Biodegradation of Petroleum

BIODEGRADATION OF PETROLEUM

HYDROCARBONS BY KERATINOLYTIC FUNGI

Krzysztof Ulfig1, Wioletta Przysta 1, Gra yna P aza2 and Korneliusz Miksch1

1Environmental 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; oil-contaminated 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

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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.


3. 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 P aza, 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.


5. 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 P aza, 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.

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


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.

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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


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 non-supplemented 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%).


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).

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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


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 1-mL 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.


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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