Species diversity improves the efficiency of mercury-reducing biofilms under changing environmental conditions

Abstract

Six mercury-resistant environmental proteobacterial isolates and one genetically modified mercury-resistant Pseudomonas putida strain were analyzed for physiological traits of adaptive relevance in an environment of packed-bed bioreactors designed for the decontamination of mercury-polluted chlor-alkali wastewater. The strains displayed characteristic differences in each trait (i.e., biofilm formation capability, growth rate in mercury contaminated wastewaters, and mercury reduction efficiency). Subsequently, they were immobilized either as a monoculture or as a mixed culture on porous carrier material in packed-bed bioreactors through which different batches of filter-sterilized industrial chlor-alkali wastewater were pumped. In monospecies bioreactors, the mercury retention efficiency was sensitive to rapidly increasing mercury concentrations in the wastewater. Mixed culture biofilms displayed a high mercury retention efficiency that was not affected by rapid increases in mercury or continuously high mercury concentrations. The dynamic in the community composition of the mixed culture bioreactors was determined by ribosomal intergenic spacer polymorphism analysis. Mercury-mediated selective pressure decreased the number of prevalent strains. Microbial diversity was completely restored after easing of the selective pressure. Microbial diversity provides a reservoir of strains with complementary ecological niches that results in a superior bioreactor performance under changing environmental conditions.

FIG. 1.

Biofilm formation in ECI 1 wastewater after 24 h in glass test tubes by the multispecies and pure culture bioreactor strains.

FIG. 2.

Maximum Hg(0) formation rates in chlor-alkali wastewaters. Initial wastewater conditions are shown in the top lines. The columns in each data set are, from left to right: Elb2, Spi3, Spi7, Ibu8, Bro12, Bro62, and KT2442::mer-73.

FIG. 3.

Growth of chlor-alkali wastewater batch cultures. The data are mean values of three parallel experiments.

FIG. 4.

Culturable cell numbers and mercury concentration in the bioreactor effluent. Solid lines, total mercury in bioreactor effluent; dotted lines, CFU in bioreactor effluent. Squares, bioreactor 1; circles, bioreactor 2; triangles, bioreactor 3. (A) P. putida KT2442::mer-73 monospecies bioreactors; (B) C. freundii Bro62 monospecies bioreactors; (C) P. aeruginosa Bro12 monospecies bioreactors; (D) multispecies bioreactors.

FIG. 5.

ISR fingerprints of the inoculum strains, the inoculum, and the final bioreactor effluent samples. The community marker (CM) bands consists of a mixture of the ISR-PCR products of the six inoculum strains (separately amplified). The numbers at the fingerprint bands indicate the diagnostic bands and their position in the community marker. The numbers at the size marker (SM) bands indicate their sizes in base pairs. B1 d81, bioreactor 1 (day 81); B2 d81, bioreactor 2 (day 81); B3 d81, bioreactor 3 (day 81).

FIG. 6.

Time course analyses of relative strain abundances in the multispecies bioreactor effluents. The presence of the inoculum strains in the effluxes of bioreactors 1 (A), 2 (B), and 3 (C) was based on the intensity of the diagnostic band of each strain within the community fingerprint relative to the size marker intensity. The mercury concentration of the wastewater is indicated in panel A.