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Unformatted text preview: A PPLIED AND ENVIRONMENTAL MICROBIOLOGY, Mar. 2004, p. 17771786 Vol. 70, No. 3 0099-2240/04/$08.00 1 0 DOI: 10.1128/AEM.70.3.17771786.2004 Copyright 2004, American Society for Microbiology. All Rights Reserved. Bacterial Succession in a Petroleum Land Treatment Unit Christopher W. Kaplan and Christopher L. Kitts* Environmental Biotechnology Institute, California Polytechnic State University, San Luis Obispo, California 93407 Received 14 July 2003/Accepted 10 December 2003 Bacterial community dynamics were investigated in a land treatment unit (LTU) established at a site contaminated with highly weathered petroleum hydrocarbons in the C 10 to C 32 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. A bioremediation project was undertaken at the Guadalupe oil field, which occupies nearly 2,700 acres of the larger Guada- lupe-Nipomo Dune Complex and is located on the central California coast in San Luis Obispo and Santa Barbara Coun- ties. 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, con- taminated soil was stockpiled for eventual cleanup. Prior to treatment, the stockpiled soil contained an average total pe- troleum hydrocarbon (TPH) concentration of 2,440 mg per kg....
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