ecofriendly disposal of feather waste by...

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ECOFRIENDLY DISPOSAL OF FEATHER WASTE BY USING KERATINOPHILIC SOIL FUNGI AND

FEASIBILITY OF USE OF FEATHER ASSAY MIXTURE AS MANURE IN AGRICULTURAL PRACTICES

By

Dr. (Mrs.) Y. AVASN Maruthi Principal Investigator

Department of Environmental Studies GITAM Institute of Science, GITAM University

Visakhapatnam – 530 045

FINAL REPORT SUBMITTED TO

UNIVERSITY GRANTS COMMISSION BAHADURSHAH ZAFAR MARG

NEW DELHI

December, 2012

ANNEXURE - III

UNIVERSITY GRANTS COMMISSION BAHADUR SHAH ZAFAR MARG

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NEW DELHI-110 002

Final Report of the work done on the Major Project

1. Project report No.1st/2nd/3rd/Final : Final

2. UGC Reference No : F.No 32-623/2006 (SR) Dated 02-03-2007

3. Period of Report : From 01-04-2007 to 01-11-2010

4. Title of research project : “Eco-Friendly Disposal of Feather Waste

by

Using Keratinophilic Soil Fungi and

Feasibility of Use of Feather Assay

Mixture

as Manure in Agricultural Practices.”

5. a) Name of the principal Investigator : Dr.(Mrs.)Y.AVASN Maruthi

b) Dept and University/College

Where work has progressed : Department of Environmental Studies

GITAM Institute of Science

Gandhi Institute of Technology and

Management (GITAM), Visakhapatnam-

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6. Effective date of starting of the project: 01-04-2007 (grant received date)

7. Grant approved and expenditure incurred during the period of the report:

Total amount approved in Rs.

Total amount Released in Rs Total Expenditure in Rs (Including overhead charges)

9,48,600.00 9,06,400.00 9,69,643.00

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Final Report

Objectives: The following three objectives have been taken up in this research.

i) To screen, isolate and identify the Keratinophilic fungi from soils rich with

keratin waste in and around Visakhapatnam.

ii) To screen potent Keratinophilic and Keratinolytic Fungi by conducting

degradation studies against feather.

iii) To suggest the best geophilic keratinophilic fungi that can be implemented

for degradation of feather waste in an eco-friendly way.

Methodology: Study Area: Visakhapatnam

Visakhapatnam is a major port and the second largest city in the state of Andhra

Pradesh with coordinates 17°42′N 83°15′E and the third largest city on the east coast

of India after Kolkata and Chennai, with a population of approximately 1.3 million

and area of 550 km². It is primarily an industrial city, apart from being a port city. It is

located 625 kilometres (388 mi) east of state capital, Hyderabad. The city is nestled

among the hills of the Eastern Ghats and faces the Bay of Bengal to the east. It is the

administrative headquarters of Visakhapatnam district and is also home to the Eastern

Naval Command of the Indian Navy. Visakhapatnam is often referred to as The Jewel

of The East Coast or The City of Destiny and is sometimes referred to as the "Goa of

the East Coast." Just like its west coast counterpart, it has beautiful virgin beaches,

laterite hillocks, smooth roads and stunning landscape. Health-tourism is a fast

developing industry. Visakhapatnam has seen rapid development in the past decade.

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Fig..1 Map showing sampling points in the study area Visakhapatnam (www.google.com)

Visakhapatnam experiences a tropical savanna climate according to Köppen climate

classification (Rubel & Kottek 2010) with little variation in temperature through the

year. May is the hottest month with average temperatures around 32°C (90°F), while

January is the coolest month with average temperatures near 23°C (73°F). As the city

is located on the Bay of Bengal, the humidity remains high throughout the year. The

total annual rainfall is around 945 mm (38 inches), the bulk of which is received

during the south-west monsoon. October is the wettest month with around 204 mm (8

inches) of rainfall. The months from November to February are the best times to visit

the city, with moderate temperatures and little precipitation.

Sampling Areas

Seven categories of areas were selected to study the distribution of Keratinophilic

Fungi the respective soils, in the city of Visakhapatnam. They were Slaughter houses

(S1), Meat markets (S2), Feather waste dumping sites (S3), Sewage (S4), Chicken

stalls (S5), Polluted beach sands (S6) and Poultry farms (S7). Garden soil was used as

control. Five sites for each area and ten samples from each site were studied, which

makes about three hundred and fifty samples for the current study of distribution in

and around the city of Visakhapatnam. The details of the each study area were as

follows.

Slaughter houses (S1): The city has only one official and bigger slaughter house,

‘The sheep and goat slaughter house (S1-1), Municipal Corporation, Visakhapatnam’

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located near Diary farm, Hanumanthwaka, NH-5. Here about 400 sheep and goat are

being slaughtered per day. For the study, the remaining four areas (S1-2 to S1-5) were

unofficial sites, located in some abandoned locations in the city, where about fifty

sheep and goat were slaughtered per day.

Meat Markets (S2): Meat markets with the flow of around 200 people per day were

chosen for the study. They were located at MVP colony (S2-1), Nehru Bazar –

Akkayyapalem (S2-2), Diary Farm – Ravindra Nagar – NH-5 (S2-3), Sriharipuram

(S2-4) and Old Gajuwaka (S2-5).

Feather waste dumping sites (S3): Apart from collective poultry slaughter waste

dump sites for entire city, these feather waste dumping sites were different, which

were small and where mostly dry feather along with a little slaughtered waste was

dumped regularly after processing of fowls for chicken from the surrounding chicken

vendors. Selected sites for present study were located Beside NH-5, Opp. Visakha

Valley School Road (S3-1); Beside Simhachalam Road, Near Mudasarlova (S3-2);

Beside Beach Road, Opp. Senora Beach Resorts, Sagarnagar (S3-3); Beside

Simhachalam road, Prahladapuram (S3-4) and Beside NH-5, Near Gajuwaka (S3-5).

Sewage sludge (S4): Soil samples around open sewage sludge were collected from

five locations viz. Sewage Treatment Plant, Appughar (S4-1); Near Gnanapuram (S4-

2); Two different locations in Port Area (S4-3 & S4-4) and Near Kurupam Market, I-

Town Area (S4-5).

Chicken stalls (S5): Chicken vendor stalls whose regular input of people was not less

than 100 per day were selected for the present study. Soils around those stalls located

at five different locations like Sagar Nagar (S5-1), MVP Colony (S5-2), Pedawaltair

(S5-3), Ramnagar (S5-4) and Poorna Market (S5-5).

Polluted beach sands (S6): These were the locations near sewage outlets as well as

waste accumulated sites along the beach of Visakhapatnam. They were Near Fishing

Harbour (S6-1), RK Beach (S6-2), Backside of VUDA Park (S6-3), Sagarnagar (S6-

4) and Rushikonda (S6-5).

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Poultry farm soils (S7): Visakhapatnam has considerably large poultry farms in the

city. Farms with reared fouls not less than 2000 were selected for the present study.

Soils inside as well as around five such farms were collected, which were located at

Dairy farm, Visalakshinagar (S7-1); Near beach road, MVP double road (S7-2);

Anandapuram (S7-3); Adavivaram (S7-4) and Kothavalasa (S7-5).

Soil collection at sampling sites

Early in the morning, 5:00 am to 6:30 am was selected as sampling time for all

sampling sites, as all the organism are supposed to be in actively growing stage by the

incubation and germination effect of previous night due to darkness as well as low

temperatures. Sampling at all sampling sites was done in triplicates for all samples.

Soil was collected in quantities of 250 g (approx.) each in self closed

polythene bags. Sampling was done for surface soil as well as at a depth of 10 cm by

digging with a sterile tool. Three samples were collected at each site with distance of

not less than five meters to each other. Proper care was taken while collecting the soil

by wearing hand gloves and face masks as those samples may contain highly

pathogenic fungi which cause mycoses in humans.

For sewage polluted beach sand soils, sampling was done at the places where

sand was in contact with the sewage drains as well as at the diameter of 10 meters

around the site.

While sampling at feather waste dumping sites much care was taken to avoid

direct contact with skin. The upper feather waste layers were removed to expose the

bottom soil where anaerobic degradation was observed. Both dry and wet soils were

collected from those sites.

For all sampling areas, garden soil was taken as control for the physico-

chemical analysis.

Soil samples after collection, from the site, were transported carefully to

laboratory where they were analyzed immediately as well as stored at 4°C for further

analysis. However they were stored not more than 3 days.

Physico-Chemical Analysis of Soil Samples

All collected soil samples were analyzed for the following Physico-Chemical

and microbial parameters (Jackson 1979).

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1. pH

2. Electrical conductivity (mMhos)

3. Water Holding Capacity (%)

4. Moisture content (%)

5. Organic Carbon (%)

6. Soil Humus (%)

7. Nitrates (mg/g)

8. Total Nitrogen (%)

9. Phosphorus (g/kg)

10. Potassium (g/kg)

11. Sulphates (mg/g)

Identification of Keratinophilic Fungi

Once it was ensured that the test fungus was Keratinophilic fungus, from positive

result from feather bait technique, identification was done up to species level, which

has three levels – i) Morphological and ii) Biochemical iii) Gene sequence analysis.

The followings methods were included

Morphological Identification

Colony Morphology

Lactophenol Mounts

Biochemical Identification

Rice Grain Test (McGinnis 1980)

Urea Hydrolysis

Vitamin Studies (McGinnis 1980)

Gene Sequence Analysis

Genomic DNA extraction from given fungal pellet

PCR Procedure

Agarose gel electrophoresis

Biodegradation Studies

Experimental Setup

Seven Keratinolytic fungi (Two species of non-dermatophytes and Five

species of dermatophytes) namely Gibberella intermedia (Anam. of Fusarium

proliferatum), Fusarium sp. F42, Chrysosporium tropicum, Microsporum canis, M.

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gypseum, Trichophyton mentagrophytes and T. terresre were selected to assay for

their potential for feather waste degradation. Apart from the above five species of

dermatophytes, non-dermatophytic species of Fusarium were taken as they were

prevalent in human and animal habitations and known to be associated with keratin

degradation (Oyeka & Gugnani 1998). They were selected based on their occurrence

from the distribution studies and due to their keratinolytic activity among others. Pure

cultures of three dermatophytic strains were procured from IMTECH – MTCC,

Chandigarh (Codes: MTCC1367-C.tropicum, MTCC2820-M.canis, MTCC2819-

M.gypseum) while Trichophyton sps. were not available for procurement.

Medium

Potato Dextrose Agar medium (PDA) (Potatoes (Scrubbed and Diced)-200.0g;

Dextrose-20.0g; Agar-15.0g; Distilled Water- 1000ml; pH-5.6) was used to culture

and maintain all strains while the same without agar (Potato Dextrose Broth) was

used to obtain spore suspensions from the respective cultures. All conditions used

were aerobic.

Mineral medium (Di-Potassium hydrogen O-phosphate - 1.500g; Magnesium sulfate -

0.050g; Calcium chloride - 0.025g; Ferrous sulfate - 0.015g; Zinc sulfate - 0.005g;

Distilled water - 1000ml; Sterile defatted feather - 10g; pH - 7.5), at aerobic

conditions was used for assay for keratin degradation. Antibiotic Chloramphenicol,

which is also heat resistant, was added at the rate of 100mg/L (Mueller et al. 2004) to

prevent bacterial contamination.

Substrate: Feathers were defatted by soaking them in Methanol-Chloroform mixture

(1:1) for 48 hours, washed with water and air dried. Cut strands of 3 – 4 cm length of

defatted feathers were used as substrate.

Screening for Keratin Degradation

100 ml of the prepared mineral medium with defatted feather was distributed in 250

ml Erlenmeyer’s flasks and were sterilized at 121°C for 20 min. The liquid media

were inoculated with 1 ml of spore suspension from the test fungi and incubated at

room temperature in static condition for a period of 60 days. During the period of

incubation, samples were drawn at regular intervals from their respective flasks of 10

days and analyzed to study the progress of feather degradation. All experiments were

carried out in triplicates.

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The culture was filtered with Whatman No. 1 filter paper and the collected filtrate was

used to assess –

• Changes in pH

• Estimation of total protein (Bradford 1976)

• Determination of Keratinolytic activity (Yu et al. 1968)

• Nitrate (Goldsmith et al. 1973)

• Nitrite (Hewitt et al. 1975)

• Cystein and Cystine (Rama Krishna et al. 1979)

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Final Report Enclosure II

WORK DONE

• Project fellow was appointed on 1st November 2007.

• Equipment (DIGI 3 Labomed Digital Research Microscope and Orbital

Shaking Incubator)were procured in April, 2008

• Chemicals and Glassware were procured in the month of April 2008

• Sampling: Collection of soil samples for screening of Keratinophilic

Fungi from soils of Slaughter houses (S1), Meat markets (S2), Feather

waste dumping sites (S3), Sewage (S4), Chicken stalls (S5), Polluted

beach sands (S6) and Poultry farms (S7) was carried out.

• Keratinophilic fungi were identified by using Vanbreuseghem’s Hair

Bait Technique (Orr 1969) from various isolates, which were pure

cultured for further experimentation.

• Biodegradation experimentations were set up to find potential of

keratinophilic and keratinolytic fungi to degrade feather substrate among

others based on analysis for feather substrate degradative products in the

post assay mixture

• Post degradation extracts were applied on seed germination in

combination with soil to test the effect on plant growth as well as

feasibility to use in agricultural practices.

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Research Papers Published

1. AVASN Maruthi, Y., Aruna Lakshmi, K., Ramakrishna Rao, S. & Apta Chaitanya, D., 2011. Degradation of Feather and Hair by Chrysosporium tropicum – A Potent Keratinophilic Fungus. African Journal of Biotechnology, 10(18), 3579-3584. Impact Factor: 0.565; SCI Indexed

Abstract:

The current study was aimed at the degradation of feather and hair wastes in

an eco-friendly way, which should further be helpful to make the waste dumping soils

fertile. Degradation of feathers and hair was assessed by a highly potent keratinophilic

fungi namely Chrysosporium tropicum. The 60 day experiment was set up with sterile

defatted feather and hair as substrates in a mineral medium along with the inoculum

of the organism. The culture filtrate was analyzed at every 10 days interval, for the

release of catabolic products such as protein and keratinase, along with the

concomitant increase in

pH. Maximum degradation was found at the 40th day sample, where the protein

released was 6.9 mg/ml and the keratinolytic activity was 8.56 KU/ml. There was

increase in pH (from neutral) towards alkalinity up to 40 days (9.0) of incubation and

decline thereafter, indicating the maximum release of soluble protein into the

medium. Among the two substrates used, C. tropicum had more effect on hair than

that of feather.

2. Maruthi, Y.A., Lakshmi, K.A., Rao, S.R., Hossain, K., Chaitanya, D.A. &

Karuna, K., 2008. Dermatophytes and other fungi associated with hair-scalp of Primary school children in Visakhapatnam, India: A Case Study and Literature Review. The Internet Journal of Microbiology, 5(2) – an online publication. Scopus Indexed

Abstract:

A total of 2804 primary section pupils aged 6-15 years of 12 schools located at

different places in Visakhapatnam were physically screened for hair -scalp infection.

Three hundred and thirty six (11.98%) of these children were positive for the

dermatophytic infection. The majority of the isolated dermatophytes according to

percentage of occurrence were Microsporum audouinii (18.88%), Chrysosporium

keratinophilum (16.66%), Trycophyton mentagrophytes (13.33%) and Trycophyton

terrestre (3.33%). Microsporum audouinii, Chrysosporium keratinophilum,

Trycophyton mentagrophytes were the most frequently isolated dermatophytes. Other

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skin mycoses isolated include Fusarium moniliforme (6.66%), Aspergillus flavus

(5.55%), Fusarium oxysporum (5.55%) and Penicillium funiculosum (4.44%).

Infection was mainly due to Microsporum audoinii, Chrysosporium keratinophilum

and Trichophyton mentagrophytes .Infected domestic animals constituted the apparent

source of infection for most pupils. Playgrounds of children and animal fields were

also source of infection for children and animals

RESULTS Physico-chemical Properties of Soil, Sampled for Keratinophilic Fungi

The soil that studied was initially characterized in order to assess its fertility

status before the application of post feather degradation extract. Table 4.1.1 shows the

values of total of seven soil types and one control that have chosen for the screening

for the occurrence of keratinophilic fungi. They were the soils of Slaughter Houses

(S1), Meat Markets (S2), Feather Waste Dumping Sites (S3), Sewage Sludge (S4),

Chicken stalls (S5), Polluted Beach Sands (S6), Poultry Farms (S7) and Garden Soil

(C) which was taken as control.

Among them, sewage sludge was proved to be alkaline than the others with

their pH value 9.83 ± 0.03 while feather waste dumping soils were shown to be acidic

with pH 5.96 ± 0.02. Electrical conductivity of these soils in the above said order was

0.12 ± 0.01, 0.16 ± 0.03, 0.65 ± 0.02, 0.975 ± 0.02, 0.62 ± 0.01, 0.69 ± 0.01, 0.43 ±

0.02 and 0.05 ± 0.01 milli Mohs respectively. The percentages of water holding

capacity and moisture content indicated natural texture of those samples, unaltered, by

showing higher values of water holding capacity for sewage sludge (65%) and least

for the polluted Beach sands (12 %). Their values are 36 ± 3, 48 ± 2, 31 ± 3, 65 ± 1,

56 ± 2, 12 ± 2, 40 ± 1 and 29 ± 1 % for water holding capacity and 6.3 ± 0.8, 8.3 ±

0.5, 7.5 ± 0.7, 12.6 ± 0.9, 6.8 ± 0.5, 3.4 ± 0.7, 7.5 ± 0.5 and 19.3 ± 0.6 % for moisture

content of the above soil samples respectively.

Coming to soil organic carbon and soil humus, they were well in proportional

with each other. Soil organic carbon for all the eight samples in the above order (S1,

S2, S3, S4, S5, S6, S7 and C) were as follows: 3.66 ± 0.4, 4.2 ± 0.5, 5.9 ± 0.4, 3.98 ±

0.3, 4.05 ± 0.5, 3.72 ± 0.5, 4.45 ± 0.6 and 10.5 ± 0.3 % d.w. respectively. Percentages

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of soil humus were 17.51 ± 0.2, 18.1 ± 0.1, 16.4 ± 0.1, 18.2 ± 0.1, 11.6 ± 0.2, 17, 8 ±

0.1, 17.3 ± 0.2 and 0.14 ± 0.2 respectively.

Analysis of total nitrogen, phosphorous and potassium revealed poor

nutritional status and imbalanced ratios of those polluted soils especially of feather

dumping sites and polluted beach sands. Their values in order were as follows: 2.4 ±

0.1, 2.8 ± 0.3, 2.6 ± 0.2, 2.7 ± 0.2, 2.5 ± 0.1, 1.3 ± 0.1, 2.0 ± 0.2 and 1.2 ± 0.1 % of

Total Nitrogen; 1.87 ± 0.03, 1.75 ± 0.04, 1.64 ± 0.03, 0.72 ± 0.02, 1.52 ± 0.04, 0.9 ±

0.03, 1.65 ± 0.02 and 1.18 ± 0.02 g/kg of Phosphorous; 5.6 ± 0.1, 6.3 ± 0.2, 5.9 ±

0.2, 4.2 ± 0.2, 5.0 ± 0.1, 4.0 ± 0.1, 5.3 ± 0.2 and 2.24 ± 0.2 g/kg of Potassium

respectively for all the eight samples.

Distribution of Keratinophilic Fungi in and around Visakhapatnam

Many numbers of Keratinophilic Fungi from seven types of soils were

identified based on feather baiting technique. They include two kinds of organisms –

dermatophytes and non-dermatophytes. However, all the soil types have shown

dermatophytes as dominating species than the others.

Soils of Slaugther Houses:

Total of 50 samples from 5 different Slaugther Houses (Table-4.2.1 to Table

4.2.5) were observed to be comprised of 31 species belong to 13 genera where 12

dermatophytic species of 4 genera and 19 non-dermatophytic species of 9 genera

occurred.

Among all, as shown by Fig-4.2.1, highest percentage was shown by Chrysosporium

tropicum (7%), Microsporum canis (7%) and M. gypseum (7%) while Trichophyton

mentagrophytes and T.terrestre came next with 6%. 5% occurrence was shown by

C.indicum, T.ruburm and Aspergillus niger where C. keratinophilum, A.flavus,

A.fumigatus, Fusarium solani and Pencillium citrinum occupied 4% each. Other fungi

sharing the rest of the 32% include genera of Arthroderma, Geomyces

(dermatophytes) and Cladosporium, Geotrichum, Rhizopus, Scopulariopsis (non-

dermatophytes) along with some non-sporulating fungi.

Relative Importance Values (RIV) of each and every organism, places them in

the order of dominance in each of the slaughter houses as follows. Top five species of

Slaughter House 1 were – M.gypseum (86.74), A.niger (86.45), C.tropicum (86.15),

T.mentagrophytes (86.15), F. solani (84.10) and M.canis (83.81); Slaughter House 2

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were – A.niger (86.94), C.tropicum (78.79), M.gypseum (78.33), Geotrichum

candidum (75.09) and M.canis (74.62); Slaughter House 3 were – A.fumigatus

(84.65), P.citrinum (76.51), F.solani (75.11), C. keratinophilum (73.72) and A.niger

(73.25); Slaughter House 4 were – M. canis (87.69), C.keratinophilum (73.84),

T.rubrum (65.55), C. indicum (64.70) and M. gypseum (56.41); Slaughter House 4

were – T.rubrum (66.79), C.keratinophilum (64.85), C. indicum (57.28), A.niger

(53.88), G.candidum (52.42).

Soils of Meat Markets:

26 species of 13 genera were identified in total of 50 soil samples of 5

different meat markets (Table-4.2.6 to Table-4.2.10). 11 dermatophytic species of 5

genera and 15 non-dermatophytic species of 8 genera were among them.

M.gypseum (8%), T.rubrum (8%) and T.terrestre (8%) were dominants (Fig-

4.2.2) while the others likes C.tropicum (7%), T.mentagrophytes (7%), C.indicum

(6%), C.keratinophilum (6%), M.canis (4%), A.flavus (4%), A.fumigatus (4%),

Scopulariopsis bravicaulis (4%) and Gemomyces pannorum (3%) were next to them.

The remaining 31% of others include genera Arthroderma, Cladosporium, Fusarium,

Geotrichum, Rhizopus, Penicillium and Trichoderma along with few non-sporulating

ones.

Sampling site wise study of all the 5 sites revealed the order of dominance of

each site as follows, based on their RIVs. Meat Market 1: C.indicum (88.71) >

C.tropicum (79.54) > A.niger (74.97) > M.gypseum (69.54) > T.mentagrophytes

(67.05); Meat Market 2: C.tropicum (68.01) > C. indicum (65.18) > Trichoderma

viridae (65.18) > T.mentagrophytes (59.43) and M.gypseum (59.43) > T.rubrum

(56.13) > A.flavus (54.71); Meat Market 3: T.terrestre (78.13) > A.fumigatus and

Scopulariopsis bravicaulis (74.06) > T.rubrum (68.94) > C.keratinophilum (68.94) >

Geomyces pannorum (64.87) > A.versicolor (63.65) and Trichoderma sp. (63.65);

Meat Market 4: C.keratinophilum (86.22) > C.indicum (78.29) > T.rubrum (77.05) >

A.flavus (74.97) > C.pannicola (72.90); Meat Market 5: M.gypseum (81.4) >

T.rubrum (60.4) > T.terrestre (56.96) > M.canis (55.97) and C.keratinophilum (55.97)

> A.fumigatus (54.47) and A.niger (54.47).

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Soils of Feather Dumping Sites:

Total of 50 samples from 5 different Feather Dumping Sites (Table-4.2.11 to

Table 4.2.15) were observed to be comprised of 23 species belong to 11 genera where

12 dermatophytic species of 5 genera and 11 non-dermatophytic species of 7 genera

occurred.

Among all, as shown by Fig-4.2.3, highest percentage was shown by

Chrysosporium indicum (10%), C.tropicum (9%) while C.keratinophilum,

M.gypesum, T.mentagrophytes, T.rubrum and T.terrestre came next with 8%. 7%

occurrence was shown by M.canis where A.flavus occupied 4%. Other fungi sharing

the rest of the 30% include genera of Chrysosporium state of Ctenomyces,

Gymnoascus (dermatophytes) and Fusarium, Geotrichum, Penicillium, Scopulariopsis

and Trichoderma (non-dermatophytes) along with some non-sporulating fungi.


the order of dominance in each of the Slaughter Houses as follows. Top five species

of Feather Dumping Site 1 were – C. keratinophilum and M.canis (78.25),

C.pannicola (75.28), A.flavus (74.29), T.rubrum (69.57), C.indicum and

T.mentagrophytes (68.25); Feather Dumping Site 2 were – A.fumigatus (73.83),

C.indicum (70.72), C.tropicum (69.19) M.canis (68.04), and M.gypseum (67.66);

Feather Dumping Site 3 were – T.terrestre (89.52), C. indicum (83.09), C. tropicum

and C. keratinophilum (79.12), M. gypseum (69.52) and T.mentagrophytes (67.53);

Feather Dumping Site 4 were – C. tropicum (80.76), A.flavus (75.38), C. indicum

(71.15), T.mentagrophytes (69.23) and T.terrestre (67.69); Feather Dumping Site 5

were - T.terrestre (88.60), C. tropicum and T.mentagrophytes (78.94), T.rubrum

(76.95), M. gypseum (75.96), Gymnoascus petalosporus (74.30).

Sewage Sludge:

28 species of 12 genera were identified in total of 50 soil samples of 5

different meat markets (Table-4.2.16 to Table-4.2.20). 14 dermatophytic species of 6

genera and 14 non-dermatophytic species of 6 genera were among them.

T.ajelloi (9%), T.mentagrophytes (8%), A.versicolor (7%), T.rubrum (8%) and

T.terrestre (8%) were dominants (Fig-4.2.4) while the others likes Aphanoascus

keratinophilus, C. indicum, M.gypseum, T.rubrum and Aspergillus flavus with 5%,

Arthroderma quadrifidum, C.keratinophilum, T.terrestre and Fusarium oxysporium

with 4%, C.tropicum, M. canis and Aspergillus fumigatus with 3% were next to them.

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The remaining 26% of others include genera Aspergillus, Cladosporium, Geotrichum,

Penicillium and Trichoderma along with few non-sporulating ones.

Sampling site wise study of all the 5 sites revealed the order of dominance of

each site as follows, based on their RIVs. Sewage Sludge 1: T. ajelloi (88.75) >

C.indicum (88.29) > T.mentagrophytes (80.59) > M.gypseum (78.29) and A.versicolor

(78.29) > Aphanoascus keratinophilus (77.37); Sewage Sludge 2: T.mentagrophytes

(90.12) > Fusarium oxysporium (76.88) > T.rubrum (76.47) > C.keratinophilum

(74.45) > T.ajelloi (68.90); Sewage Sludge 3: T. ajelloi (99.80) > C.keratinophilum

(85.5) > C.tropicum (84.90) > C.indicum (84.57) > T.rubrum (75.22); Sewage Sludge

4: A.versicolor (98.05) > T.terrestre (88.05) > Fusarium oxysporium (85.12) >

T.ajelloi (79.89) > T.mentagrophytes (78.42); Sewage Sludge 5: A.versicolor (86.58)

> T.rubrum (77.18) > Fusarium oxysporium (75.08) > T.ajelloi (68.38) >

T.mentagrophytes (65.38).

Soils around Chicken Vendor Stalls:

Total of 50 soil samples around 5 different Chicken stalls (Table-4.2.21 to



occurred.

Among all, as shown by Fig-4.2.5, highest percentage was shown by M.

gypseum (8%), T. rubrum (7%) while C. keratinophilum, C.tropicum,

T.mentagrophytes, and A.flavus came next with 6%. 5% occurrence was shown by

Arthroderma quadrifidum, C. indicum and A.fumigatus where C. pannicola,

T.terrestre and A.niger occupied 4%. Other fungi sharing the rest of the 26% include

genera of Aspergillus, Fusarium, Geotrichum, Penicillium, Trichoderma (non-

dermatophytes) along with some non-sporulating fungi.



of Chicken stall 1 were – A.flavus (79.39), A.fumigatus (78.05), A.versicolor (67.38),

Fusarium solani (64.69) and M. canis (55.36); Chicken stall 2 were – M.gypseum

(68.78), C.tropicum (66.75), A.flavus (66.08), A.fumigatus and Trichoderma viridae

(64.72), C.keratinophilum and M.canis (58.10); Chicken stall 3 were – T.rubrum

(79.65), T.terrestre (69.65), Arthroderma quadrifidum (65.11), A.versicolor (63.40)

and C. tropicum (57.95); Chicken stall 4 were – T.mentagrophytes (70.65), T.terrestre

16

and A.niger (67.69), T.rubrum and M.gypseum (57.10), C. tropicum (47.10) and

Arthroderma quadrifidum (45.91); Chicken stall 5 were – A.flavus (68.00),

C.keratinophilum (58.00), C. indicum (48.00), Arthroderma quadrifidum (46.66) and

C.tropicum (46.00).

Polluted Beach Sands:

24 species of 9 genera were identified in total of 50 soil samples of 5 different

meat markets (Table-4.2.26 to Table-4.2.30). 14 dermatophytic species of 6 genera

and 10 non-dermatophytic species of 3 genera were among them.

C.tropicum (8%), C.keratinophilum (7%), C.pannicola (6%), M.canis (6%), T.rubrum

(6%) and A.flavus (6%) were dominants (Fig-4.2.6) while the others likes M.gypseum

with 5%, aphanoascus keratinophilus, Aphanoascus fulvescens, Arthroderma

quadrifidum, C.indicum, T. mentagrophytes and A.nidulans with 4%, Arthroderma

curreyi, Myceliophthora vellera, T.ajelloi, A.fumigatus, Fusarium solani and

Fusarium oxysporium with 3%, Penicillim citrinum with 2% were next to them. The

remaining 13% of others include remaining species of above genera along with few

non-sporulating ones.

Sampling site wise study of all the 5 sites reveal the order of dominance of

each site as follows, based on their RIVs. Polluted Beach Sands 1 (Near Fishing

Harbour): T. rubrum (61.64) > Aphanoascus fulvescens and Aspergillus nidulans

(56.16) > M.gypseum (54.10) > C.tropicum (53.01) > M. canis (45.57); Polluted

Beach Sands 2: Myceliophthora vellera (65.98) > Aspergillus flavus (57.69) >

Fusarium oxysporium (56.83) > F. solani (55.98) > Aphanoascus keratinophilus and

A.versicolor (54.27); Polluted Beach Sands 3: C.tropicum (71.25) > C. pannicola

(68.60) > A.nidulans and F.oxysporium (63.97) > Aphanoascus fulvescens (55.29) >

Arthroderma quadrifidum (53.97); Polluted Beach Sands 4: C.keratinophilum (78.09)

> C.pannicola (70.9) > A.flavus (57.51) > T.mentagrophytes (5.78) > C.indicum, M.

canis, A.fumigatus and A.versicolor (54.62); Polluted Beach Sands 5: Aphanoascus

keratinophilus (68.51) > C.indicum (67.09) > M.gypseum, A.niger and Fusarium

oxysporium (64.25) > Arthroderma quadrifidum (57.09) > Aphanoascus fulvescens

(55.67).

17

Poultry Farm Soils:

Total of 50 soil samples around 5 different Poultry Farms (Table-4.2.31 to



occurred.

Among all, as shown by Fig-4.2.7, highest percentage was shown by

C.indicum and C.keratinophilum (9%), C.tropicum, M. canis and A.flavus (8%) while

M.gypseum came next with 7%. 6% occurrence was shown by A.fumigatus where

A.niger occupied 5%, Geomyces pannorum, A. nidulans occupied 4%.

Myceliophthora vellera and Trichoderma viridae with 3%, T.mentagrophytes with 2%

came last in occurrence. Other fungi sharing the rest of the 24% include genera of

Geomyces (dermatophytes) and Aspergillus, Fusarium, Penicillium, Rhizopus and

Scopulariopsis (non-dermatophytes) along with some non-sporulating fungi.



of Poultry Farm 1 were – Geomyces pannorum (88.83), M.gypseum (79.23), A.flavus

(78.03), C.tropicum (76.42) and C.indicum (67.63); Poultry Farm 2 were – A.flavus

(81.35), C.keratinophilum (69.72), C.tropicum (69.18), A.fumigatus (56.48), A.

nidulans (54.32); Poultry Farm 3 were – C. keratinophilum (80.09), M. canis (66.88),

A.terrus (66.42), T. mentagrophytes and A. nidulans (62.75) and C. indicum (58.25);

Poultry Farm 4 were – C. keratinophilum (69.09), C. tropicum (63.33), Penicillium

citrinum (56.06), A. fumigatus (54.24) and C. indicum (50.30); Poultry Farm 5 were –

M.canis (87.90), Geomyces pannorum (85.64), C. keratinophilum (61.86), C. indicum

(59.03) and A. niger (47.90).

Garden Soil (Control)

Garden soil which was taken as control was found with few colonies of some species

of Aspergillus and few other fungi but they were found completely negative for

keratinolysis.

Statistical Analysis of the Distribution

Poisson distribution was performed for each organism for their original

occurrence versus expected ones (0 colonies to 5 colonies per sample). All values

were perfectly satisfied goodness-of-fit with chi-square test.

18

Feather Degradation Studies

Tables 4.4.1 to 4.4.7 represent the results of the analyses of parameter pH,

total protein released, keratinolytic activity, nitrates, nitrites, cysteine and cystine

released respectively by all the seven test fungi.

Changes in pH

The changes in pH towards alkalinity were assessed during hydrolysis.

Experimental set ups with almost all the seven organisms have shown a gradual

increase in pH of the medium up to 40 days of incubation followed by decline

thereafter, except for Chrysosporium tropicum, which has the maximum value at 30

days setup (Fig-4.4.1).

pH values of the medium in experimental set up for feather hydrolysis,

observed during 0th, 10th, 20th, 30th, 40th, 50th and 60th days – 7.5, 7.72, 8.54, 10.23,

9.9, 9.12 and 8.50 for C.tropicum; 7.5, 7.59, 7.97, 8.71, 9.62, 9.1 and 8.8 for

Gibberella intermedia; 7.5, 7.60, 8.63, 9.9, 10.9, 9.64 and 9.12 for Fusarium sp.F42;

7.5, 7.61, 8.23, 8.8, 9.15, 8.92 and 8.5 for Microsporum canis; 7.5, 7.8, 8.31, 9.83,

11.3, 9.42, 9.13 for M. gypseum; 7.5, 7.72, 8.5, 10.06, 10.8, 10.13 and 9.2 for

Trichophyton mentagrophytes; 7.5, 7.68, 8.72, 9.94, 10.27, 9.6 and 8.83 for T.

terrestre.

Based on the maximum pH change, all the species can be put together in a

descending order as - M. gypseum (7.5 to 11.30) > Fusarium sp.F42 (7.5 to 10.9) > T.

mentagrophytes (7.5 to 10.8) > T. terrestre (7.5 to 70.27) > C. tropicum (7.5 to 10.23)

> Gibberella intermedia (7.5 to 9.62) > M. canis (7.5 to 9.15).

Total Protein Released

As shown in the Fig-4.4.2, maximum amount of protein was released by M.

gypseum, in 40th day sample (9.13 mg/ml) which contributes 91.3% degradation of

feather substrate, which was notably high compared with that of the respective

control.

A remarkable amount of protein was released by the keratinolytic activity by

all the organisms and the values were as follows – 1.21, 7.54, 8.51, 5.0, 4.25 and 2.52

mg/ml by C. tropicum; 0.75, 2.2, 6.0, 6.41, 3.98 and 3.5 mg/ml by Gibberella

intermedia; 0.8, 3.76, 5.7, 7.63, 3.5 and 3.25 mg/ml by Fusarium sp.F42; 0.9, 3.75,

4.25, 5.0, 4.0 and 3.5 mg/ml by M. canis; 1.92, 4.25, 5.74, 9.13, 7.21 and 3.32 by M.

19

gypseum; 1. 8, 3.9, 5.97, 9.01, 7.83 and 3.5 mg/ml for T. mentagrophytes; 1.1, 3.2,

6.13, 8.4, 5.6 and 3.8 mg/ml for T. terrestre for 10th, 20th, 30th, 40th, 50th and 60th days

respectively.

Order of the seven test organisms, based on the maximum total protein

released by them, can be as follows: M. gypseum (91.3%) > T. mentagrophytes

(90.1%) > C. tropicum (85.1%) > T. terrestre (84%) > Fusarium sp.F42 (76.3%) >

Gibberella intermedia (60%) > M. canis (50%)

Keratinolytic Activity

Among all the setup samples, maximum keratinolytic activity registered was

12.26 KU/ml at the 40th day by M. gypseum.

It is evident from the results (Fig-4.4.3) that gradual increase in the

keratinolytic activity of all the seven organisms up to 40 days and decline there after

(except C. tropicum which has the maximum at 30th day) was occurred. The values

were – 2.31, 5.12, 9.26, 8.07, 4.71 and 3.8 KU/ml by C. tropicum; 3.2, 4.62, 5.05,

5.94, 5.53 and 4.02 KU/ml by Gibberella intermedia; 4.63, 6.5, 7.9, 8.69, 6.31 and

6.10 KU/ml by Fusarium sp.F42; 1.59, 2.9, 3.47, 5.18, 4.27 and 3.88 KU/ml by M.

canis; 3.76, 5.2, 8.92, 12.26, 10.64 and 9.5 KU/ml by M. gypseum; 3.6, 5.89, 9.1,

11.97, 11.2 and 10.5 KU/ml by T. mentagrophytes; 2.1, 3.6, 5.2, 9.13, 7.6 and 4.52

KU/ml by T. terrestre for 10th, 20th, 30th, 40th, 50th and 60th days respectively.

Values of maximum keratinolytic activity, by each and every organism, enable

to arrange them in a descending order as - M. gypseum (12.26 KU/ml) > T.

mentagrophytes (11.97 KU/ml) > C. tropicum (9.26 KU/ml) > T. terrestre (9.13

KU/ml) > Fusarium sp.F42 (8.69 KU/ml) > Gibberella intermedia (5.94 KU/ml) > M.

canis (5.18 KU/ml) (Fig-4.4.3).

Nitrates (NO3) released

Estimation of the Nitrates released into the growth medium was performed

after the hydrolysis, where the samples were analyzed at regular interval of 10 days

for the whole set up of 60 days (Fig-4.4.4).

Results respectively for 10th, 20th, 30th, 40th, 50th and 60th day were as follows

(Fig-4.4.4): 5.31, 14.03, 34.65, 26.72, 25.89 and 19.04 µM by C. tropicum; 4.69,

12.33, 20.71, 25.03, 23.86 and 22.90 µM by Gibberella intermedia; 12.07, 20.54,

20

25.50, 30.55, 28.03 and 16.68 µM by Fusarium sp.F42; 3.41, 8.85, 17.86, 20.61,

13.33 and 11.27 µM by M. canis; 6.88, 19.07, 32.6, 43.52, 28.39 and 20.61 µM by

M. gypseum; 6.21, 17.4, 29.72, 41.31, 29.17 and 24.92 µM by T.mentagrophytes;

5.01, 13.83, 21.72, 35.1, 27.27 and 17.56 µM by T. terrestre.

The potential of all the test fungi, to release NO3 from the substrate, can be put

in an order as M. gypseum (43.52 µM) > T.mentagrophytes (41.31 µM) > T. terrestre

(35.1 µM) > C. tropicum (34.65 µM) > Fusarium sp.F42 (30.55 µM) > Gibberella

intermedia (25.03 µM) > M. canis (20.61 µM).

Nitrites (NO2) released

Results of the analysis for total Nitrite released explain the trends in

degradation by all the seven organisms. The values include not only the nitrite

released directly from the substrate but also the converted nitrate to nitrite.

Results (as shown in Table-4.4.5) were – 6.04, 14.6, 33.87, 26.09, 25.33and

19.33 µM by C. tropicum; 5.5, 12.96, 20.81, 24.39, 23.12 and 22.47 µM by

Gibberella intermedia; 12.70, 20.64, 24.72, 31.34, 28.79 and 17.1 µM by Fusarium

sp.F42; 4.06, 9.69, 18.3, 20.72, 13.9 and 12 µM by M. canis; 7.73, 19.4, 32.63, 41.56,

28.79 and 20.55 µM by M. gypseum; 7.42, 20.1, 30.32, 39.47, 30.06 and 22.13 µM by

T. mentagrophytes; 6.21, 13.98, 21.6, 31.79, 25.9 and 18.72 µM by T. terrestre for

10th, 20th, 30th, 40th, 50th and 60th days respectively.

The maximum values of Nitrite released by these seven species can be

summarized (Fig-4.4.5) as - M. gypseum (41.56 µM) > T. mentagrophytes (31.79 µM)

> C. tropicum (33.87 µM) > T. terrestre (31.79 µM) > Fusarium sp.F42 (31.34 µM)

> Gibberella intermedia (24.39 µM) > M. canis (20.72 µM).

Cysteine Released

As shown in the Table-4.4.6, maximum amount of Cysteine was released by

M. gypseum, in 50th day sample (13.22 µg/ml).

A significant amount of cysteine was released by all the organisms and the

values were as follows – 1.81, 2.33, 5.72, 11.91, 10.63 and 9.89 µg/ml by C.

tropicum; 1.21, 2.11, 3.65, 8.06, 10.01 and 9.83 µg/ml by Gibberella intermedia;

1.66, 1.91, 2.42, 8.87, 11.51 and 11.03 µg/ml by Fusarium sp.F42; 0.9, 1.82, 2.17,

7.91, 9.27 and 7.68 µg/ml by M. canis; 2.84, 3.25, 4.52, 9.86, 13.22 and 11.43 µg/ml

by M. gypseum; 2.72, 3.16, 4.07, 10.69, 13.03 and 12.01 µg/ml for T. mentagrophytes;

21

1.7, 2.63, 5.42, 11.3, 11.07 and 9.19 µg/ml for T. terrestre for 10th, 20th, 30th, 40th, 50th

and 60th days respectively.

Array of the seven test organisms, based on the maximum cysteine released by

them, can be as follows (Fig-4.4.6): M. gypseum (13.22 µg/ml) > T. mentagrophytes

(13.03 µg/ml) > C. tropicum (11.91 µg/ml) > Fusarium sp.F42 (11.51µg/ml) > T.

terrestre (11.3 µg/ml) > Gibberella intermedia (10.01µg/ml) > M. canis (9.27µg/ml).

Cystine Released

During the process of degradation the quantitative analysis for released cystine

yielded strange values in relation with that of the cysteine. The reason behind the

strangeness was discussed in later chapters.

Maximum cystine registered was 7.95 µg/ml at the 50th day by T.

mentagrophytes. As shown in the Table-4.4.7 the values were as follows – 1.69, 4.92,

6.73, 5.95, 5.22 and 4.91 µg/ml by C. tropicum; 0.92, 2.35, 3.81, 5.1, 5.79 and 5.02

µg/ml by Gibberella intermedia; 1.41, 2.83, 4.21, 6.33, 6.58 and 6.17 µg/ml by

Fusarium sp.F42; 0.89, 1.97, 2.73, 3.41, 4.68 and 4.5 µg/ml by M. canis; 2.73, 3.62,

5.23, 6.9, 7.4 and 7.11 µg/ml by M. gypseum; 3.13, 3.40, 4.97, 6.72, 7.95 and 7.38

µg/ml for T. mentagrophytes; 0.94, 4.68, 5.7, 6.81, 5.68 and 4.73 µg/ml for T.

terrestre for 10th, 20th, 30th, 40th, 50th and 60th days respectively.

The utmost values of cystine released by these seven species can be

summarized (Fig-4.4.7) as: T. mentagrophytes (7.95 µg/ml) > M. gypseum (7.4

µg/ml) > C. tropicum (6.73 µg/ml) > Fusarium sp.F42 (6.58 µg/ml) > Gibberella

intermedia (5.79 µg/ml) > T. terrestre (5.68 µg/ml) > M. canis (4.63 µg/ml).

Statistical Analysis of Degradation Studies

Regression analysis was done for all the results of seven parameters by seven

test fungi, to test their goodness-of-fit. As shown in the Fig-4.5.1 to Fig-4.5.7, all

were perfectly fitted in 6th degree polynomial regression curves, with P value

perfectly 0 and R squared is 1.

Seed germination studies

The effect of post feather degradation extract of all the seven test fungi on

seed germination along with respective controls was studied by using fenugreek

seeds. Extract taken from all samples of 60 days experimental setups (of intervals of

10 days each) were used on the seed germination in concentrations of 5, 10, 15 and 20

22

%. Resulting growth was measured based on Number of seeds germinated, Relative

seed germination (when compared with that of control), Mean root length and

Relative root growth (when compared with that of control) and finally Germination

index.

Here in this section, results and their corresponding values are narrated based

on Germination index only, while the other values were represented in Table-4.6.1 to

Table-4.6.7.

Extracts of Fusarium sp.F42 exerted significant results on germination index.

30 days extract was shown to be effective than the others (Fig-4.6.1) yielding almost

9.5 times more germination than that of control and among all the four concentrations

used, 10 % was shown to be the best. Nevertheless, 10% concentration was proved to

be the best among the four not only for the 30th day extract but in the rest of the

extracts. 5 and 15 % concentration came next leaving the 20 % behind. Germination

index values were increased upto 30 days (maximum was 951.44 of 10%

concentration) and then declined gradually, showing 60th day extract was completely

fatal when compared to that of control.

While control (with 0% extract) has the GI 100, the rest of the study was as

follows (Table-4.6.1): 178.96, 204.21, 175.00 and 167.39 for 5, 10, 15 and 20%

respectively of the 10th day extract; 493.69, 541.93, 512.97 and 483.91 for 20th day

extract; 885.03, 951.44, 840.88 and 817.99 for 30th day; 546.18, 635.34, 541.51 and

484.71 for 40th day; 247.83, 299.23, 260.31 and 189.34 for 50th day; 90.603, 112.66,

67.88 and 53.65 for 60th day of 5, 10, 15 and 20 % respectively.

Gibberella intermedia unlike the previous one, showed 40th day extracts were

effective while 15 % concentration of them was the best. Level of GI increased

considerably upto the 40th day, but dropped sharply for 50th and even more decline

thereafter (Fig-4.6.2). Among four concentrations used, except in 40th day, 10% was

shown to be the best among all other extracts. Their values were as follows – 10th day:

119.84, 146.79, 113.26 and 107.47; 20th day: 241.67, 284.82, 216.07 and 176.09; 30th

day: 277.68, 307.41, 256.22 and 228.17; 40th day: 283.03, 327.02, 350.2 and 287.04;

50th day: 147.88, 165.18, 120.93 and 90.97; 60th day: 76.65, 119.41, 61.31 and 43.65

for 5, 10, 15 and 20 % concentrations respectively (Table-4.6.2). It was clearly

observed that increased concentrations more than 10 % all extracts effected negatively

except for that of 40th day where 15% was the best and result of 20% concentration

was even lower than that of 5%.

23

Fig-4.6.3 illustrates the patterns of the effect of extracts on GI, from the

feather degradation setups by Chrysosporium tropicum. The best concentration was

10% of 30th days extract, where it touched the 1200 grid line on the graph revealing

the 12 times more growth than the control. All other extracts never crossed 700 which

show that the GI level rose up to 30th day extract and declined there after reaching

really low levels for 60th day one, indicating a stunted growth with GI values even

lower than that of control. 10% concentrations were highlighted to be the best ones

among all extracts, followed by 5 and 15 %, leaving the 20 % behind with decreased

GI values.

Values as shown in the Table-4.6.3, were as follows – 254.30, 296.48, 304.69

and 247.16 for 10th day extract of all four concentrations; 618.78, 676.56, 596.59 and

549.82 for 20th day extract; 1076.53, 1202.06, 1132.78 and 1005.15 for 30th day

extract; 571.02, 673.40, 546.59 and 489.70 for 40th day extract; 199.43, 288.42,

188.92 and 176.67 for 50th day extract and 105.15, 118.75, 70.03 and 52.27 for 60th

day extract of all four concentrations respectively.

Coming to the test fungi Microsporum canis, which reported not much values

like the previous ones, but it showed 5 times better growth than that of control,

showing the GI 511.84 for 15 % concentration of 30th day extract. Here the curve,

unlike that of previous ones, has shown sharp increase up to 30th day extract and

gradual decline there after (Fig-4.6.4). 10 % concentration of all the extracts except

30th day, were shown to be effective, but GI levels of 60th day extract were still lower

than that of control as usual. Results were as follows – 10th day: 150.59, 168.48,

134.40 and 121.60; 20th day: 206.50, 260.16, 196.74 and 166.40; 30th day: 379.94,

447.17, 511.84 and 380.93; 40th day: 348.19, 374.78, 289.54 and 272.38; 50th day:

197.18, 216.86, 168.96 and 135.87; 60th day: 80.00, 114.82, 65.09 and 49.50 for all

four concentrations respectively (Table-4.6.4).

Extracts of Microsporum gypseum exerted noteworthy results on germination

index. 40th day extract was the best than the others (Fig-4.6.5) reporting almost 7

times more germination than that of control and among all the four concentrations

used, 15 % was shown to be the best. However, 10% concentration was proved to be

the best among the four for all the extracts except that of 40th day. 5 and 15 %

concentration came next leaving the 20 % behind. Germination index values were

increased upto 40 days (maximum was 717.63 of 15% concentration) and then

24

decreased very sharp, showing 60th day extract was completely fatal when compared

to that of control.

Considering control (with 0% extract) has the GI 100, the rest of the study was

as follows (Table-4.6.5): 123.55, 141.32, 132.95 and 146.35 for 5, 10, 15 and 20%

respectively of the 10th day extract; 270.63, 319.18, 263.43 and 204.41 for 20th day

extract; 453.99, 595.01, 528.03 and 398.45 for 30th day; 576.10, 662.12, 717.630 and

577.17 for 40th day; 204.96, 261.53, 198.72 and 154.89 for 50th day; 86.78, 103.30,

71.90 and 57.37 for 60th day of 5, 10, 15 and 20 % respectively.

Trichophyton mentagrophytes strangely showed 50th day extracts were

effective while 10 % concentration of them was the best (showing maximum GI

961.60). Level of GI increased considerably up to the 50th day, but dropped sharply

for 60th day (Fig-4.6.6). Among four concentrations used, 10% was shown to be the

best among all extracts. Their values as shown in the Table-4.6.6, were as follows –

10th day: 104.80, 132.17, 122.71 and 103.20; 20th day: 170.43, 195.16, 180.31 and

161.74; 30th day: 442.02, 523.37, 507.55 and 478.75; 40th day: 598.96, 674.54, 508.35

and 479.58; 50th day: 895.30, 961.60, 812.87 and 790.26; 60th day: 236.87, 289.25,

251.23 and 181.77 for 5, 10, 15 and 20 % concentrations respectively. Uniquely this

genus returned strange values of GI which stood still more than that of control even

for the 60th day extract.

Fig-4.6.7 demostrates the trends of the effect of extracts on GI, from the

feather degradation setups by Trichophyton terrestre. The best concentration was 15%

of 50th days extract, where it reached the 736.98 revealing the 7.36 times more growth

than the control. All other extracts except 60th day never crossed 350 which show that

the GI level rose suddenly at 50th day extract and showed little lower levels for 60th

day. 10% concentrations were highlighted to be the best ones among all extracts,

except for 50th day, followed by 5 and 15 %, leaving the 20 % behind with decreased

GI values.

Values as shown in the Table-4.6.7, were as follows – 103.30, 118.80, 112.77

and 101.65 for 10th day extract of all four concentrations; 127.38, 145.59, 136.91 and

154.89 for 20th day extract; 260.33, 328.37, 271.14 and 212.7 for 30th day extract;

212.67, 252.89, 205.58 and 161.78 for 40th day extract; 593.80, 680.89, 736.98 and

594.08 for 50th day extract and 469.32, 580.16, 544.76 and 382.78 for 60th day extract

of all four concentrations respectively.

25

Altogether, based on the maximum values of GI, effect of organisms can be

summarized as - Chrysosporium tropicum (1202.06) > Trichophyton mentagrophytes

(961.60) > Fusarium sp.42 (951.44) > Trichophyton terrestre (736.98) >

Microsporum gypseum (717.63) > Microsporum canis (511.84) > Gibberella

intermedia (350.2).

Soil Amendment Studies

Results of each of the test fungi for their effects on pH, N, P, K and Soil

Organic Carbon of soil were shown in Table-4.7.1 to Table-4.7.7. Four concentrations

of extracts viz. 5, 10, 15 and 20 % from all the 6 extracts of 60 days (10 days intervals

each) experimental setup have been tested for efficiency, along with a control which

bears 0% of extract.

Organism wise results were shown in the above mentioned tables; however, all

the seven test fungi were comparatively discussed together for their influence on each

parameter.

And as each parameter showed number of values for each test fungi, only the

maximum values for each day’s extract were described here, while all the data can be

accessed from result tables.

pH

After the employment of various extracts, significant changes in pH values of

soils were observed for all the extracts. The change was considered from pH around 7

for control soil. Levels shown graphically in Fig-4.7.1 indicated that 20%

concentration of 40th day extract from M. gypseum was having the maximum effect by

hitting the highest pH value 8.21. On the other hand, all seven tests extracts yielded

increased pH values up to 40th day and declined there after.

Maximum values yielded by 20% concentration up to 40th day extract and 5%

concentration for 50th and 60th day extracts of all the test fungi were as follows – 6.91,

7.14, 7.39, 7.65, 7.92, 7.81 and 7.46 for Chrysosporium tropicum; 6.98, 7.26, 7.43,

7.79, 7.87, 7.79 and 7.63 for Gibberella intermedia; 7.01, 7.19, 7.28, 7.62, 7.95, 7.89

and 7.31 for Fusarium sp.F42; 6.89, 7.15, 7.21, 7.59, 7.83, 7.41, 7.73 and 7.37 for

Microsporum canis; 6.95, 7.22, 7.59, 7.9, 8.21, 8.06 and 7.65 for M. gypseum; 7.05,

7.22, 7.36, 7.54, 7.89, 7.82 and 7.29 for Trichophyton mentagrophytes; 6.86, 7.18,

26

7.47, 7.88, 8.02, 8.05 and 7.45 for T. terrestre for Control, 10th, 20th, 30th, 40th, 50th

and 60th day extracts.

Based on the maximum effect on pH change of the soil, all the species can be

put together in a descending order as - M. gypseum (8.21) > T. terrestre (8.05) >

Fusarium sp.F42 (7.95) > Chrysosporium tropicum (7.92) >T. mentagrophytes (7.89)

> Gibberella intermedia (7.87) > M.canis (7.83).

Total Nitrogen

Maximum amount of Total Nitrogen (3.92 %) was recorded when 20%

concentration of 40th day extract of T.mentagrophytes was used in the soil. As shown

in Fig-4.7.2, highest peaks were observed for 40th day extracts of all the seven test

fungi. Maximum levels which were shown up by 20% concentrations in the soil

indicate that concentration of the extract in the soil was directly proportional to

Nitrogen levels. However, gradual decline was observed after 40th day till 60th day

extracts, where in the 5% concentration of extract yielded maximum Total Nitrogen

values in the soil for 50th and 60th day extracts.

Compared with the corresponding control (0%) soils, remarkable amounts of

Total Nitrogen was recorded and the maximum values were as follows – 1.2, 1.82,

2.41, 3.29, 3.81, 3.58 and 3.06 % for Chrysosporium tropicum; 1.4, 1.92, 2.37, 2.43,

2.49, 2.39 and 1.97 % for Gibberella intermedia; 1.3, 1.89, 2.07, 2.86, 3.41, 3.32 and

2.44 % for Fusarium sp.F42; 1.3, 1.46, 1.61, 1.75, 1.92, 1.9 and 1.72 % for

Microsporum canis; 1.4, 2.09, 2.27, 2.88, 3.18, 3.01 and 2.87 % for M. gypseum; 1.4,

1.59, 1.97, 2.81, 3.92, 3.81 and 3.42 % for Trichophyton mentagrophytes; 1.3, 1.59,

1.77, 2.18, 3.67, 3.21 and 2.37 % for T. terrestre for Control, 10th, 20th, 30th, 40th, 50th

and 60th day extracts.

Order of the seven test organisms, based on the maximum Total Nitrogen

recorded in the soil, can be as follows: T. mentagrophytes (3.92%) > C. tropicum

(3.81%) > T.terrestre (3.67%) > Fusarium sp.F42 (3.41%) > M. gypseum (3.18%) >

Gibberella intermedia (2.49%) > M. canis (1.92%)

Phosphorous

Among all the amendments of soil with four concentrations of six extracts

from seven test fungi, maximum effect on Phosphorous yield (5.94 g/kg) was

observed when 20% concentration of 40th day extract of C. indicum was mixed with

soil.

27

It was evident from the results (Fig-4.7.3) that soil mixed with various

concentrations has shown increased levels of phosphorous up to 40th day extract of all

the seven test fungi and gradual decrease there after. The maximum values for soils

with Control, 10th, 20th, 30th, 40th, 50th and 60th day extracts were – 1.18, 2.53, 3.11,

4.32, 5.94, 5.12 and 4.32 g/kg for Chrysosporium tropicum; 1.13, 1.98, 2.99, 3.76,

4.06, 4.01 and 3.43 g/kg for Gibberella intermedia; 1.12, 2.41, 3.88, 4.63, 5.18, 4.98

and 3.79 g/kg for Fusarium sp.F42; 1.16, 1.93, 2.54, 2.81, 3.22, 3.13 and 2.92 g/kg

for Microsporum canis; 1.15, 1.91, 3.08, 3.74, 4.33, 4.31 and 3.58 g/kg for M.

gypseum; 1.15, 2.23, 3.56, 4.91, 5.68, 4.78 and 3.72 g/kg for Trichophyton

mentagrophytes; 1.2, 1.81, 3.38, 4.14, 4.73, 4.52 and 2.78 g/kg for T. terrestre.

Values of maximum effect on soil in yielding maximum phosphorous, by

extracts of each and every organism, enable to arrange them in a descending order as -

C. tropicum (5.94 g/kg) > T. mentagrophytes (5.68 g/kg) > Fusarium sp.F42 (5.18

g/kg) > T. terrestre (4.73 g/kg) > M. gypseum (4.33 g/kg) > Gibberella intermedia

(4.06 g/kg) > M. canis (3.22 g/kg).

Potassium

Analysis of the Potassium in the soil after extracts were added was performed

where the samples were treated with four concentrations (5, 10, 15 and 20 %) of 6

feather degradation extracts at regular interval of 10 days for the whole set up of 60

days.

Highest values of potassium respectively for Control, 10th, 20th, 30th, 40th, 50th

and 60th day were as follows (Fig-4.7.4): 2.24, 2.92, 5.31, 8.74, 10.57, 10.03 and 8.06

g/kg by C. tropicum; 2.31, 3.17, 4.48, 5.12, 5.83, 5.74 and 4.72 g/kg by Gibberella

intermedia; 2.29, 3.06, 3.94, 6.41, 8.57, 7.91 and 4.92 g/kg by Fusarium sp.F42; 2.21,

2.52, 2.83, 3.11, 3.51, 3.32 and 2.76 g/kg by M. canis; 2.27, 3.01, 4.53, 6.08, 7.61,

7.09 and 5.11 g/kg by M. gypseum; 2.31, 3.17, 4.24, 7.81, 8.13, 5.32 and 4.82 g/kg by

T.mentagrophytes; 2.41, 3.35, 5.13, 6.17, 7.21, 7.19 and 5.31 g/kg by T. terrestre.

The capacities of all the test fungi, to raise the levels of Potassium in the soil

when their respective feather degradation extracts were added, can be put in an order

as C. tropicum (10.57 g/kg) > Fusarium sp.F42 (8.57 g/kg) > T.mentagrophytes (8.13

g/kg) > M. gypseum (7.61 g/kg) > T. terrestre (7.21 g/kg) > Gibberella intermedia

(5.83 g/kg) > M. canis (3.51 g/kg).

28

Soil Organic Carbon

Results of the analysis of the soil for Soil Organic Carbon after amending with

post feather derivative extracts of seven test fungi in four concentrations were clear

indications that the fertility of soil was increased by number of folds (Fig-4.7.5).

Values were – 10.5, 11.55, 15.47, 18.36, 21.73, 20.7 and 17.3 % d.w by C.

tropicum; 10.4, 11.61, 12.34, 13.52, 14.44, 14.13 and 13.5 % d.w by Gibberella

intermedia; 10.6, 11.19, 12.11, 16.57, 19.83, 19.18 and 16.9 % d.w by Fusarium

sp.F42; 10.3, 10.64, 10.92, 11.25, 11.7, 11.61 and 10.93 % d.w by M. canis; 10.6,

11.89, 13.8, 15.03, 16.45, 16.11 and 14.05 % d.w by M. gypseum; 10.7, 12.21, 13.12,

15.56, 18.84, 18.19 and 15.98 % d.w by T. mentagrophytes; 10.5, 11.08, 12.81, 14.63,

16.05, 15.91 and 14.15 % d.w by T. terrestre for extracts of Control, 10th, 20th, 30th,

40th, 50th and 60th days respectively.

The maximum values of Soil Organic Carbon caused by extracts of these test

fungi can be summarized as - C. tropicum (21.73 % d.w) > Fusarium sp.F42 (19.83

% d.w) > T. mentagrophytes (18.84 % d.w) > M. gypseum (16.45 % d.w) > T.

terrestre (16.05 % d.w) > Gibberella intermedia (14.44 % d.w) > M. canis (11.7 %

d.w).

Statistical Analysis of the Soil Amendment Studies

Two way Analysis of Variance (ANOVA) with replication was done to test

the significant effect of the various post feather derivative extracts on 5 parameters

(pH, Total Nitrogen, Phosphorous, Potassium and Soil Organic Carbon). As explained

by Table-4.7.8 to Table-4.7.15, all values of all seven organisms showed significance

with P values very near to 0 and calculated F value was more than the table F value,

which lead finally to rejection of null hypothesis.

CONCLUSION & SUGGESTIONS OUT OF THE WORK

A total of 31 species of keratinophilic fungi were isolated from soils rich in

keratin in Visakhapatnam, using feather bait technique. Five dermatophytic fungal

species (Chrysosporium tropicum, Microsporum gypseum, M. canis, Trichophyton

mentagrophytes and T. terrestre) which have shown luxuriant growth with maximum

occurrence and two non-dermatophytic fungi (Fusarium sp.F42, Gibberella

intermedia anam. of Fusarium proliferatum) were selected for the further feather

29

degradation studies. The use of degradation experimentation procedure as studied

here enables the reduction of pathogenesis of Microsporum gypseum, Trichophyton

mentagrophytes and Trichophyton terrestre which would not normally occur in native

environments.

The feather substrate used in this study was, regular Indian poultry chicken

feathers (Gallus gallus), which was defatted prior to experimentation. Feathers of

different fowls would be desirable to determine keratin degradation other than

chemical process against few micro organisms. Efficiency of feather degradation by

individual species was carried out for 60 days. The degradation studies were

supported by periodic analysis of parameters like changes in pH, Total Protein

released, Keratinolytic Activity, Release of Nitrates, Nitrites, Cysteine and Cystine

etc. Further the studies were extended towards applicability of the post feather

degradation extracts on the seed germination and also on soil where in precisely the

effect on pH, Total Nitrogen, Phosphorous, Potassium and Soil Organic Carbon in

soil were studied. From the study, the following conclusions can be drawn.

All the seven selected fungi have shown the keratinolytic activity providing

proof that the feather waste (keratin rich) can be effectively degraded by these fungi.

The results give way to the native promising technology where in conversion of

feather substrate to nutrients was far more efficient and faster when compared to the

natural process. In addition to this, the technology also forms a basis for conversion of

waste to wealth.

In spite of many decades, research on geophilic keratinophilic fungi, there still

remains a large untapped potential in the discovery of keratinolytic fungi with

possibility of producing keratin derived bi-products. The degradation efficiencies of

the seven fungi have shown varied range among which 3 species (Microsporum

gypseum, Trichophyton mentagrophytes and Trichophyton terrestre) have proved to

be promising with greater competence while the other fungi (Chrysosporium

tropicum, Gibberella internedia and Fusarium spF42) remain as potential source for

the future discovery of keratin derived bi-products with other bio activity.

The study when extended to application came up with interesting and potential

results both for seed germination and effect on soil. Feather degradation extracts of

the species that had resulted in finest seed germination was Chrysosporium tropicum.

The extracts of C. tropicum, Fusarium sp.F42 and Trichophyton mentagrophytes have

30

resulted in a shoot up in Total Nitrogen as well as Potassium and Phosphorous than

the normal levels, when applied to soil.

In conclusion, keratinolytic fungi remain as an important source for degrading

a wide range of potentially useful feather waste. The investigation of versatile habitats

and utilizing selective techniques such as hair baiting and feather degradation assay

should yield handsome rewards. The overall study states that the degradation of

feather waste is not only feasible but also eco friendly as it improves soil fertility.

More over the technology is economical, available and adaptable conditions. Hence,

this can be taken up at large scale to far better results and products.

Suggestions

• Keratinolytic fungi are incredible tools to degrade as well as to extract

nutrients from abundant keratin material like feather waste.

• Understanding the enzymatic mechanism of degradation of feather waste by

keratinophilic fungi isolated from polluted areas, is the future prospect.

31

FIGURES

Fig-4.2.1: Distribution of Keratinophilic Fungi in Slaugther Houses

5% 4% 7%7%

7%

6%5%6%4%4%5%4%

4%

32%

Chrysosporium indicum C. keratinophilum C. tropicum

Microsporum canis M. gypseum Trichophyton mentagrophytes

T. rubrum T. terrestre Aspergillus flavus

A. fumigatus A. niger Fusarium solani

Penicillium citrinum Others

Fig-4.2.2: Distribution of Keratinophilic Fungi in Soils of Meat Markets

6% 6% 7%

3%

4%

8%7%8%8%4%4%

4%

31%


Geomyces pannorum Microsporum canis M. gypseum

Trichophyton mentagrophytes T. rubrum T. terrestre

Aspergillus flavus A. fumigatus Scopulariopsis brevicaulis

Others

32

Fig-4.2.3: Distribution of Keratinophilic Fungi in Feather Dumping Site Soils

10%8%

9%

7%8%

8%8%8%4%

30%


Microsporum canis M. gypseum Trichophyton mentagrophytes


Others

Fig-4.2.4: Distribution of Keratinophililc Fungi in Sewage Sludge

5% 4% 5%4%

3%

3%

5%

9%8%5%4%5%3%7%

4%

26%

Aphanoascus keratinophilus Arthroderma quadrifidum Chrysosporium indicum

C. keratinophilum C. tropicum Microsporum canis

M. gypseum Trichophyton ajelloi T. mentagrophytes


A. fumigatus A. versicolor Fusarium oxysporum

Others

33

Fig-4.2.5: Distribution of Keratinophilic Fungi in Soils of Chicken Stalls

5% 5%6%

4%

6%

6%8%

6%7%4%6%5%

4%

2%

2%

24%

Arthroderma quadrifidum Chrysosporium indicum C. keratinophilum

C. pannicola C. tropicum Microsporum canis

M. gypseum Trichophyton mentagrophytes T. rubrum

T. terrestre Aspergillus flavus A. fumigatus

A. niger Fusarium solani Trichoderma viridae

Others

Fig-4.2.6: Distribution of Keratinophilic Fungi in Polluted Beach Sands

4% 4%

4%

3%

4%

7%

6%

8%6%5%3%4%6%3%

6%

3%

4%

3%

3%

2%

13%

Aphanoascus keratinophilus Aphanoascus fulvescens Arthroderma quadrifidum

Arthroderma curreyi Chrysosporium indicum C. keratinophilum

C. pannicola C. tropicum Microsporum canis

M. gypseum Myceliophthora vellerea Trichophyton mentagrophytes

T. rubrum T. ajelloi Aspergillus flavus

A. fumigatus A. nidulans Fusarium solani

Fusarium oxysporum Penicillium citrinum Others

34

Fig-4.2.7: Distribution of Keratinophilic Fungi in Poultry Farm Soils

9%9%

8%

4%8%

7%3%2%8%6%4%

5%

3%

24%


Geomyces pannorum Microsporum canis M. gypseum

Myceliophthora vellerea Trichophyton mentagrophytes Aspergillus flavus

A. fumigatus A. nidulans A. niger

Trichoderma viridae Others

35

Fig-8: Changes in pH during feather degradation by keratinolytic fungi

Fig-9: Total protein released (mg/ml) during feather degradation by keratinolytic fungi

6

7

8

9

10

11

12

0 10 20 30 40 50 60 Days

pH

Chrysosporium tropicum Gibberella intermedia Fusarium sp.F42 Microsporum canis M. gypseum Trichophyton mentagrophytes T. terrestre

0 1 2 3 4 5 6 7 8 9

10

0 10 20 30 40 50 60 Days

Total Protein


36

Fig-10: Keratinolytic activity (KU/ml) of keratinolytic fungi on feather

Fig-11: Nitrate (NO3) released (µM) during feather degradation by keratinolytic fungi

Fig-12: Nitrite (NO2) released (µM) during feather degradation by keratinolytic fungi

0

2

4

6

8

10

12

14

0 10 20 30 40 50 60 Days

Keratinolytic Activity (KU/ml)


0 5

10 15 20 25 30 35 40 45 50

0 10 20 30 40 50 60 Days

Nitrate (Micro Molar)


37

Fig-13: Cysteine released (µg/ml) during feather degradation by keratinolytic fungi

Fig-14: Cystine released (µg/ml) during feather degradation by keratinolytic fungi

0 5

10 15 20 25 30 35 40 45

0 10 20 30 40 50 60 Days

Nitrite (Micro Molar)


0 2

4 6

8 10

12 14

0 10 20 30 40 50 60 Days

Cystiene (micrograms/ ml)


38

0 1 2 3 4 5 6 7 8 9

0 10 20 30 40 50 60 Days

Cystine (micrograms/ ml)


39

Fig-4.6.1: Effect of post feather degradation extract of Fusarium sp.F42 on seed germination

0

200

400

600

800

1000

0 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20

C 10 Days 20 Days 30 Days 40 Days 50 Days 60 Days

Days of Feather Degradation and Concentration of Extract (%)

Ger

min

atio

n In

dex

Fig-4.6.2: Effect of post feather degradation extract of Gibberella intermedia on seed germination

050

100150200250300350400

0 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20



Ger

min

atio

n In

dex

Fig-4.6.3: Effect of post feather degradation extract of Chrysosporium tropicum on seed

germination

0200400600800

100012001400

0 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20 5 10 15 20



Ger

min

atio

n In

dex

40

PHOTOGRAPHS

Plate-1: Photographs showing sampling areas

Plate-1.: Slaugther house soil filled with sheep and goat fur

Plate-1.2: Polluted beach sands

Plate-1.3: Positive feather bait plate

41

Plate-2.: Photograph showing visible degradation of feather after 60 days which were inoculated with spore suspension of keratinolytic fungi

ecofriendly disposal of feather waste by...

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