BIODEGRADATION OF PETROLEUM

HYDROCARBONS BY KERATINOLYTIC FUNGI

Krzysztof Ulfig1, Wioletta Przysta1, Grayna Paza2 and Korneliusz

Miksch1
1

Environmental Biotechnology Department, Silesian University of Technology, Gliwice,

Poland; 2Environmental Microbiology Department, Institute for Ecology of Industrial Areas,
Katowice, Poland

Abstract:

The chapter reviews available data on the ability of keratinolytic fungi to

remove hydrocarbons from different media and on the ecology of these fungi
in oil-contaminated environments. In pure culture, these fungi were able to
remove hexane, toluene, hexadecane, pristane and autoclaved crude oil from
mineral media. The hydrocarbon removal process was much more effective
when hair or peptone was added to the media. Thus, the process was
dependent on fungal proteolytic or keratinolytic activity, specifically on the
readily available protein content in the media. The ability for hydrocarbon
removal was found to be species- and strain-specific. In pure culture,
keratinolytic fungi removed polar products of petroleum biodegradation from
the media. In a soil environment, the degradation process was slowed down
due to the accumulation of these polar products.

Key words:

keratinolytic fungi; oil hydrocarbon removal; survival; ecology; oilcontaminated environments

1.

INTRODUCTION

Two major reasons to examine keratinolytic fungi in the environment can

be named. First, the abundance of these microorganisms is observed in
environments rich in keratinous remnants of human and animal origin and in
other substrata needed for fungal growth, e.g., in soils of highly populated
and animal-inhabited areas, sewage sludge and municipal waste (Garg et al.,
1985; Deshmukh and Agrawal, 1998; Ulfig, 2000). Keratinolytic fungi play
553
I. Twardowska et al. (eds.),
Soil and Water Pollution Monitoring, Protection and Remediation, 323.
2006 Springer.

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Krzysztof Ulfig et al.

the main role in the decomposition of these substrata and can be used for
biotechnological applications (Kushwaha, 1998; Onifade et al., 1998).
Second, keratinolytic fungi display potential pathogenic properties to
animals, including humans (Filipello-Marchisio, 2000). Therefore, studies of
these fungi in the environment are of epidemiological importance.
Keratinolytic fungi also occur in abundance in highly industrialized and
polluted areas, in which organic and inorganic contaminants considerably
affect microbial populations. Therefore, an essential problem is an
evaluation of the effects of these contaminants, including oil hydrocarbons
on fungal distribution in these areas. The role of keratinolytic fungi in
biodegradation of these hydrocarbons is also to be explained.
Extensive studies have recently been carried out to explain the factors
influencing keratinolytic fungi in the environment (Ali-Shtayeh and Jamous,
2000; Ulfig, 2000; Ulfig et al., 2003a). Special attention has been paid to
those fungi inhabiting environments heavily contaminated with oil
hydrocarbons. The present chapter reviews available data on the ability of
keratinolytic fungi to remove the hydrocarbons from different media and on
the ecology of these microorganisms in the above-mentioned environments.

2.

KERATINOLYTIC FUNGI AS BIOINDICATORS

OF TOXICITY AND BIOREMEDIATION
PROGRESS

Ecotoxicity bioassays for bioremediation have comprised a great number

of organisms (Salanitro et al., 1997), but the available literature indicates
that microscopic fungi have not been used for toxicological studies during
bioremediation. However, antifungal properties of oils, fatty acids, and some
volatile compounds have been known for almost one hundred years (Garg et
al., 1985). During biopile and column experiments within a bioremediation
study at one of the Polish oil refineries, it was observed that keratinolytic
fungi reacted to the changes in TPOC (Total Petroleum Organic Carbon that
includes non-polar hydrocarbons and their polar derivatives) concentrations
in leachates. This finding inspired us to examine the inhibitory effect of
leachates on radial growth and dry weight (biomass) production of these
fungi (Ulfig et al., 1998).
Based on the results obtained, the bioremediation process could be
divided into three stages. The first stage included the first six months of the
process and was characterized by a high inhibition of fungal growth. The
second stage was three-month long and showed a considerable decrease of
the inhibition. The last stage (after 9 months) was associated with low
inhibition. The inhibition of fungal growth and dry weight production

Biodegradation of Petroleum Hydrocarbons by Keratinolytic Fungi

555

inhibition correlated with TPOC values in leachates (Figure 1). Polar

compounds prevailed in these leachates. The dry weight production was
more sensitive to the leachates than the radial growth of the fungi. It was
evident that below certain TPOC levels (concentrations of polar compounds)
the leachates stopped inhibiting the growth of fungi and started stimulating
them to grow. Since the leachates were sterilized by autoclaving, the data did
not include volatile hydrocarbons.

Figure 1. The relationship between the inhibition of the growth of the Trichophyton ajelloi
strain R66 and TPOC concentration in leachate.

In another study (Ulfig et al., 2003b), keratinolytic fungi were found to

occur frequently in a biopile with the geophilic dermatophyte, Trichophyton
ajelloi (teleomorph Arthroderma uncinatum), as the predominating species.
The fungal growth was dependent on the concentration of oil contaminants
in soil. Negative correlations between fungal growth indices and TPH (Total
Petroleum Hydrocarbons that includes non-polar compounds), TPOC and
PAHs were found. No other data on the influence of PAHs on keratinolytic
fungi were found in available literature. Overall, the keratinolytic fungi,
especially T. ajelloi, were found to be a useful tool for a rough assessment of
oil hydrocarbon contamination and associated bioremediation progress of
soils heavily contaminated with hydrocarbons.

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3.

Krzysztof Ulfig et al.

SURVIVAL AND VIABILITY OF

KERATINOLYTIC FUNGI IN AUTOCLAVED
CRUDE OIL

Geophilic dermatophytes possess the ability to survive for a long time in

autoclaved crude oil (Ulfig et al., 2002). These fungi were also able to attack
hair in contact with conidia suspensions in oil. The survival time and growth
on hair varied between species and strains. Overall, Trichophyton terrestre
displayed the longest survival time (>6 months) in oil and the best growth on
hair, while Microsporum sp. only survived for 3 months in oil and had the
worst growth on hair. In comparison with the dermatophyte growth on hair
laid on soil, fungal growth on hair partly submerged in conidia suspension in
oil was retarded for about one month. Subsequently, coating the hair with
autoclaved crude oil limited the growth of T. ajelloi. Broadly, the geophilic
dermatophytes isolated from a refinery site were found to be resistant to
autoclaved crude oil (non-volatile hydrocarbons). The mechanism of this
resistance is unknown.

4.

THE INFLUENCE OF VOLATILE OIL

HYDROCARBONS ON KERATINOLYTIC FUNGI

A study was performed to determine the effect of volatile oil

hydrocarbons on keratinolytic fungi (Ulfig and Paza, unpublished data).
Petri dishes with a refinery soil covered by hair were placed in desiccators
with open side tubes. On the bottom of one desiccator, dishes with crude oil
were placed at the beginning of the experiment. In another desiccator, dishes
with oil were replaced every week. A desiccator without oil served as
control. The experiment lasted for two months. In the desiccator
environment, BTEXs adsorbed on activated carbon were measured with a
GC/MS method. In comparison to the control, the single oil application
stimulated keratinolytic fungi to grow on hair. Subsequently, the repeated oil
application considerably restricted the growth of the fungi, except for T.
ajelloi strains (including the strain R66). These strains were found to be
resistant to continuous high concentrations of volatile oil hydrocarbons. The
mechanism of this resistance is unknown.

Biodegradation of Petroleum Hydrocarbons by Keratinolytic Fungi

5.

557

UPTAKE OF HEXANE AND TOLUENE BY

KERATINOLYTIC FUNGI

Selected dermatophytes such as Microsporum gypseum, M. canis,

Trichophyton terrestre, T. ajelloi, T. mentagrophytes var. mentagrophytes
and T. rubrum and two other keratinolytic fungi, e.g. Aphanoascus
reticulisporus and Scopulariopsis brevicaulis were surveyed for hexane and
toluene uptake (Ulfig and Paza, 2004). The strains examined were isolated
from oil-contaminated sites and skin lesions. The closed serum bottle and
headspace GC/MS methods were used. It was found that the average hexane
uptake was comparable to the mean toluene uptake by the fungi examined.
However, the mean dry weight production was much higher for hexane
uptake. The highest hexane uptake was observed for S. brevicaulis, followed
by T. ajelloi, M. canis, T. mentagrophytes and other species. Subsequently,
the highest toluene uptake was observed for T. terrestre, followed by S.
brevicaulis, T. mentagrophytes, T. ajelloi and other species. The lowest
hydrocarbon uptakes were found in A. reticulisporus. The M. canis hexane
uptake was much higher than its toluene uptake.
0,40

Hexane

Hydrocarbon uptake (mg/sample/24 h)

0,35

Toluene

0,30

0,25

0,20

0,15

0,10

0,05

0,00
Bacteria

Yeasts

Keratinolytic fungi

Other fungi

Microorganisms

Figure 2. Hexane and toluene uptakes by bacteria, yeasts, keratinolytic fungi and other fungi.

Krzysztof Ulfig et al.

558

Generally, the geophilic dermatophytes showed higher hydrocarbon

uptakes than zoophilic and anthropophilic species did. The results were
compared with the data obtained for other filamentous fungi, yeasts and
bacteria isolated from oil-contaminated sites. Keratinolytic fungi had the
lowest hexane uptake but the uptake of toluene by these fungi was higher
than that of bacterial strains (Figure 2). Keratinolytic fungi produced much
higher dry weight than the other microorganisms did.

6.

REMOVAL OF OIL HYDROCARBONS BY

TRICHOPHYTON AJELLOI STRAIN R66 DURING
HAIR BIODEGRADATION

In a preliminary study (Ulfig et al., 2000), the efficiency of oil

hydrocarbon removal from a mineral medium by the T. ajelloi strain R66
was determined.
100
90

Hydrocarbon removal (%)

80
70
60
50
40
30
20
10
0
Oil + strain
R66

Oil + hair +
strain R66

Pristane +
strain R66

Pristane +
hair + strain
R66

Hexadecane Hexadecane
+ strain R66
+ hair +
strain R66

Hydrocarbons & treatment

Figure 3. Hydrocarbon removal by the Trichophyton ajelloi strain R66 from the mineral
medium supplemented or non-supplemented with hair.

The considerable amount of losses of pristane and hexadecane from

autoclaved crude oil, were observed during the biodegradation of hair by this

Biodegradation of Petroleum Hydrocarbons by Keratinolytic Fungi

559

strain. Without an addition of hair to the medium, the strain did not decrease,
or decreased to a small degree, the amount of these hydrocarbons.
The highest hydrocarbon losses were determined for crude oil, followed
by the losses for pristane and hexadecane (Figure 3). The study confirmed
the processes of proteolysis, sulfitolysis, and alkalization of the mineral
medium during hair biodegradation. The presence of hydrocarbons made the
process of hair biodegradation by the strain R66 more effective.

7.

REMOVAL OF OIL HYDROCARBONS BY

KERATINOLYTIC FUNGI DURING PROTEIN
BIODEGRADATION

7.1

Pure Culture Experiments

The removal of autoclaved crude oil from mineral medium containing

NH4NO3 supplemented or non-supplemented with hair by keratinolytic fungi
strains was examined (Miksch et al., 2002). The strains were isolated from
bottom sediments, sewage sludge and oil-contaminated soil and belonged to
the species as follows: A. reticulisporus, Chrysosporium keratinopilum,
Chrysosporium sp., M. gypseum, T. ajelloi and T. terrestre. The mean TPH
and TPOC removals from the medium without addition of hair were 18 and
14.5%, respectively. The addition of hair to the medium did not significantly
change the mean hydrocarbon removals, which were 18.6 and 17.1% for
TPH and TPOC, respectively. Among the strains examined, the T. terrestre
strain PS22 was exceptional. In this case, the addition of hair to the medium
increased TPH and TPOC removals from 13.1 and 10.8% to 45.4 and 45.6%,
respectively. A relatively high increase of TPH/TPOC removals after
addition of hair to the medium was also obtained for some M. gypseum
strains.
In a subsequent experiment, the removal of autoclaved crude oil from
mineral medium containing peptone (4 g/L) supplemented or nonsupplemented with hair (100 mg/sample) by keratinolytic fungi strains was
determined. The mean TPH and TPOC removals from the medium without
addition of hair were 53.4 and 54%, respectively. The addition of hair to the
medium considerably increased the mean hydrocarbon removals, which
were 71.8 and 71.7% for TPH and TPOC, respectively. The highest
hydrocarbon removal from the medium without addition of hair was
observed for T. ajelloi (67.2%) and from the medium with hair addition for
T. terrestre (88.9%).

Krzysztof Ulfig et al.

560

Two strains, i.e. C. keratinopilum PS14 and T. ajelloi PS16, were used
for determination of the influence of increasing peptone concentrations (0, 2,
4 and 8 g/L) and hair addition (100 mg/sample) to the medium on fungal
hydrocarbon removals. The results are illustrated in Figure 4. The strain
PS14 increased TPH removal with increasing peptone concentrations. The
addition of hair caused further THP removal increase up to the peptone
concentration of 4 g/L. However, the statistically significant TPH removal
increase caused by addition of hair was observed for the medium without
peptone and at a peptone concentration of 2 g/L. The strain PS16 increased
TPH removal up to the peptone concentration of 4 g/L. The addition of hair
caused TPH increase at the peptone concentrations of 4 and 8 g/L. However,
the differences observed were not statistically significant. The TPOC
removals were found to be similar to TPH removals. The TPH and TPOC
removals were positively correlated with the increase of NH4-N (r = 0.83
and 0.86), dry weight (0.71 and 0.73) and pH (0.57 and 0.58).
90

Without hair
80

With hair

TPH removal (%)

70
60
50
40
30
20
10
0
0

PS14

PS16

Strain number & peptone quantity (g/L)

Figure 4. Oil hydrocarbon removal by the Chrysosporium keratinophilum strain PS14

and Trichophyton ajelloi strain PS16 from the media containing increasing peptone
concentration supplemented or non-supplemented with hair.

The results confirmed that the oil hydrocarbon removal process was
associated with fungal keratinolytic activity. Proteins (hair or peptone) were
the main source of nitrogen, carbon and sulfur for the growth of these fungi,
whereas oil hydrocarbons were the additional but essential source of carbon

Biodegradation of Petroleum Hydrocarbons by Keratinolytic Fungi

561

for some strains examined. However, the biochemical mechanism of this

phenomenon requires explanation in further studies.

7.2

Soil Experiments

In a soil experiment, the influence of increasing autoclaved crude oil

amounts (0, 0.5, 1, 2 and 5 mL per Petri dish) on the growth of keratinolytic
fungi on hair laid on clayey, organic and sandy soils was examined (Miksch
et al., 2002; Przysta et al., 2002). TPH and TPOC removals from samples
covered and uncovered by hair being decomposed by the fungi were also
determined. T. ajelloi was found to be the only keratinolytic fungus in clayey
and sandy soils and predominated in organic soil. The addition of 0.5- and 1mL oil portions to the clayey soil stimulated T. ajelloi to grow on hair.
Higher oil amounts restricted the growth of this fungus. The addition of oil
(even in the smallest portion of 0.5-mL) to the organic soil restricted the
growth of keratinolytic fungi. The addition of 5-mL oil portions to this soil
eliminated the fungi. T. ajelloi grew in all Petri dishes with sandy soil.
However, the amount of the observed mycelium decreased with increasing
oil amounts added to this soil.
The TPH removal from the clayey soil covered by hair being
decomposed by fungi decreased with increasing oil supplementation. In the
organic soil, the highest TPH removal (89.7%) was observed in samples
supplemented with 1-mL oil portions. This was also the highest TPH
removal in the whole experiment. The TPH removals considerably decreased
with increasing oil amounts added to the organic soil. In the sandy soil, the
highest TPH removal was observed in samples supplemented with 1-mL oil
portions. The TPH removals from the sandy soil decreased with increasing
oil supplementation. Broadly, the TPOC removals were found to be lower
than the TPH removals. This was well observed in clayey and organic soils.
Except for the organic soil, the addition of hair did not significantly change
TPH and TPOC removals from soils. The hair on the organic soil
considerably decreased the TPOC removal. This indicated that polar
products of the oil biodegradation process were accumulated in the samples.
The acidification of the soils examined was also observed during a 5-month
incubation.
In another soil experiment (Miksch et al., 2002; Przysta et al., 2002), the
hair cover on cattle farm soil generally decreased TPH and TPOC removals.
The accumulation of polar compounds (TPOC) and acidification of this soil
were again evident. It can be stated that both the factors slowed down the oil
hydrocarbon biodegradation process in the soils examined.

Krzysztof Ulfig et al.

562

8.

CONCLUSIONS

The inhibitory effect of polar compounds (products of the oil

hydrocarbon biodegradation process) in leachates on the radial growth and
dry weight (biomass) production by T. ajelloi and the qualitative and
quantitative composition of keratinolytic fungi can be used as indicators of
bioremediation progress and associated contamination of a biopile soil with
oil hydrocarbons. The geophilic dermatophytes possess the ability to survive
for a long time (over six months) in autoclaved crude oil. These fungi are
also able to attack hair in contact with conidia suspension in oil. The survival
time and growth on hair varies between species and strain. Geophilic
dermatophytes isolated from an oil-polluted site are resistant to non-volatile
oil hydrocarbons. When applied once, volatile oil hydrocarbons (measured
as BTEXs) enrich the composition of keratinolytic fungi in soil. However,
continuous high volatile oil hydrocarbon concentrations inhibit fungal
growth. Only some strains of T. ajelloi were able to survive long exposition
to these compounds. No data on the impact of PAHs on keratinolytic fungi
are available. The resistance of fungal strains to high oil hydrocarbon
concentrations requires explanation in further studies.
In pure culture, oil hydrocarbon removal depends on fungal proteolytic or
keratinolytic activity and is associated with high dry weight (biomass)
production. The fungal ability for hydrocarbon removal from the medium
during protein biodegradation is species- and strain-specific. The
hydrocarbon addition to the medium stimulates, to a certain degree, fungal
keratinolytic activity. These abilities can be used in biotechnologies, in
which protein addition accelerates hydrocarbon biodegradation or vice versa.
Proteins, including keratin are the main source of nitrogen, carbon and sulfur
for the growth of these fungi, whereas oil hydrocarbons are the additional
but essential source of carbon for some strains examined. The biochemical
mechanism of this phenomenon requires explanation in further studies.
In pure culture, keratinolytic fungi are able to remove polar compounds
from the medium. In soil, however, these compounds are accumulated,
which may intoxicate the soil environment and slow down the hydrocarbon
biodegradation process. The TPH removal decrease caused by addition of
hair to the soil, accumulation of polar compounds, soil acidification and
weak abilities of keratinolytic fungi to compete with other soil hydrocarbon
degraders question a wide use of keratinous waste or biosolids containing
high amounts of keratinous substrata, e.g. sewage sludge, for bioremediation
purposes. Oil and keratinous substrata also support the growth of some
strains of pathogenic M. gypseum in the environment.

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