Molecular quantification of genes encoding for green-fluorescent proteins.

A quantitative PCR approach is presented to analyze the amount of recombinant green fluorescent protein (gfp) genes in environmental DNA samples. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively coamplified DNA standard constructed by point mutation PCR. A single base difference was introduced to achieve a suitable migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to monitor recombinant DNA in environments like the efflux of a biotechnological plant.

Long-term performance of bioreactors cleaning mercury-contaminated wastewater and their response to temperature and mercury stress and mechanical perturbation.

The long-term performance of bioreactors retaining mercury from contaminated industrial wastewater was analyzed at the laboratory scale, and its response to mechanical perturbations (gas bubbles and shaking) as well as to physical (increased temperature and hydraulic load) and chemical stresses (increased mercury concentration) likely to occur during on site operation was studied. Two packed-bed bioreactors with 80-cm(3) lava chips as biofilm carrier were inoculated with nine Hg(II)-resistant natural isolates of alpha- and gamma-proteobacteria. Chloralkali wastewater containing ionic mercury (3.0 to 9.7 mg/L Hg(2+)), amended with sucrose and yeast extract, flowed through the bioreactors at 160 mL/h. During the 16-month investigation the bioreactors showed no sign of depleted performance in terms of mercury-retaining capacity. After 16 months, both bioreactors still retained 96% of the mercury load. The performance of the bioreactors was sensitive to mechanical perturbations (e.g., sheer forces of gas bubbles). Shifts to higher Hg(2+) inflow concentrations initially decreased the mercury retention efficacy slightly. However, the bioreactors could adapt to Hg(2+) concentrations of up to 7.6 mg/L within several days. Old biofilms were less affected than the younger ones. The performance of the bioreactors was not affected by an increase in temperature up to 41 degrees C and an increased volumetric load (up to 240 mL/h). The bioreactors regained activity spontaneously after the stress had stopped. Recovery could be accelerated by increased nutrient concentration, although this may lead to blocking of the packed bed.

Detection of small sequence differences using competitive PCR: molecular monitoring of genetically improved, mercury-reducing bacteria.

A quantitative PCR approach is presented to detect small genomic sequence differences for molecular quantification of recombinant DNA. The only unique genetic feature of the mercury-reducing, genetically improved Pseudomonas putida KT2442::mer73 available to distinguish it from its native mercury-resistant relatives is the DNA sequence crossing the border of the insertion site of the introduced DNA fragment. The quantification assay is a combination of specific PCR amplification and temperature gradient gel electrophoresis (TGGE). Gene quantification is provided by a competitively co-amplified DNA standard constructed by point mutation PCR. After computing the denaturation behavior of the target DNA stretch, a single base difference was introduced to achieve maximum migration difference in TGGE between the original target DNA and the modified standard without altering the PCR amplification efficiency. This competitive PCR strategy is a highly specific and sensitive way to detect small sequence differences and to monitor recombinant DNA in effluxes of biotechnological plants.

Structure and species composition of mercury-reducing biofilms.

Mercury-reducing biofilms from packed-bed bioreactors treating nonsterile industrial effluents were shown to consist of a monolayer of bacteria by scanning electron microscopy. Droplets of several micrometers in diameter which accumulated outside of the bacterial cells were identified as elemental mercury by electron-dispersive X-ray analysis. The monospecies biofilms of Pseudomonas putida Spi3 initially present were invaded by additional strains, which were identified to the species level by thermogradient gel electrophoresis (TGGE) and 16S rDNA sequencing. TGGE community fingerprints of the biofilms showed that they were composed of the effluent bacteria and did not contain uncultivable microorganisms. Of the 13 effluent bacterial strains, 2 were not mercury resistant, while all the others had resistance levels similar to or higher than the inoculant strain.

Removal of mercury from chloralkali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain.

A mercury-resistant bacterial strain which is able to reduce ionic mercury to metallic mercury was used to remediate in laboratory columns mercury-containing wastewater produced during electrolytic production of chlorine. Factory effluents from several chloralkali plants in Europe were analyzed, and these effluents contained total mercury concentrations between 1.6 and 7.6 mg/liter and high chloride concentrations (up to 25 g/liter) and had pH values which were either acidic (pH 2.4) or alkaline (pH 13.0). A mercury-resistant bacterial strain, Pseudomonas putida Spi3, was isolated from polluted river sediments. Biofilms of P. putida Spi3 were grown on porous carrier material in laboratory column bioreactors. The bioreactors were continuously fed with sterile synthetic model wastewater or nonsterile, neutralized, aerated chloralkali wastewater. We found that sodium chloride concentrations up to 24 g/liter did not inhibit microbial mercury retention and that mercury concentrations up to 7 mg/liter could be treated with the bacterial biofilm with no loss of activity. When wastewater samples from three different chloralkali plants in Europe were used, levels of mercury retention efficiency between 90 and 98% were obtained. Thus, microbial mercury removal is a potential biological treatment for chloralkali electrolysis wastewater.