Microbially Induced Calcium Carbonate Precipitation on Surface or in the Bulk of Soil

Microbial precipitation of calcium carbonate takes place in nature by different mechanisms. One of them is microbially induced carbonate precipitation (MICP), which is performed due to bacterial hydrolysis of urea in soil in the presence of calcium ions. The MICP process can be adopted to reduce the permeability and/or increase the shear strength of soil. In this paper, a study on the use of Bacillus sp., which was isolated from tropical beach sand, to perform MICP either on the surface or in the bulk of sand is presented. If the level of calcium salt solution was below the sand surface, MICP took place in the bulk of sand. On the other hand, if the level of calcium salt solution was above the sand surface, MICP was performed on the sand surface and formed a thin layer of crust of calcium carbonate. After six sequential batch treatments with suspension of urease-producing bacteria and solutions of urea and calcium salt, the permeability of sand was reduced to 14 mm/day (or 1.6×10^?7 m/s) in both cases of bulk and surface MICP. Quantities of precipitated calcium after six treatments were 0.15 and 0.60 g of Ca per cm² of treated sand surface for the cases of bulk or surface MICP, respectively. The stiffness of the MICP treated sand also increased considerably. The modulus of rupture of the thin layer of crust was 35.9 MPa which is comparable with limestone.     

Impact of Oxygen Availability on Microbially Induced Calcite Precipitation (MICP) Treatment

Most microbially induced calcite precipitation (MICP) processes are induced by aerobic bacteria; thus, oxygen availability plays an important role in MICP treatment. To determine the effects of oxygen supply on MICP treatment catalyzed by Sporosarcina pasteurii, contrast tests under an aerated condition, air-restricted condition, and open air condition were conducted. The results showed that dissolved oxygen (DO) in the air-restricted reactor decreased with time and was almost exhausted within 7 days; DO in the open box decreased by 50% after 7 days of treatment because of the superficial air supply; and DO in the aerated box maintained an initial high level because the consumed oxygen was supplied immediately by adequate air bubbles in the treatment solution. Unconfined compressive strength (UCS) and CaCO₃ content are high under the aerated condition, moderate under the open condition, and poor under the air-restricted condition. The UCS can be 100 times different depending on the different oxygen supply conditions. The overall influence process is as follows: oxygen is dissolved to supply DO for life and activity of the aerobic urea hydrolysis bacteria; then, urea is hydrolyzed to carbonate anions for CaCO₃ precipitation in the presence of Ca²⁺; and finally, CaCO₃ precipitation results in the strengthening of sand. The results indicate that a sufficient air supply is essential to improve MICP processes catalyzed by aerobic bacteria.     

Carbonate Mineral Precipitation for Soil Improvement Through Microbial Denitrification

Microbially induced carbonate precipitation (MICP) and associated biogas production may provide sustainable means of mitigating a number of geotechnical challenges associated with granular soils. MICP can induce interparticle soil cementation, mineral precipitation in soil pore space and/or biogas production to address geotechnical problems such as slope instability, soil erosion and scour, seepage of levees and cutoff walls, low bearing capacity of shallow foundations, and earthquake-induced liquefaction and settlement. Microbial denitrification has potential for improving the mechanical and hydraulic properties of soils because it promotes precipitation of calcium carbonate (CaCO₃) and produces nitrogen (N₂) gas without generating toxic by-products. We evaluated the potential for inducing carbonate precipitation in soil via bacterial denitrification using bench-scale experiments with the facultative anaerobe Pseudomonas denitrificans. Bench-scale experiments were conducted (1) without calcium in an N-rich bacterial growth medium in 2.0 L glass batch reactors and (2) with a source of calcium in sand-filled acrylic columns. Changes of pH, alkalinity, NO₃^? and NO₂^? in the batch reactors and columns, quantification of biogas production and observations of calcium-carbonate precipitation in the sand-filled columns indicate that denitrification led to carbonate precipitation and particle cementation in the pore water as well as a substantial amount of biogas production in both systems. These results document that bacterial denitrification has potential as a soil improvement mechanism.     

Fixation and distribution of bacterial activity in sand to induce carbonate precipitation for ground reinforcement

The mechanical properties of soil (cohesion, friction, stiffness and permeability) are important parameters for engineering constructions and ecosystems in sedimentary environments. BioGrout is an in situ soil strengthening technique involving microbial-induced carbonate precipitation (MICP). This process involves hydrolysis of urea by bacteria containing the enzyme urease in the presence of dissolved calcium ions, resulting in calcium carbonate precipitation. In order to control the BioGrout process for engineering applications, it is necessary to improve understanding of the relevant phenomena and develop efficiencies to enable up-scaling of the technology to suit commercial applications. Control of a homogeneous distribution of bacterial activity in a sand bed is considered crucial in order to prevent clogging during injection and provide homogeneous reinforcement results. This paper describes a methodology to distribute and fix bacteria (with their enzyme activity) relatively homogeneously in a sand bed, before supplying cementation reagents. The methodology is based on a two-phase injection procedure: a bacterial suspension is injected into the sand body, immediately followed by a fixation fluid (i.e. a solution with high salt content). It is proposed that bacteria are retarded by adsorption and filtration processes and are permanently adsorbed to the sand grains when overtaken by the fixation fluid. The presented experimental approach for optimizing bacterial fixation in porous media can be used as a tool to design the treatment protocol for engineering applications in practice.     

Engineered applications of ureolytic biomineralization: a review

Microbially-induced calcium carbonate (CaCO₃) precipitation (MICP) is a widely explored and promising technology for use in various engineering applications. In this review, CaCO₃ precipitation induced via urea hydrolysis (ureolysis) is examined for improving construction materials, cementing porous media, hydraulic control, and remediating environmental concerns. The control of MICP is explored through the manipulation of three factors: (1) the ureolytic activity (of microorganisms), (2) the reaction and transport rates of substrates, and (3) the saturation conditions of carbonate minerals. Many combinations of these factors have been researched to spatially and temporally control precipitation. This review discusses how optimization of MICP is attempted for different engineering applications in an effort to highlight the key research and development questions necessary to move MICP technologies toward commercial scale applications.     

Functional gene diversity of oolitic sands from Great Bahama Bank

Despite the importance of oolitic depositional systems as indicators of climate and reservoirs of inorganic C, little is known about the microbial functional diversity, structure, composition, and potential metabolic processes leading to precipitation of carbonates. To fill this gap, we assess the metabolic gene carriage and extracellular polymeric substance (EPS) development in microbial communities associated with oolitic carbonate sediments from the Bahamas Archipelago. Oolitic sediments ranging from high‐energy ‘active’ to lower energy ‘non‐active’ and ‘microbially stabilized’ environments were examined as they represent contrasting depositional settings, mostly influenced by tidal flows and wave‐generated currents. Functional gene analysis, which employed a microarray‐based gene technology, detected a total of 12 432 of 95 847 distinct gene probes, including a large number of metabolic processes previously linked to mineral precipitation. Among these, gene‐encoding enzymes for denitrification, sulfate reduction, ammonification, and oxygenic/anoxygenic photosynthesis were abundant. In addition, a broad diversity of genes was related to organic carbon degradation, and N₂ fixation implying these communities has metabolic plasticity that enables survival under oligotrophic conditions. Differences in functional genes were detected among the environments, with higher diversity associated with non‐active and microbially stabilized environments in comparison with the active environment. EPS showed a gradient increase from active to microbially stabilized communities, and when combined with functional gene analysis, which revealed genes encoding EPS‐degrading enzymes (chitinases, glucoamylase, amylases), supports a putative role of EPS‐mediated microbial calcium carbonate precipitation. We propose that carbonate precipitation in marine oolitic biofilms is spatially and temporally controlled by a complex consortium of microbes with diverse physiologies, including photosynthesizers, heterotrophs, denitrifiers, sulfate reducers, and ammonifiers.     

Factors Controlling Microbially Induced Desaturation and Precipitation (MIDP) via Denitrification during Continuous Flow

Three continuous-flow column experiments were conducted to investigate the effects of nitrate loading rate and input concentrations of acetate and calcium on the effectiveness of denitrification to promote microbially induced desaturation and precipitation (MIDP). MIDP differs from microbially induced carbonate precipitation (MICP) in that it relies upon desaturation by biogenic gas production, along with carbonate precipitation, to improve the behavior of saturated granular soil. Denitrification is a stepwise process that is susceptible to inhibition and accumulation of intermediates, like nitrite, due to unfavorable chemical conditions (e.g., low pH). Therefore, nitrate reduction, nitrite accumulation, calcite saturation, and formation of carbonate precipitates were monitored in the columns. Factors investigated in these experiments included the effects of nitrate loading rate and input concentrations of calcium and acetate. Low nitrate-loading rates (i.e., ≤0.7?mol/m²-day) generally led to favorable outcomes (i.e., less accumulation of intermediates and more efficient carbonate precipitation). However, faster precipitation rates associated with low nitrate-loading rates led to smaller carbonate crystals and a less uniform precipitation pattern (i.e., precipitation focused near the nutrient source). Input concentrations of calcium and acetate also affected MIDP, but to a lesser extent than nitrate loading rate. The most important stimulated denitrifying bacteria, identified via 16S rDNA sequencing of suspended and dissolved DNA in the soil columns, were Bacillus species, Pseudomonas species, Brevundimonas species, and members of the Rhizobiaceae family. The results support that lower nitrate loading rates can be beneficial for MIDP although higher nitrate loading rates might be useful for some soil improvement applications.     

Efficacy of Fe3O4/Starch Nanoparticles on Sporosarcina pasteurii Performance in MICP Process

The microbial induced calcite precipitation (MICP) has been explored using well-known urease producer bacterium Sporosarcina pasteurii for many applications including soil stabilization. Urease enzyme hydrolyzes urea and in the presence of calcium chloride causes calcium carbonate precipitation between sand particles increasing sand stiffness and strength. In this study, the liquefied soil samples from Anzali coast were positioned inside injection columns by standard positioning technique. The columns were treated by injecting S. pasteurii suspension and cementation solution (CaCl₂ and urea). The effect of different conditions consisting of number of injections, injection intervals, flow rate, and ratio of injection solution on unconfined compression strength (USC) of sands formed inside the columns were evaluated. The results indicated that soil strength was increased when ratio of reactant solutions and injection time were elevated. Moreover, the maximum Ca-precipitation in MICP reaction in liquid medium was obtained while Fe₃O₄/starch concentration and time of addition of nanoparticle to culture medium were 10.8?mg/L and 1.4?h, respectively. The USC results showed that the columns injected by bacterial suspension treated by Fe₃O₄/starch under optimized conditions improved the soil strength up to 1200?kPa in comparison to the control column as 220?kPa.     

Effects of Different Clay’s Percentages on Improvement of Sand-Clay Mixtures with Microbially Induced Calcite Precipitation

Abstract

Microbially induced calcite precipitation (MICP) is currently appraised for the improvement of sandy soils, but only few studies use it to improve sand-clay mixtures. The effect of contents of kaolin clay and the effect of ions in kaolin clay on bacterial urease activity and productive rates for calcium carbonate were studied. Moreover, sand solidification tests were conducted and the solidifying effects of MICP for sand-clay mixtures were evaluated. The results show that adding kaolin clay has an inhibitory effect on the urease activity of bacteria, and adding too many kaolin clays also decrease the productive rates for calcium carbonate. With adding Al₂O₃ or FeCl₃, urease activity both decreases and it becomes lower with adding more Al₂O₃ or FeCl₃. The permeability of sand columns all decreased gradually with MICP curing. With more kaolin clay, the increasing range of bacterial utilization rates of those with larger particle sizes is bigger. The maximum productive rate for calcium carbonate of samples with smaller particle sizes exists in sample with 5% of kaolin clay while other samples with 7.5% of clay have more calcium carbonate. Sand columns with different sand particle sizes have different suitable amounts of added kaolin clays for MICP solidification.     

Non-ureolytic calcium carbonate precipitation by <Emphasis Type="Italic">Lysinibacillus</Emphasis> sp. YS11 isolated from the rhizosphere of <Emphasis Type="Italic">Miscanthus sacchariflorus</Emphasis>

Although microbially induced calcium carbonate precipitation (MICP) through ureolysis has been widely studied in environmental engineering fields, urea utilization might cause environmental problems as a result of ammonia and nitrate production. In this study, many non-ureolytic calcium carbonate-precipitating bacteria that induced an alkaline environment were isolated from the rhizosphere of Miscanthus sacchariflorus near an artificial stream and their ability to precipitate calcium carbonate minerals with the absence of urea was investigated. MICP was observed using a phase-contrast microscope and ion-selective electrode. Only Lysinibacillus sp. YS11 showed MICP in aerobic conditions. Energy dispersive X-ray spectrometry and X-ray diffraction confirmed the presence of calcium carbonate. Field emission scanning electron microscopy analysis indicated the formation of morphologically distinct minerals around cells under these conditions. Monitoring of bacterial growth, pH changes, and Ca²⁺ concentrations under aerobic, hypoxia, and anaerobic conditions suggested that strain YS11 could induce alkaline conditions up to a pH of 8.9 and utilize 95% of free Ca²⁺ only under aerobic conditions. Unusual Ca²⁺ binding and its release from cells were observed under hypoxia conditions. Biofilm and extracellular polymeric substances (EPS) formation were enhanced during MICP. Strain YS11 has resistance at high pH and in high salt concentrations, as well as its spore-forming ability, which supports its potential application for self-healing concrete.     

Formate Oxidation-Driven Calcium Carbonate Precipitation by Methylocystis parvus OBBP

Microbially induced carbonate precipitation (MICP) applied in the construction industry poses several disadvantages such as ammonia release to the air and nitric acid production. An alternative MICP from calcium formate by Methylocystis parvus OBBP is presented here to overcome these disadvantages. To induce calcium carbonate precipitation, M. parvus was incubated at different calcium formate concentrations and starting culture densities. Up to 91.4% ± 1.6% of the initial calcium was precipitated in the methane-amended cultures compared to 35.1% ± 11.9% when methane was not added. Because the bacteria could only utilize methane for growth, higher culture densities and subsequently calcium removals were exhibited in the cultures when methane was added. A higher calcium carbonate precipitate yield was obtained when higher culture densities were used but not necessarily when more calcium formate was added. This was mainly due to salt inhibition of the bacterial activity at a high calcium formate concentration. A maximum 0.67 ± 0.03 g of CaCO₃ g of Ca(CHOOH)₂⁻¹ calcium carbonate precipitate yield was obtained when a culture of 10⁹ cells ml⁻¹ and 5 g of calcium formate liter⁻¹ were used. Compared to the current strategy employing biogenic urea degradation as the basis for MICP, our approach presents significant improvements in the environmental sustainability of the application in the construction industry.     

A correlation study on optimum conditions of microbial precipitation and prerequisites for self-healing concrete

Microbial induced CaCO₃ precipitation (MICP) based upon enzymatic urea hydrolysis has been verified as an effective way for crack treatment, especially for self-healing of concrete cracks. This paper aimed at correlating optimum conditions of MICP with prerequisites for self-healing concrete. Orthogonal experiments on a combination of factors contributing to the MICP process were firstly performed. Initial cell density and Ca²⁺ concentration were highly significant factor and significant factor respectively. High initial cell density (1×10⁸ cells·mL^-1) together with relatively low Ca²⁺ concentration (50 mM) favored microbial precipitation. The second part of this study was associated with dissolution tests to simulate the dissolving behavior of urea and calcium, since the dissolving of healing agents in cracks is a prerequisite of self-healing. By an addition of urea and Ca(NO₃)₂ with constant mass ratio of 2:3 in concrete, the highest values of the estimated urea concentration (345 mM) and Ca²⁺ concentration (44 mM) dissolved in cracks were close to the optimal values found by orthogonal studies. Although the addition of urea and Ca(NO₃)₂ would not have a negative impact on the mechanical properties of concrete, direct mixing is not recommended due to the low utilization efficiency of incorporating healing agents for self-healing.     

微生物水泥相关酶的研究进展

微生物诱导碳酸钙沉积(microbial induced calcium carbonate precipitation,MICP)是微生物通过新陈代谢在其周围微环境中形成碳酸钙沉积的一种自然现象,根据其原理目前研发出了"微生物水泥".因其具有绿色环保、经济高效的特点,已成为生物、土木、环境等领域的研究热点.文中就与微...     

Construction of two ureolytic model organisms for the study of microbially induced calcium carbonate precipitation

Two bacterial strains, Pseudomonas aeruginosa MJK1 and Escherichia coli MJK2, were constructed that both express green fluorescent protein (GFP) and carry out ureolysis. These two novel model organisms are useful for studying bacterial carbonate mineral precipitation processes and specifically ureolysis-driven microbially induced calcium carbonate precipitation (MICP). The strains were constructed by adding plasmid-borne urease genes (ureABC, ureD and ureFG) to the strains P. aeruginosa AH298 and E. coli AF504gfp, both of which already carried unstable GFP derivatives. The ureolytic activities of the two new strains were compared to the common, non-GFP expressing, model organism Sporosarcina pasteurii in planktonic culture under standard laboratory growth conditions. It was found that the engineered strains exhibited a lower ureolysis rate per cell but were able to grow faster and to a higher population density under the conditions of this study. Both engineered strains were successfully grown as biofilms in capillary flow cell reactors and ureolysis-induced calcium carbonate mineral precipitation was observed microscopically. The undisturbed spatiotemporal distribution of biomass and calcium carbonate minerals were successfully resolved in 3D using confocal laser scanning microscopy. Observations of this nature were not possible previously because no obligate urease producer that expresses GFP had been available. Future observations using these organisms will allow researchers to further improve engineered application of MICP as well as study natural mineralization processes in model systems.     

Dairy manure pellets and palm oil mill effluent as alternative nutrient sources in cultivating Sporosarcina pasteurii for calcium carbonate bioprecipitation

Microbially induced carbonate precipitation (MICP) is a process that hydrolysis urea by microbial urease to fill the pore spaces of soil with induced calcium carbonate (CaCO₃) precipitates, which eventually results in improved or solidified soil. This research explored the possibility of using dairy manure pellets (DMP) and palm oil mill effluent (POME) as alternative nutrient sources for Sporosarcina pasteurii cultivation and CaCO₃ bioprecipitation. Different concentrations (20–80 g l⁻¹) of DMP and POME were used to propagate the cells of S. pasteurii under laboratory conditions. The measured CaCO₃ contents for MICP soil specimens that were treated with bacterial cultures grown in DMP medium (60%, w/v) was 15·30 ± 0·04 g ml⁻¹ and POME medium (40%, v/v) was 15·49 ± 0·05 g ml⁻¹ after 21 days curing. The scanning electron microscopy showed that soil treated with DMP had rhombohedral structure-like crystals with smooth surfaces, whilst that of POME entailed ring-like cubical formation with rough surfaces Electron dispersive X-ray analysis was able to identify a high mass percentage of chemical element compositions (Ca, C and O), whilst spectrum from Fourier-transform infrared spectroscopy confirmed the vibration peak intensities for CaCO₃. Atomic force microscopy further showed clear topographical differences on the crystal surface structures that were formed around the MICP treated soil samples. These nutrient sources (DMP and POME) showed encouraging potential cultivation mediums to address high costs related to bacterial cultivation and biocementation treatment.     

微生物成因的碳酸盐矿物研究进展

微生物诱导碳酸盐类矿物沉淀是地质微生物学的研究热点之一.微生物主要通过其代谢活动促进细胞周围微环境pH值及水体[CO32-]的升高,最终表现为碳酸盐类矿物饱和指数的增加.此外,微生物及其分泌的胞外聚合物可作为碳酸盐晶核的成核位点,为碳酸盐矿物晶体的生长进一步提供有利条件.微生物成因与纯化学成因的碳酸盐类矿物相比具有不同的矿物特征(如形貌、微量元素含量及碳同位素等).深入了解微生物诱导碳酸钙沉淀的行为对理解地质时期微生物活动及其在二氧化碳的地质封存中的潜在应用具有指导意义.本文综述了微生物诱导碳酸盐矿物沉淀的机理、代谢过程,总结了该领域的最新进展,探讨了生物成因以及化学成因碳酸盐矿物的区别,最后指出了该项研究在微生物岩以及CO2地质封存上的一些可能的拓展方向.     

Optimization of growth medium for <Emphasis Type="Italic">Sporosarcina pasteurii</Emphasis> in bio-based cement pastes to mitigate delay in hydration kinetics

Microbial-induced calcium carbonate precipitation has been identified as a novel method to improve durability and remediate cracks in concrete. One way to introduce microorganisms to concrete is by replacing the mixing water with a bacterial culture in nutrient medium. In the literature, yeast extract often has been used as a carbon source for this application; however, severe retardation of hydration kinetics has been observed when yeast extract is added to cement. This study investigates the suitability of alternative carbon sources to replace yeast extract for microbial-induced calcium carbonate precipitation in cement-based materials. A combination of meat extract and sodium acetate was identified as a suitable replacement in growth medium for Sporosarcina pasteurii; this alternative growth medium reduced retardation by 75 % (as compared to yeast extract) without compromising bacterial growth, urea hydrolysis, cell zeta potential, and ability to promote calcium carbonate formation.     

产脲酶芽胞杆菌的微波诱变育种

巴氏芽胞杆菌是目前微生物诱导碳酸钙沉淀(MICP)方法中应用最为热门的一种细菌。为提高巴氏芽胞杆菌尿素分解以及矿化能力,以巴氏芽胞杆菌YB-B为出发菌株,采用微波诱变育种技术,通过诱变菌株特性筛选及其遗传稳定性检测,成功选育出2株突变菌株YB-3和YB-4。与出发菌株相比,诱变菌株尿素分解能力较原菌株提高1.5倍左右,矿化能力提高114%。诱变菌株具有生长速度快,环境适应性强,矿化能力高等优点,这为MICP更广层次的应用奠定了坚实的基础。     

Comparison of microbially induced calcium carbonate precipitation eligibility using sporosarcina pasteurii and bacillus licheniformis on two different sands

The use of biological means for ground improvement have become popular, which generally works through the process called microbially-induced calcium carbonate precipitation (MICP). Many studies indicate successful application of MICP based improvement with multiple bacteria and on several soils. Given the proven performance of MICP, this study aims to examine the MICP process by comparing the calcium carbonate precipitation ability of widely studied bacteria, i.e., Sporosarcina pasteurii and relatively under-recognized bacteria, i.e., Bacillus licheniformis to outline the formation success. For this purpose, two different sands were tested for observing precipitation behavior using a series of syringe tests. Furthermore, the effect of concentration and inclusion of calcium chloride for nutrition of bacteria, saturation with water, and hybrid use of two bacteria were investigated in some tests for diversification. X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive x-ray spectroscopy (EDS) were used for the interpretation of results. Results indicated that Sporosarcina pasteurii had performed superior over Bacillus licheniformis when achieving calcium carbonate precipitation in tests for both sands. In addition, many intriguing SEM images contributed to the literature of MICP monitoring, highlighting the effects of the variables investigated.     

A Novel Approach to Enhance the Urease Activity of Sporosarcina pasteurii and its Application on Microbial-Induced Calcium Carbonate Precipitation for Sand

Abstract

Urease is involved in the formation of carbonate sediments by microbial-induced calcium carbonate precipitation (MICP), and Sporosarcina pasteurii used extensively in this technique owing to its high urease production. In this study, a simple two-step culture method with the appropriate medium was developed to enhance the urease activity of S. pasteurii. Urea played an important role in the culture process, particularly during the pre-cultivation step and the newly developed method improved both urease activity and specific urease activity. Furthermore, the increase in urease activity by MICP resulted in increased production of calcium carbonate and better strength of bio-cemented sand.