Lee and Park AMB Expr (2019) 9:49 https://doi.org/10.1186/s13568-019-0773-x ORIGINAL ARTICLE Open Access Enhanced calcium carbonate-bioflm complex formation by alkali-generating Lysinibacillus boronitolerans YS11 and alkaliphilic Bacillus sp. AK13 Yun Suk Lee and Woojun Park* Abstract Microbially induced calcium carbonate (CaCO3) precipitation (MICP) is a process where microbes induce condition favorable for CaCO3 formation through metabolic activities by increasing the pH or carbonate ions when calcium is near. The molecular and ecological basis of CaCO3 precipitating (CCP) bacteria has been poorly illuminated. Here, we showed that increased pH levels by deamination of amino acids is a driving force toward MICP using alkalitoler- ant Lysinibacillus boronitolerans YS11 as a model species of non-ureolytic CCP bacteria. This alkaline generation also facilitates the growth of neighboring alkaliphilic Bacillus sp. AK13, which could alter characteristics of MICP by chang- ing the size and shape of CaCO3 minerals. Furthermore, we showed CaCO3 that precipitates earlier in an experiment modifes membrane rigidity of YS11 strain via upregulation of branched chain fatty acid synthesis. This work closely examines MICP conditions by deamination and the efect of MICP on cell membrane rigidity and crystal formation for the frst time. Keywords: Alkaline generation, Dual species CaCO3 precipitation, Bacteria-CaCO3 interaction, Branched chain fatty acid synthesis, Membrane rigidity Introduction exopolysaccharide (EPS) formation, eventually leading to Calcium carbonate precipitating (CCP) bacteria con- microbially induced CaCO3 precipitation (MICP). Bac- tribute to the geochemical cycle as they precipitate car- terial metabolic pathways can create compounds that bonate minerals, including calcium carbonate in nature increase the solution pH. Tese include photosynthe- (Douglas and Beveridge 1998). Formation of stromato- sis, ureolysis, ammonifcation, denitrifcation, sulphate lite is an example of calcium carbonate precipitation by reduction, and formate oxidation are known to induce cyanobacterial-bacterial mat communities (Paerl et al. calcium carbonate precipitation (Hammes and Verstraete 2001). Tere are four main environmental parameters 2002; Ganendra et al. 2014). When resultant pH and DIC that govern reaction kinetics for calcium carbonate pre- increases are accompanied by the aforementioned path- cipitation: (1) pH, (2) calcium (Ca2+) ion concentration, ways in an environment with calcium ions, bacterial cell (3) dissolved inorganic carbon (DIC) concentration, and walls or their extracellular polymeric substances (EPSs) [4] nucleation site availability (Hammes and Verstraete can serve as a nucleation site for precipitation reaction 2002). Bacteria infuence these parameters through (Hammes and Verstraete 2002). Despite the fact that their metabolic activity, the production of bioflm, and alkaline generating bacteria are ubiquitous in the natural environment, much of their microbial ecology remains to *Correspondence: [email protected] be investigated. Laboratory of Molecular Environmental Microbiology, Department Bacteria can form bioflms in most environmental of Environmental Science and Ecological Engineering, Korea University, niches, and nearly all bioflm communities in nature Seoul 02841, Republic of Korea © The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Lee and Park AMB Expr (2019) 9:49 Page 2 of 17 comprise a variety of species (Elias and Banin 2012). of Ca2+ inside bacteria remains far from clear (Nor- Bioflms provide protection to cells when bacteria face ris et al. 1996; Dominguez 2004). Functions of Ca 2+ in fuctuating and harsh environmental conditions. Tey eukaryotes are well-established compared to those in facilitate complex interactions between individual cells prokaryotic cells. Ca2+ can act as a signaling molecule and an environment (Hall-Stoodley et al. 2004; Liu et al. and a secondary messenger that is tightly regulated by 2016). Te bioflm matrix is composed of multiple spe- the gradient between intracellular and extracellular cies and a mixture of components such as EPS, protein, Ca2+ concentrations (Islam 2012). It has been recently nucleic acids, and other substances. Te best studied one found that Ca2+ also possesses signaling functions in of these components is EPS (Davey and O’Toole 2000). prokaryotic cells, including bacteria. Ca 2+ concentra- Te co-occurrence pattern of bacteria embedded in this tion inside cell is tightly controlled. It is much lower matrix is determined by species interaction (Nadell et al. in concentration than that in the outside environment 2016). Bioflms not only contain biogenic substances, (Dominguez 2004; Islam 2012; Dominguez et al. 2015). but also contain granules trapped from sediments (Rid- Furthermore, Ca2+-binding proteins pertaining EF- ing 2000). EPSs generally possess metal binding proper- hand motifs are found to be distributed among various ties, including Ca2+, because EPSs retain high molecular bacterial species (Dominguez et al. 2015; Zhou et al. weight compounds that have charged functional groups 2006; Rigden et al. 2011). Recent studies suggest that (Bhaskar and Bhosle 2006). Terefore, multi-species bio- the delicate Ca2+ signaling found in eukaryotes might flm in nature can act as a foundation for MICP. Previ- have evolved from prokaryotic signaling (Shemarova ous studies have shown that EPS formed by bioflms is and Nesterov 2005; Case et al. 2007). Recent studies are crucial when investigating bacterial physiological func- unveiling specifc roles of calcium in bacteria, such as tions and activities (Giufre et al. 2013; Braissant et al. Ca2+-signaling, Ca2+-binding proteins, motility, spore 2003). Tese ex situ research eforts have also found that germination, quorum sensing, EPS production, and EPS could regulate spatial position of precipitation dur- bioflm formation (Bertrand et al. 2010; Bilecen and ing mineralization. In the absence of microscale chemi- Yildiz 2009; Cruz et al. 2014; Guragain et al. 2013; John- cal gradients, nucleation models have estimated that son et al. 2011; Porsch et al. 2013; Sarkisova et al. 2005). crystals produced by bioflms can distribute randomly It has been found that Ca2+-transporting ATPases, in EPS (Arp et al. 2001). Most current studies of bioflms motility proteins, and two-component systems in bac- and biomineralization suggest that precipitation of min- teria can act in calcium concentration dependent ways eral appears on the bioflm surface primarily (Zhang and (Bertrand et al. 2010; Bilecen and Yildiz 2009; Cruz Klapper 2010). However, these studies and models are et al. 2014; Guragain et al. 2013; Johnson et al. 2011; not suitable for in situ observation since information of Porsch et al. 2013; Sarkisova et al. 2005). Because MICP mineral formation in bioflms, such as spatial patterns, occurs when Ca2+ exists in surrounding, CCP bacteria are lacking. A recent study has developed a method for a can provide a physiological model to examine bacterial real time, in situ biomineralization examination by imag- response to Ca2+ when they produce calcium carbonate ing the biomineralized calcium carbonate within bioflms minerals. produced by Pseudomonas aeruginosa (Bai et al. 2017). In our previous study, a novel CCP bacterium Lysini- However, interdisciplinary research studies of multi-spe- bacillus sp. YS11 was isolated and characterized for cies embedded bioflm, and its resultant MICP, have not its ability to produce calcium carbonate in solution been reported yet. Exploring the relationship between (Lee et al. 2017). Unlike increase in pH level via ure- pH increasing bacteria involved in multispecies bioflm olysis which has been broadly studied regarding MICP, and calcium carbonate formation is important for under- strain YS11 was found to utilize pathways other than standing integral mechanisms of microbially induced cal- urea hydrolyses for pH increase of a solution (Yan cium carbonate formation (MICP). et al. 2017). Here, we found that alkaline generation All living organisms, including bacteria, require of YS11 was through ammonia production via deami- metal elements for metabolic activities as metals have nase activity. Alkaline generation from YS11 facilitated structural or catalytic roles (Ehrlich 1997). Among the growth of alkaliphilic bacterium Bacillus sp. AK13, many metal ions, iron (Fe 2+), magnesium (Mg 2+), man- which was isolated from the identical environment. It ganese (Mn2+), zinc (Zn2+), copper (Cu2+), and Ca2+ altered the morphology and amount of bioflm-bound are reported to possess important cellular functions calcium carbonate. Furthermore, we demonstrate that in microorganisms (Roane et al. 2009). Various func- the precipitated calcium carbonate can modify mem- tions of metals, including Fe2+, Mg2+, Zn2+, and Mn2+, brane rigidity in bacteria by upregulating branched in bacterial cells have been well scrutinized (Andrews chain amino acid (BCAA) and branched chain fatty et al. 2003; Diaz-Ochoa et al. 2014). However, the role acid (BCFA) synthesis. Lee and Park AMB Expr (2019) 9:49 Page 3 of 17 Materials and methods boronitolerans YS11) were