Feather Degradation Research Articles

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.

In vitro synergistic effects of three enzymes from Bacillus subtilis CH-1 on keratin decomposition.

Extracellular protease Vpr (Vpr), gamma-glutamyltranspeptidase (GGT; EC 2.3.2.2) and glyoxal/methylglyoxal reductase (YvgN; EC 1.1.1.21) are extracellular enzymes involved in feather degradation, which were identified by secretome analyses from an efficient feather-degrading strain Bacillus subtilis CH-1. The encoding sequences corresponding to the three secretory enzymes were cloned into vector pET22b for recombinant expression in Escherichia coli strain BL21 (DE3). Afterward, the proteins containing the C-terminal His-tag were purified using a Ni-IDA column. The optimal temperatures and pH values for protease activity of recombinant Vpr, GGT, and YvgN were identified as 45°C/pH 7.0, 40°C/pH 8.0, and 50°C/pH 6.0 respectively when casein is the substrate. Furthermore, the synergistic effects of the three enzymes were studied using feather powder as substrate. Vpr was the core enzyme to hydrolyze keratin, while GGT and YvgN were coenzymes providing reducing activities for keratin decomposition. The keratinolytic activity was enhanced to about 1.4-folds when YvgN and Vpr applied together in comparison to Vpr alone. And the keratinolytic activity almost reached to 1.5-folds when all the three enzymes were combined to use. The study provides a novel perspective of the mechanism of keratin degradation by microorganisms, and thereby may also be of relevance for the design of an industrial process for enzymatic keratin degradation; however, additional experiments must be done to substantiate this conclusion.

Progress in Microbial Degradation of Feather Waste.

Feathers are a major by-product of the poultry industry. They are mainly composed of keratins which have wide applications in different fields. Due to the increasing production of feathers from poultry industries, the untreated feathers could become pollutants because of their resistance to protease degradation. Feathers are rich in amino acids, which makes them a valuable source for fertilizer and animal feeds. Numerous bacteria and fungi exhibited capabilities to degrade chicken feathers by secreting enzymes such as keratinases, and accumulated evidence shows that feather-containing wastes can be converted into value-added products. This review summarizes recent progress in microbial degradation of feathers, structures of keratinases, feather application, and microorganisms that are able to secrete keratinase. In addition, the enzymes critical for keratin degradation and their mechanism of action are discussed. We also proposed the strategy that can be utilized for feather degradation. Based on the accumulated studies, microbial degradation of feathers has great potential to convert them into various products such as biofertilizer and animal feeds.

CHARACTERIZATION AND OPTIMIZATION OF KERATINOLYTIC BACTERIA Pseudomonas stutzeri C3 ISOLATED FROM POULTRY WASTE SOIL SAMPLE

Microbial degradation of keratinous wastes is preferred over physicochemical methods, because it’s a cost effective and eco-friendly. Novel habitats are promising for discovery of new microbial strains. Towards isolate a keratinolytic bacteria, screening of bacterial strains from a poultry farm soil in Namakal district, Tamil Nadu, India was done and a promising the isolate C3 was found to degrade native chicken feather efficiently. It could grow over a pH range 7 at 40°C and in presence of potassium nitrate as a nitrogen source and citrate as a carbon source. Based on phenotypic characterization and 16S rRNA gene sequence analysis, the keratinolytic isolate was identified as Pseudomonas stutzeri C3. It produced 30.86±1.08 U/ml keratinase in raw feather medium and complete feather degradation at 5 days incubation periods.

Microbial degradation of poultry feather biomass in a constructed bioreactor and application of hydrolysate as bioenhancer to vegetable crops.

Bioconversion of recalcitrant keratinous biomass is one of the greatest ways to utilize products of feather hydrolysis and recycle them into bionetwork. Present study revealed 87% degradation of poultry feathers within 48 h in a constructed bioreactor using Chryseobacterium sp. RBT. The resulting feather hydrolysate (FH) was rich in soluble protein (3.56 ± 0.18 mg/ml), amino acids (3.83 ± 0.20 mg/ml), and macro and micro nutrients like N (8.0302%), P (0.3876%), K (0.5532%), Cu (0.0684%), Mg (0.8078%), Mn (0.2001%), Ca (0.4832%), Zn (0.0442%), and Fe (0.0330%). HPTLC analysis of FH revealed presence of tryptophan, cysteine, methionine, phenylalanine, glycine, valine, tyrosine, lysine, leucine, and serine as the primary amino acids. Field studies were conducted to apply FH as the bioenhancer to commercially important crops like brinjal and chilli through root drenching (20%, v/v). FH showed positive impact on the growth and development of plants along with early flowering and improved crop yield. In addition, nutritional quality of brinjal and chilli in terms of protein, amino acids, reducing sugars, phenolics, flavonoids, and antioxidant was elevated. Therefore, promotion and utility of by-products generated in feather degradation would be an effective strategy focusing on sustainable agricultural practices and problems associated with the waste management.

Improving the catalytic performance of Proteinase K from Parengyodontium album for use in feather degradation

Proteinase K (PROK) from Parengyodontium album hydrolyzes keratin, a major protein component of poultry feathers, which are an inexpensive and renewable protein resource. Based on structural studies for analysis of amino acid flexibility near the catalytic center, identification of highly conserved residues, and experimental screening, we obtained a mutant R218S with residual activity 1.6-fold higher than that of PROK after incubation at 60 °C for 1 h. Molecular dynamics simulation indicated that substitution of Arg218 with Ser leads to three hydrogen bonds being introduced into the structure, stabilizing the β-sheet in which Ser218 is located, and thus improvement of thermostability. Additionally, the mutant R218S had a 15% increase in specific activity compared to PROK and improvement in the rate and thoroughness of feather degradation compared with PROK. We confirmed the positive effects of enhancing catalytic center rigidity on enzyme thermostability, a finding which may have broad applications.

Microbial Degradation of Poultry Feather Wastes under the Influence of Temperature and pH – A Review

Poultry feathers require different approaches of disposal. Feather waste represents a huge resource of protein and the rational utilization of millions of tons of waste feathers will not only ease the shortage of protein resources but will also improves the environment. Conventional methods used for disposing or converting them to protein hydrolysates such as land filling, incineration, burning or the use of chemicals are costly, not environmentally friendly, and pose a danger to living organisms. Microbial hydrolysis is considered an alternative environmental-friendly method for recycling. As physiological and nutritional factors greatly affect feather degradation and keratinase production, the effects of various factors such as incubation temperature and pH on feather degradation by various microorganisms were reviewed in this study.

Selective biodegradation of recalcitrant black chicken feathers by a newly isolated thermotolerant bacterium Pseudochrobactrum sp. IY-BUK1 for enhanced production of keratinase and protein-rich hydrolysates.

Black chicken feathers generated in large amount from poultry and slaughter houses are highly recalcitrant to microbial degradation due to their tough structural nature. A novel keratinolytic bacterium that possessed high affinity for black feather was isolated from chicken manure and identified as Pseudochrobactrum sp. IY-BUK1. Keratinase and feather soluble protein were effectively produced by the free living cells of the bacterium in media containing only black feathers and a mixture of equal amount of black-, brown- and white-coloured feathers. Complete degradation of 5g/L of black feathers was completed in 3days following optimisation of physico-chemical conditions. However, the bacterium selectively completed the degradation of black feather in a medium containing mixture of feathers in 144h leaving behind approximately 33% and 45% of brown and white feathers in the medium respectively. Gellan gum-immobilised cells of strain IY-BUK1 enhanced the keratinase production by about 150% and were used repeatedly for ten cycles to degrade 5g/L of black feather in a semi continuous fermentation of 18h per cycle with enhanced and stable production of soluble protein. The study demonstrated the potential use of Pseudochrobactrum sp. IY-BUK1 not only in biodegradation of highly recalcitrant black feathers, but also in producing keratinase enzymes and valuable soluble proteins for possible industrial usage.

Effective biodegradation of chicken feather waste by co-cultivation of keratinase producing strains

BackgroundChicken feather, a byproduct of poultry-processing industries, are considered a potential high-quality protein supplement owing to their crude protein content of more than 85%. Nonetheless, chicken feathers have been classified as waste because of the lack of effective recycling methods. In our previous studies, Bacillus licheniformis BBE11-1 and Stenotrophomonas maltophilia BBE11-1 have been shown to have feather-degrading capabilities in the qualitative phase. To efficiently recycle chicken feather waste, in this study, we investigated the characteristics of feather degradation by B. licheniformis BBE11-1 and S. maltophilia BBE11-1. In addition, in an analysis of the respective advantages of the two degradation systems, cocultivation was found to improve the efficiency of chicken feather waste degradation.ResultsB. licheniformis BBE11-1 and S. maltophilia BBE11-1 were used to degrade 50 g/L chicken feather waste in batches, and the degradation rates were 35.4% and 22.8% in 96 h, respectively. The degradation rate of the coculture system reached 55.2% because of higher keratinase and protease activities. Furthermore, cocultivation was conducted in a 3 L fermenter by integrating dissolved oxygen control and a two-stage temperature control strategy. Thus, the degradation rate was greatly increased to 81.8%, and the conversion rate was 70.0% in 48 h. The hydrolysates exhibited antioxidant activity and contained large quantities of amino acids (895.89 mg/L) and soluble peptides.ConclusionsCocultivation of B. licheniformis BBE11-1 and S. maltophilia BBE11-1 can efficiently degrade 50 g/L chicken feather waste and produce large amounts of amino acids and antioxidant substances at a conversion rate of 70.0%.

Cosmetic colouring by Bearded Vultures Gypaetus barbatus: still no evidence for an antibacterial function.

Bearded Vultures regularly visit ferruginous springs for cosmetic purposes to obtain their reddish plumage colouration. Different hypotheses have been proposed to explain this deliberate application of adventitious colouration: (1) to signal individual dominance status; (2) to exploit an anti-bacterial effect of iron oxides or ochre to reduce feather degradation by bacteria and, in parallel (3) to enable incubating birds to transfer this protection to their developing embryos to increase hatching success. Here, we re-evaluate the antibacterial hypothesis using three experimental approaches: (a) by applying feather-degrading bacteria to stained and unstained bearded vulture feathers; (b) by assessing the antibacterial activity of ochre; and (c) by comparing the breeding success of orange individuals with pale ones. Our findings suggest that the in vitro addition of feather degrading Bacillus licheniformis to naturally stained Bearded Vulture feathers did not retard feather degradation compared to controls. Iron particles from red soil (ochre) or iron salts had no antibacterial effect on the growth of three species of bacteria (Escherichia coli, Kocuria rhizophila and Bacillus licheniformis), incubated either in the dark or under visible light. Finally, breeding success did not differ between territories occupied by pale individuals versus orange ones. These results run counter to the hypothesis that iron oxides have an antibacterial role in Bearded Vultures. The use of red soils by Bearded Vultures may function as a territorial status signal, but may also be involved in other processes, such as pair formation and the long-term maintenance of the pair bond, as suggested for the closely related Egyptian vulture Neophron percnopterus.

Enhanced stability of immobilized keratinolytic protease on electrospun nanofibers

Feathers from poultry industries are considered a major pollutant and its degradation is a challenging problem due to its recalcitrant nature. The high cost of energy and loss of essential amino acids by conventional methods have paved a way for an environmentally benign approach using microbial keratinolytic proteases. The widespread application of keratinolytic proteases is limited due to autolysis and denaturation of the enzyme upon storage. Immobilization overcomes these disadvantages by adsorbing the enzyme onto a solid support. Recently, electrospun nanofibers have been used due to their increased surface area and porous structure. The biocompatible and hydrophilic polyvinyl alcohol (PVA) has been blended with biodegradable chitosan for immobilization in electrospinning. The present study focuses on feather degradation by immobilized keratinolytic proteases on electrospun nanofibers. The keratinolytic protease production was enhanced by using a media containing hydrolyzed feather under optimized conditions. The immobilized keratinolytic protease on electrospun PVA chitosan (PVA-Ch) nanofibers (100–150 nm diameter) degraded the chicken feathers with 88% efficiency at the end of 72 hr.

A comparative study about decomposable ability of two thermophilic bacteria to keratin

Keratin is a refractory protein which widely exists as animal hairs, feathers and so on in nature. Because the structure of keratin becomes loose and easy to be degraded at high temperatures, thermophilic bacteria and keratinases have attracted extensive attention. Bacillus sp. WF146 and Thermoactinomyces sp. CDF are two thermophilic bacteria isolated from soil in our previous studies. They can both degrade keratin well. By changing the nitrogen source in the medium to cause nutritional stress, and monitoring protease degradation of feather substrate, we found that the expressed protease is the key to the degradation of keratin. The results showed that there were some differences in the feather degradation process of two strains, they may related to damaging disulphide bond of keratin with reducing power provided by the cells, and physics destroy the structure of feather. That is what led to their different feather-degrading characteristics.

Screening and Characterisation of Keratin-Degrading Bacillus sp. from Feather Waste

Aim: This study focuses on the screening and characterisation of keratin-degrading Bacillus species from feather waste.
 Methods: Nine bacteria were isolated from feather waste obtained from a poultry layout at Egbeda local government secretariat, Ibadan, Nigeria. These bacteria were grown in basal medium with feather as primary source of carbon, nitrogen, sulfur and energy. Feather degrading bacteria were screened for both proteolytic activity and keratin degradation on skimmed milk agar and keratin azure medium respectively. They were also screened for their ability to degrade other keratin substrates such as hair and nail.
 Results: Three of the isolates with higher feather degradation levels also showed high proteolytic activity and release of azure dye. They were selected and identified phenotypically and genotypically using 16S rRNA sequencing as Bacillus licheniformis-K51, Bacillus subtilis-K50 and Bacillus sp.-K53. The bacteria were capable of degrading other keratin-containing substrates such as nail and hair. Bacillus subtilis-K50 and Bacillus licheniformis-K51 showed significant difference (P) in degradation among the three different keratin sources used yielding higher degradation with feather as keratin source with respective optical densities of 0.07 and 0.11 followed by hair and least in nails with optical densities of 0.05 and 0.07 respectively. Highest degradation of all the three keratin substrates was observed in Bacillus licheniformis-K51.
 Conclusion: The three isolated bacteria possess the ability to degrade keratin and utilize feather as keratin substrate. As a result, these can be considered as potential candidates for degradation and utilization of feather keratin.

Synthesis of Keratin Nanoparticle and Characterization using FTIR

Chicken feathers consists of more amount of proteins in it. Each year in poultry farms the chicken feathers are wasted and dumped into the environment, because degradation of chicken feathers is more complicated to do, even though some ways like burying and burning are available to degrade the chicken feather it is not environmental friendly and because of this the environment gets polluted. Keratin is a group of fibrous protein and has many useful applications. Considering this advantage, the wasted chicken feathers are collected and the keratin is extracted using sodium sulfite as the solvent. The keratin extraction is confirmed by biuret test. The characterization studies are done using FTIR analysis. Further the keratin nanoparticles are synthesized using Silver Nitrate.

Keratinolytic activity of Bacillus subtilis LFB-FIOCRUZ 1266 enhanced by whole-cell mutagenesis.

Discarded feathers represent an important residue from the poultry industry and are a rich source of keratin. Bacillus subtilis LFB-FIOCRUZ 1266, previously isolated from industrial poultry wastes, was used in this work and, through random mutation using ethyl methanesulfonate, ten strains were selected based on the size of their degradation halos. The feather degradation was increased to 115% and all selected mutants showed 1.4- to 2.4-fold increase in keratinolytic activity compared to their wild-type counterparts. The protein concentrations in the culture supernatants increased approximately 2.5 times, as a result of feather degradation. The mutants produced more sulfide than the wild-type bacteria that produced 0.45µg/ml, while mutant D8 produced 1.45µg/ml. The best pH for enzyme production and feather degradation was pH 8. Zymography showed differences in the intensity and molecular mass of some bands. The peptidase activity of the enzyme blend was predominantly inhibited by PMSF and EDTA, suggesting the presence of serine peptidases. HPTLC analysis evidenced few differences in band intensities of the amino acid profiles produced by the mutant peptidase activities. The mutants showed an increase in keratinolytic and peptidase activities, demonstrating their biotechnological potential to recycle feather and help to reduce the environmental impact.

Strategical isolation of efficient chicken feather-degrading bacterial strains from tea plantation soil sample.

Chicken feather waste is generally insufficiently utilized despite its high content of protein, constituting an environmental issue. Biodegradation of the waste with enabling microbes provides an advantageous option among the available solutions. In this study, an efficient whole feather-degrading strain was strategically isolated from a soil sample taken from a local tea plantation that has little or nothing to do with feathers. The strain was identified as Bacillus thuringiensis (designated as FDB-10) according to the cloned complete 16S rRNA sequence. The FDB-10 could efficiently degrade briefly heat-treated whole feather (102°C, 5min; up to 90% of a maximum concentration of 30g/L) in a salt medium supplemented with 0.1g/L yeast extract within 24h (37°C, 150rpm). Addition of carbon sources (glycerol, glucose, starch, Tween 20, Tween 80, 1.25g/L as glycerol) to the fermentation medium could improve the degradation. However, significant inhibition could be observed when the added carbon source reached the amount usually adopted in the investigation of carbon source preference (1%). Nitrogen source (NH4Cl, (NH4)2SO4, peptone) adversely influenced the performance of the strain. When the molar concentrations of NH4+ were equal for the two salt, the inhibitory effect on degradation of whole feathers was similar. Entirely different from other reported feather-degrading strains showing a preference to melanin-free feather substrates, the strain isolated in this study could degrade melanin-containing feather equally efficiently, and higher protease activity could be detected in the digest mix. As a plus, the strain could degrade feathers in rice wash produced in daily cooking, indicating its potential use in the simultaneous treatment of rice cooker wastewater produced by a rice processing plant. All these results imply that the FDB-10 is a strain with great potential in the biodegradation of feather waste.

Feather degradation by keratinolytic bacteria and biofertilizing potential for sustainable agricultural production.

Feathers account for 5-7% of the total weight of chicken have become one of the major pollutants due to their recalcitrant nature. Feather which is constituted of 90% keratin can be a good source of peptides, amino acids, and minerals for use as organic fertilizer. Traditional feather degradation methods consume large amount of energy and reduces the overall quality of the proteins. However, degradation of keratin by keratinolytic bacteria may represent as an alternative for the development of cheap, cost effective, eco-friendly, and easily available nitrogen (N) and minerals rich source as potential organic fertilizers. Keratinase enzymes from bacteria are serine-type proteases showing optimal activity at pH 6 to 9 and 30 to 50 °C. Mechanism of degradation includes, sulfitolysis, proteolysis, followed by deamination. Keratinolytic bacteria showing antagonism against important plant pathogens may act as biocontrol agent. Feather hydrolyzate can also be employed as nitrogenous fertilizers for plant growth. Tryptophan release from the feather degradation can act as precursor for plant phytohormone, indole-3-acetic acid (IAA). Solubilization of inorganic phosphate (P) by keratinolytic bacteria may further elevate the growth of plant. Application of hydrolyzate increases the water holding capacity, N, carbon (C) and mineral content of the soil. It elevates protein, amino acids, and chlorophyll content of plant. Feather hydrolyzate enhances seed germination and growth of plant. Soil application further increases the population of beneficial bacteria. The use of keratinolytic bacteria having antagonistic and plant growth promoting activities, and feather hydrolyzate can emerge as sustainable and alternative tools to promote and improve organic farming, agro-ecosystem, environment, human health, and soil biological activities.

Isolation of a feather-degrading strain of bacterium from spider gut and the purification and identification of its three key enzymes.

A novel feather-degrading bacterium named CA-1 was isolated from the gut of the spider Chilobrachys guangxiensis, which degrades native whole chicken feathers within 20h. The CA-1 was confirmed to belong to Stenotrophomonas maltophilia based on morphologic and molecular analysis. Maximum feather degradation activity of the bacterium was observed at 37°C in basal feather medium (NaCl 0.5g/L, KH2PO4 0.3g/L, K2HPO4 0.4g/L, feather powder 10.0g/L, pH 8.0), which was inhibited when glucose and ammonium nitrate were added in the medium. Furthermore, the purified enzymes under the optimal and suppressive conditions were analyzed respectively by SDS-PAGE and LC-MS/MS. Three enzymes, namely alkaline serine protease (29.1kDa), ABC transporter permease (27.5kDa), and alkaline phosphatase (40.8kDa), were isolated and identified from the supernatant of the optimal culture and were considered to play principal roles. On the other hand, the potential synergic effects of the three proteins in S. maltophilia CA-1 feather degradation system were analyzed theoretically. CA-1 may product outer-membrane vesicles comprised of membranes and periplasmic proteins in the feather medium. The newly identified CA-1 and its synergic enzymes provide a new insight into further understanding the molecular mechanism of feather degradation by microbes. They also have potential application in cost-effectively degrading feathers into feeds and fertilizers through careful optimization and engineering of the three newly identified enzymes.

Bioconversion of chicken feathers by Bacillus aerius NSMk2: A potential approach in poultry waste management

About 73 bacterial strains were isolated from poultry dump sites in Punjab and an isolate showing high keratinolytic activity was identified as Bacillus aerius NSMk2. Strain showed prompt hydrolysis of white chicken feathers on minimal salt medium (MSM) after 5 days of cultivation. Melanized chicken, pigeon and duck feathers could also be degraded by bacteria with high activity to duck and pigeon feathers. MSM supplemented with fructose and beef extract supported higher feather keratinase production and improved degradation of white chicken feathers. Feather concentration 1.375%, pH 7.5 and temperature 35 °C showed maximum feather keratinase activity (127.63 U/ml) after 2 days which was 7-fold higher than activity on minimal salt medium. Time for complete degradation of white chicken feathers was reduced to 3 days on optimized medium. The results of present investigation showing potential keratinolytic ability of Bacillus aerius NSMk2 could provide an environmental friendly approach for bioconversion of poultry waste.

Production of Keratinase from a Newly Isolated Feather Degrading Bacillus cereus VITSDVM4 from Poultry Waste

A new feather-degrading bacterium was isolated from soil near poultry waste. Based on the morphological, physiochemical, and phylogenetic characteristics, the keratinolytic bacteria was identified as Bacillus cereus VITSDVM4 (Accession number KJ725087).To enhance the yield of keratinase, the effect of various parameters such as pH, temperature, carbon source, nitrogen source, substrate concentration, inoculum size and incubation time were optimized. The maximum keratinase activity was found to be 134 U ml−1 at pH 7. At 25 °C, it was slightly increased and showed 174 U ml−1 activity. The carbon source, sucrose revealed the maximum enzyme activity of 226 U ml−1. Yeast extract was found to be the altered nitrogen source in the production medium with 271 U ml−1 of enzyme activity. 10 mg of feathers as substrate, 3% of the microbial inoculum and 72 h of incubation enhanced the enzyme production and exhibited a maximum activity of 378, 408 and 453 U ml−1 respectively. The partially purified keratinase showed maximum specific activity of 528.7 U mg−1 as well as visible degradation of feather, hair and nail after 48 h of incubation confirms the keratinolytic activity.