Bacterial in situ-reproduction and colonization of sediments occurring in a limestone mine

A 3-m thick sediment was found in a limestone mine located in the southern part of the Gargano Promontory, Apulia region (south of Italy), at a depth of ca. 25-30 m from the current ground level.

Samples from 5 layers were analysed by X-ray diffraction (XRD), elementar analysis (CHNS), and Inductively Coupled Plasma Mass Spectrometry (ICP-MS). Microbial DNA was also extracted and bacterial diversity analysed by PCR amplification and Illumina High-Throughput Sequencing (HTS) of the V3-V4 hypervariable regions of 16S rRNA.

Preliminary data showed that these sediments formed by subsequent weathering of carbonates and silicates, either by in situ oxidation or by dissolution followed by migration and reprecipitation, rather than during the accumulation of shallow marine sediments occurring between the middle Pliocene and the lower Pleistocene, when the extreme western sectors of the Apulian foreland underwent strong subsidence.

The main mineral compounds occurring in the 5 layers, from the top to the bottom, were the following: calcite (80%) and clay minerals in sample #1, goethite (75%) and hematite in sample #2, manganese (66%) and iron oxides in sample #3, almost exclusively goethite in sample #4, and calcite (71%) and clay minerals in sample #5.

From the microbiological point of view, drawn from a 16S metabarcoding amplicons sequencing analysis, these 5 layers appear to cluster in three groups: a) the uppermost layer (sample #1), dominated by a single and abundant taxon of Arthrobacter sp., which includes species known for  the capability of calcite precipitation; b) a middle layer (including samples #2 and #3), without prevailing abundances and less consistent occurrences across replicates, which featured members of the Oxalobacteraceae family and of the Methylophilus genus. Their closest matches in Genbank subjects included isolates from habitats such as calcium carbonate (moonmilk) muds in percolating waters within caves, mine tailings and other groundwater microcosms; c) a bottom layer (samples #4 and #5), showing an oligarchic situation and high abundances of bacteria but different from the ones that prevailed in the top layer and including members of the Nocardioidacaeae family. Also for these sequence queries, the closest GenBank subjects include cases with calcium carbonate-precipitating capabilities isolated from cave and groundwater sediments or former mining sites in studies on iron oxidizers in creek sediments at pH 4.4 or at high heavy metal concentrations.

Overall, such a distribution suggests that, both in the top and bottom layer, different communities would have undergone in situ-reproduction and colonization exploiting metabolically the substrate, whereas the mid layers would have received bacterial convection by passive transport of percolating waters.