Technical paper Fungi and Bacteria Isolated from Two Highly Polluted Soils for Hydrocarbon Degradation M. E. Mancera-López,a M. T. Rodríguez-Casasola,a E. Ríos-Leal,a F. Esparza-García,a B. Chávez-Gómez,b R. Rodríguez-Vázquez,a J. Barrera-Cortésa* a CINVESTAVIPN, Depto. Biotecnología y Bioingeniería, Av. IPN 2508, Col. San Pedro Zacatenco, C.P. 07360, México D.F. Tel.: +52 55 50613800/4380, Fax: +52 55 50613313, E-mail: jbarrera@cinvestav.mx. b Instituto Mexicano del Petróleo. Depto Biotecnología, Eje Central Lázaro Cárdenas 152, Col. San Bartolo Atepehuacan, C.P. 07730, México D.F. Received: 03-08-2006 Abstract Cultivable fungi and bacteria were isolated from two highly contaminated soils with total petroleum hydrocarbon (TPH) concentrations of 60,600 and 500,000 mg kg-1. The aim of the study was to determine the capacity of these bacteria and fungi to degrade TPH, specifically the aliphatic hydrocarbon (AH) and polycyclic aromatic hydrocarbon (PAH) fractions, when cultivated in a mineral liquid culture (modified Rennie medium) together with a complex mixture of TPH as the unique source of carbon. Thirty-seven hydrocarbon-degrading strains were isolated, but only six strains showed a high ability to degrade PAHs, AHs and TPH. These strains were identified as Pseudomonas pseudoalcaligenes, Bacillus firmus, Bacillus alvei, Penicillium funiculosum, Aspergillus sydowii and Rhizopus sp., and they removed 79%, 80%, 68%, 86%, 81% and 67% of TPH, respectively. P. pseudoalcaligenes and P. funiculosum removed 75% of PAHs, while B. firmus and P. funiculosum removed 90% and 92% of AHs, respectively. The highest TPH removal was observed by P. funiculosum, which was isolated from the soil with a high TPH concentration. A. sydowii was also isolated from this soil; no reports were found regarding its capacity to remove PAHs, but it was able to degrade five- and six-ring aromatic compounds. Keywords: Bacteria, bioremediation, fungi, polycyclic aromatic hydrocarbons, aliphatic hydrocarbons. 1. Introduction Petroleum spillage is a source of severe water and soil pollution that, in addition to the known environmental problems, reduces microbial diversity through the phenomenon of selectivity.1 The microorganisms capable of surviving in such a polluted environment are those that develop specific enzymatic and physiological responses that allow them to use the hydrocarbon compounds as substra-tes.1 This response of microorganisms to organic contaminants has been studied for many years. It has been found that individual microorganisms can mineralize only a limited range of hydrocarbon substrates, so assemblages of mixed populations with an overall broad of enzymatic capacities are required to increase the rate and extent of petroleum biodegradation.2 At present, various microbial genera have been detected in petroleum-contaminated soil or water, which strongly suggests that each has a role in the hydrocarbon transformation process.2 The most frequently found microorganisms are bacteria and fungi, with bacteria assuming the dominant role in marine ecosystem and fungi in terrestrial environments.3 It has been reported that adapted communities previously exposed to hydrocarbons exhibit higher biodegradation rates than communities with no history of hydrocarbon contamination.4 Organic compounds of low molecular weight and simple molecular structure are preferred by many microorganisms.3 Compounds with more complex structures, such as polycyclic aromatic hydrocarbons (PAHs), with more than five benzene rings, are more resistant to microbial breakdown.5,6 According to Atlas, the most prevalent bacterial hydrocarbon degraders, in decreasing order, belong to the genera Pseudomonas, Achromobacter, Flavobacterium, Nocardia, Arthrobacter and other coryneforms, Vibrio, Bacillus, Micrococus and Acinetobacter. Other genera of bacteria able to degrade hydrocarbons include Actinomyces, Aeromonas and Alcaligenes.3 Fewer fungi are known to degrade hydrocarbons, because of reduced fungal growth in the soil due to factors such as competition with bacteria and the toxicity of the pollutants;7 other mechanisms that limit fungal growth are antibiotic production, nutrient competition, and mycoparasitism. Fungi are of interest because of their ability to synthesize relatively unspecific enzymes involved in cellulose and lignin decay that can degrade high molecular weight, complex and more recalcitrant toxic compounds, including aromatic struc-tures.8 In some Environmental Protection Agency reports, PAHs with more than five benzene rings have been considered as harbingers of cancer.9 The impact of these compounds on human health has stimulated great interest in the identification of microbial strains with specific PAH-degrading activity.10 The objective of this research was to study the cultivable hydrocarbon strains (bacteria and fungi) isolated from two highly polluted soils (60,600 and 500,000 mg kg-1 of TPH) to determine whether AH, PAH, and TPH degradation efficiency was associated with the concentration of hydrocarbons in the soils from which the strains were isolated. 2. Materials and Methods 2.1. Soils Contaminated with Hydrocarbons Native hydrocarbonoclast microorganisms were isolated from aged soils obtained from two different sites polluted with total petroleum hydrocarbon (TPH). The first soil was obtained from a 30-year-old waste lagoon in Poza Rica (PR), Veracruz, Mexico. The second was taken from the Santa Alejandrina (SA) swamp site near an oil refinery in Tabasco, Mexico, and had been contaminated with hydrocarbons for more than 40 years. Both soils were characterized according to their TPH concentration and hydrocarbon fractions: aliphatics (AHs), PAH, saturates (SHs) and asphaltenes (ASPHs). The following characteristics were also measured and recorded: pH, density (gravimetric method), water holding capacity, total organic carbon, assimilated phosphorus, nitrogen content and cultivable microorganism count.11,12 2.2. Hydrocarbon Analysis of Soil A 1 g sample of polluted soil was mixed with 0.5 g of NaSO4 anhydrous and extracted with dichloromethane for 8 h in a Soxhlet apparatus (EPA method 3540C). The extract was evaporated to dryness and measured for TPH quantification by difference in weight.13 Next, the hydrocarbon extract was fractionated into asphaltene, aliphatic, polycyclic aromatic and saturate hydrocarbon fractions. Asphaltenes were first precipitated with n-pentane from 1 g TPH extracts. The asphaltene fraction was recuperated by filtration and was dried and weighed (until constant weight) for its quantification. Next, n-pentane was evapo- rated from the filtrate for fractionation of the residual hydrocarbons. The fractionation was carried out in samples of 0.1 g in a silica gel (60-200 mesh) column of 20 1.5 cm, where the separation was accomplished by successive elution with hexane (aliphatics), benzene/hexane 1:3 (aromatics) and acetone/methanol 1:1 (saturates). The hydrocarbon fractions eluted were quantified by difference in weight. Analyses of hydrocarbons were carried out in triplicate. A stock of TPH extracted from the Poza Rica Veracruz soil was prepared as described above for its use in the study of identification of hydrocarbonoclast strains. 2.3. Isolation of Strains from Contaminated Soils Cultivable native microorganisms were isolated from the severely, long-term contaminated soils described above. Strains were isolated from polluted soil previously biostimulated with a mineral solution specific to bacterial or fungal cultivation (see below). Biostimulation was performed in 125 mL Erlenmeyer flasks, where 10 g of polluted soil was humidified with 10 mL of mineral medium. The Erlenmeyer flasks were incubated at environmental temperature for 7 days. The microorganisms were separated from the soil particles by gentle shaking of 1 g soil (dry weight) with 10 mL of sterile water for 30 min. After sedimentation, the supernatant suspension was used to prepare appropriate dilutions (from 1 x 10-1 to 1 x 10-7) with sterile water. Aliquots of 0.2 mL were spread-plated on the appropriate medium (solid nutrient agar medium, Sabouraud maltose agar, or Czapek agar). The plates were incubated at 28 °C for 7 days. The microorganisms were allowed to spread until purification and were then conserved in a refrigerator ready for use in the production of inocula for the hydrocarbon degradation experiments. 2.4. Production of Bacterial Inocula A loop of bacterial inoculum was placed in 25 mL of sterile nutritive broth in a 125 mL Erlenmeyer flask and incubated at 30 °C and 130 rpm for 3 days. One millilitre of the bacterial culture (1 x 105 cell mL-1) was used as the inoculum in a medium containing mineral salts and a complex mixture of hydrocarbons (20,000 mg L-1) extracted from the polluted soil of Poza Rica.14 The morphology and purity of the bacterial strains was determined by Gram staining, and they were then streaked in a Petri dish.15 The isolated bacterial cultures were characterised morphologically and biochemically using the crystal-BBL® system and complementary tests.16 2.5. Production of Fungal Inocula Fungal spores cultivated in Petri dishes with Sabouraud maltose agar were suspended in 10 mL of sterile di- stilled water. The spore suspension was then washed and filtered through sterile glass wool and kept in amber vials at 4 °C.17 The morphology of the fungi was identified by microculture. Microcultures were prepared by setting up a small Petri dish chamber containing a V-shaped piece of glass tubing resting on several layers of moistened filter paper. A sterile block of agar medium about 1 cm square was placed on a flame-sterilized microscope slide and the slide was then placed in the moist chamber on the tubing (all material was sterilized). The fungus was inoculated near the four edges of the agar block and a sterile coverslip was put over it. After a few days, the slide was observed under the microscope, and the undisturbed fungal structures were viewed as they grew.18 2.6. Hydrocarbon Degradation in Liquid Culture A 500 mg sample of TPH extracted from the Poza Rica soil was dissolved in 2 mL of hexane and added to a vial of 120 mL. After solvent evaporation at 25 °C, 25 mL of a sterile mineral medium, specific for bacteria or fungi, was added in order to obtain a TPH concentration of 20,000 mg L-1. Next, 1 mL of the respective strain was inoculated onto each vial to be incubated at 30 °C and 150 rpm for 15 days. Mineral medium (1 L) for bacteria consisted of two solutions: solution A (0.9 L) was composed of: 0.8 g K2HPO4, 0.2 g KH2PO4, 0.1 g KCL, 0.025 g Na2MoO4 • 2H2O, 0.014 g Na2FeEDTA and 1.0 g NH4NO3, and solution B (0.1 L) consisted of 0.2 g MgSO4 • 7H2O and 0.06 g CaCl2 • H2O.19 Both mineral solutions, previously prepared and fixed at pH 7, were sterilized separately at 121 °C for 18 min, before being mixed under sterile conditions. Mineral medium (1 L) for fungi was prepared with: 0.5 g (NH4)2SO4, 0.5 g KH2PO4, 0.2 g KCL, 0.2 g Mg-SO4 • 7H2O and 0.1 g CaCl2 • H2O.19 The mineral solution was adjusted to a pH of 6 and sterilized at 121 °C for 18 min. 2.7. Analysis of Residual Hydrocarbons in Liquid Culture Vials were sealed for AH and PAH analysis by headspace solid-phase microextraction (HS-SPME), combined with gas chromatography (GC).20,21 Both analyses were carried out on a Varian 3800 gas chromatograph (GC) implemented with a flame ionization detector (FID) and a non-polar 007 fused silica capillary (Quadrex Corporation) column: 25 m, i.d. 0.53 mm and film thickness 1 pm. Carrier gas was nitrogen at 0.7 kg cm-2. Detector and injector temperatures were fixed to 250 and 220 °C, respectively. The AH fraction was microextracted with fibre coated with 85 pm polydimethylsiloxane, and previous heating of TPH samples (in a hermetically sealed vial) to 80 °C. The time required for absorption was 3 min, with 30 s for desorption, on gas chromatography (GC). The temperature regimen for the GC column was: 50 °C, 6 °C min-1 up to 230 °C, maintained for 36 min. A standard mixture of AHs in the range C12-C30 was used to identify the range of hydrocarbons present in the TPH samples; the removed AHs were quantified on the basis of chromatograms obtained before the hydrocarbon biotreatment. PAH fraction microextraction was carried out with fibre coated with 100 pm polydimethylsiloxane. In this case, TPH samples were heated to 100 °C (vial hermetically sealed) for 15 min. The required adsorption and desorption times were 10 and 1 min, respectively. In order to obtain a good separation and quantification of the PAH compounds, two different sequential temperature regimes were applied to the capillary column: (a) 80 °C for 7 min, with increments of 4 °C min-1 up to 228 °C, maintained for 5 min; (b) 4 °C min-1 up to 270 °C, maintained for 3 min. Quantification of PAH was carried out with a mixture of known hydrocarbons to form ten standard PAH: naphthalene, acenaphthylene, phenanthrene, anthracene, fluoranthene, pyrene, chrysene, benzo(k)fluoranthene, benzo(a)pyrene, inden[1,2,3-cd]pyrene; the mixture was analysed under the same conditions as the gas samples. TPH were extracted with hexane from the liquid culture and deposited in 120-mL vials. The hexane was evaporated to dryness and the vials were measured for TPH quantification by difference in weight.13 3. Results 3.1. Hydrocarbon Composition Table 1 shows the characteristics of the two highly polluted soils: SA with 500,000 mg of TPH per kg-1 of dry soil, approximately 90% more TPH than PR soil. As shown in the table, SA soil had low AH and PAH concentrations (32 ± 4% and 5 ± 2%, respectively) and a high ASPH concentration (53 ± 5%) compared with PR soil. Considering the relatively high PAH concentration in PR (21%) compared with SA (5%), PR soil was selected for extraction of the complex hydrocarbon mixture to be used in the identification of hydrocarbonoclasts. An additional advantage of this TPH extract was the AH and PAH composition, as shown in Figures 1 and 2. For AHs, the following composition was determined: 11% 0.8 indicates the efficiency of these fungi in removing PAHs. The three fungi identified as the most efficient at removing hydrocarbons have already been reported as being able to degrade organic compounds. Rhizopus sp. degraded 60% of pentachlorophenol (12.5 mg L1) in a solid Table 4. TPHs removed by bacteria in terms of carbon or benzene ring number. AH PAH < C12 C12-C20 C20-C30 2-rings 3-rings 4-rings 5-rings 6-rings P. pseudoalcaligenes 0.84 0.78 0.83 0.92 0.8 0.7 0.26 0.25 B. firmus 0.95 0.83 0.92 0.82 0.75 0.4 0.11 0.17 B. alvei 0.72 0.59 0.78 0.78 0.75 0.21 0.1 0.03 Table 5. Hydrocarbon removal by fungi. Strain Identified as AH (%) PAHs (%) PAHs (%)/AH (%) TPHs (%) H02SA Penicillium funiculosum 92 ± 5a 75 ± 0.5a 0.81 86 ± 6a H03SA Aspergillus sydowii 88 ± 4b 70 ± 2a 0.93 81 ± 5b H01PR Rhizopus sp. 72 ± 4b 60 ± 4b 0.83 67 ± 3b Conditions of culture: Modified culture media, Rennie (1981), for hydrocarbonoclastic fungi; temperature 30 °C, 150 rpm, treatment duration: 15 days. PR and SA: origin of the polluted soil (PR, Poza Rica; SA, Santa Alejandrina swamp). Different letters are significant different (P(F)=0.05). Table 6. TPHs removed by fungi in terms of carbon or benzene ring number. AH PAH < C12 C12-C20 C20-C30 2-rings 3-rings 4-rings 5-rings 6-rings P. funiculosum 0.89 0.9 0.93 0.9 0.85 0.75 0.3 0.22 A. sydowii 0.63 0.73 0.78 0.85 0.81 0.65 0.25 0.2 Rhizopus sp 0.8 0.72 0.71 0.8 0.61 0.51 0.06 0.1 fermentation incubated at 30 °C for 24 h, with sugarcane bagasse as the solid matrix.31 A P. funiculosum strain isolated from the soil at a former gasworks site degraded 57% of the pyrene contained in a liquid culture (50 mg L-1) at 22°C over 28 days.27 A. sydowii isolated from pesticide-treated wheat straw was able to hydrolyze (in 3 weeks) 300 mg kg-1 of organophosphate pesticides contained in a soil treated with 1,000 mg kg-1 of these contaminants.28 There have been internal reports on the isola- tion of A. sydowii from marine sediments, but none were found on its capacity to remove PAHs.32 P. funiculosum and A. sydowii, which showed the greatest capacity (81-86%) to remove 20,000 mg of TPH L-1, were isolated from soil containing 500,000 mg kg-1 of TPH. Their greater capacity to remove TPHs could possibly be due to adaptation of these fungi to the pollutant composition, as well as to the enzymatic systems of the fungi. Alexander reported that microorganisms isolated Figure 3. Chromatogram of the residual aliphatic hydrocarbon (AH) fraction analysed after biotreatment with A. sydowii. GC implemented with an FID and a non-polar 007 fused silica capillary column: 25 m, i.d. 0.53 mm and film thickness 1 |im. from highly polluted soils could degrade organic compounds of high molecular weight.33 Atlas reported that, in pure cultures, specific AH and PAH fractions have been removed by up to 90% and 75%, respectively.34 However, it is well known that these hydrocarbon removal percentages can diminish or increase depending on the fermentation type (solid, liquid or slurry) and microorganisms or microflora involved (pure cultures or co-cultures; bacteria-bacteria, fungi-fungi or bacteria-fungi), as well as on the characteristics and concentration of the pollutant involved.35 In the present work, the results seem to be satisfactory regarding the high complexity and high concentration of the hydrocarbon mixture used (Figures 1 and 2). Figures 3 and 4 show the chromatograms of the residual AH and PAH concentrations determined by GC after complex hydrocarbon mixture biotreatment with A. sydowii. Comparison of these two chromatograms with those obtained before the biotreatment (Figures 1 and 2), shows clear diminishment of the peaks, principally of