Stabnikov V. Scientific concepts for the development of biocementation biotechnology and its applications in environmental engineering.

Biocementation biotechnology is developing intensively for last decade. It is using microorganisms for new material such as biocement that is useful for environmental engineering and industry. Biocement can replace cement in soil sealing and strengthening because the size of the biocement particles is just few microns and viscosity of the biocement is significantly than viscosity of cement, so biocement suspension is penetrating porous materials deeper than the cement suspension. The thesis is devoted to the development of biocement and biocementation biotechnologies that are based on the activity of urease-producing bacteria. Phylogenetic analysis by nucleotide sequences of 16S rRNA gene of strains from enrichment culture of urease-producing bacteria has been done. Pure bacterial cultures should be used to avoid the presence of opportunistic pathogens. Urease-active halotolerant and alkaliphilic strains of Bacillus sp. VS1and Yaniella sp. VS8 have been isolated, identified, and studied as the bioagents for biocementation.Phylogenetic and physiological similarity of the strains of Bacillus sp., which were isolated for ureolytic precipitation of calcium carbonate from different climate world zones was shown. To increase biosafety of biocementation, a method for production of inactivated but urease-active bacterial cells of urease-producing bacteriawas developed. It was shown that two types of urease-producing bacteria with constitutive urease (Bacillus sp. VS1) and inducible urease (Yaniella sp. VS8) can be cultivated for large scale biocementation using hydrolysate of activated sludge of municipal wastewater treatment plant. It is possible to increase greatly urease activity and biocementation rate by concentration of urease-producing bacterial biomass. It was shown that mechanism of biocementationincludes adsorption of bacterial cells onto the surface of sand grains, formation of the calcite, aragonite, and vaterite crystals, and binding with surface and between themselves.Adsorption efficiency of bacterial cells on the sand particles can be increased from 29 to 37% by pretreatment of sand with cations of either calcium, aluminum, or iron.To obtain material with needed properties, the following microbial biocementation technologies have been developed: 1) the formation of the strong biocemented layer with a thickness of a few mm by precipitation on the surface of material; 2) the formation of the layer with a thickness of a few cm by spraying biocement solution onto the surface, and 3) the formation of the biocemented volume of the porous material by percolation or injection of the biocement solution. To increase efficiency of microbial biocementation two new methods have been proposed and studied. First method is the replacement of calcium ions with ions of ferrous, which were produced by iron-reducing bacteria from hematite of iron ore. This method decreased the hydraulic conductivity of sand to 1.4∙10-7 m/s. Second method includes bioimmobilization of the gas bubbles, which were produced in the sand pores. This method can decrease the hydraulic conductivity of sand to 2∙10-7 m/s. Biotechnology of the surface biocementation has been proposed. This process can be used for the sealing of the ponds or landfills in sandy soil. It was shown that biocementation can be used to control wind and water erosion of soil and release of associated with dust chemical and bacteriological model pollutants. An application of biocementation for the coating of the surfaces of concrete, stone, glass, plastic, and wood with the layer of calcite was studied. This biocoating can be used for the surface protection of coastal concrete structures and creation of artificial reefs to promote environmental sustainability in the coastal area. The pilot test of biotechnological biocementation for the sealing of the cracks in the rocks with inactivated but urease active cells Yaniella sp. VS8 showed the applicability of the method in environmental engineering for prevention of pollutants migration to groundwater and surface water from the landfills. The pilot biocementation of the mixture of sand and limestone in by the injection of urease-producing bacteria Bacillus sp. VS1 significantly decreased hydraulic conductivity. The pilot tests confirmed applicability of biocementation to control the wind erosion of soil and for the manufacturing of sand filters for microfiltration of water.Selected microorganisms, discovered mechanisms of microbial biocementation, and the developed biotechnologies of the surface and bulk biocementation give the opportunity for further scientific development and practical applications of new biotechnological discipline – biotechnology of biocement production and application.