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

ABSTRACT: 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.

SUBMITTER: Reeksting BJ

PROVIDER: S-EPMC7082560 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundMicrobially 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.ResultsIn 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.ConclusionsOur 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.

Project description:Mineral precipitation via microbial activity is a well-known process with applications in various fields. This relevance of microbially induced calcite precipitation (MICP) has pushed researchers to explore various naturally occurring MICP capable bacterial strains. The present study was performed to explore the efficiency of microbially induced calcite precipitation (MICP) via locally isolated bacterial strains and role of guar gum, which is a naturally occurring polymer, on the MICP process. The strains were isolated from local soil and screened for urease activity Further, the urease positive strain was subjected to urea and calcium chloride based medium to investigate the efficacy of isolated strain for microbial induced precipitation. Among screened isolates, the soil bacterium that showed urease positive behaviour and precipitated calcium carbonate was subjected to 16S rRNA gene sequencing. This strain was identified as Bacillus velezensis. Guar gum-a natural polymer, was used as a sole carbon source to enhance the MICP process. It was observed that the isolated strain was able to breakdown the guar gum into simple sugars resulting in two-fold increase in calcium carbonate precipitate. Major bio-chemical activities of isolated strain pertaining to MICP such as ammonium ion concentration, pH profiling, and total reducing sugar with time were explored under four different concentrations of guar gum (0.25%, 0.5%, 0.75% and 1% w/v). Maximum ammonium ion concentration (17.5 ?g/ml) and increased pH was observed with 1% guar gum supplementation, which confirms augmented MICP activity of the bacterial strain. Microstructural analysis of microbial precipitation was performed using scanning electron microscopy (SEM) and X-ray diffraction (XRD) techniques, which confirmed the presence of calcium carbonate in different phases. Further, XRD and SEM based studies corroborated that guar gum supplemented media showed significant increase in stable calcite phase as compared to media without guar gum supplementation. Significant diverse group of nitrogenous compounds were observed in guar gum supplemented medium when subjected to Gas Chromatography-Mass spectrometry (GC-MS) profiling.

Project description:Microbial-Induced Calcite Precipitation (MICP) stimulates soil microbiota to induce a cementation of the soil matrix. Urea, calcium and simple carbon nutrients are supplied to produce carbonates via urea hydrolysis and induce the precipitation of the mineral calcite. Calcium chloride (CaCl2) is typically used as a source for calcium, but basic silicate rocks and other materials have been investigated as alternatives. Weathering of calcium-rich silicate rocks (e.g., basalt and dolerite) releases calcium, magnesium and iron; this process is associated with sequestration of atmospheric CO2 and formation of pedogenic carbonates. We investigated atmospheric carbon fluxes of a MICP treated sandy soil using CaCl2 and dolerite fines applied on the soil surface as sources for calcium. Soil-atmosphere carbon fluxes were monitored over 2 months and determined with an infrared gas analyser connected to a soil chamber. Soil inorganic carbon content and isotopic composition were determined with isotope-ratio mass spectrometry. In addition, soil-atmosphere CO2 fluxes during chemical weathering of dolerite fines were investigated in incubation experiments with gas chromatography. Larger CO2 emissions resulted from the application of dolerite fines (116 g CO2-C m-2) compared to CaCl2 (79 g CO2-C m-2) but larger inorganic carbon precipitation also occurred (172.8 and 76.9 g C m-2, respectively). Normalising to the emitted carbon to precipitated carbon, the environmental carbon cost was reduced with dolerite fines (0.67) compared to the traditional MICP treatment (1.01). The carbon isotopic signature indicated pedogenic carbonates (δ13Cav = -8.2 ± 5.0‰) formed when dolerite was applied and carbon originating from urea (δ13Cav = -46.4 ± 1.0‰) precipitated when CaCl2 was used. Dolerite fines had a large but short-lived (<2 d) carbon sequestration potential, and results indicated peak CO2 emissions during MICP could be balanced optimising the application of dolerite fines.

Project description:Biomineralization plays a key role in modifying the geological properties of soil, thereby stabilizing it against wind erosion, especially in areas characterized by harsh weather and harsh soil (calcareous and arid); i.e. Arabic Gulf region. Among soil microorganisms, ureolytic bacteria are capable of modifying soil characteristics and thus, inducing biomineralization. This research investigated the occurrence and diversity of ureolytic bacteria in Qatari soils, specifically to study their acquired potential to adapt to harsh conditions exhibiting ureolytic activity. Soil samples were collected from various locations in Qatar and were used to isolate the indigenous ureolytic bacteria. It was noticed that most of the ureolytic bacteria in Qatari soil belong to the genus Bacillus mainly Bacillus cereus. Identification and differentiation of 18 ureolytic isolates were performed using MALDI-TOF MS techniques while ribotyping (16S rRNA) molecular technique was used mainly for 6 selected strains. This study not only shows the diversity of species of ureolytic bacteria in Qatari soil but also shows the diversity in their protein profiles, which confirms that bacteria have adapted well to the harsh environment. In addition, the strains were evaluated based on a newly modified screening method in this work; i.e. production of arbitrary urease activity (AUA). Thus, the strains showing the highest AUA, exhibited the highest capability to produce urease enzymes induced by urea. Analysis of calcium carbonate precipitation utilizing SEM-EDX showed that the ureolytic bacteria also play a significant role in the precipitation of minerals such as CaCO3, in the presence of urea in soil. Therefore, this research showed a high occurrence of indigenous Bacillus bacteria in Qatari soil that can perform biomineralization and thus can be helpful, if properly stimulated, in enhancing soil stabilization, and for other local applications as well, since they are adapted to these soil and weather conditions.

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

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