Characterization of pyrene utilizing Bacillus spp. from crude oil contaminated soil.

Pyrene, a high molecular weight polycyclic aromatic hydrocarbon (PAH), is a priority pollutant present in soil contaminated with crude oil, coal-tar and complex PAHs. Bacterial consortium CON-3 developed from crude oil contaminated soil of Patiala, Punjab (India) cometabolized 50 μg ml(-1) pyrene in the presence of glucose (0.5 %; w/v) at 30 °C, as determined by reverse-phase high performance liquid chromatography (HPLC). Bacillus sp. PK-12, Bacillus sp. PK-13 and Bacillus sp. PK-14 from CON-3, identified by 16S rRNA gene sequence analysis, were able to cometabolize 64 %, 55 % and 53 % of pyrene in 35 days, respectively. With the increase in glucose concentration to 1.0 % (w/v) in growth medium isolates PK-12, PK-13 and PK-14 showed 19 - 46 % uptake of 50 μg ml(-1) pyrene in 4 days, respectively. Uptake of pyrene was correlated with growth and biosurfactant activity, which is suggestive of the potential role of members of Bacillus genera in pyrene mobilization and its uptake.

INTRODUCTION

A variety of polycyclic aromatic hydrocarbons (PAHs) are formed as a result of anthropogenic activities such as incomplete combustion of coal, oil, gas and wood (13), use of creosote as wood preservatives (34), generation of wastes from petrochemical industries (14), oil refining (43) and coal gasification plants (17). PAHs e.g. anthracene, phenanthrene and pyrene have been identified as biohazardous chemicals by different State and Central Pollution Control Boards due to their toxic, carcinogenic and tetragenic effects on living systems (23). Physico-chemical properties of PAHs such as low water solubility, high adsorption coefficient and high stability of the complex aromatic ring structure limit the application of conventional remediation techniques (16, 26). In situ bioremediation is an attractive process due to its cost effectiveness, versatility and the benefit of pollutant mineralization to carbon dioxide and water (12). Bacterial degradation of petroleum and xenobiotic aromatic hydrocarbon contaminants in natural ecosystems may result from catabolism by individual strains such as Mycobacterium (9; 13), Bacillus, Pseudomonas (10), Aeromonas, Beijerinckia, Flavobacterium, Nocardia, Corynebacterium, Burkholderia (7), Paracoccus (43), Stenotrophomonas (4) and Sphingomonas (29, 37) or from combined metabolism by mixed communities called 'consortia' (4, 16, 24).

The metabolic pathways, enzymatic reactions, and genetic control of the catabolism of lower-molecular-weight (LMW) PAHs (naphthalene, phenanthrene, and anthracene) have been well documented (9, 12, 13). However a low rate of bacterial degradation of higher-molecular-weight (HMW) PAHs (pyrene, and benzo[a]pyrene) has been reported which is attributed to their inability to degrade chemicals that are insoluble in water. The aqueous solubility of PAHs decreases almost logarithmically with increasing molecular mass, therefore HMW PAHs ranging in size from four to seven rings are of special environmental concern (17). Some hydrocarbon-degrading bacteria respond to these non soluble carbon sources by producing surface-active compounds (10, 22) which help to pseudo-solubilize the hydrocarbons (32) and promote their bioavailability in the environment (6, 35).

Pyrene, a model compound for HMW PAH degradation studies, is commonly found in Indian soils contaminated with crude oil, coal tar and other complex mixtures of PAHs (10, 27). It has a chemical structure found in several carcinogenic PAHs and is included in the list of 129 'Priority Pollutants' compiled by the U.S. Environmental Protection Agency (12, 14). Pyrene degradation at the metabolic, genomic and proteomic level by actinomycetes group of bacteria has been documented (18; 19 and references therein). However, information about the prevalence of PAH-catabolic nonactinomycetes bacterial genotypes (3, 23, 33) in PAH-contaminated Indian soils is meagre and according to Habe and Omori (12) there may still be many unidentified PAH-degrading bacteria. The aim of the present study was to isolate an aerobic, mesophilic bacterial consortium and its monoculture bacterial isolates from crude oil-contaminated soil with ability to grow in aqueous medium and utilize pyrene.

MATERIALS AND METHODS

Development of bacterial consortium

Crude oil contaminated soil samples were collected from a refinery located in Patiala (Punjab), India. Ten gram of soil was added to 100 ml Bushnell-Haas (BH) liquid medium containing 1 % (v/v) crude oil (Bombay High, India) and shaken on a rotary shaker (120 rpm) at 30 °C. After 3 weeks of incubation the mixed culture (CON-3) of crude oil utilizing microorganisms was obtained. For selective enrichment 10 ml of the consortium was grown in 100 ml BH medium supplemented with 0.5 % (v/v) crude oil and 10 μg ml-1 pyrene (Merck - Schuchardt, Germany) as carbon sources with orbital shaking (120 rpm) at 30 °C. BH medium (Himedia labs, India) was composed of (g L-1 of deionized water): MgSO4.7H2O, 0.2; CaCl2.2H2O, 0.02; KH2PO4, 1; K2HPO4, 1; NH4NO3.6H2O, 1 and FeCl3, 0.05; pH 7.0 ± 0.2 (39). The medium was sterilized by autoclaving at 121 °C for 20 min in all tests in vitro and pyrene dissolved in acetone solvent (5 %; w/v stock solution) was aseptically added to the media flasks followed by shaking on a rotary shaker (120 rpm) at 30 °C for 12 hrs before inoculation to allow acetone evaporation as described by Vila et al. (41). After 4 weeks of incubation, 10 ml of enriched CON-3 culture was transferred into another flask containing 100 ml fresh sterile BH medium with 0.4 % (v/v) crude oil, 0.2 % (w/v) glucose and 20 μg ml-1 pyrene and incubated. Glucose was added to increase the biomass level of enriched culture (38). Every 4 weeks, in fresh 100 ml BH medium, the concentration of crude oil was decreased in steps of 0.1 % (v/v) upto 0.2 % while the concentration of glucose was increased in steps of 0.1 % (w/v) upto 0.5 %. A total of five gradual enrichments of pyrene in steps of 10 μg ml-1 upto 50 μg ml-1 were carried out to develop the HMW-aromatic hydrocarbon degradation phenotype and to selectively enrich the pyrene-utilizing bacterial isolates in consortium.

Isolation of pyrene utilizing bacterial isolates

The finally developed bacterial consortium CON-3 was serially diluted in 0.85 % (w/v) saline (NaCl) solution and plated on pyrene coated BH agar plates containing 0.25 % (w/v) glucose, in duplicate and incubated for 48-72 hr at 30 °C. Pyrene coating of BH agar plates was done according to Kiyohara et al. (20) by uniformly spreading 0.1 ml of pyrene stock solution over the surface of the media plate. The acetone immediately vaporized at ambient temperature and a white, thin layer of pyrene remained on the entire surface. Morphologically different, discrete bacterial colonies were picked, purified by repetitive streaking on the same medium (16) and Luria-Bertani (LB) agar medium (24) and evaluated for growth on 25, 50 and 75 μg ml-1 pyrene in 100 ml BH medium containing glucose in 250-ml capacity flasks with orbital shaking (120 rpm) at 30 °C for 30 days. Glucose was provided at 0.25 %, 0.5 % and 0.75 % (w/v), respectively. Growth was measured spectrophotometrically (Hitachi model U-2900, Japan) at 600 nm. Pyrene utilization efficiency was determined by high performance liquid chromatography (HPLC) analysis.

Molecular characterization by 16S ribosomal RNA gene analysis

The phylogenetic affiliation of bacterial isolates with maximum pyrene utilization efficiency greater than 50 % was determined. Genomic DNA was extracted by modified boiling lysis method of Krivobok et al. (21). Briefly, bacterial pellet from 2 ml monoculture (cells harvested in eppendorf tube by centrifugation at 10,000 x g, 15 min) was suspended in 0.1 ml of 0.22 μm filter-sterilized milli-Q water (Millipore, Germany) by vigorous vortexing. The bacterial cells were lysed by incubating the cell suspension in water bath set at 95 °C for 10 minutes followed by immediate chilling to 5 °C. Cell debris was pelleted down by centrifugation at 10,000 x g for 15 min. The supernatant (2 μl) containing genomic DNA (~ 10 ng) was used along with Taq DNA polymerase (1 unit reaction-1), 4 deoxyribonucleoside triphosphates (200 μM each), MgCl2 (1.5 mM final concentration; MBI Fermentas Life sciences, USA) and universal bacterial primers E8F (E. coli position 8-27, 5'-AGA GTT TGA TCC TGG CTC AG-3'; 25) and E1492R (E. coli position 1492-1513, 5'-GGT TAC CTT GTT ACG ACT T-3'; 42) (0.5 μM each; Qiagen Operon GmBH, Germany) to amplify the 16S rRNA gene in a PCR thermal cycler (GeneAmpÒ 9700, Applied Biosystems). The template DNA in the reaction mixture underwent initial denaturation at 92 °C for 2 min and 10 sec followed by 36 cyclic episodes of denaturation (92 °C for 1 min and 10 sec), renaturation (48 °C for 30 sec) and extension (72 °C for 2 min and 10 sec). Final extension occurred at 72 °C for 6 min and 10 sec. The PCR product was visualized on 0.8 % agarose gel, ligated with pGEM-T Easy vector (Promega, Wisconsin, USA) and sequenced by Bangalore Genei Pvt Ltd, Bangalore, India. Sequence analysis and nucleotide identity (similarity) search was performed with BLAST program (1) using the nucleic acid sequences deposited in multiple databases like National Centre for Biotechnology Information (NCBI) GenBank database and Ribosomal Database Project release 10 (RDP X).

The 16S rRNA gene sequences obtained in present study have been deposited with the NCBI GenBank database under accession numbers EU685814 to EU685816. Bacterial isolates Bacillus sp. PK-12 and Bacillus sp. PK-14 have been deposited at Microbial Type Culture Collection library at IMTECH, Chandigarh (India) with MTCC number 1002 and 1003 respectively.

Pyrene uptake studies

Pyrene uptake by bacterial consortium: The capacity of the developed consortium CON-3 to uptake pyrene was evaluated in BH medium (100 ml) containing 50 μg ml-1 of pyrene with and without 0.5 % (w/v) glucose in triplicate flasks with orbital shaking (120 rpm) at 30 °C for 30 days. In another experiment the effect of media optimization on rate of pyrene uptake and absolute / optional requirement of glucose for pyrene metabolism by consortium CON-3 was studied in BH medium (100 ml) supplemented with 50 μg ml-1 pyrene at interval of 10 days for 30 consecutive days at 30 °C in presence of either 0.5 % glucose, 2 ml of trace elements stock solution and 0.1 ml of trace vitamins stock solution (TEV), or 1.0 % glucose. Trace element 50 X stock solution (40) contained (mg ml-1): Nitrilotriacetic acid, 15; MgSO4, 5; FeSO4.7H2O, 1; CoCl2, 1; CaCl2.2H2O, 1; ZnSO4, 0.1; CuSO4.5H2O, 0.1; AlK(SO4), 0.1; H3BO3, 0.1 and Na2MoO4, 0.1 and was autoclaved. Trace vitamins 1000 X stock solution (28) contained (mg ml-1): Pyridoxine HCl, 10; Thiamine HCl, 5; Riboflavin, 5; Nicotinic acid, 5; Calcium pentothenate, 5; DL-α-Lipoic acid, 5; Biotin, 2 and Folic acid, 1 (Himedia labs, India), was filter-sterilized and stored at 4 °C. Batch cultures of CON-3 consortium and uninoculated media (control) flasks were withdrawn at 10 day interval, solvent extracted and quantified by HPLC for pyrene utilization.

Time course uptake of pyrene by bacterial isolates: Time course studies were conducted for determining utilization percentage of pyrene by monoculture isolates. For each bacterial isolate, a batch of sixteen flasks containing BH medium (100 ml) with 50 μg ml-1 pyrene, 0.5 % (w/v) glucose and 5 % culture inoculum was incubated at 30 °C with orbital shaking (120 rpm). One culture flask was withdrawn at zero time (control) to determine initial pyrene concentration. Another flask containing the same amount of pyrene but uninoculated was used as control to determine abiotic losses. Culture flasks (in triplicate) from each batch were withdrawn every 7th day upto 35 days, solvent-extracted and quantified by HPLC.

Effect of glucose concentration on pyrene uptake by bacterial isolates: Glucose concentration in 100 ml BH medium was increased from 0.5 to 1 % (w/v) and the pyrene metabolized (utilized) by the selected bacteria was determined after every 24 hr interval. Total protein content in culture (mg ml-1) was estimated using biuret method of Itzhaki and Gil (15) and glucose utilization (%) was determined by 3, 5-Dinitrosalicylic acid (DNS) assay as given by Plummer (30).

Biosurfactant activity: Standard emulsification assay / index of Barkay et al. (2) and Jacques et al. (16) was followed to monitor biosurfactant activity of bacterial isolates with maximum pyrene utilization efficiency greater than 50 %. The isolates were grown in duplicate flasks containing BH medium (100 ml) with 50 μg ml-1 pyrene plus 1.0 % (w/v) glucose and incubated at 30 °C for 4 days with orbital shaking (120 rpm). From one flask withdrew a 5 ml aliquot of the culture supernatant (cells were removed by centrifugation at 10,000 x g for 30 min, at 4 °C) and mixed with 2 % (v/v) Mobil oil (Racer 2T, HP Corporation Ltd., Mumbai) in a glass tube (150 × 18 mm) by vortexing for 1 min (16). The tubes were rested for 10 min and then the degree of dispersion of mobil oil and stability of the emulsion were measured spectrophotometrically at 550 nm (2) against a blank of uninoculated medium with 2 % mobil oil. Contents of the duplicate flasks were estimated for pyrene uptake by HPLC.

Estimation of pyrene by HPLC: Residual concentration of pyrene was determined by liquid-liquid extraction of residual pyrene from BH - glucose medium (inoculated with culture or noninoculated control) followed by spectrophotometric and reverse phase HPLC (PerkinElmer) analysis. The growing culture was acidified to pH 2.0 with 6 N HCl (13; 16) and extracted thrice with 50 ml hexane solvent (Merck; purity > 99.8 %). The extracted material was dried in fume-hood, resuspended in 5 ml acetonitrile and measured spectrophotometrically at 254 nm for pyrene (5). Extraction efficiency was found to be 87 % ± 3 %. Spectrophotometric results were confirmed by quantifying the amount of pyrene in a reversed phase high-performance liquid chromatograph using C18 column (33 x 4.6 mm). A linear gradient of 50 - 95 % methanol in MQ-water was developed over 20 min at a flow rate of 1 ml min-1. A 0.02 ml aliquot of acetonitrile extract was injected. Pyrene was identified by comparing characteristic absorption spectra (at 254 nm) and retention times to authentic pyrene, using a PerkinElmer diode array detector with data display, and analyzed using PerkinElmer Total Chrome Ver 6.0 software.

RESULTS AND DISCUSSION

Boonchan et al. (4), Johnsen et al. (17), Jacques et al. (16) and Lin and Cai (24) have reported microbial consortia to possess multiple metabolic capacities that increase the efficiency of the bioremediation process. In present study bacterial consortium CON-3 was developed from refinery waste contaminated soil by selective enrichment technique, by providing pyrene as co-carbon source in the concentration range 10 - 50 μg ml-1 in five successive transfers over a period of five months. The finally developed CON-3 consortium showed 49 % removal of 50 μg ml-1 pyrene from BH mineral salt medium containing 0.5 % (w/v) glucose during 30 days of growth (Fig 1). Utilization of pyrene by bacterial consortium CON-3 could be due to the synergistic effect of various bacterial isolates (4) or the consortium might contain predominantly certain class of bacteria that have wide range of substrate specificity (34). The capability of CON-3 consortium to remove pyrene was quite different from those of PAH-degrading consortia enriched by Jacques et al. (16) and Lin and Cai (24) from petrochemical sludge landfarming site and mangrove sediment samples which degraded 22 and 92 % of pyrene in mineral medium, after 30 and 21 days of incubation, respectively.

Figure 1

Pyrene uptake by bacterial consortium CON-3 in BH medium supplemented with 50 µg ml-1 pyrene at 10, 20 and 30 days interval at 30 oC. * TEV indicates trace elements and trace vitamins.

Minerals and vitamins are required for the enhanced growth and activity of bacteria (26). Addition of trace elements and vitamins (TEV) in basal medium stimulated pyrene uptake by CON-3 in 30 days to 58 %, both in the absence and presence of 0.5 % (w/v) glucose (Fig. 1). When glucose concentration was doubled to 1.0 % (w/v) it stimulated the pyrene uptake to 63 %. This result favoured the requirement of glucose for maximum uptake of pyrene from basal BH medium. According to Cerniglia, (8) pyrene cannot be utilized as a sole carbon and energy source, so a growth substrate must be supplied to initiate growth of the microorganisms and to induce the production of catabolic enzymes. Therefore the growth of CON-3 consortium in BH medium in presence of pyrene is cometabolic in nature. Churchill et al. (9) and Boonchan et al. (4) also reported cometabolic reaction in Pseudomonas, Acinetobacter, Nocardia, Bacillus, Mycococcus, Methylosinus and Arthrobacter bacterial species. De-Sisto et al. (11) grew bacteria on waste electrical transformer oil in basal medium supplemented with 1 % yeast peptone glucose. Das and Mukherjee (10) observed enhancement of pyrene utilization and bacterial growth in medium containing 0.01 % glucose.

Ten bacterial isolates (PK-12 to PK-21) were obtained from the selectively enriched bacterial consortium CON-3. In the initial screening it was observed that no isolate was capable of utilizing pyrene as sole carbon source. All isolates utilized pyrene as a co metabolite and removed 6 - 98 % of 25 μg ml-1 pyrene in presence of 0.25 % (w/v) glucose after 30 days of incubation (Table 1). Five isolates PK-12, PK-13, PK-14, PK-15 and PK-16 showing more than 50 % uptake of pyrene as 98 %, 61 %, 55 %, 54 % and 51 %, respectively, were further exposed to higher concentrations of pyrene in BH plus glucose medium. It was found that 75 μg ml-1 pyrene concentration inhibited the growth of all five isolates; however 50 μg ml-1 pyrene could support good growth in presence of 0.5 % and 1 % (w/v) glucose respectively. Therefore extent of pyrene uptake by bacterial isolates decreased with increasing concentrations of pyrene. In a 35 day time-course study it was observed that bacterial isolates PK-12, PK-13, PK-14, PK-15 and PK-16 cometabolized 50 μg ml-1 pyrene (in presence of 0.5 %; w/v glucose) by 18, 13, 18, 11 and 17 %, respectively, after 14 days of incubation which increased to 59, 53, 50, 50 and 47 %, respectively, as compared to negligible change in abiotic control after 28 days (Fig 2). Thereafter pyrene uptake for two bacterial isolates PK-15 and PK-16 remained stable till 35 days, while for three isolates PK-12, PK-13 and PK-14 it (uptake) increased upto 64, 55 and 53 %, respectively (Fig 2). Results demonstrate that maximum uptake of pyrene by isolates occurred between 14 and 28 days of growth. Lin and Cai (24) also observed 66 % and 34 % utilization of 50 μg ml-1 pyrene by Bacillus cereus and B. megaterium, respectively, in three weeks.

Table 1

Pyrene (25 mg l-1) uptake efficiency of bacterial isolates from CON-3 consortium, in presence of 0.25 % (w/v) glucose at 30 oC in 30 days.

Bacterial isolates	Pyrene uptake (%)
PK12	98
PK13	61
PK14	55
PK15	54
PK16	51
PK17	8
PK18	16
PK19	6
PK20	17
PK21	10

Figure 2

Pyrene (50 µg ml-1) uptake by bacterial isolates PK-12 (- - ▪ - -), PK-13 (- - ◊ - -), PK-14 (- -δ- -), PK-15 (…○…) and PK-16 (– × –), from CON-3 consortium, in presence of 0.5 % (w/v) glucose at 30 oC over a time course of 35 days.

Molecular characterization of three maximum pyrene utilizing bacterial isolates by analysis of approx. 1500 bp long 16S rRNA gene sequence and similarity searches revealed isolates PK-12, PK-13 and PK-14 belonged to the genus Bacillus (Table 2). The Bacillus strain PK-12 was genetically very close to type strain B. pumilus (T) FO-036b (99 % identity). Isolate PK-13 was most homologous to the type specie B. flexus (T) IFO-15715 and isolate PK-14 clustered nearest with the type strain B. firmus (T) IAM-12464 (both 98 % identity). Members of the genus Bacillus have been used in past studies for PAH biodegradation (10; 16; 24, 39). Toledo et al. (39) have majorly attributed Bacillus strains with the property to colonize environments contaminated with PAHs. They isolated eight B. pumilus strains from waste crude oil capable of growth on naphthalene, phenanthrene or pyrene as sole carbon source. B. flexus is not reported to be involved in the degradation of any polycyclic aromatic hydrocarbons till date. Mohamed et al. (27) have isolated B. firmus as bacterial degraders of petroleum hydrocarbons from contaminated soils in Kuwait. All the low molecular weight (LMW) PAH dioxygenase genes were in gram-negative bacteria, while the high molecular weight (HMW) PAH dioxygenase genes were in gram-positive strains was suggested by Habe and Omori (12). Our findings are in agreement as the three pyrene utilizing isolates, Bacillus spp. PK-12, PK-13 and PK-14, isolated from CON-3 consortium belong to gram-positive category.

Table 2

Nucleotide identity (BLAST search output) of bacterial isolates 16S rRNA gene sequence to sequences deposited in the GenBank database.

Bacterial isolate (GenBank Accession No.)	NCBI match (Accession no)	Nucleotide identity (%)
PK-12 (EU685814)	Bacillus pumilus (T) FO-036b (AF234854)	99 %
PK-13 (EU685815)	Bacillus flexus (T) IFO-15715 (AB021185)	98 %
PK-14 (EU685816)	Bacillus firmus (T) IAM-12464 (D16268)	98 %

Pyrene uptake (PU) by the selected bacteria Bacillus spp. PK-12, PK-13 and PK-14 in presence of glucose and its correlation with total protein in basal growth medium was studied (Fig. 3). In time course study (Fig. 2) Bacillus spp. PK-12, PK-13 and PK-14 showed 14, 7 and 18 % uptake of 50 μg ml-1 pyrene respectively, from BH medium containing 0.5 % (w/v) glucose in 7 days. When glucose concentration in BH medium was doubled to 1.0 % (w/v) Bacillus spp. PK-12, PK-13 and PK-14 showed increased and rapid cometabolic uptake of pyrene which was 46, 19 and 37 % respectively, in 4 days (Fig 3). Tao et al. (37) also observed enhanced cell growth of Sphingomonas sp. GY2B on 100 μg ml-1 phenanthrene upon addition of glucose at concentration 0.001 to 0.02 % in minimal medium. However glucose concentration greater than 0.05 % inhibited the bacterial growth. In contrast we have found that glucose concentration, as high as 1.0 % (w/v) exerted a positive and stimulatory effect on pyrene utilization, by CON-3 consortium and its three member isolates. A corresponding increase in total culture protein was also observed. Trend in total protein produced in four days of growth was similar to percent pyrene uptake (Fig. 3). Total protein content estimated was maximum for Bacillus sp. PK-12 and minimum for Bacillus sp. PK-13.

Figure 3

Pyrene (50 µg ml-1) uptake, (– ▲ –), Biosurfactant activity (B.A; –♦–), Total protein (T.P.; –x–), Glucose utilization (G.U.; –δ–) and growth (- - ▪ - -) profile of bacteria Bacillus spp. (A) PK-12, (B) PK-13 and (C) PK-14 in presence of 1.0 % (w/v) glucose at 30 oC.

Bacillus sp. are capable of using numerous carbohydrates, but glucose is the most preferred carbon source according to Stulke and Hillen (36) and it often represses the expression and activity of catabolic systems that enable the utilization of secondary substrates (3). Our results however support that glucose supplementation stimulates pyrene metabolism. Glucose is believed to act as an inducer and co-source of carbon, energy and reducing power for microbes that leads to substantial increase in bacterial biomass with a corresponding increase in metabolic activities (10). Das and Mukherjee (10) have shown 48 % utilization of 2500 μg ml-1 pyrene as sole carbon source by B. subtilis DM-04 in 4 days of growth at 55 °C incubation temperature. In present study Bacillus sp. PK-12 was able to cometabolize 46 % of 50 μg ml-1 pyrene as a co-carbon source in same time interval of 4 days but at 30 °C, which is optimum for lab-scale and in situ bioremediation applications.

It is known that microorganisms growing on hydrocarbons frequently produce biopolymers with emulsifying (6) or surfactant activity (39) so as to improve their ability to utilize these compounds (31). In this study, emulsification assays were carried out to know the capacity of the genetically identified, pyrene utilizing bacteria to produce biosurfactant activity (BA). The supernatants of exponentially growing cultures of Bacillus spp. PK-12, PK-13 and PK-14 were evaluated for residual glucose concentration and biosurfactant activity by their ability to emulsify mobil oil at 24 hour interval (Fig. 3). Almost half of the 1.0 % (w/v) glucose provided in the growth medium was utilized by Bacillus sp. PK-14 (Fig. 3C) at the end of 4 days, while Bacillus spp. PK-12 (Fig. 3A) and PK-13 (Fig. 3B) consumed less glucose. Interestingly the trend for biosurfactant activity was also similar with maximum biosurfactant activity (1.5 units) for Bacillus sp. PK-14 (Fig. 3C) followed by Bacillus sp. PK-12 (1.1 units; Fig. 3A) and least for Bacillus sp. PK-13 (0.7 units; Fig. 3B) after 4 days of incubation. It was further observed that in Bacillus sp. PK-14 pyrene uptake occurred during exponential phase of growth and biosurfactant activity (Fig 3 C), while in Bacillus spp. PK-12 and PK-13 (Fig. 3A and B, respectively) major uptake of pyrene was observed during the stationary phase in the culture medium.

At the end of the biosurfactant assay, emulsification of mobil oil resulted from the presence of biosurfactant activity in the culture medium. And this may be the reason that biosurfactant activity corresponded to the change in pyrene concentration in four days. Our proposed assay for biosurfactant activity is quite practical and convenient to use, since it permits preliminary prediction of production of extracellular biopolymers with biosurfactant or bioemulsifier activities by bacteria on the basis of a simple and rapid test. Jacques et al. (16) have used a similar emulsification index for diesel oil to estimate surfactant activity produced by bacterial and fungal isolates capable of utilizing a variety of PAHs. In another report Barkey et al. (2) tested for emulsifying activity of alasan preparations of Acinetobacter radioresistens KA53 by measuring emulsion formation spectrophotometrically at 600 nm.

The dynamics of glucose utilization and biosurfactant activity in the cultures were consistent with that of pyrene concentration change, indicating that the pyrene uptake by the bacterial isolates PK-12, PK-13 and PK-14 may be correlated with biosurfactant activity. In present study pyrene uptake by Bacillus sp. PK-14 occurred during the log phase of growth and biosurfactant activity. Glucose utilization and biosurfactant activity of Bacillus sp. PK-14 were highest among the three isolates. Therefore, it may be said that the uptake of pyrene is logarithmically associated with biosurfactant activity in Bacillus sp. PK-14. In contrast Bacillus sp. PK-12 displayed 1.4 times less biosurfactant activity and 1.2 times more pyrene uptake during stationary phase of growth. Correspondingly glucose utilization was also 1.6 times less. This result suggests that biosurfactant activity of Bacillus sp. PK-12 was more efficient than Bacillus sp. PK-14 in mobilizing pyrene uptake from growth medium. It has also been found by Das and Mukherjee (10) that the biosurfactant secreted by B. subtilis DM-04 was more efficient than P. aeruginosa strains in enhancing the solubility of pyrene in aqueous media resulting in a higher uptake and utilization of pyrene by the former bacteria. They also formulated the hypothesis that a minor variation in biosurfactant isoforms between P. aeruginosa NM and M strains may result in a large variation of the emulsification property and specificity.

There is little literature on uptake of pyrene by Bacillus species and its link to biosurfactant activity and glucose utilization. Different members of same genera showing different pyrene uptake capabilities in this study may be related to the biosurfactant activities and glucose utilization ability of respective bacterial species. The present study showing increased pyrene uptake and enhanced emulsification capacities of the bacterial isolates Bacillus sp. PK-12 and Bacillus sp. PK-14 indicates that these bacteria can be used for biotreatment and bioaugmentation of soils contaminated with PAHs.

CONCLUSION

PAHs occur at relatively high concentrations in crude oil-contaminated soils and sediments (14), so there is an increasing need in the use of PAH-acclimatized microbial consortia and monoculture isolates for the bioremediation of PAHs in the environment. The present study shows 3 different bacteria isolated from the same bacterial consortium (CON-3) from crude oil-contaminated soil and belonging to the same Bacillus genera, but they show different pyrene uptake, biosurfactant activity and glucose utilization profiles. Bacillus spp. PK-12, PK-13 and PK-14 exhibited 46 %, 19 % and 37 % cometabolic uptake of 50 μg ml-1 pyrene in presence of 1.0 % (w/v) glucose in 4 days. High glucose utilization (0.4 - 0.6 %) and enhanced biosurfactant activities (OD550nm > 1.0) of bacterial isolates Bacillus spp. PK-12 and PK-14 may be related to their enhanced pyrene uptake and subsequent utilization abilities. Therefore, soils contaminated with crude oil from refinery wastes, serve as an abode for pyrene metabolizing bacterial microorganisms. Increased pyrene uptake and enhanced emulsification capacities of the soil bacteria Bacillus spp. PK-12 and PK-14 in the present study indicates that these bacteria can be used for biotreatment and bioaugmentation of soils contaminated with PAHs.