Appl Environ Microbiol 70(3): 1777-1786

Bacterial Succession in a Petroleum Land Treatment Unit

Environmental Biotechnology Institute, California Polytechnic State University, San Luis Obispo, California 93407

^{Corresponding author. Mailing address: Environmental Biotechnology Institute, California Polytechnic State University, San Luis Obispo, CA 93407. Phone: (805) 756-2949. Fax: (805) 756-1419. E-mail:}ude.yloplac@sttikc.

Received 2003 Jul 14; Accepted 2003 Dec 10.

Abstract

Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C₁₀ to C₃₂ range. The treatment plot, 3,000 cubic yards of soil, was supplemented with nutrients and monitored weekly for total petroleum hydrocarbons (TPH), soil water content, nutrient levels, and aerobic heterotrophic bacterial counts. Weekly soil samples were analyzed with 16S rRNA gene terminal restriction fragment (TRF) analysis to monitor bacterial community structure and dynamics during bioremediation. TPH degradation was rapid during the first 3 weeks and slowed for the remainder of the 24-week project. A sharp increase in plate counts was reported during the first 3 weeks, indicating an increase in biomass associated with petroleum degradation. Principal components analysis of TRF patterns revealed a series of sample clusters describing bacterial succession during the study. The largest shifts in bacterial community structure began as the TPH degradation rate slowed and the bacterial cell counts decreased. For the purpose of analyzing bacterial dynamics, phylotypes were generated by associating TRFs from three enzyme digests with 16S rRNA gene clones. Two phylotypes associated with Flavobacterium and Pseudomonas were dominant in TRF patterns from samples during rapid TPH degradation. After the TPH degradation rate slowed, four other phylotypes gained dominance in the community while Flavobacterium and Pseudomonas phylotypes decreased in abundance. These data suggest that specific phylotypes of bacteria were associated with the different phases of petroleum degradation in the LTU.

Abstract

A bioremediation project was undertaken at the Guadalupe oil field, which occupies nearly 2,700 acres of the larger Guadalupe-Nipomo Dune Complex and is located on the central California coast in San Luis Obispo and Santa Barbara Counties. Due to the viscous nature of the oil at the site a light petroleum distillate, referred to as diluent, was pumped into the wells to thin the oil for more efficient removal. This diluent was inadvertently released into the environment as pipes and storage tanks began to degrade. During site remediation, contaminated soil was stockpiled for eventual cleanup. Prior to treatment, the stockpiled soil contained an average total petroleum hydrocarbon (TPH) concentration of 2,440 mg per kg. A 3,000-cubic yard land treatment unit (LTU) was set up to investigate the feasibility of bioremediation at the site. Soil at the site is coastal dune sand and contained negligible carbon, nitrogen, and phosphorous. Therefore, basic nutrients consisting of phosphate and ammonia were added to obtain a C/N/P ratio of 100:6:1, and soil was periodically watered and tilled to a depth of 18 in. to aerate and mix nutrients. Details of LTU construction and maintenance were listed by Kaplan et al. (19).

Land treatment, an alluring method of remediation due to its effectiveness, low cost, and minimal environment impact, is a form of bioremediation whereby autochthonous soil bacteria degrade undesirable environmental waste. Three types of bioremediation are predominant in the industry today: natural attenuation, biostimulation, and bioaugmentation. The simplest method of bioremediation to implement is natural attenuation, where contaminated sites are only monitored for contaminant concentration to assure regulators that natural processes of contaminant degradation are active. Biostimulation is the process of providing bacterial communities with a favorable environment in which they can effectively degrade contaminants. When nutrients are low and the speed of contaminant degradation is an issue, the addition of nitrogen and phosphorous as well as aeration of soil will speed up the bioremediation process (17, 32, 34). In cases where natural communities of degrading bacteria are at low levels or not present, the addition of contaminant-degrading organisms, known as bioaugmentation, can speed up the process (3). Although significant research is being performed in this area, bioaugmentation is generally not practiced, since introduced bacteria usually can't compete with well-adapted autochthonous bacterial communities (24).

Many studies have looked at the chemical degradation process associated with land treatment (2, 4, 29). A common phenomenon in land treatment is a two-phase pattern of degradation characterized by an initial fast degradation phase followed by a slow degradation phase. To explain the change in degradation rates, it has been suggested that the initial fast degradation phase is mediated by bacterial utilization of bioavailable compounds and is governed by enzyme kinetics. In contrast, the slow phase may be governed by the rate of petroleum dissolution from soil particles (2, 4).

Although significant work has been published discussing the bacterial community structure and degradation kinetics associated with bioremediation of environmental contaminants, few have focused on a detailed description of bacterial community dynamics during this process. A recent report described the structure and dynamics of bacterial communities involved in bioremediation of crude oil (24). In this study, a few groups of bacteria were observed to increase in abundance in response to oil contamination, but the paucity of samples analyzed left gaps during the first 3 weeks, when key events in bioremediation are known to occur (2, 4).

Because the fast degradation phase is where most petroleum is degraded, determining the key bacteria in this phase compared to the later, slower phase of degradation is important to a complete understanding of the degradation process. In this study, we present the characterization of an autochthonous bacterial community capable of degrading petroleum hydrocarbons after biostimulation by aeration and the addition of nitrogen and phosphorous nutrients. The sampling frequency was increased compared to other studies to better understand how the bacterial community changed during land treatment. A combination of 16S rRNA gene terminal restriction fragment (TRF; also known as TRF length polymorphism [T-RFLP]) analysis of bacterial communities using multiple enzymes and a clone library constructed from study samples allowed monitoring of relative bacterial abundance and the identification of bacterial phylotypes associated with the phases of TPH degradation.

Acknowledgments

This work was supported by the generous contributions of the UNOCAL Corporation, whom we thank for the opportunity to conduct research at their site and for the support of their staff in accomplishing the common goal of a cleaner environment.

We also thank everyone at the Environmental Biotechnology Institute, past and present, who contributed to successful completion of the LTU project.

Acknowledgments

REFERENCES

References

1. Abed, R. M. M., N. M. D. Safi, J. Köster, K. de Beer, Y. El-Nahhal, J. Rullkötter, and F. Garcia-Pichel. 2002. Microbial diversity of a heavily polluted microbial mat and its community changes following degradation of petroleum compounds. Appl. Environ. Microbiol.68:1674-1683.
2. Admon, S., M. Green, and Y. Avnimelech. 2001. Biodegradation kinetics of hydrocarbons in soil during land treatment of oily sludge. Bioremediat. J.5:193-209. [PubMed]
3. Al-Awadhi, N., R. Al-Daher, A. ElNavavy, and M. T. Balba. 1996. Bioremediation of oil-contaminated soil in Kuwait: landfarming to remediate oil-contaminated soil. J. Soil Contam.5:243-260. [PubMed]
4. Alexander, M. 2000. Aging, bioavailability, and overestimation of risk from environmental pollutants. Environ. Sci. Technol.34:4259-4265. [PubMed]
5. Atlas, R. M., and R. Bartha. 1972. Degradation and mineralization of petroleum by two bacteria isolated from coastal waters. Biotechnol. Bioengin.14:297-308. [[PubMed]
6. Baggi, G., and MZangrossi. 1999. Degradation of chlorobenzoates in soil suspensions by indigenous populations and a specialized organism: interaction between growth and non-growth substrates. FEMS Microbiol. Ecol.29:311-318. [PubMed][Google Scholar]
7. Busse, H. J., P. Kampfer, E. R. Moore, J. Nuutinen, I. V. Tsitko, E. B. M. Denner, L. Vauterin, M. Valens, R. Rossello-Mora, and M. S. Salkinoja-Salonen. 2002. Thermomonas haemolytica gen. nov., sp. nov., a γ-proteobacterium from kaolin slurry. Int. J. Syst. Bacteriol.52:473-483. [[PubMed]
8. Carvalho, M. F., C. C. T. Alves, M. I. M. Ferreira, P. De Marco, and P. M. L. Castro. 2002. Isolation and initial characterization of a bacterial consortium able to mineralize fluorobenzene. Appl. Environ. Microbiol.68:102-105.
9. Chakrabarty, A. M., G. Chou, and I. C. Gunsalus. 1973. Genetic regulation of octane dissimulation plasmids in Pseudomonas. Proc. Natl. Acad. Sci. USA70:1137-1140.
10. Chaudhry, G. R., and G. H. Huang. 1998. Isolation and characterization of a new plasmid from a Flavobacterium sp. which carries the genes for degradation of 2,4-dichlorophenoxyacetate. J. Bacteriol.170:3897-3902.
11. Clement, B. G., L. E. Kehl, K. L. DeBord, and C. L. Kitts. 1998. Terminal restriction fragment patterns (TRFPs), a rapid, PCR-based method for the comparison of complex bacterial communities. J. Microbiol. Methods31:135-142. [PubMed]
12. Esteve-Nunez, A., A. Caballero, and J. L. Ramos. 2001. Biological degradation of 2,4,6-trinitrotoluene. Microbiol. Mol. Biol. Rev.65:335-352.
13. Felsenstein, J. 1989. PHYLIP—phylogeny inference package (version 3.2). Cladistics5:164-166. [PubMed]
14. Fries, M. R., J. Zhou, J. Chee-Sanford, and J. M. Tiedje. 1994. Isolation, characterization, and distribution of denitrifying toluene degraders from a variety of habitats. Appl. Environ. Microbiol.60:2802-2810.
15. Greene, E. A., J. G. Kay, K. Jaber, L. G. Stehmeier, and G. Voodouw. 2000. Composition of soil microbial communities enriched on a mixture of aromatic hydrocarbons. Appl. Environ. Microbiol.66:5282-5289.
16. Horn, M., M. Wagner, K.-D. Muller, E. N. Schmid, T. R. Fritsche, K.-H. Schleifer, and R. Michel. 2000. Neochlamydia hartmannellae gen. nov, sp. nov. (Parachlamydiaceae), an endoparasite of the amoeba Hartmannella vermiformis. Microbiology146:1231-1239. [[PubMed]
17. Huesemann, M. H., and M. J. Truex. 1996. The role of oxygen diffusion in passive bioremediation of petroleum contaminated soils. J. Hazard. Mater.51:93-113. [PubMed]
18. Kaplan, C. W., J. C. Astaire, M. E. Sanders, B. S. Reddy, and C. L. Kitts. 2001. 16S ribosomal DNA terminal restriction fragment pattern analysis of bacterial communities in feces of rats fed Lactobacillus acidophilus NCFM. Appl. Environ. Microbiol.67:1935-1939.
19. Kaplan, C. W., B. G. Clement, A. Hamrick, R. W. Pease, C. Flint, R. J. Cano, and C. L. Kitts. 2003. Complex co-substrate addition increases initial petroleum degradation rates during land treatment by altering bacterial community physiology. Remediation13:61-78. [PubMed]
20. Kaplan, C. W., and C. L. Kitts. 2003. Variation between observed and true terminal restriction fragment (TRF) length is dependent on true TRF length and purine content. J. Microbiol. Methods54:121-125. [[PubMed]
21. Kato, K., K. Ohtsuki, Y. Koda, T. Maekawa, T. Yomo, S. Negoro, and I. Urabe. 1995. A plasmid encoding enzymes for nylon oligomer degradation: nucleotide sequence and analysis of pOAD2. Microbiology141:2585-2590. [[PubMed]
22. Kitts, CL. 2001. Terminal restriction fragment patterns: a tool for comparing microbial communities and assessing community dynamics. Curr. Issues Intest. Microbiol.2:17-25. [[PubMed][Google Scholar]
23. Lai, B., and SKhanna. 1996. Degradation of crude oil by Acintobacter calcoaceticus and Alcaligenes oderans. J. Appl. Bacteriol.81:355-362. [[PubMed][Google Scholar]
24. MacNaughton, S. J., J. R. Stephen, A. D. Venosa, G. A. Davis, Y. Chang, and D. C. White. 1999. Microbial population changes during bioremediation of an experimental oil spill. Appl. Environ. Microbiol.65:3566-3574.
25. Maidak, B. L., J. R. Cole, T. G. Lilburn, C. T. Parker, Jr., P. R. Saxman, R. J. Farris, G. M. Garrity, G. J. Olsen, T. M. Schmidt, and J. M. Tiedje. 2001. The RDP-II (Ribosomal Database Project). Nucleic Acids Res.29:173-174.
26. Mannisto, M. K., M. A. Tiirola, M. S. Salkinoja-Salonen, M. S. Kulomaa, and J. A. Puhakka. 1999. Diversity of chlorophenol-degrading bacteria isolated from contaminated boreal groundwater. Arch. Microbiol.171:189-197. [[PubMed]
27. Mulbry, W. W., P. C. Kearney, J. O. Nelson, and J. S. Karns. 1987. Physical comparisons of parathion hydrolase plasmids from Pseudomonas diminuta and Flavobacterium sp. Plasmid18:173-177. [[PubMed]
28. Nalin, R., P. Simonet, T. M. Vogel, and P. Normand. 1999. Rhodanobacter lindaniclasticus gen. nov., sp. nov., a lindane-degrading bacterium. Int. J. Syst. Bacteriol.49:19-23. [[PubMed]
29. Olivera, F. L., R. C. Loehr, B. C. Coplin, H. Eby, and M. T. Webster. 1998. Prepared bed land treatment of soils containing diesel and crude oil hydrocarbons. J. Soil Contam.7:657-674. [PubMed]
30. Pelz, O., A. Chatzinotas, N. Anderson, S. M. Bernasconi, C. Hesse, W.-R. Abraham, and J. Zeyer. 2001. Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations. Arch. Microbiol.175:270-281. [[PubMed]
31. Rojas-Avelizapa, N. G., R. Rodriguez-Vazquez, R. Enriquez-Villanueva, J. Martinez-Cruz, and H. M. Poggi-Varaldo. 1999. Transformer oil degradation by an indigenous microflora isolated from a contaminated soil. Resour. Conserv. Recycl.27:15-26. [PubMed]
32. Salanitro, J. P., P. B. Dorn, M. H. Huesemann, K. O. Moore, I. A. Rhodes, L. M. Rice Jackson, T. E. Vipond, M. M. Western, and H. L. Wisniewski. 1997. Crude oil hydrocarbon bioremediation and soil ecotoxicity assessment. Environ. Sci. Technol.31:1769-1776. [PubMed]
33. Thompson, J. D., D. G. Higgins, and T. J. Gibson. 1994. CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucleic Acids Res.22:4673-4680.
34. Venosa, A. D., M. T. Suidan, B. A. Wrenn, K. L. Strohmeier, J. R. Haines, B. L. Eberhart, D. King, and E. Holder. 1996. Bioremediation of an experimental oil spill on the shoreline of Delaware Bay. Environ. Sci. Technol.30:1764-1775. [PubMed]
35. Whiteley, A. S., and M. J. Bailey. 2000. Bacterial community structure and physiological state within an industrial phenol bioremediation system. Appl. Environ. Microbiol.66:2400-2407.
36. Yeom, S.-H., and A. J. Daugulis. 2001. Benzene degradation in a two-phase partitioning bioreactor by Alcaligenes xylosoxidans Y234. Process Biochem.36:765-772. [PubMed]