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To be presented at 1999 Petroleum Hydrocarbons Conference, Houston, Texas the last two sample times. No significant loss of hexane or MTBE was noted in the sterile controls. In the isopentane systems (Figure 4), no significant degradation was noted in the contaminated systems. Rapid degradation of both isopentane and MTBE was observed in the background systems. Isopentane was completely mineralized between day 1 and 12, and MTBE was not detected after day 12. Cyclohexane was degraded very slowly in the contaminated soils, but no cometabolic degradation of MTBE was detectable (Figure 5). Cyclohexane was also slowly degraded in the background sediments, and some MTBE 9000 8000 7000 6000 n 5000 4000 G 3000 2000 1000 0 cyclohexane - MtBE systems - �- - cyclohexane - bg _ 0 MtBE - bg cyclohexane - con El-_ MtBE - con *- _ cyclohexane - sc _ *-_ MtBE - sc 10 20 30 40 50 60 70 80 90 elapsed time (days) Figure 5. Concentration history of cyclohexane and MTBE in sacrificed batch microcosms. bg = background sediments, con = contaminated sediments, sc = sterilized controls. cometabolism may be occurring but the decrease in concentration is not significantly different from the sterile controls to make a strong argument. It is possible that the slow rate of cyclohexane degradation did not result in sufficient biomass growth to allow cometabolism or that the appropriate enzyme was not expressed. In either case, cyclohexane does not appear to be a good primary substrate candidate for cometabolism in the VAFB sediments. Enumeration and Growth Studies After completion of these batch tests, the last set of vials from the contaminated soil systems demonstrating bioactivity were sent to North Carolina State University (NCSU) for further examination. The aim was to determine the numbers of microorganisms in the microcosms and whether differences in these numbers could account for the variations in cometabolic activity observed in the microcosm samples. The bacterial numbers were established using conventional serial dilution and spread plate techniques. Samples (-5 g) of microcosm sediment was mixed with sterile mineral salts medium (10 mL) in sterile plastic tubes (50 mL). The tubes were shaken and then briefly (3 x 15 s) immersed in an ultrasonic water bath. The microcosm sediment was then allowed to settle and samples (100 pL) of the supernatant were then transferred into sterile plastic tubes containing mineral salts medium. The samples were then serially diluted to a final dilution of 104. Samples of each dilution (100 pL) were spread on mineral salts agar plates and incubated in black plastic paint pots (5 L). The pots contained a small conical flask (100 mL) in which pure hydrocarbon growth substrates (n -hexane or iso -pentane) were added. The plates were incubated in the pots for 8 days at room temperature. The colony forming units (cfus) were then visually scored and counted. Three separate serial dilutions were made for each microcosm sample and three spread plates were generated for each sample. The microbial numbers reported below represent average values obtained from plates containing between 30-300 clearly resolvable microbial Page 7 4-1 - �- - cyclohexane - bg _ 0 MtBE - bg cyclohexane - con El-_ MtBE - con *- _ cyclohexane - sc _ *-_ MtBE - sc 10 20 30 40 50 60 70 80 90 elapsed time (days) Figure 5. Concentration history of cyclohexane and MTBE in sacrificed batch microcosms. bg = background sediments, con = contaminated sediments, sc = sterilized controls. cometabolism may be occurring but the decrease in concentration is not significantly different from the sterile controls to make a strong argument. It is possible that the slow rate of cyclohexane degradation did not result in sufficient biomass growth to allow cometabolism or that the appropriate enzyme was not expressed. In either case, cyclohexane does not appear to be a good primary substrate candidate for cometabolism in the VAFB sediments. Enumeration and Growth Studies After completion of these batch tests, the last set of vials from the contaminated soil systems demonstrating bioactivity were sent to North Carolina State University (NCSU) for further examination. The aim was to determine the numbers of microorganisms in the microcosms and whether differences in these numbers could account for the variations in cometabolic activity observed in the microcosm samples. The bacterial numbers were established using conventional serial dilution and spread plate techniques. Samples (-5 g) of microcosm sediment was mixed with sterile mineral salts medium (10 mL) in sterile plastic tubes (50 mL). The tubes were shaken and then briefly (3 x 15 s) immersed in an ultrasonic water bath. The microcosm sediment was then allowed to settle and samples (100 pL) of the supernatant were then transferred into sterile plastic tubes containing mineral salts medium. The samples were then serially diluted to a final dilution of 104. Samples of each dilution (100 pL) were spread on mineral salts agar plates and incubated in black plastic paint pots (5 L). The pots contained a small conical flask (100 mL) in which pure hydrocarbon growth substrates (n -hexane or iso -pentane) were added. The plates were incubated in the pots for 8 days at room temperature. The colony forming units (cfus) were then visually scored and counted. Three separate serial dilutions were made for each microcosm sample and three spread plates were generated for each sample. The microbial numbers reported below represent average values obtained from plates containing between 30-300 clearly resolvable microbial Page 7