Bioconversion of chicken feather wastes by keratinolytic bacteria

Journal Pre-proof Bioconversion of Chicken Feather Wastes by Keratinolytic Bacteria Samira Alahyaribeik, Seyed davood Sharifi, Fatemeh Tabandeh, Shirin Honarbakhsh, Shokoufeh Ghazanfari

PII:

S0957-5820(19)31898-1

DOI:

https://doi.org/10.1016/j.psep.2020.01.014

Reference:

PSEP 2072

To appear in:

Process Safety and Environmental Protection

Received Date:

25 September 2019

Revised Date:

3 January 2020

Accepted Date:

7 January 2020

Please cite this article as: Alahyaribeik S, Sharifi Sd, Tabandeh F, Honarbakhsh S, Ghazanfari S, Bioconversion of Chicken Feather Wastes by Keratinolytic Bacteria, Process Safety and Environmental Protection (2020), doi: https://doi.org/10.1016/j.psep.2020.01.014

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2020 Published by Elsevier.

Bioconversion of Chicken Feather Wastes by Keratinolytic Bacteria Given name (Samira) and family name (Alahyaribeik)a* a

Department of Animal and Poultry Science, College of Aburaihan, University of

Tehran, Pakdasht 33916–53755 E-mail address: [email protected] Given name (Seyed davood) and family name (Sharifi)b b

Department of Animal and Poultry Science, College of Aburaihan, University of

[email protected]

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Given name (Fatemeh) and family name (Tabandeh)c c

E-mail address:

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Tehran, Pakdasht, Tehran, P.O.Box: 11365/7117, Iran

Industrial and Environmental Biotechnology Department, National Institute of E-mail address:

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Genetic Engineering and Biotechnology (NIGEB), Tehran, Iran

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[email protected]

Given name (Shirin) and family name (Honarbakhsh)d Department of Animal and Poultry Science, College of Aburaihan, University of

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d

Tehran, Pakdasht, Tehran, P.O.Box: 11365/7117, Iran.

E-mail address

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[email protected]

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Given name (Shokoufeh) and family name (Ghazanfari)e e

Department of Animal and Poultry Science, College of Aburaihan, University of

Tehran, Pakdasht, Tehran, P.O.Box: 11365/7117, Iran

E-mail address:

[email protected] *

E-mail address of Corresponding author: [email protected]

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Graphical abstract

Highlights

An affordable source of digestible protein by biodegradation of chicken feather waste.

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Rhodococcus erythropolis a novel feather-degrading bacterial strain

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Among bacterial strains Bacillus licheniformis showed significant proteolytic activity

Abstract

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Rhodococcus erythropolis, Geobacillus stearothermophilus and two Bacillus species (Bacillus pumilis, and Bacillus licheniformis) were evaluated for protease production

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using feathers as the sole carbon and nitrogen source. The B. licheniformis produced enzymes optimally at 40 °C and pH 10.0, while B. pumilis performed optimally at 37 °C and pH 7.0. Furthermore, the optimum conditions for enzyme production by G. stearothermophilus were 55 °C and pH 7.0-8.0, while for R. erythropolis they were 37 °C and pH 7.0. The maximum proteolytic activities of the protease produced by B. licheniformis, G. stearothermophylus and B. pumilis were 50.41, 9.91 and 35.41 2

U/ml after 48, 72 and 48 h of culture, respectively. With R. erythropolis, the maximum enzyme activity was 33.39 U/ml after 96 h of culture. Also, the production of soluble protein showed the same pattern as that of proteolytic protease. When the initial pH value was 10.0, culturing the feathers (30 g l-1) for four days at 40 °C resulted in the maximum production of soluble proteins (8.28 mg/ml) by B. licheniformis. These results suggest that new strategies for waste management have emerged after the introduction of keratinases in sustainable material development, which has industrials

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applications in green technologies.

Keywords: Protease activity, Biodegradation, Poultry waste, Rhodococcus

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erythropolis, Bacillus licheniformis

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1. Introduction

The consumption of poultry meat increases each year as one of the most economical

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and healthiest protein's sources. Due to rearing and processing procedures tremendous amounts of feather waste are generated. The accumulation of these wastes in the

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environment is regarded as a substantial source of pollution which can cause healthrelated concerns. Feather waste can be considered as an enormous source of protein

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when discussing large-scale productions, and insufficient attention has been given to the use or recycle of these wastes, despite the growth of technological advancements that can provide this realm with more pragmatic opportunities (Donner et al., 2019; Esparza, Y.; Ullah, A.; Bolak, Y., Wu, 2017). Nonetheless, the physiochemical processes and the treatments that convert the feather waste into value-added products are costly. They are not environmentally friendly and 3

have the disadvantage of destroying essential amino acids including lysine, methionine and cysteine (Rajput and Gupta, 2013). A considerable decrease in environmental contamination and increase in feather waste nutritional value can be achieved through the degradation of feather keratin by microorganisms (Holkar et al., 2018; Kumawat et al., 2017; Peng et al., 2019). On the other hand, the feather keratin has the disadvantage of containing disulfide and hydrogen bonds that make it extremely stable and resistant to proteolytic hydrolysis

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(Coulombe and Omary, 2002; Eslahi et al., 2014). However, the available literature cites a number of microorganisms capable of producing proteases that own an effective activity on insoluble and fibrous proteins like keratin(Tao et al., 2018;

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Valeika et al., 2019).

Thus, identification of keratinolytic microorganisms can be important in two aspects:

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first, in producing various keratinases for industrial applications(Ghaffar et al., 2018; Santha Kalaikumari et al., 2019), and second, the production of keratin

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hydrolysates(Fontoura et al., 2019). The role of microorganisms in producing feather protein hydrolysate (FPH) can be explored even further, since there is an increasing

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popularity of FPH as an alternative source of protein in animal feed formulation

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(Fakhfakh et al., 2011; Holkar et al., 2018) and some other industrial applications (Fontoura et al., 2019). Various physical and medium-related factors affect the amount of protease being produced by microorganisms. Higher growth rate of bacteria, capability of feather degradation and maximum protease production are obtained when these factors are optimized. The one-factor-at-a-time (OFAT) technique is a conventional procedure

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for research in this regard (Okoroma et al., 2012). In the present study, an initial step involved selecting four proteolytic bacteria to find the most efficient strain for the disintegration of chicken feathers. Then, the culture conditions of the selected strain were optimized for protease production in order to enhance the efficiency of feather degradation. 2. Materials and methods

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2.1. Starting material Chicken feathers were collected from a poultry slaughterhouse in Khansar, Iran. After washing and drying at 30 °C for 72 h, the feathers were chopped into smaller parts,

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powdered using a grinding mill and kept sealed at room temperature. The feathers were the sole carbon and nitrogen source throughout this work.

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2.2. Microorganisms

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Four strains of bacteria were evaluated for protease production. These were Bacillus pumilus PTCC 1733 (ATCC 7061), Geobacillus stearothermophilus PTCC 1713

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(ATCC 7953), Rhodococcus erythropolis PTCC 1767 (isolated from Iranian soil (Haghighat et al., 2003)), and Bacillus licheniformis (Accession Number: FN678352)

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(isolated from Iranian soil).

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2.3. Culture medium The feathers were utilized as a constituent of feather meal powder broth (FMPB). The basal medium for growth had the following composition: 10 g l-1 of the keratinous residue, 0.5 g l-1 NaCl, 0.3 g l-1 K2HPO4, 0.3 g l-1 KH2PO4 and 0.1 g l-1 MgCl·6H2O. The pH was adjusted to the designed pH prior to sterilization. Aliquots of 200 ml of this culture medium in 1000 Erlenmeyer flasks were dispensed, sterilized and

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incubated for 96 h under shaking conditions (200 rpm) at 37 °C. For shake-flask culture, bacterial strain (B. licheniformis, B. pumilis, and R. erythropolis, and G. stearothermophilus) were initially grown overnight in Luria-Bertani (LB) at 37°C, 37°C, 37°C, and 55°C respectively and finally washed three times with 0.1 M phosphate-buffered saline (pH 7.5), and 5% (v/v) inoculum (108 cells/ml) added to 200 ml medium. The growth of various strains on nutrient agar was determined by the plate count method. Also, protease production and soluble protein experiments

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were performed under optimum pH values, ranging from 7 to 10 (i.e. 7, 8, 9, and 10). The pH adjustment was done by the use of 0.1 N HCl or 0.1 N NaOH solutions. All cultures were set in triplicate.

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2.4. Effects of temperature and feather concentration on protease production

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and soluble proteins

The suitable temperature for enzyme production was determined by means of

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incubation. Accordingly, the culture media of B. licheniformis were incubated at temperatures ranging from 31 to 40 °C (i.e. 31, 35, 37, and 40°C). Also, the

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experimental feather concentration for protease production ranged from 10 to 30 g l(i.e. 10, 15, 20, 25, and 30 g l-1). Each concentration was tested for 96 h separately.

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Soluble proteins were released as a result of the biodegradation of chicken feathers.

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The amounts of these proteins were determined as described by Bradford (Marion M, 1976) using bovine serum albumin (BSA) as a standard. 2.6. Determination of proteolytic activity Proteolytic activity measurement, employing casein as a substrate, were performed according to a method reported by Kembhavi et al. (Kembhavi et al., 1993) (Sigma– Aldrich). A solution of 1% (w/v) casein in 0.5 ml of 100mM Tris–HCl buffer (pH 6

8.5) was thoroughly mixed with 0.5 ml of culture supernatants with suitable dilution. The resultant solution was incubated in a water bath for 15 min at 60 °C. When necessary, the enzyme reaction was stopped by the addition of 0.5 ml 20% (w/v) trichloroacetic acid. The solution was then centrifuged for 15 min at 17,000×g. The absorbance was measured against an appropriate blank at 280 nm (T70, UV/VIS Spectrophotometer, and UK). A standard curve was created using different solutions of tyrosine (0–50 m g l-1). The amount of enzyme necessary for releasing 1 µg of

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tyrosine per minute was consider as unit of protease activity.

2.7. Analytical methods and assessing the digestibility in vitro

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Dry matter (culture broths after filtering ) was obtained by oven-drying at 85°C (AOAC, 2000). Crude protein content was analyzed by the Kjeldahl method (AOAC,

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2000). Fat content was extracted by Soxhlet extraction with hexane for 8 h. The ash content was estimated by burning the sample at a temperature of 550ºC for 8 hours.

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Also, the proteolytic digestion of samples was determined in vitro by pepsin and pancreatin, as already reported by Ikeda et al. (Ikeda et al., 1995). After five days of

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cultivation to measuring the percentage of feather hydrolysis by different strains, the cultures were filtered by filter paper. Thoroughly washed and dried feathers residuals

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at 105 °C, to reach a stable mass, were collected and stored for further use. Ultimately,

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the filtrate was autoclaved and dried at 85 °C overnight to acquire FPH. 2.8. Fourier transform infrared spectroscopy (FTIR) The FTIR technique was used to study functional groups of raw feathers and treated chicken feathers using a Thermo Scientific Nicolet iS5 FTIR in the transmission mode with the 500 and 4500cm-1 wavenumber range. 2.9. Differential scanning calorimetry (DSC) measurement 7

The melting temperatures of all samples were measured using DSC (Pyris 1, Norwalk, CT, USA) under a continuous nitrogen purge at heating rate of 10 °C/min over a temperature range of 0 °C to 250 °C. 2.10. Electron microscopy After filtering and double washing the culture broths containing feathers, the substrates were lyophilized and plated with Eiko IB-3 ion coater. The samples were

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then detected using a Hitachi S-2700 scanning electron microscope at an advancing voltage of 30 kV. 2.11. Statistical analysis

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Analysis of variance was conducted using the general linear models procedure of SAS version 9.1 (SAS Institute, 2012) including the strain in the model as a classification

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factor. Differences among strains were analyzed with Duncan’s multiple range test.

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Data pertaining to different periods were analyzed as repeated measures using the MIXED procedure of SAS (SAS Institute, 2012) with a model containing the

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continuous effect of covariate (as a measure of the same variable), the random effect of strain, and the fixed effects of pH, temperature and feather concentration. By the use of the Akaike’s information criterion the best covariance structure was specified

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among first-order autoregressive, unstructured, and compound symmetry. Tukey’s

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multiple comparison method was used for estimating the differences among groups and their significance at the 5% level. All data represent the mean values of three replicates. 3. Results and Discussion 3.1. Growth of microorganisms and production of protease and soluble protein

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The proteolytic abilities of tested bacteria was assessed by culturing on FMPB. The proteolytic strain with the highest enzyme production on FMPB was then selected. The measurements involved monitoring the protease production and the level of soluble proteins during an incubation period of 4 days (Fig. 1. a). The initial pH of the feather medium not only affected the production course of protease, but also had a great impact on the enzymatic process, probably the transportation of nutrients through the cell membrane of bacteria and the environment in which the reaction

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occurred (Fig. 1. a). Generally, a suitable pH in culture media is a prerequisite for the maximum production of protease. The highest amount of enzyme production in B. licheniformis was achieved at starting pH of 10.0, whereby maximum levels were

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reached after 48 hours of culture. According to Gupta and Ramnani (Gupta and Ramnani, 2006), alkaline pH values facilitate keratin degradation. Temperature, pH

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and other parameters in relation to culture play vital roles in the production of

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enzymes. Accordingly, an optimal level of enzyme production by B. pumilis was found in a pH of 7.0 and maximum amounts were reached after 48 hours. When the initial pH of the culture was 7.0, the final pH of the media ranged from 8.5 to 9.0 after

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4 days (data not shown). Alkaline compounds and ammonia production caused an increase in pH value. In the feather medium, the best pH for the production of

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protease by G. stearothermophilus and R. erythropolis was 7.0 and the maximum

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amounts of enzyme preparation produced by each bacterial species were reached after 48 and 96 hours of culture, respectively. However, a further rise in pH resulted in a loss in protease production. These results are in accordance with previous reports which indicated that the keratinase enzyme produced by Bacillus species could be classified as an alkaline protease and that it is most active under neutral or alkaline conditions (Jayalakshmi et al., 2011; Mohamedin, 1999; Suntornsuk and Suntornsuk,

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2003). As shown in Fig. 1. a. the maximum protease activities of B. licheniformis, B. pumilis, G. stearothermophilus and R. erythropolis were approximately 50.58±2.45, 35.74±1.85, 9.13±1.27 and 33.39±2.35 U/ml, respectively. Based on these observations, the newly isolated strain of R. erythropolis is considered to be a novel

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keratin-degrading strain.

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Fig. 1. Effects of medium pH on the production of proteolytic enzymes (a) and the content of soluble proteins (b). B. licheniformis, B. pumilis and R.

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erythropolis were cultured for 4 days on feather powder medium (1%) which

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was adjusted to different pH values at 37 °C. G. stearothermophilus was cultured in the same conditions at 55 °C.

The initial pH values of the medium (ranging from 7.0 to 10.0) affected the contents of soluble proteins by the activities of B. licheniformis, B. pumilis, G. stearothermophilus and R. erythropolis in media containing 10 g l_1 feathers (Fig. 1.b). It was shown that soluble proteins had the same tendency to be produced as

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protease did. For instance, the production of soluble proteins was considerably reduced in the lower pH range, concurrent with the decrease in enzyme production in the lower pH range in B. licheniformis. This suggests that as the enzyme production increases under optimal conditions, the production of soluble protein also increases due to the degradation of more feather. The level of soluble proteins being released into the culture fluid varied among the various strains. After 48h of culture, the maximum content of soluble proteins produced by B. licheniformis, B. pumilis and G.

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stearothermophilus was 5.68±0.25, 4.85±0.18 and 2.14±0.23 mg/ml in conditions where the initial pH values were 10.0, 7.0 and 7.0, respectively (Fig. 1. b). Rhodococcus.erythropolis produced a maximum content of soluble proteins

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(2.96±0.183 mg/ml) after 96 h of culture in the condition where the starting pH was

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7.0.

Figure. 2 shows the growth curves of B. licheniformis, B. pumilis, G.

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stearothermophilus and R. erythropolis at 37, 37, 55 and 37°C, respectively. Maximum enzyme activities were observed at the beginning of the stationary phase

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for all strains except R. erythropolis (Fig. 2). In the late logarithmic growth phase, enzyme activities remained with little changes. These results are in agreement with a

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previous report which indicated that, similar to B. pumilis, the B. licheniformis PDW1 is able to secrete the enzyme in the late logarithmic growth phase (Lin et al., 1992).

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Sangali and Brandelli (Sangali and Brandelli, 2000) also reported that keratinolytic activity tends to increase with a longer culture time, even though the maximum activity was observed at the beginning of the initiation phase. Similar results were recorded when experimenting with Streptomyces fradiae (Shama and Berwick, 1991). In the current research, however, maximum enzyme production was found in the mid-

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logarithmic phase by R. erythropolis, and protease production was not measured until the beginning of the stationary phase.

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G. stearothermophilus B. pumilus

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B. licheniformis

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Log(CFU ml -1)

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R. erythropolis

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48

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120

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Cultivation Time (hr)

Fig. 2. Cell growth of strains. Culturing the B. licheniformis, B. pumilis, and R.

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erythropolis on feather powder medium (1%) which was adjusted to pH 7.0 at 37 °C. G. stearothermophilus was cultured in the feather medium adjusted to pH 7.0

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at 55 °C.

3.3. Optimization of the feather-degrading efficiency by B. licheniformis 3.3.1. The effect of temperature on the production of protease and soluble protein Among all tested strains, the best protease-producing bacterium (i.e. B. licheniformis) was selected based on its enzyme production. Extracellular protease production by B. 13

licheniformis and the subsequent release of soluble proteins were regarded as a function of culture time in the media with various temperatures (Fig. 3. a.b). With respect to protease production, maximal amount were achieved after 48 hours of culture, regardless of the temperature of the culture (Fig. 3.a). Interestingly, the protease that comes from B. licheniformis is catalytically active at high temperature (40°C) and in alkaline pH (pH 10.0). As the temperature rose from 31 to 40 °C, the production of protease increased. Meanwhile, Bacillus sp. showed optimal protease

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production at temperatures moving around 30 to 50 °C. Previous reports described the maximal protease production of Bacillus sp. FK 46 at 37 °C (Suntornsuk and Suntornsuk, 2003) and B. licheniformis PWD-1 at 50 °C (Williams et al., 1990). As

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shown in Fig.3. b, the production of soluble proteins by B. licheniformis decreased significantly in the lower temperature range, but increased in the higher range.

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Accordingly, after 48h of culture, the maximum content of soluble proteins were

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found to be 5.97 ± 0.56 mg/ml where the initial pH and the constant temperature were 10.0 and 40 °C, respectively. It was also confirmed by SEM that B. licheniformis gradually degraded chicken feathers and altered the main structure of feather keratin

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(Fig. 4). This may change the stability of keratin to the digestive enzymes of animals that consume it. These details signify a possible bio-technique for the degradation and

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utilization of feather keratin.

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3.3.2. Effect of feather concentration The effects of various feather concentrations on the production of protease and soluble proteins are shown in Fig 3(c-d). Bacterial growth (data not shown) and protease production increased with the increment in feather concentration (Fig. 3.c). However, the percentage of feather hydrolysis (data not shown) correlated inversely with the feather concentration. Highest protease production and feather hydrolysis were 14

observed at 1% feather concentration which was used at optimal concentration although, maximum protease production occurred at 2.5% feather concentration (Fig. 3.c). Kainoor and Naik (Kainoor and Naik, 2010) obtained maximum keratinase production in the presence of 1% feather meal with Bacillus sp. JB99. In the mentioned research, protease production and soluble proteins were produced at a similar level between 25-30 g l-1 concentrations. Generally, presence of keratin and its concentration effects the production of keratinase (Navone and Speight, 2018).

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Enzyme production could decline in the presence of higher concentrations of feather meal, showing catabolite repression (Saibabu et al., 2013). Similar results were reported when researchers experimented with Bacillus sp. FK46 (Suntornsuk and

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Suntornsuk, 2003), where higher feather concentrations (3 and 5%) were observed to cause suppression of keratinase production and/or substrate inhibition.

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The maximum content of soluble proteins was 5.47 ±0.422 mg/ml after 48h of culture,

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where the initial pH value was 10.0 in the medium including a feather concentration of 10 g l-1 (Fig. 3.d). In this context, Kim et al. (10) found 0.7 mg mL-1 soluble protein by having Bacillus cultured on 1% feather medium. The soluble protein values

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achieved by B. licheniformis in the presence of 10 g l-1 feathers after 48h of culture were higher than those achieved by several other microorganisms grown on chicken

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feathers, consist of Bacillus species (Nagal and Jain, 2010) which are considered as

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strong feather-degrading strains and keratinase producers (Gupta and Ramnani, 2006). In the current research, the feather rachises continued to exist until the 4th day of culture (data not shown) which accords closely with previous results reported by Riffel et al in which the feather barbules were attacked at first by Chryseobacterium sp. kr6, and then the feather rachea degraded slowly after 36 hours of culture (Riffel et al., 2011). It is probable that the intractable feather b-keratin hampers the

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degradation of feather since b-keratin degrades slowly, thereby releasing available nitrogen and upholding microbial growth. During 120 h of aerobic growth, making use of scanning electron microscopy (SEM) actualized the monitoring of feather degradation as it occurred due to the activity of B. licheniformis (Fig. 4). The surface of feather keratin cracked after the first 24 h of culture, and then degraded severely after 48 h of culture. After 72 h, the feather keratin started up to decompose, until it was no longer feasible to discern the whole structure of keratin after 120 h. Also, the

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surface of feather keratin remained attached to microorganisms in their mid-

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exponential growth phase.

Fig. 3. Effect of culture temperature (a-b) and feather concentration (10, 15, 20, 25 and 30 gl-1) (c-d) on the production of soluble protein and protease by B.

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licheniformis. The bacteria were cultured at the designated temperatures and the best temperatures (40 °C) was selected to evaluate feather concentration. The initial pH was 10.0 and the culture was maintained in a medium containing

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feather powder (1%) for 4 days.

Fig. 4a–d Scanning electron micrographs of thin sections of degraded native feathers during aerobic growth of B. licheniformis. a 24 h, b 48 h, c 72 h, d 120 h.

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The strains grew on basal medium supplemented with 1% feathers (Initial pH 10.0), incubated at 40°C at 200 rpm for five days.

3.4. Fourier transform infrared spectroscopy (FTIR)

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Figure. 5 shows the FTIR spectra of raw feather and feather protein hydrolysate (FPH) in the region 400–4000 cm−1 where the characteristic

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absorption peaks correspond mainly to the peptide bonds (-CO-NH).

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Amides I–III are created by vibrations in the peptide bonds(Sun et al.,

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2009). The range of 1700–1600 cm−1 wavenumbers show the amide I band which is connected typically with the C=O stretching vibration

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(A.K. Mohanty, M. Misra, 2005). The amide II band which comes in

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1520cm−1 region, is related to N-H bending and C-H stretching

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vibration (N. Eslahi, F. Dadashian, 2013). The transmission band in the range of 1220 cm−1 to 1300 cm−1 is attributed to C-N stretching and C=O bending rations which are identified as amide III(A. Vasconcelos, G. Freddi, 2008). It represented the carboxylic acids groups in the sample at wave numbers 1021, 1070, 1237, 1398, 2874, 2958, 3071 and 18

3285 cm-1 (Fig. 5). In the protein hydrolyses process the reaction of sulfites and cysteine created the S=O stretching vibrations (cysteine-Ssulfonated residues), which comes in 1124 cm−1 absorption band(A. Aluigi, C. Tonetti, F. Rombaldoni, D. Puglia, E. Fortunati, I. Armentano, C.Santulli, L. Torre, 2014).

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The achieved results confirm that after microbial hydrolysis of chicken feather, free functional groups such as carboxylic acids have been

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increased significantly compared to the raw feather, which can relate to

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degradation of raw feather and turn it into short chain polypeptides

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according to the protease activity of each microorganism. Therefore the

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samples can be identified as true polypeptides mainly without damage

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of amino acids during hydrolyses process.

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Fig. 5 FTIR spectra of degraded feathers during the aerobic growth of

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strains (where the blue line represents B. pumilis, the dark green line

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B. licheniformis, the yellow line R. erythropolis, the red line G.

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stearothermophilus, and light blue line raw feather) in the 1% feather

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powder medium which was adjusted to the pH of 7.0 at 37 °C for 5 days. G. stearothermophilus was cultured at 55 °C in the same

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conditions.

3.5. Differential scanning calorimetry (DSC) of feather protein hydrolysate (FPH) by various microorganisms

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The thermal behavior of raw feather and feather protein hydrolysate (FPH) by various microorganisms were investigated by DSC and are shown in Figure 6. The first endothermic peak at 99.51 °C in raw feather assigns to the dehydration of keratin matrix, and the second broad peak at 235.67 °C refers to the crystalline melting of the protein belongs to deformation of α- helices network (Spei and Holzem, 1987). The DSC diagrams of feather protein hydrolysate (FPH) by difference microorganisms displayed crystalline melting at lower temperature (from 90.85 to 200.5 °C) when

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compared with raw feather (235.67 °C). Diminution of melting temperature could explain with denaturation of a-helix structures and gain of amorphous form in all FPH (Kadokawa, J.-i.; Takegawa, A.; Mine, S.; Prasad, 2011). Feather protein

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hydrolysate (FPH) by B. licheniformis and B. pumilis illustrated lower crystalline melting temperatures (Tm) in comparison to G. stearothermophilus and R.

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erythropolis which are in general compromise with the changes as confirmed by

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degradation and in vitro digestibility results. DSC plots of feather protein hydrolysate (FPH) by G. stearothermophilus showed additional peak in the area of 138.36 °C, which was related to the loss of water. Above results showed that bioconversion has

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significant effect on the thermal behavior of raw chicken feathers.

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Fig. 6. DSC heat flow signals of degraded feathers during the aerobic growth

of strains (where the dark blue line represents G. stearothermophilus, the

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green line B. licheniformis, the light blue line R. erythropolis, the red line

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B. pumilis, and black line raw feather) in the 1% feather powder medium which was adjusted to the pH of 7.0 at 37 °C for 5 days. G.

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stearothermophilus was cultured at 55 °C in the same conditions.

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3.6. Biochemical and physical characterization of feather protein hydrolysate (FPH) and feather degradation by microorganisms The physiochemical characterization of the feather protein hydrolysate indicated the suitability of different temperatures, i.e. 37, 37, 50, and 37 °C, for culturing B. licheniformis, B. pumilis, G. stearothermophilus and R. erythropolis, respectively, in the 1% feather powder medium adjusted to pH 7.0 (Table 1). Among all strains, B. licheniformis caused maximum degradation, i.e. 83.58 ± 3.02 % within 120 h (Fig.

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7). Furthermore, B. pumilis and G. stearothermophilus caused feather degradation by 58 ± 3.59% and 30.85 ± 4.27%, respectively. El-Refai et al. (El-Refai et al., 2005) found 87.2% degradation as caused by B. licheniformis and 49.4% weight loss caused

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by Bacillus subtilis when grown on basal medium complemented with 1% hen

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feathers. As shown in Table 1, the FPH obtained by B. licheniformis displays high digestibility (83.04%), in comparison with raw feathers (12.39%) and also compared

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with those caused by other microorganisms. This enhanced digestibility and a higher digestible content in FPH may be caused by the action of B. licheniformis in breaking

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cross-linking disulfide bonds of feathers, which show high levels of consistency and resistance to proteolytic enzymes for instance papain, pepsin and trypsin (Jaouadi et

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al., 2013; Riffel et al., 2011). The composition of FPH was in contradistinction to that of raw feathers (p < 0.05) (Table1). The crude protein content of FPH by B.

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licheniformis (79.91%) was less than the amount detected in raw feathers (90.108%). This reduced protein content in FPH by B. licheniformis may be a result of the absence of non-degraded feathers, which were removed by filtration. Also, the conversional organic content to biomass during microbial hydrolysis cannot be ignored. The fat content of FPH as caused by B. licheniformis (2.33%) was greater than the amount detected in raw feathers (1.38%) but less than the FPH caused by other 23

microorganisms. This difference may be explained by the amount of biomass in the FPH. This value was similar to a previous report about fermented feather meal using a strain of Kocuria rosea (Bertsch and Coello, 2005). In this study, the ash content was higher (P < 0.05) in FPH caused by B. licheniformis (16.05%) than in raw feathers (1.65%). The culture medium used for B. licheniformis included mineral salts, which may entail an enhanced ash content in FPH (16.05%) or it might be related to the mineral formation by bacteria (Douglas and Beveridge, 1998). This is in agreement

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with a previous report by Fakhfakh et al. (Fakhfakh et al., 2011) about B. pumilus A1 and its action on chicken feathers. Due to reduction of melting point of FPHs by B. licheniformis (90. 85 °C), applying 85 °C of heat for measuring dry matter may end

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in the loss of volatile amino acids and peptides (ammonia) and complete dehydration. Also, DSC curve displays because of dehydrated peaks (138.36°C) in FPHs by G.

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stearothermophilus may 85 °C temperature for measuring dry matter did not

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completely remove moisture so it shows more than dry matter. The microbial conversion of feather wastes is a potential technique for the degradation and utilization of feathers as a value-added product, especially in the scale of industrial

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applications.

24

Table 1. Physiochemical composition of FPH and raw feathers. Composition of FPH (%) Protein

Fat

Ash

Dry matter

In vitro digestibility (%)

Raw feathers

90.11a

1.38e

1.65e

96.88a

12.39e

G. stearothermophilus

45.13d

4.79b

4.61d

90.08b

46.47d

Bacillus pumilus

77.58b

7.92a

7.50c

96.16a

71.44b

Bacillus licheniformis

79.91b

2.33d

16.05b

88.02c

83.04a

R. erythropolis

60.65c

6.80b

19.10a

87.83c

65.99c

P- Value1 Treat

0.0004

<.0001 <.0001 <.0001

SEM2

1.732

0.169

0.207

<.0001 0.806

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0.376

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Strain

Different superscript letters in each column indicate significant differences (P<0.05).

2

Standard error of mean values.

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1

25

100

a Feather hydrolysis (%)

80

b 60

c 40

d

20

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B. l

ic he ni

fo rm R. is er yt hr op ol is B. G .s pu te m ar ilu ot s he rm op hi lu s

0

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Fig. 7. Effects of different strains on feather

hydrolysis (%). B. licheniformis, B. pumilis

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and R. erythropolis were cultured on 1%

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feather powder medium adjusted to pH 7.0 at 37 °C for 5 days. G. stearothermophilus was

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cultured at 55 °C in the same conditions. Different superscript letters in each column

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indicate significant differences (p<0.05).

4. Conclusions These results strongly suggest that feather-degrading bacteria and their proteolytic enzymes could be used for production of value-added materials. Specifically, B. licheniformis appeared to be the most efficient strain in causing keratin degradation. Also, Rhodococcus erythropolis possess proteolytic activity and is effective in feather 26

degradation. For future research, the commercial application of the keratinolytic proteases created by R. erythropolis can be particularized by more precise studies on enzyme purification using genetic engineering techniques.

Conflict of Interest

Acknowledgments

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The authors would like to thank the National Science Foundation for the partial financial support of this work (Grant No. 97006008). We are thankful to the National Institute of Genetic Engineering and Biotechnology (NIGE) for providing laboratory facilities for this work. The authors would also like to thank Mr. M. Alahyaribeik for all-out support of this project.

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The authors would like to thank the National Science Foundation for the partial financial support of this work (Grant No. 97006008). We are thankful to the National

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Institute of Genetic Engineering and Biotechnology (NIGE) for providing laboratory facilities for this work. The authors would also like to thank Mr. M. Alahyaribeik for

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all-out support of this project.

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