Bacillus methanolicus is a thermophilic methylotroph able to overproduce amino acids from methanol, a substrate not used for human or animal nutrition. Based on our previous RNA-seq analysis a mannitol inducible promoter and a putative mannitol activator gene mtlR were identified. The mannitol inducible promoter was applied for controlled gene expression using fluorescent reporter proteins and a flow cytometry analysis, and improved by changing the -35 promoter region and by co-expression of the mtlR regulator gene. For independent complementary gene expression control, the heterologous xylose-inducible system from B. megaterium was employed and a two-plasmid gene expression system was developed. Four different replicons for expression vectors were compared with respect to their copy number and stability. As an application example, methanol-based production of cadaverine was shown to be improved from 11.3 to 17.5 g/L when a heterologous lysine decarboxylase gene cadA was expressed from a theta-replicating rather than a rolling-circle replicating vector. The current work on inducible promoter systems and compatible theta- or rolling circle-replicating vectors is an important extension of the poorly developed B. methanolicus genetic toolbox, valuable for genetic engineering and further exploration of this bacterium.

Objective of this study was the selection and adaptation of mixed microbial cultures (MMCs), able to ferment crude glycerol generated from animal fat-based biodiesel and produce building-blocks and green chemicals. Various adaptation strategies have been investigated for the enrichment of suitable and stable MMC, trying to overcome inhibition problems and enhance substrate degradation efficiency, as well as generation of soluble fermentation products. Repeated transfers in small batches and fed-batch conditions have been applied, comparing the use of different inoculum, growth media, and Kinetic Control. The adaptation of activated sludge inoculum was performed successfully and continued unhindered for several months. The best results showed a substrate degradation efficiency of almost 100% (about 10 g/L glycerol in 21 h) and different dominant metabolic products were obtained, depending on the selection strategy (mainly 1,3-propanediol, ethanol, or butyrate). On the other hand, anaerobic sludge exhibited inactivation after a few transfers. To circumvent this problem, fed-batch mode was used as an alternative adaptation strategy, which led to effective substrate degradation and high 1,3-propanediol and butyrate production. Changes in microbial composition were monitored by means of Next Generation Sequencing, revealing a dominance of glycerol consuming species, such as Clostridium, Klebsiella, and Escherichia.

Oil biodegradation studies have mainly focused on microbial processes in dispersions, not specifically on the interfaces between the oil and the seawater in the dispersions. In this study, a hydrophobic adsorbent system, consisting of Fluortex fabrics, was used to investigate biodegradation of n-alkanes and microbial communities on oil-seawater interfaces in natural non-amended seawater. The study was performed over a temperature range from 0 to 20 °C, to determine how temperature affected biodegradation at the oil-seawater interfaces. Biodegradation of n-alkanes were influenced both by seawater temperature and chain-length. Biotransformation rates of n-alkanes decreased by reduced seawater temperature. Low rate coefficients at a seawater temperature of 0 °C were probably associated with changes in physical-chemical properties of alkanes. The primary bacterial colonization of the interfaces was predominated by the family Oceanospirillaceae at all temperatures, demonstrating the wide temperature range of these hydrocarbonoclastic bacteria. The mesophilic genus Oleibacter was predominant at the seawater temperature of 20 °C, and the psychrophilic genus Oleispira at 5 and 0 °C. Upon completion of n-alkane biotransformation, other oil-degrading and heterotrophic bacteria became abundant, including Piscirickettsiaceae (Cycloclasticus), Colwelliaceae (Colwellia), Altermonadaceae (Altermonas), and Rhodobacteraceae. This is one of a few studies that describe the biodegradation of oil, and the microbial communities associated with the degradation, directly at the oil-seawater interfaces over a large temperature interval.

Microbial assemblages were sampled from an offshore deep sub-surface petroleum reservoir 2.5 km below the ocean floor off the coast of Norway, providing conditions of high temperature and pressure, to identify new thermostable enzymes. In this study, we used DNA sequences obtained directly from the sample metagenome and from a derived fosmid library to survey the functional diversity of this extreme habitat. The metagenomic fosmid library containing 11,520 clones was screened using function- and sequence-based methods to identify recombinant clones expressing carbohydrate-degrading enzymes. Open reading frames (ORFs) encoding carbohydrate-degrading enzymes were predicted by BLAST against the CAZy database, and many fosmid clones expressing carbohydrate-degrading activities were discovered by functional screening using Escherichia coli as a heterologous host. Each complete ORF predicted to encode a cellulase identified from sequence- or function-based screening was subcloned in an expression vector. Five subclones was found to have significant activity using a fluorescent cellulose model substrate, and three of these were observed to be highly thermostable. Based on phylogenetic analyses, the thermostable cellulases were derived from thermophilic Archaea and are distinct from known cellulases. Cellulase F1, obtained from function-based screening, contains two distinct cellulase modules, perhaps resulting from fusion of two archaeal cellulases and with a novel protein structure that may result in enhanced activity and thermostability. This enzyme was found to exhibit exocellulase function and to have a remarkably high activity compared to commercially available enzymes. Results from this study highlight the complementarity of hybrid approaches for enzyme discovery, combining sequence- and function-based screening.Electronic supplementary materialThe online version of this article (doi:10.1186/s13568-017-0485-z) contains supplementary material, which is available to authorized users.

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