There are exploration and production (EP) sites in Oklahoma that are contaminated with both oil and salt. This poses a problem in cleaning up these sites through bioremediation since the harsh conditions will not support externally added bacteria. The purpose of this project is to use the native halophilic and halotolerant bacteria for the remediation of these sites. This would provide a cost-effective approach for cleaning up oil at these EP sites that have been contaminated with high concentrations of salt.
This project was initiated with soil samples obtained from five contaminated sites in Oklahoma including Stephens and Seminole counties. They are labeled Stephens, Sem-1, Sem-2, Sem-3, and Sem-4. Table 1 shows analytical results for benzene, toluene, ethylbenzene, total petroleum hydrocarbon (TPH), and chloride for each oil-brine soil used in this study.
| Soil Analysis* | Method (EPA) |
Stephens | Sem-1 | Sem-2 | Sem-3 | Sem-4 |
| Sample depth (ft) | 0-1.5 | 4 | 0-3 | 4 | 4 | |
| Benzene (mg/Kg) | 8021B | BDL | 4.48 | 3.41 | 1.72 | 0.149 |
| Toluene (mg/Kg) | 8021B | 0.241 | 9.72 | 10.4 | 7.48 | 0.715 |
| Ethylbenzene (mg/Kg) | 8021B | BDL | 30.2 | 7.95 | 14.1 | 3.04 |
| TPH (mg/Kg) | 8015M | 125580 | 72744 | 6071 | 25198 | 120276 |
| Chloride (mg/Kg) | 325.3 | 1270 | 42200 | 14800 | 3740 | 220 |
Since, Sem-2 has considerably less TPH, it is being used for evaluating aerobic degradation of benzene and naphthalene, while soil from the other four sites are being used to assess biodegradation under anaerobic conditions.
Microcosms were prepared using 70-ml capacity serum bottles containing 10 g (wet weight) soil and 40-ml mineral salts medium (MSM) containing high concentrations of NaCl (2.5 M). The bottles for the aerobic studies were supplemented with 100 mg of magnesium peroxide (MgO2) as a source of oxygen. Anaerobic microcosms were prepared similarly in an anaerobic glove box filled with N2. Each bottle was spiked with 100 µL stock 14C-benzene (specific activity of 14C-benzene = 33.2 mCi/mmole). This amounts to 27500 dpm/ml. The bottles were closed with Teflon-coated septa and aluminum caps and are being incubated static in the dark. At the end of 4-6 weeks, triplicate active and duplicate control bottles were sacrificed and stored at -200C until analyzed for radioactive CO2. Microcosms were also prepared as described above to evaluate the effect of osmolytes on the rate of biodegradation of 14C-benzene under aerobic and anaerobic conditions.
Currently we are modifying GC and HPLC analytical methods to detect both parent and water-soluble degradation products produced under aerobic and anaerobic conditions.
Microcosms will also be established using the oil-brine soil to evaluate the biodegradation of 14C-naphthalene under aerobic and anaerobic conditions. In addition, the stimulatory of effect of osmolytes will also studied.
Evidence for complete mineralization of radiolabeled benzene or naphthalene will be assayed by measuring the production 14CO2 from 14C-radiolabled benzene or naphthalene.
bioremediation, halophilic, halotolerant, benzene, naphthalene