Transforming Chicken Feather Waste into Feather

1 downloads 0 Views 3MB Size Report
1 3. Waste Biomass Valor. DOI 10.1007/s12649-017-0037-4. ORIGINAL PAPER. Transforming Chicken Feather Waste into Feather Protein. Hydrolysate Using a ...

Transforming Chicken Feather Waste into Feather Protein Hydrolysate Using a Newly Isolated Multifaceted Keratinolytic Bacterium Chryseobacterium sediminis RCM-SSR-7 Pintubala Kshetri, Subhra Saikat Roy, Susheel Kumar Sharma, Thangjam Surchandra Singh, Meraj Alam Ansari, Narendra Prakash, et al. Waste and Biomass Valorization ISSN 1877-2641 Waste Biomass Valor DOI 10.1007/s12649-017-0037-4

1 23

Your article is protected by copyright and all rights are held exclusively by Springer Science+Business Media B.V.. This e-offprint is for personal use only and shall not be selfarchived in electronic repositories. If you wish to self-archive your article, please use the accepted manuscript version for posting on your own website. You may further deposit the accepted manuscript version in any repository, provided it is only made publicly available 12 months after official publication or later and provided acknowledgement is given to the original source of publication and a link is inserted to the published article on Springer's website. The link must be accompanied by the following text: "The final publication is available at link.springer.com”.

1 23

Author's personal copy Waste Biomass Valor DOI 10.1007/s12649-017-0037-4

ORIGINAL PAPER

Transforming Chicken Feather Waste into Feather Protein Hydrolysate Using a Newly Isolated Multifaceted Keratinolytic Bacterium Chryseobacterium sediminis RCM-SSR-7 Pintubala Kshetri1 · Subhra Saikat Roy1   · Susheel Kumar Sharma1 · Thangjam Surchandra Singh1 · Meraj Alam Ansari1 · Narendra Prakash1 · S. V. Ngachan2 

Received: 3 May 2017 / Accepted: 28 July 2017 © Springer Science+Business Media B.V. 2017

Abstract  Accumulation of feather waste is becoming a major issue in solid waste management. Towards discovery of keratinolytic bacteria, screening of bacterial strains from feather dumping sites in North East, India was performed and 26 keratinolytic bacterial strains were isolated. Out of these, one isolate RCM-SSR-7 was found to be most promising strain exhibiting feather degradation as well as antioxidant and indole-3-acetic acid production. The strain was identified as Chryseobacterium sediminis RCM-SSR-7. The strain could use chicken feather as sole carbon and nitrogen source for growth. Three parameters (feather concentration, pH and incubation time) were studied to optimize feather protein hydrolysate (FPH) preparation using response surface methodology (RSM). The optimum condition for FPH preparation was achieved at 5% (w/v) feather concentration, pH 7.5, 30 °C and 84 h incubation time upon optimization by RSM. FPH was found to be rich in essential amino acids and trace elements (phosphorous, potassium, calcium, and iron). FPH exhibited radical scavenging activity with an ­IC50 value of 0.102 mg ml−1. In vitro digestibility showed that FPH is 86% digestible with pepsin and trypsin treatment. This study revealed that FPH produced by C. sediminis RCM-SSR-7 has the potential to be used as animal feed and organic fertilizer.

* Subhra Saikat Roy [email protected] 1

ICAR Research Complex for NEH Region, Manipur Centre, Imphal 795004, India

2

ICAR Research Complex for NEH Region, Umiam, Meghalaya, India



Keywords  Chryseobacterium sediminis · Keratinolytic bacteria · Feather degradation · Feather protein hydrolysate · Antioxidant · Indole-3-acetic acid

Introduction Feathers are the major waste by-product of poultry industry and several million tons of feather wastes are produced annually worldwide [1, 2]. Today, such wastes form a problematic issue in solid waste management as they are hard to degrade due to the highly rigid structures of complex protein rendered by extensive disulfide bonds and crosslinkages [3]. As 90% of feather weight is pure keratin, feather waste represents a good source of nitrogen, protein and amino acids. Towards valorization of this waste product into animal feed and fertilizer, many researchers have adopted different methods of processing. Conventionally, feather meal was prepared by using harsh physical and chemical treatments [4, 5]. These processes not only require significant energy inputs but also destroy certain essential amino acids such as methionine, lysine and tryptophan [6]. Limitations to feather utilization as animal feed arise from its poor digestibility and minimal biological value due to the deficiencies of nutritionally essential amino acids, such as methionine, lysine, histidine and tryptophan [7]. Nutritional enhancement of feathers can be achieved by hydrolysis with feather-degrading microorganisms [8, 9]. Hence, hydrolysis of keratinous wastes by microorganisms possessing keratinolytic activity presented an attractive alternative method for the efficient bioconversion of feather into animal feed. The relationship between nutrition and animal health has long been recognized. Nutritional deficiencies increase morbidity and mortality which can be explained partly by impaired immune responsiveness [10].

13

Vol.:(0123456789)

Author's personal copy

Waste Biomass Valor

Among many dietary factors, natural antioxidants have special importance in animal health. It protects the body from free radicals either by directly scavenging free radicals or by inhibiting the activity of oxidizing enzymes. Recently, hydrolysed proteins from many animal and plant sources including feather meal have been reported to possess antioxidant activity [11, 12]. Another simplest and most appropriate application of recycled feather wastes is as cheap soil amendments and fertilizers. Feather hydrolysates have been reported to act as slow-release N fertilizers [13, 14]. Recently researchers are focused on use of microbes having both feather degradation and plant growth promoting activity as biofertilizer [15, 16]. Present study aimed at isolating native feather degrading microorganisms from the soil habitats of North East (NE) India and preparation of feather protein hydrolysate using most promising strain. A multifaceted Chryseobacterium sediminis RCM-SSR-7 strain isolated in present study was found more efficient in biodegradation of chicken feather. To the best of our knowledge this is the first report of a rapid feather degrading bacterium C. sediminis RCM-SSR-7 from North Eastern India, one of the Mega Biodiversity Hotspots in the world. The results of this study should be useful in wide array of fields such as organic crop production, soil enrichment, manufacturing of animal feed additives and amino acids.

albumin (BSA) as standard. The most promising strain was selected for further studies.

Materials and Methods

The factors studied included initial pH of medium (6.0–9.5) adjusted by 1  M HCl or 1  M NaOH, incubation temperatures (20–35 °C), feather concentration (1–12%) and incubation time (12–84 h).

Isolation and Screening of Keratinolytic Bacteria The soil sample was collected from feather waste dumping sites and air-dried for a week. 1 g finely powdered soil sample was dissolved in 100 ml sterile distilled water and kept incubated in a shaker (25 °C, 150 rev min−1, 30 min). The soil suspension was serially diluted ­(10−3–10−7) and 0.1 ml diluted soil suspension was spread plated on nutrient agar plates and were kept incubated at 30 °C for a week. Morphologically distinct bacterial colonies were selected and subculture till pure cultures was obtained. The purified cultures were preserved as slants at 4 °C and as glycerol stocks (20%, v/v) at −20  °C. Bacterial strains were screened for feather degradation in chicken feather medium (CFM) containing chicken feather, 1% (w/v); ­ KH2PO4, 0.1% (w/v); K ­ 2HPO4, 0.3% (w/v) and ­MgSO4, 0.02% (w/v), pH 7. One loopful of bacterial culture was inoculated in to the CFM medium and incubated at 30 °C for 48 h, 200 rev m−1. Extent of feather degradation was studied by monitoring soluble peptide production and feather weight loss. Feather weight loss was measured according to Kshetri and Ningthoujam [17]. Soluble peptide content was determined according to Lowry method [18] using bovine serum

13

Identification of Bacteria The strain RCM-SSR-7 was identified on the basis of morphological descriptors [19] and 16S rRNA gene sequence analysis. The 16S rRNA gene was amplified using the primers 27F (5-AGA​GTT​TGATCMTGG​CTC​AG-3) and 1492R (5-CGG​TTA​CCT​TTG​TTA​CGA​CTT-3). The PCR mixture contained 12.5 µl 2X Mastermix (GCC, Biotech India), 1 µl of each primer (100  µM) and the final volume was made up to 25  µl with deionized water. PCR was carried out with initial denaturation at 94 °C for 5 min followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 55 °C for 30  s, extension at 72  °C for 90  s and final extension at 72  °C for 10  min. The amplified 16S rRNA gene was sent for sequencing (1st Base Sequencing service, Malaysia). The partial 16S rRNA gene sequence of the strain was identified using the EzTaxon-e server database [20] and NCBI GenBank databases and aligned with the 16S rRNA gene sequences of other related species using CLUSTAL X v2.1. Phylogenetic analyses were performed using the software package MEGA v5.2 [21]. Sequence was deposited under NCBI GenBank Accession No. KX776189. Factors Affecting Feather Degradation

Optimization of Feather Degradation by Response Surface Methodology (RSM) Optimization of feather degradation was performed using RSM. Three parameters viz. feather concentrations, initial pH and incubation time were studied at five levels (−α, −1, 0, +1, +α) using Central Composite Design (CCD). The experimental levels of the three parameters used in RSM in terms of actual and coded forms are listed in Table  1. A set of 20 experiments with six replicates at the center point was generated using Design Expert 6.0.1 Software. A multiple regression analysis of the data was carried out to obtain an empirical model that defines the response (soluble peptide production) in terms of the independent variables. The model equation is represented as:

Y = β0 +



βi xi +



βi x2i +



βij xi xj

where, Y is the predicted response, β0 is the intercept, βi is the linear coefficient and βij is the interaction coefficient.

Author's personal copy Waste Biomass Valor Table 1  Experimental ranges of four different variables used in CCD

Run

1 2 3

Variables

Range coding

Incubation time (h) pH Feather concentration (%)

Analysis of variance (ANOVA) of the CCD was then analyzed. Indole‑3‑acetic Acid (IAA) Production For quantitative assay of IAA production, strains were allowed to grow on chicken feather medium (CFM) with or without tryptophan (2  mg  ml−1) supplementation and incubated in a shaker (150  rev  min−1, 30  °C). 5  ml aliquot was withdrawn periodically from each culture flask at 24  h intervals, centrifuged (8000×g, 10  min) and 1  ml of the supernatant was mixed with 2 ml of Salkowski reagent. Solutions were kept for 20 min at room temperature. Absorbance was measured at 530  nm and the amount of IAA produced was calculated by comparing with the standard IAA curve [22]. Antioxidant Activity DPPH (2,2-diphenyl-1-picrylhydrazyl) radical-scavenging activity of the hydrolysates was determined as described by Thaipong et al. [23]. A volume of 100 µl of sample at different concentrations was reacted with 1.9 ml of DPPH in methanol ­(A517 = 1.1 ± 0.02). The mixture was then kept at room temperature in the dark for 60 min, and the reduction of DPPH radical was measured at 517 nm using a UV–visible spectrophotometer. The percentage inhibition of the DPPH radical (scavenging activity) was calculated according to the formula:

Scavanging activity (%) = [Ac − As ÷ Ac] × 100 where Ac is the absorbance of the control reaction and As is the absorbance of the sample. Sample concentration providing 50% inhibition ­(IC50) was calculated from the graph plotting inhibition percentage against feather protein hydrolysate (FPH) concentration. A lower absorbance of the reaction mixture indicated a higher DPPH radical-scavenging activity. Ascorbic acid was used as positive control. The test was carried out in triplicate. Preparation of FPH The bacterium was grown in raw feather medium containing 50 g l−1 feathers (pH 7.5). The fermentation was carried out for 84 h at 30 °C under agitation in an orbital

−α (−1.68)

−1

0

+1

+α (1.68)

1 5.8 0.01

24 6.5 1

72 7.5 4

120 8.5 7

152 9.18 9

shaker at 200  rev  min−1 and filtered through a sieve (1 mm mesh size) to remove the undigested feathers. The culture filtrate was air dried overnight in an incubator at 60  °C. The product was ground to powder form using a grinder. Proximate Composition and Nutrient Analysis of FPH Total nitrogen content in FPH was estimated using microKjeldahl digestion method [24]. Dry matter, ash content, ether extract was determined according to AOAC methods [25]. Samples for nutrient analysis was prepared as described by Chapman and Pratt [26]. One gram of the sample was digested in 550  °C muffle furnace for 4  h (Dry ashing method). Then the sample was dissolved in 2  mol  l−1 HCl and filtered through a Whattman No. 42 filter paper. Potassium and phosphorus was estimated using flame photometer and spectrophotometer respectively. Whereas other nutrients such as calcium, manganese, iron, copper and zinc were analysed using atomic absorption spectrophotomer (Perkin Elmer 200 series). Amino acid profile of feather meal was performed as described by Bruckner et  al. [27] with some modifications. 0.2 g FPH and native feather was hydrolyzed with 5 ml of 6 mol l−1 HCl in a boiling water bath for 24 h and centrifuged (8000×g, 15  min) and filtered (0.2  µm). For determination of free amino acid in the cell free supernatant, the fermentation broth was centrifuged (8000×g, 20 min.) and removed the undigested feather and microbial biomass. The filtrates were used for determination of amino acid profile by HPLC (Agilent 1100 RP-HPLC). The sample was derivatized with orthopthalaldehyde (OPA) to make the amino acid more volatile and analyzed with HPLC using C-18 column (5.0  µm particle size, 150 mm × 4.6–ID). The mobile phase consist of eluent A (100 mM sodium acetate containing 0.018% Triethylamine, pH 7.20 ± 0.05) and eluent B (100 mM sodium acetate-methanol-acetonitrile; 20:40:40,v/v). A linear elution gradient programme from 0% B to 100% B for 25 min followed by 100% A for 5 min with a flow rate of 0.5 ml ­min−1 at 40 °C was performed. Amino acid peaks were detected at 338  nm using the VW detector. Amino acids were quantified using standard amino acid solutions (Hewlett Packard).

13

Author's personal copy

In Vitro Digestibility of FPH In vitro proteolytic digestion of feather meal was performed using pepsin and trypsin as described by Grazziotin et  al. [28]. 1  g feather meal was suspended in 10  ml 2  mol  l−1 HCl; 2 mg pepsin was then added and the suspension was kept incubated at 37  °C for 2  h. After incubation, the pH was adjusted to 8.0 with 2 mol l−1 ­NaHCO3 and incubated for an additional 16  h in presence of 2  mg trypsin. The sample was centrifuged (8000×g, 30  min) and release of peptides was monitored at 280 nm.

Results Isolation and Identification All total 102 morphologically distinct bacterial strains were recovered from soil samples collected from different feather waste dumping sites in North East India. The isolates were investigated for possible degradation of chicken feather in chicken feather medium. Of these, 26 strains were found to be positive for keratinolytic activity. One isolate, RCMSSR-7, could degrade feather completely in 48 h therefore it was selected for further studies. RCM-SSR-7 was a gram negative, rod shaped, non-motile bacterium. The organism forms translucent yellow colonies on nutrient agar medium; colonies were regular in shape. Based on 16S rRNA gene sequence analysis, RCM-SSR-7 was identified as a Chryseobacterium sediminis RCM-SSR-7 showing 99.92% sequence similarity. Phylogenetic tree of the strain with other closest Chryseobacterium spp. is shown in Fig. 1. Factors Affecting Feather Degradation The effect of temperature, pH, substrate concentration and incubation time on feather degradation by C. sediminis RCM-SSR-7 was studied and results are presented in Table  2. Incubation temperature profoundly influenced feather degradation. Incubation temperature of 30 °C resulted in maximal feather weight loss (81%) and

Fig. 1  Phylogenetic analysis of RCM-SSR-7 with closely related Chryseobacterium spp. based on 16S rRNA gene sequences. The tree was constructed with MEGA 5.2 using neighbour-joining method.

13

Waste Biomass Valor

soluble peptide production (120  mg  g−1 feather) upon 24  h incubation. Feather degradation was drastically reduced at 35 °C. Initial pH of the medium also affected feather degradation and soluble peptide production. Maximal feather degradation (86%) and soluble peptide

Table 2  Factors effecting feather degradation by RCM-SSR-7 Factors

pH  6  6.5  7  7.5  8  8.5  9  9.5 Temperature (°C)  20  25  30  35 Incubation time (h)  12  24  48  72  96  120  144 Feather concentration (%)  1  3  5  7  10  12

Soluble peptides (mg g−1 feather)

Feather weight loss (%)

33 ± 3 90 ± 2 140 ± 9 209 ± 15 137 ± 4 122 ± 6 106 ± 8 92 ± 8

36 ± 2 65 ± 1 81 ± 1 86 ± 3 78 ± 3 73.5 ± 2 69 ± 4 59 ± 4

61 ± 3 97 ± 7 140 ± 9 47 ± 1

50 ± 7 65 ± 1 81 ± 1 26 ± 2

102 ± 6 140 ± 9 150.±2 202 ± 6 180 ± 9 170 ± 4 160 ± 3

50 ± 5 81 ± 1 97 ± 1 98 ± 1 97 ± 1 98 ± 0 98 ± 1

140 ± 9 187 ± 6 208 ± 7 218 ± 1 206 ± 3 183 ± 17

97 ± 1 94 ± 3 88 ± 6 84 ± 3 70 ± 1 62 ± 4

Numbers at nodes are levels of bootstrap support (%) for branch points (1000 resamplings). Bar 0.001 substitutions per nucleotide position

Author's personal copy Waste Biomass Valor

released (2  mg  g−1 feather) was observed at pH 7.5 in 24  h of incubation. Optimal incubation time for feather degradation was observed at 72  h. The strain RCMSSR-7, interestingly, could degrade very high concentrations of feathers within 72 h of incubation at 30 °C. Even at 10% (w/v) feather concentration, 74% feather weight loss was achieved. Maximum soluble peptide production (218  mg  g−1 feather) was observed at 7% (w/v) feather concentration and further increase in feather concentration retarded feather weight loss and soluble peptide production. Optimization of Feather Degradation The effects of three parameters (incubation time, pH and feather concentration) on feather degradation were studied using RSM. A set of 20 experiments with different combinations of the three selected factors were performed. Both observed and predicted values were found to be similar, suggesting the authenticity of the model. The results of the RSM experiments are presented in Table  3. The soluble peptide production (Y) by the strain RCM-SSR-7 can be expressed in terms of the following regression equation:

IAA Production

Y = + 288.65 + 43.01 × A + 5.37 × B + 25.73 × C − 67.36 × A2 − 68.78 × B2 − 71.96 × C2 − 14.13

Strain RCM-SSR-7 could produce IAA from 24 h of incubation in CFM with or without tryptophan supplementation

× A × B + 23.38 × A × C − 15.63 × B × C

Table 3  Results of RSM studies for soluble peptide production by strain RCMSSR-7

where A, B and C represent incubation time, pH and feather concentration respectively. The model coeficients determined by multiple linear regression and analysis of variance (ANOVA). The model has F- value of 102 and implies that the model is significant. The model has high ­R2 value (0.99) indicating good agreement between the experimental results and theoretical values predicted by the model. The predicted R ­ 2 of 0.98 is also high and in reasonable agreement with the adjusted ­R2 of 0.94 indicating a high significane of this model. In addition, the model has an “Adequate Precision” value of 26.6. It measures the signal to noise ratio. A ratio >4 is desirable. One factor, contour, and 3D response surface curves were plotted to study the interactions among the three factors in order to determine the optimum condition for maximum soluble peptide production. The 3D interaction plots are shown in Fig.  2a–c. Release of soluble peptide was increased upto 92  h of incubation. Between the two widely spaced concentration levels, the concentration of feather at 5% (w/v) results in maximum maximal soluble peptide production. After overall analysis it was found that optimum soluble peptide released (295 mg g−1 feather) was occurred at pH 7.5, 5% (w/v) feather concentration and 84 h of incubation.

Run

Incubation time (h)

pH

Feather concen- Actual soluble peptide protration (%) duction (mg g−1 feather)

Predicted soluble peptide production (mg g−1 feather)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

+1 +1 0 0 0 −α −1 0 0 −1 +α 0 −1 −1 +1 0 0 0 0 +1

+1 −1 +α 0 0 0 +1 0 0 −1 0 0 −1 +1 −1 0 −α 0 0 +1

+1 −1 0 -α 0 0 −1 0 0 +1 0 0 −1 +1 +1 +α 0 0 0 −1

148.3 67.5 103.1 41.8 288.6 25.8 70.3 288.6 288.6 36.03 170.4 288.6 0.068 43.8 197 128.4 85.1 288.6 288.6 81.3

145 80 100 20 268 14 85 300 278 40 170 298 12 40 191 138 76 288 302 36

13

Author's personal copy

13

Waste Biomass Valor

Author's personal copy Waste Biomass Valor ◂Fig. 2  3D response curves showing interactions among variables

on soluble peptide production by C. sediminis RCM-SSR-7 (a–c): a Interaction between feather concentration and pH, b interaction between feather concentration and incubation time and c interaction between pH and incubation time

(Fig.  3). Maximum IAA production was observed at 72  h incubation in both the medium. However IAA production is higher in tryptophan supplemented medium (64.26 ± 1.1  µg  ml−1) than the unsupplemented one (44.4 ± 2.6 µg ml−1). Antioxidant Activity The DPPH radical-scavenging activity was investigated at different concentrations (0.02–0.2  mg  ml−1) of the FPH. The results presented in Fig. 4 clearly showed that the FPH exhibited an interesting radical scavenging activity with an ­IC50 value of 0.102 mg ml−1. FPH Preparation and Proximate Analysis From 100  g of feather 80 ± 3  g of FPH (i.e. 80% recovery) could be obtained after final processing. The proximate composition of FPH is shown in Table 4. It contains 75%, crude protein, 12% Nitrogen, 11%, ash and 3%, fat. FPH contains trace elements such as phosphorous (840 mg 100  g−1), potassium (450  mg 100  g−1), calcium (114  mg 100 g−1), iron (13.7 mg 100 g− 1), zinc (10.3 mg 100 g−1), manganese (0.52  mg 100  g−1) and copper (0.48  mg 100 g−1). Amino acid profile of raw feather, FPH and cell free fermentation broth is shown in Table 5. FPH prepared by C. sediminis RCM-SSR-7 is rich in essential amino acids such as histidine (90 mg g−1), leucine (106.3 mg g−1), lysine (43.1  mg  g−1), threonine (86.1  mg  g−1), isoleucine (17.9 mg g− 1) and methionine (56.3 mg g−1) as compared with raw feather. Essential amino acids are also detected as free soluble amino acids in cell free fermentation broth. In  vitro digestibility results indicated that feather meal prepared with C. sediminis RCM-SSR-7 was digestible by pepsin and trypsin releasing 650 mg peptides per gram of FPH. Digestibility of the present FPH is 86%.

Discussion Microbial bioconversion of feather waste into valuable products such as animal feed, fertilizer, amino acids, glues and foils is an ideal approach for management of such recalcitrant environmental wastes [29]. This present study reports an efficient keratinolytic C. sediminis RCM-SSR-7 isolated from feather waste dumping site in Manipur. Feather degrading Chryseobacterium spp. has been

reported earlier and most of the strains were isolated from poultry waste deposit sites [30–32]. The strain RCM-SSR-7 could degrade feather (81% feather weight loss) in 24  h incubation at 30  °C, this is comparatively faster than the other previously reported keratinolytic strains. For example, Park and Son [6] reported complete degradation of chicken feather by Bacillus megaterium upon 7 days postinoculation and Williams et  al. [33] showed that Bacillus licheniformis PWD-1 degraded chicken feather completely in 10 days. Hong et al. [31] reported that Chryseobacterium sp. P1-3 took 2 days incubation for 65% feather weight loss. Feather degradation is influenced by many nutritional and cultural conditions. Medium pH and incubation temperature are important factors for efficient feather degradation and it varies from strain to strain. The optimum pH and temperature for feather degradation by strain RCMSSR-7 was observed at neutral pH (pH 7.0–7.5) and 30 °C respectively, however, at higher pH and temperature feather degradation was retarded. Ability of this strain to degrade feather at neutral pH and moderate temperature is attractive from biotechnological perspectives. Because, digestion of feather at higher alkaline values (>pH 9) may destroy some of the essential amino acids [34]. Incubation time also affects the feather degradation even though the strain degrade feather within 24  h incubation, release of soluble peptide was increased with incubation time attaining optimum at 72  h incubation. Riffel et  al. [35] observed that maximum soluble peptide released at 44  h incubation by Chryseobacterium sp. Kr6. However, extent of soluble peptide released (202  mg  g−1 feather) in this present study is higher than the earlier reported value i.e. 90 mg g−1 feather. Feather concentration also found to be an important factor for enhanced soluble peptide production by RCM-SSR-7. Maximum soluble peptide production (218 mg g−1 feather) was observed at 7 with 84% feather weight loss. At higher substrate concentration feather degradation was decreased. Presence of high concentrations of feather in fermentation medium may leads to drastic decrease in aeration and increase in viscosity which, in turn, retards microbial growth and feather degradation [36, 37]. Optimization of feather degradation by C. sediminis RCM-SSR-7 was performed using RSM. RSM is a widely accepted statistical approach for modeling and analysis of problems in which a response, e.g. level of extracellular enzyme production, is influenced by several variables such as temperature, pH, incubation time and media components [38]. In this study three parameters viz. pH, incubation time and feather concentration was selected to study their effect on soluble peptide production. Based on the RSM study, final feather degradation condition was optimized at 5% feather concentration, pH 7.5, 84  h incubation and 30  °C. After optimization soluble peptide production by C. sediminis RCMSSR-7 was enhanced to 295 mg g−1 feather.

13

Author's personal copy

Waste Biomass Valor Table 4  Proximate composition of feather protein hydrolysate

Fig. 3  IAA production by C. sediminis RCM-SSR-7. (−▲−) represents IAA production in CFM supplemented with trypthophan and (−●−) represents IAA production in CFM without tryptophan. Data shown are mean ± standrad deviation of triplicate

Fig. 4  Antioxidant activity of FPH (−▲−) and ascorbic acid (−●−). The ­IC50 of ascorbic acid was recorded as 0.015  mg  ml−1. Data shown are mean ± standrad deviation of triplicate

The application of microbial technology for feather processing holds nutritional significance. Fermentation of feather waste by microbes not only modified the structure of keratin but also enriched the feather meal from microbial biomass. Feather protein hydrolysate prepared with C. sediminis RCM-SSR-7 contains 75%, crude protein, 11%, ash and 3%, fat. FPH was also found to be rich in trace elements such as phosphorous, potassium, calcium, iron and zinc which are required for growth of animal. Moreover, the FPH also possesses the antioxidant activity with an ­IC50 value of 0.102  mg  ml−1. Antioxidant activity of this FPH is higher as compared to other FPH prepared with Bacillus pumilus A1(IC50 = 0.3  mg  ml−1) and Streptomyces sp.

13

Contents

% Dry matter FPH

Dry matter Crude protein Ash Ether extract

92 ± 6 75 ± 4 11 3

Macronutrients

mg 100 g− 1

Nitrogen Phosphorus Potassium Calcium

12,000 ± 275 840 ± 24 450 ± 12 114 ± 8

Micronutrients

mg 100 g− 1

Iron Zinc Manganese Copper

13.7 ± 0.8 10.3 ± 0.2 0.52 0.48

MAB18 ­(IC50 = 78 mg ml−1) [12, 39]. Presence of antioxidants in animal feed not only enhanced the nutritive value of feed but also protects feed from deterioration. Animal feeds containing polyunsaturated fatty acids are susceptible to lipid peroxidation. Hence, animal feed manufacturing industries used synthetic antioxidants such as ethoxyquin (EQ, 6-ethoxy-1,2-dihydro-2,2,4-trimethylquinoline) as feed additive in order to protect lipid peroxidation [40]. The important limitation for using feather meal as animal feed is its poor digestibility hence researchers are focusing in improvement of digestibility by adopting different treatment methods. Feather meal prepared with whole cell has higher digestibility than the chemical or enzymatic treatment. In vitro digestibility results showed that digestibility of the present FPH is 0.86 i.e., 86% which is better than commercial feather meal (57.8%) or enzyme hydrolysed feather meal (73%) [41]. Amino acid analysis revealed that FPH prepared with strain RCM-SSR-7 is rich in essential amino acids such as histidine, leucine, lysine, threonine, isoleucine and methionine as compared with raw feather. Enhancement of methionine and lysine contents in FPH fermented with keratinolyic bacteria have been reported earlier. These results suggest that it is not only the feather keratin that can be used as protein source but also the microbial biomass as well [42]. Strain RCM-SSR-7 could also release soluble amino acids including essential amino acids in cell free supernatant. There has been an increased demand for amino acids to be used in many areas such as pharmaceutical drug manufacturing, feedstuffs and feed additives. Hence, strain RCM-SSR-7 could be a good candidate for production of amino acids from feather waste.

Author's personal copy Waste Biomass Valor Table 5  Comparison of amino acid content

Amino acids

Raw feather (mg g−1)

Feather protein hydrolysate (mg g−1)

Free amino acid in cell free fermentation broth (µg ml−1)

Aspartic acid Glutamic acid Serine Histidine Glycine Threonine Arginine Alanine Tyrosine Methionine Valine Phenylalanine Isoleucine Leucine Lysine

20 34.3 34.2 14.9 44.69 4.08 31.82 26.9 10.1 2.2 17.7 17.6 13.66 45.3 5.86

102.2 33.8 108.1 90.3 71.0 86.1 115.4 77.0 64.9 56.3 45.1 100.5 71.9 106.0 43.1

42.6 85.2 48.1 109.2 29.9 3.0 55.3 47.6 27.4 17.7 36.7 31.5 32.4 50.5 43.6

As FPH has nitrogen content (12%), these could be a good source of slow releasing N fertilizer. Keratinolytic microorganism, which is able to produce plant growth promoting activities, could offer a number of economic and environmental advantages over chemical-based fertilizers [15]. Keeping this in view the strain RCM-SSR-7 was studied for IAA production. IAA is an important plant hormone which exhibits a pronounced effect on plant growth and development. IAA is responsible for the cell division, cell elongation, cell differentiation and pattern formation in the plants [43]. The strain C. sediminis RCM-SSR-7 could produce IAA in feather medium with or without tryptophan supplementation. Hydrolysis of feather may results in tryptophan production and serves as the precursor for IAA synthesis. Supplementation of tryptophan in feather medium further enhances IAA production. These findings is consistent with the results reported by Bhange et al. [44] who also observed that Bacillus subtilis PF1 produce higher amount of IAA in CFM supplemented with tryptophan.

Conclusion The present study demonstrated that bacterial strain C. sediminis RCM-SSR-7 is a multifaceted bacteria which not only degrade chicken feather with higher digestibility but also produced antioxidant and plant growth promoting hormone, Indole-3-acetic acid. The most favorable condition for FPH production by C. sediminis RCM-SSR-7 as optimized using response surface methodology was achieved at 5% (w/v) feather concentration, pH 7.5, 30 °C and 84 h incubation time. Moreover, feather protein hydrolysate

prepared with this organism is rich in essential amino acids and nutrients. Hence, the strain C. sediminis RCM-SSR-7 could be a good candidate for valorization of chicken feather waste into useful products such as animal feed ingredients or as organic fertilizer. Acknowledgements  The authors gratefully acknowledges the award of DBT-Research Associateship by Department of Biotechnology (DBT), Government of India, which facilitated the completion of this research work. Authors also acknowledge Shankara Nethrayala, Chennai, India for providing amino acid analysis. Compliance with Ethical Standards  Conflict of interest  The authors declare that they have no conflict of interest.

References 1. Agrahari, S., Wadhwa, N.: Degradation of chicken feather a poultry waste product by keratiniolytic bacteria isolated from dumping site at Ghazipur poultry processing plant. Int. J. Poul. Sci.9, 482–489 (2010) 2. Xu, B., Zhong, Q., Tang, X., Yang, Y., Huang, Z.: Isolation and characterization of a new keratinolytic bacterium that exhibits significant feather-degrading capability. Afr. J. Biotechnol. 8, 4590–4596 (2009) 3. Sangali, S., Brandelli, A.: Feather keratin hydrolysis by a Vibrio sp. strain kr 2. J. Appl. Microbiol. 89, 735–743 (2000) 4. Papadopolous, M.C., El-Boushy, A.R., Roodbeen, A.E., Ketelaars, E.H.: Effects of processing time and moisture content on amino acids composition and nitrogen characteristics of feather meal. Anim. Feed Sci. Technol. 14, 279–290 (1986) 5. Steiner, R.J., Kellms, R.O., Church, D.C.: Feather and hair meals for ruminants. IV. Effects of chemical treatments of

13

Author's personal copy feathers and processing time on digestibility. J. Anim. Sci. 57, 495–502 (1983) 6. Park, G.T., Son, H.J.: Keratinolytic activity of Bacillus megaterium F7-1, a feather-degrading mesophilic bacterium. Microbiol. Res. 164, 478–485 (2009) 7. Moran, E., Summers, J., Slinger, S.: A source of protein for the growing chick. I. amino acid imbalance as the cause for inferior performance of feather meal. Poult. Sci. 45, 1257–1266 (1966) 8. Williams, C.M., Shih, J.C.H: Enumeration of some microbial groups in thermophilicn poultrywaste digesters and enrichment of a feather-degrading culture. J. Appl. Bacteriol. 67, 25–35 (1989) 9. Bertsch, A., Coello, N.: A biotechnological process for treatment and recycling poultry feathers as a feed ingredient. Bioresour. Technol. 96, 1703–1708 (2005) 10. Chew, B.P.: Importance of antioxidant vitamins in immunity and health in animals. Anim Feed Sci Technol. 59, 103–114 (1966) 11. Salami, S.A., Guinguina, A., Agboola, J.O., Omede, A.A., Agbonlahor, E.M., Tayyab, U.: Review: in vivo and postmortem effects of feed antioxidants in livestock: a review of the implications on authorization of antioxidant feed additives. Animal. 10, 1375–1390 (2016) 12. Fakhfakh, N., Ktari, N., Siala, R., Nasri, M.: Wool-waste valorization: production of protein hydrolysate with high antioxidative potential by fermentation with a new keratinolytic bacterium, Bacillus pumilus A1. J Appl Microbiol. 115, 424–433 (2013) 13. Choi, J.M., Nelson, P.V.: Developing a slow-release nitrogen fertilizer from organic sources: using poultry feathers. J. Am. Soc. Hortic. Sci. 121, 634–638 (1996) 14. Vesela, M., Friedrich, J.: Amino acid and soluble protein cocktail from waste keratin hydrolysed by a fungal keratinase of Paecilomyces marquandii. Biotechnol. Bioprocess. Eng. 14, 84–90 (2009) 15. Jeong, J.H., Lee, O.M., Jeon, Y.D., Kim, J.D., Lee, N.R.: Production of keratinolytic enzyme by a newly isolated feather-degrading Stenotrophomonas maltophilia that produces plant growthpromoting activity. Process Biochem. 45, 1738–1745 (2010) 16. Paul, T., Halder, S.K., Das, A., Bera, S., Maity, C.: Exploitation of chicken feather waste as a plant growth promoting agent using keratinase producing novel isolate Paenibacillus woosongensis. Biocatal. Agric. Biotechnol. 2, 50–57 (2013) 17. Kshetri, P., Ningthoujam, S.: Keratinolytic activities of alka liphilic Bacillus sp. MBRL 575 from a novel habitat, limestone deposit site in Manipur, India. Springerplus. 5, 1–16 (2016) 18. Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J.: Protein measurement with the folin phenol reagent. J. Biol. Chem. 193, 267–275 (1951) 19. Cappuchino, J.G., Sherman, N.: Microbiology: a laboratory manual. Addison-Wesley Longman Inc, England (1999) 20. Kim, O.S., Cho, Y.J., Lee, K., Yoon, S.H., Kim, M., Park, S.C., Jeon, Y.S., Lee, J., Yi, H., Won, S., Chen, J.: Introducing EZ Taxon-e: a prokaryotic 16 S rRNA gene sequence database with phylotypes that represent uncultural species species. Int. J. Syst. Evol. Microbiol. 62, 716–721 (2012) 21. Tamura, K., Peterson, D., Peterson, N., Steche, R.G., Nei, M., Kumar, S.: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance and maximum parsimony and methods. Mol. Bio. Evol. 28, 2731–2739 (2011) 22. Bano, N., Musarrat, J.: Characterization of a new Pseudomonas aeruginosa strain NJ-15 as a potential biocontrol agent. Curr. Microbiol. 46, 324–328 (2003) 23. Thaipong, K., Boonprakob, U., Crosby, K., Cisneros-Zevallos, L., Byrne, D.H.: Comparison of ABTS, DPPH, FRAP, and ORAC assays for estimating antioxidant activity from guava fruit extracts. J. Food. Comp. Anal. 19, 669–675 (2006)

13

Waste Biomass Valor 24. Jackson, M.L.: Soil Chemical Analysis Prentice Hall of India Private Limited, New Delhi, India (1973) 25. AOAC International Official methods of analysis of AOAC International. AOAC International, Gaithersburg, MD (1997) 26. Chapman, H. D., Pratt, P. F.: Methods of analysis for soils, plants and waters. Division of Agricultural Sciences, University of California, Riverside, USA (1961) 27. Bruckner, H., Wittner, R., Godel, H.: Fully automated high performance liquid chromatographic separation of DL amino acids derivatized with OPA together with N-isobutyrl-cystine, applications to food samples. Chromatographia. 32, 383–388 (1991) 28. Grazziotin, A., Pimentel, F.A., Jeong, E.V.D: Brandelli, A. Nutritional improvement of feather protein by treatment with microbial keratinase. Anim. Feed. Sci. Technol. 126, 135–144 (2006) 29. Gupta, R., Ramani, P.: Microbial keratinases and their prospective applications:an overview. Appl. Microbiol. Biotechnol. 70, 21–33 (2006) 30. Riffel, A., Lucas, F.S., Heeb, P., Brandelli, A.: Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Arch. Microbiol. 179, 258–265 (2003) 31. Hong, S.J., Park, G.S., Jung, B.K., Khan, A.R., Park, Y., Lee, C.H., Shin, J.H.: Isolation, identification, and characterization of a keratin-degrading bacterium Chryseobacterium sp. P1-3. J. Appl. Biol. Chem. 58, 247–251 (2015) 32. Gurav, R.G., Tang, J., Jadhav, J.P.: Sulfitolytic and keratinolytic potential of Chryseobacterium sp. RBT revealed hydrolysis of melanin containing feathers. 3 Biotech (2016). doi:10.1007/ s13205-016-0464-0 33. Williams, C.M., Richter, C.S., Mackenzie, J.M., Shih, J.C.H.: Isolation identification and characterization of a feather degrading bacterium. Appl. Environ. Microbiol. 56, 1509–1515 (1990) 34. Brandelli, A.: Bacterial keratinases: useful enzymes for bio processing agroindustrial wastes and beyond. Food. Bioprocess. Technol. 1, 105–116 (2008) 35. Riffel, A., Dariot, D.J., Brandelli, A.: Nutritional regulation of protease production by the feather-degrading bacterium Chryseobacterium sp. kr6. New Biotechnol. (2011). doi:10.1016/j. nbt.2010.09.008 36. Suntornsuk, W., Suntornsuk, L.: Feather degradation by Bacillus sp. FK 46 in submerged cultivation. Bioresour. Technol. 86, 239–243 (2003) 37. Rajput, R., Gupta, R.: Thermostable keratinase from Bacillus pumilus KS12: production, chitin crosslinking and degradation of Sup35NM aggregrates. Bioresour. Technol. 133, 118–126 (2013) 38. Dutta, J.R., Dutta, P.K., Banerjee, R.: Optimization of culture parameters for extracellular protease production from a newly isolated Pseudomonas sp. using response surface and artificial neural network models. Process Biochem. 39, 2193–2198 (2004) 39. Manivasagan, P., Venkatesan, J., Sivakumar, K., Kim, S.K.: Production, characterization and antioxidant potential of protease from Streptomyces sp. MAB18 using poultry wastes. BioMed. Res. Int. (2013). doi:10.1155/2013/496586 40. Błaszczyk, A., Augustyniak, A., Skolimowski, J.: LEthoxyquin: an antioxidant used in animal feed. Int. J. Food Sci. (2013). doi:10.1155/2013/585931 41. Tiwary, E., Gupta, R.: Rapid conversion of chicken feather to feather meal using dimeric keratinase from Bacillus licheniformis ER-15. J. Bioprocess Biotechniq. 4, 1–5 (2012) 42. Onifade, A.A., Al-Sane, N.A., Al-Musallam, A.A., Al Zarban, S.: A review: potentials for biotechnological applications of keratin-degrading microorganisms and their enzymes for nutritional improvement of feathers and other keratins as livestock feed resources. Bioresour. Technol. 66, 1–11 (1998)

Author's personal copy Waste Biomass Valor 43. Dastager, S.G., Deepa, C.K., Pandey, A.: Isolation and characterization of novel plant growth promoting Micrococcus sp NII0909 and its interaction with cowpea. Plant Physiol. Biochem. 48, 987–992 (2010)

44. Bhange, K., Chaturvedi, V., Bhatt, R.: Ameliorating effects of chicken feathers in plant growth promotion activity by a keratinolytic strain of Bacillus subtilis PF1. Bioresour. Bioprocess. 13, 2–10 (2016)

13