EPA Grant Number: R827015-01-0
Title: Biodegradation of Petroleum Hydrocarbons in Salt-Impacted Soil by Native Halophiles or Halotolerants and Strategies for Enhanced Degradation
Investigator: Babu Fathepure
Institution: Oklahoma State University
EPA Project Officer: Bala Krishnan
Project Period: January - November 2002
Project Amount: $20,000
Research Category: Bioremediation of oil spills
There are exploration and production (EP) sites in Oklahoma and other oil producing states that are contaminated with both oil and salt. This poses a problem for cleaning up these sites using bioremediation technologies since the harsh conditions caused by high salinity will not support externally added bacteria. The main purpose of this project was to demonstrate the potential of indigenous halophilic and halotolerant bacteria to degrade petroleum hydrocarbons present in oil-brine soil and optimize conditions for enhanced degradation. We have evaluated several oil-brine-impacted soil for their ability to degrade benzene. Further, we assessed the effects of various osmolytes on the rate and extent of biodegradation. Under hypersaline conditions, most halophilic and halotolerant bacteria synthesize or accumulate in cells elevated levels of osmolytes (also known as compatible solutes), which confer tolerance to the cells under stressed conditions. In addition, we have enriched microbial populations from a brine-contaminated soil that have the ability to completely degrade benzene. Our future goals are to isolate pure cultures of halophiles and assess their ability to degrade petroleum compounds under high salinity for the development of bioaugmentation technologies. This would provide a cost-effective approach for cleaning up oil at EP sites that have been contaminated with high concentrations of salt.
As mentioned in the first quarterly report, five soil samples from Oklahoma- four from Seminole County and one from Stephens County-that were contaminated with both petroleum and brine were obtained and analyzed to determine the levels of contamination. From this data, one soil, named Sem- 2, was used for aerobic work, while the others were obtained from greater depths and had contamination levels warranting anaerobic remediation.
Using the Sem-2 soil, we have obtained a highly enriched aerobic culture that has the ability to rapidly degrade benzene as the sole carbon and energy source when added to the mineral salt media containing 2.5M NaCl. The benzene concentrations in the headspace of the microcosms were analyzed by gas chromatography. After more than four months of continuous enrichment, our Sem-2 culture consistently degraded benzene completely within 2.5 weeks at room temperature.
Figure 1 shows the degradation of benzene after repeated spikes by the Sem- 2 enrichment culture (results from only one of the two enrichment bottles are shown; results from the other bottle were similar).
In order to determine conclusively that benzene was mineralized to CO2, microcosms were set up for each of the anaerobic soils obtained from the Seminole county. Also, microcosms were setup using the Sem-2 enrichment to test if the culture mineralized the added benzene to CO2. The microcosms were prepared with 10 g soil and 40 g mineral salts medium containing 2.5 M NaCl. Each bottle was amended with 14C-benzene and incubated under anaerobic conditions for 0, 4, 8, or 12 weeks. The bottles were analyzed for 14C-CO2. As seen in Table 1, at the end of three months of incubation, about 5 to 10% of the radiolabled benzene was mineralized over the control bottles in all of the anaerobic soil samples obtained from the Seminole county. However, no degradation was seen under same growth conditions in soil obtained from the Stephens County, OK. Although, the removal of benzene under anaerobic conditions seem poor, the rate and extent of degradation could be enhanced under more optimal growth conditions. Results show that significant amount of benzene was mineralized by the Sem- 2 enrichment culture.
Table 1. Mineralization of 14C-benzene
| Site | Soil Type | Time incubated | Percent¹ 14C-benzene Mineralized to 14C-CO2 |
| Sem-1 | Anaerobic | > 12 weeks | 5.93 ± 0.94 |
| Sem-2 | Aerobic | 4 weeks | 46.83 ± 14.09 |
| Sem-3 | Anaerobic | 12 weeks | 10.16 ± 5.87 |
| Sem-4 | Anaerobic | 12 weeks | 4.6 ± 0.12 |
| Steph | Anaerobic | 12 weeks | < 1% |
Microcosms were also established to evaluate the effects of different osmolytes on the rate of benzene degradation. Our data reveal that none of the tested osmolytes showed enhanced degradation activity. On the contrary, the rates are lower than when no osmolytes was present, as seen in Table 2. The exact reason for the lack of stimulation in the presence of tested osmolytes is not known. However, it is possible that perhaps bacteria were able to synthesize their own osmolytes. Alternatively, the tested osmolytes were not preferred by halophiles present in the soil. Our studies also showed that the addition of 0.02% yeast extract markedly enhance the benzene degradation. These results are very encouraging from the point of bioremediation of oilbrine sites.
Table 2. Effect of Osmolytes on Benzene Degradation
| Additive Concentration | % Degradation | # of days | |
| Autoclaved Control | NA* | 43.50 ± 12.95 | 61 |
| No osmolyte | NA | 97.77 ± 0.87 | 12 |
| Glycine | 1M | 62.69 ± 0.22 | 61 |
| Proline | 1M | 42.30 ± 4.20 | 61 |
| Betain | 1M | 67.25 ± 2.38 | 61 |
| KCl | 1M | 56.41 ± 2.11 | 61 |
| Yeast Extract | 0.02% | 97.92 ± 1.87 | 12 |
| Aspartic Acid | 0.1M | 51.41 ± 3.37 | 61 |
| Glutamic Acid | 0.1M | 61.32 ± 0.46 | 61 |
Microcosms will be set up to explore the degradation of the related petroleum hydrocarbons including toluene, ethylbenzene, and o-, m-, and p- xylene by the Sem-2 enrichment cultures.
Other work with the enrichment culture includes determining its ability to survive and degrade BTEX at varied NaCl concentrations. In addition, microbial diversity and the dominant populations present in the Sem-2 enrichment will be characterized using the 16S rDNA and DGGE (denaturing gradient gel electrophoresis) techniques. The DNA bands from the dominant populations can then be excised and sequenced. Databases, such as BLAST, can then be used to identify the microbes or related microbes, if they are known.
bioremediation, halophilic, halotolerant, benzene, Sem-2 enrichment culture, 14C-benzene, osmolytes