The goal of this study was to further develop an incubation-quantitative polymerase chain reaction (qPCR) method for quantifying viable Ascaris eggs by characterizing the detection limit and number of template copies per egg, determining the specificity of the method, and testing the method with viable and inactivated larvated eggs. The number of template copies per cell was determined by amplifying DNA from known numbers of eggs at different development stages; the value was estimated to be 32 copies. The specificity of the method was tested against a panel of bacteria, fungi, protozoa and helminths, and no amplification was found with non-target DNA. Finally, fully larvated eggs were inactivated by four different treatments: 254 nm ultraviolet light, 2,000 ppm NH 3 -N at pH 9, moderate heat (48 W C) and high heat (70 W C). Concentrations of treated eggs were measured by direct microscopy and incubation-qPCR. The qPCR signal decreased following all four treatments, and was in general agreement with the decrease in viable eggs determined by microscopy. The incubation-qPCR method for enumerating viable Ascaris eggs is a promising approach that can produce results faster than direct microscopy, and may have benefits for applications such as assessing biosolids.

This study explores the effect of microbial consortium composition and reactor configuration on methyl tert-butyl ether (MTBE) biodegradation in the presence of benzene, toluene, ethylbenzene and p-xylenes(BTEX). MTBE biodegradation was monitored in the presence and absence of BTEX in duplicate batch reactors inoculated with distinct enrichment cultures: MTBE only (MO-originally enriched on MTBE) and/or MTBE BTEX (MB-originally enriched on MTBE and BTEX). The MO culture was also applied in a semi-batch reactor which received both MTBE and BTEX periodically in fresh medium after allowing cells to settle. The composition of the microbial consortia was explored using a combination of 16S rRNA gene cloning and quantitative polymerase chain reaction targeting the known MTBE-degrading strain PM1T. MTBE biodegradation was completely inhibited by BTEX in the batch reactors inoculated with the MB culture, and severely retarded in those inoculated with the MO culture (0.18+/-0.04 mg/L-day). In the semi-batch reactor, however, the MTBE biodegradation rate in the presence of BTEX was almost three times as high as in the batch reactors (0.48+/-0.2 mg/L-day), but still slower than MTBE biodegradation in the absence of BTEX in the MO-inoculated batch reactors (1.47+/-0.47 mg/L-day). A long lag phase in MTBE biodegradation was observed in batch reactors inoculated with the MB culture (20 days), but the ultimate rate was comparable to the MO culture (0.95+/-0.44 mg/L-day). Analysis of the cultures revealed that strain PM1T concentrations were lower in cultures that successfully biodegraded MTBE in the presence of BTEX. Also, other MTBE degraders, such as Leptothrix sp. and Hydrogenophaga sp. were found in these cultures. These results demonstrate that MTBE bioremediation in the presence of BTEX is feasible, and that culture composition and reactor configuration are key factors.

The aim of this study was to enrich cultures capable of anaerobic methyl tert-butyl ether (MTBE) biodegradation and to determine their compositions using biomolecular tools. MTBE biodegradation as the sole carbon source was observed under Fe(III) and SO42–reducing conditions and with both electron acceptors present together. The estimated MTBE biodegradation rates ranged from 3.80 to 9.20 mg·L–1·d–1when Fe(III) was the sole electron acceptor, from 1.46 to 1.70 mg·L–1·d–1when Fe(III) and SO42–were present together, and from 1.13 to 1.71 mg·L–1·d–1when SO42–was the sole electron acceptor. Five to eight members were identified in the three consortia, and their characteristics were congruent with the electron acceptor conditions. A clone 99% similar to a recently described MTBE degrader, Ochrobactrum cytisi, was detected in both cultures containing Fe(III). Other 16S rRNA gene sequences detected were highly similar at the species or genus level to additional known MTBE degraders, including Pseudomonas spp. (cometabolic), Sphinogomonas, Achromobacter, and Rhodococcus. Results suggest that the buildup of intermediates and (or) the presence of sulfides had an inhibitory effect on biodegradation. Anaerobic MTBE biodegradation remains poorly understood, and degrading strains still have not been identified to date. This work represents the first detailed 16S rRNA gene profiling of highly enriched iron- and sulfate-reducing MTBE-degrading consortia. These results may provide useful biomarkers to support current efforts for confirming anaerobic MTBE biodegradation in the field.

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