Ravi Kolhatkar

Analysis of a Microbial Community Oxidizing Inorganic Sulfide and Mercaptans

Successful treatment of refinery spent‐sulfidic caustic (which results from the addition of sodium hydroxide solutions to petroleum refinery waste streams) was achieved in a bioreactor containing an enrichment culture immobilized in organic polymer beads with embedded powdered activated carbon (Bio‐Sep). The aerobic enrichment culture had previously been selected using a gas mixture of hydrogen sulfide and methyl mercaptan (MeSH) as the sole carbon and energy sources. The starting cultures for the enrichment consisted of several different Thiobacillispp. (T. thioparus, T. denitrificans, T. thiooxidans, and T. neopolitanus), as well as activated sludge from a refinery aerobic wastewater treatment system and sludge from an industrial anaerobic digester. Microscopic examination (light and SEM) of the beads and of microbial growth on the walls of the bioreactor revealed a great diversity of microorganisms. Further characterization was undertaken starting with culturable aerobic heterotrophic microorganisms (sequencing of PCR‐amplified DNA coding for 16S rRNA, Gram staining) and by PCR amplification of DNA coding for 16S rRNA extracted directly from the cell mass, followed by the separation of the PCR products by DGGE (denaturing gradient gel electrophoresis). Eight prominent bands from the DGGE gel were sequenced and found to be closest to sequences of uncultured Cytophagales (3 bands),Gram‐positive cocci (Micrococcineae), α proteobacteria (3 bands), and an unidentified β proteobacterium. Culturable microbes included several genera of fungi as well as various Gram‐positive and Gram‐negative heterotrophic bacteria not seen in techniques using direct DNA extraction.

Biotreatment of Refinery Spent-Sulfidic Caustic Using an Enrichment Culture Immobilized in a Novel Support Matrix

Sodium hydroxide solutions are used in petroleum refining to remove hydrogen sulfide (H2S) and mercaptans from various hydrocarbon streams. The resulting sulfide-laden waste stream is called spent-sulfidic caustic. An aerobic enrichment culture was previously developed using a gas mixture of H2S and methylmercaptan (MeSH) as the soleenergy source. This culture has now been immobilized in a novel support matrix, DuP ont BIO-SEPTM beads, and is used to biotreat a refinery spent-sulfidic caustic containing both inorganic sulfide and mercaptans in a continuous flow, fluidized-bed column bioreactor. Complete oxidation of both inorganic and organic sulfur to sulfate was observed with no breakthrough of H2S and <2 ppmv of MeSH produced in the bioreactor outlet gas. Excessive buildup of sulfate (>12 g/L) in the bioreactor medium resulted in an upset condition evidenced by excessive MeSH breakthrough. Therefore, bioreactor performance was limited by the steady-state sulfate concentration. Further improvement in volumetric productivity of a bioreactor system based on this enrichment culture will be dependent on maintenance of sulfate concentrations below inhibitory levels.

Diagnostic Tools to Assess Mass Removal Processes During Pulsed Air Sparging of a Petroleum Hydrocarbon Source Zone

During remediation of contaminated aquifers, diagnostic tools can help evaluate whether an intended mass removal process was successfully initiated and acted on specific contaminants of concern. In this study, several diagnostic tools were tested in a controlled-release in situ air sparging experiment that focused on the treatment of target hydrocarbons (e.g., benzene, toluene, ethylbenzene, and xylenes). The tools included compound-specific isotope analysis (CSIA), expression of functional genes (mRNA), and metabolites characteristic of aerobic and anaerobic biodegradation. Total and compound-specific mass balances were established and used, along with traditional monitoring parameters, to validate the results from the various tools. CSIA results indicated biodegradation as the main process contributing to benzene and toluene removal. Removal process-specific isotope shifts were detected in groundwater as well as in the system effluent gas. CSIA, metabolite, and mRNA biomarkers consistently indicated that both aerobic and anaerobic biodegradation of benzene and toluene occurred, but that their relative importance evolved over time and were related to the treatment system operation. While the indicators do not allow quantif cation of the mass removed, they are particularly useful to identify if a removal process has been initiated, and to track relative changes in the predominance of in situ contaminant attenuation processes resulting from remediation efforts.