Mycopathologia 143: 155159, 1999.
1999 Kluwer Academic Publishers. Printed in the Netherlands.
Impact of some ecological factors on the occurrence of poultry
soil-inhabiting keratinophiles
Sanjana Kaul & Geeta Sumbali
Department of Botany, University of Jammu, Jammu J&K 180004, India
Received 3 July 1998; accepted in revised form 22 December 1998
Abstract
Investigations were conducted to assess the ecological factors governing distribution and survival of keratinophilic
fungi in poultry farm soils. All the poultry farm soils were rich in humus and the keratinophilic fungi were gen-
erally found to be proportional to the soil organic matter. These soils were nearly neutral to weakly alkaline and
organically rich with a high content of organic carbon, nitrogen, phosphorus, potassium, magnesium, calcium and
iron.
Key words: ecological factors, fungi, keratinophilic, poultry soil
Introduction
Keratinophilic fungi represent an important compo-
nent of soil microflora where they decompose the
highly resistant keratin, a proteinaceous substrate.
This unique group may exist and proliferate actively in
soil under favourable conditions, particularly in places
where they can utilize various forms of keratin. Fre-
quency of occurrence of keratinophilic fungi in the
soil is influenced by a number of biotic and abiotic
factors [1]. Among the abiotic factors, physical effects
of importance are soil pH, soil moisture and chemi-
cal composition and quantity of organic matter in the
soil. The ecology of keratinophilic fungi of the soil
has not been sufficiently studied so far. In view of this,
the present investigations were conducted to assess the
impact of ecological factors on the distribution and
survival of keratinophilic fungi in poultry farm soils
and to discern correlation between them.
Materials and methods
Isolation of fungi
A total of 100 soil samples were collected from ten
poultry farms of Jammu district and were screened for
the keratinophilic fungi by employing the keratin-bait
155
technique introduced by Vanbreuseghem [2]. Each
sterilized petri dish was filled with 2030 g of the soil
sample which was moistened with sterile water, baited
with pre-sterilized poultry feathers and incubated at
28 C for 2025 days. The baits were examined pe-
riodically after 6 days of incubation for any mycelial
growth. Isolations were done by direct transfer of
the fungal mycelium from the bait to Sabourauds
dextrose agar medium supplemented with chloram-
phenicol (50 mg/1000 ml) and cycloheximide (500
mg/1000 ml).
Identification of the isolated fungal species was
done using relevant literature and various keys [3
10]. Identity of the fungal isolates was confirmed by
the International Mycological Institute, UK, and by
the Indian Type Culture collection, IARI, New Delhi,
India.
Analysis of essential macro and micro elements
The essential macro and micro elements for which the
poultry farm soils were analysed were total nitrogen,
total phosphorus, postassium, magnesium, calcium
and iron. Determination of organic carbon and humus
were also made.
Organic carbon was determined by a rapid titration
method [11]. Poultry soil samples were digested with
156
Table 1. Prevalence of keratinophilic fungi in different poultry farm soils of Jammu

No. of soil samples examined 100 (10 samples from each poultry farm)
No. of soil samples positive 100
Occurrence (%) 100.0

Fungi recorded Number of positive soil samples Occurrence

A B C D E F G H I J (%)

Chrysosporium keratinophilum (ITCC No. 382) 6 7 6 5 10 42 42

C. queenslandicum (IMI No. 364408) 9 8 5 7 29
C. tropicum (ITCC No. 4264) 7 9 5 7 10 6 5 5 54
C. pannorum (JUCC No. 101) 8 3 4 3 18
Malbranchea flava (IMI No. 364404) 6 10 7 10 33
Scopulariopsis brevicaulis (IMI No. 343497) 5 7 6 7 6 4 35
Microascus manganii (IMI No. 364406) 4 3 5 12
Gliocladium virens (ITCC No. 4278) 6 5 7 8 8 4 38
Circinella muscae (ITCC No. 4491) 5 2 4 4 15
Fusarium moniliforme (ITCC No. 391) 3 4 2 6 15
F. solani (ITCC No. 386) 3 5 3 11
Aspergillus candidus (IMI No. 343502) 6 6 5 7 4 5 33
A. deflectus (ITCC No. 294) 5 4 5 3 5 22
A. flavus (ITCC No. 830) 5 6 6 5 6 28
A. orchraceus (ITCC No. 4275) 6 4 6 3 19
A. sydowii (ITCCNO. 308.94) 5 5 4 14
A. versicolor (ITCC No. 306.94) 4 3 4 5 4 5 3 28
Pencillium aurantiogriseum (ITCC No. 275) 5 4 4 13
P. citrinum (ITCC No. 17.94) 5 5 7 3 3 5 4 6 38
P. griseofulvum (ITCC No. 4376) 4 4 4 5 17

Fungal species recorded 8 8 10 12 12 8 10 8 13 8

from each poultry farm

Total no. of fungal species 20

Poultry farms
A. Machlian F. Belicharana
B. Prem Nagar G. Muthi
C. Nihalpur Simbal H. Nagbani
D. Kothi Shahdaula I. Maralia
E. Akhnoor J. Ranbir Singh Pura

chromic and sulphuric acids making use of the heat
of dilution of sulphuric acid. The excess of chromic
acid not reduced by the soil carbon was determined by
titration with standard ferrous sulphate solution.
Humus (organic matter) was calculated by multi-
plying the Walkley Black value (i.e. percent of carbon
in the soil) by a factor of 1.724:
percent of organic matter in soil = percent carbon
in soil 1.724.
Total nitrogen was determined by Kjeldahls method
[12]. The nitrogen of the nitrogenous substance is
converted into ammonia by boiling with excess of
concentrated sulphuric acid. The ammonia is fixed
as ammonium sulphate which is then determined by
adding an excess of caustic alkali and distilling off the
liberated ammonia into standard acid.
Phosphorus determination was done colorimetri-
cally [13]. This method is based on the extraction of
available phosphorus from the soil by shaking with 0.5
N sodium bicarbonate solution adjusted to pH 8.5. The
blue colour was compared colorimetrically at 640 nm
 157
Table 2. Ecological characteristics of the poultry farm soils investigated for the prevalence of keratinophilic fungi.

Poultry farms pH/H2 O Organic Organic Nitrogen C/N Phosphorus Potassium Magnesium Calcium Iron
matter carbon (Kjeldahl) value (%) (%) (%) (%) (%)
(%) (%) (%)

Machlian 7.2 8.44 4.90 1.57 3.1 0.0026 0.024 1.00 2.11 1.68
Prem Nagar 8.0 10.13 5.88 1.62 3.6 0.0019 0.024 0.50 1.62 1.82
Nihalpur Simbal 7.7 11.89 6.90 1.41 4.6 0.0022 0.023 1.08 3.80 1.47
Kothi Shahdaula 7.6 12.72 7.38 1.40 4.3 0.0025 0.023 0.53 1.76 1.96
Akhnoor 7.2 11.99 6.96 1.56 6.3 0.0012 0.024 0.57 1.40 1.47
Belicharana 8.0 10.96 6.36 1.30 4.9 0.0016 0.024 0.21 0.51 1.61
Muthi 7.6 11.06 6.42 1.81 3.5 0.0016 0.021 0.10 0.30 1.30
Nagbani 8.0 12.61 7.32 1.34 5.5 0.0031 0.023 0.50 1.02 1.40
Maralia 7.5 13.03 7.56 1.70 5.4 0.0046 0.023 0.60 1.80 1.54
Ranbir Singh Pura 7.9 4.55 2.64 1.10 1.7 0.0033 0.023 0.41 0.87 2.10

in a balanced cell colorimeter 102 (Systronics) and the
phosphorus was calculated from the standard curve.
Magnesium was determined by using Titan yel-
low (sodium salt of dihydrothio-p-toluidine sulphonic
acid) as the dye [12]. In this method, magnesium hy-
droxide is precipitated with sodium hydroxide solution
in the presence of Titan yellow resulting in the forma-
tion of a red lake whose colour intensity was measured
at 535 nm with a spectrophotometer (Perkin Elmer
model).
Available potassium was extracted from the soil
samples by shaking with Morgans reagent containing
sodium acetate and acetic acid. The extract on treat-
ment with sodium cobalt nitrite and alcohol produces
a turbidity whose colour represents the concentration
of potassium, which is then compared with stan-
dard potassium chloride solution and the amount of
potassium present in the soil sample calculated [13].
Calcium was determined titrimetrically with
EDTA [13]. The method is based on calcium form-
ing a stable complex with EDTA. A known volume of
the sample is titrated with standard versenate 0.01 N
solution using murexide indicator in the presence of
NaOH solution. The endpoint is the change of colour
from orange red to purple at pH 12, when the calcium
forms a complex with EDTA.
Iron was determined spectrophotometrically [12]
at 525 nm. Salicylic acid and ferric ions form a deep
coloured complex with a minimum absorption at about
525 nm. This complex is used as the basis for the pho-
tometric titration of ferric ions with standard EDTA
solution (0.01 M).
Soil pH was determined with a pH meter.
Results and discussion
All the poultry farm soils were rich in humus (4.5
13.0%) and the occurrence of keratinophilic fungi
was generally found to be directly proportional to the
soil organic matter (Table 1). Maralia poultry farm
soil, which was richest in humus content (13%), was
the source of the maximum number of keratinophilic
species, i.e. 13. Poultry farm soil of Ranbir Singh
Pura contained the lowest humus content (4.5%) and
only eight fungal species were recovered from it (Ta-
ble 1). A higher number of keratinophilic fungi in soil
with high humus content has also been observed by
other researchers [1418]. Contradictory to these ob-
servations, the distribution of a few keratinophiles is
reported to be irrespective of soil humus [4, 15, 19,
20]. There may be two groups of keratinophilic fungi
based on their preference for humus, one exclusively
associated with humus and second lacking such a dis-
tinct relationship. The individual soils of the poultry
farms differed both as to fungal species representa-
tion and the percentage of types of keratinophilic fungi
(Table 1).
Total nitrogen present in the investigated samples
was also very high, ranging from 1.11.8%. This high
content of nitrogen is probably due to continuous en-
richment of the soil with poultry bird droppings and
also due to feather keratin which is a rich source of
nitrogen that can be decomposed by keratinophilic
fungi [21]. The amount of nitrogen present in the soil
samples did not vary much, thus correlation between
nitrogen content and fungal genera could not be es-
tablished. However, recovery of 813 keratinophilic
158
species from each poultry farm indicates the impor-
tance of appropriate nitrogen and carbon source in
growth and distribution of these fungi.
The ratio of nitrogen to carbon in the soils of poul-
try farms varied from 1.76.3. A close correlation
between the occurrence of keratinophilic fungi and the
C/N ratio is not indicated as the values of C/N ratio
differed from soil to soil (Table 2). Mercantini et al.
[22] also reported that no specific conclusions about
the biological activity of these soils could be made
because the nitrogen supply was partly from the de-
composition of other organic residues and partly from
poultry bird excrements.
Poultry farm soils were also analysed for the pres-
ence of macro (phosphorus, potassium and magne-
sium) and micro (calcium and iron) elements. These
elements were present in adequate amounts with
phosphorus ranging from 0.00120.0046%; potassium
from 0.0210.024%; magnesium from 0.101.08%;
calcium from 0.023.8%; iron from 1.32.1%. These
elements, in general, favour distribution and luxuriant
growth of fungal species by playing a vital role in their
structural and metabolic processes.
The pH of these soils varied from 7.28.0 (Ta-
ble 2). Thus, the poultry soils were nearly neutral
to weakly alkaline and were favourable for the ker-
atinophilic fungi. A similar range of pH was the most
favourable for production of proteolytic enzymes (ker-
atinase) by these fungi, necessary for their growth
on an insoluble substrate like keratin [23]. Therefore,
presence of keratinophilic fungi in alkaline soils may
be due to activation of these enzymes under these
conditions. On this basis, all the keratinophilic fungi
recovered in the present investigation can be catego-
rized as alkalophilic. These results are in agreement
with the findings of others [2526]. Very acid soils
are known to inhibit fungi even if organic matter is
abundant, but weakly acid to weakly alkaline soils
are optimal for keratinophilic fungi regardless of soil
organic content [27, 28].
Results of the present investigation indicate that
ecological factors of soil such as a weakly alkaline
pH and the presence of high organic matter along with
minerals in optimal concentrations influence the oc-
currence of soil keratinophiles. However, it is difficult
to determine the relative importance of these factors
as they are often closely related and have a collective
effect.
Acknowledgements
The authors are thankful to the International Mycolog-
ical Institute, UK and Indian Type Culture Collection,
IARI, New Delhi for confirming identity of the fun-
gal isolates. The financial assistance from CSIR, New
Delhi is gratefully acknowledged by the senior author.
References
1. Otsenasek M. Ecology of the dermatophytes. Mycopathologia
1978; 65: 6772.
2. Vanbreuseghem R. Keratin digestion by dermatophytes; a
specific diagnostic method. Mycologia 1953; 44: 176182.
3. Hesselltine CW, Dorothy IF. The genus Circinella. Mycologia
XLVII 1955; 193212.
4. Carmichael JW. Chrysosporium and other aleuriosporic hy-
phomycetes. Can J Bot 1962; 40: 11371173.
5. Raper KB, Fennel DI. The genus Aspergillus. Baltimore:
Williams and Wilkins, 1965.
6. Booth C. The genus Fusarium. Commonwealth Mycological
Institute, Kew, Surrey. England, 1971.
7. Ellis MB. Dematiaceous Hyphomycetes. Commonwealth My-
cological Institute, Kew Surrey, England, 1971.
8. Sigler L and Carmichael JW. Taxonomy of Malbranchea
and some other hyphomycetes with arthroconidia. Mycotaxon
1976; 4: 349488.
9. Van Oorschot CAN. A revision of Chrysosporium and allied
genera. Studies in Mycol 1980; 20: 189.
10. Chabasse D. Taxonomic study of keratinophilic fungi iso-
lated from soil and some mammals in France. Mycopathologia
1988; 101: 133140.
11. Walkley A, Black CA. An examination of methods for deter-
mining organic carbon and nitrogen in soils. J Agri Sci 1935;
1935; 25: 589609.
12. Vogel AI. A Text Book of Quantitative Inorganic Analysis.
The English Language Book Society and Longmans, Green
and Co. Ltd. 1964: 11216.
13. Chopra SL, Kanwar JS. Analytical Agricultural Chemistry.
New Delhi: Kalyani publishers, 1976.
14. Chmel S, Hasilikova A, Hrasco J, Vlasilikova A. The influence
of some ecological factors on keratinophilic fungi in the soil.
Sabouraudia 1972; 10: 2634.
15. Ajello L, Padhye AA. Keratinophilic fungi of the Galapagos
islands. Mykosen 1974; 17: 239243.
16. Chmel L, Vlasilikova A. The ecology of keratinophilic fungi
at different depths of soil. Sabouraudia 1975; 13: 185191.
17. Abdel Fattah HM, Moubasher AH, Maghazy SM. Kerati-
nolytic fungi in Egyptian soils. Mycopathologia 1982; 79:
4953.
18. Schonborn C. Zur Vorkommen Von. Chrysosporium ker-
atinophilum (Frey) Carmichael und einer thermophilen vari-
ante in Erdboden und bei Tieren, Mykosen 1983; 11: 847864.
19. Schonborn C. Incidence of Chrysosporium keratinophilum in
soil. Mykosen 1968; 329.
20. Muhammed SI, Lalji N. The distribution of geophilic dermato-
phytes in Kenyan soils. Mycopathologia 1978; 63: 9597.
21. Kuester E, Safwat. Untersuchungen zur Kompostierung von
Federn. (Studies on the composting of feathers.) Zbl. Batch.

22. Mercantini R, Marsella R, Caprilli F. Dovgiallo G. Isolation of
dermatophytes and correlated species from the soil of public
gardens and parks in Rome. Sabouraudia 1980; 18: 123128.
23. Kaul S, Sumbali G. Keratinolysis by poultry farm soil fungi.
Mycopathologia 1997; 139: 137140.
24. Pugh GJF, Mathison GE. Studies on fungi in coastal soils III
An ecological survey of keratinophilic fungi. 1962; Trans Brit
Mycol Soc 1962; 4: 567572.
25. Bohme H, Ziegler H. The distribution of geophilic dermato-
phytes and other keratinophilic fungi in relation to the pH of
the soil. Mycopath Mycol Appl 1968; 38: 247255.
26. Hubalek Z. Fungi associated with free living birds in
159
Czechoslovakia and Yugoslavia. Acta Sci Nat Acad Sci Bo-
hem Bmo 1974; 8: 171.
27. Blaschke-Helimesson R. Verbreitung Keratinophiler Boden
pilze in Dresdener Raun in Abangigkeit von pH Wert des
Standortes. Mykosen 1969; 12: 551556.
28. Bohme H, Ziegler H. The distribution of geophilic
dermatophytes and other keratinophilic fungi in relation
to the ph of the soil. Mycopath Mycol Appl 1968; 38:
247255.
Address for correspondence: Department of Botany, University of
Jammu, Jammu J&K 180004, India