Time-Course Transcriptome of Parageobacillus thermoglucosidasius DSM 6285 Grown in the Presence of Carbon Monoxide and Air.

Parageobacillus thermoglucosidasius is a metabolically versatile, facultatively anaerobic thermophile belonging to the family Bacillaceae. Previous studies have shown that this bacterium harbours co-localised genes coding for a carbon monoxide (CO) dehydrogenase (CODH) and Ni-Fe hydrogenase (Phc) complex and oxidises CO and produces hydrogen (H2) gas via the water-gas shift (WGS) reaction. To elucidate the genetic events culminating in the WGS reaction, P. thermoglucosidasius DSM 6285 was cultivated under an initial gas atmosphere of 50% CO and 50% air and total RNA was extracted at ~8 (aerobic phase), 20 (anaerobic phase), 27 and 44 (early and late hydrogenogenic phases) hours post inoculation. The rRNA-depleted fraction was sequenced using Illumina NextSeq, v2.5, 1x75bp chemistry. Differential expression revealed that at 8 vs 20, 20 vs 27 and 27 vs 44 hours post inoculation, 2190, 2118 and 231 transcripts were differentially (FDR < 0.05) expressed. Cluster analysis revealed 26 distinct gene expression trajectories across the four time points. Of these, two similar clusters, showing overexpression at 20 relative to 8 hours and depletion at 27 and 44 hours, harboured the CODH and Phc transcripts, suggesting possible regulation by O2. The transition between aerobic respiration and anaerobic growth was marked by initial metabolic deterioration, as reflected by up-regulation of transcripts linked to sporulation and down-regulation of transcripts linked to flagellar assembly and metabolism. However, the transcriptome and growth profiles revealed the reversal of this trend during the hydrogenogenic phase.

Effects of different operating parameters on hydrogen production by Parageobacillus thermoglucosidasius DSM 6285.

Hydrogen gas represents a promising alternative energy source to dwindling fossil fuel reserves, as it carries the highest energy per unit mass and its combustion results in the release of water vapour as only byproduct. The facultatively anaerobic thermophile Parageobacillus thermoglucosidasius is able to produce hydrogen via the water-gas shift reaction catalyzed by a carbon monoxide dehydrogenase-hydrogenase enzyme complex. Here we have evaluated the effects of several operating parameters on hydrogen production, including different growth temperatures, pre-culture ages and inoculum sizes, as well as different pHs and concentrations of nickel and iron in the fermentation medium. All of the tested parameters were observed to have a substantive effect on both hydrogen yield and (specific) production rates. A final experiment incorporating the best scenario for each tested parameter showed a marked increase in the H2 production rate compared to each individual parameter. The optimised parameters serve as a strong basis for improved hydrogen production with a view of commercialisation of this process.

Acetogenic Fermentation From Oxygen Containing Waste Gas.

The microbial production of bulk chemicals from waste gas is becoming a pertinent alternative to industrial strategies that rely on fossil fuels as substrate. Acetogens can use waste gas substrates or syngas (CO, CO2, H2) to produce chemicals, such as acetate or ethanol, but as the feed gas often contains oxygen, which inhibits acetogen growth and product formation, a cost-prohibitive chemical oxygen removal step is necessary. Here, we have developed a two-phase microbial system to facilitate acetate production using a gas mixture containing CO and O2. In the first phase the facultative anaerobic carboxydotroph Parageobacillus thermoglucosidasius was used to consume residual O2 and produce H2 and CO2, which was subsequently utilized by the acetogen Clostridium ljungdahlii for the production of acetate. From a starting amount of 3.3 mmol of CO, 0.52 mmol acetate was produced in the second phase by C. ljungdahlii. In this set-up, the yield achieved was 0.16 mol acetate/mol CO, a 63% of the theoretical maximum. This system has the potential to be developed for the production of a broad range of bulk chemicals from oxygen-containing waste gas by using P. thermoglucosidasius as an oxygen scrubbing tool.

Comparative genomic analysis of Parageobacillus thermoglucosidasius strains with distinct hydrogenogenic capacities.

BACKGROUND: The facultatively anaerobic thermophile Parageobacillus thermoglucosidasius produces hydrogen gas (H2) by coupling CO oxidation to proton reduction in the water-gas shift (WGS) reaction via a carbon monoxide dehydrogenase-hydrogenase enzyme complex. Although little is known about the hydrogenogenic capacities of different strains of this species, these organisms offer a potentially viable process for the synthesis of this alternative energy source. RESULTS: The WGS-catalyzed H2 production capacities of four distinct P. thermoglucosidasius strains were determined by cultivation and gas analysis. Three strains (DSM 2542T, DSM 2543 and DSM 6285) were hydrogenogenic, while the fourth strain (DSM 21625) was not. Furthermore, in one strain (DSM 6285) H2 production commenced earlier in the cultivation than the other hydrogenogenic strains. Comparative genomic analysis of the four strains identified extensive differences in the protein complement encoded on the genomes, some of which are postulated to contribute to the different hydrogenogenic capacities of the strains. Furthermore, polymorphisms and deletions in the CODH-NiFe hydrogenase loci may also contribute towards this variable phenotype. CONCLUSIONS: Disparities in the hydrogenogenic capacities of different P. thermoglucosidasius strains were identified, which may be correlated to variability in their global proteomes and genetic differences in their CODH-NiFe hydrogenase loci. The data from this study may contribute towards an improved understanding of WGS-catalysed hydrogenogenesis by P. thermoglucosidasius.

CO-dependent hydrogen production by the facultative anaerobe Parageobacillus thermoglucosidasius.

BACKGROUND: The overreliance on dwindling fossil fuel reserves and the negative climatic effects of using such fuels are driving the development of new clean energy sources. One such alternative source is hydrogen (H2), which can be generated from renewable sources. Parageobacillus thermoglucosidasius is a facultative anaerobic thermophilic bacterium which is frequently isolated from high temperature environments including hot springs and compost. RESULTS: Comparative genomics performed in the present study showed that P. thermoglucosidasius encodes two evolutionary distinct H2-uptake [Ni-Fe]-hydrogenases and one H2-evolving hydrogenases. In addition, genes encoding an anaerobic CO dehydrogenase (CODH) are co-localized with genes encoding a putative H2-evolving hydrogenase. The co-localized of CODH and uptake hydrogenase form an enzyme complex that might potentially be involved in catalyzing the water-gas shift reaction (CO + H2O → CO2 + H2) in P. thermoglucosidasius. Cultivation of P. thermoglucosidasius DSM 2542T with an initial gas atmosphere of 50% CO and 50% air showed it to be capable of growth at elevated CO concentrations (50%). Furthermore, GC analyses showed that it was capable of producing hydrogen at an equimolar conversion with a final yield of 1.08 H2/CO. CONCLUSIONS: This study highlights the potential of the facultative anaerobic P. thermoglucosidasius DSM 2542T for developing new strategies for the biohydrogen production.

High Quality Draft Genomes of the Type Strains Geobacillus thermocatenulatus DSM 730T, G. uzenensis DSM 23175T And Parageobacillus galactosidasius DSM 18751T.

The thermophilic 'Geobacilli' are important sources of thermostable enzymes and other biotechnologically relevant macromolecules. The present work reports the high quality draft genome sequences of previously unsequenced type strains of Geobacillus uzenensis (DSM 23175T), G. thermocatenulatus (DSM 730T) and Parageobacillus galactosidasius (DSM 18751T). Phylogenomic analyses revealed that DSM 18751T and DSM 23175T represent later heterotypic synonyms of P. toebii and G. subterraneus, respectively, while DSM 730T represents the type strain for the species G. thermocatenulatus. These genome sequences will contribute towards a deeper understanding of the ecological and biological diversity and the biotechnological exploitation of the 'geobacilli'.

CRISPR/Cas9 targeting events cause complex deletions and insertions at 17 sites in the mouse genome.

Although CRISPR/Cas9 genome editing has provided numerous opportunities to interrogate the functional significance of any given genomic site, there is a paucity of data on the extent of molecular scars inflicted on the mouse genome. Here we interrogate the molecular consequences of CRISPR/Cas9-mediated deletions at 17 sites in four loci of the mouse genome. We sequence targeted sites in 632 founder mice and analyse 54 established lines. While the median deletion size using single sgRNAs is 9 bp, we also obtain large deletions of up to 600 bp. Furthermore, we show unreported asymmetric deletions and large insertions of middle repetitive sequences. Simultaneous targeting of distant loci results in the removal of the intervening sequences. Reliable deletion of juxtaposed sites is only achieved through two-step targeting. Our findings also demonstrate that an extended analysis of F1 genotypes is required to obtain conclusive information on the exact molecular consequences of targeting events.

Substitution of the native srfA promoter by constitutive Pveg in two B. subtilis strains and evaluation of the effect on Surfactin production.

The genetic enhancement of Surfactin production increasingly gained attention in the last years, since relatively low product yields limit the industrial application of this biosurfactant. The natural quorum sensing regulation of the srfA operon (coding for the Surfactin synthetase) can reasonably be assumed to be the bottleneck of Surfactin synthesis. Therefore, the replacement of the naturally quorum sensing regulated, and herewith cell density dependent, promoter PsrfA against the Bacillus subtilis endogenous and constitutive promoter Pveg was hypothesized to generally enhance Surfactin yields. The markerless promoter replacement was conducted in the two B. subtilis Surfactin producer strains 3A38 and DSM 10(T). The promoter substitution led to an enhancement of Surfactin concentrations in the producer strain 3A38, initially producing only minor amounts of Surfactin (0.07g/L increased to 0.26g/L). In contrast, promoter exchange in B. subtilis DSM 10(T) (wild-type strain producing 0.62g/L Surfactin) did not achieve an enhancement of Surfactin concentrations (detrimental reduction to 0.04g/L). These findings implicate that Surfactin synthesis is differently regulated in minor and strong Surfactin producer strains. The hypothesized general enhancement of Surfactin yields after substitution of the native promoter was therefore not confirmed.

Enhancement of Surfactin yield by improving the medium composition and fermentation process.

Surfactin is one of the most promising biosurfactants due to its extraordinary surface activity. Commonly, the well-established Cooper medium, a glucose-based mineral salt medium, is utilized for the microbial production of Surfactin. The current study investigated the enhancement of Surfactin yields by analyzing the effects of different glucose concentrations, next to the introduction of an alternative chelating agent and nitrogen source. The utilization of 8 g/L glucose, 0.008 mM Na3citrate and 50 mM (NH4)2SO4 increased Surfactin yields from 0.7 to 1.1 g/L during shake flask experiments applying Bacillus subtilis DSM10(T). Consequentially conducted shake flask experiments, employing five other Surfactin producer strains during cultivation in the former and enhanced version of the Cooper medium, suggest a general enhancement of Surfactin yields during application of the enhanced version of the Cooper medium. The enhancement of the medium composition is therefore most likely independent from the employed producer strain. The following utilization of the enhanced medium composition during fed-batch fermentation with integrated foam fractionation yielded 30 % more Surfactin in comparison to batch fermentations with integrated foam fractionation employing the former version of the Cooper medium.

Evaluation of different Bacillus strains in respect of their ability to produce Surfactin in a model fermentation process with integrated foam fractionation.

Biosurfactants increasingly gain attention due to the manifold of possible applications and production on the basis of renewable resources. Owing to its various characteristics, Surfactin is one of the most studied biosurfactants. Since its discovery, several Surfactin producers have been identified, but their capacity to produce Surfactin has not been evaluated in a comparison. Six different Bacillus strains were analyzed regarding their ability to produce Surfactin in model fermentations with integrated foam fractionation, for in situ product enrichment and removal. Three of the investigated strains are commonly used in Surfactin production (ATCC 21332, DSM 3256, DSM 3258), whereas two Bacillus strains are described for the first time (DSM 1090, LM43a50°C) as Surfactin producers. Additionally, the Bacillus subtilis type strain DSM 10(T) was included in the evaluation. Interestingly, all strains, except DSM 3256, featured high values for Surfactin recovered from foam in comparison to other studies, ranging between 0.4 and 1.05 g. The fermentation process was characterized by calculating procedural parameters like substrate yield Y X/S, product yield Y P/X, specific growth rate µ, specific productivity q Surfactin, volumetric productivity q Surfactin, Surfactin and bacterial enrichment as well as Surfactin recovery. The strains differ most in specific and volumetric productivity; nevertheless, it is evident that it is not possible to name a Bacillus strain that is the most appropriate for the production of Surfactin under these conditions. In contrast, it becomes apparent that the choice of a specific strain should depend on the applied fermentation conditions.