Waste Management 31 (2011) 16891701

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Waste Management
journal homepage: www.elsevier.com/locate/wasman

Review

Biodegradation of keratin waste: Theory and practical aspects

Korniowicz-Kowalska Teresa, Bohacz Justyna
University of Life Sciences in Lublin, Faculty of Agrobioengineering, Department of Environmental Microbiology, Mycological Laboratory, Leszczynskiego 7, 20-069 Lublin, Poland

a r t i c l e i n f o a b s t r a c t

Article history: Keratin-rich by-products, i.e. bristles, horns and hooves, chicken feathers and similar, are a source of
Received 2 December 2010 nutrients for animals (amino acids) and plants (N, S). Contemporary developments in the management
Accepted 30 March 2011 of keratin waste in feeds and fertilizers comply with human and animal health protection regulations
Available online 6 May 2011
and respect the principles of ecological development. Biotechnological methods employing keratinolytic
bacteria and microscopic fungi play a key role in processing keratin waste.
Keywords: This study reviews the current knowledge on the ecology and physiology of keratinolytic microorgan-
Keratin
isms and presents the biodegradation mechanism of native keratin. The structure and chemical compo-
Keratinolytic microorganisms
Keratin waste management
sition of keratin proteins are described, and methods of keratin waste biotransformation into products of
practical industrial and natural value, especially composts, are discussed.
2011 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1689
2. The occurrence, structure and chemical composition of keratins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1690
3. Keratinolytic microorganisms and their activity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1691
4. The mechanism of keratin degradation by microorganisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1695
5. Keratin waste management . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1696
6. Conclusions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1698
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1698
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1698

1. Introduction cle in native keratin processing. Animal remains rich in a-keratin,

such as animal skin and hair, human hair, horns, or claws, are in
Keratins are valuable but unavailable brous animal proteins. nature relatively quickly biodegraded by keratinolytic microorgan-
They are components of a range of by-products occurring isms represented by some Procaryota and so-called keratinophilic
especially abundantly in slaughterhouses and meat and poultry fungi. They use native keratin as the sole source of C, N, S and en-
plants: skin remains, bristle, animal hair, horns and hooves, feath- ergy (Noval and Nickerson, 1959; Korniowicz-Kowalska, 1997a;
ers, etc. Keratin waste is classied as category 3 material in regula- Burtt and Ichida, 1999). Studies show that the enzymatic apparatus
tion (EC) 1774/2002 of the European Parliament and Council of 3rd of keratinolytic microorganisms, and in the case of fungi also the
October 2002 laying down health rules concerning animal form of the growth of the mycelium (formation of the so-called
by-products not intended for human consumption. It is not eroding mycelium), are adapted to the chemical and physical
suitable for consumption but does not transmit diseases to humans structure of native keratin (English, 1963, 1969; Kunert, 1989;
or animals and is obtained from carcasses of animals suitable for Yamamura et al., 2002). Keratinolytic microorganisms colonize dif-
human consumption. ferent biotopes. Species parasitizing the skin and its products (hair,
The high number of disulde bonds in the structure of a-keratin nails, hooves, feathers), such as anthropo- and zoophilic dermato-
(Fraser et al., 1972; Filipello Marchisio, 2000) makes it insoluble phytes, that is fungi pathogenic to humans and animals, as well as
and resistant to enzymatic lysis (proteases). This is a major obsta- many keratinolytic species are saprotrophes. Keratinolytic sapro-
trophic microorganisms are a natural component of soil microbio-
Corresponding author. Tel.: +48 815248104; fax: +48 815248106. coenoses and a range of microhabitats rich in keratin matter, e.g.
E-mail address: justyna.bohacz@up.lublin.pl (J. Bohacz).
birds nests, their plumage, mammal hair and waste (Hublek,

0956-053X/$ - see front matter 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.wasman.2011.03.024
1690 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
1974, 2000; Korniowicz-Kowalska and Bohacz, 2002a,b; Kor-
niowicz-Kowalska and Kitowski, 2009; Korniowicz-Kowalska
et al., 2011; Burtt and Ichida, 1999). Those are biotopes constitut-
ing a potential source of acquisition of keratinolytic microorgan-
isms for biotechnological purposes.
New approaches to waste management of category 3 material
in the European Union member states advocating environmentally
friendly technologies have encouraged an interest in saprotrophic
keratinolytic microorganisms and biodegradation methods of na-
tive keratin. Previous studies on keratinolytic microorganisms
and native keratin conversion mostly focused on the pathogenesis
of dermatophytes conditioned by, for instance, the ability to pro-
duce keratinolytic protease (Tsuboi et al., 1987; Kunert, 2000). Re-
cently, considerable attention has been paid also to microbial
keratinolytic proteases in the context of their application for bio-
technological purposes (Brandelli et al., 2010; Gupta and Ramnani,
2006). Whereas, in the literature published so far there is a lack of
review papers addressing the problems of microbiological degra-
dation of keratin wastes in a comprehensive manner, i.e. taking
into account the various taxonomic, ecological, biochemical and
biotechnological aspects.
The ecology and physiology of keratinolytic microorganisms are
discussed in this study. Special emphasis is placed on as broad as
possible discussion of the indices of keratinolytic activity, on the
analysis of the products of biodegradation and biotransformation
of native keratin, on the mechanism of keratinolysis and on the
contemporary trends in the microbial management of keratin
waste.
2. The occurrence, structure and chemical composition of
keratins
Keratins are highly specialized brous proteins known as scle-
roproteins. All keratins are produced in epithelial cells in higher
vertebrates (reptiles, birds and mammals) and in humans. Their
function is mechanical and protective. Keratin, or cytokeratin, la-
ments (known as tonolaments or tonobrils in the past) are two
of six types of intermediary laments forming the cytoskeleton,
that is an intracellular system of protein bres and microtubules.
Intermediary laments are more rigid than other types of la-
ments. Keratin laments can consist of approximately 15 acidic
classes and 15 neutral and alkaline keratin polypeptides. Keratins
occur particularly abundantly in epithelia exposed to considerable
mechanical forces such as keratinized epidermis and corneous skin
products: hair (including wool), nails, claws, hooves, horns, scales,
beaks and feathers. These proteins are resistant to physical and
chemical environmental factors. They are insoluble in water, weak
acids and alkalis, organic solvents, and are insensitive to the attack
of common proteolytic enzymes such as trypsin or pepsin. Keratin
insensitivity and resistance are closely related to the adaptation of
vertebrates to living on the land. A high cystine content is the most
important property that differentiates keratins from other struc-
tural proteins such as collagen and elastin. Numerous disulde cys-
tine bonds present in keratin permanently bind peptide chains
making it resistant to enzymatic lysis. Both a high cystine content
as well as a high content of glycin, proline, serine and acidic amino
acids, and a low content of lysine, histidine and methionine (or
their lack) as well as the absence of tryptophan are also character-
istic of keratins (Fraser et al., 1972; Fraser and Parry, 2003; Jones,
2001).
Keratins are classied as heterogeneous proteins due to the
amino acid structure and composition. Polypeptide chains in kera-
tins can occur in two major congurations: a-helix and b-sheet,
also known as a pleated sheet (Fraser et al., 1972; Lee and Baden,
1975). This corresponds to the division of keratins into: a-keratin,
b-keratin, feather keratin and amorphic keratin (Fraser et al.,
1972). Hill et al. (2010) report that keratin proteins extracted from
hair can be divided into 3 large groups: a, b and c-keratins. That
last group contains non-structural keratins which in the classica-
tion by Fraser et al. (1972) are dened as amorphous keratins. Ker-
atins can also be divided into soft and hard keratin depending on
the content of sulphur amino acids. Soft keratin has a lower cystine
content (up to 2%), weak cross-linking and a smaller resistance to
chemical factors. It is found in the outer layer of the epidermis
and hair core (Fraser et al., 1972). Hard keratin which contains
a-keratin has a high cystine content. It is present in hair, nails,
claws, horns, feathers, beaks and the skin. Cystine content can be
as high as 22% in brittle and stiff hard keratin forms occurring in
horns and nails, and ranges between 10% and 14% in soft and ex-
ible forms occurring in hair, wool and the skin (Filipello Marchisio,
2000). Hard keratin has a diversied morphological structure and a
high resistance to the inuence of chemical factors (Fraser et al.,
1972; Fraser and Parry, 2003).
X-ray studies of hair keratin show an a-helical conformation of
the polypeptide chain. The polypeptide chain is a spiral coil in the
a-helix. Intracellular hydrogen bonds that are the principal inter-
actions make hair elastic. Hair can stretch by 300% of its initial
length and ex back to its original size. An analysis of a-keratin
X-ray diffraction in hair also shows that individual polypeptide
chains in the a-helix are right-handed and form a left-handed
superhelix (Fig. 1) stabilised by hydrogen bonds running parallel
to the main axis and by disulde bridges formed by cystine radicals
between adjacent chains (Filipello Marchisio, 2000; Jones, 2001).
The superhelix known as the protobril is composed of four a-heli-
ces and is the main unit of a-keratin. Protobrils form microbrils;
nine microbrils are arrayed in the periphery and two in the centre
(Fraser et al., 1972; Lee and Baden, 1975; Filipello Marchisio,
2000).
Microbrils visible under an electron microscope form macro-
brils visible under a light microscope (Fraser et al., 1972; Fraser
and Parry, 2003; Lee and Baden, 1975; Jones, 2001). Both proto-
brils and microbrils are xed by disulde bridges, which deter-
mines their stiffness (Filipello Marchisio, 2000) and resistance to
microbial degradation. Individual microbrils and macrobrils
are embedded in an anamorphic matrix. The matrix surrounding
microbrils contains low-molecular keratins with high cystine
content. Conversely, microbrils are formed by high-molecular
and low-cystine keratins (Fig. 1). The matrix surrounding macro-
brils contains little keratin and mostly has non-keratin proteins
(Fraser et al., 1972).
b-keratin occurs in reptiles and birds in scales, claws, beaks and
feathers and cuticule hair. It lacks cystine and has a high content of
serine, glycin, proline, alanine and low levels of lysine, histidine
and tryptophan (Fraser et al., 1972; Hill et al., 2010). Dalev
(1994), analysing the amino acid composition of feathers, detected
the largest amounts of serine, glutamic acid and proline, and the
least of methionine, histidine and lysine. The notable variation in
the content of the particular amino acids in keratin protein may
be due to a variety of factors, among others by food consumed
by birds. Intercellular hydrogen bonds are present in the conforma-
tion of the b-type polypeptide chain. They x antiparallel peptide
chains stabilising the protein structure (Filipello Marchisio,
2000). Polypeptide chains in b-keratin can be parallel as well as
perpendicular (cross-b) to the lament axis (Lee and Baden, 1975).
Feather keratin, the third type of keratin, occurs in both cong-
uration systems of the polypeptide chain: a-helix and b-sheet (Fra-
ser et al., 1972; Lee and Baden, 1975). It probably arose from a
mutation in reptiles, triggering a line from which birds developed
(Lee and Baden, 1975). Feather keratin is composed of b-conforma-
tion proteins in 1/3 and a-keratin in 2/3 (Fraser et al., 1972). Like in
akeratin in hair, microbrils of a-keratin in feathers are
 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
Fig. 1. A schematic of a wool ber drawn by Bruce Fraser and Tom MacRae [Copyright CSIRO Australia 1996. Reproduced with permission from The Lennox Legacy (Rivett DE,
Ward SW, Belkin LM, Ramshaw JAM and Wilshire JFK). Published by CSIRO PUBLISHING, Melbourne, Australia] (Hill et al., 2010).
immersed in the matrix. Unlike hair keratin, feather keratin does
not stretch and is not elastic. This makes feathers light and durable,
which is important during ight (Cameron et al., 2003). Feather
keratin is in 90% formed by homogeneous proteins with molecular
weight of 10.4 kDa as compared to hair keratin consisting of differ-
ent molecular weight proteins ranging from 9 kDa to 51 kDa
(Fraser et al., 1972). The cystine content in feather keratin is lower
than that in nail and hair keratin, and is ca. 8% (Akhatar and
Edwards, 1997). Amorphic keratin is a part of the matrix and is
usually rich in cystine (Fraser et al., 1972). Large amounts of kera-
tin rich in cystine, as so-called c-keratins, occur in the hair cuticle
(especially in its external layer) (Jones, 2001; Hill et al., 2010).
Gamma-keratins are globular proteins with molecular weight of
approximately 15 kDa (Hill et al., 2010).
3. Keratinolytic microorganisms and their activity
Only some bacteria, actinomycetes and keratinophilic fungi and,
of macro-organisms, larvae of the common clothes moth (Tineola
bisselliella Hummel) are capable of using keratin as the sole source
of C, N, S and energy.
Keratinolytic bacteria, particularly from the genus Bacillus and
actinomycetes, were most often isolated from the plumage and
bird feathers (Burtt and Ichida, 1999; Ichida et al., 2001), feather
waste processed by fermentation (Williams and Shih, 1989;
Williams et al., 1990) or composting (Kim et al., 2001). In bacteria,
feather keratin-degrading abilities are observed mostly in strains
of Bacillus licheniformis (Burtt and Ichida, 1999; Lin et al., 1999),
less frequently in populations of Bacillus pumilis, B. cereus and B.
subtitis (Kim et al., 2001) and non-sporeforming bacteria belonging
to, for instance, Stenotrophomonas sp. (Yamamura et al., 2002), Fer-
vidobacterium pannavorans (Friedrich and Antranikian, 1996) and F.
islandicum (Nam et al., 2002).
Keratinolytic species of actinomycetes intensively degrading
native keratin in feathers, hair, nails and horns are mostly repre-
sented by the genus Streptomyces, including S. fradiae (Noval and
Nickerson, 1959; Nickerson et al., 1963), S. pactum (Bckle et al.,
1995) S. thermoviolaceus (Chitte et al., 1999), or other genera, e.g.
Thermoactinomyces (Gousterova et al., 2005).
Fungi known as keratinophilic are a nutrient-specialized group
that utilizes keratin as a rich-nutrient substrate. The term keratino-
philicity is an ecological equivalent to microbial keratinolycity
(Korniowicz-Kowalska, 1997a). Keratinolytic fungi are repre-
1691
sented by dermatophytes and related species of the genus Chrysos-
porium. Dermatophytes, next to species pathogenic to humans and
animals, include saprotrophs preferring to live in the soil (geophilic
dermatophytes). An exception is Microsporum gypseum which
causes inammatory tinea corporis and tinea capitis in humans
(Hayashi and Toshitani, 1983; Ofdani et al., 1998) and dermato-
phytoses in animals (Caretta et al., 1992). Geophilic dermatophytes
include some species of Trichophyton and Microsporum and their
teleomorphs, Arthroderma and Nannizzia, respectively. Non-der-
matophytic keratinophilic fungi consist of two genera: Chrysospori-
um and Myceliophthora (anamorphs). Their teleomorphs belong to
the genera Arthroderma, Aphanoascus and Ctenomyces, or remain
unknown (Oorschot van, 1980; Currah, 1985).
Some authors (Malvija et al., 1992; Santos et al., 1996; Filipello
Marchisio et al., 2000; Gupta and Ramnani, 2006) also classify
different species of ubiquitous moulds as keratinophilic fungi,
e.g. Aspergillus fumigatus and Scopulariopsis brevicaulis, Paecilomy-
ces sp., Penicillium sp., Fusarium sp. and other. Studies by
Korniowicz-Kowalska (1997a) show that ubiquitous moulds in
liquid cultures with native feather keratin as the sole source of C,
N, S and energy degrade only 2040% of the substrate. On the other
hand, typically keratinolytic species representing geophilic derma-
tophytes and Chrysosporium fully solubilize native feather keratin
in the same conditions (Korniowicz-Kowalska, 1997a). According
to Kunert (2000), fungi are weakly keratinolytic if they degrade no
more than 40% of keratin in liquid cultures after 8 weeks and are
non-keratinolytic if they degrade less than 20%. Growth ability
on native keratin observed in many different species of fungi that
are de facto non-keratinolytic (Grifn, 1960; Korniowicz, 1991/
1992; Vries de, 1962) is determined by the presence of organic
substrates other than keratin. Mercer (1958) reports that non-
keratin compounds make up 10% of dry hair mass. Free amino
acids, urea, phenols, soluble proteins, lipid compounds, carbohy-
drates (glycogen, pentosans) were identied in water extracts
and organic extracts of hair (Bolliger, 1951; Safranek and Goos,
1982).
Keratinophilic geophilic fungi colonize natural environments
that have a regular supply of keratin matter, i.e. the soil in areas
inhabited by humans, birds and mammals (Battelli et al., 1978;
Caretta and Piontelli, 1975; Marcantini et al., 1980; Vollenkov,
1984; Nooruddin and Singh, 1987), arable soils (Korniowicz-
Kowalska and Bohacz, 2002a) and avian nests (Hublek, 1974,
2000; Korniowicz-Kowalska and Kitowski, 2009; Korniowicz-
1692 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
Kowalska et al., 2011). These fungi also colonize bird plumage and
mammal hair (Pugh, 1965; Pugh and Evans, 1970; Rees, 1967;
Hublek, 1974, 2000), and display secondary occurrence in com-
munal waste water, waste sediments, communal waste and pol-
luted water (Ulg and Korcz, 1983, 1994; Ulg and Ulg, 1990;
Ulg et al., 1996; Ulg, 2000).
Garg et al. (1985) report that environment animalization and
the subsequent enrichment in keratin are major factors that deter-
mine the occurrence and development of these microorganisms. As
well as the nutrient availability, high humus content, neutral or
slightly alkaline pH, high content of CaCO3 are favourable for the
development of keratinophilic fungi in the soil (Chmel et al.,
1972; Chmel and Vlcilikov, 1977; Garg et al., 1985; Kaul and
Sumbali, 1999; Korniowicz-Kowalska and Bohacz, 2002a).
The distribution of populations of keratinophilic fungi in natural
environments is not uniform and mostly depends on pH differ-
ences. Geophilic dermatophytes colonize more abundantly envi-
ronments with a neutral or slightly acidic reaction, while
representatives of Chrysosporium more frequently colonize slightly
alkaline biotopes (Korniowicz-Kowalska and Bohacz, 2002a;
Korniowicz-Kowalska et al., 2011). Humidity is also a selective
factor in keratinomycete communities (Chmel et al., 1972; Hublek
and Balt, 1974; Korniowicz-Kowalska et al., 2011). Some species,
e.g. Ch. keratinophilum, are classied as hydrophilic species in the
ecological classication, and other, such as e.g. Trichophyton terres-
tre, are xerophiles (Garg et al., 1985).
The majority of keratinophilic fungi are mesophiles. Some, such
as Ch. keratinophilum, Ch. tropicum A. fulvescens, M. gypseum, are
thermotolerant (Garg et al., 1985), less frequently evidently ther-
mophilic (Dozie et al., 1994). A high frequency of thermotolerant
keratinomycetes is recorded in such microhabitats as bird plumage
and nests, or mammal hair (Hublek, 1974; Hublek and Balt,
1974; Pinowski et al., 1999; Korniowicz-Kowalska et al., 2011).
Microorganisms can be divided into two types based on their
keratinolytic abilities: true keratinolytic and potentially keratino-
lytic microorganisms. True keratinolytic microorganisms degrade
hard keratin and fully solubilize keratin structures (Korniowicz-
Kowalska, 1997a). Potentially keratinolytic microorganisms have
strong proteolytic properties. They can play an important role in
the conversion of non-keratin proteins (Korniowicz-Kowalska,
1997a; Kunert, 2000) occurring together with hard keratin in hair,
feathers, or nails, and soft keratin (callus).
The use of native keratin as a substrate is also an important cri-
terion of keratinolytic abilities. Noval and Nickerson (1959), who
investigated wool and feather degradation by a keratinolytic strain
of Streptomyces fradiae, were the rst to notice this. Denatured
(autoclaved) wool was solubilized at a faster rate by the strain they
investigated than undenatured wool (ethylene oxide sterilization).
As Martin and So (1979) showed, denatured keratin proteins could
be degraded even by non-keratinolytic slime bacteria
(myxobacteria).
The mass loss of the keratin substrate is the most reliable indi-
cator of microbial keratinolytic abilities (Korniowicz-Kowalska,
1997a). Other indicators include a release of peptides, amino
groups, amino acids, ammonia, sulfhydryl groups, sulfate excre-
tion, keratinase activity and substrate alkalinization (Chesters
and Mathison, 1963; Korniowicz, 1994; Korniowicz-Kowalska,
1997a; Nickerson et al., 1963; Nickerson and Durand, 1963; Noval
and Nickerson, 1959). The substrate mass loss, a release of peptides
and ammonia, sulphate excretion and substrate alkalinization
should be recognized as homogenised (due to mutual correlation)
assessment parameters of the keratinolytic activity of fungi (Fig. 2).
Keratinophilic fungi caused a 6585% mass loss of the substrate,
solubilization of 50% of peptides and 2560% of amino groups after
21 days of culture in the medium containing feathers as the sole
source of C, N, S and energy. These changes were accompanied
by an accumulation of NNH 4 in the medium correlated with a
pH increase up to 8.59.0 (Korniowicz-Kowalska, 1997a). A mass
loss of the keratin substrate and a related release of peptides were
not correlated with the extracellular proteolytic enzymes (extra-
cellular peptidases acc. to MEROPS database; http://merops.san-
ger.ac.uk) activity of these microorganisms (Korniowicz-
Kowalska, 1997a).
Keratinolytic proteases, known as keratinases (EC 3.4.21/24/
99.11), are produced by some bacteria, actinomycetes and fungi
(Asahi et al., 1985; Bckle et al., 1995; Chattaway et al., 1963;
Gessesse et al., 2003; Kim et al., 2001; Korniowicz-Kowalska,
1999; Lal et al., 1999; Noval and Nickerson, 1959; Sanyal et al.,
1985; Suh and Lee, 2001; Syed et al., 2009; Takiuchi et al., 1982,
1984; Wang and Shih, 1999; Yu et al., 1969, 1971, 1972). They cat-
alyze the release of peptides from keratin (Nickerson and Durand,
1963). Keratinolytic proteases are primarily extracellular induction
enzymes, and their maximum activities were recorded in keratin-
containing substrates (Asahi et al., 1985; Brandelli et al., 2010;
Chattaway et al., 1963; Gupta and Ramnani, 2006; Meevootison
and Niederpruem, 1979; Sanyal et al., 1985; Takiuchi et al., 1982,
1984; Yu et al., 1969). They can also be excreted constitutionally
when keratin is absent in the substrate (Apodaca and McKerrow,
1989; Sanyal et al., 1985). Some bacteria and fungi also produce
intracellular keratinolytic enzymes (Brandelli et al., 2010; Gupta
and Ramnani, 2006). Studies by Yu et al. (1968, 1971) conducted
in the zoophilic dermatophyte T. mentagrophytes cultures contain-
ing guinea pig hair showed that these enzymes were present both
in the substrate and in the mycelium. As Korniowicz-Kowalska
(1999) observed, extracellular and mycelial keratinolytic proteases
were produced by geophilic keratinophilic fungi, i.e. Arthroderma
quadridum, A. curreyi and Chrysosporium pruinosum, in cultures
containing native feather keratin. On the other hand, Wawrzkie-
wicz et al. (1987) showed that Trichophyton gallinae, a zoophilic
dermatophyte, did not produce extracellular keratinase and only
produced intracellular keratinase, deposited in the cell surface
structures of the fungus. The production of microbial keratinolytic
protease was inhibited when sources of carbon and energy, such as
glucose, were easily available (Apodaca and McKerrow, 1990;
Grzywnowicz et al., 1989; Korniowicz-Kowalska and Ziaja,
1999; Meevootison and Niederpruem, 1979; Noval and Nickerson,
1959). The effect was caused by a catabolic repression of the bio-
synthesis of the enzymes (Meevootison and Niederpruem, 1979).
The repression prolongs the process of keratinolysis as the biosyn-
thesis of keratinolytic enzymes is depressed when easily assimila-
ble sources of carbon are used up (Apodaca and McKerrow, 1989;
Meevootison and Niederpruem, 1979). A reverse effect was ob-
served by Syed et al. (2009) in Streptomyces gulbargensis cultures
on a substrate containing feathers and enriched with starch. The
addition of starch increased keratinase secretion by S. gulbargensis.
Phosphates, on the other hand, are a factor activating fungal kera-
tinolysis (Page and Stock, 1974). Korniowicz-Kowalska (2002)
showed that the addition of phosphates and magnesium ions in-
creased the decomposition rate of native feathers by Arthroderma
quadridum.
Microbial keratinolytic proteases chiey represent serine pro-
teinases, less frequently metalloproteinases (Brandelli et al.,
2010; Gupta and Ramnani, 2006). Based on activity inhibition by
phenylmethanesulfonyluoride (PMSF) and other inhibitors of ser-
ine proteases and the optimum values of the activity at pH ranging
between 6.0 and 9.0, these enzymes are classied as neutral or
alkaline proteinases, including serine proteinases, dependent on
Ca2+ ions (Apodaca and McKerrow, 1989; Asahi et al., 1985; Bckle
et al., 1995; Friedrich and Antranikian, 1996; Gessesse et al., 2003;
Korniowicz-Kowalska, 1999; Lin et al., 1999; Nickerson et al.,
1963; Sanyal et al., 1985; Suh and Lee, 2001; Takiuchi et al.,
1984). Some alkaline keratinases of actinomycetes reach the
 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
Fig. 2. Products of feather protein catabolism and changes in the medium pH in cultures of keratinolytic fungi. (A) Peptide release; (B) NNH4 accumulation in the medium;
(C) Sulfate release; (D) pH of medium; R2-determination coefcients (Korniowicz-Kowalska, 1997a).
activity optimum at pH 10 (Letourneau et al., 1998). An exception
are serine proteases isolated from a culture of Trichophyton ment-
agrophytes whose activities are recorded at pH 4.5 (Tsuboi et al.,
1987). Intracellular keratinases of Trichophyton gallinae and Micros-
porum canis are the most important metal-dependent keratinolytic
fungal proteases sensitive to chelating agents (Brouta et al., 2001;
Grzywnowicz et al., 1989). Among bacteria, keratinolytic enzymes
sensitive to chelating agents, i.e. EDTA, are produced by some acti-
nomycetes, e.g. Streptomyces albidoavus (Bressollier et al., 1999).
Metal-dependent keratinolytic enzymes are neutral proteases
(Grzywnowicz et al., 1989; Brouta et al., 2001).
Keratinolytic proteases of microorganisms have a broad sub-
strate range (Brandelli et al., 2010). They usually easily hydrolyze
both a variety of soluble (globular) proteins, such as albumin, glob-
ulin, hemoglobin, casein, and insoluble (brous) proteins: collagen,
elastin, hair, feather and wool keratin (Apodaca and McKerrow,
1990; Asahi et al., 1985; Bckle et al., 1995; Chitte et al., 1999;
Lin et al., 1992; Shih, 1993; Syed et al., 2009; Yu et al., 1969). Dozie
et al. (1994) reported that keratinolytic proteases of the thermo-
philic strain Chrysosporium keratinophilum hydrolyze only keratin
(Table 1).
Some bacterial keratinases, such as Streptomyces gulbargensis
keratinase, are able to fully solubilize native keratin (hard keratin)
of hair, feathers, nails (Gradisar et al., 2000; Syed et al., 2009).
Some, like keratinolytic proteases of Streptomyces pactum (Bckle
et al., 1995) and Fervidobacterium pennavorens (Friedrich and
Antranikian, 1996), do not solubilize native keratin. Bckle et al.
(1995) observed that post-culture ltrates of S. pactum growing
on native keratin solubilize entire chicken feathers while keratin-
ase isolated from these ltrates and then puried solubilizes less
than 10% of the native substrate. Both crude and puried prepara-
tions of keratinases (Brandelli et al., 2010; Gupta and Ramnani,
2006; Korniowicz-Kowalska, 1999; Takiuchi et al., 1982; Yu
et al., 1969) are also unable to fully solubilize hard keratin. The en-
zymes key role in biodegradation of keratinized tissues by fungi
was criticized due to the low activity of keratinase in respect of
1693
crude keratin (Chattaway et al., 1963; Kunert, 1976, 2000; Takiuchi
et al., 1982). According to Takiuchi et al. (1982), the term keratin-
ase is confusing as it does not reect the actual substrate specic-
ity of the enzyme. According to many authors (Chattaway et al.,
1963; Grzywnowicz et al., 1989; obarzewski et al., 1990; Letour-
neau et al., 1998; Korniowicz-Kowalska, 1999; Meevootison and
Niederpruem, 1979; Sanyal et al., 1985; Asahi et al., 1985), the ker-
atinolytic activity of microorganisms is conditioned by the pres-
ence of different proteases. Letourneau et al. (1998) showed that
a keratinolytic strain of Streptomyces sp. produced a mix of prote-
ases with a high keratinolytic activity in cultures containing feath-
er meal. Their activity was partly inhibited by PMSF or EDTA. Based
on research into native feather keratin degradation by 34 strains of
different species of geophilic dermatophytes and Chrysosporium,
Korniowicz-Kowalska (1997a) believes that a cascade of en-
zymes with an incremental afnity to different keratin proteins
acts in cultures with keratinolytic fungi. According to Hose and
Evans (1977), enzymes taking part in keratin decomposition are
more specialized than those that degrade fractions of non-keratin
proteins in keratin structures (hair). Large differences in the molec-
ular weight of these enzymes, produced by one fungal species, are
also indicative of a complex nature of keratinolytic protease
(Apodaca and McKerrow, 1989; Asahi et al., 1985). The molecular
weight of a puried keratinase preparation of a range of microor-
ganisms, bacteria and fungi, determined using PAGESDS elecro-
phoresis, ranges from 27 kDa (Apodaca and McKerrow, 1989) to
200 kDa (Nam et al., 2002). These are mostly enzymes with the
molecular weight 3050 kDa (Brouta et al., 2001; Sanyal et al.,
1985; Syed et al., 2009; Takiuchi et al., 1983; Yu et al., 1969).
Screening tests of keratinolytic microorganisms aim to select
active strains and to identify products of native keratin conversion
(Korniowicz-Kowalska, 1997a). This is not only of cognitive
importance but also has practical implications as both inuence
the development of keratin waste management methods using
microorganisms. Due to a high N and S content, keratin substrates
have narrow C/N and C/S ratios. Studies by Korniowicz-Kowalska
1694 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701

Table 1
Characteristic of keratinolytic properties of proteinase some microorganisms.

No. Species and strain Types of Molecular pH (optimum) Temperature Substrate References
proteinase mass (optimum)
(kDa) (C)
1 Bacillus licheniformis PWD-1 Serine 33 (7.5) (50) Bovine serum albumin (BSA), casein, Lin et al. (1992) and Wang
collagen, elastin, feather, keratin, and Shih (1999)
azokeratin
2 Bacillus pumilis Serine 31 10.010.5 (60) Keratin, caseine Han and Damodaran (1998)
3 Stenotrophomonas sp. D-1 Serine 40 7.010.0 (40) Keratin, collagen, elastine Yamamura et al. (2002)
4 Streptomyces pactum DSM Serine 30 7.010.0 (8.0) 4075 (65) Keratin azure, feather meal, native, Bckle et al. (1995)
40530 autoclaved chicken feather
5 Streptomyces 40 6.58.5 (8.0) (55) Fibrin, muscle collagen, nail, hair, Chitte et al., 1999
thermoviolaceus SD8 feather
6 Fervidobacterium islandicum Serine >200, 97 7.010.0 (9.0) (100) 70-90 Caseine, keratin Nam et al. (2002)
AW-1 (subunits)
7 Streptomyces fradiae Serine 24 8.012.0 (8.0) (50) 3050 _
Galas and Kauzewska
(1992)
8 Streptomyces gulbargensis 46 7.09.0 (9.0) 3045 (45) Gelatin, casein, BSA, soluble keratin, Syed et al. (2009)
elastin, human hair, nail, chicken
feather
9 Chrysosporium Alcaline 69 (9.0) 7.010.0 (90) Chicken feather, guinea pig hair, cow Dozie et al. (1994)
keratinophilum hair, human scalp hair
10 Trichophyton rubrum Serine 3690 (8.0) Keratin, elastin, denaturated type I Asahi et al. (1985)
collagen (azocool), casein
11 Trichophyton rubrum Serine 27>200 (8.0) Azocoll, elastin, keratin syntenic Apodaca and McKerrow
peptide, collagen (1990)
12 Microsporum canis Serine 33 (8.0) 3545 Azokeratin, hair keratin Descamps et al. (2003)
13 Microsporum gypseum 33 7.09.0 (8.0) BSA, human hair Raju et al. (2007)

(1997a) show that chicken feathers contain 49.09% organic C,
14.72% total N, 3.67% total S, and the C/N and C/S ratios of the
waste are 3:1 and 13:1, respectively. Microbial cells are made up
of no more than 6% N and 0.51% S (Kunert, 1973; Kjller and
Struwe, 1982). Excess amounts of these components are excreted
into the environment during keratin degradation. The chemical
composition of nitrogen and sulfur products released and the ratios
between them depend on the type of the native keratin destruent.
The substrate conversion caused by fungi differs from the conver-
sion induced by actinomycetes and bacteria.
The prole of nitrogen products released in the course of native
keratin degradation by fungi and some actinomycetes, such as
Streptomyces fradiae, is similar. Products of S-keratin conversion
by these microorganisms, however, are different. Saprotrophic ker-
atinolytic fungi, represented by different species of geophilic der-
matophytes and Chrysosporium representatives in the process of
complete solubilization of native keratin (feathers), used as the
only source of C, N, S and energy convert up to 75% of nitrogen
to the ammonium form (Korniowicz-Kowalska, 1997a). Between
80 and 100 mg NNH4 g1 of the substrate accumulated in the cul-
ture medium of these microorganisms during the peak period of
feather degradation, i.e. after 21 days. Due to substrate alkaliniza-
tion caused by the release of ammonia in the total nitrogen bal-
ance, up to 60% of nitrogen volatilized in gas form (Korniowicz-
Kowalska, 1997a). Streptomyces fradiae exhibited a similar, high
mineralization potential. It converted 7075% of organic nitrogen
to ammonia during wool solubilization in the mineral medium
(Noval and Nickerson, 1959). Fungi and S. fradiae release the
remaining nitrogen on native keratin as peptides and amino acids.
Only a few percent of it is built into the mycelium (Korniowicz-
Kowalska, 1997a). No more than 20% of the substrate nitrogen is
released as peptides and amino acids in cultures of feather-degrad-
ing keratinophilic fungi, among which high-molecule peptides
with a weight of P10 kDa predominated (Korniowicz-Kowalska,
1997a,b). The concentration of high-molecule peptides did not ex-
ceed 20% of total peptides released during hair keratinolysis by the
geophilic dermatophyte Microsporum gypseum. Oligo- and poly-
peptides with a molecular weight of 12 kDa prevailed (Kunert,
1976). The amino acid composition of fungal lysates of feathers
was mostly characterized by a high content of serine and cys-
tine/cysteine, 1020% and 2030%, respectively, of the total of ami-
no acids released (Korniowicz-Kowalska, 1997b). The organic
composition of nitrogen products produced during native keratin
(wool) degradation by S. fradiae also included two fractions. The
high-molecule fraction was from 6% to 12% of soluble nitrogen,
and the low-molecule fraction was ca. 20% (Noval and Nickerson,
1959).
Fermentation was the dominant mode of native keratin conver-
sion by bacteria and thermophilic actinomycetes (Gousterova
et al., 2005; Kim et al., 2001; Nam et al., 2002; Williams et al.,
1990, 1991). Small amounts of ammonia and large amounts of or-
ganic N-products are produced during this type of N-keratin catab-
olism (Hussein and Swelin, 1987). In their studies on native feather
degradation by the thermophilic anaerobic rod-bacterium Fervido-
bacterium islandicum AW-1, Nam et al. (2002) showed that free
amino acids were the main product of N biotransformation. Among
them, alanine, serine, cysteine and proline, i.e. the most abundant
amino acids occurring in feather keratin, dominated. Another ther-
mophilic bacterium of Thermoactinomyces conducting enzymatic
wool hydrolysis produced amino acids as well as considerable
amounts of low-molecule peptides (Gousterova et al., 2005).
Ammonia, however, constituted only 10% of the balance of N prod-
ucts in hydrolyzates of chicken feathers obtained after their full
solubilization in mineral medium by a thermophilic strain of Micr-
opolyspora keratynolytica. Feathers were mostly converted to solu-
ble protein compounds and free amino acids. The latter constituted
31% of nitrogen products (Hussein and Swelin, 1987). Whereas,
Matsui et al. (2009) demonstrated that more than 55% of keratin
proteins nitrogen was released in the form of free amino acids
and/or oligopeptides during the decomposition of intact chicken
feathers by the thermophilic bacterium Meiothermus ruber H328.
Sulfates are the main products of fungal conversion of sulfur
occurring in native keratin (Kunert, 1973; Korniowicz-Kowalska,
1997a). Keratinolytic fungi also produce some amounts of sultes
in the presence of S-keratin (Kunert, 1972a). Different species of
geophilic dermatophytes and keratinolytic non-dermatophytic
fungi produced from 12 to 18 mg SSO2 4 g
1
of feathers, which
corresponded to between 30% and 50% of S-feathers, in the peak
 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
mineralization period of native feather keratin (Korniowicz-Kow-
alska, 1997a). Conversely, only slight amounts of sulfates were re-
corded in S. fradiae cultures degrading wool in mineral medium.
Hyposulte, not detected during native keratin degradation by fun-
gi, was the main product containing inorganic sulfur (Kunert,
1989). Hydrogen sulde was not detected either in cultures with
keratinophilic fungi or S. fradiae metabolizing S-keratin (Noval
and Nickerson, 1959; Kunert, 1989; Korniowicz, 1994).
Sulfhydryl compounds were also recorded among keratinolysis
products containing organic sulfur (Noval and Nickerson, 1959;
Kunert, 1989; Korniowicz, 1994). Noval and Nickerson (1959)
showed that the concentration of sulfhydryl compounds released
during wool decomposition by S. fradiae corresponded to 25% of
cysteine content in the substrate.
Kunert (1989), however, observed that S. fradiae converted up
to 75% of cystine of the substrate to sulfhydryl compounds, mostly
cysteine-containing peptides, during growth on wool in mineral
medium. In contrast to actinomycetes, keratinolytic fungi produce
very few sulfhydryl compounds during native keratin solubiliza-
tion (Kunert, 1989; Korniowicz, 1994). Sulfhydryl compounds dif-
fer from those produced by S. fradiae in containing sulfocysteine
(Kunert, 1989). Kunert (1973, 1976, 1989) found both cysteine
and peptides containing sulfocysteine in cultures with Microspo-
rum gypseum decomposing wool and hair. Sulfur amino acids are
products of S-keratin catabolism in cultures of bacteria. Williams
et al. (1990) showed that the concentration of soluble sulfhydryl
compounds in cultures of the strain Bacillus licheniformis PWD-1
decomposing feathers was high when B. licheniformis developed
in aerobic conditions and low when the cultures were conducted
in anaerobic conditions. Puried keratinase of B. licheniformis did
not reduce disulde bonds with a release of sulfhydryl compounds
(Lin et al., 1992).
4. The mechanism of keratin degradation by microorganisms
The mechanism of native keratin degradation by microorgan-
isms is not fully known. A number of hypotheses, especially in rela-
tion to keratin biodegradation by fungi, have been proposed and
veried over time. A sole mechanical keratin breakdown proposed
by Raubitschek (1961) was one of the rst theories but was dis-
carded after the discovery of keratinase. This was followed by a
theory of sole enzymatic keratin digestion by keratinolytic en-
zymes (Yu et al., 1968, 1969). Another important hypothesis, sup-
ported by experimental evidence, was proposed by Kunert
(1972a,b, 1973, 1976). Based on results of long-term research into
native wool degradation by the geophilic dermatophyte Microspo-
rum gypseum and sulfuric amino acid metabolism, Kunert proposed
a two-stage process of keratin degradation divided into sultolysis
and proteolysis. The cleaving of disulde bonds between polypep-
tide keratin chains by means of inorganic sulte produced by the
fungus would occur during sultolysis and lead to protein denatur-
ation. A schematic of cystine splitting by sultes (sultolysis)
according to Kunert (1976) can be presented as follows:
cys-SS-cys cystine HSO03 $ cySH cysteine cyS  SO03 S-sulfocysteine
The presence of bound sulte and sulfocysteine, the product of
keratin cystine reduction by the compound, in cultures of M. gyp-
seum containing wool as the sole source of C, N and S, provided evi-
dence to support Kunerts theory (1976). Sultolysis would cause
keratin structure denaturation facilitating an attack of keratinolyt-
ic proteases. Denatured keratin proteins would next undergo pro-
teolysis by extracellular proteases that are endopeptidases (Kunert,
1976). Some authors (Rufn et al., 1976) believe that both pro-
cesses, sultolysis and proteolysis, occur at the same time during
fungal keratinolysis. Alkalinisation of the reaction environment is
1695
an important factor conditioning sultolysis. It is connected with
the process of deanimation of simpler proteins and ammonia re-
lease (Kunert, 1976).
Micromorphological investigations of hair destruction by kera-
tinophilic fungi (English, 1963, 1969, 1976; Filipello Marchisio
et al., 1994; Hsu and Volz, 1975; Kunert and Krajci, 1975) showed
new light on the hypothesis regarding the participation of a
mechanical factor, i.e. the destructive inuence of the mycelium
in the biodegradation of native keratin.
Micromophological, cytochemical and biochemical research
conducted so far shows that the process of degradation of native
keratin by fungi is an enzymatic lysis coupled with mechanical
destruction (Filipello Marchisio, 2000; Kunert, 1989, 2000).
The eroding mycelium complex takes part in the mechanical
destruction of hair by fungi. Kunert (1972b) showed that the erod-
ing mycelium complex participates in sultolysis. Decomposition
of cytoplasmatic membrane proteins induces sultolysis and ex-
poses disulde bonds of keratin located intracellularly (Page and
Stock, 1974). It is composed of structures causing substrate surface
erosion, known as frond mycelium, and penetrating inside perpen-
dicularly to its surface (radial penetration). In the case of dermato-
phytes, these are wedge-shaped perforating organs (Fig. 3)
described by English (1963, 1969). Fungi producing perforating or-
gans are strong destructors of native keratins rapidly degrading
this substrate. Non-dermatophytic keratinolytic fungi, such as rep-
resentatives of Chrysosporium, do not produce perforating organs
but only swollen hyphae known as boring hyphae (Fig. 3) (English,
1969, 1976). Non-keratinolytic fungi growing on hair produce sin-
gle thin boring hyphae (English, 1965). According to English (1965,
1969), the evolutionary transition from borers to perforating or-
gans was coupled with the development of lysis abilities in keratin.
Fungi producing thin boring hyphae decompose non-keratin pro-
teins and soft keratin. Fungi producing swollen boring hyphae,
e.g. Chrysosporium keratinophilum, exhibit keratinolytic abilities to-
wards hard keratin. The keratinolytic activity of these fungi in rela-
tion to fungi producing perforating organs is, however, denitely
weaker (Filipello Marchisio, 2000). Filipello Marchisio (2000) re-
ports that simpler structures penetrate less compact keratin areas
and with a smaller number of disulde bonds. It is also known that
some typically keratinolytic fungi, i.e. Chrysosporium tropicum and
Myceliophtora vellerea, are not able to produce structures penetrat-
ing hair inwards. They attack the substrate only supercially,
which is often accompanied by a mass loss of the substrate (Filip-
ello Marchisio et al., 1994). Based on this, Filipello Marchisio
(2000) distinguishes two types of attack: surface erosion and radial
penetration. Gradual hair destruction occurs from outside inwards
during surface erosion and consists of the spread of hyphae along
the hair cuticle, the erection of the cuticle and its dissolution. These
hyphae either retain their original shape or branch off, forming
palm-like or hand-like structures (English, 1963). They form a at-
tened, branching fern-shaped mycelium, called the eroding myce-
lium by English (1963), in the hair cortex. Transformed hyphae
form a coat-like structure surrounding the hair, making the erosion
surface uniform. The attack produces hollows in the hair cortex,
and the destruction zone expands as the eroding mycelium devel-
ops further. During radial penetration which is perpendicular to
the hair axis, areas of lysis form around boring hyphae and perfo-
rating organs. They cover a greater surface area than penetrating
hyphae. Both attack forms, surface erosion and radial penetration,
are not mutually exclusive and usually co-occur (Filipello
Marchisio, 2000).
Prokaryotic organisms decompose native keratin enzymatically
and the process of digestion of disulde bonds occurs by reduction.
During studies on wool decomposition by S. fradiae, Kunert (1989)
recorded considerable amounts of sulfhydryl compounds in the
form of cysteine-containing peptides. Disulde bond reduction in
1696
Fig. 3. (A) Perforating organs of Trichophyton mentagrophytes; in vitro hair infection; x650, according to English, 1963; (B) Boring hyphae of Chrysosporium sp.; in vitro hair
infection; x1320, according to English (1976).
S. pactum cultures growing on chicken feathers was observed by
Bckle et al. (1995). Sangali and Brandelli (2000) suggest that the
enzyme disulte reductase is responsible for the reduction of
disulde bonds of feather keratin by the strain Vibrio Kr2. The
occurrence of an enzyme similar to disulte reductase in cultures
of keratin-decomposing bacteria was conrmed by Yamamura
et al. (2002). Yamamura et al. (2002) isolated a keratin-decompos-
ing strain of the bacterium Stenotrophomonas sp. D-1 from deer fur.
The strain produced two types of extracellular enzymatic proteins:
serine protease and a disulte reductase-like protein. Neither en-
zyme exhibited keratinolytic activity on its own. Yamamura et al.
(2002) proposed a mechanism of native keratin (hair) degradation
by the bacterium that relies on the co-operation of both enzymatic
proteins:
Disulfide reductase like protein
K-S-S-K ! K-SH
Native Keratin Reduced keratin
In the rst stage, the disulte reductase-like protein would
cause a reduction of disulde bonds and the production of dena-
tured keratin which would later be decomposed by protease, and
soluble products (peptides/amino acids) would be released
(Yamamura et al., 2002).
5. Keratin waste management
Small amounts of keratin are usually observed in natural envi-
ronment such as the soil. High concentrations of keratin proteins
are recorded during animal production and processing, and on live-
stock and poultry farms. These proteins constitute keratin by-prod-
ucts: bristles, hair, down, horns and hooves, and contain from
15% to 18% nitrogen, 25% sulfur, 1.27% fat and 3.20% mineral
elements, and ca. 90% protein (Gessesse et al., 2003; Kunert,
2000; Sangali and Brandelli, 2000; Korniowicz-Kowalska,
1997a). Chicken feathers are by far the most common keratin
waste product globally with high amounts of chicken feathers
being produced in poultry slaughterhouses and on poultry farms.
Wojtowicz and Moscicki (1998) report that waste produced on
one poultry farm (Poland) is estimated at ca. 1.25 Mg of carrion
per two-week production cycle. Poultry mortality on farms or in
processing plants in the US may reach between 0.1% and 0.25%
per day (after Ichida et al., 2001). Dalev (1994) reports that
50,000 chicken produce from 2 to 3 Mg of feathers. In Poland, at
least 77,000 Mg feathers annually are obtained only from broilers
(Bohacz and Korniowicz-Kowalska, 2007). Approximately 1 mil-
lion Mg of waste feathers and 1 million Mg human and animal hair
are produced annually in the US (Ichida et al., 2001; Shih, 1993).
According to Frazer (2004), as much as 3 billion pounds of waste
feathers, i.e. 1.36 billion Mg, can be produced annually in the US.
Due to high-protein content, feather waste was largely con-
verted to feedstuffs. A variety of processing methods using ther-
T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
mal, chemical and enzymatic factors have been described in the
literature (Papadopoulos, 1985). Thermohydrolysis was a popular
method of waste feathers and bristle conversion to meal in Poland
(Jeske et al., 1976; Orzeszko and Sutarzewicz, 1979). Feather meal
was mostly employed in feedstuffs for poultry and swine. Such
feedstuffs had a high content of protein nitrogen, fat and mineral
substances. However, they were decient in lysine, methionine
and histidine. Feather meals also had a lower coefcient of digest-
ibility of amino acids crucial for domestic fowl, i.e. lysine, methio-
nine, threonine and cysteine, in comparison with other industrial
protein feedstuffs. Thermal or chemical processing of feather waste
additionally destroys some amino acids, including cystine (Papad-
opoulos, 1985;Papadopoulos et al., 1986).
Acidic or alkaline hydrolysis was used to solubilize insoluble
keratin and release sulfur amino acids for dietary purposes
Protease D-1
! Piptide=Amino Acid (Orzeszko and Sutarzewicz, 1979). A physicochemical method of
Products
heating keratin material with some organic solvents, e.g. dimethyl-
formamide (DMF) and dimethyl sulfoxide (DMSO), was also ap-
plied to obtain soluble keratin proteins for use in animal feeding
(Wolski, 1985). DMSO a compound with low toxicity, was suc-
cessfully applied by Wolski (1985) for the dissolution of chicken
feathers. After the solubilization of those wastes in DMSO, followed
by precipitation of dissolved protein with acetone and distillation
removal of both solvents, a sediment with the taste of lean cottage
cheese was obtained. When dried, that sediment constituted a pro-
tein preparation (Wolski, 1985). Studies by Coward-Kelly et al.
(2006) show that a liquid hydrolysate rich in amino acids and poly-
peptides is obtained by chemical-thermal hydrolysis of chicken
feathers. Its composition is similar to the protein in soybeans and
cotton seeds, and the hydrolysate can be used as a diet supplement
in feeding ruminants. It is not suitable for use in feedstuffs for
monogastric animals due to its low content of arginine, histidine,
lysine, methionine and threonine. Ammonolysis was also used to
process waste feathers and bristles, obtaining keratin-urea granu-
lated products recommended as supplementary additions in feed-
ing ruminants (Orzeszko and Sutarzewicz, 1979; Wolski, 1979). All
physical and chemical methods of processing waste feathers and
other native keratins require considerable energy investments.
Both poor assimilability of amino acids present in keratin products
as well as high production costs considerably discouraged interest
in microbiological methods of processing waste feathers and other
keratin waste (Korniowicz-Kowalska, 1997a).
At present technologies aimed at obtaining protein hydroly-
sates with keratinolytic micro-organisms are used in many coun-
tries worldwide (India, Brazil, Venezuela). As reported by
Vasileva-Tonkova et al. (2009), Syed et al. (2009), Grazziotin
et al. (2006), feather degradation by microorganisms is an ecolog-
ically safe and cost-efcient form of waste utilization. Microorgan-
isms suitable for obtaining nutrients from native keratin for
animals include keratinolytic strains of Bacillus spp. (Kim et al.,
2001; Suntornsuk and Suntornsuk, 2003; Gessesse et al., 2003;
 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
Mc Govern, 2000; Park and Son, 2009), Vibrio spp. (Grazziotin et al.,
2006), Kocuria rosea (Bertsch and Coello, 2005), Serratia sp. (Khard-
enavis et al., 2009), Meiothermus ruber H328 (Matsui et al. (2009)),
mesophilic and thermophilic actinomycetes (Vasileva-Tonkova
et al., 2009; Syed et al., 2009), and fungi belonging to geophilic der-
matophytes Microsporum gypseum (Raju et al., 2007) and the genus
Aspergillus (Santos et al., 1996; Farag and Hassan, 2004). Feather
conversion to high-protein feedstuffs for poultry using the thermo-
philic keratinolytic bacterium Bacillus licheniformis strain PWD-1 or
its keratinase is especially interesting. The technology, being im-
proved in a number of research centres in the US, China and Tai-
wan, yields a so-called feather lysate, a product considered to be
nutritionally equivalent to soya protein (Williams et al., 1990).
The nutrient product is assimilated by poultry in 65% (Shih,
1999). Vasileva-Tonkova et al. (2009) report that feather hydroly-
sates obtained from cultures of a mix of thermophilic strains of
actinomycetes (Thermoactinomyces) after 72 h are rich in soluble
proteins and amino acids, including lysine, threonine, leucine, iso-
leucine and valine, and, in smaller quantities, proline and serine.
Likewise, Khardenavis et al. (2009) observed that feather hydroly-
sis by proteases secreted by Serratia sp. HPC1383 was a source of
highly digestible feedstuffs for animals.
Enzymatic-chemical hydrolysis have also been used to obtain
feedstuffs from feathers. Elmayergi and Smith (1971) as well as
Papadopoulos (1985) obtained improved digestibility of feather
meal produced with the method of thermohydrolysis, and even
an increase in the content of essential amino acids by enzymatic
processing using, e.g. microbial keratinolytic proteases. Dalev
(1994) and Dalev et al. (1996) applied enzymatic-alkaline hydroly-
sis (feather keratin) to obtain protein concentrates for feed. He
demonstrated also (Dalev et al., 1997) that enzymatic modication
of hydrolysates of feather keratin through enrichment with lysine
caused an increase in their nutritive value.
The application of processed animal by-products, including
feather meals, in feedstuffs for animals has been declining in recent
years. The use of meat and bone meal produced from processed
animal by-products, including feather meal, obtained from slaugh-
tered and processed animals, also poultry, has not been allowed in
the EU member states since 1st January 2000. The ban aims to cre-
ate an efcient system of protecting human health within the EU
from infections caused by pathogenic microorganisms of animal
origin (http://www.kipdip.org.pl). Regulation number 999/2001
of the European Parliament and of the Council of 22 May 2001 lays
down rules for the prevention, control and eradication of by-prod-
ucts highly harmful to humans, and Regulation (EC) No. 1774/2002
of the European Parliament and of the Council of 3rd October 2002
stipulates health rules concerning the collection, treatment and
utilization of all types of by-products in animal production. Quotas
imposed on the use of bioconversion products of native keratin in
foodstuffs for animals stimulated research into other uses of native
keratin.
An interest in practical applications of enzymatic keratin
hydrolysates in the cosmetic and pharmaceutical industries has
also been growing (Rodziewicz and aba, 2006). Microbial kera-
tinase can be used in the textile, cosmetic and leather industries
and in medicine (Farag and Hassan, 2004; Gupta and Ramnani,
2006; Brandelli et al., 2010). Brandelli et al. (2010) and Gupta
and Ramnani (2006) indicate the possibility of using keratinases
secreted by microorganisms for the degradation of prions and
prion-like proteins and of their application in detergents. Inves-
tigations by Poopathi and Abidha (2008) demonstrate that native
keratin can be used as a substrate for the production of an insec-
ticidal endotoxin by strains of Bacillus sphaericus and B. thuringi-
ensis serovar israelensis. These bioinsecticides can be used to
control larvae of members belonging to the subfamily
Phlebotominae.
1697
Research into keratin-based biomaterials in medical technolo-
gies as gels, lms, coatings or bres have also been carried out in
the last few years (Hill et al., 2010). Hill et al. (2010) tested two
biomaterials obtained from keratin by chemical processing, such
as kerateines or keratoses (oxidatively or reductively derived from
keratin), as medical supplements in wound healing, bone regener-
ation, peripheral nervous system repair and hemostasis. Aluigi
et al. (2008) used native wool keratin to produce nanobres, so-
called keratin/PEO (poly/ethylene oxide) blend nanobres, with
electrospinning. Products obtained in these studies have a high
bioadaptability and susceptibility to degradation both in vivo and
in vitro, and are recommended as biomedications equal to collagen
and brinogen (Aluigi et al., 2008).
Limits imposed on the processing of animal by-products for
feedstuffs and the subsequent increase in the unused amount of
keratin waste have enhanced an interest in methods of their natu-
ral and agricultural management. Attempts to use waste feathers
in the production of fertilizing agents, such as keratin-bark-urea
granulates, were made in Poland as early as in the 1980s (Debicki
et al., 1989; Dechnik and Wiater, 1989). Microplot experiments
showed that such fertilizing agents favourably inuenced soil
properties and some crop plants (Dechnik and Wiater, 1989;
Debicki et al., 1989; Nurzynski, 1989; Styk, 1989; Wolski et al.,
1996), at the same time limiting patch weeding (Pawowski
et al., 1989). However, their inuence on the microbiological activ-
ity and nitrogen transformation in the soil was not uniformly posi-
tive. Gostkowska et al. (1989) showed that the fertilizer caused a
reduction in the respiratory activity of light soils, disturbances in
the nitrication process and nitrogen losses from this
environment.
Relevant legal provisions harmonized with the EU legislation
provide the framework for organic waste management (the act
on waste of 27th April 2001 in Poland and the Council Directive
of 15 July 1975 on waste (75/442/EEC), a so-called framework
directive, with later amendments). At present, they dene two
types of organic waste management: aerobic digestion, including
composting, and anaerobic methods (biomethanation) using meth-
ane-producing bacteria (Bar, 2001). Biogas composed largely of
methane and solid material with fertilizing values is produced dur-
ing anaerobic feather digestion by methane fermentation (Jiang
et al., 1987). Composting is the most popular rational keratin waste
management method. It is biological conversion of organic waste
to hygienic, humus-rich, relatively biostable, mature product that
improves soil properties and serves nutritional needs of plants,
and that occurs under controlled conditions (Mathur et al., 1993).
Feather composting is a safe, sanitary and cost-effective tech-
nology that yields products (compost) applicable as fertilizers
(Ichida et al., 2001; Orzeszko and Sutarzewicz, 1979; Shih, 1993;
Bohacz and Korniowicz-Kowalska, 2005b). Both keratinolytic bac-
teria and fungi are proposed for use in composting. The active con-
tribution of keratinolytic bacteria of the genus Bacillus and
actinomycetes to keratin waste composting was discussed by
Ichida et al. (2001), Kster and Sofwat (1978), Letourneau et al.
(1998), Lin et al. (1999), Tiquia (2005), and Tiquia et al. (2005).
Intensive mineralization of feather nitrogen and sulfur by active
keratinolytic fungi makes such fungi useful in the management
of keratin waste (feathers) by composting (Korniowicz-Kowalska,
1997a). Composting of chicken feathers (in a model system) to-
gether with plant waste rich in ligninocellulose (grain straw and
pine bark) yields a product that, after introduction into the soil
(pot experiment), has a stimulating effect on biochemical pro-
cesses occurring in the soil, causing an increase in organic C, N
NO3 and SSO4 (Bohacz and Korniowicz-Kowalska, 2005a,b) and
improves the phytosanitary condition of the soil by limiting the
development of potentially phytopathogenic fungi of the genus
Fusarium (Korniowicz-Kowalska and Bohacz, 2002b). As studies
1698 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
by Korniowicz-Kowalska and Bohacz (2010) show, biodegrada-
tion of chicken feathers mixed either with straw or with straw
and bark occurs by taxonomically, ecologically and physiologically
different groups of microorganisms. The composting process of this
waste can be divided into three stages during which bioecological
groups of microorganisms occur in succession. In the rst stage
(weeks 13), an intensive growth of meso- and termophilic bacte-
ria and actinomycetes decomposing available organic substrates is
observed. They are replaced by more nutritionally specialized
groups of microorganisms such as bacteria and cellulolytic fungi
(weeks 26). Keratinophilic fungi that decompose native keratin
become active in the nal stage (weeks 46). Composting of this
waste with high-carbon ligninocellulose waste not only provides
valuable compost but also limits nitrogen losses by volatilization
of ammonia, which occurs in pure fungal cultures (Korniowicz-
Kowalska, 1997a; Bohacz and Korniowicz-Kowalska, 2009b).
Feather waste is fully biodegraded and bioconverted by fungi to
nitrogen and sulfur forms, NNH 4 , NNO3 and SSO4, that are eas-
ily absorbed by plants, in the process of colonization and succes-
sion of physiologically different microbial communities and
changes in their metabolic and enzymatic activity associated with
it (Bohacz and Korniowicz-Kowalska, 2007, 2009a,b). As reported
by Bohacz and Korniowicz-Kowalska (2007, 2009a,b), reliable
indicators of the maturity of these composts include: a decrease
in the activity of dehydrogenases, cellulose and protease, and an
accumulation of sulfates, the nal product of S-keratin transforma-
tion by keratinophilic fungi, in the composted mass, similarly to
the observed increase in nitrates in the compost water fraction
and the solid material, and a decrease in NNH4 content in mature
composts consisting of keratin and lignionocellulose waste (Bohacz
and Korniowicz-Kowalska, 2009b), which is also an indicator of
compost maturity (Mathur et al., 1993; Iwegbue et al., 2006). Con-
trary to composts obtained from communal waste whose pH is
close to neutral (Bertoldi et al., 1983; Jimenez and Garcia, 1989;
Miller, 1993; Parr and Hornic, 1993), composts derived from feath-
er, bark and straw were acidic (pH 4). They were also characterized
by a good phytosanitary condition. According to Bohacz and
Korniowicz-Kowalska (2009a), the acidic pH of composts ob-
tained from feather, bark and straw is a great asset, especially in
horticultural production and the dynamically developing sector
of ornamental plants which have a high demand for this type of
compost. The production of such composts on a larger scale can
also contribute to peat protection by limiting peat extraction.
6. Conclusions
New regulations concerning the management of category 3 ani-
mal by-products stress the need for environment-friendly technol-
ogies. They are an incentive to employ effective keratinolytic
microorganisms and to develop biotechnologies of keratin waste
processing. Two major developments in keratin waste treatment
can be distinguished in the current approaches to native keratin
biodegradation: the fermentation method that provides amino
acids and the mineralization method that leads to keratin con-
version to inorganic compounds. Solubilization of native keratin
by enzymes of keratinolytic bacteria such as Bacillus licheniformis
strain PWD-1 is an example of the fermentation technology. That
method is considered to be the most suitable for the purposes of
acquisition of animal feed preparations. As well as feather utiliza-
tion in feedstuffs, feather waste composting with plant waste is an
especially promising method. Feather composting with ligninocel-
lulose material (bark, straw) occurs with a high participation of
keratinolytic fungi and actinomycetes, and causes mineralization
and biotransformation of nitrogen and sulphur into forms easily
absorbed by plants. Over time, large-scale composting of keratin
wastes, especially poultry feathers that are produced in the great-
est amounts, may provide a solution to the problem of their utilisa-
tion. It will also prevent wasting of the material and the ecological
hazards resulting from keeping those wastes on waste dumps.
However, further research is required, especially focused on accel-
eration of the process of composting and on achievement of earlier
maturity of composts.
Acknowledgements
This work has been supported by the Ministry of Science and
Higher Education No. N N523213737.
References
Akhatar, W., Edwards, H.G.M., 1997. Fourier-transform Raman spectroscopy of
mammalian and avian keratotic biopolymers. Spectocim. Acta A 53, 8190.
Aluigi, A., Vineis, C., Varesano, A., Mazzuchetti, G., Ferrero, F., Tonin, C., 2008.
Structure and properties of keratin/PEO blend nanobres. Euro. Polym. J. 44,
24652475.
Apodaca, G., McKerrow, J.H., 1989. Purication and characterization of a 27,000-Mr
extracellular proteinase from Trichophyton rubrum. Infect. Immun. 57, 3072
3080.
Apodaca, G., McKerrow, J.H., 1990. Expression of proteolytic activity by cultures of
Trichophyton rubrum. J. Med. Vet. Mycol. 28, 159171.
Asahi, M.R., Lindquist, K., Fukuyama, G., Apocarda, W., Epstein, W.L., Mc Kerrow,
J.H., 1985. Purication and characterization of major extracellular proteinases
from Trichophyton rubrum. Biochem. J. 232, 139144.
Bar, M., 2001. New legal regulations in organic waste management and EU
regulations. In: Conference proceedings waste composting: a protable
business or a nuisance? Osieczany near Krakw, 1921 September, http://
www.tnz.most.org.pl/kompost/spis.htm (in Polish).
Battelli, G., Banched, M., Frigo, W., Amorati, P., Mantovani, A., Pegli, A., 1978. Survey
of keratinophilic fungi alpine marmot (Marmota marmota) burrow soil in
adjoining soil. Sabouraudia 16, 8386.
Bertoldi, M., de Vallini, G., Pera, A., 1983. The biology of composting: a review.
Waste Manag. Res. 1, 157176.
Bertsch, A., Coello, N., 2005. A biotechnological process for treatment and recycling
poultry feathers as a feed ingredient. Biores. Technol. 95, 17031708.
Bckle, B., Galunsky, B., Muller, R., 1995. Characterization of keratinolytic serine
protease from Streptomyces pactum DSM 40530. Appl. Environ. Microbiol. 61,
37053710.
Bohacz, J., Korniowicz-Kowalska, T., 2005a. Inuence of keratin-bark and keratin-
bark-straw composts on properties of selected soils. Part I. Biochemical
properties. Prob. Notes Adv. Agric. Sci. 505, 5564 (in Polish).
Bohacz, J., Korniowicz-Kowalska, T., 2005b. Inuence of keratin-bark and keratin-
bark-straw composts on properties of selected soils. Part II. Chemical
properties. Prob. Notes Adv. Agric. Sci. 505, 5564 (in Polish).
Bohacz, J., Korniowicz-Kowalska, T., 2007. Choice of maturity indexes for feathers-
plant material composts on a base of selected microbiological and chemical
parameters-preliminary research. Pol. J. Environ. Stud. 16 (2A), 719725.
Bohacz, J., Korniowicz-Kowalska, T., 2009a. Changes in enzymatic activity in
composts containing chicken feathers. Biores. Technol. 100, 36043612.
Bohacz, J., Korniowicz-Kowalska, T., 2009b. Nitrogen and sulfur transformations in
composts containing chicken feathers. Comput. Sci. Util. 17, 180188.
Bolliger, A., 1951. Water extractable constituents of hair. J. Investigat. Dermatol. 11,
7984.
Brandelli, A., Daroit, D.J., Riffel, A., 2010. Biochemical features of microbial
keratinases and their production and applications. Appl. Microbiol.
Biotechnol. 85, 17351750.
Bressollier, P., Letourneau, F., Urdaci, M., Verneuil, B., 1999. Purication and
characterization of a keratinolytic serine proteinase from Streptomyces
albidoavus. Appl. Environ. Microbiol. 65, 25702576.
Brouta, F., Descamps, F., Fett, T., Losson, B., Gerday, Ch., Mignon, B., 2001.
Purication and characterization a 43.5 kDa keratinolytic metalloprotease
from Microsporum canis. Med. Mycol. 39, 269275.
Burtt Jr., E.H., Ichida, J.M., 1999. Occurrence of feather- degrading Bacilli in the
plumage of birds. The Auk 116, 364372.
Cameron, G.J., Wess, T.J., Bonser, R.H.C., 2003. Youngs modulus varies with
differential orientation of keratin in feathers. J. Struct. Biol. 143, 118123.
Caretta, G., Piontelli, E., 1975. Isolation of keratinophilic fungi from soil Pavia Italy.
Sabouraudia 13, 3337.
Caretta, G., Mangiarotti, A.M., Piontelli, E., 1992. Keratinophilic fungi isolated from
soil of Italian parks in the province of Pavia. Eur. J. Epidemiol. 8, 330339.
Chattaway, F.W., Ellis, D.A., Barlow, A.J.E., 1963. Peptidases of dermatophytes. J.
Invest. Dermatol. 43, 3137.
Chesters, C.G.C., Mathison, G.E., 1963. The decomposition of wool keratin by
Keratinomyces ajelloi. Sabouraudia 2, 225237.
Chitte, R.R., Nalawade, V.K., Dey, S., 1999. Keratinolytic activity from the broth of a
feather-degrading thermophilic Streptomyces thermoviolaceus strain SD8. Lett.
Appl. Microbiol. 28, 131136.
Chmel, L., Vlcilikov, A., 1977. Keratinophilne huby v niektorych pochodnych
typoch a factory ovplyvnujece ich zastupenie. Biologia (Bratislava) 32, 5359.
 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
Chmel, L., Hasilkov, B., Hraco, J., Vlacikov, A., 1972. The inuence of some Han, X., Damodaran, S., 1998. Purication and characterization of protease Q: A
ecological factors on keratinophilic fungi in the soil. Sabouraudia 10, 2634.
Coward-Kelly, G., Chang, V.S., Agbogbo, F.K., Holtzapple, M.T., 2006. Lime treatment
of keratinous materials for the generation of highly digestible animal feed:
Chicken feathers. Biores. Technol. 97, 13371343.
Currah, R.S., 1985. Taxonomy of the onygenales: arthodermataceae, gymnoasceae,
myxotrichaceae and onygenaceae. Mycotaxon 24, 1216.
Dalev, G.P., 1994. Utilisation of waste feathers from poultry slaughter for
production of a protein concentrate. Biores. Technol. 48, 265267.
Dalev, P., Ljubomirova, A., Simeonova, L., Ivanov, I., 1996. Protein hydrolysates from
waste feathers for feed and their enrichment with lysine through enzyme
catalyzed covalent binding. Med. Fac. Landbouww. Univ. Gent. 61/4a.
Dalev, P., Ivanov, I., Liubomirova, A., 1997. Enzymic modication of feather keratin
hydrolysates with lysine aimed at increasing the biological value. J. Sci. Food
Agric. 73, 242244.
Debicki, R., Rejman, J., Wontroba, J., 1989. Effect of keratin-bark-urea granulate on
the physico-chemical properties of soils. Prob. Notes Adv. Agric. Sci. 370, 3948
(in Polish).
Dechnik, I., Wiater, J., 1989. Effect of fertilization with various organic materials on
the chemical composition of rye during the tillering and owering phases. Prob.
Notes Adv. Agric. Sci. 370, 119128 (in Polish).
Descamps, F., Brouta, F., Vermout, S., Monod, M., Losson, B., Mignon, B., 2003.
Recombinant expression and antigenic properties of a 31.5-kDa keratinolytic
subtilisin-like serine protease from Microsporum canis. FEMS Immun. Med.
Microbiol. 38, 2934.
Dozie, I.N.S., Okeke, C.N., Unaeze, N.C., 1994. A thermostable, alkaline-active,
keratinolytic proteinase from Chrysosporium keratinophilum. World J. Microbiol.
Biotechnol. 10, 563567.
Elmayergi, H.H., Smith, R.E., 1971. Inuence of growth of Streptomyces fradiae on
pepsin-HCl digestibility and methionine content of feather meal. Can. J.
Microbiol. 17, 10671072.
English, M.P., 1963. The saprophytic growth of keratinophilic fungi on keratin.
Sabouraudia 2, 115130.
English, M.P., 1965. The saprotrophytic growth of non-keratinophilic fungi on
keratinized substrata, and a comparison with keratinophilic fungi. Trans. Brit.
Mycol. Soc. 48, 219235.
English, M.P., 1969. Destruction of hair by Chrysosporium keratinophilum. Trans. Br.
Mycol. Soc. 52, 247255.
English, M.P., 1976. Destruction of hair by two species of Chrysosporium. Trans. Br.
Mycol. Soc. 66, 357358.
Farag, A.M., Hassan, M.A., 2004. Purication, characterization and immobilization of
a keratinase from Aspergillus oryzae. Enz. Microb. Technol. 34, 8593.
Filipello Marchisio, V., 2000. Karatinophilic fungi: their role in nature and
degradation of keratinic substrates. In: Kushawaha, R.K.S., Guarro, J. (Eds.),
Biology of dermatophytes and other keratinophilic fungi. Rev. Iber. Micol., 17,
8692.
Filipello Marchisio, V., Fusconi, A., Rigo, S., 1994. Keratinolysis and its morphological
expression in hair digestion by airborne fungi. Mycopathologia 127, 103115.
Filipello Marchisio, V., Fusconi, A., Querio, F.L., 2000. Scopulariopsis brevicaulis: a
keratinophilic or a keratinolytic fungus? Mycoses 43, 281292.
Fraser, R.D.B., Parry, D.A.D., 2003. Macrobril assembly in trichocyte (hard a-)
keratins. J. Struct. Biol. 142, 319325.
Fraser, R.B.D., Mac Rae, T.P., Rogers, G.E., 1972. Keratins- their Composition,
Structure and Biosynthesis. Charles C. Thomas, Illinois, 283.
Frazer, L., 2004. Chicken electronics. A technology plucked from waste. Environ.
Health Persp. 112, 565567.
Friedrich, A.B., Antranikian, G., 1996. Keratin degradation by Fervidobacterium
pennavorans a novel thermophilic anaerobic species of the order Termotogales.
Appl. Environ. Microbiol. 62, 28752882.
_
Galas, E., Kauzewska, M., 1992. Proteinases of Streptomyces fradiae III. Catalytic and
some physic-chemical properties of keratynolytic proteinase. Acta Microbiol.
Polon. 41, 169177.
Garg, A.P., Gandotra, S., Mukerji, K.G., Pugh, G.J.F., 1985. Ecology of keratinophilic
fungi. Proc. Indian Acad. Sci. (Plant Sci.) 94, 149163.
Gessesse, A., Hatti-Kaul, R., Gashe, B.A., Mattiasson, B., 2003. Novel alkaline
proteases from alkaliphilic bacteria grown on chicken feather. Enz. Microbial.
Technol. 32, 519524.
Gostkowska, K., Woytowicz, B., Szember, A., Furczak, J., JezierskaTys, S., Jaskiewicz,
W., 1989. Effect of various fertilizers on the microbiological activity of sandy
soil. Prob. Notes Adv. Agric. Sci. 370, 7584 (in Polish).
Gousterova, A., Braikova, D., Goshev, I., Christov, P., Tishinov, K., Vasileva-Tonkova,
E., Haertl, T., Nedkov, P., 2005. Degradation of keratin and collagen containing
wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis. Lett.
Appl. Microbiol. 40, 335340.
Gradisar, H., Kern, S., Friedrich, J., 2000. Keratinase of Doratomyces microsporus.
Appl. Microbiol. Biotechnol. 53, 196200.
Grazziotin, A., Pimentel, F.A., Jong de, E.V., Brandelli, A., 2006. Nutritional
improvement of feather protein by treatment with microbial keratinase.
Anim. Feed Sci. Technol. 126, 135144.
Grifn, D.M., 1960. Fungal colonization of sterile hair in contact with soil. Trans. Br.
Mycol. Soc. 43, 583596.
Grzywnowicz, G., obarzewski, J., Wawrzkiewicz, K., Wolski, T., 1989. Comparative
characterization of proteolytic enzymes from Trichophyton gallinae and
Trichophyton verrucosum. J. Med. Vet. Mycol. 27, 319328.
Gupta, R., Ramnani, P., 2006. Microbial keratinases and their prospective
applications: an overview. Appl. Microbiol. Biotechnol. 70, 2133.
1699
detergent-and stable serine endopeptidase from Bacillus pumilus. J. Agaric. Food
Chem. 46, 35963603.
Hayashi, N., Toshitani, S., 1983. Human infections with Microsporum gypseum in
Japan. Mycosen 26, 337345.
Hill, P., Brantley, H., Dyke, M.V., 2010. Some properties of keratin biomaterials:
Kerateines. Biomaterials 31, 585593.
Hose, H., Evans, G., 1977. Degradation of native keratin by dermatophytes. J. Invest
Dermatol. 68, 245246.
Hsu, Y.C., Volz, P.A., 1975. Penetration of Trichophyton terrestre in human hair.
Mycopathol. 55, 179183.
Hublek, Z., 1974. Fungi associated with free-living birds in Czechoslovakia and
Yuogoslavia. Acta Sci. Nat. Brno. 8, 162.
Hublek, Z., 2000. Keratinophilic fungi associated with free-living mammals and
birds. In: Kushawaha, R.K.S., Guarro, J. (Eds.), Biology of dermatophytes and
other keratinophilic fungi. Rev. Iber. Micol. 17, 8692.
Hublek, Z., Balt, F., 1974. The survival of microfungi in the nests of tree-sparrow
[Passer montanus L.] in the nest-boxes over the winter season. Mycopathol.
Mycol. Applicat. 54, 517530.
Hussein, M.M., Swelin, M., 1987. Bioconversion of chicken feather into
soluble nitrogenous product. Microbe-86. Int. Congr. Microbiol. 14 Meet.:
85.
Ichida, J.M., Krizova, L., Lefevre, C.A., Keener, H.M., Elwell, D.L., Burtt Jr., E.H., 2001.
Bacterial inoculum enhances keratin degradation and biolm formation in
poultry compost. J. Microbiol. Methods 47, 199208.
Iwegbue, C.M.A., Egun, A.C., Emuh, F.N., Isirimah, N.O., 2006. Compost maturity
evaluation and its signicance to agriculture. Pakistan J. Biol. Sci. 9, 2933
2944.
Jeske, J., Doruchowski, W., Wciso, H., 1976. The assessment of by-product
management in the poultry industry. Food Industry 30, 255257 (in Polish).
Jiang, Z.H., Steinsberger, C., Shih, J.C.H., 1987. In situ utilization of biogas on a
poultry farm: heating, drying and animal brooding. Biomass 14, 269281.
Jimenez, E.I., Garcia, V.P., 1989. Evaluation of city refuse compost maturity: a
review. Biol. Wastes 27, 115142.
Jones, L.N., 2001. Hair structure anatomy and comparative anatomy. Clin. Dermatol.
19, 95103.
Kaul, S., Sumbali, G., 1999. Production of extracellular keratinases by keratinophilic
fungal species inhabiting feathers of living poultry birds (Gallus domesticus): a
comparison. Mycopathology 146 (1), 1924.
Khardenavis, A.A., Kapley, A., Purohit, H.J., 2009. Processing of poultry feathers by
alkaline keratin hydrolyzing enzyme from Serratia sp. HPC 1383. Waste Manag.
29, 14091415.
Kim, J.M., Lim, W.J., Suh, H.J., 2001. Feather-degrading Bacillus species from poultry
waste. Process Biochem. 37, 287291.
Kjller, A., Struwe, S., 1982. Microfungi in ecosystems: fungal occurrence and
activity in litter and soil. Oikos 39, 389422.
Korniowicz, T., 1991/1992. Investigations into the microora colonizing raw
keratin waste in the soil. Acta Mycol. 27, 231245 (in Polish).
Korniowicz, T., 1994. Methods for determing keratinolytic activity of saprophytic
fungi. Acta Mycol. 29, 169178.
Korniowicz-Kowalska, T., 1997a. Studies on the decomposition of keratin wastes
by saprotrophic microfungi. P.I. Criteria for evaluating keratinolytic activity.
Acta Mycol. 32, 5179.
Korniowicz-Kowalska, T., 1997b. Studies on the decomposition of keratin wastes
by saprotrophic microfungi. II. Sulfur and nitrogen balance. Acta Mycol. 32, 81
93.
Korniowicz-Kowalska, T., 1999. Studies on the decomposition of keratin waste by
saprotrophic microfungi. III. Activity and properties of keratinolytic enzymes.
Acta Mycol. 34, 6578.
Korniowicz-Kowalska, T., 2002. Optimalisation in the conditions of feathers
decomposition in fungi cultures. Acta Agrophys. 73, 175187 (in Polish).
Korniowicz-Kowalska, T., Bohacz, J., 2002a. Some correlations between the
occurrence frequency of keratinophilic fungi and selected soil properties. Acta
Mycol. 37, 101116.
Korniowicz-Kowalska, T., Bohacz, J., 2002b. An attempt at composting feather
waste using fungi inoculum. III. Evaluation of the phytosanitary condition. Acta
Agroph. 73, 199212 (in Polish).
Korniowicz-Kowalska, T., Bohacz, J., 2010. Dynamics of growth and succession of
bacterial and fungal communities during composting of feather waste. Biores.
Technol. 101, 12681276.
Korniowicz-Kowalska, T., Kitowski, I., 2009. Diversity of fungi in nests and pellets
of montagus harrier (Circus pygargus) from eastern Poland-importance of
chemical and ecological factors. Ecol. Chem. Eng. S 16, 453471.
Korniowicz-Kowalska, T., Kitowski, I., Iglik, H., 2011. Geophilic dermatophytes and
other keratinophilic fungi in the nests of wetland birds. Acta Mycol. 46, 1 (in
press).
Korniowicz-Kowalska, T., Ziaja, J., 1999. Inuence of glucose on feather wastes
mineralization by saprophytic keratinolytic fungi.. Fol. Univ. Agaric. Stetin. 200
Agric. 77, 167172.
Kunert, J., 1972a. Thiosulphate esters in keratin attacked by dermatophytes in vitro.
Sabouraudia 10, 613.
Kunert, J., 1972b. Keratin decomposition by dermatophytes: evidence of the
sulphitolysis of the protein. Experientia 28, 10251026.
Kunert, J., 1973. Keratin decomposition by dermatophytes. 1: sulte production as a
possible way of substrate denaturation. Zeitschrift fr Allg. Mikrobiologie 13,
489498.
1700 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
Kunert, J., 1976. Keratin decomposition by dermatophytes. II: presence of S-
sulfocysteine and cysteic acid in soluble decomposition products. Zeitschrift fr
Allg. Mikrobiologie 16, 97105.
Kunert, J., 1989. Biochemical mechanisms of keratin degradation by actinomycete
Streptomyces fradiae and fungus Microsporum gypseum, a comparison. J. Basic
Microbiol. 29, 597604.
Kunert, J., 2000. Physiology of keratinophilic fungi. In: Kushawaha, R.K.S., Guarro, J.
(Eds.), Biology of dermatophytes and other keratinophilic fungi. Rev. Iber. Micol.
17, 7785.
Kunert, J., Krajci, D., 1975. An electron microscopy study of keratin degradation by
the fungus Microsporum gypseum in vitro. Mycosen 24, 485496.
Kster, E., Sofwat, M.S.A., 1978. Studies on the composting of feathers. Zbl. Bakt. II.
Abt. Bd. 133 (Suppl.), 385393.
Lal, S., Rajak, R., Hasijia, C., 1999. In vitro degradation of keratin by two species of
Bacillus. J. Gen. Appl. Microbiol. 45, 283287.
Lee, D.L., Baden, H.P., 1975. Chemistry and composition of the keratins. J. Dermatol.
14, 161171.
Letourneau, F., Soussotte, V., Bressollier, P., Branland, P., Verneuil, B., 1998.
Keratinolytic activity of Streptomyces sp. S.K.1-02: a new isolated strain. Lett.
Appl. Microbiol. 26, 7780.
Lin, X., Lee, C.G., Casale, E.S., Shih, J.C.H., 1992. Purication and characterization of a
keratinase from a feather-degrading Bacillus licheniformis strain. Appl. Environ.
Microbiol. 58, 32713275.
Lin, X., Inglis, G.D., Yanke, L.J., Cheng, K.J., 1999. Selection and characterization of
feather-degrading bacteria from canola meal compost. J. Industr. Microbiol.
Biotech. 23, 149153.
obarzewski, J., Grzywnowicz, K., Wawrzkiewicz, K., Staszczak, M., Wolski, T., 1990.
Feather keratin as a ligand in an afnity chromatographic technique for
isolation of protease from Trichophyton verrucosum. J. Chromatogr. 520, 223
235.
Malvija, H.K., Hasija, S.K., Rajak, R.C., 1992. In vitro utilization of L-cystine by
keratinophilic fungi inhabiting a gelatin factory. Mycopathology 188, 147
152.
Marcantini, R., Marsella, F., Caprilli, F., Dovgiallo, G., 1980. Isolation of
dermatophytes and correlated species from the soil of public gardens and
park of Roma. Sabouraudia 18, 123128.
Martin, S.M., So, V., 1979. Solubilization of autoclaved feathers and wool by
myxobacteria. Can. J. Microbiol. 15, 13931397.
Mathur, S.P., Owen, G., Dinel, H., Schnitzer, M., 1993. Determination of compost
biomaturity. I: literature review. Biol. Agric. Hortic. 10, 6585.
Matsui, T., Yamanda, Y., Mitsuya, H., Shigeri, Y., Yoshida, Y., Saito, Y., Matsui, H.,
Watanabe, K., 2009. Sustainable and practical degradation of intact chicken
feathers by cultivating a newly isolated thermophilic Meiothermus ruber H328.
Appl. Microbiol. Biotechnol. 82, 941950.
Mc Govern, V., 2000. More bang for the cluck. Environ. Health Perspect. 108, 366
369.
Meevootison, V., Niederpruem, D.J., 1979. Control of exocellular proteases in
dermatophytes and especially Trichophyton rubrum. Sabouraudia 17, 91106.
Mercer, E.H., 1958. The ne structure keratin. Textile Res. J. 28, 860866.
Miller, F.C., 1993. Composting as a process based on the control of ecologically
selective factors. In: Metting F.B. Jr. (Ed.), Soil Microbial Ecology, Dekker, New
York, pp. 515545.
Nam, G.-W., Lee, D.-W., Lee, H.S., Lee, N.J., Kim, B.C., Choe, E.A., Hwang, J.K.,
Suhartono, M.T., Pyun, Y.R., 2002. Native-feather degradation by
Fervidobacterium islandicum AW-1, a newly isolated keratinase-producing
thermophilic anaerobe. Arch. Microbiol. 178, 538547.
Nickerson, W.J., Durand, S.C., 1963. Keratinase II. Properties of the crystalline
enzyme. Biochim. Biophys. Acta 77, 8799.
Nickerson, W.J., Noval, J.J., Robison, R.S., 1963. Keratinase I. Properties of the
enzyme conjugate elaborated by Streptomyces fradiae. Biochim. Biophys. Acta
77, 7379.
Nooruddin, M., Singh, B., 1987. Prevalence of dermatophytes and other
keratinophilic fungi in soil and dried dung of the premises of buffalo sheds.
Mycoses 30, 589593.
Noval, J.J., Nickerson, W.J., 1959. Decomposition of native keratin by Streptomyces
fradiae. J. Bacteriol. 77, 251259.
Nurzynski, J., 1989. The usability of keratin-bark-urea granulates in fertilizing
greenhouse tomatoes. Prob. Notes Adv. Agric. Sci. 370, 237243 (in Polish).
Ofdani, A., Simoncini, C., Arzeni, D., Cellini, A., Amerio, P., Scalise, G., 1998. Tinea
capitis due to Microsporum gypseum in an adult. Mycoses 41, 239241.
Oorschot van, C.A.N., 1980. A revision of Chrysosporium and allied genera. Stud.
Mycol. 20, 189.
Orzeszko, G., Sutarzewicz, D., 1979. Bristle utilization. Meat Manag. 48, 51.
Page, W.J., Stock, J.J., 1974. Phosphate-mediated alternation of the Microsporum
gypseum germination protease specicity for substrate enhanced keratinase
activity. J. Bacteriol. 117, 422431.
Papadopoulos, M.C., 1985. Processed chicken feathers as feedstuff for poultry and
swine: a review. Agric. Wastes 14, 275290.
Papadopoulos, M.C., El Boushy, A.R., Roodbeen, A.E., Ketelaars, E.H., 1986. Effects of
processing time and moisture content on amino acid composition and nitrogen
characteristics of feather meal. Animal Feed Sci. Technol 14, 279290.
Park, G.T., Son, H.J., 2009. Keratinolytic activity of Bacillus megaterium F7-1, a
feather-degrading mesophilic bacterium. Microbiol. Res. 164, 478485.
Parr, J.F., Hornic, B., 1993. Utilization of municipal wastes. In: Metting, F.B. Jr. (Ed.),
Soil microbial ecology: applications in agricultural and environmental
management. Dekker, New York, pp. 545559.
Pawowski, F., Wesoowski, M., Styk, B., 1989. The usability of keratin-bark-urea
granulates in weeding grains and Brassica napus var. Arvensis f. annua. Prob.
Notes Adv. Agric. Sci. 370, 217228 (in Polish).
Pinowski, J., Pinowska, B., Haman, A., 1999. Fungi in birds plumage and nests
(review article). Abstract Dept. Vertebrate Ecology. Intern. Stud. Sparrows 26,
328.
Poopathi, S., Abidha, S., 2008. Biodegradation of poultry waste for the production of
mosquitocidal toxins. Inter. Biodeter. Biodegrad. 62, 479482.
Pugh, G.J.F., 1965. Cellulolytic and keratinophilic fungi recorded on birds.
Sabouraudia 4, 8591.
Pugh, G.J.F., Evans, M.D., 1970. Keratinophilic fungi associated with birds. I: fungi
isolated from feathers nests and soil. Trans. Br. Mycol. Soc. 54, 233240.
Raju, K.C., Neogi, U., Saumya, R., Goud, N.R., 2007. Studies on extra cellular enzyme
keratinase from dermatophyte Microsporum gypseum. Inter. J. Biol. Chem. 1,
174178.
Raubitschek, F., 1961. Mechanical versus chemical keratolysis by dermatophyes.
Sabouraudia 1, 87.
Rees, R.G., 1967. Keratinophilic fungi from Queesland II. Isolation from animal hair
and scales. Sabouraudia 5, 165172.
Rodziewicz, A., aba, W., 2006. Keratins and their biodegradation. Biotechnology 2,
130147 (in Polish).
Rufn, P., Andrieu, S., Biserte, G., Biguet, J., 1976. Sulphitolysis in keratinolysis.
Biochemical proof. Sabouraudia 14, 181184.
Safranek, W.W., Goos, R.D., 1982. Degradation of wool by saprotrophic fungi. Can. J.
Microbiol. 28, 137140.
Sangali, S., Brandelli, A., 2000. Isolation and characterization of a novel feather-
degrading bacterial strain. Appl. Biochem. Biotechnol. 87, 1724.
Santos, R.M.D.B., Firmino, A.A.P., de S, C.M., Felix, C.R., 1996. Keratinolytic activity
of Aspergillus fumigatus Fresenius. Current Microbiol. 33, 364370.
Sanyal, A.K., Das, S.K., Banerjee, A.B., 1985. Purication and partial characterization
of an exocellular proteinase from Trichophyton rubrum. Sabouraudia 23, 165
178.
Shih, J.C.H., 1993. Recent development in poultry waste and feather utilization a
review. Poultry Sci. 72, 16171620.
Styk, B., 1989. Response of wheat and barley to fertilization with keratin-bark-urea
granulate. Prob. Notes Adv. Agric. Sci. 370, 179197 (in Polish).
Suh, H.J., Lee, H.K., 2001. Characterization of a keratinolytic serine protease from
Bacillus subtilis KS-1. J. Protein Chem. 20, 165169.
Suntornsuk, W., Suntornsuk, L., 2003. Feather degradation by Bacillus sp. FK46 in
submergaded cultivation. Biores. Technol. 86, 239243.
Syed, D.G., Lee, J.C., Li, W.-J., Kim, C.-J., Agasar, D., 2009. Production, characterization
and application of keratinase from Streptomyces gulbargensis. Biores. Technol.
100, 18681871.
Takiuchi, J., Higuchi, D., Sei, Y., Koga, M., 1982. Isolation of an extracellular
proteinase (keratinase) from Microsporum canis. Sabouraudia 20, 281288.
Takiuchi, I., Higuchi, D., Yoshihiro, S., Koga, M., 1983. Immunological studies of an
extra-cellular keratinase. J. Dermatol. 10, 327330.
Takiuchi, I., Sci, Y., Tagagi, H., Negi, M., 1984. Partial characterization of the
extracelluralar keratinase from Microsporum canis. Sabouraudia 22, 219224.
Tiquia, S.M., 2005. Microbiological parameters as indicators of compost maturity. J.
Appl. Microbiol. 99, 816828.
Tiquia, S.M., Ichida, J.M., Keener, H.M., Elwell, D.L., Burtt Jr., E.H., Michel Jr., F.C.,
2005. Bacterial community proles on feathers during composting as
determined by terminal restriction fragment length polymorphism analysis of
16S rDNA genes. Environ. Biotechnol. 67, 412419.
Tsuboi, R., Ko, I.J., Matsuda, K., Ogawa, H., 1987. A new keratinolytic proteinase from
clinical isolates of Trichophyton mentagrophytes. J. Dermatol. 14, 506508.
Ulg, K., 2000. The occurrence of keratinolytic fungi in waste and waste
contaminated habitats. In: Kushawaha, R.K.S., Guarro, J. (Eds.), Biology of
dermatophytes and other keratinophilic fungi. Rev. Iber. Micol. 17, 4450.
Ulg, K., Korcz, M., 1983. Isolation of keratinolytic fungi from sewage sludge.
Sabouraudia 21, 247250.
Ulg, K., Korcz, M., 1994. Keratinolytic fungi in sewage sludge applied to devastated
urban soil. A preliminary experiment. Inter. J. Environ. Health Res. 4, 244253.
Ulg, K., Ulg, A., 1990. Keratinophilic fungi in bottom sediments of surface waters.
J. Med. Vet. Mycol. 28, 419422.
Ulg, K., ukasik, W., Paza, G., 1996. Further statistical evaluation of the occurrence
of keratinolytic fungi in dam sediments. Inter. J. Environ. Health Res. 6, 3947.
Vasileva-Tonkova, E., Gousterova, A., Neshev, G., 2009. Ecologically safe method for
improved feather wastes biodegradation. Inter. Biodeter. Bioderad. 63, 1008
1012.
Vollenkov, A., 1984. Microsporum persicolor a ine keratinolne huby v pode v nore
hladovcov. Biologia (Bratislava) 39, 899904.
Vries de, G.A., 1962. Keratinophilic fungi and their action. Antonie van
Leeuwenhoek 28, 122133.
Wang, J.J., Shih, J.C.H., 1999. Fermentation production of keratinase from Bacillus
licheniformis PWD-1 and B. subtilis FDB-29. J. Ind. Microbiol. Biotechnol. 22,
608616.
Wawrzkiewicz, K., obarzewski, J., Wolski, T., 1987. Intracellular keratinase of
Trichophyton gallinae. J. Med. Vet. Mycol. 25, 261268.
Williams, C.M., Shih, J.C.H., 1989. Enumeration of some microbial groups in
termophilic poultry waste digestors and enrichment of a feather-degrading
culture. J. Appl. Bacteriol. 67, 2535.
Williams, C.M., Richter, C.S., MacKenzie Jr., J.M., Shih, J.C.H., 1990. Isolation,
identication and characterization of a feather-degrading bacterium. Appl.
Environ. Microbiol. 56, 15091515.
 T. Korniowicz-Kowalska, J. Bohacz / Waste Management 31 (2011) 16891701
Williams, C.M., Lee, C.G., Garlich, J.D., Shih, J.C.H., 1991. Evaluation of a bacterial
feather fermentation product, feather-lysate as a feed protein. Poult. Sci. 70, 85
94.
Wojtowicz, A., Moscicki, L., 1998. The American method of the utilization of
production waste of animal origin. Industrial Feeds 4, 1718 (in Polish).
Wolski, T., 1979. Urea granulates obtained from feathers for use in feeds. Food
Industry 33, 302303 (in Polish).
Wolski, T., 1985. Modied keratin proteins, their physicochemical properties,
analysis and application. DSc Dissertation. Medical Academy, Lublin, Poland (in
Polish).
Wolski, T., Orlikowski, L.B., Glinski, J., 1996. The use of processed lignino-cellulose
and keratin waste in plant protection. Prob. Notes Adv. Agric. Sci. 437, 359364.
1701
Yamamura, S., Morita, Y., Hasan, Q., Yokoyama, K., Tamiya, E., 2002. Keratin
degradation: a cooperative action of two enzymes from Stenotrophomonas sp..
Biochem. Biophys. Res. Commun. 294, 11381143.
Yu, R.J., Harmon, S.R., Blank, F., 1968. Isolation and purication of an extracellular
keratinase of Trichophyton mentagrophytes. J. Bacteriol. 96, 14351436.
Yu, R.J., Harmon, S.R., Blank, F., 1969. Hair digestion by keratinase of Trichophyton
mentagrophytes. J. Invest. Dermatol. 53, 166171.
Yu, R.J., Harmon, S.R., Grappel, S.F., Blank, F., 1971. Two cell bound keratinase of
Trichophyton mentagrophytes. J. Invest. Dermatol. 56, 2732.
Yu, R.J., Harmon, S.R., Grappel, S.F., Blank, F., 1972. Keratinases. Hydrolysis
keratinous substrates three enzymes of Trichophyton mentagrophytes.
Experientia 28, 15121513.