Application of denaturing high-performance liquid chromatography for monitoring sulfate-reducing bacteria in oil fields.

Sulfate-reducing bacteria (SRB) participate in microbially induced corrosion (MIC) of equipment and H2S-driven reservoir souring in oil field sites. Successful management of industrial processes requires methods that allow robust monitoring of microbial communities. This study investigated the applicability of denaturing high-performance liquid chromatography (DHPLC) targeting the dissimilatory sulfite reductase ß-subunit (dsrB) gene for monitoring SRB communities in oil field samples from the North Sea, the United States, and Brazil. Fifteen of the 28 screened samples gave a positive result in real-time PCR assays, containing 9 × 10(1) to 6 × 10(5) dsrB gene copies ml(-1). DHPLC and denaturing gradient gel electrophoresis (DGGE) community profiles of the PCR-positive samples shared an overall similarity; both methods revealed the same samples to have the lowest and highest diversity. The SRB communities were diverse, and different dsrB compositions were detected at different geographical locations. The identified dsrB gene sequences belonged to several phylogenetic groups, such as Desulfovibrio, Desulfococcus, Desulfomicrobium, Desulfobulbus, Desulfotignum, Desulfonatronovibrio, and Desulfonauticus. DHPLC showed an advantage over DGGE in that the community profiles were very reproducible from run to run, and the resolved gene fragments could be collected using an automated fraction collector and sequenced without a further purification step. DGGE, on the other hand, included casting of gradient gels, and several rounds of rerunning, excising, and reamplification of bands were needed for successful sequencing. In summary, DHPLC proved to be a suitable tool for routine monitoring of the diversity of SRB communities in oil field samples.

Biotests for environmental quality assessment of composted sewage sludge.

The quality of sewage sludge-based products, such as composts and growth media, is affected by the contamination of sewage sludge with, potentially, hundreds of different substances. Therefore, it is difficult to achieve the reliable environmental quality assessment of sewage sludge-based products solely based on chemical analysis. In the present work, we demonstrate the use of the kinetic luminescent bacteria test (ISO 21338) to evaluate acute toxicity and the Vitotox™ test to monitor genotoxicity of sewage sludge and composted sewages sludge. In addition, endocrine-disrupting and dioxin-like activity was studied using yeast-cell-based assays. The relative contribution of industrial waste water treated at the Waste Water Treatment Plants led to elevated concentrations of polyaromatic hydrocarbons (PAHs), polychlorinated biphenyls (PCBs), and polychlorinated dibenzo-p-dioxins and -furans (PCDD/F) in sewage sludge. The effect of elevated amounts of organic contaminants could also be identified with biotests able to demonstrate higher acute toxicity, genotoxicity, and potential for endocrine-disruptive properties. Additional extraction steps in kinetic luminescent bacteria test with DMSO and hexane increased the level of toxicity detected. Composting in a pilot-scale efficiently reduced the amounts of linear alkylbenzensulphonates (LASs), nonylphenols and nonylphenolethoxylates (NPE/NPs) and PAH with relative removal efficiencies of 84%, 61% and 56%. In addition, decrease in acute toxicity, genotoxicity and endocrorine-disrupting and dioxin-like activity during composting could be detected. However, the biotests did have limitations in accessing the ecotoxicity of test media rich with organic matter, such as sewage sludge and compost, and effects of sample characteristics on biotest organisms must be acknowledged. The compost matrix itself, however, which contained a high amount of nutrients, bark, and peat, reduced the sensitivity of the genotoxicity tests and yeast bioreporter assays.

Characterization of bacterial diversity to a depth of 1500 m in the Outokumpu deep borehole, Fennoscandian Shield.

This paper demonstrates the first microbiological sampling of the Outokumpu deep borehole (2516 m deep) aiming at characterizing the bacterial community composition and diversity of sulphate-reducing bacteria (SRB) in Finnish crystalline bedrock aquifers. Sampling was performed using a 1500-m-long pressure-tight tube that provided 15 subsamples, each corresponding to a 100-m section down the borehole. Microbial density measurements, as well as community fingerprinting with 16S rRNA gene-based denaturing gradient gel electrophoresis, demonstrated that microbial communities in the borehole water varied as a function of sampling depth. In the upper part of the borehole, bacteria affiliated to the family Comamonadaceae dominated the bacterial community. Further down the borehole, bacteria affiliated to the class Firmicutes became more prominent and, according to 16S rRNA gene clone libraries, dominated the bacterial community at 1400-1500 m. In addition, the largest number of bacterial classes was observed at 1400-1500 m. The dsrB genes detected in the upper part of the borehole were more similar to the dsrB genes of cultured SRBs, such as the genus Desulfotomaculum, whereas in the deeper parts of the borehole, the dsrB genes were more closely related to the uncultured bacteria that have been detected earlier in deep earth crust aquifers.

Functional genes reveal the intrinsic PAH biodegradation potential in creosote-contaminated groundwater following in situ biostimulation.

A small-scale functional gene array containing 15 functional gene probes targeting aliphatic and aromatic hydrocarbon biodegradation pathways was used to investigate the effect of a pilot-scale air sparging and nutrient infiltration treatment on hydrocarbon biodegradation in creosote-contaminated groundwater. Genes involved in the different phases of polycyclic aromatic hydrocarbon (PAH) biodegradation were detected with the functional gene array in the contaminant plume, thus indicating the presence of intrinsic biodegradation potential. However, the low aerobic fluorescein diacetate hydrolysis, the polymerase chain reaction (PCR) amplification of 16S rRNA genes closely similar to sulphate-reducing and denitrifying bacteria and the negligible decrease in contaminant concentrations showed that aerobic PAH biodegradation was limited in the anoxic groundwater. Increased abundance of PAH biodegradation genes was detected by functional gene array in the monitoring well located at the rear end of the biostimulated area, which indicated that air sparging and nutrient infiltration enhanced the intrinsic, aerobic PAH biodegradation. Furthermore, ten times higher naphthalene dioxygenase gene copy numbers were detected by real-time PCR in the biostimulated area, which was in good agreement with the functional gene array data. As a result, functional gene array analysis was demonstrated to provide a potential tool for evaluating the efficiency of the bioremediation treatment for enhancing hydrocarbon biodegradation in field-scale applications.

Comparative use of bacterial, algal and protozoan tests to study toxicity of azo- and anthraquinone dyes.

Toxicity of two azo dyes (Reactive Orange 16 (RO16); Congo Red (CR)) and two anthraquinone dyes (Remazol Brilliant Blue R (RBBR); Disperse Blue 3 (DB3)) were compared using bacterium Vibrio fischeri, microalga Selenastrum capricornutum and ciliate Tetrahymena pyriformis. The following respective endpoints were involved: acute toxicity measured as bacterial luminescence inhibition, algal growth inhibition, and the effects on the protozoa including viability, growth inhibition, grazing effect and morphometric effects. In addition, mutagenicity of the dyes was determined using Ames test with bacterium Salmonella typhimurium His(-). DB3 dye was the most toxic of all dyes in the bacterial, algal and protozoan tests. In contrast to other dyes, DB3 exhibited mutagenic effects after metabolic activation in vitro in all S. typhimurium strains used. Of the methods applied, the algal test was the most sensitive to evaluate toxicity of the dyes tested.

Biodegradation of lactic acid based polymers under controlled composting conditions and evaluation of the ecotoxicological impact.

The biodegradability of lactic acid based polymers was studied under controlled composting conditions (CEN prEN 14046), and the quality of the compost was evaluated. Poly(lactic acids), poly(ester-urethanes), and poly(ester-amide) were synthesized and the effects of different structure units were investigated. The ecotoxicological impact of compost samples was evaluated by biotests, i.e., by the Flash test, measuring the inhibition of light production of Vibrio fischeri, and by plant growth tests with cress, radish, and barley. All the polymers biodegraded to over 90% of the positive control in 6 months, which is the limit set by the CEN standard. Toxicity was detected in poly(ester-urethane) samples where chain linking of lactic acid oligomers had been carried out with 1,6-hexamethylene diisocyanate (HMDI). Both the Flash test and the plant growth tests indicated equal response to initial HMDI concentration in the polymer. All other polymers, including poly(ester-urethane) chain linked with 1,4-butane diisocyanate, showed no toxicological effect.