convert these wastes into valuable products. Ever since primitive feathers first appeared on dinosaurs, microorganisms have evolved to degrade this most recalcitrant keratin. In this study, we identified a promising keratinolytic bacterial strain for bioconversion of poultry solid wastes. A true keratinolytic bacterium was isolated from the slaughterhouse soil and was identified and designated as Bacillus pumilus AR57 by 16S rRNA sequencing. For enhanced keratinase production and rapid keratin degradation, the media components and substrate concentration were optimized through shake flask culture. White chicken feather (1% w/v) was found to be the good substrate concentration for high keratinase production when supplemented with simple medium ingredients. The biochemical characterization reveals astounding results which makes the B. pumilus AR57 keratinase as a novel and unique protease. Optimum activity of the crude enzyme was exhibited at pH 9 and 45 °C. The crude extracellular keratinase was characterized as thermo-and-solvent (DMSO) stable serine keratinase. Bacillus pumilus AR57 showed complete degradation (100%) of white chicken feather (1% w/v) within 18 h when incubated in modified minimal medium supplemented with DMSO (1% v/v) at 150 rpm at 37 °C. Keratinase from modified minimal medium supplemented with DMSO exhibits a half-life of 4 days. Whereas, keratinase from the modified minimal medium fortified with white chicken feather (1% w/v) was stable for 3 h only. Feather meal produced by B. pumilus AR57 was found to be rich in essential amino acids. Hence, we proposed B. pumilus AR57 as a potential candidate for the future application in eco-friendly bioconversion of poultry waste and the keratinase could play a pivotal role in the detergent industry. While feather meal may serve as an alternative to produce animal feed and biofertilizers.

Valorization of feather via the microbial production of multi-applicable keratinolytic enzyme

The keratinous structure of feathers is an attractive proposition for its incorporation in various biotechnological applications. In the current study, feather was used for the bacterial production of keratinase under submerged fermentation using Bacillus haynesii ALW2 with optimum productivity of 92.9U/mL. Additionally, the enzyme activity after partial purification was optimized in respect to the effects of temperature and pH in which the optimum activity was achieved at 70οC and over a pH ranged from 8 to 9. Thermal stability study indicated that the half-lives of the enzyme were 234.41, 75.24, 37.48 and 31.87 min-1 at 40, 45, 50 and 55οC, respectively. By studying the effects of different substrate concentrations, the values of Km, Vmax and Kcat were indicated as 10 mg/mL, 2500U/mg protein/min and 1.45min-1, respectively. The potential application of the partial pure enzyme in leather and detergent industry was examined. Results indicated that the enzyme achieved complete dehairing of cow-hide after 6 h incubation at 37οC in addition to its efficiency in removing protein-rich stains within 30 min at 50οC. Moreover, the enzyme activity in the degradation of feather indicated the degradation percentage of 60% with the release of a combination of amino acids as confirmed by thin layer chromatography analysis. Finally, the amount of the soluble protein was indicated to be 0.379 mg/mg dry hydrolysate with antioxidant activity of 70.8 μg Trolox Equivalents/mg.

Statistical optimisation of protease production using a freshwater bacterium Chryseobacterium cucumeris SARJS-2 for multiple industrial applications.

Feathers, rich in keratin, are usually the unused by-products of poultry industries. In addition, the cast-off X-ray films serve as secondary sources of silver, and the traditional method of silver extraction from this source is costly and not eco-friendly. Therefore, the current study focuses on protease production using a freshwater bacterium Chryseobacterium cucumeris SARJS-2, aiming to convert these wastes into useful products. The protease production was optimized by one-factor-at-a-time (OFAT), followed by Plackett Burman design (PBD) and response surface methodology (RSM). The protease production got enhanced by more than two folds after the statistical optimisation. Upon partial purification, the enzyme activity increased by approximately three folds. The protease was active in the range of temperatures from 25 to 75°C, but the optimum temperature was recorded as 35°C. The protease exhibited detergent compatibility and organic solvent stability. The detergent compatibility suggests the protease could be a detergent additive. It was also found that the presence of Fe+2 enhanced protease activity. The protease was tested for stain removal, feather degradation and silver recovery applications. It was found that the protease could efficiently remove stains of blood and tomato sauce. In addition, the protease was found to be a successful candidate for feather degradation, thereby feather-hydrolysate production which has prominent roles as nature-friendly fertilizer and animal feed ingredient. The protease also degraded gelatin from the X-ray films to release the silver-halides for silver recovery. The results recommend that the SARJS-2 protease is a potential candidate for use in eco-friendly applications in various industrial sectors.

Proteolytic bacteria isolated from agro-waste dumpsites produced keratinolytic enzymes

Microbial bioconversion of carbonoclastic materials is an efficient tool for the exploitation and valorization of underutilized agro-industrial wastes. The agro-industrial sector accumulates tones of keratinous wastes biomass which may be valorized into high value products. Consequently, the keratinolytic potentials of some bacteria isolated from terrestrial milieu was evaluated. Soil samples were collected from dumpsites, keratinase producing bacteria were isolated. Bacterial species were identified through 16S rRNA gene sequences. The keratinase activity was assessed in relation to thiol formation, percentage feather degradation and quantitation of keratinase produced. Keratinolytic bacteria were identified as Bacillus spp. (accession numbers: MG214989 – MG214992, MG214997, MG214998, MG215000, MG215002–MG215005) and Arthrobacter sp. (accession numbers; MG215001). The degree of chicken feather degradation ranged from 61.5 ± 0.71 % to 85.0 ± 1.41 %. Similarly, the activity of keratinase, total protein and thiol group ranged from 198.18 ± 15.43–731.83 ± 14.14 U/mL; 0.09 ± 0.01–0.87 ± 0.05 mg/mL; and 0.69 ± 0.12–2.89 ± 0.11 mM respectively. Notably, Bacillus sp. Nnolim-K1 displayed the best keratinolytic potential with extracellular keratinase activity and feather degradation of 731.83 ± 14.14 U/mL and 85.0 ± 1.41 % respectively, and that is an indication of a potential relevance biotechnologically.

Optimization of Keratinase Production Using Pseudomonas aeruginosa SU-1 Having Feather as Substrate

In this optimization study, Pseudomonas aeruginosa SU-1 was producing keratinase at optimal condition of 4 days, pH – 7 and temperature 37 0C, where it was producing 23.7 U/mL After the one factor at a time, RSM was performed to understand the combination of the physical parameter that ends up for the maximum production of keratinase enzyme and the degradation percentage. The study involved in three variables (pH(A), temperature(B) and Incubation Design (C)) in three ranges (-1,0,+1) using Box-Behnken Design (BBD). The results of the analysis of variance and regression analysis of the second order model showed that the factorial effect if the degradation. The optima of the variables pH - 7, temperature - 30 and incubation time – 4 days. The isolated Pseudomonas species was subjected to feather degradation for 4 days and it was degrading 55.26 %. Keratinase was to be size of 56KDa.

Degradation of chicken feathers by Aphanoascus keratinophilus and Chrysosporium tropicum strains from pellets of predatory birds and its practical aspect

The present study is the first report on the biodegradation of native keratin by fungi isolated from food pellets of birds of prey. The aim of the study was to evaluate the diversity of keratinolytic activity of 2 fungal species, Aphanoascus keratinophilus and Chrysosporium tropicum, isolated from pellets of raptors and to find a relationship between the origin of fungal strains and their keratinolytic activity. The feather weight loss ranged from 60% to 75% for A. keratinophilus and from 55% to 70% for Ch. tropicum. Strains isolated from kestrel and buzzard pellets transformed feathers after 6 weeks into a soluble protein form in the range from 11% to 17% and 12%–17%, respectively. A. keratinophilus strains transformed feather N into N–NH4+ maximally in 53% and minimally in 20%, whereas Ch. tropicum strains converted from approx. 3%–42%. Feather S was transferred to the medium as S–SO42- after 42 days in 74% by A. keratinophilus and from 2% to 68% by Ch. tropicum strains. The effectiveness of fungal strains isolated from buzzard and kestrel pellets, was generally higher than that of strains isolated from owl pellets. The passage of the tested fungal strains through the gastric barrier and their thermo-tolerance allow a conclusion that pellet-derived keratinolytic fungi (especially from diurnal raptors) are a suitable inoculum material for keratin waste composting.

The feather degradation mechanisms of a new Streptomyces sp. isolate SCUT-3

Feather waste is the highest protein-containing resource in nature and is poorly reused. Bioconversion is widely accepted as a low-cost and environmentally benign process, but limited by the availability of safe and highly efficient feather degrading bacteria (FDB) for its industrial-scale fermentation. Excessive focuses on keratinase and limited knowledge of other factors have hindered complete understanding of the mechanisms employed by FDB to utilize feathers and feather cycling in the biosphere. Streptomyces sp. SCUT-3 can efficiently degrade feather to products with high amino acid content, useful as a nutrition source for animals, plants and microorganisms. Using multiple omics and other techniques, we reveal how SCUT-3 turns on its feather utilization machinery, including its colonization, reducing agent and protease secretion, peptide/amino acid importation and metabolism, oxygen consumption and iron uptake, spore formation and resuscitation, and so on. This study would shed light on the feather utilization mechanisms of FDBs.

Bacillus sp. FPF-1 Produced Keratinase with High Potential for Chicken Feather Degradation.

Chicken feathers are predominantly composed of keratin; hence, valorizing the wastes becomes an imperative. In view of this, we isolated keratinase-producing bacteria and identified them through the 16S rDNA sequence. The process condition for keratinase activity was optimized, and electron micrography of the degradation timelines was determined. Keratinolytic bacteria were isolated and identified as Bacillus sp. FPF-1, Chryseobacterium sp. FPF-8, Brevibacillus sp. Nnolim-K2, Brevibacillus sp. FPF-12 and Brevibacillus sp. FSS-1; and their respective nucleotide sequences were deposited in GenBank, with the accession numbers MG214993, MG214994, MG214995, MG214996 and MG214999. The degree of feather degradation and keratinase concentration among the isolates ranged from 62.5 ± 2.12 to 86.0 ± 1.41(%) and 214.55 ± 5.14 to 440.01 ± 20.57 (U/mL), respectively. In the same vein, 0.1% (w/v) xylose, 0.5% (w/v) chicken feather, an initial fermentation pH of 5.0, fermentation temperature of 25 °C and an agitation speed of 150 rpm, respectively, served as the optimal physicochemical conditions for keratinase activity by Bacillus sp. FPF-1. The time course showed that Bacillus sp. FPF-1 yielded a keratinase concentration of 1698.18 ± 53.99(U/mL) at 120 h. The electron microscopic imaging showed completely structural dismemberment of intact chicken feather. Bacillus sp. FPF-1 holds great potential in the valorization of recalcitrant keratinous biomass from the agro sector into useful products.

Efficient keratinase expression via promoter engineering strategies for degradation of feather wastes

Keratinases are promising alternatives over ordinary proteases in several industrial applications due to their unique properties compared with their counterparts in the protease categories. However, their large-scale industrial application is limited by the low expression and poor fermentation efficiency of keratinase. Here, we demonstrate that the expression level of keratinase can be improved by constructing a more efficient enzyme expression system hereby enables the highest production titer as regarding recombinant keratinase production to date. Specially, ten promoters were evaluated and the aprE promoter exhibits a significant promotion of keratinase (kerBv) titer from 165 U/mL to 2605 U/mL in Bacillus subtilis. The batch fermentation mode resulted in a maximum keratinase activity of 7176 U/mL at 36 h in a 5-L fermenter. Furthermore, the extracellular keratinase activity attained up to 16,860 U/mL via fed-batch fermentation within 30 h. The combination of keratinase with l-cysteine brings about 66.4 % degree of degradation of feather. Our work provides a new insight into the development of efficient keratinase fermentation processes with B. subtilis cell factory.