Abstract: Addition of nitrogen sources as supplementary nutrient into MSM medium to enhance biodegradation by stimulating the growth four isolates, Acinetobacter faecalis, Staphylococcus sp., Pseudomonas putida and Neisseria elongata isolated from petroleum contaminated groundwater, wastewater aeration pond and biopond at the oil refinery Terengganu Malaysia was investigated. The organic nitrogen sources tested not only supported growth but also enhances biodegradation of 1% Tapis crude oil. All four isolates showed good growth especially when peptone was employed as the organic nitrogen compared to growth in the basal medium. Gas chromatography showed that more then 91, 93, 94 and 95% degradation of total hydrocarbon was observed after 5 days of incubation by isolates Pseudomonas putida, Neisseria elongate, Acinetobacter faecalis and Staphylococcus sp., respectively.
INTRODUCTION
Biodegradation as the core process of bioremediation technologies depends on culture conditions, aeration and nutrients addition to enhance hydrocarbon breakdown. Rahman et al. (2002) reported that optimal rates of growth and hydrocarbon biodegradation can be sustained by ensuring that adequate concentrations of nutrients are present (Atlas and Bartha, 1992). Biodegradation conditions within contaminated aquifers are often found to be limited by the availability of nutrients including nitrogen. Consequently, microorganisms that are capable of degrading contaminants as well as fixing molecular nitrogen as their sole nitrogen could have a growth advantage in fixed-nitrogen-deficient environments would be favorable for promoting in situ bioremediation (Westrick et al., 1994). Heterotrophic populations require a group of other nutrients in addition to the organic compounds that serves as a source of carbon (C) and energy and thus in the study of biodegradation, much attention has been given to the roles of nitrogen (N) and phosphorus (P). In any environment, the ratio of C: N: P needs to be maintained at about 120:10:1 to sustain microbial activity (Thomas et al., 1992). Nutrient such as nitrogen from urea, ammonium or nitrate ions and phosphorus from orthophosphate are required to stimulate growth and biodegradation (Atlas, 1981). Biostimulation involves introduction of additional nutrients in organic or inorganic form into a contaminated environment which then increases the population of the indigenous microorganisms (Pankrantz, 2001). Therefore, the goal of the present study is to examine the effect of nitrogen source addition for enhancement of biodegradation of crude petroleum oil of four potential local isolate.
MATERIALS AND METHODS
Source of microorganisms, media and culture condition: Bacterial strains were isolated from samples collected (Jun 2005) from groundwater and wastewater aeration pond and biopond located at the Terengganu oil refinery, Malaysia. Ten milliliter of each sample were washed with 90 mL saline and filtered with membrane. Incubation was carried out for 24 h at 37°C on nutrient agar plate. Mineral Salts Medium (MSM) (Zajic and Supplisson, 1972) was prepared by dissolving 1.8 g K2HPO4, 4.0 g NH4CI, 0.2 g MgSO4.7H2O, 0.1 g NaCl, 0.01 g FeSO4.7H2O in 1 L of distilled water. Bacteriological agar was added (15 g L-1) to the solution where solid basal medium was required. The pH was adjusted to 6.90 and the medium was autoclaved at 121°C for 15 min. 1.0% (v/v) Tapis crude oil was added as sole carbon source and complex vitamin solution (1.0 ml L-1) according to manufacturer`s suggestion.
Inoculum preparation: Bacterial inoculums were prepared in 50 mL nutrient broth by inoculating a loopful of cells from nutrient agar plate. The cultures were incubated for 24 h at 37°C at agitation of 150 rpm. The cells were then harvested by centrifugation (Appendorf) at 4000 rpm for 10 min at 4°C. The bacterial pellet was resuspended in 10 mL saline to give the inoculum suspension at absorbance of 0.5 and wavelength at 550 nm. Unless specified otherwise, bacterial inoculums was added to give a final concentration of 10% (v/v). The cultures were incubated at 37°C for 5 days. Growth was evaluated by the resulting colony forming unit (cfu mL-1).
Isolation and identification of microorganisms: All colonies that had grown on crude oil agar were subcultured on Nutrient Agar (NA) and were incubated at 37°C for 24 h. A colony on NA was recultured to obtain pure culture. All the pure strains were conserved on glycerol and stored at -20°C. Screening of the isolates was carried out based on colony morphology, size, shape, color, Gram staining and biochemical tests, methyl red-Voges proskauer (MR-VP) tests, indole, catalase and oxidase. The isolates were identified according to the descriptions in the Bergeys manual of Systematic Bacteriology, vol. 1 (1984), vol. 2 (1986). API 24 E (Commercial Kit) was used for identification of Gram negative bacteria.
Effect of nitrogen source on growth and biodegradation: To determine effect of organic and inorganic nitrogen source on biodegradation, the inorganic nitrogen source of the basal medium were substituted by the addition of the following; peptone, yeast extract and ammonium sulfate at concentrations of 0.30, 0.50, 1 and 1.50% (v/v). Cultivations were carried out after 5 days and the growth was measured as CFU count.
Extraction of residual crude oil for Gas Chromatography (GC) analysis: Crude oil was extracted with a pre-cleaned separating funnel, following the modified methods of Chaillan et al. (2004). Fifty milliliter of sample (BM + crude oil + bacteria), then centrifuged at 4000 rpm for 15 min. The separating funnel was stoppered with a glass stopper and it was shaken vigorously. Vapour was carefully vented out through the stopcock. The process of venting and vigorous shaking was repeated for several minutes and the mixture was allowed to separate into two phases. After the phases have been separated in the funnel, chloroform was collected in a 250 mL pre-cleaned bottle. The extraction was repeated with 25 mL chloroform twice, then filtered and dried by passing it through of 3 g pre-combusted Na2SO4 (150°C for 3 h in incubation) and collected in a round bottom flask. The extract was then evaporated by using a rotary evaporator. The crude oil extracted washed with 2 mL chloroform. The solvents were removed under a gentle stream (dried in fume chamber) for 7 days. The residue was reconstituted with 1 mL of chloroform and analyzed by GC.
Analysis of extract: A detailed analysis of the hydrocarbon extract was performed by Gas Chromatography (GC). The clarus GC 500 was equipped with a split injector (split ratio 50/1) and a Flame Ionization Detector (FID) both set at 300°C; carrier gas was nitrogen 1.50 mL min-1; the column was fused silica capillary column (30.0 mx0.32 mm, film thickness 0.25 μm); temperature programming was 60-320°C, 5°C min-1, injection volume 1 μL.
RESULTS AND DISCUSSION
Identification of bacteria: Biochemical and growth characteristics of four isolates (WD2, DD3, TDA4.2 and TAM4.4) that exhibited good potential were further investigated. The results showed that two isolates were short rod Gram Negative Acinetobacter faecalis from groundwater sample and Pseudomonas putida from aeration pond sample. The other two isolates were coccus Gram Positive Staphylococcus sp. from groundwater sample and Neisseria elongata from biopond sample (Table 1).
Effect of peptone on growth and degradation: The bacterial growths in different percentages of peptone as nitrogen source were studied (Table 2). All four isolate showed good growth at different peptone concentration 0.3, 0.5, 1.0 and 1.50%. Optimum growths were observed at percentages 1% Tapis crude oil on the 5 day of incubation. Among the four isolates, isolate WD2 and DD3 showed a maximum growth with 1% peptone while, isolates TDA4.2 and TAM4.4 showed a maximum growth with 0.5% peptone. From this study, 1% peptone was the best nitrogen source tested for growth compared to the basal medium, yeast extract and ammonium sulfate. However, increasing the concentration of peptone from 1 to 1.50% significantly decreases growth and degradation. This is in agreement with the finding by Tano-Debrah et al. (1999) that due to enhanced growth; degradability of oil was better in a medium containing 1% peptone, compared to medium containing either ammonium sulfate or potassium nitrate. Zeng et al. (2004) also reported cell growth increase in the medium with 1% peptone for Pseudomonas fluoresences FS1.
| Table 1: | Biochemical and growth characteristics of isolated bacterial cultures |
-: Negative, +: Positive, K/K: Alkaline
no fermentation, A/A: Acid glucose, lactose and/or sucrose fermentation,
O: Oxidative, F: Fermentive |
|
| Table 2: | Growth (cfu mL-1) of isolates WD2, DD3, TDA4.2 and TAM4.4 with different concentration of nitrogen sources in the MSM medium with 1% Tapis crude oil at pH 7.0 after 5 days |
Control: MSM medium without nitrogen
sources |
|
Effect of yeast extract on growth and degradation: Medium containing different concentrations of yeast extract as nitrogen source promotes cell growth and hydrocarbon degradation (Table 2). Good growth was observed with all the four isolates when yeast extract were present in the medium. However the four isolates isolate WD2 and DD3 showed high growth with 1% yeast extract while, isolates TDA4.2 and TAM4.4 showed high growth with 0.5% yeast extract. However, increasing the concentration of yeast extract from 0.5 to 1% significantly lowered the amount of growth hydrocarbon degradation. Khleifat (2006) reported that the nitrogen sources supplied, except yeast extract and casein, led to the enhancement of the phenol biodegradation of Ewingella americana. In another report, Okeke and Frankenberger (2003) demonstrated that good growth and degradation of crude oil in medium containing 1% yeast extract. Optimal amount of yeast extract that should be supplemented for phenol biodegradation was reported by Loh and Tan (2000).
Effect of ammonium sulfate on growth and degradation: Different inorganic nitrogen concentration sources can be used by the microorganisms, the effect of ammonium sulfate for the study of the influence of inorganic nitrogen source on growth hydrocarbon biodegradation of the four strains (Table 2). The addition of ammonium sulfate to the medium of all the four isolates showed good growth but isolate DD3 showed high growth with 0.3% while, isolates WD2, TDA4.2 and TAM4.4 showed high growth with 0.5%. The cell growth values obtained with inorganic nitrogen sources as ammonium sulfate were considerably lower than when peptone and yeast extract were used as organic nitrogen sources. However, increasing the concentration of ammonium sulfate from 0.5 to 1% significantly decrease hydrocarbon degradation because the addition of ammonium sulfate greater than the optimal amount would result in slow growth and consequently biodegradation by Lee et al. (2003) reported that the growth kinetics of Pseudomonas putida G7 on pyruvate was determined from a chemostat culture of Pseudomonas putida G7 using pyruvate as carbon and ammonium source.
Biodegradation of crude oil with MSM medium containing peptone: The biodegradation in MSM containing peptone was studied using four bacteria Staphylococcus sp., Acinetobacter faecalis, Neisseria elongata and Pseudomonas putida following enhanced biodegradation of 1% Tapis crude oil polluted water after 5 day of incubation. GC results showed 95, 94, 93 and 91% degradation of Tapis crude oil by Staphylococcus sp., Acinetobacter faecalis, Neisseria elongata and Pseudomonas putida, respectively. Maximum degradation was achieved after 5 days of incubation of Staphylococcus sp. Aliphatic compounds in the range of C8-C19 was degraded completely followed by C20-C38 with percentage degradations of 100, 96 and 97%, respectively. Compared to control (Fig. 1), between 3 and 2% of C20-C38 remained after 5 days. The medium-chain aliphatic and aromatic compounds C8-C19 were almost fully degradation where less than 5% of these compounds remained. More then 94% of these aliphatic compounds have been removed; a percentage of hydrocarbon degradation of approximately 100% C8-C37 and between 95 and 97% of C20-C37 was degraded by Acinetobacter faecalis (Fig. 1) after 5 days of incubation compared to control, with 4 and 2% of C20-C37 remained. Short-chain and medium-chain aliphatic and aromatic compounds C8-C20 were degraded up to 94% where less than 6% of these compounds remained. Gas chromatographic analysis showed degradation of all aliphatic in the range of C8-C38. Degradation of approximately 100% of C8-C16. and between 80 to 96% of C17-C26 and between 95 and 87% of C27-C35 was recorded by Neisseria elongata after 5 days of incubation (Fig. 2). However less than 7% of short-chain and medium-chain aliphatic and aromatic compounds C8-C20 remained. Pseudomonas putida was able to degrade alkanes in the range of C8-C13 completely followed by C14-C29 and C30-C36 with percentage degradation of 100, 90 and 91%, respectively after 5 days of incubation with only 9 and 8% of these compounds remained (Fig. 2). Rahman et al. (2003) reported that the percentage of hydrocarbon degradation of approximately 100% of (C8-C11), 83-98% of (C12-C21), (80-85%) of (C22-C31) and 57-73% of (C32-C40) using bacterial consortium. In other report by Verma et al. (2006), the chromatograms of oily sludge inoculated with three isolates for 5 days, indicate that the Acinetobacter sp., SV9 degrades the aliphatic fraction to a greater extent than the aromatic fraction, Bacillus sp., SV9 consumed almost all aliphatic and aromatic, while the Pseudomonas sp., SV9 degraded 60% of both fractions of oily sludge. Among 368 isolates belonging to the genus Bacillus isolated by Sorkhoh et al. (1993) from desert soils, two Bacillus stearothermophilus strains degraded 80-89% of crude oil within 5 days at 60°C. Ijah (1998) reported crude oil components of chain length C12-C32 were extensively degraded by Serratia marcescens OCS-21 after 16 days of incubation while Acinetobacter calcoaceticus COU-27 was able to degrade C22-C30 components of the crude oil. All four isolates showed maximum removal of short-chain and medium-chain aliphatic compounds compared to longer-chain aliphatic compounds. This seems logical as short-chain and medium-chain alkanes are generally more easily degraded due to their lower hydrophobicity.
| Fig. 1: | (a) Chromatograph of Tapis crude oil before microbial degradation (b) Chromatograph of Tapis crude oil after microbial degradation using Staphylococcus sp. and (c) using Acinetobacter faecalis after 5 days incubation |
| Fig. 2: | (a) Chromatograph of Tapis crude oil before microbial degradation (a) Chromatograph of Tapis crude oil after microbial degradation using Neisseria elongate and (b) using Pseudomonas putida after 5 days incubation |
CONCLUSION
The best nitrogen source was peptone. Two isolates (Acinetobacter faecalis WD2 and Staphylococcus sp. DD3) showed maximum growth in 1% peptone and two isolates (Neisseria elongate TDA4.2 and Pseudomonas putida TAM4.4) showed maximum growth in 0.5% peptone. Degradation of total hydrocarbon of 91, 93, 94 and 95% in Tapis crude oil after 5 days of incubation with MSM + peptone was achieved by isolates Pseudomonas putida TAM4.4, Neisseria elongate TDA2.4, Acinetobacter faecalis WD2 and Staphylococcus sp. DD3, respectively.
ACKNOWLEDGMENT
The authors would like to thank UKM Zamalah (S 1.14/32/3 Jilid XI) and PETRONAS (No. CRO50833), for the financial support provided to carry out this study.