Development of Microbiome Biobanks - Challenges and Opportunities.

The microbiome research field is rapidly evolving, but the required biobanking infrastructure is currently fragmented and not prepared for the biobanking of microbiomes. The rapid advancement of technologies requires an urgent assessment of how biobanks can underpin research by preserving microbiome samples and their functional potential.

Evaluation of the microbial community in industrial rye sourdough upon continuous back-slopping propagation revealed Lactobacillus helveticus as the dominant species.

AIMS: To assess the structure and stability of a dominant lactic acid bacteria (LAB) population during the propagation of rye sourdough in an industrial semi-fluid production over a period of 7 months. METHODS AND RESULTS: The sourdough was started from a 6-year-old freeze-dried sourdough originating from the same bakery. A unique microbial consortium consisting mainly of bacteria belonging to species Lactobacillus helveticus, Lactobacillus panis and Lactobacillus pontis was identified based on culture-dependent (Rep-PCR) and culture-independent [denaturing gradient gel electrophoresis (DGGE)] methods. Three of the isolated Lact. helveticus strains showed remarkable adaptation to the sourdough conditions. They differed from the type strain by the ability to ferment compounds specific to plant material, like salicin, cellobiose and sucrose, but did not ferment lactose. CONCLUSION: We showed remarkable stability of a LAB consortium in rye sourdough started from lyophilized sourdough and propagated in a large bakery for 7 months. Lactobacillus helveticus was detected as the dominant species in the consortium and was shown to be metabolically adapted to the sourdough environment. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of an established and adapted microbial consortium as a starter is a good alternative to commercial starter strains.

Growth characterization of individual rye sourdough bacteria by isothermal microcalorimetry.

AIMS: The present work tests the feasibility of the isothermal microcalorimetry method to study the performance of individual lactic acid bacteria during solid-state fermentation in rye sourdough. Another aim was to elucidate the key factors leading to the formation of different microbial consortia in laboratory and industrial sourdough during continuous backslopping propagation. METHODS AND RESULTS: Strains of the individual LAB isolated from industrial and laboratory sourdough cycle were grown in 10 kGy irradiated rye dough in vials of an isothermal calorimeter and the power-time curves were obtained. Sugars, organic acids and free amino acids in the sourdough were measured. The OD-time curves of the LAB strains during growth in flour extract or MRS (De Man, Rogosa and Sharpe) broth were also determined. The maximum specific growth rates of Lactobacillus sakei, Lactobacillus brevis, Lactobacillus curvatus and Leuconostoc citreum strains that dominated in backslopped laboratory sourdough were higher than those of Lactobacillus helveticus, Lactobacillus panis, Lactobacillus vaginalis, Lactobacillus casei and Lactobacillus pontis strains originating from industrial sourdough. Industrial strains had higher specific growth rates below pH 4·8. It was supposed that during long-run industrial backslopping processes, the oxygen sensitive species start to dominate because of the O(2) protective effect of rye sourdough. CONCLUSIONS: Measurements of the power-time curves revealed that the LAB strains dominating in the industrial sourdough cycle had better acid tolerance but lower maximum growth rate and oxygen tolerance than species isolated from a laboratory sourdough cycle. SIGNIFICANCE AND IMPACT OF THE STUDY: Isothermal microcalorimetry combined with chemical analysis is a powerful method for characterization of sourdough fermentation process and determination of growth characteristics of individual bacteria in sourdough.

Role of the DmpR-mediated regulatory circuit in bacterial biodegradation properties in methylphenol-amended soils.

Pathway substrates and some structural analogues directly activate the regulatory protein DmpR to promote transcription of the dmp operon genes encoding the (methyl)phenol degradative pathway of Pseudomonas sp. strain CF600. While a wide range of phenols can activate DmpR, the location and nature of substituents on the basic phenolic ring can limit the level of activation and thus utilization of some compounds as assessed by growth on plates. Here we address the role of the aromatic effector response of DmpR in determining degradative properties in two soil matrices that provide different nutritional conditions. Using the wild-type system and an isogenic counterpart containing a DmpR mutant with enhanced ability to respond to para-substituted phenols, we demonstrate (i) that the enhanced in vitro biodegradative capacity of the regulator mutant strain is manifested in the two different soil types and (ii) that exposure of the wild-type strain to 4-methylphenol-contaminated soil led to rapid selection of a subpopulation exhibiting enhanced capacities to degrade the compound. Genetic and functional analyses of 10 of these derivatives demonstrated that all harbored a single mutation in the sensory domain of DmpR that mediated the phenotype in each case. These findings establish a dominating role for the aromatic effector response of DmpR in determining degradation properties. Moreover, the results indicate that the ability to rapidly adapt regulator properties to different profiles of polluting compounds may underlie the evolutionary success of DmpR-like regulators in controlling aromatic catabolic pathways.

Effect of inoculation of a TOL plasmid containing mycorrhizosphere bacterium on development of Scots pine seedlings, their mycorrhizosphere and the microbial flora in m-toluate-amended soil.

The purpose of this study was to evaluate the influence of introduced bacteria containing a contaminant degrading plasmid on the growth and survival of pine seedlings and mycorrhizosphere microbial flora in contaminated soil. The Pseudomonas fluorescens strain OS81, originally isolated from fungal hyphae in contaminated soil, was supplied with the TOL plasmid pWW0::Km (to generate OS81(pWW0::Km)) and inoculated in humus-soil microcosms with and without pine seedlings mycorrhized with Suillus bovinus. After 3 months of regular treatment with m-toluate (mTA) solutions, the introduced catabolic plasmid was found to be disseminated in the indigenous bacterial population of both mycorrhizosphere and soil uncolonized by the fungus. Transconjugants were represented by bacteria of the genera Pseudomonas and Burkholderia and their number correlated positively with the concentration of mTA applied. Indigenous mTA degrading bacteria with low similarity to Burkholderia species were also enriched in microcosms. They were mostly associated with mycorrhizal soil or fungal structures and virtually absent in microcosms without pines. The total number of Tol(+) bacteria was higher in mycorrhizospheric soil compared with bulk soil. Inoculation with P. fluorescens OS81(pWW0::Km) had a positive effect on the development of roots and fungus in contaminated soil. Both inoculation with the P. fluorescens OS81(pWW0::Km) and mTA contamination as well as the presence of mycorrhized pine roots and fungal hyphae had an effect on the microbial community structure of soil as measured by carbon source oxidation patterns. However, the impact of mTA on the microbial community was more prominent. The study indicates that an effect on plant and fungal development can be obtained by manipulating the mycorrhizosphere. Both introduction of the bacterium carrying the degradative plasmid and the plasmid itself are likely to have a positive effect not only on the organisms involved, but also on bioremediation of contaminated soil, a factor that was not directly monitored here.

Means to improve the effect of in situ bioremediation of contaminated soil: an overview of novel approaches.

Different aspects of bacterial degradation of organic contaminants in soil, and how to improve the efficiency and reproducibility is discussed in this review. Although bioremediation in principle includes the use of any type of organism in improving the condition of a contaminated site, most commonly bacteria are the degraders and other organisms, such as soil animals or plant roots, play a role in dissemination of bacteria and, indirectly, plasmids between bacteria, and in providing nutrients and co-substrates for the bacteria active in the degradation process. There are a number of different procedures that have been tested more-or-less successfully in attempts to improve reliability, cost efficiency and speed of bioremediation. The methods range from minimal intervention, such as mere monitoring of intrinsic bioremediation, through in situ introduction of nutrients and/or bacterial inocula or improvement of physico-chemical conditions, all the way to excavation followed by on site or ex situ composting in its different varieties. In the past the rule has been that more intervention (leading to higher costs) has been more reliable, but novel ideas are continuously tried out, both as a means to come up with new truly functional applications and also as a line of studies in basic soil microbial ecology. Both approaches generate valuable information needed when predicting outcome of remediation activities, evaluating environmental risks, deciding on cleaning-up approaches, etc. The emphasis of this review is to discuss some of the novel methods for which the value has not been clearly shown, but that in our view merit continued studies and efforts to make them work, separately or in combination.

Tolerance and biodegradation of m-toluate by Scots pine, a mycorrhizal fungus and fluorescent pseudomonads individually and under associative conditions.

The tolerance to, and degradation of m-toluate by Scots pine (Pinus sylvestris), a symbiotic mycorrhizal fungus (Suillus bovinus) and Pseudomonas fluorescens strains, with or without m-toluate-degrading capacity, was determined individually and in all symbiotic/associative plant-microbe combinations. Fungal survival on medium with m-toluate was increased in co-culture with the degradative bacterial strains on agar plates (up to 0.02%, w/v). When fungi were grown in mycorrhizal association with Scots pine seedlings in test-tube microcosms containing expanded clay pellets and growth media, the fungus was able to withstand m-toluate concentrations up to 2.0%, w/v in all treatments. The seedling tolerance remained unaltered regardless of the presence or absence of mycorrhizal fungi or biodegradative bacteria. Reduction in m-toluate levels was only detected in treatments inoculated with bacterial strains harbouring TOL catabolic plasmids. The plant and fungus, alone or in mycorrhizal symbiosis, were unable to cleave m-toluate. The presence of easily available plant-derived carbon sources did not impede m-toluate degradation by the bacteria in the mycorrhizosphere.

New derivatives of TOL plasmid pWW0.

Two new segregants, PPW1-1 and PPW161-1, of Pseudomonas putida were isolated from the stock cultures PaW85(pWW0) and PaW85(pWW0-161). Strain PPW1-1 had lost its ability to grow on m-xylene but was able to grow on m-toluate. A deletion of the left-hand of transposon Tn4651, including the upper-operon genes, had taken place in plasmid pWW0mut1, isolated from strain PPW1-1. Additional deletions were observed in pWW0mut1 after 'benzoate-curing' (plasmids pWW0mut15, pWW0mut19, pWW0mut27). The genes of the upper-operon and beginning of the meta-operon were deleted from pWW0-161mut1, isolated from strain PPW161-1. Despite this deletion, cells of PPW161-1 grew on all normal TOL plasmid substrates. The Tol+ phenotype was stable in cells of PPW161-1 growing on benzoate. We propose that this is because in cells of strain PPW161-1 the catabolic genes deleted from pWW0-161mut1 were integrated into the chromosome at the site where the (chromosomally encoded) ortho-pathway genes are located, resulting in the inability of the cells to use this pathway.