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.

Biogenic calcium improved Cd2+ and Pb2+ immobilization in soil using the ureolytic bacteria Bacillus pasteurii.

Bioremediation based on microbial-induced carbonate precipitation (MICP) was conducted in cadmium and lead contaminated soil to investigate the effects of MICP on Cd and Pb in soil. In this study, soil indigenous nitrogen was shown to induce MICP to stabilize heavy metals without inputting exogenous urea. The results showed that applying Bacillus pasteurii coupled with CaCl2 reduced Cd and Pb bioavailability, which could be clarified through the proportion of exchangeable Cd and Pb in soil decreasing by 23.65 % and 12.76 %, respectively. Moreover, B. pasteurii was combined separately with hydroxyapatite (HAP), eggshells (ES), and oyster shells (OS) to investigate their effects on soil heavy metals' chemical fractions, toxicity characteristic leaching procedure (TCLP)-extractable Cd and Pb as well as enzymatic activity. Results showed that applying B. pasteurii in soil significantly decreased the heavy metals in the exchangeable fraction and increased them in the carbonate phase fraction. When B. pasteurii was combined with ES and OS, the content of carbonate-bound Cd increased by 114.72 % and 118.81 %, respectively, significantly higher than when B. pasteurii was combined with HAP, wherein the fraction of carbonate-bound Cd increased by 86 %. The combination of B. pasteurii and biogenic calcium effectively reduced the leached contents of Cd and Pb in soil, and the TCLP-extractable Cd and Pb fractions decreased by 43.88 % and 30.66 %, respectively, in the BP + ES group and by 52.60 % and 41.77 %, respectively, in the BP + OS group. This proved that MICP reduced heavy metal bioavailability in the soil. Meanwhile, applying B. pasteurii and calcium materials significantly increased the soil urease enzyme activity. The microstructure and chemical composition of the soil samples were studied, and the results from scanning electron microscope, Fourier transform infra-red spectroscopy, and X-ray diffraction demonstrated the MICP process and identified the formation of CaCO3, Ca0.67Cd0.33CO3, and PbCO3 in heavy metal-contaminated soil.

Biotrapping Ureolytic Bacteria on Sand to Improve the Efficiency of Biocementation.

Microbially induced calcium carbonate precipitation (MICP) has emerged as a novel technology with the potential to produce building materials through lower-temperature processes. The formation of calcium carbonate bridges in MICP allows the biocementation of aggregate particles to produce biobricks. Current approaches require several pulses of microbes and mineralization media to increase the quantity of calcium carbonate minerals and improve the strength of the material, thus leading to a reduction in sustainability. One potential technique to improve the efficiency of strength development involves trapping the bacteria on the aggregate surfaces using silane coupling agents such as positively charged 3-aminopropyl-methyl-diethoxysilane (APMDES). This treatment traps bacteria on sand through electrostatic interactions that attract negatively charged walls of bacteria to positively charged amine groups. The APMDES treatment promoted an abundant and immediate association of bacteria with sand, increasing the spatial density of ureolytic microbes on sand and promoting efficient initial calcium carbonate precipitation. Though microbial viability was compromised by treatment, urea hydrolysis was minimally affected. Strength was gained much more rapidly for the APMDES-treated sand than for the untreated sand. Three injections of bacteria and biomineralization media using APMDES-treated sand led to the same strength gain as seven injections using untreated sand. The higher strength with APMDES treatment was not explained by increased calcium carbonate accrual in the structure and may be influenced by additional factors such as differences in the microstructure of calcium carbonate bridges between sand particles. Overall, incorporating pretreatment methods, such as amine silane coupling agents, opens a new avenue in biomineralization research by producing materials with an improved efficiency and sustainability.

Structural and compositional diversity of biosurfactants produced by a novel strain of Sporosarcina luteola ME44 from oil reservoir.

The urealytically active microorganism Sporosarcina luteola induces the precipitation of metals, which has attracted attention in biomineralization, bioremediation, and industrial waste recycling. Herein, we report a novel biosurfactant-producing strain of S. luteola ME44 isolated from Chinese Oilfield. The structure, composition, and surface activity of the biosurfactants produced by S. luteola ME44 were investigated by using a combination of the high-performance liquid chromatography, time-of-flight mass spectrometry, and surface tensiometer. The biosurfactant extracted by strain ME44 was identified as surfactin with five variants and the yield was 1010 ± 60 mg⋅L-1 . This is the first report on the structural composition and surface activity of biosurfactants isolated from the S. luteola. It extended our knowledge about the role of the species S. luteola in the ecosystem of extreme natural environments such as oil reservoir. In addition, S. luteola ME44 showed bioprecipitation properties for metal ions Cd(II), Cu(II), Zn(II), and Ag(I), which indicated the application potential of S. luteola in the field of bioremediation.

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.

Bioreduction and mineralization of Cr(VI) by Sporosarcina saromensis W5 induced carbonate precipitation.

The microbial reduction of Cr(VI) to Cr(III) is widely applied, but most studies ignored the stability of reduction products. In this study, the Cr(VI)-reducing bacterium of Sporosarcina saromensis combined with microbially induced carbonate precipitation (MICP) was used to explore the reduction and mineralization mechanisms of Cr(VI). The results indicated that the high concentration of Ca2+ could significantly enhance the reduction and mineralization of Cr(VI). The highest reduction and mineralization efficiencies of 99.5% and 55.9% were achieved at 4 g/L Ca2+. Moreover, the urease activity of S. saromensis in the experimental group was up to 13.28 U/mg NH3-N. Besides, the characteristic results revealed that Cr(VI) and reduced Cr(III) were absorbed on the surface or got into the interspace of CaCO3, which produced a new stable phase (Ca10Cr6O24(CO3)). Overall, the combination of S. saromensis and MICP technology might be a high-efficiency and environmentally friendly strategy for further application in the Cr(VI)-containing groundwater.

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.

Study on MICP dust suppression technology in open pit coal mine: Preparation and mechanism of microbial dust suppression material.

With the recent increases in energy demands, the dust hazards of coal mining caused by transportation, loading and unloading and other processes are becoming increasingly serious. To control dust in open pit coal mines more environmentally friendly and efficiently, and to promote the use and development of non-in situ high-yield urease microorganisms for dust suppression in coal mines, Bacillus pasteurii was selected for dust suppression experiments in this article. Additionally, the growth of microorganisms in the coal dust microenvironment was simulated, and the effect of microbial mineralization products on the calorific value of upper coal dust was further studied. Our findings indicated that Bacillus pasteurii induced dust suppression by forming a calcite precipitate with non-uniform particle size to coal dust cementation. Moreover, after a single spray, the wind erosion resistance efficiency was 84% when the wind speed was set at 10 m/s. The growth of microorganisms and urease activity in the coal dust leachate were largely equal to those in the control group, reaching a peak at approximately 24 h, that the maximum growth quantity of OD600 was about 1.5, and the maximum urease activity was 11 mmol·L-1·min-1. The difference between the peak heat release rate of mixed coal dust and pure coal was only 4.82 kW/m2, which would not affect the value of coal products. Non in-situ Bacillus pasteurii can be growth metabolized normally in the microenvironment of coal dust. Finally, the mechanism of coal dust suppression by mineralization of microbial bacterial solution to form calcium carbonate was described by a reaction equation, which is important for further application and development of microbial dust suppressants.

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.

Application of urease-producing microbial community in seawater to dust suppression in desert.

In order to solve the dust problem caused by sandstorms, this paper aims to propose a new method of enriching urease-producing microbial communities in seawater in a non-sterile environment. Besides, the difference of dust suppression performance of enriched microorganisms under different pH conditions was also explored to adapt the dust. The Fourier-transform infrared spectrometry (FTIR) and Scanning electron microscopy (SEM) confirmed the formation of CaCO3. The X-ray diffraction (XRD) further showed that the crystal forms of CaCO3 were calcite and vaterite. When urease activity was equivalent, the alkaline environment was conducive to the transformation of CaCO3 to more stable calcite. The mineralization rate at pH = 10 reached the maximum value on the 7th day, which was 97.49 ± 1.73%. Moreover, microbial community analysis results showed that the relative abundance of microbial community structure was different under different pH enrichment. Besides, the relative abundance of Sporosarcina, a representative genus of urease-producing microbial community, increased with the increase of pH under culture conditions, which consistent with the mineralization performance results. In addition, the genus level species network diagram also showed that in the microbial community, Sporosarcina was negatively correlated with another urease-producing genus Bacillus, and had a reciprocal relationship with Atopostipes, which means that the urease-producing microbial community was structurally stable. The enrichment of urease-producing microbial communities in seawater will provide empirical support for the large-scale engineering application of MICP technology in preventing and controlling sandstorms in deserts.

Taxonomic and genomic characterization of Sporosarcina cyprini sp. nov., moderately tolerant of Cr+6 and Cd+2 isolated from the gut of invasive fish Cyprinus carpio var. communis (Linn., 1758).

A Gram-stain-positive, motile, and rod-shaped bacterium, designated as strain MB25T, was isolated from the gut of Cyprinus carpio from the highly polluted river Yamuna, India. Phylogenetic analysis based on 16S rRNA gene sequence revealed that strain MB25T belonged to the genus Sporosarcina, sharing the highest sequence similarity with S. luteola Y1T (98.98%) and S. koreensis S-K12T (98.91%). Digital DNA-DNA hybridization and average nucleotide identity values of strain MB25T with strain Y1T and S-K12T were 18.9, 77.69, and 18.2, 76.80 respectively. Genome analysis of strain MB25T revealed its biotechnological properties such as tolerance to potent heavy metals, genes for the production of carbohydrate-active enzymes, antimicrobial compounds, and also degradation of aromatic compounds. The G + C content of strain MB25T genome was 45%. Growth observed at 10-40 °C (optimum, 28-30 °C), pH 6.0-8.5 (optimum pH 7.5-8.0); NaCl concentrations up to 6.0% (w/v). The dominant respiratory quinone was MK-7, cell wall peptidoglycan is of the A-4 type containing amino acids Lys-Glu and the major fatty acids are anteiso-C11:0 and iso-C15: 0. The major polar lipids of strain MB25T are diphosphatidylglycerol, phosphatidylglycerol, and phosphatidylethanolamine. On the basis of phenotypic, chemotaxonomic, phylogenetic, and phylogenomic data, strain MB25T represents a novel species of the genus Sporosarcina, for which the name Sporosarcina cyprini sp. nov. is proposed. The type strain is MB25T (= MCC 4366 T = JCM 34521 T = CCM 9113 T).

Experimental study on tailings cementation by MICP technique with immersion curing.

The filling mining method is an effective method for controlling ground stress and preventing surface subsidence in the mining field during exploitation of underground resources. Tailings can be utilized as the filling material, so as to realize the reuse of industrial waste. However, utilization of the traditional Portland cement as the cementing material for tailings leads to groundwater pollution. In addition, production of Portland cement results in consumption of a great amount of ore and air pollution. In this paper, a tailings cementation method by using the microbial induced calcite precipitation (MICP) technique with immersion curing is proposed. Tailings are cemented by the MICP technique with aerobic bacteria (Sporosarcina pasteurii) under a soaked curing environment. The variable control method is applied to investigate the factors influencing the cementation effects by the MICP technique with Sporosarcina pasteurii, including the bacterial solution concentration, the cementing solution concentration, the particle size of tailings, and the curing temperature. The results indicate that: when OD600 of the Sporosarcina pasteurii solution is 1.6, the urea concentration in the cementing solution is 0.75 mol/L, the tailings are raw materials without grinding, and the curing temperature is 30°C, the cementation effect is the best. In view of uneven calcification during MICP with Sporosarcina pasteurii, mixed Sporosarcina pasteurii and Castellaniella denitrificans are used for tailings cementation. Higher strength of cemented tailings is achieved. It is proved that the MICP technique with mixed aerobic bacteria and facultative anaerobes is an effective method for tailings cementation.

Microdroplet-Based In Situ Characterization Of The Dynamic Evolution Of Amorphous Calcium Carbonate during Microbially Induced Calcium Carbonate Precipitation.

Amorphous calcium carbonate (ACC) plays an important role in microbially induced calcium carbonate precipitation (MICP), which has great potential in broad applications such as building restoration, CO2 sequestration, and bioremediation of heavy metals, etc. However, our understanding of ACC is still limited. By combining microscopy of cell-laden microdroplets with confocal Raman microspectroscopy, we investigated the ACC dynamics during MICP. The results show that MICP inside droplets can be divided into three stages: liquid, gel-like ACC, and precipitated CaCO3 stages. In the liquid stage, the droplets are transparent. As the MICP process continues into the gel-like stage, the ACC structure appears and the droplets become opaque. Subsequently, dissolution of the gel-like structure is accompanied by growth of precipitated CaCO3 crystals. The size, morphology, and lifetime of the gel-like structures depend on the Ca2+ concentration. Using polystyrene colloids as tracers, we find that the colloids exhibit diffusive behavior in both the liquid and precipitated CaCO3 stages, while their motion becomes arrested in the gel-like ACC stage. These results provide direct evidence for the formation-dissolution process of the ACC-formed structure and its gel-like mechanical properties. Our work provides a detailed view of the time evolution of ACC and its mechanical properties at the microscale level, which has been lacking in previous studies.

Crystal transformation and self-assembly theory of microbially induced calcium carbonate precipitation.

Microbially induced calcium carbonate precipitation (MICP) is ubiquitous in the earth's lithosphere and brings the inspiration of bionic cementation technology. Over recent years, MICP has been proposed as a potential solution to address many environmental and engineering issues. However, the stability of cemented precipitations generated via MICP technology, especially the characteristics and change mechanism of crystal forms, is still unclear, which substantially hindered the understanding of biomineralization and prohibited the application and upscaling of MICP technology. Here, Sporosarcina pasteurii was selected as a model microbe to induce calcium carbonate mineralization in a series of standard nutrient solutions. The authors studied the process of precipitation from amorphous calcium carbonate to calcite crystal form and revealed the assembly behavior and mechanism of precipitations by FTIR, SEM, TEM and EDS. In the two crystal forms of induced calcium carbonate, the relative position and content of C, O, N, P and Ca elements were only slightly different. The molecular attachment and structural match of organic matrix made the crystals form change. Finally, a self-assembly theory was proposed to MICP, and it provided a solid theoretical basis for the technical specification of MICP technology in engineering application. KEY POINTS: • Organic matrix is intensively involved in MICP by forming functional groups. • Molecular attachment and structural match cause calcite crystal evolution. • A self-assembly theory is proposed for MICP.

Best-performing Bacillus strains for microbiologically induced CaCO3 precipitation: Screening of relative influence of operational and environmental factors.

The microbiologically induced calcite precipitation (MICP) can be an emerging approach that could tap onto soil bacterial diversity and use as a bioremediation technique. Based on the concept that bacteria with biomineralization capacity could be effective CaCO3 inductance agents, this study aimed to evaluate the simultaneous influence of 11 operational and environmental factors on the MICP process, for the first time. Therefore, Bacillus muralis, B. lentus, B. simplex, B. firmus, and B. licheniformis, isolated from alkaline soils, were used in the selection of the best performing bacterium compared with a well-known MICP bioagent Sporosarcina pasteurii DSM 33. Plackett-Burman's experimental design was labouring to screen all independent variables for their significances on five outputs (pH value, number of viable cells and spores, amount of urea and CaCO3 precipitate). According to experimentally obtained data, an artificial neural network model based on the Broyden-Fletcher-Goldfarb-Shanno algorithm showed good prediction capabilities, while differences in the relative influences were observed at the bacterial strain level. B. licheniformis turn out to be the most potent bioagent, with a maximum amount of CaCO3 precipitate of 3.14 g/100 mL in the optimal conditions.

Multiple heavy metal immobilization and strength improvement of contaminated soil using bio-mediated calcite precipitation technique.

Bio-mediated calcite precipitation potential for multiple heavy metal immobilization in contaminated soils at industrial, waste dump, abandoned mine, and landfill sites is not explored yet. This study includes investigation of bio-mediated calcite precipitation for strength improvement and immobilization of heavy metals, specifically lead (Pb), zinc (Zn), and hexavalent chromium (Cr(VI)), in contaminated soils. Firstly, the toxicity resistance of bacteria against different concentrations (1000, 2000, 3000, 4000, and 5000 mg/l) of each heavy metals was investigated and observed that Pb and Cr were less toxic to Sporosarcina pasteurii than Zn. The poorly graded sand was spiked with 333-2000 mg/kg concentrations of a selected individual or mixed metal solutions, i.e., 1000 mg/kg and 2000 mg/kg individual concentrations of Pb, Zn, and Cr(VI); 500 mg/kg and 1000 mg/kg concentration of each metal in "Pb and Zn," "Pb and Cr(VI)," and "Zn and Cr(VI)" mixture of heavy metals; and 333 mg/kg and 666 mg/kg concentration of each metal in "Pb, Zn, and Cr(VI)" mixed metal concentration. Contaminated soil was biotreated with Sporosarcina pasteurii and cementation (a solution of urea and calcium chloride dihydrate) solutions for 18 days. Biocemented sand specimens were subjected to testing of hydraulic conductivity, ultrasonic pulse velocity (UPV), unconfined compressive strength (UCS), calcite content, pH, toxicity characteristic leaching procedure (TCLP), X-ray diffraction (XRD), and scanning electron microscopy (SEM). The heavy metal contaminated samples showed decrease in hydraulic conductivity and increase in UPV and UCS after biotreatment; however, the changes in engineering properties were found more moderate than clean biocemented sand. The conversion of Cr(VI) to Cr(III) followed by Cr2O3 precipitation in calcite lattice was observed. Zn was precipitated as smithsonite (ZnCO3), while no Pb precipitate was identified in XRD results. TCLP leaching showed Pb and Cr immobilized proportional to calcite precipitated amount, and higher calcite amounts yielded levels within regulatory limits. Pb and Cr(VI) immobilization up to 92 % and 94 % was achieved, respectively, in contaminated biocemented sand. Zn was found completely leachable as smithsonite is only stable down to pH~5, and strongly acidic TCLP solution reversed all immobilization at natural soil pH~8-9.

Column study of enhanced Cr(VI) removal by bio-permeable reactive barrier constructed from novel iron-based material and Sporosarcina saromensis W5.

In this study, the feasibility of Cr(VI) removal from synthetic groundwater by bio-permeable reactive barrier constructed from novel iron-based material (SiO2/nano-FeC2O4 composite, SNFC) and Sporosarcina saromensis W5 was investigated. According to breakthrough study, an enhanced Cr(VI) removal was found in Bio-SNFC column. The Cr(VI) removal performances of biotic column with 0.2 g biomass and 0.4 g biomass were 16.2 mg/g and 17.9 mg/g, respectively, which were 19.6% and 32.1% higher than that of abiotic column (13.5 mg/g). However, excessive biomass (0.9 g) would cause pore clogging and have a negative impact on the Cr(VI) removal performance of the biotic column, whose removal capability (29.1%) was lower than that of abiotic column. The introduction of proper microorganisms enhanced the utilization of iron and enabled a higher proportion of Fe(II) in biotic column, which provided more reactive sites for Cr(VI) removal. The solid phase characterization indicated the generation of Fe(III) oxide/hydroxide on SNFC surface. The removal of Cr(VI) in Bio-SNFC column was depended on reduction-precipitation, and the final products related to chromium were mainly Cr(OH)3 and Cr2O3. The present work provides a new and sustainable remediation technology for in situ bioremediation of Cr(VI)-contaminated groundwater.

Sporosarcina jiandibaonis sp. nov., isolated from saline soil.

A Gram-stain-positive, aerobic, motile, rod-shaped bacterium, designated strain LAM9210T, was isolated from a saline soil sample collected from Lingxian County, Shandong Province, PR China. Analysis of the 16S rRNA gene sequence of the isolate revealed highest sequence similarities to the type strain of Sporosarcina pasteurii NCIMB 8841T (97.6 % sequence similarity). The genomic G+C content was 40.4 mol%. The average nucleotide identity and in silico DNA-DNA hybridization values between strain LAM9210T and the type strain of the most closely related species S. pasteurii NCIMB 8841T were 73.6 and 20.6 %, respectively. Strain LAM9210T was found to grow at 10-40 °C (optimum, 30 °C), at pH 6.0-10.0 (optimum, pH 9.0) and with 0-6 % (w/v) NaCl (optimum, 0.5 %), respectively. The major fatty acids were anteiso-C15 : 0 and iso-C14 : 0. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and one unidentified phospholipid. Menaquinone-7 was detected as the predorminant respiratory quinone. Strain LAM9210T contained glycine, lysine, alanine and glutamic acid as the diagnostic amino acids in the cell-wall peptidoglycan. On the basis of phenotypic, phylogenetic and genotypic data, strain LAM9210T is considered to represent a novel species of the genus Sporosarcina, for which the name Sporosarcina jiandibaonis sp. nov. is proposed. The type strain is LAM9210T (=CGMCC 1.18607T=GDMCC 1.2002T=JCM 32514T).

A quantitative, high-throughput urease activity assay for comparison and rapid screening of ureolytic bacteria.

Urease is a dinickel enzyme commonly found in numerous organisms that catalyses the hydrolysis of urea into ammonia and carbon dioxide. The microbially induced carbonate precipitation (MICP) process mediated by urease-producing bacteria (UPB) can be used for many applications including, environmental bioremediation, soil improvement, healing of cracks in concrete, and sealing of rock joints. Despite the importance of urease and UPB in various applications, a quantitative, high-throughput assay for the comparison of urease activity in UPB and rapid screening of UPB from diverse environments is lacking. Herein, we reported a quantitative, 96-well plate assay for urease activity based on the Christensen's urea agar test. Using this assay, we compared urease activity of six bacterial strains (E. coli BL21, P. putida KT2440, P. aeruginosa PAO1, S. oneidensis MR-1, S. pasteurii DSM 33, and B. megaterium DSM 319) and showed that S. pasteurii DSM 33 exhibited the highest urease activity. We then applied this assay to quantify the inhibitory effect of calcium on urease activity of S. pasteurii DSM 33. No significant inhibition was observed in the presence of calcium at concentrations below 10 mM, while the urease activity decreased rapidly at higher concentrations. At a concentration higher than 200 mM, calcium completely inhibited urease activity under the tested conditions. We further applied this assay to screen for highly active UPB from a wastewater enrichment and identified a strain of S. pasteurii exhibiting a substantially higher urease activity than DSM 33. Taken together, we established a 96-well plate-based quantitative, high-throughput urease activity assay that can be used for comparison and rapid screening of UPB. As UPB and urease activity are of interest to environmental, civil, and medical researchers and practitioners, we envisage wide applications of the assay reported in this study.

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.