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Article Calcifying Flexibility in Induction of CaCO3 Mineralization

Darya A. Golovkina 1,2, Elena V. Zhurishkina 1,2, Lyubov A. Ivanova 1,2 , Alexander E. Baranchikov 3 , Alexey Y. Sokolov 1, Kirill S. Bobrov 1,2 , Alexey E. Masharsky 4 , Natalia V. Tsvigun 5, Gennady P. Kopitsa 1 and Anna A. Kulminskaya 1,2,* 1 Petersburg Nuclear Physics Institute Named by B.P. Konstantinov of National Research Centre “Kurchatov Institute”, 188300 Gatchina, Russia; [email protected] (D.A.G.); [email protected] (E.V.Z.); [email protected] (L.A.I.); [email protected] (A.Y.S.); [email protected] (K.S.B.); [email protected] (G.P.K.) 2 Kurchatov Genome Centre-PNPI, 188300 Gatchina, Russia 3 Kurnakov Institute of General and Inorganic Chemistry of the Russian Academy of Sciences, 119991 Moscow, Russia; [email protected] 4 Core Facility Centre for Molecular and Technologies, St. Petersburg State University, 198504 St. Petersburg, Russia; [email protected] 5 Federal Scientific Research Centre “Crystallography and Photonics”, Russian Academy of Sciences, 119333 Moscow, Russia; [email protected] * Correspondence: [email protected]; Tel./Fax: +7-81-3713-2014

 Received: 29 October 2020; Accepted: 26 November 2020; Published: 28 November 2020 

Abstract: Microbially induced CaCO3 precipitation (MICP) is considered as an alternative green technology for cement self-healing and a basis for the development of new biomaterials. However, some issues about the role of bacteria in the induction of biogenic CaCO3 crystal nucleation, growth and aggregation are still debatable. Our aims were to screen for ureolytic calcifying and analyze their MICP abilities during their growth in urea-supplemented and urea-deficient media. Nine candidates showed a high level of specific activity, and a sharp increase in the urea-containing medium pH resulted in efficient CaCO3 biomineralization. In the urea-deficient medium, all ureolytic bacteria also induced CaCO3 precipitation although at lower pH values. Five strains (B. licheniformis DSMZ 8782, B. cereus 4b, S. epidermidis 4a, M. luteus BS52, M. luteus 6) were found to completely repair micro-cracks in the cement samples. Detailed studies of the most promising strain B. licheniformis DSMZ 8782 revealed a slower rate of the polymorph transformation in the urea-deficient medium than in urea-containing one. We suppose that a ureolytic retains its ability to induce CaCO3 biomineralization regardless the origin of carbonate ions in a cell environment by switching between mechanisms of urea-degradation and metabolism of calcium organic salts.

Keywords: calcium carbonate; biomineralization; ureolytic bacteria; polymorph

1. Introduction Biomineralization is known as a process of induction of mineral formation by living organisms within their metabolic reactions with the environment. To date, formations of calcium carbonate crystals of various morphology have been reported to be induced by different bacterial strains. In addition to numerous representatives of , Sporosarcina pasteurii [1,2], Synechococcus sp. as well as species of Arthrobacter crystallopoietes, Rhodococcus qingshengii, Psychrobacillus psychrodurans [3,4] are known to induce calcium carbonate crystallization. The most common polymorph is calcite that was formed during the growth of B. megaterium, B. licheniformis, B. flexus and B. pasteurii strains [5–7]. B. sphaericus

Life 2020, 10, 317; doi:10.3390/life10120317 www.mdpi.com/journal/life Life 2020,, 10,, 317x FOR PEER REVIEW 2 of 14 sphaericus have been shown to induce crystallization of various types of carbonates under alkaline have been shown to induce crystallization of various types of carbonates under alkaline conditions conditions and at low temperatures [8]. Notably, only the fungal strain of Aspergillus nidulans capable and at low temperatures [8]. Notably, only the fungal strain of Aspergillus nidulans capable of growing of growing on concrete slabs [9] was shown to induce calcium carbonate mineralization. on concrete slabs [9] was shown to induce calcium carbonate mineralization. During the last decade, an alternative innovative biotechnological approach based on microbial During the last decade, an alternative innovative biotechnological approach based on microbial systems providing CaCO3 crystallization inside of microcracks on the surface of concrete has been systems providing CaCO crystallization inside of microcracks on the surface of concrete has been actively developing [10–13].3 Microbially induced precipitation seems to be an environmentally actively developing [10–13]. Microbially induced precipitation seems to be an environmentally friendly friendly process [14] and has the potential to launch inside the cracks spontaneously [7,10–13,15,16]. process [14] and has the potential to launch inside the cracks spontaneously [7,10–13,15,16]. Beside the Beside the attempts to develop an effective technology for cement self-healing using biomineraliza- attempts to develop an effective technology for cement self-healing using biomineralization, biogenic tion, biogenic CaCO3 is being used to create new biomaterials based on different polymorphs of the CaCO is being used to create new biomaterials based on different polymorphs of the mineral [17,18]. mineral3 [17,18]. The induction of CaCO3 precipitation by microorganisms is known to be able to accompany The induction of CaCO3 precipitation by microorganisms is known to be able to accompany two two fundamentally different metabolic routes: heterotrophic and autotrophic. Three mechanisms fundamentally different metabolic routes: heterotrophic and autotrophic. Three mechanisms are dis- are distinguished in autotrophy: non-methylotrophic methanogenesis, oxygenic and anoxygenic tinguished in autotrophy: non-methylotrophic methanogenesis, oxygenic and anoxygenic photosyn- photosynthesis [19,20]. In all three pathways, CO2 is used as a source for the production of thesis [19,20]. In all three pathways, CO2 is used as a carbon source for the production of organic organic compounds yielding in a local deficit of CO2 in the medium or the bacterial cell environment 2 compounds yielding in a local deficit of CO in the medium or the bacterial cell environment2+ and, as and, as a consequence, the induction of CaCO3 precipitation in the presence of Ca ions near the cell. a consequence, the induction of CaCO3 precipitation in the presence of Ca2+ ions near the cell. In the In the heterotrophic pathway, organic compounds are used as a source of energy following the oxidation heterotrophic pathway, organic compounds are used as a source of energy following the oxidation of compounds, or organic acid salts or the cycle as the main mechanism. In turn, of sulfur compounds, or organic acid salts or the nitrogen cycle as the main mechanism. In turn, the the nitrogen cycle includes the ammonification of amino acids, the dissimilatory decomposition nitrogen cycle includes the ammonification of amino acids, the dissimilatory decomposition of ni- of nitrates, and the urea degradation pathway [21]. The most studied and easily detectable trates, and the urea degradation pathway [21]. The most studied and easily detectable laboratory laboratory process of microbial-based induction of CaCO3 precipitation is the production of large process of microbial-based induction of CaCO3 precipitation is the production of large amounts of amounts of carbonates during urea degradation by ureolytic bacteria [22–24] and many others. carbonates during urea degradation by ureolytic bacteria [22–24] and many others. Urease (or ure- Urease (or ureaaminohydrolase, E.C. 3.5.1.5) catalyzes the hydrolysis of urea to carbon dioxide aaminohydrolase, E.C. 3.5.1.5) catalyzes the+ hydrolysis of urea to carbon dioxide and ammonia. The and ammonia. The appearance of NH4 ions in the reaction medium increases its pH rapidly. appearance of NH4+ ions in the reaction medium increases its pH rapidly. Carbon dioxide combines Carbon dioxide combines with water to form carbonic acid, which then dissociates yielding carbonate with water to form carbonic acid, which then dissociates yielding carbonate ions (Figure 1). ions (Figure1).

Figure 1. Schematic illustration of urease-driven calcium carbonate precipitation induced by an Figure 1. Schematic illustration of urease-driven calcium carbonate precipitation induced by an ure- ureolytic microorganism. olytic microorganism. The less studied mechanism of induction of calcium carbonate crystallization through the oxidation The less studied mechanism of induction of calcium carbonate crystallization through the oxi- of Ca organic acid salts represents an alternative to the urease-aided method. This mechanism is based dation of Ca organic acid salts represents an alternative to the urease-aided method. This mechanism on the oxidation of the organic component to CO2 and OH− ions catalyzed by microbial carbonic is based on the oxidation of the organic component to CO2 and OH− ions catalyzed by microbial car- anhydrases (Figure2). As a result of the medium alkalization due to the presence of hydroxyl ions, bonic anhydrases (Figure 2). As a result of the medium alkalization due to the presence+ of hydroxyl CO2 is transformed into carbonic acid followed by conversion into HCO3− and H ions. Furthermore, ions, CO2 is transformed into carbonic acid followed by conversion into HCO3− and H+ ions. Further- in the presence of free calcium ions in the medium, calcium carbonate is formed [25]. more, in the presence of free calcium ions in the medium, calcium carbonate is formed [25].

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Figure 2. Chemical reactions leading to CaCO3 crystallization through the oxidation of Ca acetate Figure 2. Chemical reactions leading to CaCO3 crystallization through the oxidation of Ca acetate induced by heterotrophic bacteria. salt induced by heterotrophic bacteria. Our initial goals were to find microorganisms capable of inducing mineral formation among culturesOur from initial our collectiongoals were and to tofind identify microorganisms candidates for capable the development of inducing of technologymineral formation for autogenous among crackcultures healing from in our cement collection samples. and to To identify address cand the requirementsidates for the applieddevelopment to microorganisms of technology that for autog- could beenous used crack as concrete healing self-healing in cement samples. agents, theTo addre screeningss the strategy requirements included applied a search to microorganisms for Gram-positive that ureolyticcould be bacteria used as capable concrete of self-healing surviving in agents, alkaline the conditions. screening We strategy were focusing included on a ureolyticsearch for bacteria Gram- sincepositive they ureolytic are the mostbacteria studied capable and of widelysurviving applied in alkaline for such conditions. technologies. We were However, focusing for on practicalureolytic purposes,bacteria since the use they of are urease-driven the most studied biomineralization and widely applied is not always for such suitable technologies. due to aHowever, negative eforffect prac- of thetical by-products purposes, formedthe use duringof urease-driven urea degradation biomineralization and some other is not disadvantages always suitable like due the to temperature a negative dependenceeffect of the ofby-products the enzyme formed catalytic during activity, urea which degradation influences and the some process other effi ciencydisadvantages [24]. Therefore, like the moretemperature efficient dependence and environmentally of the enzyme friendly catalytic methods activity, of which using microorganismsinfluences the process in the efficiency processes [24]. of autogenousTherefore, more healing efficient of cementitous and environmentally structures arefriendly needed. methods The reportedof using microorganisms effect of both in the andpro- cesses of autogenous healing of cementitous structures are needed. The reported effect of both ure- carbonic anhydrases on microbially induced CaCO3 precipitation (MICP) [26,27] prompted us to compareases and the carbonic kinetics anhydrases of calcite precipitation on microbially by the induced selected CaCO ureolytic3 precipitation bacteria in (MICP) media with[26,27] and prompted without urea.us to Ascompare a result, the our kinetics research of calcite has provided precipitation new insights by the selected into the understandingureolytic bacteria of thein media processes with that and underliewithout MICP.urea. As This a result, could leadour research to the development has provided of new tools insights to control into the the course understanding of biomineralization of the pro- cesses that underlie MICP. This could lead to the development of tools to control the course of bio- in order to produce certain CaCO3 polymorphs required for the creation of new biomaterials. mineralization in order to produce certain CaCO3 polymorphs required for the creation of new bio- 2.materials. Materials and Methods

2.1.2. Materials Bacterial Strains,and Methods Screening and Cultivation Conditions Bacterial strains used in the study were from the in-house collection of the Enzymology laboratory of2.1. B.P. Bacterial Konstantinov Strains, Screen Petersburging and Nuclear Cultivation Physics Conditions Institute NRC “Kurchatov Institute” and were maintainedBacterial on strains LB used medium in the (peptone study were 10 gfrom/L, the in-house extract 5 collection g/L and NaCl of the 10 Enzymology g/L). For screening labora- purposes,tory of B.P. bacterial Konstantinov stains were Peters transferredburg Nuclear onto Physics LB-agar Institute with pH NRC 9 (adjusted “Kurchatov with 5 Institute” M NaOH and solution) were andmaintained grown for on 3LB days agar at medium 37 ◦C. Gram-positive (peptone 10 g/L, cultures capable 5 ofg/L growing and NaCl on 10 an g/L). alkaline For screening medium werepurposes, then transferredbacterial stains to starvationwere transferred agar (30 onto g agarLB-agar and with 1 L pH of distilled9 (adjusted water) with and 5 M cultured NaOH solu- for 2tion) days and at 37grown◦C followed for 3 days by at the 37 selection°C. Gram-positiv of bacteriae cultures that formed capable a of large growing number on an of endosporesalkaline me- observeddium were microscopically. then transferred to starvation agar (30 g agar and 1 L of distilled water) and cultured for 2 daysFor at assessment 37 °C followed of the abilityby the of selection selected of bacterial bacteria cultures that formed to induce a large calcium number carbonate of endospores precipitation, ob- eachserved bacterial microscopically. strain was grown in 10 mL of medium YPG containing 0.5% (w/v) peptone, 0.5% , 0.05%For yeast assessment extract [28 of] the for ability one day of atselected a temperature bacterial of cultures 37 ◦C with to induce shaking calcium (at 100 carbonate rpm). Then precipi- the tubetation, with each each bacterial culture strain was heatedwas grown at 80 in◦ C10 for mL 10 of min medium and cells YPG were containing separated 0.5% by (w centrifugation/v) peptone, 0.5% at 2000glucose,g for 0.05% 10 min. yeast The extract precipitate [28] for was one suspended day at a temperature in 2 mL of 0.9%of 37 NaCl °C with solution shaking and (at transferred 100 rpm). × intoThen 100 the mL tube of with liquid each medium culture B4-AC was heated or B4-U. at 80 Medium °C for 10 B4-AC min and contained: cells were calcium separated acetate, by centrifu- 2.5 g/L; yeastgation extract, at 2000× 2 gg/ L;for glucose, 10 min. The 10 g /precipitateL, pH 7.2. was Medium suspended B4-U contained:in 2 mL of 0.9% yeast NaCl extract, solution 2 g/L; and glucose, trans- 10ferred g/L; urea,into 100 2.5 mL g/L; of CaCl liquid2, 2.5medium g/L, pH B4-AC 5.3. Molaror B4-U. calcium Medium concentrations B4-AC contained: were approximatelycalcium acetate, the 2.5 sameg/L; yeast in each extract, medium 2 g/L; (1.6 glucose, mM in 10 B4-AC g/L, andpH 7.2. 2.2 Medium mM in B4-U). B4-U Pre-sterilizedcontained: yeast urea, extract, CaCl2 2or g/L; Ca glucose, acetate solutions10 g/L; urea, were 2.5 added g/L; separatelyCaCl2, 2.5to g/L, the pH medium 5.3. Molar after autoclaving.calcium concentrations Bacteria were were cultivated approximately for 14 days the atsame 37 ◦ inC witheach medium shaking at(1.6 110 mM rpm. in B4-AC The resulted and 2.2 calciummM in B4-U). carbonate Pre-sterilized precipitates urea, were CaCl collected2 or Ca acetate on a solutions were added separately to the medium after autoclaving. Bacteria were cultivated for 14 days at 37 °C with shaking at 110 rpm. The resulted calcium carbonate precipitates were collected on

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filter membrane with a pore size of 0.2 µm, washed two times with distilled water to remove cells and culture medium, oven dried at 50 ◦C for 48 h and sent for analysis. For each selected bacterium, changes in cultivation medium pH and urease activity, biomass accumulation and precipitation yields during 14-day growth in both media were monitored. A sample without bacteria was used as a chemical control and E. coli DH5α was used as a negative control. Periodically withdrawn 5-mL aliquots were cooled to the room temperature followed by measurements of pH, optical density at 595 nm and urease activity. In each sample, insoluble precipitates were treated as above and weighed. To evaluate the mineralizing ability of the strain B. licheniformis DSMZ 8782, the inoculate was transferred into the flasks with 100 mL of the liquid medium B4-U or B4-AC and cultivated for 14 days at 37 ◦C with shaking at 110 rpm with periodic removing of 5-mL aliquots. Withdrawn aliquots were cooled to the room temperature followed by measurements of pH, optical density at 595 nm and urease activity. In each sample, insoluble precipitates were treated as above and analyzed.

2.2. Strain Identification Genomic DNA was isolated from the overnight bacterial cultures using GeneJET Genomic DNA Purification Kit (Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania) according to the provided protocol. The PCR was carried out with Taq DNA polymerase (Evrogen, Moscow, Russia) and universal primers 8F and 1492R (l) for the 16S rRNA [29] in the Eppendorf Mastercycle Personal. The agarose gel electrophoresis confirmed the formation of only one product with the length about 1500 bases for all strains (data not shown). The PCR products were purified with the phenol-chloroform extraction [30] and sequenced. The sequencing was performed at the Core Facility Center for Molecular and Cell Technologies. Resulting sequences were aligned against the database of 16S ribosomal RNA sequences using the program BLASTN 2.9.0+ [31]. Multiple sequence alignment by ClustalW and phylogenetic analysis were performed using the MEGA X (version 10.1.8) software.

2.3. Nucleotide Sequence Accession Numbers The nucleotide sequence data reported are available in the GenBank database under the accession number(s): MW251744 (Micrococcus luteus 6), MW251742 (B. cereus 4b), MW251741 ( epidermidis 4a); MW251743 (B. cereus 168), MW251746 (M. luteus BS52), MW251745 (B. cereus BSP), MW251747 (B. subtilis K51), and MW251748 (B. subtillus 170).

2.4. Urease Assay Urease activity was determined according to the method described in [28] by evaluation of the conductivity of a culture medium. To assess the urease activity, bacterial strains were grown in 5 mL of beef broth containing 3 g/L of beef extract and 5 g/L of peptone for 24 h at 37 ◦C with shaking (100 rpm). The resulting bacterial cells were separated by centrifugation at 3000 rpm for 5 min. The precipitate was suspended in 0.9% NaCl solution, adjusted to optical density 0.5 at 600 nm, and 0.5 mL of the suspension was added to 40 mL of B4-U medium. The medium did not contain CaCl2 to avoid CaCO3 crystal formation that makes the conductivity assay difficult. Bacteria were cultivated for 14 days at 37 ◦C with shaking (100 rpm) and aliquots (5 mL) were withdrawn daily for pH and urease assays. For the latter, the aliquot was cooled to 25 ◦C and the conductivity was measured using a conductometer DFRobot DFR0300-H Gravity: Analog Electrical Conductivity Sensor/Meter (K = 10). The formation of ionic particles of non-ionic substrates led to an increase in the total conductivity of the solution and the rate at which the conductivity increased was proportional to the concentration of the active urease presented in the reaction mixture. The conductivity of the medium with growing E. coli DH5α strain was taken as a negative control. Molar concentration of degraded urea was calculated using the Equation (1) with the coefficient taken from [32]:

Ureahydrolysed (mM) = Conductivity (mS) 11.11 (1) × Life 2020, 10, 317 5 of 14

One unit of urease activity corresponded to the enzyme amount capable of catalyzing the conversion of 1 µM urea per minute under the standard assay conditions (pH 5.5, 37 ◦C, 20 min). The data points are presented as the means of at least three independent experiments, and the errors were calculated for each data point using Excel Solver add-in (Microsoft, Redmond WA, USA). The time-dependent curves reported below for pH- and urease activity assays during the bacterial growth were generated with ORIGIN 8.0 software (OriginLab, Northampton, MA, USA).

2.5. XRD and SEM Analysis Powder X-ray diffraction (XRD) analysis of all samples was performed on a Bruker D8 Advance diffractometer (Bragg-Brentano geometry) with Ni-filtered CuKα (λ = 1.5418 Å) radiation and a LYNXEYE detector. Diffraction patterns were recorded in the 10–70◦ 2θ range, with a step of 0.02◦ and collection time of 0.3 s/step. Rietveld analysis of the patterns was carried out with a help of FullProf Suite [33]. Structure models were obtained from Crystallography Open Database [34]. The microstructure of the samples was investigated using a Carl Zeiss NVision 40 high resolution scanning electron microscope at 1 kV acceleration voltage.

2.6. Restoration of Concrete Cracks with Selected Bacteria Dry grade M300 non-sterilized cement was diluted in distilled water and poured into silicone molds. After the cement had completely solidified, the samples (Ø 4.5 cm, h 1.0 cm) were removed and micro-cracks were made using a marble pestle. Each selected bacterial strain was grown in 10 mL of YPG medium for one day and centrifuged at 2000 g for 10 min. Cells were re-suspended in 5 mL × of 0.9% NaCl and sterile transferred to 100 mL of the medium B4-U. Then 30 mL of each bacterial suspension were applied to a cement sample with cracks. Cement samples were put at 37 ◦C with periodical addition of the nutrient medium. A sample without the addition of bacteria was taken as a control. After a month of observation, the samples were dried and visually analyzed using a MSP-1 optical microscope.

3. Results and Discussion

3.1. Screening and Characterization of CaCO3 Mineralyzing Bacteria A laboratory microbial collection containing 71 bacterial strains that have been gathered from various locations and purchased from different microbial collections were used to screen for ability to induce CaCO3 precipitation. Considering the requirements applied to microorganisms that could be used as concrete self-healing agents, we used a specific screening strategy. At the first stage, bacteria able to survive in alkaline conditions were selected. This stage was necessary because freshly prepared concrete has a pH value in the range of 8 to 11. As a result, 28 strains capable of growing on LB medium with a pH 9 were found among 71 strains taken into the study. The next task was to determine Gram-positive bacteria as potentially able to form spores, since the strains should retain their viability for several years to be used as a restoring material in the place of formation of the opening crack. As a result, 9 cultures out of 13 Gram-positive bacterial strains appeared to be able to withstand starvation growth conditions including six strains with the ability to produce abundant endospores that were determined microscopically. A 16S rRNa gene of eight earlier non-identified microorganisms were partially sequenced and the phylogenetic analysis of these sequences was performed (Figure3), whereby these microorganisms were identified as: Micrococcus luteus 6, 4b, Staphylococcus epidermidis 4a; B. cereus 168, M. luteus BS52, B. cereus BSP, B. subtilis K51, and B. subtillus 170. Three strains (S. epidermidis 4a, M. luteus 6 and M. luteus BS52) did not produce endospores but due to the production of cyst-like complexes during the starvation stage they were included into the list of studied microorganisms. LifeLife2020 2020, 10, 10, 317, x FOR PEER REVIEW 6 of 14 6 of 14 Life 2020, 10, x FOR PEER REVIEW 6 of 14

Figure 3.FigurePhylogenetic 3. Phylogenetic tree constructedtree constructed with with the the neighbor-joining neighbor-joining methodmethod from from partial partial 16S 16S rDNA rDNA gene sequencesFiguregene 3. ofPhylogenetic sequences the eight of isolates treethe eight constructed and isolates reference andwith reference the strains neighbor strains (accession-joining (accession numbers method numbers are from are indicated). partial indicated). 16S Species rDNASpecies used as thegene outgroup sequencesused as wasthe of outgroupPseudomonas the eight was isolates aeruginosa and reference .aeruginosa Bootstrap strains. Bootstrap probabilities (accession probabilities numbers as determined as determinedare indicated). for for 1000 1000Species replicates rep- are used aslicates the outgroup are given was as a Pseudomonaspercentage. aeruginosa. Bootstrap probabilities as determined for 1000 rep- given as a percentage. licates are given as a percentage. The ability of the selected cultures to induce precipitation of calcium carbonate was monitored The ability of the selected cultures to induce precipitation of calcium carbonate was monitored Theduring ability their of growththe selected in a liquid cultures urea-supplemen to induce precipitationted medium of B4-U. calcium The carbonatevalues for wasthe medium monitored pH duringduring theirhave their been growth growth increasing in in a a liquid liquid from 5 urea-supplementedurea to 7–-8.5supplemented during the first medium medium two days B4 B4-U. -forU. alThel The cultures values values and for for remainedthe the medium medium constant pH pH have beenhave increasing beenuntil increasingthe end from of thefrom 5 to experiment 7–8.55 to 7– during8.5 (Figure during the 4). the first first two two days days for for allall culturescultures and and remained remained constant constant until theuntil end the of end the of experiment the experiment (Figure (Figure4). 4).

Figure 4. Time-dependence of the pH values during the bacteria growth in B4-U medium. Blue line represents data for a sample without bacteria used as a chemical control and green line represents data for E. coli DH5α used as a negative control. Data points are presented as the means of three independent experiments, the errors were calculated for each data point using Excel Solver add-in (Microsoft, Redmond, WA, USA). Life 2020, 10, x FOR PEER REVIEW 7 of 14

Figure 4. Time-dependence of the pH values during the bacteria growth in B4-U medium. Blue line represents data for a sample without bacteria used as a chemical control and green line represents data for E. coli DH5α used as a negative control. Data points are presented as the means of three Life 2020independent, 10, 317 experiments, the errors were calculated for each data point using Excel Solver add-in7 of 14 (Microsoft, Redmond, WA, USA).

AnalysisAnalysis ofof thethe time-dependenttime-dependent curves curves of of the the specific specific urease urease activity activity for for each each strain strain revealed revealed two twotypes types of behavior: of behavior: the majority the majority of the of strains the strains showed showed the maximal the maximal activity activity after afterthe first the day first of day culti- of cultivationvation following following by bysome some decrease decrease during during the the next next two two days, days, whereas whereas urease activity ofofB. B. subtilis subtilis K51K51 and andB. B. cereus cereus168 168 reached reached maximum maximum after after 44–50 44–50 h h and and then then remained remained constant constant (Figure (Figure5). 5).

FigureFigure 5. 5.Time-dependence Time-dependence of of the the specific specific urease urease activity activity of of each each tested tested strain strain during during the the growth growth in in thethe B4-U B4-U medium. medium. The The specific specific urease urease activity activity was was assessed assessed using using measured measured value value for for conductivity conductivity of of thethe reaction reaction mixture mixture according according toto thethe Formulaformula (1). (1). G Greenreen line line represents represents data data for for E.E. coli coli DH5αDH5α usedused as asa anegative negative control. control. The The data data points points are are presented presented as asthe the means means of thre of threee independent independent experiments, experiments, and andthe theerrors errors were were calculated calculated for foreach each data data point point as described as described for forFigure Figure 2. 2.

AllAll selectedselected ureolytic strains strains appeared appeared to to induce induce calcium calcium carbonate carbonate mineralization mineralization in the in urea the - urea-deficientdeficient medium medium (B4-AC) (B4-AC) showing showing almost almost the same the same precipi precipitatetate yields yields as in as the in medium the medium with with urea ureaB4-U B4-U (see (seeTable Table 1). 1We). We observed observed that that after after 14 14days days of ofcultivation cultivation the the highest highest amount amount of ofCaCO CaCO3 in3 B4in - B4-UU medium medium was was produced produced by by the the strains strains M.M. luteus luteus 6,6, S.S. epidermidis epidermidis 4a, B. cereus 4b4b whilewhile inin thethe B4-AC B4-AC mediummediumM. M. luteus luteusBS52 BS52 and andB. B. cereus cereusBSP BSP showed showed the the best best results results (Table (Table1). 1).

Table 1. CaCO3 precipitates produced during the bacteria growth in two media. Table 1. CaCO3 precipitates produced during the bacteria growth in two media. a Quantity of Precipitates aProduced by Studied Strains in 100 mL of Microorganism Quantity of Precipitates Produced by Studied Strains in 100 mL Microorganism B4-U Medium, mg b of B4-AC Medium, mg b B. licheniformis DSMZ 8782B4-U Medium, 180.9 8.1 mg b B4-AC 113.0 Medium,4.7 mg b ± ± S. epidermidis 4a 234.5 1.5 121.2 3.8 B. licheniformis DSMZ 8782 180.9 ±± 8.1 113.0± ± 4.7 B. cereus 4b 217.8 12.7 110.3 4.1 S. epidermidis 4a 234.5 ± 1.5 121.2± ± 3.8 M. luteus 6 207.5 8.3 187.6 5.4 ± ± B.B. cereus subtilis 4bK51 217.8 120.2 ± 12.74.5 167.2110.3 ±9.3 4.1 ± ± M.B. subtilisluteus 1706 207.5 151.9 ± 8.32.8 170.6187.6 ±6.8 5.4 ± ± B. B.subtilis cereus K51168 120.2 180.6 ± 4.55.9 120.0167.2 15.5± 9.3 ± ± M. luteus BS52 157.7 0.1 233.5 12.9 B. subtilis 170 151.9 ±± 2.8 170.6± ± 6.8 B. cereus BSP 155.5 5.4 220.6 6.9 ± ± B.E. cereus coli DH5 168α 180.6 0± 5.9 120.0 0 ± 15.5 M. luteus BS52 157.7 ± 0.1 233.5 ± 12.9 a Insoluble precipitates were thoroughly collected from the flask walls with a scraper, separated from the planktonic b cells andB. cereus the medium BSP by filtration, washed with155.5 distilled ± 5.4 water, oven dried at 50 ◦C, and weighed.220.6 ± 6.9Errors bars representE. coli average DH5α and standard deviation of three replicates.0 0 a Insoluble precipitates were thoroughly collected from the flask walls with a scraper, separated from 3.2. CaCOthe planktonic3 Mineral Analysiscells and the medium by filtration, washed with distilled water, oven dried at 50 °C, and weighed. b Errors bars represent average and standard deviation of three replicates. All samples of bacterial CaCO3 precipitates obtained after the two-week growth of selected strains in B4-U and B4-AC media were analyzed by powder X-ray diffraction (XRD) spectrometry using the Rietveld method. Some samples were monophase but the majority represented the mixture of calcite [35], vaterite [36] and non-identified phase in various combinations (Figure S1a,b, in the Supplementary Materials). Neither aragonite [37], nor hydrated forms (monohydrocalcite and Life 2020, 10, 317 8 of 14 ikaite) [38] were detected. Here, calcite formation was recorded in six of nine studied strains after their growth in urea-containing medium B4-U (Table2). Vaterite, in addition to calcite, was detected for B. subtilis 170 and B. cereus 168 grown in B4-U medium and for all strains except B. subtilis K51 and B. licheniformis DSMZ 8782 during their growth in B4-AC. Surprising was the appearance of an unidentified phase in some samples (Figure S1c).

Table 2. Results of powder X-ray diffraction (XRD) analysis of CaCO3 polymorphs formed by bacterial strains under study.

B4-U Medium B4-AC Medium Microorganism Calcite Vaterite Unidentified Phase Calcite Vaterite Unidentified Phase B. licheniformis DSMZ 8782 + -- + - + S. epidermidis 4a - - + + + - B. cereus 4b - - + + + - M. luteus 6 + - + + + + B. subtilis K51 + -- + - + B. subtilis 170 + + - + + + B. cereus 168 + + - + + + M. luteus BS52 - - + - + + B. cereus BSP + -- + + +

We found only one recent report by Ghosh with co-authors [39] where an unidentified phase was mentioned with respect to the bacterium Sporosarcina pasteurii biomineralization. Most probably, the appearance of peaks corresponding to the unidentified phase may indicate the appearance of a crystalline substance due to the interaction of calcium ions with organic species present in the culture media. It was observed for four strains grown in B4-U medium and for seven strains grown in calcium acetate medium B4-AC (Table2). In the B4-AC medium, calcite was formed by all microorganisms except M. luteus BS52, for which only vaterite and the unidentified phase were detected. It may indicate that thermodynamically unstable ACC and metastable vaterite phases have not been transformed into stable calcite by the end of the M. luteus BS52 growth in urea-deficient medium. For the strains S. epidermidis 4a and B. cereus 4b calcite and vaterite were detected in B4-AC medium and only the unidentified phase was observed in B4-U medium. SEM analysis of the precipitates produced by the selected strains after a 2-week growth in both media also confirmed that the samples consisted of strongly aggregated particles having various shapes (Figure S2, Supplementary Materials). To evaluate the potential of the selected strains for practical applications, we have tested them in cement-sand microcracks healing. It should be taken into account that the microcracks in the cement-sand samples were made manually, so their size and distribution could also affect the rate of the crack repair process. Since we used this method only for screening purposes, we have been considering the results after a month of treatment by bacteria to exclude the influence of the difference in the lesions size on healing. Thus, the selected strains demonstrated different abilities to restore micro-cracks on the cement-sand surface. The strains B. licheniformis DSMZ 8782 (Figure6a,b), B. cereus 4b (Figure6c,d), S. epidermidis 4a (Figure6k,l), M. luteus BS52 (Figure6q,r), and M. luteus 6 (Figure6m,n) appeared to repair microcracks in the samples while the effect of cultivation of B. cereus 168 (Figure6e,f), B. cereus BSP (Figure6g,h), B. subtilis 170 (Figure6o,p) and B. subtilis K51 (Figure6s,t) was negligible. E. coli DH5α, negative control (Figure6i,j). Life 2020, 10, 317 9 of 14 Life 2020, 10, x FOR PEER REVIEW 9 of 14

Figure 6. TypicalTypical micrographs micrographs of of microcrack microcrack filling filling by by the the selected selected strains strains bef beforeore and and after after a month a month of theof the growth growth on onthethe cement cement surface: surface: B. licheniformisB. licheniformis DSMZDSMZ 8782 8782 (a,b); (a B.,b cereus); B. cereus 4b (c,4bd); ( B.c,d cereus); B. cereus 168 (e168,f); α (B.e, fcereus); B. cereus BSP (BSPg,h); ( gE.,h );coliE. DH5α, coli DH5 negative, negative control control (i,j); (iS.,j );epidermidisS. epidermidis 4a (4ak,l); (k M.,l); M.luteus luteus 6 (m6, (nm);, nB.); B. subtillus 170 (o,p); M. luteus BS52 (q,r); B. subtilis K51 (s,t). Cement samples were 4.5 1 cm in size; subtillus 170 (o,p); M. luteus BS52 (q,r); B. subtilis K51 (s,t). Cement samples were 4.5 ×× 1 cm in size; microcracks were 3.5–4 cm in length and 0.12–0.4 mm in width). microcracks were 3.5–4 cm in length and 0.12–0.4 mm in width). 3.3. Analysis of Precipitation Process Induced by B. licheniformis DSMZ 8782 3.3. Analysis of Precipitation Process Induced by B. licheniformis DSMZ 8782 The strain B. licheniformis DSMZ 8782 was the fastest in microcracks healing. Considering the observationThe strain that B. it producedlicheniformis only DSMZ the most 8782 stable was polymorph,the fastest in calcite, microcracks during itshealing. growth Considering in liquid media, the observationthis strain was that further it produced studied only in more the details.most stable During polymorph, a two-weeks calcite, cultivation during ofits the growthB. licheniformis in liquid media,DSMZ 8782this strain in liquid was B4-U further and studied B4-AC in media more with details. sequential During aliquot a two-weeks sampling cultivation at regular of intervals,the B. li- cheniformis DSMZ 8782 in liquid B4-U and B4-AC media with sequential aliquot sampling at regular the changes in the growth media pH values, biomass accumulation, CaCO3 precipitation, and the intervals,specific urease the changes activity in were the monitoredgrowth media (Figure pH7 values,). biomass accumulation, CaCO3 precipitation, and theFigure specific7 shows urease that activity during thewere growth monitored in B4-U (Figure medium, 7). pH values increased rapidly after the first day andFigure then 7 remainedshows that constant; during the the growth exponential in B4 growth-U medium, phase pH of thevalues bacterium increased corresponded rapidly after to the firstintense day crystal and then formation remained and constant; a linear increasethe exponential in the urease growth rate. phase As expected, of the bacterium no urease corresponded activity was todetected the intense in the crystal B4-AC formation medium, indicatingand a linear that increase crystal in formation the urease is rate. proceeded As expected, by a diff noerent urease mechanism. activity was detected in the B4-AC medium, indicating that crystal formation is proceeded by a different mechanism.

Life 2020, 10, 317 10 of 14 Life 2020, 10, x FOR PEER REVIEW 10 of 14

FigureFigure 7. 7.Time-dependences Time-dependences ofofB. B. licheniformislicheniformisgrowth growth parametersparameters inin 100100 mLmL ofof B4-UB4-U medium medium (black(black lines)lines) and and B4-AC B4-AC medium medium (red (red lines): lines): ( aa)) pH (filled (filled squares) and specificspecific urease activity (open(open squares);squares);

(bb)) CaCO 33 precipitateprecipitate amount amount (filled (filled squares) squares) and and bacterial bacterial biomass biomass accumulation accumulation (open (open squares). squares). Er- Errorsrors bars bars represent represent average average and and standard standard dev deviationiation of of three three replicates. replicates.

B. licheniformis Finally,Finally, cell suspensions suspensions of of B. licheniformis DSMZDSMZ 8782 8782 were wereapplied applied to completely to completely dried cement dried- cement-sandsand samples. samples. A month later, A month the sample later, thetreated sample with treatedthe bacterium with the in B4 bacterium-U medium in was B4-U completely medium wasrepaired, completely while repaired,in the case while of usin in theg the case B4- ofAC using medium, the B4-AC the crack medium, was not the closed crack up was totally not closed but a upwhite totally coating but aby white calcium coating carbonate by calcium appeared carbonate (Figure appeared S3, Supplemental (Figure S3, Supplementarymaterial). This agrees Materials). with Thisour agreesresults withobtained our results during obtained the bacterial during growth the bacterial in liquid growth media in (Figure liquid media 7). When (Figure using7). Whenthe urea using-rich the urea-rich medium, urease was induced and, accordingly, an intense CaCO precipitation occurred. medium, urease was induced and, accordingly, an intense CaCO3 precipitation3 occurred. Conversely, Conversely,in the B4-AC in medium, the B4-AC the medium, peak of theprecipitate peak of precipitateproduction production was reached was much reached later, much at the later, beginning at the beginningof the second of the week. second week. XRDXRD andand SEMSEM analysesanalyses ofof samplessamples takentaken fromfrom thethe liquidliquid B4-UB4-U mediummedium withwith growinggrowing strainstrain allowed us to follow the CaCO biomineralization during 14 days. After the first day of the growth, allowed us to follow the CaCO33 biomineralization during 14 days. After the first day of the growth, wewe detected detected peaks peaks characteristic characteristic toto calcitecalcite andand hardlyhardly observableobservable peakspeaks ofof vateritevaterite (Figure(Figure8 ,8, red red line) line) followedfollowed byby accumulationaccumulation ofof bothboth vateritevaterite andand calcitecalcite phasesphases afterafter 4848 hh (Figure(Figure8 ,8, green, green, blue blue and and violetviolet lines).lines). SEMSEM analysisanalysis confirmedconfirmed thatthat thethe amorphousamorphous phasephase ofof calciumcalcium carbonatecarbonate consistedconsisted ofof µ ultrasmallultrasmall particlesparticles (10 (10–20–20 µm)m) with with irregular irregular shape shape and and was was formed formed on the on first the firstday (Figure day (Figure 9). Then9). µ Thenthe precipitates the precipitates were were transformed transformed into intoellipsoid ellipsoid or spherical or spherical particles particles (30– (30–5050 µm) m)characteristic characteristic to the to thepolymorph polymorph vaterite; vaterite; on on the the second second day day they they increased increased in in both both size size and and quantity. quantity. Closer toto thethe 14th14th µ dayday we we observed observed appearance appearance of of large large particles particles (80–120 (80–120m) µm) with with well-defined well-defined faceting faceting typical typical to calcite to cal- (Figurecite (Figure9). Note 9). Note that non-biogenicthat non-biogenic and biogenicand biogenic processes processes of calcium of calcium carbonates carbonates crystallization crystallization are ratherare rather complex complex and and proceed proceed through through various various intermediates intermediates [17, 38[17,38,40],40] depending depending on various on various factors fac- liketors reaction like reaction and culture and culture medium medium composition, composition, bacteria bacteria species, species, aeration aeration process process and others and [ 41 others,42], so[41,42], that it so can that be it considered can be considered typical for typical both bacteriallyfor both bacterially induced vateriteinduced or vaterite calcite or microcrystals. calcite microcrys- tals. The ability of microorganisms to induce calcium carbonate precipitation through different mechanismsThe ability is well of microorganisms known. However, to induce most calcium reports carbonate on MICP precipitation processes describe through only different one ofmech- the mechanismsanisms is well inducing known. microbial However, biomineralization most reports on MICP of calcium processes carbonate describe [6,11 only,22– 24one,43 of] andthe mecha- others. Fornisms some inducing bacteria (microbialBacillus, Arthrobacter biomineralizationand Rhodococcus of calcium) the inductioncarbonate of[6,11,22 Ca carbonate–24,43] crystallization and others. For in asome medium bacteria containing (Bacillus, calcium Arthrobacter organic and acid Rhodococcus salts (acetate,) the lactate, induction citrate, of Ca succinate) carbonate was crystallization described [44 in]. a medium containing calcium organic acid salts (acetate, lactate, citrate, succinate) was described [44].

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FigureFigure 8. 8.Typical Typical X-ray X-ray di diffractionffraction patterns patterns ofof CaCOCaCO3 precipitatesprecipitates formed formed by by the the strain strain B. B.licheniformis licheniformis DSMZDSMZFigure 8287 8287 8. Typical during during 14-daysX-ray 14-days diffraction growth: growth: patterns after 29 h, of red CaCO lin line;e;3 precipitates 72 72 h, h, green green formedline; line; 9 9days, by days, the blue bluestrain line; line; B. 14 licheniformis 14days, days, violetvioletDSMZ line. line. 8287 during 14-days growth: after 29 h, red line; 72 h, green line; 9 days, blue line; 14 days, violet line.

FigureFigure 9. Representative9. Representative scanning scanning electron electron micrographs micrographs of of CaCO CaCOprecipitates3 precipitates appeared appeared during during 14-days 14- 3 of daysB. licheniformis of B. licheniformis DSMZ DSMZ8782 growth 8782 growth in B4-AC in B4-AC medium. medium. Figure 9. Representative scanning electron micrographs of CaCO3 precipitates appeared during 14- HelmiHelmidays withof with B. licheniformis coauthorscoauthors DSMZshowed showed 8782 the the growth induction induction in B4-AC of ca oflcium medium. calcium carbonate carbonate precipitation precipitation in different in di urea-fferent urea-containingcontaining media media during during the growth the growth of the bacterium of the bacterium B. licheniformisB. licheniformis [45]. Barabesi[45 et]. al. Barabesireported the et al. reportedrelationshipHelmi the relationship withbetween coauthors the activity between showed of thecell the activitygenes induction resp ofonsible of cell calcium for the carbonate responsible induction precipitation of for the the biogenic induction in different minerali- of theurea- biogeniczationcontaining process mineralization media and duringthe metabolism process the growth and of the fatty of metabolism the acids bacterium [46]. ofOnly fattyB. licheniformisthe acids recent [46 publication]. [45]. Only Barabesi the of recent Reeksting et al. publication reported et al. the described the ability of microorganisms to induce CaCO3 precipitation through two different mech- ofrelationship Reeksting et between al. described the activity the ability of cell of genes microorganisms responsible tofor induce the induction CaCO3 ofprecipitation the biogenic through minerali- anisms depending on the growth conditions [47]. The aforementioned authors showed that different twozation different process mechanisms and the metabolism depending of on fatty the acids growth [46]. conditions Only the [recent47]. The publication aforementioned of Reeksting authors et al. polymorphs were formed with a dependence on different nutrients added into the showeddescribed that the diff erentability polymorphs of microorganisms were formed to induce with CaCO a dependence3 precipitation on diff througherent nutrients two different added intomech- and, accordingly, the process depended on the mechanism that led to the CaCO3 precipitation. Our theanisms growth depending medium and, on the accordingly, growth conditions the process [47]. depended The aforementioned on the mechanism authors that showed led to thethat CaCO different results confirm that this phenomenon is more widespread than previously thought. All selected ure- 3 precipitation.polymorphs Our were results formed confirm with thata dependence this phenomenon on different is more nutrients widespread added than into previously the growth thought. medium olytic bacteria retained their ability to induce CaCO3 mineralization in urea-deficient medium (Tables 3 Alland, selected accordingly, ureolytic the bacteria process retained depended their on ability the mechanism to induce CaCO that led3 mineralization to the CaCO in precipitation. urea-deficient Our 1 and 2) although the transformation of CaCO3 morphologies occurred at a slower rate than along results confirm that this phenomenon is more widespread than previously thought. All selected ure- mediumthe ureolytic (Tables pathway.1 and2) althoughMost likely, the this transformation behavior cannot of CaCObe explained3 morphologies only by the occurred species-specificity at a slower rateolytic than bacteria along theretained ureolytic their pathway. ability to induce Most likely, CaCO this3 mineralization behavior cannot in urea-deficient be explained medium only by (Tables the species-specificity1 and 2) although of the bacteria transformation or non-biogenic of CaCO crystallization3 morphologies due occurred to elevated at a contentslower rate of Ca than2+ and along the ureolytic pathway. Most likely, this behavior cannot be explained only by the species-specificity

Life 2020, 10, 317 12 of 14

carbonate ions in the media that was verified in experiments without bacteria. We suppose that the explanation is in switching the genes coding enzymes (at least, carbonic anhydrases and ureases as was reported for various representatives of the genus Bacillus [26,27]) that catalyze reactions resulting in the formation of carbonate ions. Since all the ureolytic strains that we selected were capable of inducing CaCO3 precipitation regardless of the presence of urea in the medium, such flexibility of bacteria can be considered the rule rather than the exception. Thus, our findings make it possible to control the production of calcium carbonate polymorphs. It is important not only in view of MICP application in civil engineering for the restoration of building constructions, but for drug delivery, regenerative medicine and tissue engineering as bone cements and substitute materials, dental implants and scaffolds as well as for the design of new materials reviewed in [18,48]. In conclusion, for each of the selected and characterized bacterial candidates, the transformation “ACC-to-vaterite-to-calcite” occurs by different ways, yielding different amounts of precipitates and variable morphologies of carbonate minerals in media with and without urea. In urea-containing medium, all selected bacteria showed a high level of specific urease activity accompanied by the process of biomineralization. In the urea-free medium, all ureolytic bacteria also induced the CaCO3 precipitation, although at lower pH values. We found that only five strains (B. licheniformis DSMZ 8782, B. cereus 4b, S. epidermidis 4a, M. luteus BS52, M. luteus 6) were able to completely repair the original shape of the cement-sand samples. Detailed studies of the most potent strain B. licheniformis DSMZ 8782 revealed the slower rate of the polymorph transformation in the urea-deficient medium than in urea-containing one. This remarkable property could be used in various industrial applications where a specific polymorph (ACC, vaterite or calcite) is required.

Supplementary Materials: The following are available online at http://www.mdpi.com/2075-1729/10/12/317/s1, Figure S1: Typical x-ray diffraction patterns of CaCO3 precipitates formed by representative bacterial strains under study: (a) B. subtilis K51 after the growth in B4-U (calcite), (b) B. cereus 4b after the growth in B4-AC (calcite+vaterite), (c) M. luteus 6 after the growth in B4-AC (calcite + vaterite + non-identified phase). Figure S2: Representative scanning electron micrograhs of CaCO3 precipitates formed by some bacterial strains under study: (a) vaterite (ellipsoid particles) produced by S. epidermidis 4a in B4-AC medium; (b) calcite, B. licheniformis DSMZ 8782, B4-U medium; (c) vaterite, B. subtilis 170, B4-AC medium. Bacterial cells and imprints are seen on the surface of the crystal; (d) calcite (large faceted crystal) and vaterite (ellipsoid particles), B. subtilis 170, B4-AC medium. Figure S3: Typical images of micro-crack filling by B. licheniformis DSMZ 8782 before (a and c) and after a month of the growth on the cement surface in B4-U medium (a and b) and in B4-AC medium (c and d). Cement samples were 4.5 1 cm in size; microcracks were 3.5–4 cm in length and 0.12–2.0 mm in width). × Author Contributions: Conceptualization, A.A.K.; methodology, D.A.G., E.V.Z.; investigation, D.A.G., E.V.Z., A.Y.S., L.A.I., K.S.B., A.E.M., N.V.T.; resources, A.Y.S., N.V.T., A.E.M. and G.P.K.; data curation, A.E.B., A.Y.S., L.A.I., G.P.K., and A.A.K.; writing—original draft preparation, D.A.G., E.V.Z., A.E.B., K.S.B. and A.A.K.; visualization, D.A.G., E.V.Z., A.E.B. and L.A.I.; supervision, G.P.K. and A.A.K.; funding acquisition, D.A.G., E.V.Z., L.A.I., K.S.B. and A.A.K. All authors have read and agreed to the published version of the manuscript. Funding: This research was funded by the Genome Research Centre development program “Kurchatov Genome Centre–PNPI” (agreement No. 075-15-2019-1663). Acknowledgments: This work was performed using the equipment of the Shared Research Centre FSRC “Crystallography and Photonics” RAS and was supported by the Russian Ministry of Education and Science (project RFMEFI62119 0035). The SEM measurements were performed using shared experimental facilities supported by IGIC RAS× state assignment. Conflicts of Interest: The authors declare no conflict of interest.

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