Calcium Carbonate Precipitation by Bacillus and Sporosarcina Strains

J. Microbiol. Biotechnol. (2016), 26(3), 540–548 http://dx.doi.org/10.4014/jmb.1511.11008 Research Article Review jmb

Calcium Carbonate Precipitation by Bacillus and Sporosarcina Strains Isolated from Concrete and Analysis of the Bacterial Community of Concrete S Hyun Jung Kim1, Hyo Jung Eom1, Chulwoo Park1, Jaejoon Jung1, Bora Shin1, Wook Kim2, Namhyun Chung2, In-Geol Choi2, and Woojun Park1*

1Laboratory of Molecular Environmental Microbiology, Department of Environmental Science and Ecological Engineering, and 2Department of Biotechnology, Korea University, Seoul 02841, Republic of Korea

Received: November 6, 2015 Revised: December 3, 2015 Microbially induced calcium carbonate precipitation (CCP) is a long-standing but re-emerging Accepted: December 14, 2015 environmental engineering process for production of self-healing concrete, bioremediation,

and long-term storage of CO2. CCP-capable bacteria, two Bacillus strains (JH3 and JH7) and one Sporosarcina strain (HYO08), were isolated from two samples of concrete and

First published online characterized phylogenetically. Calcium carbonate crystals precipitated by the three strains December 23, 2015 were morphologically distinct according to field emission scanning electron microscopy.

*Corresponding author Energy dispersive X-ray spectrometry mapping confirmed biomineralization via extracellular Phone: +82-2-3290-3067; calcium carbonate production. The three strains differed in their physiological characteristics: Fax: +82-2-953-0737; growth at alkali pH and high NaCl concentrations, and urease activity. Sporosarcina sp. HYO08 E-mail: [email protected] and Bacillus sp. JH7 were more alkali- and halotolerant, respectively. Analysis of the community from the same concrete samples using barcoded pyrosequencing revealed that the S upplementary data for this relative abundance of Bacillus and Sporosarcina species was low, which indicated low paper are available on-line only at culturability of other dominant bacteria. This study suggests that calcium carbonate crystals http://jmb.or.kr. with different properties can be produced by various CCP-capable strains, and other novel pISSN 1017-7825, eISSN 1738-8872 isolates await discovery.

Introduction vaterite depend on microbial metabolic processes, with calcite being the most common form of bacterial carbonate Biomineralization is a common biochemical process that [3, 11, 12, 45]. Emerging evidence also suggests that the represents the synthesis of inorganic crystalline or amorphous physical and chemical compositions of the surrounding minerals containing calcium, silicon, iron, magnesium, and environment determine the efficiency of production, shape, zinc [3, 40, 43]. A variety of microorganisms, including and size of carbonate mineral polymorphs [9, 10, 34, 45]. bacteria, fungi, algae, and lichens, may be responsible for This phenomenon has been observed in many CCP-capable the production of biominerals such as silicate, carbonate, bacteria in the genus Bacillus, Sporosarcina, Pseudomonas, and calcium phosphate [14, 15, 24]. In particular, calcium Cyanobacteria, Pantoea, Myxococcus, and Halobacteria, and carbonate (CaCO3) precipitation (CCP) appears to have sulfate-reducing bacteria. [4, 8, 45]. geobiochemical significance because it shapes soil structure Many bacteria are known to promote extracellular CCP by changing its porosity, compressibility, and shear strength [11, 12, 17, 45]. Microbial extracellular polymeric substances [5, 9]. CCP is linked to global carbon cycling and global (EPS) consisting of polysaccharides, proteins, nucleic acids, warming because of CO2 capture during the CCP process and lipids serve as nucleation sites for CCP owing to their [32, 45]. Carbonate minerals such as calcite, aragonite, and anionic chemical characteristics that attract the surrounding

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metal ions such as Ca2+ [3, 47]. Many physiological activities manufactured are highly alkaline. such as photosynthesis, denitrification, sulfur reduction, The 16S rRNA gene was amplified with primers 27F (5’- ammonification, and ureolysis can produce ammonium AGAGTTTGATCMTGGCTCAG-3’) and 1492R (5’-CGGTTACCT and hydroxide ions, which create alkaline conditions, thus TGTTACGACTT-3’). Polymerase chain reactions (PCRs) were accelerating CCP [5, 9, 11, 14, 45]. On the other hand, performed on a MyCycler Thermal Cycler (Bio-Rad, USA), and the cycling conditions were as follows: 94°C for 90 sec, followed intracellular CCP is widespread among cyanobacteria, and by 25 cycles consisting of 94°C, 60°C, and 72°C, 45 sec each. The sulfur-oxidizing chemolithotrophic Achromatium cells can extension/hold step lasted 5 min at 72°C. Taxonomic affiliation store CaCO granules inside the cells, although the 3 was determined by sequencing the 16S rRNA and by construction physiological role of CCP remains unclear [27, 35]. of phylogenetic trees via sequence similarity by means of EzTaxon The contribution of CCP-capable bacteria to atmospheric [6]. The 16S rRNA sequences of the three isolates have been

CO2 fixation and removal is substantial, and the use of deposited in National Center for Biotechnology Information (NCBI) CCP-capable bacteria for solving environmental and GenBank under the accession numbers KU168425, KU168426, industrial problems is a promising approach. Therefore, KU168427 for strains JH3, JH7, and HYO08, respectively. The research on CCP-capable bacteria has been a steadily optimal liquid medium for strains JH3 and JH7 was Luria-Bertani growing field [10, 20, 29, 31], and the following are some of broth, and for strain HYO08 was tryptic soy broth with 2% urea the popular topics: plugging and increasing durability of (w/v). The incubation temperature for each strain was 37°C. soil, biosorption of calcium from water, filling cracks in Various pH (pH range: pH 5, 7, 9, 10, and 11) and NaCl concentrations (0.5, 1, 2, 5, and 10% (g/v)) were used to test alkali concrete (self-healing concrete) [9, 12, 20, 36], bioremediation tolerance and halotolerance. of heavy metals [1, 32], and biomining of metals [33]. Nonetheless, the reasons for mineralization by CCP-capable FE-SEM and EDS Mapping Analysis bacteria, regulation of the possible CCP-specific genetic The strains JH3, JH7, and HYO08 were each incubated in a system, and the role or effects of CCP-capable bacteria in urea-CaCl2 medium (3 g of nutrient broth, 20 g of urea, 2.12 g of ecosystems are still poorly understood. The purpose of this NaHCO3, 10 g of NH4Cl, 4 g of CaCl2·2H2O, per liter of distilled study was to isolate and characterize novel CCP-capable water, adjusted to pH 6) for 12 h. Depending on the opacity of the bacteria from concrete. We sampled concrete from two culture, up to 2 ml of the cells was harvested by centrifugation locations (Jingyo and Seoul, South Korea) and characterized (1 min, 13,000 ×g) and the pellet was washed twice with PBS. them by field emission scanning electron microscopy Primary fixation was performed overnight at 4°C (Karnovsky’s (FE-SEM), energy dispersive X-ray spectrometry (EDS) fixation method). After that, we performed three washes with mapping, urease activity assays, and EPS quantification. 0.05 M potassium phosphate buffer for 10 min each at 4°C. The cells were additionally fixed with a mixture of 0.1 M potassium We also performed bacterial community analysis of the phosphate buffer and 2% osmium tetroxide at 4°C for 2 h. Two concrete samples using pyrosequencing. additional washes with distilled water were conducted briefly at room temperature. Dehydration by increasing ethanol concentrations Materials and Methods (30, 50, 70, 80, and 90%, and three times in 100% ethanol, 10 min each step) was performed at room temperature. The samples were Isolation of CCP-Capable Bacteria from Concrete and Their coated with platinum prior to examination by FE-SEM (FEI, Growth Conditions Japan). EDS mapping analysis was conducted on a NORAN Bacterial strains were isolated from two concrete walls: a 60- System 7: Silicon Drift Detector (Thermo Fisher Scientific, USA). year-old concrete from Jingyo (35°00'29.9"N 127°53'02.3"E) and a The mapping elements were depicted as counts. freshly mortared concrete from Seoul (37°35'06.9"N 127°01'33.5"E), South Korea. The surface of the concrete samples was gently Urease Activity wiped with 70% ethanol for sterilization. Then, the inner parts Urease activity was measured by a colorimetric assay [30]. were aseptically hammered and ground to isolate calcium Briefly, the strains JH3, JH7, and HYO08 and Escherichia coli carbonate-precipitating bacteria. The ground parts were washed MG1655 (negative control) were grown to the stationary phase. with phosphate-buffered saline (PBS, pH 7.5) and spread first onto The cell suspensions were then diluted with PBS to adjust the a minimal salts basal medium containing 0.1% cycloheximide to optical density at 600 nm (OD600) to 0.5. The cells (20 µl) were suppress the growth of eukaryotic organisms. The colonies were inoculated into 200 µl of Stuart’s urea broth in a 96-well plate subcultured onto Difco nutrient broth with pH set to 11. The (total final volume: 220 µl). The plate was incubated at 28°C for colonies that were capable of growing on the alkaline nutrient 24 h in a Sunrise microplate reader (TECAN, Austria), and OD550 broth were selected as possible calcium carbonate-precipitating was measured at 1 h intervals. During the incubation, the growth bacteria because the conditions under which concrete is of the three strains was negligible; hence, their urease activity was

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measured in the same amount of cells. remove short and low-quality sequences [37]. Classification and rarefaction curves were analyzed in RDPipeline of the Ribosomal EPS Quantification and Biofilm Formation Database Project (RDP Release 11) [44]. The NGS data for EPS quantification was conducted according to previous studies, community analysis was deposited at NCBI under the accession with slight modifications [25]. One milliliter of cell culture was number SRA312453. mixed with Congo red at the final concentration of 3.5 mg/l and was incubated for 30 min at 37°C with inversion every 10 min. The Results culture was centrifuged (10 min, 13,000 ×g) and OD480 of the clear colored supernatant was measured. A standard curve of Congo Isolation of CCP-Capable Bacteria from Concrete Samples red was plotted after measurement of OD at the concentration 480 The concrete samples were obtained from two locations gradient from 0.5 to 15 mg/l. The fraction of Congo-red bound (Jingyo and Seoul, South Korea). The concrete sample from cells was calculated from the difference between Congo red Jingyo (JC) was taken from a part of a 60-year-old building remaining in the supernatant and Congo red in the medium that had not experienced significant weathering. The without cells and from the obtained equation of the linear curve. sample from Seoul (SC) was freshly mortared concrete. The calculation results were normalized to OD600 of 1 ml of cell culture. Three possible CCP-capable isolates were selected on A biofilm formation assay was conducted by the crystal violet alkaline medium; two isolates from JC, named strains JH3 staining method [16]. Briefly, the four strains, including and JH7, and one isolate from SC, named strain HYO08. Escherichia coli MG1655 as a control, were incubated overnight Based on their phylogenetic relationship (sequence similarity with aeration and washed twice with PBS. Approximately 106 and a neighbor-joining tree), strains JH3 and JH7 were CFU/ml of each strain were inoculated into a 48-well microplate assigned to the genus Bacillus (Fig. 1). Strain JH3 shared in a static state, and OD was measured on a multi-detection 595 99.93% 16S rRNA gene similarity with alkali-tolerant microplate reader (HIDEX Sense, Finland). OD was normalized 595 B. aryabhattai, and 99.33% with the calcite-precipitating to OD . 600 bacterium B. megaterium [25]. On the other hand, the close phylogenetically species of strain JH7 were B. toyonensis Endospore Formation (99.98%) and B. thuringiensis (99.80%), respectively; both The strains JH3, JH7, and HYO08 were incubated for 5 days to induce sporulation. They were also incubated on TYE medium species are known to be alkali-tolerant calcium carbonate- [42] to verify endospore formation. The spores were either stained biomineralizing polymorphs [12, 18]. Strain HYO08 shared with malachite green or unstained for visualization under a light 98.83% 16S rRNA gene similarity with Sporosarcina soli and microscope (Zeiss, Germany). 98.54% with S. contaminans, both having an optimal pH of 9.0 and a well pronounced ability to precipitate calcium Soil Aggregate Visualization carbonate [23]. Strain HYO08 was incubated overnight, and 7 × 106 CFU/ml was inoculated into 50 ml of autoclaved soil with 25 mM urea and FE-SEM/EDS Mapping Analysis of CCP CaCl as sources for precipitation of calcium carbonate. The same 2 To test whether the isolated strains can precipitate volume of distilled water was inoculated as a negative control, crystals in a calcium carbonate-inducing medium, all three every 5 days. The soil condition was visualized by FE-SEM after strains were visualized by phase contrast microscopy (data 10 days of incubation at room temperature. not shown) and FE-SEM (Fig. 2). The cells of each strain Pyrosequencing Analysis were aggregated with crystals. Further EDS mapping To analyze the bacterial community inside the concrete samples confirmed that those crystals are composed of calcium where strains JH3, JH7, and HYO08 were isolated, DNA was carbonate (Fig. 3, Fig. S1). It is worth noting that both extracted using the NucleoSpin Soil kit (Macherey-Nagel, Bacillus and Sporosarcina species formed extracellular Germany). The DNA yield was quantified by a Nanodrop calcium carbonate and their crystals were morphologically Spectrophotometer ND-1000 (Thermo Fisher Scientific, USA). A distinct (Fig. 2). sequencing library was prepared by amplification of the 16S rRNA gene using primers 27F-AMP (5’-Adapter-Barcode-GAG Alkali Tolerance and Halotolerance of the Strains TTTGATCMTGGCTCAG-3’) and 518R (5’-Adapter-WTTACC The ability to tolerate high pH, temperature, and a high- GCGGCTGCTGG-3’) targeting the region V1 to V3. Sequencing salt environment is crucial for CCP-capable bacteria because was performed on a GS-FLX (Roche, Switzerland). All sequencing concrete is exposed to many environmental conditions and procedures were conducted by Macrogen (Republic of Korea). is made under these conditions [20]. The optimal growth Raw sequencing data were filtered in the mothur software to

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Fig. 1. A neighbor-joining tree based on the 16S rRNA gene sequence of the bacterial strains capable of calcium carbonate precipitation. The strains are depicted in boldface. Numbers at nodes indicate bootstrap values for 1,000 replicates. temperature of each strain was 37°C. The strains JH3, JH7, temperature (40°C) was strain HYO08. and HYO08 showed alkali tolerance and halotolerance near their optimal growth temperature (25-37°C); however, Physiological Characteristics of the CCP-Capable Bacteria: HYO08 was the only strain that preferred alkaline conditions Urease Activity, EPS Production, and Endospore Formation (up to pH 11) at all temperatures because it showed smaller Ureolysis is a key process for accelerating major CCP [5]. differences in the lag phase and maximum growth at high The urease activities of the three strains using the same pH levels. Nonetheless, strains JH3 and JH7 also grew well number of cells were measured. The colorimetric changes at 30°C and pH 9 and 10, respectively (Fig. S2). On the of phenol red revealed that all three strains showed urease other hand, strain JH7 was the most halotolerant because it activity (Fig. 4). Strain HYO08 had the strongest urease was capable of growing at the NaCl concentration of 5%. In activity, followed by strains JH7 and JH3. Strain HYO08 addition, the only strain that could not tolerate high started to increase urease activity after 1 h of incubation,

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Fig. 3. Energy dispersive X-ray spectrometry mapping of the strains JH3, JH7, and HYO08. Two calcium ion peaks indicate the presence of calcium in Fig. 2. Field emission scanning electron microscopy (10,000× biomineralized crystals produced by these calcium carbonate magnification) of calcium carbonate crystals produced by the precipitation-capable strains. (A) Strain JH3. (B) Strain JH7. (C) Strain strains JH3, JH7, and HYO08 after 12 h of incubation in a HYO08. urea-CaCl2 medium. (A) Strain JH3. (B) Strain JH7. (C) Strain HYO08. [41, 42]. Strain JH3 formed subterminal endospores, whereas strains JH7 and HYO08 produced central endospores with the maximum activity being within 7 h. Strain JH7 (Fig. S4). Among the three strains, strain JH3 showed the showed urease activity after 4 h with a double-sigmoid fastest spore formation cycle and as strain JH3 showed pattern. The reason for this pattern is not straightforward. mature spore state (seed-like spores) much earlier than other The urease activity of strain JH3 appeared after 17 h and strains within the same time interval (data not shown). The increased slowly. EPS might be an important key factor for soil-aggregation effect of strain HYO08 was analyzed CCP because it can bind to calcium ion, which facilitates because this strain was found to be the most alkali tolerant CCP [3]. It has been known that the EPS production and and had the highest urease activity. As shown in Fig. S5, a biofilm formation are often co-related [25]. EPS and biofilm large amount of minerals were drawn to and surrounded formation were quantified using Congo red and crystal the cell wall of strain HYO08 in the presence of urea and violet, respectively. Strain JH3 produced the greatest amount CaCl2, whereas sporulation occurred when there were no of EPS (1.001 mg/l) and showed higher biofilm formation CCP sources available. Soil aggregation assay under the (Fig. S3). two tested conditions showed that formation of the Endospores were observed in all three strains because aggregated strain HYO08-soil particle complex occurred in the genera Bacillus and Sporosarcina can form endospores the presence of urea and calcium chloride, whereas there

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Fig. 4. Urease activity of strains JH3, JH7, and HYO08 according to a colorimetric assay in Stuart’s Broth. Escherichia coli MG1655 was used as a negative control. were no signs of soil aggregation without CCP.

Bacterial Community Analysis in the Concrete Samples The DNA yield from the duplicate samples of JC and SC was 2.60 ± 2.78 and 0.11 ± 0.01 ng/mg (mean ± SD), respectively. Different DNA yields from the two samples suggest that there could be substantial variation in the community size among concrete samples. The large standard deviation of the DNA yield from JC was suggestive of Fig. 5. Bacterial community analysis of two concrete samples: significant variation in bacterial colonization depending on Jingyo concrete (JC) and Seoul concrete (SC). the concrete structure, such as cracks and pores. The Phyla and genera with relative abundance less than 1% were included bacterial community of JC and SC showed similar structure, as well. (A) Phyla. (B) Genera. with Actinobacteria, Proteobacteria, and Cyanobacteria being the major phyla (Fig. 5). The three phyla accounted JC, a number of taxonomically ambiguous groups of for >70% of cells in the two samples. Although the relative bacteria were noticed, such as Gp1, Gp8, unidentified abundance of Actinobacteria was similar in the two Rhodobacteraceae, unidentified Geodermatophilaceae, and communities (40.33% in JC and 37.7% in SC), Proteobacteria unidentified Micrococcaceae. Another characteristic of the showed different relative abundance in the two samples two communities was that many prevalent genera in the SC (23.1% in JC and 34.7% in SC). Cyanobacteria was the third community had low relative abundance in JC. Those genera dominant phylum, accounting for 11.9% and 8.8% of cells included Methylobacterium, Sphaerobacter, Propionibacterium, in JC and SC, respectively. There were a large proportion Truepera, Pseudomonas, Lactobacillus, and Microbacterium. of unidentified bacteria, pointing to unexpectedly high bacterial diversity inside the concrete. The community Discussion structure at the genus level was substantially different between the two samples. JC did not contain a genus with Application of biomineralization to fields such as relative abundance >10% and the predominant genus was environmental and civil engineering and geology [4, 8, 10] Blastococcus (9.6%), whereas the predominant genus of SC, is now a popular ecofriendly strategy; biomineralization is Methylobacterium, showed a relative abundance of 23.5%. In also known to participate in the carbon cycle because this

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process sequesters a large amount of carbon [3, 4, 7, 29, 31]. none of the strains had calcium carbonate on their cell wall In fact, this phenomenon is a natural process that occurs because whole cells were clearly visible (Fig. S5). Although almost ubiquitously; it exists in terrestrial and aquatic the nucleation effect of CCP-capable bacteria is still a habitats such as shells, coral reefs, and sands [7, 11, 12] and hypothesis [3, 13], the exact location triggering CCP in our even in the human body as mineralo-organic forms [46]. three strains needs further research. Biomineralization usually involves metabolic activities of Formation of calcium carbonate crystals requires a high microorganisms and can occur in a biologically controlled, concentration of calcium and carbonate sources. In a biologically influenced, or biologically induced manner (or habitat abundant with calcium sources such as a cave, a combination of the three) [8, 9, 32]. Utilization of this limestone, and soil [7, 8, 9, 31], utilization and production natural process could be a turning point in the field of of carbonates through metabolic activities such as urease- environmental engineering; therefore, it is important to driven hydrolysis may be the cornerstone of mineralization select bacterial strains with high efficiency of CCP. of calcium carbonates [2]. Here, all three strains exhibited The ability to exist in such an alkaline, high-salt urease activity that drove the biologically influenced environment is crucial for calcium carbonate-precipitating and biologically induced CCP under optimal alkaline bacteria because concrete itself represents such environments environmental conditions. Recent studies suggest that urea [9, 26]. Therefore, the three strains, Bacillus sp. JH3, Bacillus is abundant in CCP environments because of input from sp. JH7, and Sporosarcina sp. HYO08, could be promising various sources: mammal urine, water infiltration from the candidates for ecofriendly industrial applications. surface, human activities, and agricultural waste [19, 31]. The parameters of a bacterial culture environment such Therefore, these findings suggest that the concrete samples as temperature and aeration can influence the types of from which we isolated the CCP-capable bacteria might calcium carbonate produced by CCP-capable bacteria [11, have contained urea that the isolates could use for 17, 26]. Because all three strains were incubated under the metabolism and formation of minerals. same conditions, the different structure (shape) of the Our results of community analysis are consistent with a resulting precipitated crystals could be due to differences previous community analysis of a sample from a deteriorated in their innate mechanisms of utilization of calcium. monument containing bacteria harboring carbonatogenic Therefore, each strain probably has a distinct optimal activity [21]. Geodermatophilaceae is a family of stone-dwelling microenvironment to induce CCP, such as a specific pH bacteria containing the genera Blastococcus, Geodermatophilus, level and production of (or the level of expression of) a and Modestobacter, all of which were detected in our analysis. specific protein of EPS [11, 13, 21]. Thus, the properties of Proteogenomic analysis of Blastococcus, Geodermatophilus, EPS associated with the strains may also be a factor in the and Modestobacter revealed their characteristic stress distinct morphology of calcium carbonate crystals [13, 40]. resistance-related features such as starvation-inducible, The magnitude of EPS production and the composition and biofilm-related, and DNA-protecting proteins; they could electrical charge of glycoproteins and proteins may differ be an important physiological requirement for an endolithic among CCP-capable strains. The composition of EPS may lifestyle [39]. The presence of Cyanobacteria and free-living affect the efficiency of collection of calcium cations and the nitrogen-fixing Actinobacteria such as Arthrobacter and efficiency of binding of calcium ions to carbonate [3, 11]. In Propionibacterium may play an important role by supplying fact, the magnitude of CCP by the three strains was altered nutrients to a concrete-dwelling community because an when we replaced the calcium sources in the original urea- endolithic lifestyle involves extreme starvation [15, 38]. For

CaCl2 medium with calcium lactate, calcium acetate, or practical applications of CCP-capable bacteria, the relation calcium sulfate (data not shown). Understanding the distinct between the capacity for CCP and other physiological mechanisms by which Bacillus and Sporosarcina strains features of CCP-capable bacteria as well as the interaction utilize different calcium sources could be the key to the of CCP-capable bacteria with other species should be production of crystals of specific shape. studied in more detail. Many studies have suggested that the cell wall is a main The results of community analysis also showed that the location of CCP because of its nucleation effect for crystal relative abundance of Bacillus and Sporosarcina was low, formation [5, 9, 25]. It seems likely that the strains JH3, JH7, even though we successfully isolated those strains. This and HYO08 generate a special macro- or microenvironment discrepancy could be due to a culturing bias that possibly for precipitation of calcium carbonate regardless of provided favorable conditions for Bacillus and Sporosarcina, nucleation. The cells and crystals were aggregated, but or due to our focused screening strategy targeting alkali-

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tolerant strains. Nevertheless, identification of CCP-capable Cupriavidus isolates induce calcium carbonate precipitation bacteria from other taxa such as Acinetobacter and Myxococcus for biorestoration of ornamental stone. J. Appl. Microbiol. is to be expected, as shown in one study on bioconsolidation 115: 409-423. of historical limestone [22]. 9. De Muynck W, De Belie N, Verstraete W. 2010. Microbial Thus, we analyzed three alkali- and halotolerant CCP- carbonate precipitation in construction materials: a review. Ecol. Eng. 36: 118-136. capable bacterial strains, Bacillus sp. JH3, Bacillus. sp. JH7, 10. De Muynck W, Leuridan S, Van Loo D, Verbeken K, and Sporosarcina sp. HYO08, isolated from two concrete Cnudde V, De Belie N, Verstraete W. 2011. Influence of samples. Our data on urease activity, EPS production, pore structure on the effectiveness of a biogenic carbonate biofilm formation, and soil aggregation suggest that our surface treatment for limestone conservation. Appl. 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March 2016 ⎪ Vol. 26⎪ No. 3