Isolation of pleomorphic bacterial phyllobacterium sp. from enhydro agates

Abstract

Enhydro agates are round to egg-shaped nodules composed of banded microcrystalline to cryptocrystalline quartz that have a water-filled internal cavity. Enhydro agates form as agate material precipitates from silica-rich groundwater flowing through volcanic rock to form concentric layers inside vesicles. As vesicles fill with successive agate layers, portions of groundwater from which the agate precipitates can become sealed in the cavity, thus preserving a volume of ancient water inside the agate. Microorganisms present in groundwater at the time of agate formation were trapped in the agate’s internal cavity producing a “microbial time capsule.” These microbes survived by adapting to near-starvation conditions at a subsistence level.
Enhydro agates used in this study are from the agate geode deposits in volcanic rocks of the Serra Geral Magmatic Province, Rio Grande do Sul, Brazil. Each enhydro agate was aseptically drilled to obtain a sample of trapped water. Direct microscopic analysis of extracted water revealed cell-like shapes including small (~ 1μm) diplococcoid and coccobacillus forms displaying erratic movement consistent with bacterial motility.

Basalt geochemistry and SEM/EDX analyses of agate accessory minerals (manganite) were used to develop a chemoautotrophic enrichment media that simulated the natural environment of groundwater microbes in a volcanic province. A basal inorganic medium amended with supplements including olivine, basalt, and Fe- and Mn-oxyhydroxides were used for initial enrichments at various temperatures under a CO2 atmosphere. Based on growth in the enrichments, cultures were transferred to a complex medium supplemented with olivine. Microscopy yielded Gram-negative motile cells of pleomorphic nature that ranged from rod-shaped to ovoid (~ 1-5μm).

Pure cultures were subjected to DNA isolation and PCR amplification of 16S rRNA genes. (BLAST) DNA sequence analysis results of isolates using GenBank indicate the bacterial DNA are most closely related to the Alphaproteobacterium Phyllobacterium myrsinacearum. These species are most commonly found in the soil rhizosphere associated with the roots of plants native to tropical regions. Related Phyllobacterium sp. have also been discovered associated with basaltic lava flows in caves and catacombs of Europe.