Calcium carbonate precipitation by heterotrophic bacteria isolated from biofilms formed on deteriorated ignimbrite stones: influence of calcium on EPS production and biofilm formation by these isolates

Abstract

Heterotrophic CaCO₃-precipitating bacteria were isolated from biofilms on deteriorated ignimbrites, siliceous acidic rocks, from Morelia Cathedral (Mexico) and identified as Enterobacter cancerogenus (22e), Bacillus sp. (32a) and Bacillus subtilis (52g). In solid medium, 22e and 32a precipitated calcite and vaterite while 52g produced calcite. Urease activity was detected in these isolates and CaCO₃ precipitation increased in the presence of urea in the liquid medium. In the presence of calcium, EPS production decreased in 22e and 32a and increased in 52g. Under laboratory conditions, ignimbrite colonization by these isolates only occurred in the presence of calcium and no CaCO₃ was precipitated. Calcium may therefore be important for biofilm formation on stones. The importance of the type of stone, here a siliceous stone, on biological colonization is emphasized. This calcium effect has not been reported on calcareous materials. The importance of the effect of calcium on EPS production and biofilm formation is discussed in relation to other applications of CaCO₃ precipitation by bacteria.

Keywords:

• ignimbrite stones
• CaCO₃ precipitation
• heterotrophic bacteria
• calcium effect
• EPS production
• biofilm formation

Abbreviations:

• BG-11, Blue Green mineral medium
• BLAST, basic local alignment search tool
• CPS, capsular polysaccharides
• CR, Congo Red
• EPS, extracellular polymeric substances
• MEGA, molecular evolutionary genetics analysis
• NCBI, National Center for Biotechnology Information
• OD, optical density
• PBS, phosphate buffered saline
• PGY-BG11, BG-11 medium supplemented with peptone, glucose and yeast extract
• rRNA, ribosomal ribonucleic acid
• SEM, scanning electron microscopy
• XRD, X-ray diffraction

Acknowledgments

The authors would like to thank Paul Gaytan and Jorge Arturo Yañez from the Instituto de Biotecnología (UNAM) for DNA sequencing procedures, Manuel Aguilar from the Instituto de Física (UNAM) for crystals analysis by XRD, and Jorge Gutiérrez from the Licenciatura en Ingeniería Biológica for his help in developing the Congo Red protocol for EPS analysis in liquid media. Maribel Hernández Guerrero is acknowledged for her critical review of the manuscript. Angélica López Moreno received a scholarship for PhD students from the CONACyT (CVU number 42640).