Isolation and screening of thermophilic bacilli from compost for electrotransformation and fermentation: characterization of Bacillus smithii ET 138 as a new biocatalyst

Abstract

Thermophilic bacteria are regarded as attractive production organisms for cost-efficient conversion of renewable resources to green chemicals, but their genetic accessibility is a major bottleneck in developing them into versatile platform organisms. In this study, we aimed to isolate thermophilic, facultatively anaerobic bacilli that are genetically accessible and have potential as platform organisms. From compost, we isolated 267 strains that produced acids from C5 and C6 sugars at temperatures of 55°C or 65°C. Subsequently, 44 strains that showed the highest production of acids were screened for genetic accessibility by electroporation. Two Geobacillus thermodenitrificans isolates and one Bacillus smithii isolate were found to be transformable with plasmid pNW33n. Of these, B. smithii ET 138 was the best-performing strain in laboratory-scale fermentations and was capable of producing organic acids from glucose as well as from xylose. It is an acidotolerant strain able to produce organic acids until a lower limit of approximately pH 4.5. As genetic accessibility of B. smithii had not been described previously, six other B. smithii strains from the DSMZ culture collection were tested for electroporation efficiencies, and we found the type strain DSM 4216(T) and strain DSM 460 to be transformable. The transformation protocol for B. smithii isolate ET 138 was optimized to obtain approximately 5 × 10(3) colonies per μg plasmid pNW33n. Genetic accessibility combined with robust acid production capacities on C5 and C6 sugars at a relatively broad pH range make B. smithii ET 138 an attractive biocatalyst for the production of lactic acid and potentially other green chemicals.

FIG 1

Neighbor-joining tree of 16S rRNA gene sequences of selected isolates and type strains. The first number indicates the isolation round, whereas a T at that position indicates a type strain. Numbers at the nodes represent bootstrap values out of 1,000 replicates.

FIG 2

Fermentation of B. smithii ET 138 on xylose and glucose. Fermentation was carried out in 1 liter TVMY supplemented with 20 g/liter xylose or glucose at 55°C, pH 6.5, 150 rpm, and without any gas addition. Gray line with plus symbol, OD₆₀₀; open circles, xylose or glucose; filled diamonds, lactate; closed squares, acetate; crosses, malate and succinate; closed triangles, pyruvate. During the xylose fermentation, the browning of the medium was observed after approximately 70 h.