Remediation of copper-contaminated soil by Kocuria flava CR1, based on microbially induced calcite precipitation

An indigenous calcifying bacterial strain CR1, identified as Kocuria flava, was isolated from soil of a mining area, Urumqi, China. An extensive copper bioremediation capacity of this isolate was studied based on microbially induced calcite precipitation (MICP). K. flava CR1 removed 97% of copper when initial Cu concentration was 1000 mg L⁻¹. The isolate produced significant amount of urease (472 U mL⁻¹), an enzyme that leads to calcite precipitation. The isolate removed 95% of copper from contaminated soil. The MICP process in bioremediation was further confirmed by FTIR and XRD analyses. FTIR analysis showed two different forms of calcium carbonate, i.e., calcite and aragonite, and the results were well supported by XRD. For the first time, the ability of K. flava has been documented in the bioremediation of polluted soil. This study showed that MICP-based bioremediation by K. flava is a viable, environmental friendly technology for cleaning-up the copper-contaminated site.     

Sporosarcina pasteurii can be used to print a layer of calcium carbonate

When using microbiologically induced calcium carbonate precipitation (MICP) to produce calcium carbonate crystals in the cavities between mineral particles to consolidate them, the inhomogeneous distribution of the precipitated calcium carbonate poses a problem for the production of construction materials with consistent parameters. Various approaches have been investigated in the literature to increase the homogeneity of consolidated samples. One approach can be the targeted application of ureolytic organisms by 3D printing. However, to date, this possibility has been little explored in the literature. In this study, the potential to use MICP to print calcium carbonate layers on mineral particles will be investigated. For this purpose, a dispensing unit was modified to apply both a suspension of Sporosarcina pasteurii and a calcination solution containing urea and calcium chloride onto quartz sand. The study showed that after passing through the nozzle, S. pasteurii preserved consistent cell vitality and therefore its potential of MICP. Applying cell suspension and calcination solution through a printing nozzle resulted in a layer of calcium carbonate crystals on quartz sand. This observation demonstrated the proof of concept of printing calcium carbonate by MICP through the nozzle of a dispensing unit. Furthermore, it was shown that cell suspensions of S. pasteurii can be stored at 4°C for a period of 17 days while maintaining its optical density, urease activity and cell vitality and therefore the potential for MICP. This initial concept could be extended in further research to printing three‐dimensional (3D) objects to solve the problem of homogeneity in consolidated mineral particles.     

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

Abstract

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

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

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

Plasmid encoded antibiotics inhibit protozoan predation of Escherichia coli K12

Bacterial plasmids and phages encode the synthesis of toxic molecules that inhibit protozoan predation. One such toxic molecule is violacein, a purple pigmented, anti-tumour antibiotic produced by the Gram-negative soil bacterium Chromobacterium violaceum. In the current experiments a range of Escherichia coli K12 strains were genetically engineered to produce violacein and a number of its coloured, biosynthetic intermediates. A bactivorous predatory protozoan isolate, Colpoda sp.A4, was isolated from soil and tested for its ability to ‘graze’ on various violacein producing strains of E. coli K12. A grazing assay was developed based on protozoan “plaque” formation. Using this assay, E. coli K12 strains producing violacein were highly resistant to protozoan predation. However E. coli K12 strains producing violacein intermediates, showed low or no resistance to predation. In separate experiments, when either erythromycin or pentachlorophenol were added to the plaque assay medium, protozoan predation of E. coli K12 was markedly reduced. The inhibitory effects of these two molecules were removed if E. coli K12 strains were genetically engineered to inactivate the toxic molecules. In the case of erythromycin, the E. coli K12 assay strain was engineered to produce an erythromycin inactivating esterase, PlpA. For pentachlorophenol, the E. coli K12 assay strain was engineered to produce a PCP inactivating enzyme pentachlorophenol-4-monooxygenase (PcpB). This study indicates that in environments containing large numbers of protozoa, bacteria which use efflux pumps to remove toxins unchanged from the cell may have an evolutionary advantage over bacteria which enzymatically inactivate toxins.     

Engineered applications of ureolytic biomineralization: a review

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

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

Abstract

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

Bacterial-based membrane protein production

Escherichia coli is by far the most widely used bacterial host for the production of membrane proteins. Usually, different strains, culture conditions and production regimes are screened for to design the optimal production process. However, these E. coli-based screening approaches often do not result in satisfactory membrane protein production yields. Recently, it has been shown that (i) E. coli strains with strongly improved membrane protein production characteristics can be engineered or selected for, (ii) many membrane proteins can be efficiently produced in E. coli-based cell-free systems, (iii) bacteria other than E. coli can be used for the efficient production of membrane proteins, and, (iv) membrane protein variants that retain functionality but are produced at higher yields than the wild-type protein can be engineered or selected for. This article is part of a Special Issue entitled: Protein trafficking and secretion in bacteria. Guest Editors: Anastassios Economou and Ross Dalbey.     

Development of multiplex PCR assay for rapid detection of Riemerella anatipestifer, Escherichia coli, and Salmonella enterica simultaneously from ducks

Three pathogens, Riemerella anatipestifer, Escherichia coli, and Salmonella enterica, are leading causes of bacterial fibrinous pericarditis and perihepatitis in ducks in China and worldwide. It is difficult to differentiate these pathogens when obtaining a diagnosis on clinical signs and pathological changes. The aim of this research was to develop a multiplex polymerase chain reaction (m-PCR) that could discriminate R. anatipestifer, E. coli, and S. enterica rapidly in field isolates, or detect the three bacteria in clinical samples from diseased ducks. We selected the DnaB helicase (dnaB) gene of R. anatipestifer, alkaline phosphatase (phoA) gene of E. coli and invasion protein (invA) gene of S. enterica as target genes. In optimized conditions, the limitation of detection was approximately 10³ colony forming units (CFU) of each of these three bacterial pathogens per PCR reaction tube. The m-PCR method showed specific amplification of respective genes from R. anatipestifer, E. coli, and S. enterica. Using the m-PCR system, bacterial strains isolated from diseased ducks in our laboratory were categorized successfully, and the pathogens could also be detected in clinical samples from diseased ducks. Therefore, the m-PCR system could distinguish the three pathogens simultaneously, for identification, routine molecular diagnosis and epidemiology, in a single reaction.     

Carbonate Mineral Precipitation for Soil Improvement Through Microbial Denitrification

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

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

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

Potential role for microbial ureolysis in the rapid formation of carbonate tufa mounds

Modern carbonate tufa towers in the alkaline (~pH 9.5) Big Soda Lake (BSL), Nevada, exhibit rapid precipitation rates (exceeding 3 cm/year) and host diverse microbial communities. Geochemical indicators reveal that carbonate precipitation is, in part, promoted by the mixing of calcium-rich groundwater and carbonate-rich lake water, such that a microbial role for carbonate precipitation is unknown. Here, we characterize the BSL microbial communities and evaluate their potential effects on carbonate precipitation that may influence fast carbonate precipitation rates of the active tufa mounds of BSL. Small subunit rRNA gene surveys indicate a diverse microbial community living endolithically, in interior voids, and on tufa surfaces. Metagenomic DNA sequencing shows that genes associated with metabolisms that are capable of increasing carbonate saturation (e.g., photosynthesis, ureolysis, and bicarbonate transport) are abundant. Enzyme activity assays revealed that urease and carbonic anhydrase, two microbial enzymes that promote carbonate precipitation, are active in situ in BSL tufa biofilms, and urease also increased calcium carbonate precipitation rates in laboratory incubation analyses. We propose that, although BSL tufas form partially as a result of water mixing, tufa-inhabiting microbiota promote rapid carbonate authigenesis via ureolysis, and potentially via bicarbonate dehydration and CO₂ outgassing by carbonic anhydrase. Microbially induced calcium carbonate precipitation in BSL tufas may generate signatures preserved in the carbonate microfabric, such as stromatolitic layers, which could serve as models for developing potential biosignatures on Earth and elsewhere.     

Effect of biosurfactant and fertilizer on biodegradation of crude oil by marine isolates of Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa

This study was conducted to investigate the effects of fertilizers and biosurfactants on biodegradation of crude oil by three marine bacterial isolates; Bacillus megaterium, Corynebacterium kutscheri and Pseudomonas aeruginosa. Five sets of experiments were carried out in shake flask and microcosm conditions with crude oil as follows: Set 1-only bacterial cells added (no fertilizer and biosurfactant), Set 2-with additional fertilizer only, Set 3-with additional biosurfactant only, Set 4-with added biosurfactant + fertilizer, Set 5-with no bacterial cells added (control), all the above experimental sets were incubated for 168 h. The biosurfactant + fertilizer added Set 4, resulted in maximum crude oil degradation within shake flask and microcosm conditions. Among the three bacterial isolates, P. aeruginosa and biosurfactant produced by this strain resulted in maximum crude oil degradation compared to the other two bacterial strains investigated. Interestingly, when biosurfactant and bacterial cells were used (Set 3), significant oil biodegradation activity occurred and the difference between this treatment and that in Set 4 with added fertilizer + biosurfactant were only 4-5% higher degradation level in shake flask and 3.2-7% in microcosm experiments for all three bacterial strains used. It is concluded that, biosurfactants alone capable of promoting biodegradation to a large extent without added fertilizers, which will reduce the cost of bioremediation process and minimizes the dilution or wash away problems encountered when water soluble fertilizers used during bioremediation of aquatic environments.     

Effects of environmental factors on microbial induced calcium carbonate precipitation

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

产脲酶芽胞杆菌的微波诱变育种

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

Formate Oxidation-Driven Calcium Carbonate Precipitation by Methylocystis parvus OBBP

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

Halotolerant, alkaliphilic urease-producing bacteria from different climate zones and their application for biocementation of sand

Microbially induced calcium carbonate precipitation (MICP) is a phenomenon based on urease activity of halotolerant and alkaliphilic microorganisms that can be used for the soil bioclogging and biocementation in geotechnical engineering. However, enrichment cultures produced from indigenous soil bacteria cannot be used for large-scale MICP because their urease activity decreased with the rate about 5 % per one generation. To ensure stability of urease activity in biocement, halotolerant and alkaliphilic strains of urease-producing bacteria for soil biocementation were isolated from either sandy soil or high salinity water in different climate zones. The strain Bacillus sp. VUK5, isolated from soil in Ukraine (continental climate), was phylogenetically close in identity (99 % of 16S rRNA gene sequence) to the strain of Bacillus sp. VS1 isolated from beach sand in Singapore (tropical rainforest climate), as well as to the strains of Bacillus sp. isolated by other researchers in Ghent, Belgium (maritime temperate climate) and Yogyakarta, Indonesia (tropical rainforest climate). Both strains Bacillus sp. VS1 and VUK5 had maximum specific growth rate of 0.09/h and maximum urease activities of 6.2 and 8.8 mM of hydrolysed urea/min, respectively. The halotolerant and alkaliphilic strain of urease-producing bacteria isolated from water of the saline lake Dead Sea in Jordan was presented by Gram-positive cocci close to the species Staphylococcus succinus. However, the strains of this species could be hemolytic and toxigenic, therefore only representatives of alkaliphilic Bacillus sp. were used for the biocementation studies. Unconfined compressive strengths for dry biocemented sand samples after six batch treatments with strains VS1and VUK5 were 765 and 845 kPa, respectively. The content of precipitated calcium and the strength of dry biocemented sand at permeability equals to 1 % of initial value were 12.4 g Ca/kg of dry sand and 454 kPa, respectively, in case of biocementation by the strain VS1. So, halotolerant, alkaliphilic, urease-producing bacteria isolated from different climate zones have similar properties and can be used for biocementation of soil.     

Potential soil reinforcement by biological denitrification

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

Immobilization of cadmium in soil by microbially induced carbonate precipitation with Exiguobacterium undae at low temperature

Microbially induced carbonate precipitation (MICP) is an advanced biological treatment technology to immobilize heavy metals in form of carbonate salts. In this MICP study, ureolytic Exiguobacterium undae was employed for immobilization of cadmium in contaminated soil at low temperature (10 °C). The sequential extraction test revealed conversion of more than 90% of cadmium in the tested soil from the soluble-exchangeable fraction to carbonate-bound fraction in 14 days of treatment. The cadmium may be precipitated in a separate CdCO₃ phase or be co-precipitated in calcite crystals. Activities of urease and dehydrogenase were enhanced during MICP, which were not affected by the testing temperatures. MICP with E. undae is a biological approach that may be worth investigating further to immobilize cadmium in soils of cold regimes.     

Efficient production of polylactic acid and its copolymers by metabolically engineered Escherichia coli

Polylactic acid (PLA) is one of the promising biodegradable polymers, which has been produced in a rather complicated two-step process by first producing lactic acid by fermentation followed by ring opening polymerization of lactide, a cyclic dimer of lactic acid. Recently, we reported the production of PLA and its copolymers by direct fermentation of metabolically engineered Escherichia coli equipped with the evolved propionate CoA-transferase and polyhydroxyalkanoate (PHA) synthase using glucose as a carbon source. When employing these initially constructed E. coli strains, however, it was necessary to use an inducer for the expression of the engineered genes and to feed succinate for proper cell growth. Here we report further metabolic engineering of E. coli strain to overcome these problems for more efficient production of PLA and its copolymers. This allowed efficient production of PLA and its copolymers without adding inducer and succinate. The finally constructed recombinant E. coli JLXF5 strain was able to produce P(3HB-co-39.6 mol% LA) having the molecular weight of 141,000 Da to 20 g l⁻¹ with a polymer content of 43 wt% in a chemically defined medium by the pH-stat fed-batch culture.