Isolation & Purification of Feather Degrading Keratinolytic
Bacteria
1.1 general introduction:
Feathers are waste of poultry industry. They are very rich in structural protein keratin. Accumulation of feathers can lead to environmental pollution which will cause a number of pathogenic diseases to human being and animal. Williams heatal (1990) isolated a number of feather degrading bacteria and other microorganisms. Subsequently many workers have isolated feather degrading keratinase producing fungi, actinomycetes and microorganisms (Williams et al.1990; Riffel et.al.2003; Manezinger et. al. 2003; El-Refai et.al. 2005; Gradisar et.al. 2000; Friedrich et.al.2005; Ignatova et.al.1999; Gousterova et.al. 2005).
Gupta and Ramnani, in the year2006 have shown many applications of keratinases in food and poultry industries. For example, the feather hydrolysates of Bacillus licheniformis PWD-1 and Vibrio sp. strain kr2 (Williams et al., <href=”#B30″>1991; Grazziotin et al., <href=”#B8″>2006) can be used as feed additives, while the keratinase from Bacillus subtilis S14 exhibits remarkable dehairing capabilities (Macedo et al., 2005). Moreover, keratinase from B. licheniformis PWD-1 can degrade the infectious form of prion, PrPsc, in the presence of detergents and heat treatment (Langeveld et al., 2003), which is very important for the utilization of animal meal as feed. Usually, it is important to improve the enzyme yield for application purposes and so various methods including the optimization of cultural conditions and medium composition, or heterologous gene expression have been applied (Ramnani and Gupta, 2004; Anbu et al., <href=”#B1″>2005). Given the effectiveness of traditional mutagenesis approach for isolating mutants that produce improved yields of various microbial enzymes such as lipase and ?-galactosidase (Tan et al., <href=”#B25″>2003; Wang et al., 2004), it is conceivable that a similar strategy may be successfully applied to improve the ability of keratinase-producing strains for the production of this important enzyme.
Almost all keratinases are inducible and different keratin-containing materials such as feathers, hair and wool can be used as substrates for keratinase production (Gupta and Ramnani, 2006).
Keratinase producing bacteria are Bacillus licheniformis(Zerdani et al., 2004; Ramnani et al.,2005; Korkmaz et al., 2004; Manczinger et al., 2003;Williams et al., 1990; P.Tamilmani et al., 2008)..Burkholderia, Chryseobacterium, Pseudomonas, Microbacterium sp (Brandell and Riffel, 2006) wereisolated and studied with respect to differentparameter.
The enzyme is a potential enzyme for removing hair and feather in the poultry industry (Takami et al., 1992), for nutritional upgrading of feather meal and conversion of feathers into a feed protein in feed industry (Williams et al., 1991), and for clearing obstructions in the sewage system during waste water treatment and eco-friendly de-hairing process in leather industry.
The aim of this study was to isolate purify and characterized feather degrading bacteria. The far reaching aim of the project was to isolate and purigy the agent/enzyme responsible for feather degradation. The objective of the work was to characterize the organism using conventional as well as modern molecular markers. Willams et al(19890 enumerated a number of microbial groups in thermophillic poultry wastes digests and enriched feather degrading cultures with feather as a soul source of carbon and nitrogen.
1.2. LITERATURE REVIEW OF KERATINASE ENZYME
Williams et al (1989) enumerated some microbial groups in thermophilic poultry waste digesters and enriched a feather degrading culture with feather as a sole source of carbon.
Mukhopadhay et al (1989) reported keratinase production by Streptomyces sp. He isolated an inducible extracellular homogenous enzyme which resulted 7.5 fold increases in its activity after DEAE cellulose column chromatography. The enzyme activity was inhibited by reduced glutathione, PMSF and 2- Mereaptaethanol.
Williams et al (1990) continued his work on enriched feather degrading culture and characterized the organism to its species level for the first time. The microorganism was identified as Bacillus licheniformis. The organism was gram staining variable, catalase positive and demonstrated facultative growth at thermophilic temperatures.
Lin et al (1992) purified and characterized keratinase from feather degrading Bacillus lichniformis strain isolated by Williams et al with the help of membrane ultra filtration and C-75 gel chromatography. He purified enzyme with 70 fold increased activity. SDS-PAGE analysis revealed the purified keratinase had a molecular weight of 33 KDa.
Dozie et al (1994) reported a thermostable, alkaline-activity, keratinolytic proteinase from fungi Chrysosporium keratinophylun, which was able to solubilize keratin in lactose/mineral salt medium with DMSO. Optimum ph of the enzyme was 9 and optimum temperature for activity was 90C.
Lin et al (1995) reported about Ker A gene on Bacillus licheniformis PWD-1.Ker A gene shares 97% sequence identity with the gene encoding Subtilisin Carlsberg from Bacillus licheniformis. Northern (RNA) blotting analysis revealed transcriptional control of Ker A expression on different growth media.
Letourneau et al (1997) cultured Streptomyces sp (SK1-02) on feather medium and found high keratinolytic activity. Comparing its activity with commercial protease he found this enzyme could be a useful biotechnological tool for valorization of keratin containing waste or the depletion process in leather industry.
Bressollier et al (1999) characterized this Streptomyces sp (SK1-02) as Streptomyces albidoflavus.
Taha et al (1998) isolated, identified several keratinolytic feather degrading isolates from Egyptain soils. Taha et al (1998) isolated,idendifed several keratinolytic feather degrading isolates from Egyptian soils .They idendified the isolates according morphological, biochemical and API 50 CBH Bacillus system.They reported keranolytic activity by bacillus pumili and Bacillus cereus and Bacillus subtilis.
Later Taha et al encoded apr A gene on Bacillus subtilis and found high level of keratinolytic activity in Bacillus subtilis, which resulted considerable amounts of soluble proteins and amino acids.
Chitte et al (1998) reported karatinolytic activity in the broth of thermophilic Streptomyces thermoviolaceus SD-8.Optimum temperature and pH for production was 8and 55o C
Lin et al (1999) described a feather degrading isolate selected from canola meal compost.From eighty isolates present in the canola meal compost; he selected the proteolytic enzyme producing bacteria on skim milk agar and later used keratin for secondary selection of keratinolytic isolates.
Regina et al (1999) investigated the production of keratinase by Aspergillus fumigatus fresenius, which was an opportunistic air borne pathogen affecting human, birds and other animal.With this pathogenic microorganism they produced keratinase enzyme. The nature of this keratinase was thermostable and showed remarkable ability to degrade feather into feather meal.
Wang et al (1999) scaled up- the fermentation condition of keratinase to a pilot scale fermentar. They optimized the fermentation condition to a level of 10 fold increase in enzyme production.
1.3 Keratin
Keratins are albuminoids or scleroproteins, which are insoluble in water, salt solution, dilute acids or alkalis or alcohols that can exits either ? or ? conformation. (West, E.S. textbook of biochemistry)
1.4 Structure of keratin
Keratin refers to a family of fibrous structural proteins. Keratin is the key of structural material making up the outer layer of human skin. It is also the key structural component of hair and nails. Keratin monomers assemble into bundles to form intermediate filaments, which are tough and insoluble and form strong unmineralized tissues found in reptiles, birds, amphibians, and mammals. The only other biological matter known to approximate the toughness of keratinized tissue is chitin.
Figure-1.1: Protein structure of keratins and immunofluorescent staining
1.5 General description of keratinase
keratinase are produced only in the presence of keratin containing substrate. It mainly attacks on the disulfide (S-S) bond of the keratin substrate (Bockelet et al.1995).
Figure1.2: Structure of keratinase
1.6 Source of keratinase
Keratinase occurs frequently in fungi, actinomycetes and bacteria. They are frequently excreted from cells. They also present in some plant. For both technological and environmental reasons microorganisms are preferable for the productions of this enzyme (Kelly et al, 1976). actinomycetes particullerly Sterptomyces are known to secret keratinase enzyme Sterptomyces keratinase was first isolated from Sterptomyces fradiae (Noval et al, 1959). First keratinase activity was observed in fungi, Trychophyton mentagrophytes (YU et al 1968) after this work different types of fungi both pathogen and non pathogen were used to peoduced deratinase enzyme such as Microsporum gypsum, Microsporium canis (Takiuchietal 1982).
Baceteria keratinase are prepared from culture bacteria at a suitable cultivation time, when keratinase production is maximum. Among bacteria the Bacillus mainly responsible for the production of keratinase enzyme. In 1959 Molyneaux reported at first the degradation of wool keratin by bacillus ap. Degradation of human hair keratin by a thermostable protease from alkaliphilic Bacillus sp. noAH-101 studied by (Takami et al, 1992).
Application of keratinase:
1.7.1 Feather processing in industry:
Feathers are produced in large amount as a waste byproduct of poultry processing plant. A current value-added use for feathers is the conversion to feather meal, a digestible dietary protein for animal feed, using physical and chemical treatments. These methods can destroy certain amino acids and decrease protein quality and digestibility (Riffel and Brandelli, 2006).
1.7.2 Use in leather industry:
Microbial keratinases have become biotechnologically important in recent years especially for the leather industry, where they advantageously can be used as clean technology dehairing agents. Traditional dehairing processes are some of the most pollutant operation steps in leather manufacturing. Alkaline proteases, including keratinase, collagenase, and elastase can be used to minimise the need for sulphide and reduce the organic waste load of the dehairing process. Keratinolytic proteases selectively degrade the keratin tissue in the follicles in the hides and skins and keratinolytic activity combined with mild collagenolytic and elastolytic activities could result in a proteolytic dehairing process, which is gentle towards the proteins forming the leather.
Bacterial keratinases are of particular interest because of their action on insoluble keratin substrates, and generally on a broad range of protein substrates (<href=”#13″>Lin et al. 1995). These enzymes have been studied for de-hairing processes in the leather industry (<href=”#15″>Raju et al. 1996) and hydrolysis of feather keratin (<href=”#13″>Lin et al. 1995), which is a by-product generated in huge amounts by the poultry industry. Discarded feathers are currently used to produce feather meal through thermal processing, resulting in a low nutritional value product (<href=”#26″>Wang and Parsons, 1997). Feather hydrolysates produced by bacterial keratinases have been used as additives for animal feed (<href=”#27″>Williams et al. 1991). In addition, keratin hydrolysates have potential use as organic fertilizers, production of edible films and rare amino acids (<href=”#4″>Dalev and Neitchev, 1991; <href=”#3″>Choi and Nelson, 1996).
1.7.3 Medical use:
In the medical and pharmaceutical fields, keratinase can be used to eliminate
– Acne
– Psoriasis
– Human callus
1.7.4 Other uses:
Keratinase have several potential application: in detergent formulation for eliminating horny epithelial cells adhered to textile fibers ecologically friendly leather processing, waste chicken feather degradation , nutritional improvement of waste feather for livestock feed and production of protein hydrolysis from keratinous waste materials. Further, their prospective application in the challenging field of prion degradation would revolutionize the protease world in the near future.
Insoluble feather keratins can be converted after enzymatic hydrolysis to feedstuffs, fertilizers, glues, and films or used for the production of their amino acidsseine, cysteine,and poline (Papadopoulos et al. 1986; Gupta and Ramnani2006).
The present work has been undertaken with following objectives.
– Categorization of selected isolates of previous work
– Evaluation of Keratinolytic activity of these isolates
– Production optimization by employing a suitable strain
– Some practical application of keratinase such as hide de-hairing and degradation of keratin to free amino acid in solution.
A number of bacteria have been screened for keratinolysis .In this project strain MS2 has been selected for characterization.
2.1 Materials and Methods:
The study was carried out in the Department of Microbiology, Gono Bishwabidyalay, Savar, Dhaka. It was conducted during the period from 29.02.2012 to03.05.2012 at Savar region Dhaka.
2.2 Isolation of microorganism:
Samples (soil and feather) were taken from the Savar Cantonment poultry farm in the town of Savar. Serial dilution for each sample was prepared by adding 1 g of the soil sample to 9 ml of distilled water. Then serial dilution up to 10-4 was done using sterile distilled water. All the dilutions were placed on Nutrient Agar medium and incubated at 370C for 24 hours. Then the colonies appeared was checked.
2.3 Characterization:
All the collected strains were grown on Nutrient Agar medium for fresh cultures. Spore production and localization were examined by microscopic observations.
2.4 Identification of Isolated feather degrading bacteria:
The organism was identified and confirmed by carrying out tests like Gram Staining, Spore staining, Motility test, Catalase Test, Starch hydrolysis test and casein hydrolysis test.
2.5 Equipments:
Different types of sterilized equipments were used for this examination-
Glass ware-Beaker, Test tube, Slide, Thermometer, Cover slip, Plastic ware-Gloves, Micro pipette, Tips, Rake. Others-forceps, Microscope, Immersion oil, Toothpick, Autoclave, Incubator, Bacteriological loop, Needle, Burner, 70% Alcohol etc.
2.6 Chemical reagents:
Crystal violet dye, Gram’s iodine, Alcohol, Safranin,
2.7 Media:
– Nutrient agar plates (NA) (OXOID LTD.,BASINGSTOKE, HAMPSHIRE, ENGLAND)
– Nutrient broth
– Minimal medium
2.8 Method:
2.9 Experimental design:
Media & Reagent Recommended for Bacillus spp. Isolation and purification from local soil.
Collection of soil sample
Incubated overnight at 370C
| Culture on Nutrient agar |
| Bacterial colony taken in LB (liquid broth) media and Minimal media |
Incubated at 370C
Look for degradation of feather after 72hrs
| Serial dilution up to 10-6 times
and spread in agar plate |
| Colony identification and inoculation into feather containing LB media and minimal media |
Incubation at 370C overnight
Identification of feather degrading bacteria
Flow chart of feather degrading bacteria isolation from soil.
2.10 Apparatus:
2.10.1 Balance
Weighing of all chemicals was carried out in an analytical electrical balance (Model: Setra; BL-310S).
2.10.2 Sterilization
All types of media, tips, petridish, flasks, feather, eppendrof tubes, and screw capped test tubes were sterilized at 1210C, 151bs. Psi for 30 minutes in an autoclave (Model: ALP KT402). Some other glass wares (pipette, conical flask, measuring cylinder, feather, screw capped tube, eppendrof tube, etc) were sterilized in hot box oven at 200C for 2 hours.
2.10.3 Centrifuge:
To collect the supernatant from cultured liquid media centrifugation is carried out in a Dynamica VELOCITY 14R.
2.10.4. Incubator:
Bacterial cultures were incubated in an (Model: WTB binder 78532 GERMANY) incubator for overnight growth.
2.10.5. Vortex mix:
For all types of mixing, a vortex mixer (model: es DIGISYSTEM-VM-200 made in TAIWAN R.O.C) was used.
2.10.6. Laminar flow:
All inoculation was done under sterile condition, in a laminar air flow cabinet (Model: 1504 Klenzaids/Bomby).
2.10.7. Micropipette:
All types of solution were measured by the micropipette (Model: Finnpipette-W 22027).
2.10.8. Stirrer Woven
All types of media were prepared by the woven (Model: HB502made in U.K).
2.10.9. Microscope:
Gram stain and Endospore stained were detected by the microscope (ZEISS-Primo star).
2.11 Media preparation:
2.12 liquid broth (LB) media:
Liquid broth media (the oxoid manual, 3th edition P-227)
Table1: Liquid broth media composition
| Component | Gram/Liter |
| Yeast extract | 3.0 |
| Sodium chloride | 5.0 |
| Tryptone | 5.0 |
The media were sterilized by autoclaving at 1210C 15lbs. psi for 30 minutes.
2.13 Media:
Nutrient agar media (The Oxoid manual, 3th edition P-223)
Table2: Nutrient agar media
| Component | Gram/Liter |
| Yeast extract | 3.0 |
| Sodium chloride | 5.0 |
| Tryptone | 5.0 |
| Agar | 20 |
The media were sterilized by autoclaving at 1210C 15lbs. psi for 30 minutes. After weighing the media components, the final volume was made up to 1.0 liter with distilled water and the pH of the media was adjusted to 7.2 ± 0.01. Then 2% agar was added just before autoclaving the media at 15lbs, psi at 1210C for 30 minutes. After autoclaving the media, it was immediately poured into petridishes.
2.14 Minimal media
Minimal media are those that contain the minimum nutrients possible for colony growth, generally without the presence of amino acids, and are often used by microbiologists and geneticists to grow “wild type” microorganisms. Minimal media can also be used to select for or against recombinants or exo – conjugants.
Table 3: Minimal medium typically contains:
| Component | Milligram |
| KH2PO4 | 680 |
| ZnCl2 | 180 |
| Na2CO3 | 529.5 |
| CaCl2 | 735 |
| MgCl2 | 1015 |
After weighing the media components, the final volume was made up 5µM/ml. Autoclaved the media at 15lbs, psi at 1210C for 30 minutes.
2.15 Isolation of bacteria from soil sample:
The soil sample was collected from the poultry wastes of Savar cantonment in Savar, Dhaka and part of it was transferred to a sterile petridish and mixed in sterile water by using class rod. After a few minutes when the soil settled down, the surface water (which contained the soil bacteria) was diluted serially up to 10-4 times with sterile distilled water and spread in Nutrient agar plate. This bacterial mix was grown at 370C overnight. At the next day bacterial culture was again grown on Nutrient agar plate for single colony measured. Then the single colony was inoculated to 10ml of Nutrient broth and 5µM/ml of minimal media with 5cm feather. This feather containing Nutrient broth and Minimal media kept at 370C for 72hrs and for complete degradation of feather. One media was used for as control.
2.16 Cultural characteristic observations
Bacterial cells were transferred from preserved culture to fresh nutrient agar plate. After overnight incubation growth from the plates were again transferred to second set of Nutrient agar plate to observe the actual growth patterns on culture plates.
2.17 Microscopic observation
The following technology were employed for microscopic observation –
a. Gram staining of the bacteria
Gram staining was performed by standard gram method.
b. Spore staining of bacteria
After fixation of bacterial smear on glass slides, the slides were placed on a stand over a boiling water bath. Then 5% Melachite green was added and allowed to stay on each slide for 5minutes. The slides were then washed gently with tap water and observed under microscope for presence of spore.
2.18 Morphological characterization by Gram’s staining method:
Preparation of Gram’s stain solution
Crystal violet solution
Crystal violet …………………….10.0gm
Ethyl alcohol (95%0………………1000ml
Working crystal violet solution
Twenty milliliter of crystal violet stock solution was mixed with 80 ml of stock oxalate solution. Working solution was made required.
Lugol’s iodine solution
Iodine crystal …………………..1gm
Potassium iodine……………….2gm
Dissolved completely in 10ml distilled water and then added to distilled water to make 300ml stored in amber bottle.
Acetone alcohol
Ethyl alcohol……………………250ml
Acetone…………………………250ml
Safranine stock solution
Safranine……………………..…2.5ml
Ethyl alcohol………………..….100ml
Safranine working solution
The stock safranine was diluted 1:4 with distilled water.
Gram’s staining procedure
The representative bacillus sp. colonies were characterized microscopically using Gram’s stain according to the method described by Merchant and Packer,(1967)
· The procedure was as follows:
· A small colony was picked up with a bacteriological loop, smeared on a glass slide and fixed by gentle heating.
· Cover with CRYSTAL VIOLET for one minute (PRIMARY STAIN) and then washed with running water.
· Cover with GRAM’S IODINE for one minute. (MORDENT) Pour off the Gram’s iodine.
· Run 95% ETHYL ALCOHOL down the slide until the solvent runs clear (about 10-20seconds) this step is critical! Thick smears require more time thin once (DECOLORIZING AGENT) Rinse with water to stop the action of the alcohol.
· Cover with SAFRANINE for 20 seconds. (COUNTER STAIN) Gently rinse off the stain with water. Blot with bibulous paper and clean off the bottom of the slide with 95% alcohol.
· Examined the slide under light microscope with high power objective (x100) using immersion oil.
2.18 Biochemical characteristics observation:
Following biochemical tests were performed for the identification of the strains.
2.18.1 Carbohydrate test:
Each phenol-Red carbohydrate fermentation broth contains carbohydrate, phenol-Red, Durhamn tube. The carbohydrate testes sugars were –glucose, lactose, dextrose. Glucose (10gm), lactose (1.15gm), dextrose (0.65) and 10ml D/W were added with phenol red broth base media for different sugar test. The medium was sterilized at 1210 C for 30 minutes. All carbohydrate test media were test media were inoculated with organism with transfer loop from 24-48 hours culture grown in Nutrient agar media and incubated at 370C for overnight.
Inoculate the tube of tryptone broth with a small amount of a pure culture. Incubate at 35°C (+/- 2°C) for 24 to 48 hours.
2.18.2 Indole production test:
This test determines whether the microbe produces indole from the amino acid tryptophan. There are two media that are used for this test: Sulfide-Indole-Motility (SIM) medium and Tryptone broth medium. This program uses the Tryptone broth medium.
SIM is a nutrient medium which allows the detection of three different traits in bacteria: it contains sulfates to serve as the substrate for detecting sulfide (H2S) production; abundant tryptophan as a substrate for indole production; and its content of 0.5% agar limits bacterial swimming, thereby allowing detection of motility.
Tryptone broth serves the same purpose as SIM as far as indole production is concerned, and the means for conducting the test and interpreting results remain the same as SIM.
To test for indole production, add 5 drops of Kovács reagent directly to the tube (3, 5).
A positive indole test is indicated by the formation of a pink to red color (“cherry-red ring”) in the reagent layer on top of the medium within seconds of adding the reagent
if a culture is indole negative, the reagent layer will remain yellow or be slightl cloudy.
2.18.3 Citrate Utilization Test (Simmons):
The purpose is to see if the microbe can use the compound citrate as its sole source of carbon and energy for growth. If a microbe can use citrate for carbon and energy, it will grow on Simmons citrate agar. The use of citrate leads to a rise in pH of the medium, and a pH indicator changes color.
The medium used is Simmons citrate agar slant. It contains mineral salts, sodium citrate for carbon, and ammonium phosphate for its nitrogen source. The pH indicator is bromo thymol blue, which is green at neutral pH, yellow at acidic pH <6.0 and turns blue at alkaline (basic) pH >7.6.
An inoculum from a pure culture is transferred aseptically to a sterile tube of Simmons citrate agar. The inoculated tube is incubated at 35-37 C for 24 hours and the results are determined. Abundant growth on the slant and a change from green to blue in the medium indicates a positive test for growth using citrate.
2.18.4 Nitrate reductase test:
Nitrate reduction test was carried out in nitrate broth. The freshly prepared cultures were inoculated in sterile nitrate broth containing tubes and incubated at 370C for 24 hrs. at the end of incubation 0.1 ml of solution A was added followed by solution B in equal volume. The appearance of deep pink color showed the positive result.
2.18.5 Catalase test:
Each one slide was sterile by the alcohol then one drop of 30% H2O2 was placed on a slide. One loop-full of fresh culture was taken by a sterile needle and placed on the drop of H2O2 and bubble production indicated positive result.
2.18.5 Starch hydrolysis test:
The purpose is to see if the microbe can use starch, a complex carbohydrate made from glucose, as a source of carbon and energy for growth. Use of starch is accomplished by an enzyme called alpha-amylase.
Hydrolysis of starch was carried out of 10gm soluble starch in 100 ml distilled water was heated in water bath until dissolved. 20 ml of this solution was mixed with 100 ml of melted nutrient agar and poured in the petridish after sterilization. A loop-full of fresh bacterial culture was picked up by the sterile needle and strick on to the agar plate; after 24 hrs of incubation at370C, the plate was flooded with dilute iodine solution.
2.18.6 Ninhydrin Test
Ninhydrin (2, 2-Dihydroxyindane-1,3-dione) is a chemical used to detect ammonia or primary and secondary amines. When reacting with these free amines, a deep blue or purple color known as Ruhemann’s purple is produced.
Procedure-
Add about 2 mg of the sample to 1 mL of a solution of 0.2 g of ninhydrin (1, 2, 3indanetrione monohydrate) in 50 mL of water. The test mixture is heated to boiling for 15-20 sec; this reaction is important not only because it is a qualitative test, but also because it is the source of the absorbing material that can be measured quantitatively by an automatic amino acid analyzer. This color reaction is also used to detect the presence and position of amino acids after paper chromatographic separation.
Positive Test
A blue to blue-violet color is given by a-amino acids and constitutes a positive test. Other colors (yellow, orange, red) are negative.
RESULT AND DISCUSSION
4.1 Isolation of feather degrading bacteria:
The soil sample was collected from an area in Savar, Dhaka where poultry feathers were dumped. The soil was processed according to the method described in section.
Table-1: Overall cultural, microscopic and biochemical characteristic of the isolated bacteria.
| Test performed | Observation | Result |
| Strick plate isolation | ||
| NA at 370C | White colonies | Positive |
| Microscopic observation | ||
| Gram stain | Small reddish pink colonies singly | Gram negative rods |
| Spore stain | Green color appeared | Spore forms |
| Biochemical test | ||
| Catalase test | No bubble formed | Negative for catalase production |
| Carbohydrate fermentation test: | ||
| Glucose
Lactose Dextrose |
Color change
Color change Color change |
Positive
Positive Positive |
| Indol (SIM) test | No red colored layer form | Negative for indole |
| Citrate test | No Change in color | No citrate utilization |
| Nitrate test | Change in color | Positive for nitrate reduction |
| Voges-proskauer test | Color change | Positive for acetoin production |
| Starch test | Slight clear zone of hydrolysis | Positive for starch hydrolysis |
| Ninhydrin Test | Color change | Positive for amino acid |
Collectively, these characteristics indicated that these two isolates were of genus Bacillus.
3.4 Feather – degrading activity of the organisms:
The bacteria were grown in the liquid media and minimal media 14 hours at 37C. The growth of the bacteria was very good but feather degradation activity was satisfactory even after 72 hours.
3.5 Production of Keratinase by isolates of Bacillus sp.
A comparative study on the production of keratinase was carried by one strain of Bacillus in minimal media. Here only feather was carbon source but that organism could grow in this media.
Figure: Graphical arrangement keratinase activity/day.
Figure- Ninhydrin Test
Figure2.0 – Glucose Figure2.0- dextrose
Figure 2.0- lactose
Figure 1.9 – Spore stain salt
Figur1.9 – Spore stain salt
Figure 1.3: Colonial features for keratin degrading bacteria
Figure 1.4: Colonial features from Minimal media to Nutrient agar
Figure2.1- Citrate negative
Figure 2.1 -Indole, citrate, nitrate, VP
Figure 2.2 -starch hydrolysis test
Figure 2.1- Citrate negative
Fig 1.7: Microscopic Examination of Gram stain (NB)
Fig 1.8: Microscopic Examination of Gram stain (Minimal media)
Fig1.5: Control of Nutrient broth with feather no change after 15 days
Fig 1.6: Complete degradation of feather
DISCUSSION
A bacterium isolated from poultry waste has been shown to degrade feather keratin. The bacterium isolated from anaerobic habitat, however,showed maximum growth under condition ,as would be expected of a number of the family bacilaceae (William et al.,1990). Bacillus sp. have been reported to produce keratinolytic protease (Lin et al.1992; Kim et al.2001; Lee et al.2002; Savitha G. Joshi et al.2007). The induction of keratinolytic enzyme produced by the species of Bacillus with feather powder was reported (Cheng et al.1995; Lal et al.1999). The presence of this species in a poultry waste may be that the bacterium is indigenous to the chicken gut. The most studied keratinolytic bacteria are Bacillus lichinoformis which have been described to possess feather degrading activity.
CONCLUSION
Bioconversion of feather with Bacillus sp. has great potential to protect our environment. This novel keratinolytic isolate could be a potential candidate for the degradation of feather keratin and also in dehairing process in leather industry and can be used as additives in poultry field . Through the strategy of isolation of Keratinolytic microorganisms utilized in this work, bacteria presenting high Keratinolytic activity were selected. Considering that feather protein has been showed to be an excellent source of metabolizable protein (Klemersrud et al., 1998) and that microbial Keratinases enhance the digestibility of feather keratin (Lee et al., 1991), these Keratinolytic strains could be used to produce animal feed protein. In addition, the selected isolates were able to grow and display Keratinolytic activity in keratin wastes (feathers). Utilization of these potential keratin degraders will definitely find biotechnological use in various industrial processes involving keratin hydrolysis. It would also solve the waste disposal problem of poultry waste and with limited resources recycling of Keratinacious waste would be beneficial financially and environmentally.
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Abbreviation
0C Degree Celsius
cm Centimeter
e.g. For example
et.al. And others
etc. And the rest
Fig. Figure
G Gram
Xg Centrifugal field
Hr. Hour
i.e. That is
KDa Killo Daltons
I Liter
Ib Pound
M Molar
MT Metric ton
Mg Milligram
mM Millimolar
No. Number
NA Nutrient agar
r.p.m. Rotation (Revolution) per minute
sec. Second
sp. Species
vol. Volume
w/v Weight in volume
% Percent
µg Microgram