Optimization of Physico-Chemical Parameters for Hyper Keratinase Production from a Newly Isolated Aspergillus sp . DHE 7 using Chicken Feather as Substrate-Management of Biowaste

Article history: Received on: 11/04/2017 Accepted on: 10/07/2017 Available online: 30/09/2017 Keratinases have recently gained biotechnological impetus due to their ability in degrading insoluble keratinous solid wastes into simplified forms, which used in the preparation of nitrogenous fertilizers, biodegradable films, glues and foils. Keratinases are widespread in nature and mainly produced by diverse microorganisms including bacteria, fungi and actinomycetes. Therefore, the aim of the present work was to isolate and identify a new potent fungal strain possessing extracellular keratinase production capacity using keratinous solid wastes as the sole carbon and nitrogen sources. Fifteen keratinolytic fungi, isolated from a poultry farm soil using white chicken feathers as substrate, were screened qualitatively and quantitatively for their ability to produce extracellular keratinase. Medium optimization for enzyme production was also studied under submerged fermentation conditions. The results revealed that a new fungus identified as Aspergillus sp. DHE7 was reported as the most potent fungal strain for extracellular keratinase production under submerged shaking conditions (120 rpm). The highest yield of enzyme of 199+4.2 U/mL was observed after four days of incubation at pH 6.0 and temperature 30 °C using 2 % of chicken feathers as substrate. Medium supplementation with 0.5 % sucrose as an additional source of carbon increased keratinase yield to 226+5.4 U/mL. While using additional nitrogen sources could not promote a favorable effect on keratinase productivity as compared with the basal medium. Different keratinous wastes were used as substrate in the basal medium and better keratinase production was obtained with goat hair medium (452+12.3 U/mL) followed by turkey feather (435+9.2 U/mL) and sheep wool (322+13.4 U/mL) media. The present data suggest Aspergillus sp. DHE7 as a potential keratinolytic strain for the bioconversion of keratinous wastes, through a non-polluting process.


INTRODUCTION
Keratins, the insoluble structural proteins of wool and feathers, are one of the most abundant highly rigid biopolymer in the world.They are known for their difficult degradation and high stability since the polypeptides are strongly stabilized and densely packed by hydrogen bonds and hydrophobic interactions (Brandelli, 2008).Moreover, cross-linkages between the protein chains by disulfide bonds provide high stability and resistance to degradation by the common proteases such as pepsin, trypsin and papain (Brandelli et al., 2010).Keratins are classified into -and β-types; -type is predominantly found in wool, hair, horns, claws of mammals and nails, while the harder -type is commonly found in beaks, claws and feathers of birds (Matikevičiene et al., 2011).Millions of tons of feathers are annually discarded to the environment as a waste byproduct at poultry processing plants, causing serious problems as a pollutant and in the outbreaks of H5N1 virus.
Hence, it's important to develop methods for remediation of keratin with easy processing set-up and lower cost.Feathers have been treated chemically under steam pressure to reduce the rigidity and increase the digestibility; however, this method has disadvantages because it consumes large amounts of energy and also some heat-sensitive amino acids, i.e. tryptophane and lysine are destroyed during treatment (Kumar et al., 2010).Therefore, enzymatic biodegradation is emerged as an alternative cheap treatment, which improves the nutritional value of feathers and also offers mild conditions for valuable products production.Microbial keratinases, a group of proteolytic enzymes, may meet this preference because they are capable of decomposing complex keratin-rich waste materials to simplified forms and keratinophilic microorganisms including bacteria, fungi and actinomycetes naturally occur on keratinous wastes (Ali et al., 2011;Selvam and Vishnupriya, 2012).
Keratinases from microorganisms are also used in the preparation of protein supplements, animal nutrients, cosmetics, leather manufacture, textile processing, detergent formulation and nitrogenous fertilizer for plants (Goushterova et al., 2005).In addition, keratinases have a prospective application in novel fields such as prion degradation for the treatment of dreaded mad cow disease, production of feather meal and biodegradable plastic manufacture (Gupta and Ramnani, 2006).Therefore, because of the growing interest of microbial keratinases in different industrial sectors, it is essential to search for new keratinolyic microbes that produce novel keratinases with prospective industrial applications.However, the production of keratinase by microorganisms and biodegradation of keratin, vary considerably according to species, culture conditions and keratin substrates as reported by Cai and Zheng (2009).
Thus, the aim of this study was to isolate new potent fungal strains possessing extracellular keratinase production capacity under submerged fermentation conditions using keratinous solid waste as sole carbon and nitrogen sources.Medium optimization for enzyme production and its application in keratin-rich wastes degradation were also investigated.

Substrate
Chicken feathers, kindly obtained from Alwatania Poultry Co., Egypt, were thoroughly washed with tape water, saline solution, rinsed with distilled water and dried under sunlight.The dried feathers were soaked in chloroform: methanol (1:1, v/v), extensively washed with tap water and finally with distilled water.Feathers were dried in hot air-circulation oven at 60 ºC to constant weight, cut into uniform size (1.0 cm length) and stored at 5 ºC until used.

Soil sample
Fifteen keratinophilic fungal strains tested in this study were locally isolated from different soil samples collected from poultry farms (El Fayoum, Egypt) by using a sterile scalpel, at 5 cm depth and transferred to a polythene bag.One gram of each soil was transferred in a flask containing 99 mL of sterile distilled water then shaken (Innova ® 4230 shaker incubator; New Brunswick Scientific) at 100 rpm for 15 min and the dilution procedure was performed to give up to 10 -8 dilutions (Choi et al., 1999).

Isolation of keratinolytic fungi using agar plate method
With the help of sterile micropipette, 0.1 mL of each diluted sample was transferred onto agar plates medium (contains (g/L): white chicken feather powder; 10, 10 MgSO 4 •H 2 O; 0.5, KH 2 PO 4 ; 0.1, FeSO 4 •7H 2 O; 0.01; ZnSO 4 •7H 2 O; 0.005; streptomycin, 0.005 and agar, 15.0 at pH 6.0) and evenly spread with a sterilized L-rod (Wawrzkiewicz et al., 1991).Keratinolytic activities of the pure isolates were determined as clear zones around the colonies after five days of incubation at 30 ºC.The diameter of each clear zone was measured in mm to qualify the activity.Single colonies were picked up and inoculated onto PDA media, incubated for seven days at 28 °C and finally stored at 4 °C.

Detection of aflatoxins
Determination of aflatoxins of the selected fungus was conducted according to AOAC (method 971.24, 2000) through high-performance liquid chromatography technique (1100 HPLC; Agilent Technology, Santa Clara, CA, USA).Millennium Chromatography Manager Software was used to record the observed data.

Preparation of keratin solution
Ten gm of chicken feathers in 500 mL of dimethyl sulfoxide (DMSO) were heated at 100 °C for two h in a reflux condenser.The soluble keratin was precipitated by the addition of chilled acetone (-20 °C) and then kept in refrigerator for two h, followed by cooling centrifugation (refrigerated Sigma 3-18KS centrifuge, Sigma Laborentnfugen GmbH, Osterode am Hart, Germany) at 8000×g for ten min.
The precipitate obtained was washed with distilled water and finally dried in a vacuum dryer at 40 °C (Kaul and Sumbali, 1997).

DNA extraction, PCR amplification and phylogenetic tree analysis
The selected fungus was grown in PDA medium for seven days at 28 ºC and directly used for 18S rRNA gene amplification by polymerase chain reaction (PCR) technique using the universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5′-TCCTCCGCTTATTGATATGC-3′) (Altschul et al., 1997).PCR was performed in 25 μL as a final volume, which composed of 12.5 μL of Taq PCR Master Mix (Promega, Germany), containing 0.5 U/μL of Taq DNA polymerase, 500 μM of each dNTP, 20 mM of Tris-HCl (pH 8.3), 100 mM of KCl, 3 mM of each MgCl 2 and Bromophenol Blue, 1 μL of fungal culture as a template DNA, 1 μL of each primer (10 μM) and 9.5 μL of double distilled H 2 O.
The reaction was as follows: denaturation at 95 °C for 1 min followed by annealing at 55 °C for 30 s and extension for two min at 72 °C; and final extension for ten min at 72 °C.The products of PCR were detected by using electrophoresis technique (MSMINIDUO, Clever scientific Ltd., Rugby, Warwickshire, UK) in 1 % agarose gel stained with Tris/Borate/EDTA (TBE) buffer containing ethidium bromide of 1 μg/mL and finally visualized under UV light, to confirm its purity and size.Core Bio Gel Extraction Kit (RKT13: Chromous Biotech, Karnataka, India) was used to extract DNA fragments from the gel.The fungal gene sequences were compared to other nucleotide sequences of some fungal strains in GenBank database (National Centre for Biotechnology Information) through Basic Local Alignment Search Tool (BLAST) (Benson et al., 2008) and MEGA 4.0 software was used to construct the phylogenetic tree.

Inoculum preparation
The fungal strains were cultivated on PDA slants at 28 ºC for seven days.Conidia of each slant were scraped and 5.0 mL of sterile distilled water contains 0.1 % Tween-80 were added and the final concentration of spore suspension was 1.5 x 10 6 spore/mL.
After cooling, the flasks were inoculated by five mL of spore suspension (1.5 x 10 6 spore/mL) and incubated under shaking conditions (Innova ® 4230; New Brunswick TM Scientific, Scituate, MA, USA) at 120 rpm for four days at 28 °C.After the fermentation period, the final medium pH was measured and the hydrolysates were centrifuged (refrigerated Sigma 3-18KS centrifuge, Sigma Laborentnfugen GmbH, Osterode am Hart, Germany) at 5000×g for 15 min and filtered (Whatman filter paper no. 1, Inc., Maidstone, UK).The clear supernatants obtained were used for keratinase assay.

Optimization of culture conditions
Medium optimization for extracellular keratinase production by the selected fungal strain was studied by varying one factor at a time, while keeping others as constant.

Effect of various types of keratin-rich waste on keratinase production
Different sterile keratinous wastes (duck feather, goose feather, turkey feather and sheep wool) were fragmented into uniform size with about 1 cm long and added individually in the production medium instead of chicken feather.All other optimum culture conditions were performed.At the end of fermentation period, the cultures filtrates were centrifuged at 5000×g, filtered through Whatmann filter paper no. 1 and the clear supernatants obtained were used to determine keratinase activity.

Determination of keratinase activity
The enzyme activity was determined according to the method described by Gradisar et al. (2005) but with slight modification.Briefly, 1.0 mL of properly diluted crude enzyme extracts in 0.1 M Tris-acetate buffer (pH 8.0) was transferred in a test tube containing 1 mL of soluble keratin solution and incubated for 30 min at 37 °C.The reaction was stopped by the addition of 2.0 mL of trichloroacetic acid (TCA) (15 %), centrifuged at 5000×g for 10 min and finally the absorbance of the clear supernatant was measured at 280 nm against a control, i.e. the enzyme solution with 2.0 mL of TCA in absence of the substrate.One unit of keratinolytic activity (U/mL) was defined as an increase of absorbance at A 280 with that of the control for 0.01 per min under the standard conditions and the following equation was used: U= 4×n×A 280 / (0.01×T) Where 4 is the final reaction volume (mL), n is the dilution rate and T is the incubation time (min).

Protein assay
The protein content was determined as described by Bradford (1976) using BSA as a standard.

Statistical analysis
Data reported represent the mean value of three independent trials with standard deviation.The software used to calculate the data was EXCEL 2010 (Microsoft Corp., Redmond, WA, USA).

Screening of different fungal strains for the production of extracellular keratinase
The isolation and identification of new keratinolytic fungal strains might help in finding their role in the degradation of insoluble keratin-rich wastes and development of new biotechnological methods with valuable products (Gioppo et al., 2009).In the present research, fifteen fungal strains locally isolated from poultry farm soil samples were qualitatively evaluated for their abilities to grow on chicken feathers as a sole source of carbon and nitrogen.The data reported revealed that most of the isolated fungal strains could grow under these conditions, however, isolate DHE7 was found as the highest keratinase-producing strain since it degraded chicken feathers completely (Fig. 1) and also exhibited the highest clear zone diameter (47 mm) (Table 1).All fungal strains were quantitatively screened for keratinolytic activity in submerged fermentation conditions using chicken feathers as substrate in the basal medium.Results in Table 1, shown that the highest keratinase production was observed with isolate DHE7 (168±4.7 U/mL) followed by DHE15 (154±2.8U/mL), DHE12 (127±2.7 U/mL) and DHE13 (123±3.6U/mL), which are much higher than data reported by Cai and Zheng (2009) and Mazotto et al. (2013) for extracellular keratinase production by Bacillus subtilis KD-N2 (75 U/mL) and A. niger 9D40 (21.3 U/mL), respectively.Therefore, this promising result justified the selection of strain DHE 7 for the following experimental studies.

Aflatoxins detection
Toxicity test was studied in the present work and the observed results revealed that strain DHE 7 was a non-mycotoxinproducing fungus, which confirm its selection.

Substrate Concentration
Keratinolytic microorganisms are known for their capabilities of using keratin-rich substrates as carbon and nitrogen sources, whereas, the carbon-free medium forces the fungus to utilize keratin and subsequently the percentage of solubilization increased (Goushterova et al., 2005).In the present experiment, chicken feather concentrations of 5, 10, 20, 30, 40 and 50 g/L were evaluated for maximum keratinase production by Aspergillus DHE 7 and the reported data revealed that the production of keratinase by the fungus increased with the increase in feather concentration up to 20 g/L with maximum production of 164.3±3.6 U/mL (Table 2).However, at higher concentration a gradual decrease in enzyme production was investigated.Similar results were reported by Suntornsuk and Suntornsuk (2003) for keratinase production by Bacillus sp.FK 46.They reported that higher feathers concentrations (30-50 g/L) resulted in repression of keratinase production.In contrast, Cai et al. (2008) reported that 10 g/L of feather was the optimal concentration for the production of keratinase by Bacillus subtilis.

Effect of initial pH value
The reported data in Fig. 3 represent the influence of various pH values on keratinase production by Aspergillus sp.DHE7 after 4 days of incubation at 30 °C.Enzyme production was considerably decreased at lower pH values and enhanced at higher ranges.However, the optimal pH for enzyme production (163±2.7)was observed at pH 6.0.This result is congruent with several studies reported on keratinolytic microorganisms and their biotechnological potential.In most keratinophilic microorganisms, neutral to alkaline pH values (6.0 to 9.0) have been reported as the most suitable for keratinase production and feather degradation, which might be attributed to the modification in cystine residues to lathionine at alkaline pH, which makes it accessible for keratinase action (Gupta and Ramnani, 2006;El-Gendy, 2009;Jain and Sharma, 2012).On the other hand, keratinase production by few microorganisms were found to be optimal at higher pH values (Gessesse et al., 2003).

Time course
Incubation period is considered as an essential parameter for optimal keratinase production by microorganisms when grown under submerged fermentation conditions.In the present work, the aerobic growth of Aspergillus sp.DHE7 on chicken feathers as a sole source of nutrition resulted in complete degradation of feathers after 72 h of fermentation.In addition, the analysis of culture supernatant showed that the enzyme activity increased from an initial of 23+1.3U/mL after 24 h of incubation giving its optimal activity of 179+3.3U/mL on the fourth day of fermentation (Fig. 4).However, the extension of incubation period was accompanied with a reduction of enzyme production, which could be either due to the inactivation of the enzyme in the presence of some kinds of proteolytic activities or the growth of the fungus reached a stage after which it could not balance its steady growth with the nutrient resources available.Keratinolytic enzymes are mostly synthesized by the microorganism during its late exponential phase or the stationary phase growth (Thys et al., 2004).In accordance with our result, Noronha et al. (2002) observed optimum keratinase from A. fumigatus after 72 h of incubation.Similarly, Kanchana (2012) reported optimal keratinase production from Bacillus sp. at 37 ºC after 72 h of incubation when grown under submerged culture fermentation.On the other hand, optimum keratinase production from Penicillium sp. was reported on the fifth day (El-Gendy, 2009).Matikevičiene et al. ( 2009) investigated maximum keratinase production by Bacillus sp. after 24 h of incubation.

Keratinase production in relation to incubation temperature
Most microorganisms (bacteria, actinomycetes and fungi) produce keratinase at temperature ranges from 28 to 50 °C (Nam et al. 2002).The reported results in Table 3 clearly indicated that maximum keratinolytic activity was obtained between 25-35 °C, with optimal enzyme production at 30 °C (176+3.4U/mL).This result is similar to that reported for keratinase production by Penicillium sp.(El-Gendy, 2009).On the contrary, Bockle and Muller (1997) and Anbu et al. (2008) reported optimum keratinase production by Streptomyces pactum and Trichophyton sp., respectively, at higher temperature ranges near 40 °C.

Influence of additional carbon source on keratinase production
In the search to improve the enzyme production by Aspergillus sp.DHE7, different carbon sources were added separately in the basal medium, while the medium free from the additional carbon source was served as the control.As depicted in Fig. 5, among all the carbon sources tested, sucrose proved to be the best for keratinase production yielding 226+4.6 U/mL followed by arabinose (198+2.5 U/mL) and glucose (195+3.1 U/mL), while maltose, raffinose and galactose negatively modulated keratinase production.This result is congruent with that reported by Cai and Zheng (2009) for the production of keratinase by Bacillus subtilis KD-N2. Similarly, Malviya et al. (1992) and El-Naghy et al. (1998) reported maximum keratinase production from Chrysosporium queenslandicum and C. georgiae, respectively, in the presence of keratin, which was further stimulated by the addition of glucose.On the contrary, production of keratinase by Streptomyces sp.MS-2 was highly inhibited in the presence of starch and glucose as reported by Mabrouk (2008).Thys et al. (2004) suggested that the suppression of glucose to keratinase synthesis is due to the catabolite repression.Our results also showed that culture grown in 0.5 % of sucrose exhibited maximum keratinase production by Aspergillus sp.DHE7 grown under submerged fermentation (Fig. 6), while at higher concentrations, sucrose acts as a repressor for enzyme production.

Role of nitrogen supplementation on the production of keratinase
The reported results in Fig. 7 showed that none of the tested nitrogen sources could exert a favorable effect on enzyme production by Aspergillus sp.DHE7 as compared with the basal medium (without the tested nitrogen source).Similarly, Santos et al. (1996) reported that nitrates had negative effects on keratinase production by Aspergillus fumigatus.El-Naghy et al. (1998) andSon et al. (2008) reported a partial or complete repressive effect on keratinase production by Chrysosporium georgiae and Bacillus pumilis, respectively, in cultures supplemented with different nitrogen molecules, which might be attributed to the catabolite repression exerted by these additional nitrogen sources (Ulfig et al., 2003;Gioppo et al., 2009).On the other hand, Veselal and Friedrich (2009) reported that the biodegradation of different keratinous wastes by Paecilomyces marquandii was enhanced in the presence of additional nitrogen sources.

Biodegradation of keratinous solid wastes by Aspergillus sp. DHE7
Different keratinous wastes were used as substrate in the growth medium of the tested fungal strain.A comparison of the enzyme production by the selected fungal strain when grown on different keratin-rich substrates is presented in Table 4. Aspergillus sp.DHE7 possessed better keratinase production in the presence of goat hair (452+12.3U/mL), turkey feather (435+9.2U/mL) and sheep wool (322+13.4U/mL).The degradation of such keratinous wastes by the filamentous fungus might be resulted from the combination effect of extracellular keratinase, sulphitolysis and proteolysis (Szabo et al., 2000;Gupta and Ramnani, 2006).In addition, Gioppo et al. (2009) reported that the initial attack of keratinous substrate by keratinases followed by disulfide reductases may permit other proteases to act, which results in an extensive hydrolysis of keratinous wastes.

CONCLUSIONS
Keratinolytic filamentous fungi are of great ecological importance not only in pathogenesis but also in the degradation of keratin residues.In the search for new source for keratinases, different fungi were isolated from poultry farm soil and a new isolate identified as Aspergillus sp.DHE7, was reported as the potent fungus for keratinase production under SmF.Maximum enzyme production from the fungal strain was reported when grown in a medium at an initial pH value of 6.0, at temperature 30 °C, for four days under shaking at 120 rpm using chicken feathers as substrate (20 g/L).Different carbon sources could enhance keratinase production by Aspergillus sp.DHE7, with sucrose being the optimal.On the other hand, maltose, raffinose and galactose negatively modulated keratinase production.It is worth mentioning that none of the tested nitrogen sources could exert a favorable effect on the production of keratinase.Different keratinous wastes were used as substrate in the growth medium and better keratinase production from Aspergillus sp.DHE7 was reported with goat hair medium followed by turkey feather and sheep wool containing media.The present findings revealed Aspergillus sp.DHE7 to be a potential organism for keratinase production and in the management of keratinous solid wastes.

Fig. 1 .
Fig. 1.Efficiency of Aspergillus sp.DHE7 in degrading white chicken feathers after 4 days of incubation at 30 °C under submerged fermentation (SmF) conditions

Fig. 5 :
Fig. 5: Effect of carbon source on the production of keratinase by Aspergillus sp.DHE7

Table 1
Screening of locally isolated filamentous fungi qualitatively and quantitatively for extracellular keratinase production capacity.

Table 2 :
Effect of different concentrations of feather on keratinase production by Aspergillus sp.DHE7 in submerged fermentation (SmF).Data are the mean value of independent triplicate measurements ±SD.

Table 3
Effect of incubation temperature on the production of keratinase from Aspergillus DHE7 grown under submerged fermentation (SmF) conditions.

Table 4 :
Biodegradation of various types of keratinous waste by Aspergillus sp.DHE7.Data are the mean value of independent triplicate measurements ±SD.