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.     

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.     

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.     

Biochemically Induced Carbonate Precipitation in Aerobic and Anaerobic Environments by Sporosarcina pasteurii

Microbially induced calcium carbonate precipitation (MICP) at laboratory scale for modifying the geotechnical properties of soils has been extensively investigated. The successful implementation of MICP in the field encounters many biotic and abiotic challenges. The study aimed to comprehend the role of oxygen availability on the efficacy of MICP catalyzed by S. pasteurii microbe. For this purpose, microbial growth rate, its ureolytic activities and carbonate precipitation by S. pasteurii over aerated, anoxic and anaerobic conditions were studied. The growth rate, ureolytic activity and amount of mineral precipitated were found to be insignificant under anaerobic environment compared to the remaining exposure conditions, which signifies the importance of oxygen for successful implementation of MICP process. The limited ureolytic activities and a minute amount of precipitation observed under anaerobic system were primarily attributed to the enzymes, already produced during the aerobic culture. The rise in the pH during the MICP process was not only because of ureolytic activities but also due to the breakdown of complex proteins in the stationary growth phase. As a whole, the MICP process was significantly inhibited in the absence of oxygen, or without frequent injection of S. pasteurii.     

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.     

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.     

Potential soil reinforcement by biological denitrification

Currently new ground reinforcement techniques are being developed based on microbially induced carbonate precipitation (MICP). Many studies on MICP use microbially catalyzed hydrolysis of urea to produce carbonate. In the presence of dissolved calcium this process leads to precipitation of calcium carbonate crystals, which form bridges between the sand grains and hence increase strength and stiffness. In addition to urea hydrolysis, there are many other microbial processes which can lead to the precipitation of calcium carbonate. In this study the theoretical feasibility of these alternative MICP processes for ground reinforcement is evaluated. Evaluation factors are substrate solubility, CaCO₃ yield, reaction rate and type and amount of side-product. The most suitable candidate as alternative MICP method for sand consolidation turned out to be microbial denitrification of calcium nitrate, using calcium salts of fatty acids as electron donor and carbon source. This process leads to calcium carbonate precipitation, bacterial growth and production of nitrogen gas and some excess carbon dioxide. The feasibility of MICP by denitrification is tested experimentally in liquid batch culture, on agar plate and in sand column experiments. Results of these experiments are presented and discussed.     

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。     

Effects of environmental factors on microbial induced calcium carbonate precipitation

Aims: To gain an understanding of the environmental factors that affect the growth of the bacterium Sporosarcina pasteurii, the metabolism of the bacterium and the calcium carbonate precipitation induced by this bacterium to optimally implement the biological treatment process, microbial induced calcium carbonate precipitation (MICP), in situ. Methods and Results: Soil column and batch tests were used to assess the effect of likely subsurface environmental factors on the MICP treatment process. Microbial growth and mineral precipitation were evaluated in freshwater and seawater. Environmental conditions that may influence the ureolytic activity of the bacteria, such as ammonium concentration and oxygen availability, as well as the ureolytic activities of viable and lysed cells were assessed. Treatment formulation and injection rate, as well as soil particle characteristics are other factors that were evaluated for impact on uniform induction of cementation within the soils. Conclusions: The results of the study presented herein indicate that the biological treatment process is equally robust over a wide range of soil types, concentrations of ammonium chloride and salinities ranging from distilled water to full seawater; on the time scale of an hour, it is not diminished by the absence of oxygen or lysis of cells containing the urease enzyme. Significance and Impact of Study: This study advances the biological treatment process MICP towards field implementation by addressing key environmental hurdles faced with during the upscaling process.     

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.     

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.     

Effects of different calcium salts on calcium carbonate crystal formation by Sporosarcina pasteurii KCTC 3558

We examined the effectiveness of using different calcium salts for bioconsolidation. Four calcium salts were chosen based on their applicability and solubility. Initial experiments demonstrated that the addition of any calcium salt had a negative effect on the urease activity of S. pasteurii. Microscopic examinations elucidated the morphological and structural differences of the calcium carbonate (CaCO₃) crystals induced. Calcite and vaterite are the prominent forms of CaCO₃ detected according to X-ray diffraction (XRD) analysis. Bioconsolidated sand samples were able to significantly resist water flow through a column compared to the non-treated samples. Also, in a tightness test, the differences in the ability to retain water within columns were observed among the samples tested. Moreover, despite the differences, the calcium salts tested still bound the sand together to form blocks. Our results further explain the influence of multiple factors in crystal formation and sand bioconsolidation effectiveness.     

Evaluation of Shear Strength Parameters of Sandy Soils upon Microbial Treatment

A new method for soil stabilization known as microbial-induced calcite precipitation (MICP) has been the focus of research in this area during the last decade. In this method, the reaction of microorganisms in the presence of urea and calcium chloride is used to produce calcite. Despite the large numbers of bacteria in soil, Sporobacillus pasteurii (previously known as Bacillus pasteurii) has the most capability to create cementation between soil particles in the MICP method due to its high urease activity. In this paper, the effect of MICP treatment on the shear strength characteristics of a sandy soil was studied. The change in the shear strength of sandy soil upon MICP treatment was measured using a strain-controlled direct shear test before and after treatment of the soil samples. The results showed an increase of 44–86% in the shear strength of the sandy soil after 15 days of MICP treatment compared to the untreated soil. The enhanced shear strength was the result of an increase in both the cohesion intercept and angle of internal friction. The increase in the cohesion intercept was more significant than the increase in the angle of internal friction.     

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.     

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.     

Improving piezoelectric cell printing accuracy and reliability through neutral buoyancy of suspensions

The sedimentation and aggregation of cells within inkjet printing systems has been hypothesized to negatively impact printer performance. The purpose of this study was to investigate this influence through the use of neutral buoyancy. Ficoll PM400 was used to create neutrally buoyant MCF‐7 breast cancer cell suspensions, which were ejected using a piezoelectric drop‐on‐demand inkjet printing system. It was found that using a neutrally buoyant suspension greatly increased the reproducibility of consistent cell counts, and eliminated nozzle clogging. Moreover, the use of Ficoll PM400 was shown to not affect cellular viability. This is the first demonstration of such scale and accuracy achieved using a piezoelectric inkjet printing system for cellular dispensing. Biotechnol. Bioeng. 2012; 109: 2932–2940. © 2012 Wiley Periodicals, Inc.     

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.     

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.     

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.     

A puddle: an ombrophilic cyano-bacterial community

Sequential stages of formation of an ombrophilic cyano-bacterial community on clay were determined in a laboratory model of a puddle community. In a suspension of washed clay obtained from loamy soil, with montmorillonite as the predominant phase, a bacterial neustonic film is initially formed; it acts as a support for cyanobacterial hormogonia. At the next stage, the upper layer of precipitated clay (about 1 mm) is reinforced by a cyano-bacterial structure of Phormidium sp. trichomes and develops a tissue-like structure. The hormogonia and sheathless cyanobacteria remain free from mineral particles. Subsequently, gas formation results in a separation of a dense cyano-bacterial film from the underlying loose suspension and in formation of gas swellings. The mineral component of the film is differentiated: mineral particles of quartz and feldspar grains are attached to Phormidium sp. trichomes, which act as a factor of mineral selection.