Bacterium Sporosarcina Pasteurii Research Articles

Feeding strategies for Sporosarcina pasteurii cultivation unlock more efficient production of ureolytic biomass for MICP.

The bacterium Sporosarcina pasteurii is the most commonly used microorganism for Microbial Induced Calcite Precipitation (MICP) due to its high urease activity. To date, no proper fed-batch cultivation protocol for S. pasteurii has been published, even though this cultivation method has a high potential for reducing costs of producing microbial ureolytic biomass. This study focusses on fed-batch cultivation of S. pasteurii DSM33. The study distinguishes between limited fed-batch cultivation and extended batch cultivation. Simply feeding glucose to a S. pasteurii culture does not seem beneficial. However, it was exploited that S. pasteurii is auxotrophic for two vitamins and amino acids. Limited fed-batch cultivation was accomplished by feeding the necessary vitamins or amino acids to a culture lacking them. Feeding nicotinic acid to a nicotinic acid deprived culture resulted in a 24% increase of the specific urease activity compared to a fed culture without nicotinic acid limitation. Also, extended batch cultivation was explored. Feeding a mixture of glucose and yeast extract results in OD600 of ≈70 at the end of cultivation, which is the highest value published in literature so far. These results have the potential to make MICP applications economically viable.

Microbial Biomineralization of Alkaline Earth Metal Carbonates on 3D-Printed Surfaces.

The biomineralizing bacterium Sporosarcina pasteurii has attracted considerable interest in the area of geotechnical engineering due to its ability to induce extracellular mineralization. The presented study investigated S. pasteurii's potential to induce the mineralization of alkali-earth metal carbonate coatings on different polymeric 3D-printed flat surfaces fabricated by different additive manufacturing methods. The use of calcium, barium, strontium, or magnesium ions as the source resulted in the formation of vaterite (CaCO3), witherite (BaCO3), strontianite (SrCO3), and nesquehonite MgCO3·3H2O, respectively. These mineral coatings generally exhibit a compact, yet variable, thickness and are composed of agglomerated microparticles similar to those formed in solution. However, the mechanism behind this clustering remains unclear. The thermal properties of these biologically induced mineral coatings differ from their inorganic counterpart, highlighting the unique characteristics imparted by the biomineralization process. This work seeks to capitalize on the bacterium S. pasteurii's ability to form an alkali-earth metal carbonate coating to expand beyond its traditional use in geoengineering applications. It lays the ground for a novel integration of biologically induced mineralization of single or multilayered and multifunctional coating materials, for example, aerospace applications.

Uncovering the Dynamics of Urease and Carbonic Anhydrase Genes in Ureolysis, Carbon Dioxide Hydration, and Calcium Carbonate Precipitation.

The hydration of CO2 suffers from kinetic inefficiencies that make its natural trapping impractically sluggish. However, CO2-fixing carbonic anhydrases (CAs) remarkably accelerate its equilibration by 6 orders of magnitude and are, therefore, "ideal" catalysts. Notably, CA has been detected in ureolytic bacteria, suggesting its potential involvement in microbially induced carbonate precipitation (MICP), yet the dynamics of the urease (Ur) and CA genes remain poorly understood. Here, through the use of the ureolytic bacteriumSporosarcina pasteurii, we investigate the differing role of Ur and CA in ureolysis, CO2 hydration, and CaCO3 precipitation with increasing CO2(g) concentrations. We show that Ur gene up-regulation coincides with an increase in [HCO3-] following the hydration of CO2 to HCO3- by CA. Hence, CA physiologically promotes buffering, which enhances solubility trapping and affects the phase of the CaCO3 mineral formed. Understanding the role of CO2 hydration on the performance of ureolysis and CaCO3 precipitation provides essential new insights, required for the development of next-generation biocatalyzed CO2 trapping technologies.

Experimental analysis of biocementation technique for sealing cracks in concrete water storage tanks

Water supply systems are vital infrastructures that provide an indispensable public service to society. An important percentage of water losses is caused by the development of cracks in water storage tanks, so efficient crack sealing repair works must be investigated. Biocementation has been used with good results for sealing cracks in many construction concrete infrastructures (e.g., buildings), as an alternative to standard materials, such as polymeric resins and cement mortars. This research aims at the experimental evaluation of biocimentation effectiveness in terms of watertightness for sealing cracks structures in contact with pressurized water, to be further applied to repair cracks in water storage tanks. Biocementation treatment is applied in cracks artificially created in small rectangular concrete plates 4 cm thick, with three crack widths (i.e., 0.1, 1 and 10 mm) to cover different real cases in which the repair with this technique is viable. The 10 mm width cracks were filled with sand before the treatment. Different rounds of bacteria Sporosarcina pasteurii are applied, being the efficiency of the treatment investigated by performing watertightness tests through variable water head tests starting at 10 kPa (1 m of water). Dissolution is discarded through measurements of ultrasonic pulse velocities before and after the watertightness tests and the results are explained by images of thermographic camera. The presence of biocement is confirmed by mineralogical analysis of the precipitate extracted from the cracks after breaking the plates through bending tests. Obtained results are very promising since the biocement precipitated can completely stop water flow in the 0.1 mm cracks and, for the cracks with 1 mm and 10 mm widths, the treatment can reduce the initial flow rate, on average, by 95% and 98%, respectively. The performance of the 10 mm width cracks previously filled with sand was similar to that of the 1 mm cracks, where no sand was used. The material strength recovered after the treatment is not as good as desirable: although a maximum recovery of 95.5% of the initial strength (average values) is found for the smallest crack width and it was almost null for the other two crack widths. These results demonstrate that the technique is effective when watertightness is required, but not when material strength must also be recovered. Further research is required to investigate the maximum water pressures that can be applied to each case, eventual effects of biocimentation on water quality and the durability of the treatment.

Revisiting the urease production of MICP-relevant bacterium Sporosarcina pasteurii during cultivation

The ureolytic bacterium Sporosarcina pasteurii is commonly used for Microbial Induced Calcium Carbonate Precipitation (MICP). This process has a variety of applications in the fields of construction, geotechnical engineering, and environmental remediation. However, the factors influencing urease activity of S. pasteurii during cultivation are not yet fully understood. Even though there is debate over whether higher urease activity is in fact beneficial for MICP applications, knowledge about urease production can help provide ureolytic biomass more cost-effectively, reducing overall production costs. This study revisits the effect of various cultivation conditions on growth and urease activity by using an automated high-throughput microbioreactor system combined with a novel system for high-throughput urease activity quantification. This experimental set-up allows for unprecedented data density and enables new insights into urease production of S. pasteurii.Several factors were tested, including oxygen limitation, the feeding of urea, choice of ammonium salt, the proportion of ammonium salt in the medium, and lowering the pH during cultivation; none of these had a notable impact on urease production. Only the addition of a broad spectrum of nutrients through regular feeding with highly concentrated yeast extract and glucose led to a 70 % increased specific urease activity compared to a batch culture.These results can improve the understanding of the regulatory mechanisms governing urease expression in S. pasteurii during cultivation, and allow to adapt the cultivation process accordingly. Additionally, the new system for automated high-throughput enzyme activity determination presented in this study could be applied to optimize bioprocesses involving other ion producing enzymes.

Bacterially induced calcite formation at the surface of recycled concrete

The construction industry is one of the main sources of greenhouse gas emissions, and portland cement production is responsible for approximately 8 % of anthropogenic CO2 emissions. Microbially induced calcium precipitation (MICP) has the potential to partially replace cement or modify the properties of materials that would otherwise not find use in construction, for example, in concrete recycling. MICP might be an environmentally friendly method to improve the properties of recycled aggregates and form conglomerates from the finest fractions. In this paper, factors influencing MICP’s ability to solidify recycled concrete fines are thoroughly investigated. Calcium carbonate precipitate crystals produced by the bacterium Sporosarcina pasteurii were analyzed using scanning electron microscopy and energy dispersive spectroscopy.

An approach of repairing concrete vertical cracks using microbially induced carbonate precipitation driven by ion diffusion

Microbially induced carbonate precipitation (MICP) is an eco-friendly method of repairing cracks in concrete. However, for vertical cracks appeared in concrete components, the microbial mineralization solution injected into cracks tends to flow out due to gravity, resulting in incomplete crack repair. To address this obstacle, this study presents a novel approach in repairing vertical cracks using MICP with the ingression of calcium and urea driven by ion diffusion. For this purpose, a combined injection and diffusion method (CIDM) is proposed, in which the bacterial solution (ureolytic bacterium Sporosarcina Pasteurii) was first injected into crack and then the crack surface was adhered with a sponge containing the saturated cementation solution (urea and calcium acetate) for continuous ion diffusion. In comparison, the traditional injection method (TIM) is also conducted on the test cylindrical specimens. In addition to the pre-cracked concrete cylinders, four transversely load-induced cracked concrete beams were also utilized in this study and their vertical cracks were treated with CIDM under both loading and unloading conditions. The crack repair effects are evaluated by means of surface observations, microanalysis, water absorption tests and chloride penetration tests. Test results indicate that the proposed CIDM, whereby the calcium and urea are supplied through an ion diffusion approach, is a more effective method to repair vertical cracks than TIM. The generated white precipitations in cracks are confirmed to be calcium carbonate crystals existed in form of vaterite through energy dispersive spectrum (EDS) and X-ray diffraction (XRD) examinations, which also indicate that the CIDM method tends to produce a denser calcium carbonate cluster structure. After crack repairing with CIDM, the improvement coefficients of waterproof performance for the pre-cracked cylinders with widths of 0.1, 0.2 and 0.4 mm are 84.2%, 81.2% and 73.6%, respectively, which is 6%–12% higher than those got from TIM method. Furthermore, the average improvement coefficients of resistance to chloride penetration for vertical cracks with widths from 0.085 mm to 0.162 mm are about 76.9% and 88.5% for the sustain-loaded beams and unloaded beams, respectively.

Calcite-functionalized micromodels for pore-scale investigations of CO2 storage security

Carbon capture and subsequent storage (CCS) is identified as a necessity to achieve climate commitments. Permanent storage of carbon dioxide (CO2) in subsurface saline aquifers or depleted oil and gas reservoirs is feasible, but large-scale implementation of such storage has so far been slow. Although sandstone formations are currently most viable for CO2 sequestration, carbonates play an important role in widespread implementation of CCS; both due to the world-wide abundancy of saline aquifers in carbonate formations, and as candidates for CO2-EOR with combined storage. Acidification of formation brine during CO2 injection cause carbonate dissolution and development of reactive flow patterns. Using calcite-functionalization of micromodels we experimentally investigate fundamental pore-scale reactive transport dynamics relevant for carbonate CO2 storage security. Calcite-functionalized, two-dimensional and siliconbased, pore scale micromodels were used. Calcite precipitation was microbially induced from the bacteria Sporosarcina pasteurii and calcite grains were formed in-situ. This paper details an improved procedure for achieving controlled calcite precipitation in the pore space and characterizes the precipitation/mineralization process. The experimental setup featured a temperature-controlled micromodel holder attached to an automatic scanning stage. A high-resolution microscope enabled full-model (22x27 mm) image capture at resolution of 1.1 µm/pixel within 82 seconds. An in-house developed image-analysis python script was used to quantify porosity alterations due to calcite precipitation. The calcite-functionalized micromodels were found to replicate natural carbonate pore geometry and chemistry, and thus may be used to quantify calcite dissolution and reactive flow at the pore-scale.

Crack Healing and Mechanical Properties of Bacteria-based Self-healing Cement Mortar

In this study, the improvement of mechanical properties and crack healing as a result of the calcium carbonate precipitation due to bacterial activity have been investigated in two phases. First, the optimum mix design of self-healing cement mortar has been achieved considering different amounts and concentrations of the bacterial solution of bacterium Sporosarcina pasteurii (ATCC 11859) in non-pre-cracked specimens. Some of the mechanical properties, such as compressive strength, flexural strength, energy absorption capability, and weight change in bacteria added cement mortar specimens are compared with those of control specimens. Second, using the determined optimum mix design, mechanical properties of self-healing cement mortar specimens with initial cracks are compared with those of non-pre-cracked specimens to evaluate the recovery degree. 28-day compressive and flexural strengths of cement mortar specimens through direct addition of bacterial suspension with a concentration of 5.1 × 107 cells/ml improved by 45% and 18%, respectively. These results for 7-day specimens were 78% and 24%, respectively. Experimental flexural strengths of pre-cracked specimens are higher than their theoretical values based on the reduced cross-sections, and in pre-cracks with smaller dimensions, higher recovery degrees are achieved.

Refining Concrete’s micro-structure by enzymatically-induced carbonate precipitation through urease activity of bacteria

This paper presents the mechanism of improving the microstructure of concrete by enzymatically-induced carbonate precipitation through urease activity of bacteria Sporosarcina pasteurii. This calcium carbonate or calcite crystal precipitated during the biological activity fills the pores in the concrete imparting the concrete the dense microstructure. The presence of 105 per milliliter of mixing water yields high compressive strength due to precipitation of highest amount of calcium crystals in the concrete pores thereby plugging the micro and macro pores in the concrete refining the micro-structure of the concrete. SEM images, XRD plots and thermo-gravimetric analysis confirmed the presence of calcite mineral in the bacterial mortar specimens. The presence of spores of Sporosarcina pasteurii in the bacterial mortar specimens of 365 days old are found through phase contrast microscopic images. This confirms the sporulation ability of bacteria Sporosarcina pasteurii. Concrete specimens induced with bacteria showed considerably less water absorption capacity compared to normal specimens. This decrease in water absorption capacity of bacteria induced concretes is credited to the reduction of pores in the concrete due to calcite mineral precipitation filling the pores reducing the typical pore radius of concrete. Average pore diameter in bacteria incorporated concrete decreases drastically due to calcite mineral precipitation in the pores because of microbial metabolic activity. The total pore volume also decreased in case of bacteria incorporated concrete samples.

Behaviour of bacterial concrete subjected to elevated temperatures of various exposure times

In this paper the behaviour of M25 grade bacterial concrete exposed to various elevated temperature and thermal cycles of different durations of exposure. Introducing the bacteria Sporosarcina pasteurii into the concrete will employ the bio-mineralization phenomena to precipitate calcite minerals in the cavity pores and dried gel pores of the concrete improving the internal void structure of the concrete. So the current study will try to understand the integrity of the bacterial concrete when subjected to a different elevated temperatures of 200 ºC, 400 ºC and 600 ºC exposed for 2 hrs. , 4 hrs. and 6 hrs. duration. Compressive strength loss and weight loss is evaluated and internal structure quality is also assessed using non-destructive techniques as first part. Results suggest that till 500 -600 ºC bacteria mixed concrete produces superior results beyond that the behaviour is almost similar to that of normal concrete

Revealing nutritional requirements of MICP-relevant Sporosarcina pasteurii DSM33 for growth improvement in chemically defined and complex media

Microbial induced calcite precipitation (MICP) based on ureolysis has a high potential for many applications, e.g. restoration of construction materials. The gram-positive bacterium Sporosarcina pasteurii is the most commonly used microorganism for MICP due to its high ureolytic activity. However, Sporosarcina pasteurii is so far cultivated almost exclusively in complex media, which only results in moderate biomass concentrations at the best. Cultivation of Sporosarcina pasteurii must be strongly improved in order to make technological application of MICP economically feasible. The growth of Sporosarcina pasteurii DSM 33 was boosted by detecting auxotrophic deficiencies (L-methionine, L-cysteine, thiamine, nicotinic acid), nutritional requirements (phosphate, trace elements) and useful carbon sources (glucose, maltose, lactose, fructose, sucrose, acetate, L-proline, L-alanine). These were determined by microplate cultivations with online monitoring of biomass in a chemically defined medium and systematically omitting or substituting medium components. Persisting growth limitations were also detected, allowing further improvement of the chemically defined medium by the addition of glutamate group amino acids. Common complex media based on peptone and yeast extract were supplemented based on these findings. Optical density at the end of each cultivation of the improved peptone and yeast extract media roughly increased fivefold respectively. A maximum OD600 of 26.6 ± 0.7 (CDW: 17.1 ± 0.5 g/L) was reached with the improved yeast extract medium. Finally, culture performance and media improvement was analysed by measuring the oxygen transfer rate as well as the backscatter during shake flask cultivation.

SPORULATION OF SPOROSARCENA PASTEURII (BACILLUS PASTEURII) ON DIFFERENT NUTRIENT MEDIUM IN LABORATORY CONDITIONS

The bacterium Sporosarcina pasteurii can produce spore at the adverse environmental condition. The sporulation is different in different nutrients medium. This study helps to search out the better nutrient medium that can use in laboratory experiments for the sporulation of Sporosarcina pasteurii. As a result, the seven to ten days of observation showed that the Sporulation of Sporosarcina pasteurii is good in Nutrient agar medium than tryptic soy agar and lysogeny broth agar. In this study, used a hemocytometer for counting the spore on the surface of agar medium with the help of an optical microscope. SEM is used to analyze the morphology of the Sporosarcina pasteurii and its spores. It helps the research work related to Sporosarcina pasteurii in the microbiological and environmental research area.

Beneficial factors for biomineralization by ureolytic bacterium Sporosarcina pasteurii

BackgroundThe ureolytic bacterium Sporosarcina pasteurii is well-known for its capability of microbially induced calcite precipitation (MICP), representing a great potential in constructional engineering and material applications. However, the molecular mechanism for its biomineralization remains unresolved, as few studies were carried out.ResultsThe addition of urea into the culture medium provided an alkaline environment that is suitable for S. pasteurii. As compared to S. pasteurii cultivated without urea, S. pasteurii grown with urea showed faster growth and urease production, better shape, more negative surface charge and higher biomineralization ability. To survive the unfavorable growth environment due to the absence of urea, S. pasteurii up-regulated the expression of genes involved in urease production, ATPase synthesis and flagella, possibly occupying resources that can be deployed for MICP. As compared to non-mineralizing bacteria, S. pasteurii exhibited more negative cell surface charge for binding calcium ions and more robust cell structure as nucleation sites. During MICP process, the genes for ATPase synthesis in S. pasteurii was up-regulated while genes for urease production were unchanged. Interestingly, genes involved in flagella were down-regulated during MICP, which might lead to poor mobility of S. pasteurii. Meanwhile, genes in fatty acid degradation pathway were inhibited to maintain the intact cell structure found in calcite precipitation. Both weak mobility and intact cell structure are advantageous for S. pasteurii to serve as nucleation sites during MICP.ConclusionsFour factors are demonstrated to benefit the super performance of S. pasteurii in MICP. First, the good correlation of biomass growth and urease production of S. pasteurii provides sufficient biomass and urease simultaneously for improved biomineralization. Second, the highly negative cell surface charge of S. pasteurii is good for binding calcium ions. Third, the robust cell structure and fourth, the weak mobility, are key for S. pasteurii to be nucleation sites during MICP.

Controlling the Distribution of Microbially Precipitated Calcium Carbonate in Radial Flow Environments.

Bacterially driven reactions such as ureolysis can induce calcium carbonate precipitation, a well-studied process called microbially induced calcium carbonate precipitation (MICP). MICP is of interest in subsurface applications such as sealing leaks around wells. For effective field deployment, it is important to study MICP under radial flow conditions, which are relevant to near-well environments. In this study, a laboratory-scale radial flow reactor of 23 cm diameter, with a 1 mm glass bead monolayer serving as a porous medium, was used to investigate the effects of fluid flow rates and calcium concentrations on the mass and distribution of MICP by the ureolytic bacterium Sporosarcina pasteurii. Experiments were performed at hydraulic residence times of 14, 7, and 3.5 min and calcium to urea molar ratios of 0.5:1, 1:1, and 2:1. The total amount of CaCO3 precipitated in the reactor increased with increasing residence time and with decreasing Ca2+ to urea molar ratios. Increased bacterial attachment and increased CaCO3 precipitation were observed with distance from the center inlet of the reactor in all experiments. More uniform calcium distribution was achieved at lower flow rates. The relationship between reaction and transport rate (i.e., the Damköhler number) is identified as a useful parameter for the prediction of MICP in radial flow environments.

Towards a low CO2 emission building material employing bacterial metabolism (1/2): The bacterial system and prototype production.

The production of concrete for construction purposes is a major source of anthropogenic CO2 emissions. One promising avenue towards a more sustainable construction industry is to make use of naturally occurring mineral-microbe interactions, such as microbial-induced carbonate precipitation (MICP), to produce solid materials. In this paper, we present a new process where calcium carbonate in the form of powdered limestone is transformed to a binder material (termed BioZEment) through microbial dissolution and recrystallization. For the dissolution step, a suitable bacterial strain, closely related to Bacillus pumilus, was isolated from soil near a limestone quarry. We show that this strain produces organic acids from glucose, inducing the dissolution of calcium carbonate in an aqueous slurry of powdered limestone. In the second step, the dissolved limestone solution is used as the calcium source for MICP in sand packed syringe moulds. The amounts of acid produced and calcium carbonate dissolved are shown to depend on the amount of available oxygen as well as the degree of mixing. Precipitation is induced through the pH increase caused by the hydrolysis of urea, mediated by the enzyme urease, which is produced in situ by the bacterium Sporosarcina pasteurii DSM33. The degree of successful consolidation of sand by BioZEment was found to depend on both the amount of urea and the amount of glucose available in the dissolution reaction.

Cell-free soil bio-cementation with strength, dilatancy and fabric characterization

A multi-disciplinary approach is adopted in the present work towards investigating bio-cemented geo-materials which extends from sample preparation, to microstructural inspection and mechanical behaviour characterization. We suggest a new way to induce “cell-free” soil bio-cementation along with a comprehensive description of bio-improved mechanical and microstructural properties. We utilize the soil bacterium Sporosarcina Pasteurii in freeze-dried, powder—instead of vegetative—, state and determine overall reaction rates of “cell-free” microbial-induced calcite (CaCO3) precipitation (MICP). We further investigate strength and stiffness parameters of three base geo-materials which are subjected to MICP under identical external bio-treatment conditions. Different trends in the mechanical response under unconfined and drained triaxial compression are obtained for fine-, medium- and coarse-grained sands for similar range of final CaCO3 contents. Pre- and post-yield dilatancy–stress relationships are obtained revealing the contribution of dilatancy in the achievement of peak strength. Medium-grained sand yields higher dilatancy rates and increased peak strength with respect to fine-grained sand. Further, insight into the bio-cemented material’s fabric is provided through scanning electron microscopy, time-lapse video microscopy and X-ray micro-computed tomography with subsequent 3D reconstruction of the solid matrix. A qualitative description of the observed precipitation behaviours is coupled with quantified microscopic data referring to the number, sizes, orientations and purity of CaCO3 crystals. Results reveal that MICP adapts differently to the adopted base materials. Crystalline particles are found to grow bigger in the medium-grained base material and yield more homogenous spatial distributions. Finally, a new workflow is suggested to ultimately determine the crucial contact surface between calcite bonds and soil grains through image processing and 3D volume reconstruction.

Sporosarcina pasteurii can form nanoscale calcium carbonate crystals on cell surface.

The bacterium Sporosarcina pasteurii (SP) is known for its ability to cause the phenomenon of microbially induced calcium carbonate precipitation (MICP). We explored bacterial participation in the initial stages of the MICP process at the cellular length scale under two different growth environments (a) liquid culture (b) MICP in a soft agar (0.5%) column. In the liquid culture, ex-situ imaging of the cellular environment indicated that S. pasteurii was facilitating nucleation of nanoscale crystals of calcium carbonate on bacterial cell surface and its growth via ureolysis. During the same period, the meso-scale environment (bulk medium) was found to have overgrown calcium carbonate crystals. The effect of media components (urea, CaCl2), presence of live and dead in the growth medium were explored. The agar column method allows for in-situ visualization of the phenomena, and using this platform, we found conclusive evidence of the bacterial cell surface facilitating formation of nanoscale crystals in the microenvironment. Here also the bulk environment or the meso-scale environment was found to possess overgrown calcium carbonate crystals. Extensive elemental analysis using Energy dispersive X-ray spectroscopy (EDS) and X-ray powder diffraction (XRD), confirmed that the crystals to be calcium carbonate, and two different polymorphs (calcite and vaterite) were identified. Active participation of S. pasteurii cell surface as the site of calcium carbonate precipitation has been shown using EDS elemental mapping with Scanning transmission electron microscopy (STEM) and scanning electron microscopy (SEM).

Sporosarcina pasteurii can clog and strengthen a porous medium mimic.

The bacterium Sporosarcina pasteurii can produce significant volumes of solid precipitation in the presence of specific chemical environments. These solid precipitate particles can enter a network of microscale pores and cause long-range clogging. As a result, the medium gains strength and exhibits superior mechanical properties. This concept is also known as Microbiologically Induced Calcite Precipitation (MICP). In this study, we have used sponge blocks as surrogate porous media mimics and analyzed several aspects of MICP. A synergistic approach involving electron microscopy (SEM), computerized X-Ray tomography (μCT), quasi-static compressive load testing and chemical characterization (EDX) has been used to understand several physical and chemical aspects of MICP.

Biological aspects of microbial-induced calcite precipitation

Microbially-induced calcite precipitation (MICP) is an emerging ground-modification technique. This paper presents the results of laboratory experiments that elucidate some biological factors affecting bioaugmentation and biostimulation strategies of MICP. Co-culture experiments suggest that ureolytic bacterium Sporosarcina pasteurii (DSMZ 33) might release the enzyme urease once introduced into a medium containing non-ureolytic bacterium Bacillus subtilis (DSMZ 6397) due to lysis by the latter, resulting in uncontrolled calcite precipitation. This suggests that exogenous bacteria introduced into a native soil might not survive due to adverse action by indigenous bacteria. It is shown that effective biostimulation of indigenous ureolytic bacteria in low-nutrient sand can be achieved using a stimulation medium containing 200 mM urea, complemented with a simple carbon source (molasses). Changes in microbial population following stimulation were quantified, using genetic enumeration, to show that (a) the net increase in urease activity is not accompanied by increases in the relative abundance of ureolytic bacteria, (b) nitrifying bacteria are part of the enriched indigenous population and (c) nitrifying bacteria can be stimulated by the addition of ammonium only. The use of the lowest effective urea concentration and simple carbon is advocated for sustainable biostimulated MICP, yielding lower ammonium emissions and reduced post-treatment recovery overheads.