Diversity and dynamics of bacterial and fungal communities in cider for distillation.

Bacterial and fungal population dynamics in cider for distillation have so far been explored by culture-dependant methods. Cider for distillation can be produced by the spontaneous fermentation of apples that do not undergo any intervention during the process. In this study, cider microbiomes extracted from six tanks containing ciders for distillation from four producers in Normandy were characterized at three main stages of the fermentation process: fermentation Initiation (I), end of the alcoholic Fermentation (F) and end of the Maturation period (M). Cider samples were subjected to Illumina MiSeq sequencing (rRNA 16S V1-V3 and ITS1 region targeting) to determine bacterial and fungal communities. Yeasts (YGC), Zymomonas (mZPP) and lactic acid bacteria selective media (mMRS, mMLO, mPSM) were also used to collect 807 isolates. Alcoholic levels, glycerol, sugar content (glucose, fructose and sucrose), pH, total and volatile acidity, nitrogen, malic and lactic acid contents were determined at all sampling points. Alpha diversity indexes show significant differences (p < 0.05) in microbial populations between I, F and M. Fungal communities were characterized by microorganisms from the environment and phytopathogens at I followed by the association of yearsts with alcoholic fermentation like Saccharomyces and non-Saccharomyces yeasts (Hanseniaspora, Candida). A maturation period for cider leads to an increase of the Dekkera/Brettanomyces population, which is responsible for off-flavors in cider for all producers. Among bacterial communities, the genera community associated to malolactic fermentation (Lactobacillus sp., Leuconostoc sp., Oenococcus sp.) was the most abundant at F and M. Acetic acid bacteria such as Acetobacter sp., Komagataeibacter sp. and Gluconobacter sp. were also detected during the process. Significant differences (p < 0.05) were found in fungal and bacterial populations between the four producers and during the fermentation process. The development of microorganisms associated with cider spoilage such as Zymomonas mobilis, Lactobacillus collinoides or Brettanomyces/Dekkera sp. was anticipated by a metagenomic approach. The monitoring of microbial diversity via high throughput sequencing combined with physical-chemical analysis is an interesting approach to improve the fermentation performance of cider for distillation and therefore, the quality of Calvados.

Temporal Shotgun Metagenomics Revealed the Potential Metabolic Capabilities of Specific Microorganisms During Lambic Beer Production.

Lambic beer production processes are characterized by a temporal succession of well-adapted microbial species. Temporal metagenomic analysis of a Belgian, traditional, lambic beer production process, which was examined microbiologically and metabolomically before, confirmed that the microbial diversity is limited. Moreover, it allowed to link the consumption and production of certain compounds to specific microbial groups or species. Fermentation characteristics, such as the conversion of malic acid into lactic acid and acetoin production, were retrieved and could be attributed to specific microorganisms, namely Pediococcus damnosus and Acetobacter species, respectively. Traits previously ascribed to brewery-specific Dekkera bruxellensis strains were confirmed during the lambic beer production process examined multiphasically; in particular, the higher production of 4-ethylguaiacol compared to 4-ethylphenol was further shown by mass spectrometric analysis. Moreover, the absence of phenolic acid decarboxylase in Brettanomyces custersianus was shown culture-independently and could explain its late occurrence during the maturation phase. Furthermore, the potential of maltooligosaccharide degradation could be ascribed metagenomically to not only Brettanomyces species but also Saccharomyces kudriavzevii, possibly explaining their degradation early in the lambic beer production process. Also, acetic acid bacteria (AAB) seemed to be able to consume maltooligosaccharides via their conversion into trehalose. Furthermore, these AAB possessed esterase genes, potentially capable of forming ethyl acetate, which may contribute to the flavor of lambic beer. Improved knowledge on the reasons behind certain community dynamics and the role of the different microorganisms in terms of potential functionality could improve brewery practices to assure to produce more quality-stable end-products.

Improved electroporation procedure for genetic transformation of Dekkera/Brettanomyces bruxellensis.

Yeast Dekkera/Brettanomyces bruxellensis is one of the most common contaminants in wine industry, but also one of the most promising candidates for large-scale bioethanol production. Brettanomyces bruxellensis not only produces and tolerates high ethanol concentrations, but can also ferment cellobiose and adapt to lignocellulose hydrolasate. Furthermore, genome sequences of several B. bruxellensis strains are available, and efforts have been made to develop tools for genetic transformation of this yeast. Previously, we reported a successful transformation using lithium acetate/PEG method and electroporation, however, with very low transformation efficiency (10-20 transformants µg(-1)). Here we describe an optimization of electroporation procedure which resulted in a significant increase of transformation efficiency (2.8 × 10(3) transformants µg(-1)). Several key transformation parameters were optimized including cell growth phase, density of cells in the transformation sample and electroporation settings. We determined that treating the cells with both lithium acetate (100 mM) and dithiothreitol (35 mM) synergistically improves transformation efficiency. Using the described procedure around 500 transformants can be obtained per transformation sample with 180 ng of non-homologous linear transforming fragment. Additionally, several transformants were obtained with less than 1 ng of DNA demonstrating that this procedure is adequate even when very limited amount of DNA is available.

Silicification of wood adopted for barrel production using pure silicon alkoxides in gas phase to avoid microbial colonisation.

The paper presents a new approach, covering wood with silica-based material in order to protect it from spoilage due to microbial colonisation and avoiding the loss of the natural features of the wood. Wood specimens derived from wine barrels were treated with methyltriethoxysilane in gas phase, leading to the deposition of a silica nanofilm on the surface. (29)Si and (13)C solid state Nuclear Magnetic Resonance and Scanning Electron Microscope-Energy Dispersive X-ray analysis observations showed the formation of a silica polymeric film on the wood samples, directly bonding with the wood constituents. Inductively Coupled Plasma-Mass Spectroscopy quantification of Si showed a direct correlation between the treatment time and silica deposition on the surface of the wood. The silica-coated wood counteracted colonisation by the main wine spoilage microorganisms, without altering the migration from wood to wine of 21 simple phenols measured using a HPLC-Electrochemical Coulometric Detection.

Rapid identification of the genus Dekkera/Brettanomyces, the Dekkera subgroup and all individual species.

The genus Dekkera/Brettanomyces comprises five described species: Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. naardenensis and B. nanus. Some of them, especially D. bruxellensis, are important spoilage organisms, particularly in the wine and beverage industries. Because of their economic importance many different methods have been developed to identify members of the genus in general and D. bruxellensis in particular. These methods vary in their rapidity, complexity and cost but, partly because of confidentiality issues, it is unclear which methods are used, or how widely, in the relevant industries. Building on previous work with the genera Saccharomyces and Zygosaccharomyces, a suite of eight PCR primer pairs has been designed either on the D1-D2 region of the 26S rRNA gene or translation elongation factor TEF1-α. These primers can specifically identify the genus as a whole, only Dekkera species, each one of the five recognised species as well as a significant subgroup of D. bruxellensis represented by NCYC 3426. Multiplexing has also been tried and it has been shown to be possible with some combinations of genus or Dekkera-level and species-specific primers. Using direct colony PCR amplification followed by gel electrophoresis, a clear positive result can be obtained in less than 3h, thus providing a quick, reliable and inexpensive way to identify target species.

Cinnamic acid, ethanol and temperature interaction on coumarate decarboxylase activity and the relative expression of the putative cd gene in D. bruxellensis

Dekkera bruxellensis is one of the main contaminating yeasts in wine due to its ability to metabolize cinnamic acids into volatile phenols. This yeast metabolizes p-coumaric acid into 4-vinylphenol through a coumarate decarboxylase (CD) and then transforms it into to 4-ethylphenol (EF) through a vinylphenol reductase. In this work we investigated the influence of the interaction between the concentration of p-coumaric acid, ferulic acid and ethanol as well as growth temperature on the production of CD activity and the expression of a putative gene that codes for this enzymatic activity. For this, a Box Behnken experimental design was used. The concentration of p-coumaric acid (5-26 ppm) and ferulic acid (3-9 ppm) alone did not show any significant effect on any of the studied response variables. However, the interaction between (ethanol concentration * cinnamic acid concentration) and (ethanol concentration * temperature) had a significant statistical effect on the production of CD activity. Additionally, a higher growth temperature negatively affected the expression of the putative cd gene and the production of CD activity. This is the first work that studies the effect of cinnamic acids on the production of CD activity and the relative expression of its putative gene, using natural concentrations of cinnamic acid found in wine.