Fruit and vegetable waste used as bacterial growth media for the biocementation of two geomaterials.

This paper investigates the feasibility of using randomly collected fruit and vegetable (FV) waste as a cheap growing medium of bacteria for biocementation applications. Biocementation has been proposed in the literature as an environmentally-friendly ground improvement method to increase the stability of geomaterials, prevent erosion and encapsulate waste, but currently suffers from the high costs involved, such as bacteria cultivation costs. After analysis of FV waste of varied composition in terms of sugar and protein content, diluted FV waste was used to grow ureolytic (S. pasteurii, and B.licheniformis) and also an autochthonous heterotrophic carbonic anhydase (CA)-producing B.licheniformis strain, whose growth in FV media had not been attempted before. Bacterial growth and enzymatic activity in FV were of appropriate levels, although reduced compared to commercial media. Namely, the CA-producing B.licheniformis had a maximum OD600 of 1.799 and a CA activity of 0.817 U/mL in FV media. For the ureolytic pathway, B. licheniformis reached a maximum OD600 of 0.986 and a maximum urease activity of 0.675 mM urea/min, and S. pasteurii a maximum OD600 = 0.999 and a maximum urease activity of 0.756 mM urea/min. Biocementation of a clay and locomotive ash, a geomaterial specific to UK railway embankments, using precultured bacteria in FV was then proven, based on recorded unconfined compressive strengths of 1-3 MPa and calcite content increases of up to 4.02 and 8.62 % for the clay and ash respectively. Scanning Electron Microscope (SEM) and energy dispersive X-ray spectroscopy (EDS), attested the formation of bioprecipitates with characteristic morphologies and elementary composition of calcite crystals. These findings suggest the potential of employing FV to biocement these problematic geomaterials and are of wider relevance for environmental and geoenvironmental applications involving bioaugmentation. Such applications that require substrates in very large quantities can help tackle the management of the very voluminous fruit and vegetable waste produced worldwide.

Molecular and geochemical basis of microbially induced carbonate precipitation for treating acid mine drainage: The case of a novel Sporosarcina genomospecies from mine tailings.

Microbially induced carbonate precipitation (MICP) immobilizes toxic metals and reduces their bioavailability in aqueous systems. However, its application in the treatment of acid mine drainage (AMD) is poorly understood. In this study, the genomes of Sporosarcina sp. UB5 and UB10 were sequenced. Urease, carbonic anhydrases, and metal resistance genes were identified and enzymatic assays were performed for their validation. The geochemical mechanism of precipitation in AMD was elucidated through geo-mineralogical analysis. Sporosarcina sp. UB5 was shown to be a new genomospecies, with an average nucleotide identity < 95 % (ANI) and DNA-DNA hybridization < 70 % (DDH) whereas UB10 is close to S. pasteurii. UB5 contained two urease operons, whereas only one was identified in UB10. The ureolytic activities of UB5 and UB10 were 122.67 ± 15.74 and 131.70 ± 14.35 mM NH4+ min-1, respectively. Both strains feature several carbonic anhydrases of the α, ß, or γ families, which catalyzed the precipitation of CaCO3. Only Sporosarcina sp. UB5 was able to immobilize metals and neutralize AMD. Geo-mineralogical analyses revealed that UB5 directly immobilized Fe (1-23 %), Mn (0.65-1.33 %) and Zn (0.8-3 %) in AMD via MICP and indirectly through adsorption to calcite and binding to bacterial cell walls. The MICP-treated AMD exhibited high removal rates (>67 %) for Ag, Al, As, Ca, Cd, Co, Cu, Fe, Mn, Pb, and Zn, and a removal rate of 15 % for Mg. This study provides new insights into the MICP process and its applications to AMD treatment using autochthonous strains.

Synergistic enhancement of cadmium immobilization and soil fertility through biochar and artificial humic acid-assisted microbial-induced calcium carbonate precipitation.

Microbially induced carbonate precipitation (MICP) is emerging as a favorable alternative to traditional soil remediation techniques for heavy metals, primarily due to its environmental friendliness. However, a significant challenge in using MICP for farmland is not only to immobilize heavy metals but also to concurrently enhance soil fertility. This study explores the innovative combination of artificial humic acid (A-HA), biochar (BC), and Sporosarcina pasteurii (S. pasteurii) to mitigate the bioavailability of cadmium (Cd) in contaminated agricultural soils through MICP. X-ray diffraction (XRD) and scanning electron microscope (SEM) analyses revealed that the integration of BC and A-HA significantly enhances Cd immobilization efficiency by co-precipitating with CaCO3. Moreover, this treatment also improved soil fertility and ecological functions, as evidenced by increases in total nitrogen (TN, 9.0-78.2 %), alkaline hydrolysis nitrogen (AN, 259.7-635.5 %), soil organic matter (SOM, 18.1-27.9 %), total organic carbon (TOC, 43.8-48.8 %), dissolved organic carbon (DOC, 36.0-88.4 %) and available potassium (AK, 176.2-193.3 %). Additionally, the relative abundance of dominant phyla such as Proteobacteria and Firmicutes significantly increased with the introduction of BC and A-HA in MICP. Consequently, the integration of BC and A-HA with MICP offers a promising solution for remediating Cd-contaminated agricultural soil and synergistically enhancing soil fertility.

Microbially induced calcium carbonate precipitation through CO2 sequestration via an engineered Bacillus subtilis.

BACKGROUND: Microbially induced calcium carbonate precipitation has been extensively researched for geoengineering applications as well as diverse uses within the built environment. Bacteria play a crucial role in producing calcium carbonate minerals, via enzymes including carbonic anhydrase-an enzyme with the capability to hydrolyse CO2, commonly employed in carbon capture systems. This study describes previously uncharacterised carbonic anhydrase enzyme sequences capable of sequestering CO2 and subsequentially generating CaCO3 biominerals and suggests a route to produce carbon negative cementitious materials for the construction industry. RESULTS: Here, Bacillus subtilis was engineered to recombinantly express previously uncharacterised carbonic anhydrase enzymes from Bacillus megaterium and used as a whole cell catalyst allowing this novel bacterium to sequester CO2 and convert it to calcium carbonate. A significant decrease in CO2 was observed from 3800 PPM to 820 PPM upon induction of carbonic anhydrase and minerals recovered from these experiments were identified as calcite and vaterite using X-ray diffraction. Further experiments mixed the use of this enzyme (as a cell free extract) with Sporosarcina pasteurii to increase mineral production whilst maintaining a comparable level of CO2 sequestration. CONCLUSION: Recombinantly produced carbonic anhydrase successfully sequestered CO2 and converted it into calcium carbonate minerals using an engineered microbial system. Through this approach, a process to manufacture cementitious materials with carbon sequestration ability could be developed.

Enrichment of Terbium(III) under synergistic effect of biosorption and biomineralization by Bacillus sp. DW015 and Sporosarcina pasteurii.

Biosorption and biomineralization are commonly used for the immobilization of metal ions. Biosorption is commonly used as a green method to enrich rare earth ions from wastewater. However, little attention has been paid to the facilitating role of biomineralization in the enrichment of rare earth ions. In this study, a strain of Bacillus sp. DW015, isolated from ion adsorption type rare earth ores and a urease-producing strain Sporosarcina pasteurii were used to enrich rare earth elements (REEs) from an aqueous solution. The results indicate that biomineralization accelerates the enrichment of Terbium(III) compared to biosorption alone. Kinetic analysis suggests that the main mode of action of DW015 was biosorption, following pseudo-second-order kinetics (R2 = 0.998). The biomineralization of DW015 did not significantly contribute to the enrichment of Tb(III), whereas excessive biomineralization of S. pasteurii led to a decrease in the enrichment of Tb(III). A synergistic system of biosorption and biomineralization was established by combining the two bacteria, with the optimal mixed bacteria (S. pasteurii:DW015) ratio being 1:19. This study provides fundamental support for the synergistic effect of biosorption and biomineralization and offers a new reference for future microbial-based enrichment methods. IMPORTANCE: A weak microbially induced calcium carbonate precipitation (MICP) promotes the enrichment of Tb(III) by bacteria, while a strong MICP leads to the release of Tb(III). However, existing explanations cannot elucidate these mechanisms. In this study, the morphology of the bioprecipitation and the degree of Tb(III) enrichment were analyzed by X-ray diffraction (XRD), scanning electron microscopy (SEM), and energy dispersive spectroscopy (EDS). The data revealed that MICP could drive stable attachment of Tb(III) onto the cell surface, forming a Tb-CaCO3 mixed solid phase. Excessive rapid rate of calcite generation could disrupt the Tb(III) adsorption equilibrium, leading to the release of Tb(III). Therefore, in order for Tb(III) to be stably embedded in calcite, it is necessary to have a sufficient number of adsorption sites on the bacteria and to regulate the rate of MICP. This study provides theoretical support for the process design of MICP for the enrichment of rare earth ions.

Cyanobacterial Biocrust on Biomineralized Soil Mitigates Freeze-Thaw Effects and Preserves Structure and Ecological Functions.

Biocrust inoculation and microbially induced carbonate precipitation (MICP) are tools used in restoring degraded arid lands. It remains unclear whether the ecological functions of the two tools persist when these methods are combined and subjected to freeze-thaw (FT) cycles. We hypothesized a synergetic interaction between MICP treatment and biocrust under FT cycles, which would allow both components to retain their ecological functions. We grew cyanobacterial (Nostoc commune) biocrusts on bare soil and on MICP (Sporosarcina pasteurii)-treated soil, subjecting them to repeated FT cycles simulating the Mongolian climate. Generalized linear modeling revealed that FT cycling did not affect physical structure or related functions but could increase the productivity and reduce the nutrient condition of the crust. The results confirm the high tolerance of MICP-treated soil and biocrust to FT cycling. MICP treatment + biocrust maintained higher total carbohydrate content under FT stress. Our study indicates that biocrust on biomineralized soil has a robust enough structure to endure FT cycling during spring and autumn and to promote restoration of degraded lands.

ß-tricalcium phosphate enhanced biomineralization of Cd2+ and Pb2+ by Sporosarcina ureilytica HJ1 and Sporosarcina pasteurii HJ2.

Microbiologically induced CaCO3 precipitation (MICP) has been proposed as a potential bioremediation method to immobilize contaminating metals. In this study, carbonate mineralizing bacteria HJ1 and HJ2, isolated from heavy metal contaminated soil, was employed for Cd2+ and Pb2+ immobilization with or without ß-tricalcium phosphate addition. Compared with the only treatments amended with strains, the combined application of ß-tricalcium phosphate and HJ1 improved the immobilization rates of Cd and Pb by 1.49 and 1.70 times at 24 h, and the combined application of ß-tricalcium phosphate and HJ2 increased the immobilization rates of Cd and Pb by 1.25 and 1.79 times. The characterization of biomineralization products revealed that Cd2+ and Pb2+ primarily immobilized from the liquid phase as CdCO3 and PbCO3, and the addition of ß-tricalcium phosphate facilitated the formation of Ca4.03Cd0.97(PO4)3(OH) and Pb3(PO4)2. Also, the calcium source was related to the speciation of carbonate precipitation and improved the Cd and Pb remediation efficiency. This research demonstrated the feasibility and effectiveness of MICP combined with ß-tricalcium phosphate in immobilization of Cd and Pb, which will provide a fundamental basis for future applications of MICP to mitigate soil heavy metal pollutions.

Microbiologically induced calcite precipitation (MICP) in situ remediated heavy metal contamination in sludge nutrient soil.

Microbiologically induced calcite precipitation (MICP), as a newly developing bioremediation technology, could redeem heavy metal contamination in diverse scenarios. In this study, MICP bacterium Sporosarcina ureilytica ML-2 was employed to suppress the pollution of Pb, Cd and Zn in municipal sludge nutrient soil. After MICP remediation, the exchangeable Cd and Zn in sludge nutrient soil were correspondingly reduced by 31.02 % and 6.09 %, while the carbonate-bound Pb, Cd and Zn as well as the residual fractions were increased by 16.12 %, 6.63 %, 13.09 % and 6.10 %, 45.70 %, 3.86 %, respectively. In addition, the extractable Pb, Cd and Zn either by diethylenetriaminepentaacetic acid (DTPA) or toxicity characteristic leaching procedure (TCLP) in sludge nutrient soil were significantly reduced. These results demonstrated that the bio-calcite generated via MICP helped to immobilize heavy metals. Furthermore, MICP treatment improved the abundance of functional microorganisms related to urea cycle, while reduced the overall abundance of metal resistance genes (MRGs) and antibiotic resistance genes (ARGs). This work confirmed the feasibility of MICP in remediation of heavy metal in sludge nutrient soil, which expanded the application field of MICP and provided a promising way for heavy metal pollution management.

Sporosarcina jeotgali sp. nov., Sporosarcina oncorhynchi sp. nov., and Sporosarcina trichiuri sp. nov., Isolated from Jeotgal, a Traditional Korean Fermented Seafood.

Three novel, Gram-stain-positive, obligate aerobic, catalase- and oxidase-positive bacterial strains, designated B2O-1T, T2O-4T, and 0.2-SM1T-5T, were isolated from jeotgal, a traditional Korean fermented seafood. Strains B2O-1T, T2O-4T, and 0.2-SM1T-5T exhibited distinct colony colors, characterized by pink, yellow, and red opaque circular colonies, respectively. Phylogenetic analysis revealed that three strains formed a paraphyletic clade within the genus Sporosarcina and shared < 99.0% similarity with Sporosarcina aquimarina KCTC 3840T and Sporosarcina saromensis KCTC 13119T in their 16S rRNA gene sequences. The three strains exhibiting Orthologous Average Nucleotide Identity values < 79.3% and digital DNA-DNA hybridization values < 23.1% within the genus Sporosarcina affirmed their distinctiveness. Strains B2O-1T, T2O-4T, and 0.2-SM1T-5T contained MK-7 as a sole respiratory menaquinone and A4α type peptidoglycan based on lysine with alanine, glutamic acid, and aspartic acid. The common polar lipids include diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. Strain T2O-4T contained one unidentified phospholipid, whereas strain 0.2-SM1T-5T contained two unidentified phospholipids. Cellular fatty acid profiles, with C15:0 anteiso as the major fatty acid, supported the affiliation of the three strains to the genus Sporosarcina. Based on the polyphasic characteristics, strains B2O-1T (= KCTC 43506T = JCM 36032T), T2O-4T (= KCTC 43489T = JCM 36031T), and 0.2-SM1T-5T (= KCTC 43519T = JCM 36034T) represent three novel species within the genus Sporosarcina, named Sporosarcina jeotgali sp. nov., Sporosarcina oncorhynchi sp. nov., and Sporosarcina trichiuri sp. nov., respectively.

Effect of (in)organic additives on microbially induced calcium carbonate precipitation.

AIM: This study aimed to understand the morphological effects of (in)organic additives on microbially induced calcium carbonate precipitation (MICP). METHODS AND RESULTS: MICP was monitored in real time in the presence of (in)organic additives: bovine serum albumin (BSA), biofilm surface layer protein A (BslA), magnesium chloride (MgCl2), and poly-l-lysine. This monitoring was carried out using confocal microscopy to observe the formation of CaCO3 from the point of nucleation, in comparison to conditions without additives. Complementary methodologies, namely scanning electron microscopy, energy-dispersive X-ray spectroscopy and X-ray diffraction, were employed to assess the visual morphology, elemental composition, and crystalline structures of CaCO3, respectively, following the crystals' formation. The results demonstrated that in the presence of additives, more CaCO3 crystals were produced at 100 min compared to the reaction without additives. The inclusion of BslA resulted in larger crystals than reactions containing other additives, including MgCl2. BSA induced a significant number of crystals from the early stages of the reaction (20 min) but did not have a substantial impact on crystal size compared to conditions without additives. All additives led to a higher content of calcite compared to vaterite after a 24-h reaction, with the exception of MgCl2, which produced a substantial quantity of magnesium calcite. CONCLUSIONS: The work demonstrates the effect of several (in)organic additives on MICP and sets the stage for further research to understand additive effects on MICP to achieve controlled CaCO3 precipitation.

Microbially Induced Calcium Carbonate Precipitation by Sporosarcina pasteurii: a Case Study in Optimizing Biological CaCO3 Precipitation.

Current production of traditional concrete requires enormous energy investment that accounts for approximately 5 to 8% of the world's annual CO2 production. Biocement is a building material that is already in industrial use and has the potential to rival traditional concrete as a more convenient and more environmentally friendly alternative. Biocement relies on biological structures (enzymes, cells, and/or cellular superstructures) to mineralize and bind particles in aggregate materials (e.g., sand and soil particles). Sporosarcina pasteurii is a workhorse organism for biocementation, but most research to date has focused on S. pasteurii as a building material rather than a biological system. In this review, we synthesize available materials science, microbiology, biochemistry, and cell biology evidence regarding biological CaCO3 precipitation and the role of microbes in microbially induced calcium carbonate precipitation (MICP) with a focus on S. pasteurii. Based on the available information, we provide a model that describes the molecular and cellular processes involved in converting feedstock material (urea and Ca2+) into cement. The model provides a foundational framework that we use to highlight particular targets for researchers as they proceed into optimizing the biology of MICP for biocement production.

Microbiologically induced calcite precipitation for in situ stabilization of heavy metals contributes to land application of sewage sludge.

Microbiologically induced calcite precipitation (MICP) has shed new light on solving the problem of in situ stabilization of heavy metals (HMs) in sewage sludge before land disposal. In this study, we examined whether MICP treatment can be integrated into a sewage sludge anaerobic digestion-land application process. Our results showed that MICP treatment not only prevented the transfer of ionic-state Cd from the sludge to the supernatant (98.46 % immobilization efficiency) but also reduced the soluble exchangeable Pb and Cd fractions by up to 100 % and 48.54 % and increased the residual fractions by 22.54 % and 81.77 %, respectively. In addition, the analysis of the stability of HMs in MICP-treated sludge revealed maximum reductions of 100 % and 89.56 % for TCLP-extractable Pb and Cd, respectively. Three-dimensional fluorescence, scanning electron microscopy-energy-dispersive X-ray spectroscopy, X-ray diffraction, and Fourier-transform infrared spectroscopy analyses confirmed the excellent performance of the ureolytic bacteria Sporosarcina ureilytica ML-2 in the sludge system. High-throughput sequencing showed that the relative abundance of Sporosarcina sp. reached 53.18 % in MICP-treated sludge, and the urease metabolism functional genes unit increased by a maximum of 239.3 %. The MICP technology may be a feasible method for permanently stabilizing HMs in sewage sludge before land disposal.

Role of Na-Montmorillonite on Microbially Induced Calcium Carbonate Precipitation.

The use of additives has generated significant attention due to their extensive application in the microbially induced calcium carbonate precipitation (MICP) process. This study aims to discuss the effects of Na-montmorillonite (Na-MMT) on CaCO3 crystallization and sandy soil consolidation through the MICP process. Compared with the traditional MICP method, a larger amount of CaCO3 precipitate was obtained. Moreover, the reaction of Ca2+ ions was accelerated, and bacteria were absorbed by a small amount of Na-MMT. Meanwhile, an increase in the total cementing solution (TCS) was not conducive to the previous reaction. This problem was solved by conducting the reaction with Na-MMT. The polymorphs and morphologies of the CaCO3 precipitates were tested by using X-ray diffraction and scanning electron microscopy. Further, when Na-MMT was used, the morphology of CaCO3 changed from an individual precipitate to agglomerations of the precipitate. Compared to the experiments without Na-MMT in the MICP process, the addition of Na-MMT significantly reduced the hydraulic conductivity (HC) of sandy soil consolidated.

Influence of native ureolytic microbial community on biocementation potential of Sporosarcina pasteurii.

Microbially induced calcium carbonate precipitation (MICP)/Biocementation has emerged as a promising technique for soil engineering applications. There are chiefly two methods by which MICP is applied for field applications including biostimulation and bioaugmentation. Although bioaugmentation strategy using efficient ureolytic biocementing culture of Sporosarcina pasteurii is widely practiced, the impact of native ureolytic microbial communities (NUMC) on CaCO3 mineralisation via S. pasteurii has not been explored. In this paper, we investigated the effect of different concentrations of NUMC on MICP kinetics and biomineral properties in the presence and absence of S. pasteurii. Kinetic analysis showed that the biocementation potential of S. pasteurii is sixfold higher than NUMC and is not significantly impacted even when the concentration of the NUMC is eight times higher. Micrographic results revealed a quick rate of CaCO3 precipitation by S. pasteurii leading to generation of smaller CaCO3 crystals (5-40 µm), while slow rate of CaCO3 precipitation by NUMC led to creation of larger CaCO3 crystals (35-100 µm). Mineralogical results showed the predominance of calcite phase in both sets. The outcome of current study is crucial for tailor-made applications of MICP.

Improvement of Biomineralization of Sporosarcina pasteurii as Biocementing Material for Concrete Repair by Atmospheric and Room Temperature Plasma Mutagenesis and Response Surface Methodology.

Microbially induced calcium carbonate precipitation (MICP) has recently become an intelligent and environmentally friendly method for repairing cracks in concrete. To improve on this ability of microbial materials concrete repair, we applied random mutagenesis and optimization of mineralization conditions to improve the quantity and crystal form of microbially precipitated calcium carbonate. Sporosarcina pasteurii ATCC 11859 was used as the starting strain to obtain the mutant with high urease activity by atmospheric and room temperature plasma (ARTP) mutagenesis. Next, we investigated the optimal biomineralization conditions and precipitation crystal form using Plackett-Burman experimental design and response surface methodology (RSM). Biomineralization with 0.73 mol/l calcium chloride, 45 g/l urea, reaction temperature of 45°C, and reaction time of 22 h, significantly increased the amount of precipitated calcium carbonate, which was deposited in the form of calcite crystals. Finally, the repair of concrete using the optimized biomineralization process was evaluated. A comparison of water absorption and adhesion of concrete specimens before and after repairs showed that concrete cracks and surface defects could be efficiently repaired. This study provides a new method to engineer biocementing material for concrete repair.

Contribution of bacterial cells as nucleation centers in microbiologically induced CaCO3 precipitation-A mathematical modeling approach.

The microbiologically induced calcite precipitation (MICP) has been extensively studied for geotechnical engineering through simultaneous action of natural phenomena and engineering processes. The focus of bacterial contribution to the MICP has been directed to calcium carbonate productivity, while the additional bacterial role as a crystal nucleation center was not explained especially from a mathematical prediction modeling point of view. Therefore, this study provides explanations and a mathematical modeling approach of bacterial influence on the MICP induced by newly-isolated ureolytic Bacillus strains and Sporosarcina pasteurii DSM 33. Using the obtained results of low-cost, rapid, and simple assays, artificial neural network modeling was applied for cell surface predispositions, pH changes as well as calcium-involved function in biofilm formation during the MICP, for the first time. Based on the obtained contribution of the alkalophilic/alkaloresistant bacteria, calcite precipitation can be significantly directed by the presence, of ureolytic bacterial cells as nucleation centers during CaCO3 precipitation as well as their morphology, surface characteristics, potential to form a biofilm, and/or generate pH changes.

Transcriptome analyses reveal the utilization of nitrogen sources and related metabolic mechanisms of Sporosarcina pasteurii.

In recent years, Sporosarcina pasteurii (S. pasteurii) has become one of the most popular bacteria in microbially induced calcium carbonate precipitation (MICP). Various applications have been developed based on the efficient urease that can induce the precipitation of calcium carbonate. However, the metabolic mechanism related to biomineralization of S. pasteurii has not been clearly elucidated. The process of bacterial culture and biomineralization consumes a large amount of urea or ammonium salts, which are usually used as agricultural fertilizers, not to mention probable environmental pollutions caused by the excessive use of these raw materials. Therefore, it is urgent to reveal the mechanism of nitrogen utilization and metabolism of S. pasteurii. In this paper, we compared the growth and gene expression of S. pasteurii under three different culture conditions through transcriptome analyses. GO and KEGG analyses revealed that both ammonium and urea were direct nitrogen sources of S. pasteurii, and the bacteria could not grow normally in the absence of ammonium or urea. To the best of our knowledge, this paper is the first one to reveal the nitrogen utilization mechanism of S. pasteurii through transcriptome methods. Furthermore, the presence of ammonium might promote the synthesis of intracellular ATP and enhance the motility of the bacteria. There should be an ATP synthesis mechanism associated with urea hydrolysis catalyzed by urease in S. pasteurii.

Calcite seed-assisted microbial induced carbonate precipitation (MICP).

Microbial-induced calcium carbonate precipitation (MICP) is a biological process inducing biomineralization of CaCO3. This can be used to form a solid, concrete-like material. To be able to use MICP successfully to produce solid materials, it is important to understand the formation process of the material in detail. It is well known that crystallization surfaces can influence the precipitation process. Therefore, we present in this contribution a systematic study investigating the influence of calcite seeds on the MICP process. We focus on the changes in the pH and changes of the optical density (OD) signal measured with absorption spectroscopy to analyze the precipitation process. Furthermore, optical microscopy was used to visualize the precipitation processes in the sample and connect them to changes in the pH and OD. We show, that there is a significant difference in the pH evolution between samples with and without calcite seeds present and that the shape of the pH evolution and the changes in OD can give detailed information about the mineral precipitation and transformations. In the presented experiments we show, that amorphous calcium carbonate (ACC) can also precipitate in the presence of initial calcite seeds and this can have implications for consolidated MICP materials.

Non-sterile corn steep liquor a novel, cost effective and powerful culture media for Sporosarcina pasteurii cultivation for sand improvement.

AIMS: Microbial induced calcium carbonate precipitation (MICP) is one of the bio-cementation methods for improving granular soils. This study evaluate the feasibility of obtaining a bacterial solution with high optical density and urease activity by an inexpensive corn steep liquor (CSL) medium in non-sterile conditions in order to achieve sand improvement. METHODS AND RESULTS: Corn steep liquor media with different concentrations (different dilution rates) were prepared and, without any autoclaving (non-sterile conditions), different percentage of the inoculum solutions were added to them and incubated. Effect of inoculum solution percentage and CSL dilution rates on specifications of bacterial solution was evaluated. Urease activity and scanning electron microscope (SEM) and X-Ray Diffraction (XRD) were used to efficiency of CLS media in sand improvement. The considerable urease activity was measured as 5·7 mS cm-1  min-1 using nonsterile CLS. By using CYNU (CSL-Yeast extract-NH4Cl-Urea) bacterial solution, the urease activity of 5·5 mS cm-1  min-1 for the OD600 (optical density at 600 nm) of 1·88 and, consequently, specific urease activity of 2·93 mS cm-1  min-1  OD600 -1 was obtained. The highest unconfined compressive strength (811 kPa) was obtained for the CYNU. XRD revealed new calcite peaks next to the quartz peaks. CONCLUSIONS: Production of inexpensive bacterial solution using diluted CSL as the inexpensive, effective and powerful culture media for Sporosarcina pasteurii cultivation in nonsterile conditions, allows geotechnical and biotechnological engineers to use MICP technology more widely in land improvement and field-scale bio-cementation and bioremediation projects. SIGNIFICANCE AND IMPACT OF THE STUDY: Obtaining high urease activity of inexpensive microbial solution using diluted CSL as the culture medium in nonsterile conditions, as the unique results of this study, can be significant in the field of bioremediation studies using MICP.

Beneficial factors for biomineralization by ureolytic bacterium Sporosarcina pasteurii.

BACKGROUND: The 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. RESULTS: The 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. CONCLUSIONS: Four 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.