Genetic optimisation of bacteria-induced calcite precipitation in Bacillus subtilis.

BACKGROUND: Microbially induced calcite precipitation (MICP) is an ancient property of bacteria, which has recently gained considerable attention for biotechnological applications. It occurs as a by-product of bacterial metabolism and involves a combination of chemical changes in the extracellular environment, e.g. pH increase, and presence of nucleation sites on the cell surface or extracellular substances produced by the bacteria. However, the molecular mechanisms underpinning MICP and the interplay between the contributing factors remain poorly understood, thus placing barriers to the full biotechnological and synthetic biology exploitation of bacterial biomineralisation. RESULTS: In this study, we adopted a bottom-up approach of systematically engineering Bacillus subtilis, which has no detectable intrinsic MICP activity, for biomineralisation. We showed that heterologous production of urease can induce MICP by local increases in extracellular pH, and this can be enhanced by co-expression of urease accessory genes for urea and nickel uptake, depending on environmental conditions. MICP can be strongly enhanced by biofilm-promoting conditions, which appeared to be mainly driven by production of exopolysaccharide, while the protein component of the biofilm matrix was dispensable. Attempts to modulate the cell surface charge of B. subtilis had surprisingly minor effects, and our results suggest this organism may intrinsically have a very negative cell surface, potentially predisposing it for MICP activity. CONCLUSIONS: Our findings give insights into the molecular mechanisms driving MICP in an application-relevant chassis organism and the genetic elements that can be used to engineer de novo or enhanced biomineralisation. This study also highlights mutual influences between the genetic drivers and the chemical composition of the surrounding environment in determining the speed, spatial distribution and resulting mineral crystals of MICP. Taken together, these data pave the way for future rational design of synthetic precipitator strains optimised for specific applications.

In-Depth Profiling of Calcite Precipitation by Environmental Bacteria Reveals Fundamental Mechanistic Differences with Relevance to Application.

Microbially induced calcite precipitation (MICP) has not only helped to shape our planet's geological features but is also a promising technology to address environmental concerns in civil engineering applications. However, limited understanding of the biomineralization capacity of environmental bacteria impedes application. We therefore surveyed the environment for different mechanisms of precipitation across bacteria. The most fundamental difference was in ureolytic ability, where urease-positive bacteria caused rapid, widespread increases in pH, whereas nonureolytic strains produced such changes slowly and locally. These pH shifts correlated well with patterns of precipitation on solid medium. Strikingly, while both mechanisms led to high levels of precipitation, we observed clear differences in the precipitate. Ureolytic bacteria produced homogenous, inorganic fine crystals, whereas the crystals of nonureolytic strains were larger and had a mixed organic/inorganic composition. When representative strains were tested in application for crack healing in cement mortars, nonureolytic bacteria gave robust results, while ureolytic strains showed more variation. This may be explained by our observation that urease activity differed between growth conditions or by the different natures and therefore different material performances of the precipitates. Our results shed light on the breadth of biomineralization activity among environmental bacteria, an important step toward the rational design of bacterially based engineering solutions.IMPORTANCE Biomineralization triggered by bacteria is important in the natural environment and has many applications in industry and in civil and geotechnical engineering. The diversity in biomineralization capabilities of environmental bacteria is, however, not well understood. This study surveyed environmental bacteria for their ability to precipitate calcium carbonate minerals and investigated both the mechanisms and the resulting crystals. We show that while urease activity leads to the fastest precipitation, it is by no means essential. Importantly, the same quantities of calcium carbonate are produced by nonureolytic bacteria, and the resulting crystals appear to have larger volumes and more organic components, which are likely beneficial in specific applications. Testing both precipitation mechanisms in a self-healing concrete application showed that nonureolytic bacteria delivered more robust results. Here, we performed a systematic study of the fundamental differences in biomineralization between environmental bacteria, and we provide important information for the design of bacterially based engineering solutions.

Sensing of Damage and Repair of Cement Mortar Using Electromechanical Impedance.

Lead zirconium titanate (PZT) has recently emerged as a low-cost material for non-destructive monitoring for civil structures. Despite the numerous studies employing PZT transducers for structural health monitoring, no studies have assessed the effects of both damage and repair on the electromechanical impedance response in cementitious materials. To this end, this study was conducted to assess the effects of the damage and repair of mortar samples on the electromechanical response of a surface-mounted PZT transducer. When damage was introduced to the specimen in stages, the resonance frequencies of the admittance signature were shifted to lower frequencies as the damage increased, and an increase in the peak amplitude was detected, indicating an increase in the damping and a reduction in the material stiffness properties. Also, increasing the damage in the material has been shown to decrease the sensitivity of the PZT to further damage. During the repair process, a noticeable difference between the after-damage and the after-repair admittance signatures was noted. The root-mean-square deviation (RMSD) showed a decreasing trend during the repair process, when compared to the before repair RMSD response which indicated a partial recovery for the material properties by decreasing the damping property in the material.

Permeable Nanomontmorillonite and Fibre Reinforced Cementitious Binders.

Clinker reduction in cementitious binders is of paramount importance today, and nanotechnology has extended permissible limits. In the present study, a reference binder consisting of 60% Portland cement, 20% limestone, 20% fly ash, 3% polyvinyl alcohol (PVA) fibres and 2% superplasticizer is optimized with three different types of nano-montmorillonite (nMt) dispersions; two organomodified ones and an inorganic one at different proportions (0.5% to 4%). Flexural strength, measured on day 7, 28, 56 and 90, was improved after day 28 with the addition of inorganic nMt. Thermal gravimetric analyses carried out on day 7, 28, 56 and 90 coupled with x-ray diffraction (at day 28) showed a distinctively enhanced pozzolanic reaction. Backscattered electron imaging confirmed changes in the microstructure. Late age relative density measurements of the nMt cementitious nanocomposites showed higher values than these of the reference paste, which can be attributed to better particle packing. Mercury intrusion porosimetry measurements give support to the optimal nMt dosage, being 1% by total mass of binder and water impermeability tests (modified with BS EN 492:2012) suggest that inorganic nMt can be a viable option material where permeability constitutes a prerequisite. Suggestions for further activation of the nMt-fibre reinforced cementitious nanocomposites were also made.