INTRODUCTION

One third of organically polluted sites are also contaminated with metals (Roane and Pepper, 1997). Studies on the effects of metals on organic pollutant biodegradation are not extensive, especially in aquatic environments, but the available few demonstrate that metals have the potential to inhibit pollutant biodegradation.

The impacts of metals (Cadmium, Nickel, Zinc, Mercury and Chromium) on litter decomposition, methanogenesis, acidogenesis and biomass generation have all been studied (Lin, 1993; Bardgett and Saggar, 1994; Knight et al., 1997). Benka-Coker and Ekundayo (1998) reported on the impact of zinc, lead, copper and manganese on crude oil biodegradation by a Micrococcus sp. and a Pseudomonas sp. Biodegradation, measured by microbial growth, was reduced most by zinc and least by manganese.

Biodegradation of petroleum hydrocarbons involves the participation of diverse taxonomic groups of microorganisms particularly bacteria and fungi (Atlas, 1981; Atlas and Cerniglia, 1995), but the relative importance of bacteria versus fungi in aquatic hydrocarbon degradation is a matter of controversy (Bartha and Atlas, 1977). Penicillium, Aspergillus, Rhodotorula, Candida, Saccharomyces, Sporobolomyces and Geotrichum are fungal genera frequently encountered in oil-impacted aquatic systems (Atlas, 1981; 1991; Ijah and Antai, 2003).

There have been reports on metal stimulation of bacterial biodegradation processes under favourable environmental conditions of pH, temperature and aerobiosis (Lin, 1993), but Sandrin and Maier (2003), observed that such stimulatory effects of metals on biodegradation occurred only when consortia, but not single microbial cultures were used for degradation processes. They argued that stimulation was a result of differential toxicity effects of the tested metals.

This research reports on the influence of pH on the response patterns of species of Penicillium and Rhodotorula to increasing concentrations of zinc during biodegradation of Nigerian light crude oil.

MATERIALS AND METHODS

The Nigerian light crude oil used in this study was obtained from Mobil Producing Nigeria Unlimited, Ibeno, Akwa Ibom State, Nigeria. Filter-sterilized oil was left at room temperature (28±2°C) until required.

Preparation of Metal Stock Solution
Concentrated stock solution of zinc was prepared by dissolving 1 g of zinc chloride (ZnCl₂) in 5 mL aliquot of acid mixture (H₂SO₄: HNO₃) at a ratio of 3:1 V/V, both acids having a pH of less than 2. The preparation was then diluted to 1 L with sterile deionised distilled water (ddH₂O). The stock solution was then analyzed with UNICAM 939/959 flame Atomic Absorption Spectrophotometer (Asuquo et al., 2004). Working concentrations of 1, 10, 100 and 1000 mg L^-1 of the heavy metal were prepared by serial dilutions of the stock solution (Zhang and Crow, 2001) in sterile ddH₂O.

Isolation and Screening of Fungal Cultures with Crude Oil Utilizing Abilities
The mold and yeast cultures used for the study were isolated from intertidal (epipellic) sediment samples obtained from Qua Iboe River estuary, Nigeria, on Mineral Salts agar Medium (MSM) of Zajic and Supplisson (1972) with composition as listed in Ekpenyong and Antai (2006). Fifty micrograms per milliliter (50 μg mL^-1) of each of penicillin G and streptomycin was incorporated into the medium to inhibit interfering bacteria. The medium pH was adjusted to 5.8. The vapour phase transfer method of Thijsee and van der Linden (1961) was adopted for the isolation. Discrete colonies were purified on yeast extract-malt extract agar. Fungal pure cultures were identified according to the schemes of Barnett and Hunter (1987) and Barnett and Pankhurst (1974).

The isolates were screened for utilization of Nigerian light crude oil using both the agar (for mold) and broth (for yeast) screen tests of Okpokwasili and Okorie (1988). Turbidity in tubes and early development of colonies on plates were indicative of oil utilization.

Influence of pH on Zinc Toxicity to Species of Penicillium and Rhodotorula During Biodegradation of Crude Oil
The influences of varying pH levels namely 5.5, 7.0 and 8.5, on the toxicity of varying concentrations of zinc to a species each of Penicillium and Rhodotorula during oil biodegradation were assessed by the carbon dioxide evolution technique of Cornfield (1961). The preparations and incubation were as described by Ekpenyong and Antai (2006). Amounts of CO₂ evolved during biodegradation were calculated using the formula of Stotzky (1965). The residual hydrocarbon in the flasks after 4, 8, 12 and 16 days was determined by the gravimetric method of Odu (1972). Mean rates of CO₂ evolution as well as inhibition efficiencies of the various concentrations of zinc at the different pH levels tested, were determined from the amounts of CO₂ evolved during the study.

Statistical Analysis
Data obtained from the study were subjected to Analysis of Variance (ANOVA) to ascertain the influences of the variables on CO₂ evolution at 95% confidence limit.

RESULTS

The yeast and mold cultures were identified as species of Rhodotorula and Penicillium, respectively. The yeast grew well in the broth medium and the mold also performed well in the agar screen test.

Cumulative amounts of CO₂ evolved at all the pH levels tested increased gradually, albeit not proportionately, throughout the sampling period in both the controls and metal test cultures. In control cultures of Penicillium sp., 68.4 mg of CO₂ was evolved at pH 5.5, 65.4 mg at pH 7.0 and 63.4 mg at pH 8.5, whereas in cultures of Rhodoturula sp., 61.3 mg of CO₂ was evolved at pH 5.5, 57.7 mg at pH 7.0 and 47.6 mg at pH 8.5 (Fig. 1-3).

Fig. 1:	Influence of zinc on the amount of CO2 evolved during biodegradation of crude oil at pH 5.5

Fig. 2:	Influence of zinc on the amount of CO2 evolved during biodegradation of crude oil at pH 7.0

Fig. 3:	Influence of zinc on the amount of CO2 evolved during biodegradation of crude oil at pH 8.5

Table 1:	Influence of zinc on mean rate of CO2 evolution during biodegradation of crude oil at varying pH levels
A: Penicillium sp. B: Rhodotorula sp.

In Table 1, the mean rate of CO₂ evolution in cultures containing Penicillium sp. was observed to be highest in the control at pH 5.5, nevertheless mean rates at pH 7.0 under the influence of 1 mg L^-1 and 10 mg L^-1 total zinc were higher than those in the control at pH 8.5. In cultures of Rhodotorula sp., mean rates of CO₂ evolution in test cultures containing 1 and 10 mg L^-1 total zinc were higher than their corresponding control cultures at pH 5.5 and 7.0. While the mean rate of evolution of carbon dioxide in the control culture of Rhodotorula at pH 8.5 was 4.05 mg day^-1, that at pH 5.5 in cultures containing 100 mg L^-1 of zinc was 4.35 mg day^-1. The results of the analysis of variance (ANOVA) of data, assessing the influence of pH on the mean rate of CO₂ evolution and the toxicity of zinc to the organisms are presented in Table 2. Inhibition Efficiency (IE) of zinc concentrations on CO₂ evolution process in Penicillium was most profound at pH 5.5 with as much as 35.1% inhibition in the presence of 1000 mg L^-1 zinc. The highest inhibition was observed at pH 8.5 to be 11.4% in the presence of 100 mg L^-1 total zinc (Fig. 4). Maximal inhibition of CO₂ evolution in Rhodotorula sp. was observed at pH 5.5 in the presence of 1000 mg L^-1 zinc, with an IE of 40.5%.

Table 2:	Two-way analysis of variance (ANOVA) showing effect of pH and zinc concentrations on mean rate of CO2 evolution process in (A) Penicillium sp. and (B) Rhodotorula sp.

Table 3:	Two-way analysis of variance (ANOVA) showing effect of pH on inhibition efficiency of zinc concentrations on CO2 evolution process in (A) Penicillium sp. and (B) Rhodotorula sp.

Fig. 4:	Zinc inhibition efficiency of CO2 evolution during biodegradation of crude oil at varying pH levels

Negative inhibition efficiency values were observed at pH 5.5 and 7.0 in the presence of zinc concentrations ≤10 mg L^-1. Least inhibition of -8.3% was observed at pH 5.5 in the presence of 10 mg L^-1 total zinc and IE value of -1.0% at pH 7.0.

At zinc concentration of 1 mg L^-1, IE values of -5.7 and -4.0% were observed at pH 5.5 and 7.0, respectively. Generally, efficiency of inhibition increased with increasing pH (5.5 < 7.0 < 8.5) in Rhodotorula sp. (Fig. 4). Statistical analysis showing relationship between pH, zinc concentrations and inhibition efficiency of CO₂ evolution process of both organisms is presented in Table 3.

DISCUSSION

The mold, Penicillium sp., did not grow well in mineral salts broth medium, especially when the broth screen tubes were agitated, but both the broth culture and the condition of agitation favoured the yeast, Rhodotorula sp. Minimal shredding (breakdown) of the overlaid oil was observed in tubes containing Penicillium sp. The mold grew as fuzzballs in the broth cultures producing little or no visible turbidity along the column of the medium. Smucker and Cooney (1981) reported that filamentous fungi grow as heavy mycelial mats or fuzzballs in liquid culture and so turbidimetric methods are not accurate measures of growth. The selection of the mold for toxicity studies was therefore based primarily on the number of colonies developed on Crude oil/Mineral Salts agar Medium. Rhodotorula sp. produced good visual turbidity and impressive optical density reading at 540 nm wavelength and displayed extensive shredding (breakdown) of overlaid oil resulting in loss of oil viscosity attendant upon oil utilization (Ekpenyong and Antai, 2006).

The predominant heavy metal in Qua Iboe Estuary, Nigeria, where the test organisms were isolated is zinc (data not shown). In the control cultures (without zinc) of both organisms, it was observed that Penicillium sp. was more tolerant of alkaline environments than Rhodotorula sp. This was evidenced by the fact that cumulative amounts of CO₂ evolved decreased from 61.3 (pH 5.5) to 47.6 mg (pH 8.5) in Rhodotorula sp., but only dropped from 68.4 (pH 5.5) to 63.4 mg (pH 8.5) in Penicillium sp. In test cultures containing zinc, it was observed that Rhodotorula sp. produced higher cumulative amounts of CO₂ than Penicillium sp. at pH 5.5, at least up to 100 mg L^-1 total zinc. The higher cumulative amounts of CO₂ evolved in cultures of Penicillium sp. in the presence of 1000 mgL^-1 of zinc would be attributable to the mycelial growth of the organism in culture media (Baldrian et al., 2000). Rhodotorula sp. produced higher cumulative amounts of CO₂ in test cultures containing 1 and 10 mg L^-1 of zinc than in the controls (without zinc), at pH 5.5 and 7.0. At pH 5.5, zinc concentration of 10 mg L^-1 favoured the production of higher amounts of CO₂ than 1 mg L^-1, while the reverse scenario was observed at pH 7.0.

The essence of a successful bioremediation exercise rests on the amount of time needed to restore normalcy at the pollutant-impacted area. Mean rates of CO₂ evolution were observed to increase with increasing alkalinity in the presence of 1000 mg L^-1 of zinc in cultures containing Penicillium sp., but no generalized pattern of toxicity response was observed at lower concentrations of the metal. In contrast, increasing alkalinity only resulted in reductions in the mean rates of CO₂ evolution in Rhodotorula sp. at all the tested metal concentrations. This suggests either the acidophilic nature of this species of Rhodotorula or that zinc speciated to a form more toxic to this Rhodotorula sp. at the alkaline pH (Sandrin and Maier, 2003). Franklin et al. (2000) earlier showed that even relatively small changes in pH (e.g., from 6.5 to 5.7) can reduce metal toxicity.

The higher cumulative amounts of CO₂ evolved at pH 5.5 and 7.0, coupled with higher mean rates of CO₂ evolution in test cultures of Rhodotorula sp. containing zinc concentrations ≤10 mg L^-1 resulted in negative inhibition efficiency values. This suggests stimulation rather than inhibition of the biodegradation process by small amounts of zinc at these pH values. The lack of similar stimulatory results at pH 8.5 indicates significant (p<0.05) influence of pH on such observation. Metal stimulation of organic biodegradation has been reported by Kuo and Genthner (1996), albeit, in studies involving microbial consortia. Sandrin and Maier (2003) argued that such stimulation was a result of differential toxicity effect wherein a second population more sensitive to the metal stress competes with the population expressing the activity of interest. The stimulation of crude oil biodegradation by low amounts of zinc at acidic and neutral pH levels in pure cultures of Rhodotorula sp. would probably be attributable to the trace requirement of this redox-inactive former of tight complexes (Nies, 1999) by the hydrocarbon decarboxylase enzyme systems of the yeast.

Apart from the progressive inhibition of biodegradation processes with increasing metal concentrations, low concentrations of certain metals, at appropriate pH levels, could stimulate organic pollutants biodegradation processes, even in pure cultures.

ACKNOWLEDGMENTS

The authors acknowledge with gratitude the efforts of Mobil Producing Nigeria Unlimited, Ibeno, Nigeria for providing the crude oil used for the study. They also thank Mr. Emmanuel Bassey of the Environmental department of the Institute of Oceanography, University of Calabar, Nigeria, for performing the hydrocarbon analysis of the samples.