Enhanced calcium carbonate-biofilm complex formation by alkali-generating Lysinibacillus boronitolerans YS11 and alkaliphilic Bacillus sp. AK13

Genomic DNA extraction, whole genome sequencing, and genomic analysis of YS11

Genomic DNA of Lysinibacillus boronitolerans YS11 was extracted using Wizard Genomic DNA purification Kit (Promega, USA). Whole-genome sequencing was performed using Pac Bio RSII Single Molecule Real Time (SMRT) sequencing method (Pacific Biosciences, USA) with a SMRTbell template library in Chunlab (Seoul, South Korea) according to manufacturers’ instructions. The complete genome was achieved using CLgenomics program provided by Chunlab. Gene annotation and assembly were conducted with NCBI Prokaryotic Genomes Automatic Annotation Pipeline. CLgenomics program provided by Chunlab and kegg pathway searching were used for genomic analysis of YS11.

L. boronitolerans YS11 culture conditions

For growth test of strain YS11, a seed culture was incubated in Luria-Bertani (LB) medium, which is composed of 1% tryptone (Bioshop), 1% sodium chloride (Sigma-Aldrich), .5% yeast extract (Bioshop), at 30 °C overnight with shaking at 220 rpm. Next, 1 ml of the seed culture was transferred to a 1.7-ml microtube and washed twice with phosphate buffered saline (PBS), which is composed of .08% NaCl, .02% KCl, .144% Na₂HPO₄, .024% KH₂PO₄. Next, strain YS11 at density of 1 × 10⁶ CFU/ml was transferred into 25 ml of calcium acetate (CaAc) medium (15.8 mM calcium acetate, .4% yeast extract, .5% glucose) or sodium acetate (NaAc) medium (15.8 mM sodium acetate, .4% yeast extract, .5% glucose) in a 50-ml flask to observe the growth on Ca-rich or Ca-poor condition. Growth curves were generated based on colony-forming unit (CFU). The culture was serially diluted from 10⁰ to 10⁻⁸ using PBS for dilution. Then, 200 µl of the diluted bacteria was streaked into pre-heated LB plates. The colonies were counted after 12 h incubation at 30 °C.

RNA isolation and transcriptomic analysis by RNA-seq

Total RNA was obtained from mid-exponentially grown YS11 cells (6 h) from NaAc and CaAc media using a RNeasy kit (Qiagen, Hilden, Germany) following the manufacturer’s instructions. The extracted total RNA was then sent to Chunlab (Seoul, South Korea) for RNA sequencing and alignment. Ribo‐Zero rRNA removal kit (Epicentre, Medison, WI, USA) was used for ribosomal RNA depletion according to the manufacturer’s instructions. Libraries for Illumina sequencing were constructed with TruSeq Stranded mRNA sample prep kit (Illumina, San Diego, CA, USA) following the manufacturer’s protocol. RNA sequencing was conducted on Illumina HiSeq 2500 platform using single-end 50 bp sequencing. Sequence data for the reference genome (Lysinibacillus boronitolerans YS11) were retrieved from the NCBI database. Quality-filtered reads were aligned to the reference‐genome sequence using Bowtie2. The abundance of relative transcript was shown by reads per kilobase of the exon sequence per million mapped sequence reads (RPKM) defined as total exon reads/(mapped reads in millions × exon length in kilobases). Metabolic pathways were analyzed based on KEGG pathway analysis and BLAST alignment with proteins using CLRNASeq program provided by Chunlab.

Isolation procedure for strain AK13

To attain bacteria native to the environment of L. boronitolerans YS11, soil sample was amassed from the rhizosphere of Miscanthus sacchariflorus near Seongbukcheon in South Korea (37° 34′ 44.9″ N 127° 01′ 28.8″ E) (Lee et al. 2017). A 1 g of soil sample was prepared and washed with PBS (pH 7.5). It was then spread onto pH 13 adjusted LB agar. Plates were cultured overnight at 30 °C. Colonies that appeared on the agar plate were isolated and single colonies were roughly cocultured with YS11 in LB medium with three replicates to observe any difference in optical density (OD) compared to YS11 single culture. Cells were grown in PVC 96-well microtiter plates (BD Biosciences) at 30 °C and measured optical density at 595 nm (OD₅₉₅) using biophotometer (Eppendorf, Germany). An isolate that showed a bump that had more than 1% increase in OD₅₆₅ value in the growth curve during coculture was observed and named as AK13.

Phylogenetic analysis of 16S rRNA gene sequence

The 16S rRNA gene of newly isolated alkaliphilic strain AK13 was amplified using universal bacterial 16S rRNA gene primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). Polymerase chain reaction (PCR) was conducted with the following cycling conditions: 94 °C for 90 s followed up by 25 cycles of 94 °C for 45 s, 58 °C for 45 s, and 72 °C for 45 s, and finally an extension step at 72 °C for 5 min. Sequence similarity with other bacteria was determined with EzTaxon program. A neighbor-joining tree was constructed using a distance matrix calculation method in MEGA 7. Bootstrapping was conducted with 1000 iterations. The fasta file of 16S rRNA gene sequence of strain AK13 was deposited at GenBank database (MK517564).

Cocultivation for L. boronitolerans YS11 and Bacillus sp. AK13

Strain YS11 was incubated overnight in pH 6.8 LB broth while strain AK13 was cultured in pH 8 LB broth at 30 °C overnight. Cells were then washed twice with PBS. For coculture of YS11 plus AK13, 1 × 10⁶ CFU/ml of overnight isolates were inoculated into YL medium composed of .4% yeast extract and .34% calcium-acetate at 1:1 ratio. For single culture of YS11 and AK13 each, 2x10⁶ CFU/ml of overnight isolates were inoculated into YL medium. They were incubated at 30 °C with shaking at 220 rpm.

The growth rate of coculture and single culture was measured by counting colony-forming unit (CFU). Normal neutral LB agar was used as a selective medium for strain YS11 while LB agar with pH 12 was used as a selective medium for alkaliphilic strain AK13. Alteration of pH and unbound calcium ion concentration were measured using a pH electrode (Thermo Fisher Scientific, USA) and a calcium-ion selective electrode (ISE) (Thermo Fisher Scientific, USA), respectively. The 7 ml volume of sub-samples were removed from the master volume, over time for 24 h. For these sub samples, organic matters were removed by centrifugation at 4000 rpm for 10 min prior to measurements. These experiments were conducted in triplicates.

For coculture experiment in agar plates, two types of modified B4 medium, CaAc medium and NaAc medium, were used. Then 20 µl of each YS11 and AK13 overnight cultures (1 × 10⁶ CFU/ml) were spotted on the edge and opposite sides of the plate and then cultured at 30 °C for 24 h.

FE-SEM and FTIR for morphological analysis of CaCO₃

Mineral from YS11 single culture or YS11 plus AK13 coculture in YL medium was dried in an oven drier at 100 °C overnight prior to FE-SEM analysis. The sample was placed onto a carbon tape for 1 h to be dried. After blowing unattached particles with nitrogen gas, samples were Pt-coated and analyzed with a Quanta 250 FEG FE-SEM (FEI, USA). For FTIR analysis, an oven dried sample was resuspended in distilled water and analyzed using ATR method in FTIR (Agilent).

pH measurement in pH increasing condition

To determine pH increasing mechanisms in various conditions, 10⁶ CFU/ml of overnight YS11 was inoculated into media consisting of either .8% yeast extract or .8% nutrient broth or MSB basal medium each supplemented with .1% pyruvate, .1% l-serine, or .1% pyruvate plus .1% l-serine. Total of 50 ml volume culture was incubated in 100 ml volume of Erlenmeyer flasks. OD₆₀₀ and pH were measured in 24-h interval during their incubation at 30 °C with agitation at 220 rpm. 7 ml volume of sub samples were taken out from three replicates and measured right away. For the optical density measurement, 10⁻¹ dilutions were done for each sample.

Ammonia measurement assay

Ammonia concentration during incubation of YS11 in YL medium was measured using ammonia assay kit (Sigma-Aldrich). Briefly, supernatant (50 µl) of YS11 cultured in YL medium was mixed with ammonia assay reagent (500 µl) containing α-ketoglutaric acid and NADPH. Then, 5 µl of l-glutamate dehydrogenase was added to each sample and incubated for 5 min at 30 °C. The absorbance of each solution was measured at wavelength of 340 nm.

CLSM analysis to examine biofilm and calcium carbonate development

Biofilms from YS11 and YS11 plus AK13 were stained for 30 min with FilmTracer™ SYPRO^® Ruby biofilm matrix dye at room temperature and visualized using CLSM (LSM700; Carl Zeiss, Jena, Germany). FilmTracer™ SYPRO^® Ruby biofilm matrix stained biofilm cells were used to obtain confocal images under red fluorescent light (excitation wavelength: 450 nm, emission wavelength: 610 nm). Calcium carbonate was visualized under blue fluorescent light by reflection signal of excitation between 483 and 493 nm (Bai et al. 2017). Both biofilms and precipitated calcium carbonate were evaluated for height and density of morphology (C-Apochromat 40×/1.20 W Korr M27; Carl Zeiss).

Biofilm formation assay

Overnight cultures of YS11 and AK13, with the same condition mentioned in cocultivation section, were washed with room-temperature PBS twice at room-temperature. Overnight cells were then inoculated into 1 ml volume of YL medium to contain 2 × 10⁶ CFU/ml of strain YS11 or mixture of YS11 and AK13 at cell density of 1 × 10⁶ CFU/ml each in 48-well microtiter plates. The volume of the cells inoculated was measured by converting OD₆₀₀ value of the overnight cells and converting them to the standard CFU/ml graph. Cell inoculated microtiter plates were then incubated at 30 °C with different time intervals (24 h, 48 h, 72 h) in static condition. Biofilms were stained with crystal violet dye using crystal violet staining assay (O’Toole 2011). Crystal violet attached to biofilms was dissolved in 95% ethanol solution. The optical density at 595 nm (OD₅₉₅) was then measured for biofilm quantification using a multi-detection microplate photometer (HIDEX Sense, Finland).

FAME analysis

Fatty acids from YS11 cultured in NaAc and CaAc media were extracted. Fatty acids were then saponified and methylated to be transformed into fatty acid methyl esters (FAMEs). At first, the bacterial cells washed twice with PBS, and were harvested with centrifugation. The cells were resuspended in 1 ml of saponification reagent into 15 ml falcon tube. This was vortexed for 30 s and heated at 100 °C for 5 min. The cells were once again vortexed for 10 s and heated at 100 °C for 25 min. Then, they were heated at room-temperature for 1 min and 2 ml of methylation reagent were added. This was vortexed for 10 s and heated at 80 °C for 10 min. The solution was cooled at room-temperature for 1 min. 1.25 ml of extraction solvent was added and vortexed for 10 min where the solution divides into two layers. The upper later was transferred into new 15 ml falcon tube. 3 ml of base wash solution was added and vortexed for 5 min. The upper layer of the solution was finally transferred into GC vials. These extracted FAMEs were analyzed using an Agilent 7890 GC system with a flow ionization detector and an HP-Ultra-2 capillary column (crosslinked 2.5% phenylmethyl silicone, 25 m, 200 mm i.d., film thickness: .33 mm). MIDI-2000 calibration standard was used to calibrate FAME values. FAMEs were identified and qualified using the Sherlock 6.0B MIDI software according to their equivalent chain value.

FE-SEM/EDX analysis for CaCO₃ nanoparticle formation

YS11 was incubated in CaAc medium for 6 and 12 h at 30 °C prior to FE-SEM and EDS analyses. Cells were fixed first with low-strength Karnovsky’s solution (2% paraformaldehyde, 2.5% glutaraldehyde, and .1 M phosphate buffer, final pH 7.2) for 2 h. Secondary fixation was done using 2% osmium tetroxide solution for 2 h. These fixed samples were gradually dehydrated with ethanol (30%, 50%, 70%, 100%) for 10 min each and placed onto aluminum stub for 4 days to be dried at room-temperature. These samples were then coated with platinum and analyzed using field-emission scanning electron microscope Quanta 250 FEG (FEI, USA) and energy dispersive X-ray spectrometer (EDS).

Bacterial strains culture deposition number

A strain YS11 culture was stored in Agricultural Culture Collection (KACC) under number KACC 81048BP. A strain AK13 culture was stored in KACC with a deposition number of KACC 81070BP.

Nucleotide and SRA sequence accession number

The complete genome sequence of Lysinibacillus boronitolerans YS11 has been deposited in NCBI under the GenBank accession number CP026007.1. The RNA-seq of Lysinibacillus boronitolerans YS11 has been deposited in NCBI under the SRA number SRR8083459.

Complete genome sequencing of CaCO₃ precipitating bacterium YS11

Whole genome sequencing of Lysinibacillus sp. YS11 previously reported for its non-ureolytic calcium carbonate precipitation (Lee et al. 2017) was conducted in this study. A complete genome comprising 1 circular chromosome with the genome size of 4,584,915 base pairs and GC content of 37.70% was obtained (Fig. 1a). Until now, a total of 111 genomes from species belonging to Lysinibacillus genus have been sequenced, including a CCP strain Lysinibacillus sphaericus LMG 22257 (Yan et al. 2017). The genome of Lysinibacillus sp. YS11 and genomes of other Lysinibacillus species, including L. boronitolerans NBRC 103108, L. macroides DSM 54, L. xylanilyticus DSM 23493, and L. pakistanensis JCM 18776, was compared (Table 1). Average Nucleotide Identity (ANI) of strain YS11 with other species belonging to Lysinibacillus genus revealed that YS11 belonged to boronitolerans species, showing ANI of 99.87% (Table 2). The genome size of YS11 was 4.584 kb, was 99.9% the size of L. boronitolerans NBRC 103108 (4.564 kb). L. boronitolerans YS11 possessed the highest number of tRNA genes (34) and rRNA genes (107) among the other four species. Similar to results from ANI analysis, analysis for the presence and absence of genes also verified that YS11 was closest to L. boronitolerans NBRC 103108 and the farthest from strain JCM 18776 (Additional file 1: Fig. S1A). YS11 also retained similar distributions of genes in COG category with strain DSM54, in which the most abundant genes were affiliated to amino acid metabolism and transport (E) (Additional file 1: Fig. S1B). Except L. pakistanensis JCM 18776, strain YS11 retained most abundant number of CDS related to amino acid metabolism and transport (E) COG group (Fig. 1b). Genes involved in ammonia production for possible alkaline generating pathway were searched in the genome of YS11. There are total of 35 genes that were directly involved in ammonia production (Table 3). When the number of genes involved in deamination of amino acids in the genome of L. boronitolerans YS11 was compared to that of NBRC 103108, a phylogenetically similar species, YS11 possessed about 3 times more genes of amino acid deaminases, suggesting species specific characteristics of basic compound production (Fig. 1c). Along with strain NBRC 103108, other Lysinibacillus species also showed smaller numbers of amino acid deaminases (Fig. 1c).

Features	Values
	YS11	NBRC 103108	JCM 18776	DSM 54	DSM 23493
Genome size (Mb)	4.58	4.56	5.01	4.87	5.22
GC content (%)	37.7	37.6	36.3	37.7	36.5
Contigs	1	81	73	15	13
CDS	4602	4576	8211	4713	4884
rRNAs	34	6	5	9	20
tRNAs	107	52	74	86	100

Strain YS11	Average nucleotide identity (%)
Lysinibacillus boronitolerans NBRC 103108T	99.87
Lysinibacillus pakistanensis JCM 18776T	98.93
Lysinibacillus macroides DSM 54T	98.16
Lysinibacillus xylanilyticus DSM 23493T	97.27

Coordinates	Strand	Product	Gene name
266,921–268,435	+	Histidine ammonia-lyase	hutH
318,892–320,310	−	Putative amidase AmiD
435,365–436,537	+	Lysine 6-dehydrogenase
535,842–537104	−	Phenylserine dehydratase
1,039,715–1,040,377	+	Probable l-serine dehydratase, beta chain	sdaAB
1,040,392–1,041,300	+	Probable l-serine dehydratase, alpha chain	sdaAA
1,101,650–1,102,153	+	Probable chemoreceptor glutamine deamidase CheD 1
1,318,421–1,319,788	−	Guanine deaminase	guaD
1,430,688–1,431,656	−	l-Asparaginase
1,547,319–1,548,554	+	Allantoate deiminase
1,581,552–1,582,922	+	Ethanolamine ammonia-lyase heavy chain
1,582,932–1,583,942	+	Ethanolamine ammonia-lyase light chain
1,614,306–1,615,679	−	NADP-specific glutamate dehydrogenase
1,616,318–1,617,646	+	Probable d-serine dehydratase
1,804,351–1,805,055	+	Glucosamine-6-phosphate deaminase
1852010–1852543	+	Peroxyureidoacrylate/ureidoacrylate amidohydrolase RutB
2,097,252–2,098,670	+	Aspartate ammonia-lyase	aspA
2,100,543–2,100,845	+	Urease subunit gamma	ureA
2,100,861–2,101,229	+	Urease subunit beta
2,101,226–2,102,941	+	Urease subunit alpha	ureC
2,423,272–2,424,252	−	Meso-diaminopimelate d-dehydrogenase
2,687,063–2,688,808	−	Adenine deaminase	ade
2,743,214–2,744,329	+	Cystathionine beta-lyase MetC
2,850,380–2,851,474	−	Leucine dehydrogenase
2,897,281–2,898,246	−	Glutaminase 1	glsA
2,913,990–2,915,093	−	Aminomethyltransferase	gcvT
3,177,391–3,178,323	−	Porphobilinogen deaminase
3,406,895–3,408,010	−	Alanine dehydrogenase	ald
3,539,367–3,540,581	−	Diaminopropionate ammonia-lyase
3,564,845–3,566,089	−	Catabolic NAD-specific glutamate dehydrogenase RocG
3,575,261–3,576,433	+	Cystathionine beta-lyase PatB
3,688,923–3,690,383	−	Putative amidase AmiC
3,856,477–3,857,190	−	Glucosamine-6-phosphate deaminase	nagB
3,991,464–3,993,185	−	Putative adenine deaminase PTO1085
4,002,226–4,002,909	−	Phosphoribosylformylglycinamidine synthase subunit PurQ

Alkaline generation by YS11 is suitable for CaCO₃ precipitation

Ammonia concentration was measured during the growth of YS11 in YL medium. The increase of ammonia concentration in accordance with pH increase suggested that ammonia production might be a major way for alkaline generation (Fig. 2a). Thus, alkaline generating pathways of non-ureolytic YS11 were analyzed using phenotypic and genomic approaches. Since yeast extract in YL medium is abounded with amino acids, pH change in two kinds of rich medium culture each supplemented with .8% yeast extract and .8% nutrient broth was measured (Fig. 2b). YS11 could increase the pH in both conditions, highly suggesting that deamination of rich amino acids provided by yeast extract in the medium was the key factor in ammonia production during this condition. Since yeast extract is an undefined complex component known to attain rich amino acids, transcriptomes during pH increasing conditions of CaAc and NaAc were analyzed. When data were trimmed with RPKM expressions higher than 100, genes involved in deamination of the following eight compounds were expressed: l-serine, glutamine, glutamate, aspartate, meso-diaminopimelate, porphobilinogen, alanine, cystathionine, and glucosamine-6-phosphate (Fig. 2c).

Increase in pH level by YS11 induces interspecies interaction with alkaliphilic bacterium

In order to observe interspecies interaction in biofilm mediated calcium carbonate formation, additional isolation from where YS11 had been found was conducted to explore species that is in cooccurrence with YS11. Alkaliphilic strains were targeted for screening procedure as L. boronitolerans YS11 promotes alkali condition during the growth, and strain AK13 was isolated. In the procedure, bacterial isolates with alkaliphilic properties were screened in pH 13 adjusted LB medium culture. After incubation, colonies formed in LB medium were further tested for coculture with strain YS11. The phylogenetic tree of strain AK13 was evaluated with 16S rRNA gene sequences, including strains showing high similarity (Additional file 1: Fig. S2A). Strain AK13 was phylogenetically affiliated with genus Bacillus. Its 16S rRNA gene sequence showed the highest similarity (99.88%) with that of Bacillus hunanensis JSM 091003^T, Bacillus oshimensis DSM 18940^T, and Bacillus lehensis MLB-2^T. These 16S rRNA gene sequences of strain AK13 also shared high similarities with Bacillus xiaoxiensis JSM 081004^T (99.25%) and Bacillus patagoniensis DSM 16117^T (99.13%). Strain AK13 was able to grow well without any inhibition at alkaline pH range from pH 8 to pH 11 compared to its growth in pH 7, showing an optimal growth at pH 8 (OD₆₀₀ ~ .65). Its growth was inhibited at pH 7 and pH 12. AK13 strain was unable to grow at pH 6 (Additional file 1: Fig. S2B). This implies that AK13 is an alkaliphilic bacterium (with optimal growth in alkaline condition, unable to grow or its growth is hampered in neutral condition). On the other hand, YS11 showed optimal growth rate at neutral pH 7. YS11 strain was able to grow at pH 6 and at pH 9, but not at pH 10 and pH 11 (Additional file 1: Fig. S2B).

Further experiments were performed to observe interaction among these two different bacteria during cocultivation. These two strains were cocultured in neutral growth medium of pH 6.8 to observe interspecies interaction in both liquid culture and agar plate culture (Fig. 3a, d). Yellow colony of AK13 also appeared in the agar plate culture when YS11 and AK13 were each spotted at the edge of the opposite side of the plate. To identify the co-cultivation more specifically, each species’ growth in pH varying LB selective medium, pH change, and unbound calcium concentration were measured. The growth of alkaliphilic AK13 in single culture was not detected throughout the experiment. Strain YS11 single culture grew to cellular density up to 10⁸ CFU/ml, reaching its stationary phase at 9 h after incubation (Fig. 3a). On the other hand, when these two bacteria were cultured together at 1:1 ratio of initial cellular density of 10⁶ cell/ml, strain AK13 could retrieve its growth starting from 9 h after incubation (Fig. 3a). This result suggests pH increase by strain YS11 enables the growth of alkaliphilic AK13 during their cooccurrence. The pH of the coculture and that of YS11 single culture were both increased along with bacterial growth. The pH increases in coculture (up to pH 7.6) was less than that of YS11 alone (up to pH 7.9) (Fig. 3b). Unbound calcium ion concentration gradually decreased with YS11 alone and YS11 plus AK13 coculture, although the coculture showed faster decrease in calcium concentration (Fig. 3c). No prominent decrease in calcium ion concentration was detected for AK13 single culture since there was no growth. However, a slight decrease of approximately 300 ppm of calcium ions was found. This might be due to charge–charge interaction of initially inoculated cell wall and calcium ion (Fig. 3c). For the alkali generating mechanism by YS11, deamination of amino acids is highly speculated as strain YS11 pertains to deamination pathways, contributing to its alkali generating characteristic.

Co-occurrence alters the appearance of calcium carbonate precipitation

After monitoring the growth, pH change, and calcium ion concentration, cultured cells were visualized for precipitated calcium carbonate minerals. It has been characterized that L. boronitolerans YS11 precipitates calcium carbonate when calcium source is provided in the medium (Lee et al. 2017). Resultant calcium carbonate was confirmed from both YS11 culture alone and YS11 plus AK13 coculture condition (Fig. 4a, b). MICP induced by YS11 in YL medium resulted in round shaped mineral aggregates whereas MICP induced by mixed culture depicted smaller particle size and more compact form (Fig. 4a). Besides such difference in mineral shape, the coculture showed distinct formation of EPSs within calcium carbonate-cell clusters (Fig. 4a). Minerals precipitated in both cultures were verified to be calcium carbonates as the FTIR analysis showed three peaks of samples identical to reference calcium carbonate peaks (Fig. 4b).

To verify relations between EPSs and calcium carbonate generation, cells were incubated in static condition for biofilm and calcium carbonate development periodically. Image analysis by CLSM showed similar results (Fig. 4c). Both YS11 single culture and YS11 plus AK13 coculture were in developmental stage of biofilm in 24 h, matured biofilm in 48 h, and dispersed form in 72 h. In all stages, biofilm formed under YS11 plus AK13 coculture condition was much higher (Fig. 4c). Along with biofilm formation, much more biofilm bound calcium carbonate was observed in the coculture (Fig. 4c). Quantification of biofilm formation was performed after crystal violet staining to examine equivalent results as CLSM images. Accordingly, biofilm formation in coculture was measured to be 1.5-fold higher than that in YS11 single culture (Fig. 4d). Enhanced biofilm formation in coculture by strain AK13 might give nucleation site for calcium carbonate, suggesting more surface-calcium carbonate bound cells.

Survival of YS11 in sole culture and coculture were compared under alkaline pH of 12. The survival of YS11 from the coculture was improved (Fig. 4e). This suggests that altered EPS composition and quantity of biofilm formation might have contributed to the improvement in the survival of YS11 when it is cocultured with AK13. CCP bacteria are often applied inside building materials such as concrete for repairment of crack initiations (Lee and Park 2018). Because the pH of the concrete matrix is up to 13, our result suggests that increased survival from the co-occurrence of YS11 and AK13 could also show enhanced healing rate of cracks when applied into cementitious materials.

Modification of membrane rigidity during calcium carbonate precipitation

The growth curve of YS11 in CaAc medium and NaAc medium showed similar growth rates (Fig. 5a). To determine changes in physiology of YS11 during calcium carbonate precipitation when Ca²⁺ was near, RNA-seq was conducted for mid-exponentially grown cells from both NaAc and CaAc conditions (Table 4). Interestingly, upregulation of branched chain amino acid (BCAA) and branched chain fatty acid (BCFA) synthesis was noticeable in transcriptomics of calcium carbonate precipitation (CaAc medium) compared to that of the control (NaAc medium). YS11 retained all genes involved in both BCAA and BCFA synthesis in one operon (Fig. 5b). BCAA synthesis pathway is directly connected to BCFA pathway where BCFA are produced as cellular components (Fig. 5b). Furthermore, genes involving multi-drug efflux pump, membrane protein related genes, and membrane protease-involved genes were also upregulated when Ca²⁺ was provided (at CaAc condition) (Table 4). These results suggest modification of membrane during MICP. Thus, fatty acid methyl esters (FAMEs) of YS11 from NaAc and CaAc cultures were quantified to determine any modification in BCFA composition. Noticeably higher value in total BCFA ratio including anteiso BCFA and iso BCFA ratios were measured under CaAc condition compared to that under NaAc condition after 12 h of culture (Fig. 5c). This result demonstrates alteration in phenotype of membrane rigidity from the presence of Ca²⁺. However, the BCFA ratio in 6 h CaAc condition was unsubstantial compared to that in 6 h NaAc condition. Although gene expression at CaAc condition showed considerable difference compared to that at NaAc condition at 6 h, protein difference and phenotypical changes were only evident from 12 h. For the first time, we show genomic and phenotypic characteristics of CCP bacteria during CaCO₃ precipitation through differential gene expression and FAME analyses. It was questionable whether calcium carbonate crystals were present during 6 h incubation where major transcriptomic changes were observed. FE-SEM and EDS obtained after 6 h of incubation in CaAc medium confirmed the presence of calcium carbonate (Additional file 1: Fig. S3). Presence of Ca²⁺ also affects phenotypes of Lysinibacillus boronitolerans YS11. Spore formation and biofilm formation were all increased in CaAc compared to those in NaAc (Figs. 6a, b). The increase of biofilm formation in CaAc was also observed in CLSM image where the glass surface was covered up by biofilms under CaAc condition compared to scattered biofilm under NaAc condition (Fig. 6c). Swimming motility was also measured under CaAc and NaAc conditions. Swimming of YS11 in Ca²⁺-rich condition was much higher than that under NaAc condition. It covered all area of the petri dish (Fig. 6d). Ca²⁺-specific differences in various phenotypes demonstrate that strain YS11 shows responses corresponding to Ca²⁺. Further experiments are needed to determine the mechanisms underlying these differences.

Locus taq	Gene symbol	Product	Strand	CaAc RPKM	NaAc RPKM	CaAc/NaAc
Genes upregulated by high Ca2+
Branched chain amino acid synthesis
LBYS11_13585		Acetolactate synthase small subunit	−	144.04	14	10.29
LBYS11_13570	leuB	3-Isopropylmalate dehydrogenase	−	124.41	12.67	9.82
LBYS11_13580		Ketol-acid reductoisomerase	−	263.2	27.42	9.6
LBYS11_13560	leuD	3-Isopropylmalate dehydratase small subunit	−	116.42	12.75	9.13
LBYS11_13565	leuC	3-Isopropylmalate dehydratase large subunit	−	144.46	17.55	8.23
LBYS11_13575		2-Isopropylmalate synthase	−	165.84	20.78	7.98
LBYS11_13590	ilvB	Acetolactate synthase large subunit IlvB1	−	131.49	18.13	7.25
LBYS11_13595	ilvD	Dihydroxy-acid dehydratase	−	223.6	41.14	5.44
Branched chain fatty acid synthesis
LBYS11_00205		Hypothetical protein (NCBI blast: 3-oxoacyl-(Acyl-carrier-protein (ACP)) synthase III)	+	1150.45	113.55	10.13
LBYS11_00220		Phosphopantetheine-binding protein	+	1044.75	125.49	8.33
LBYS11_00200		Hypothetical protein: holo-[acyl-carrier-protein] synthase	+	956.13	115.3	8.29
LBYS11_00230		Hypothetical protein: holo-[acyl-carrier-protein] synthase	+	1028.85	125.53	8.2
Lysine synthesis
LBYS11_00215	lysA	Diaminopimelate decarboxylase	+	1089.26	118.57	9.19
Might serve as radioprotective agent
LBYS11_06125		Glutathionylspermidine synthase	+	315.38	82.76	3.81
Protease involved genes
LBYS11_00265		Uncharacterized zinc protease YmfH	+	754.93	96.72	7.81
LBYS11_00260		Zinc protease	+	685.49	90.94	7.54
LBYS11_06700		n|Uncharacterized protein YuaG	+	560.04	124.56	4.5
LBYS11_16935	hflK	FtsH protease activity modulator HflK|Protein HflK	−	605.83	146.1	4.15
LBYS11_16930	hflC	Protease modulator HflC|Protein HflC	−	887.2	231.43	3.83
LBYS11_17070	sppA	Signal peptide peptidase SppA|Putative signal peptide peptidase SppA	−	139.57	36.6	3.81
Multidrug efflux pump and swarming motility involved genes
LBYS11_00190		Multidrug export protein AcrF Cobalt-zinc-cadmium resistance protein CzcA	+	336.58	42.35	7.95
LBYS11_00195		Swarming motility protein SwrC	+	800.18	106.83	7.49
LBYS11_00255		Hypothetical protein Multi antimicrobial extrusion protein (Na(+)/drug antiporter)	+	376.26	51.85	7.26
LBYS11_00250		Hypothetical protein Multi antimicrobial extrusion protein (Na(+)/drug antiporter)	+	265.05	37.79	7.01
TCA cycle involved genes
LBYS11_00240		Acyl-CoA dehydrogenase	+	1676.26	217.77	7.7
LBYS11_07645		NADP-specific glutamate dehydrogenase	−	453.98	112.24	4.04
Hydantoinase
LBYS11_08510		N-methyl hydantoinase	+	162.46	21.19	7.67
LBYS11_08515		Hydantoinase subunit beta	+	225.3	32.87	6.85
Membrane proteins
LBYS11_13515		Uncharacterized membrane protein YceF	+	664.23	68.7	9.67
LBYS11_06695		Uncharacterized membrane protein YuaF	+	432.69	89.78	4.82
LBYS11_17065		RDD family protein|Uncharacterized membrane protein YteJ	−	362.02	93.05	3.89
LBYS11_20665		Hypothetical protein|UPF0699 transmembrane protein YdbS	−	148.94	39.48	3.77
Hypothetical proteins
LBYS11_00210		Hypothetical protein	+	1190.22	124.33	9.57
LBYS11_00225		Hypothetical protein	+	1021.86	116.54	8.77
LBYS11_00235		Hypothetical protein	+	1193.34	140.21	8.51
LBYS11_00245		Hypothetical protein	+	1002.8	129.52	7.74
LBYS11_09000		Hypothetical protein	+	232.67	38.56	6.03
LBYS11_09005		Hypothetical protein	+	186.62	33.96	5.5
LBYS11_09815		Hypothetical protein	+	153.15	32.91	4.65
LBYS11_21200		Hypothetical protein	−	143.8	32.67	4.4
LBYS11_19760		Hypothetical protein|Uncharacterized protein YvlA	+	133.72	33.88	3.95
LBYS11_06120		Hypothetical protein|Uncharacterized serine-rich protein C21513	+	413.46	112.58	3.67
Genes downregulated by high Ca2+
LBYS11_19235		Copper amine oxidase	−	63.11	224.53	.28
LBYS11_08750		Copper amine oxidase	+	52.07	239.12	.22
LBYS11_20870		Zinc transporter ZupT	−	133.27	521.26	.26
LBYS11_01430		Divalent metal cation transporter	−	8.88	211.74	.04
LBYS11_20140		Guanine/hypoxanthine permease	−	9.31	157.38	.06