Microbial communities associated with the anthropogenic, highly alkaline environment of a saline soda lime, Poland

Abstract

Soda lime is a by-product of the Solvay soda process for the production of sodium carbonate from limestone and sodium chloride. Due to a high salt concentration and alkaline pH, the lime is considered as a potential habitat of haloalkaliphilic and haloalkalitolerant microbial communities. This artificial and unique environment is nutrient-poor and devoid of vegetation, due in part to semi-arid, saline and alkaline conditions. Samples taken from the surface layer of the lime and from the depth of 2 m (both having pH ~11 and EC_e up to 423 dS m^-1) were investigated using culture-based (culturing on alkaline medium) and culture-independent microbiological approaches (microscopic analyses and pyrosequencing). A surprisingly diverse bacterial community was discovered in this highly saline, alkaline and nutrient-poor environment, with the bacterial phyla Proteobacteria (representing 52.8% of the total bacterial community) and Firmicutes (16.6%) showing dominance. Compared to the surface layer, higher bacterial abundance and diversity values were detected in the deep zone, where more stable environmental conditions may occur. The surface layer was dominated by members of the genera Phenylobacterium, Chelativorans and Skermanella, while in the interior layer the genus Fictibacillus was dominant. The culturable aerobic, haloalkaliphilic bacteria strains isolated in this study belonged mostly to the genus Bacillus and were closely related to the species Bacillus pseudofirmus, B. cereus, B. plakortidis, B. thuringensis and B. pumilus.

Keywords: 16S rRNA gene pyrosequencing; Alkaliphiles; Halophiles; Saline soda (Solvay) lime.

Fig. 1

The surface of the saline soda lime deposited in the repository in Janikowo

Fig. 2

Physical structure of the saline soda lime a less compact surface layer, b more compact internal layer, c, d arrows point putative bacterial cells, covered with salts)

Fig. 3

Distribution of bacterial sequences retrieved by pyrosequencing from the interior and the surface saline soda lime samples. Contribution of taxa to the total bacterial community is given based on their relative abundance (a) and the distribution of species (b). Phylum-level distribution (except for Proteobacteria, where class-level distribution is shown) was expressed as a percentage of total sequences and as estimated species number based on the number of observed OTUs (assigned at 97% similarity level) per group. Species numbers were calculated assuming 97% sequence similarity threshold based on the 16S rRNA gene. Only taxa having contribution higher than 5% at least in one sample are shown

Fig. 4

Association network of major bacterial OTUs in the saline soda lime samples based on pyrosequencing data. OTUs were calculated assuming 97% species-level sequence similarity threshold based on the 16S rRNA gene. Top 50 most abundant bacterial OTUs are shown as circles with size corresponding to relative abundance in the total dataset, while the width of the edges corresponds with the relative abundance of an OTU in a particular sample. Circles are color-coded according to phylogenetic affiliation and genus-level identity of each OTU is also shown. OTUs distantly related to cultured bacteria were marked with letters as follows: a uncultured Rhodobacteraceae, b unclassified DB1-14 Alphaproteobacteria, c uncultured Rhodobacteraceae, d unclassified Elev-16S-976 Actinobacteria, e unclassified E6aC02 Sphingobacteriales, f unclassified Rhizobiales alphal cluster, g unclassified Microbacteriaceae, h unclassified ML602 M-17 Bacteriodetes, i unclassified NKB5 Gammaproteobacteria

Fig. 5

Phylogenetic tree of chloroplast genotypes obtained from the saline soda lime samples based on the pyrosequencing of the 16S rRNA gene. Tree was constructed using the Maximum Likelihood method with the General Time Reversible nucleotide substitution model and is based on 334 nucleotide positions. GenBank accession numbers are given in parentheses. Representative sequences obtained by pyrosequencing are marked with grey squares. In this case, sequences differing only in one position are represented with a single sequence, number of sequences is given in square brackets (I interior and S surface sample) and major phylotypes representing at least 0.5% of total chloroplast sequences are highlighted with bold letters

Fig. 6

Phylogenetic tree of the saline soda lime bacterial isolates and closely related genotypes from pyrosequencing data based on the 16S rRNA gene. Tree was constructed using the Maximum Likelihood method with the Kimura 2-Parameter nucleotide substitution model and is based on 356 nucleotide positions. Type strains are marked with superscript T, GenBank accession numbers are given in parentheses. Bacterial isolates are marked with black squares, while representative sequences obtained by pyrosequencing are marked with grey squares. In the latter case, sequences differing only in one position are represented with a single sequence, number of sequences is given in square brackets (I, interior and S, surface sample) and major phylotypes representing at least 0.5% of total bacterial sequences are highlighted with bold letters