New Malolactic Bacteria Strains Isolated from Wine Microbiota. Characterization and Technological Properties

Malolactic fermentation (MLF) or biological decrease of wine acidity is defined as the enzymatic bioconversion of malic acid in lactic acid, a process performed by lactic acid bacteria (LAB). The procedures for the isolation of new indigenous LAB strains from the red wines produced in Copou Iasi wine center (NE of Romania) undergoing spontaneous malolactic fermentation, resulted in the obtaining of 67 catalase-negative and Gram-positive LAB strains. After testing in the malolactic fermentative process, application of specific screening procedures and identification (API 50 CH), two bacterial strains belonging to the species Oenococcus oeni (strain 13-7) and Lactobacillus plantarum (strain R1-1) with high yield of malolactic bioconversion, non-producing biogenic amines, and with active extracellular enzymes related to wine aroma, were retained and characterized. Tested in synthetic medium (MRS-TJ) for 10 days, the new isolated LAB strains metabolized over 98% of the malic acid at ethanol concentrations between 10 and 14 % (v/v), low pH (>3.0), total SO2 doses up to 70 mg/L and temperatures between 15 and 35 °C, showing high potential for future use in the winemaking process as bacterial starter cultures, in order to obtain high quality wines with increased typicity.

Optimization of Propagation Medium for Enhanced Polyhydroxyalkanoate Production by Pseudomonas oleovorans

Polyhydroxyalkanoates (PHAs) represent a promising alternative to commercially used petroleum-based plastics. Pseudomonas oleovorans is a natural producer of medium-chain-length PHA (mcl-PHA) under cultivation conditions with nitrogen limitation and carbon excess. Two-step cultivation appears to be an efficient but more expensive method of PHA production. Therefore, the aim of this work was to prepare a minimal synthetic medium for maximum biomass yield and to optimize selected independent variables by response surface methodology (RSM). The highest biomass yield (1.71 ± 0.04 g/L) was achieved in the optimized medium containing 8.4 g/L glucose, 5.7 g/L sodium ammonium phosphate and 35.4 mM phosphate buffer. Under these conditions, both carbon and nitrogen sources were completely consumed after 48 h of the cultivation and the biomass yield was 1.7-fold higher than in the conventional medium recommended by the literature. This approach demonstrates the possibility of using two-stage PHA cultivation to obtain the maximum amount of biomass and PHA.

Selection of Three Indigenous Lebanese Yeast Saccharomyces cerevisiae with Physiological Traits from Grape Varieties in Western Semi-Desert and Pedoclimatic Conditions in the Bekaa Valley

Wine production depends on the fermentation process performed by yeasts, especially (but not solely) strains of the species Saccharomyces cerevisiae, which is a technique that has been practiced from the Middle Ages till modern days. Selecting indigenous starters offers a beneficial technique to manage alcoholic grape juice fermentation, conserving the particular sensory qualities of wine produced from specific regions. This paper investigated yeast biodiversity of four grape varieties (Carignan, Syrah, Grenache, and Aswad Karesh) grown in the pedoclimatic western semi-desert Bekaa Valley. Further research identified, characterized, and selected strains with the most industrial wine interest and economic value to Lebanon. By using molecular methods and by the ITS PCR analysis, the isolates belonging to the Saccharomyces and non-Saccharomyces genus were identified. These isolates taken from four varieties were further characterized by amplification with Interdelta and δ12/δ21 primer pairs, permitting the identification of 96 S. cerevisiae strains. Forty-five genomically homogenous groups were classified through the comparison between their mtDNA RFLP patterns. Based on physiological characterization analysis (H2S and SO2 production, killer phenotype, sugar consumption, malic and acetic acid, etc.), three strains (NL28629, NL28649, and NL28652) showed interesting features, where they were also vigorously fermented in a synthetic medium. These strains can be used as a convenient starter for typical wine production. In particular, Carignan and Syrah had the highest percentage of strains with the most desirable physiological parameters.

In vitro biodegradation of low-density polyethylene by soil microorganisms

The accumulation of recalcitrant plastics in the environment, particularly polyethylene, is a major threat to the ecology. Among the different kinds of polyethylene, low-density polyethylene (LDPE) is the most widely used polyethylene. The goal of this work was to isolate and discover a powerful polyethylene degrading microbial strain from plastic waste disposal soil. The bacterial and fungal strains were isolated by enrichment technique and were identified based on the morphological and biochemical characteristics. Further, they were screened individually for their lowdensity polyethylene (LDPE) degrading efficiency by in vitro biodegradation assay. The efficiency of the potent strain to colonize on the LDPE surface and its biodegradation ability were investigated. The degraded products of low-density polyethylene were analysed by FTIR after the biodegradation study which was conducted for a long incubation period by inoculating the selected bacterial strain in synthetic medium (SM) with LDPE as the carbon source. Totally six different bacterial and five different fungal strains were isolated from the polluted soil. Among the bacterial strains, the JSB2, JSB3 and JSB4 and among the fungal strains, JSF1, JSF3 and JSF4 showed maximum growth, more cell surface hydrophobicity and weight loss and they were selected for further studies. The incubation of LDPE films with bacteria and fungi led to the formation of new absorbance bands such as dehydrated dimer of carbonyl group (1720 cm-1), CH3 deformation (1463 cm-1) and C=C conjugation band (862 cm-1). The results inferred that the exogenous addition of these microbes to LDPE contaminated soil causes an enhanced degradation. Among the microbial isolates, Bacillus species showed high degradation.

Аcinetobacter baumannii bv Tryptophandestruens bv nov. isolated from clinical samples

The aim of the study was to determine the taxonomic status of a group consisting of atypical strains of Acinetobacter baumannii, outline relevant characteristics and methods necessary for their identification. There were examined 10 strains of A. baumannii (6 of them primary comprised) bearing similar profile of atypical features isolated from clinical samples (urine, sputum) in 2017–2019 at the Military Medical Academy. Сlinical strains of typical A. baumannii (n = 36), Acinetobacter nosocomialis (n = 14), Acinetobacter pittii (n = 9) and 1 strain of Acinetobacter calcoaceticus isolated from the external environment were used in comparative studies. Atypical strains had the characteristics of A. calcoaceticus — A. baumannii (ACB) complex bacteria and were identified as A. baumannii. The utilization of substrates as the only carbon source was studied on a dense synthetic medium added with 0.2 % substrate during incubation for 72 hours at 37°C. Carbohydrate oxidation coupled to acid formation was detected on the Hugh–Leifson medium by using a micromethod. Aromatic amino acid biotransformation was carried out in liquid and dense nutrient media assessed in chromogenic reaction. The rpoB gene was used for strain genetic characterization. Amplification of two 940 and 1210 base pair (bp)-long fragments from the rpoB gene was performed by the routine polymerase chain reaction using primers with previously described sequences. Amplification products were sequenced by Sanger using Big Dye Terminator v3.1 (Applied Biosystems, USA) and capillary electrophoresis on an automatic sequencer ABI PRISM 3130 (Applied Biosystems, USA), followed by using methods for determining the similarity levels of sequenced fragments with the rpoB gene sequences of the reference strain A. baumannii ATCC 17978 (GenBank accession no. CP053098.1). It was found that all strains belonging to atypical A. baumannii spp. had a specific set of features that distinguish them from typical strains of A. baumannii as well as other types of the ACB complex: detected biotransformation of L-tryptophan (via anthranilate pathway) and anthranilic acid under unambiguous lack of such signs in other bacteria; lack of utilized sodium hippurate and L-arabinose being unambiguously evident in other bacteria; lack of utilized L-tryptophan, putrescine, L-ornithine being utilized in the majority of strains of belonging to other bacterial species. Genetic analysis showed that the control strains of typical A. baumannii displayed 99.20–99.21% similarity within the sequenced fragments of the rpoB gene with those from the rpoB gene of the reference strain. All 10 strains of atypical A. baumannii had similar features (99.20–99.21%). At the same time, parameters of control strains from other bacterial species significantly differed: A. nosocomialis (95.10–95.97%), A. pittii (94.63–94.92%), A. calcoaceticus (93.00%). Hence, the strains of atypical and typical A. baumannii are genetically homogeneous and belong to the same species. The data presented allow us to consider this group of atypical A. baumannii strains as a new biovar. We propose the name for this new biovar — tryptophandestruens (tryptophan-destroying) stemming from the Latin word destruens — destroying. Identification of A. baumannii bv. tryptophandestruens bacteria can be carried out in laboratory of any level by using tests for L-tryptophan biotransformation as well as sodium hippurate utilization.

Antifungal activity of crude extracts of Ageratum conyzoides and Chromolaena odorata for management of Lasiodiplodia theobromae and Lasiodiplodia pseudotheobromae through in vitro evaluation

Lasiodiplodia is an important genus of fungi causing destructive diseases on perennial crops, including cocoa. Two crucial species of Lasiodiplodia that cause diseases in cocoa are Lasiodiplodia theobromae and Lasiodiplodia pseutheobromae. A variety of weeds is the potential to be applied as botanical fungicides to control the pathogens. The main objective of this study was to evaluate Ageratum conyzoides and Chromolaena odorata leaf extract to inhibit the growth of L. theobromae and L. pseudotheobromae on a synthetic medium. Solvent organic was methanol for weed extraction with a ratio of 1:5. The experiment was conducted through the poison food technique method, both in the solid and liquid medium in three different concentrations, 1, 3, and 5%. The result showed that A. conyzoides and C. odorata were significantly inhibited the colony growth of both Lasiodiplodia in all concentrations in a solid medium. A. conyzoides performed better than C. odorata in all concentrations of both Lasiodiplodia in inhibition. A. conyzoides 5% performed well to suppress the colony growth of L. pseudotheobromae (100%), followed by A. conyzoides 3% and A. conyzoides 1%. A. conyzoides 5% able to inhibit the colony growth of L. theobromae until 100%, followed by A. conyzoides 3% and 1%. Meanwhile, A. conyzoides and C. odorata extract tested on PDB medium at 1, 3, and 5% reduced the fungal biomass significantly at all concentrations. C. odorata was found most effective in inhibiting fungal biomass of both pathogens either on wet weight or on dry weight at 1, 3, and 5% %. A. conyzoides and C. odorata can manage the growth of L. theobromae and L. pseudotheobromae through in vitro conditions.

Rational Selection of Mixed Yeasts Starters for Agave Must Fermentation

Tequila and mezcal are both traditional Mexican liquors that are produced from cooked Agave spp. must fermentation and usually rely on spontaneous or pure Saccharomyces cerevisiae strain inoculation. In order to contribute to the rational selection of yeast starters for tequila and mezcal productions, we tested a collection of 25 yeasts originally isolated from mezcal musts, spanning 10 different yeast species. These strains were first characterized in a semi synthetic medium (labeled as M2, having 90 g/L fructose and 10 g/L glucose of initial hexoses) at 48 h of culture, observing a differential pattern in the consumption of sugars and productivity. Selected Saccharomyces strains left around 10 g/L of fructose and showed higher fermentation performance. However, some non-Saccharomyces strains, specifically from Torulospora (Td), Kluyveromyces (Km), and Zygosaccharomyces (Zb) genera, consumed almost all the sugar (i.e., Km1Y9 with <5 g/L) and had a high productivity of ethanol. In general, all Saccharomyces strains presented a high production of ethyl-butyrate, ethyl-decanoate, and ethyl-hexanoate with peaks of 10, 38, and 3 μg/L, respectively. In addition, some Kluyveromyces and Torulospora strains showed a high production of phenyl ethyl acetate (i.e., Km1D5 with up to 1400 μg/L); isoamyl acetate (i.e., Km1D5 and Td1AN2 with more than 300 μg/L), and hexyl acetate (i.e., Td1AN2 with 0.3 μg/L). Representative strains of the most productive genera (Saccharomyces, Torulospora, and Kluyveromyces) were selected to evaluate their fermentative performance and survival in a mixed culture on a medium based on Agave tequilana must, and their population kinetics was characterized using specific fluorescent in situ hybridization (FISH) probes in a qualitative and semi-quantitative analysis during fermentation. We observed that the mixture ratios of 0.1:1:1 or 1:1:1 (Saccharomyces:Kluyveromyces:Torulospora), maintained good fermentation productivities, with alcohol yields above 0.45 g/g, and allowed a high survival rate of the non-Saccharomyces strains during the fermentation process. Finally, mixed inoculum fermentations on A. tequilana must medium, including different Saccharomyces strains and the finally selected Torulospora and Kluyveromyces strains, showed the best production parameters in terms of ethanol, carbon dioxide, glycerol, and acetic acid values, as well as improved volatile metabolite profiles as compared to the pure cultures. All these data were used to propose a methodology of selection of strains to be used as a pure or mixed starter for tequila and mezcal fermentations, with high primary metabolite productivity and desired aromatic profile.

Enhancement of Growth and Paramylon Production of Euglena gracilis by Upcycling of Spent Tomato Byproduct as an Alternative Medium

Euglena gracilis (E. gracilis) accumulates paramylon, an immune-functional beta-glucan that can be used as a functional food. Paramylon production is strongly affected by the organic carbon source and the initial pH conditions. Food processing byproducts have attracted attention for microalgal cultivation because of their low cost and abundance of nutrients, including carbon and nitrogen. We investigated the optimal carbon source and its concentration for efficient paramylon production. A spent tomato byproduct (STB) generated from a tomato processing plant was applied for biomass and paramylon production from E. gracilis with respect to the initial pH condition. The highest paramylon concentration (1.2 g L−1) and content (58.2%) were observed with 15 g L−1 glucose. The biomass production increased when STB was used as compared with that when a synthetic medium was used (1.6-fold higher at pH 3 and 2-fold higher at pH 8). The optimal initial pH was determined according to the maximum production of biomass and paramylon. Upcycling the food processing byproduct, STB, can contribute not only to cost reduction of the biorefinery process using E. gracilis but also to environmental remediation by removing organic carbon and nitrogen from the byproducts.

Top-down, knowledge-based genetic reduction of yeast central carbon metabolism

Saccharomyces cerevisiae, whose evolutionary past includes a whole-genome duplication event, is characterised by a mosaic genome configuration with substantial apparent genetic redundancy. This apparent redundancy raises questions about the evolutionary driving force for genomic fixation of minor paralogs and complicates modular and combinatorial metabolic engineering strategies. While isoenzymes might be important in specific environments, they could be dispensable in controlled laboratory or industrial contexts. The present study explores the extent to which the genetic complexity of the central carbon metabolism (CCM) in S. cerevisiae, here defined as the combination of glycolysis, pentose phosphate pathway, tricarboxylic acid cycle and a limited number of related pathways and reactions, can be reduced by elimination of (iso)enzymes without major negative impacts on strain physiology. Cas9-mediated, groupwise deletion of 35 from the 111 genes yielded a minimal CCM strain, which despite the elimination of 32 % of CCM-related proteins, showed only a minimal change in phenotype on glucose-containing synthetic medium in controlled bioreactor cultures relative to a congenic reference strain. Analysis under a wide range of other growth and stress conditions revealed remarkably few phenotypic changes of the reduction of genetic complexity. Still, a well-documented context-dependent role of GPD1 in osmotolerance was confirmed. The minimal CCM strain provides a model system for further research into genetic redundancy of yeast genes and a platform for strategies aimed at large-scale, combinatorial remodelling of yeast CCM.

Features of the Opportunistic Behaviour of the Marine Bacterium Marinobacter algicola in the Microalga Ostreococcus tauri Phycosphere

Although interactions between microalgae and bacteria are observed in both natural environment and the laboratory, the modalities of coexistence of bacteria inside microalgae phycospheres in laboratory cultures are mostly unknown. Here, we focused on well-controlled cultures of the model green picoalga Ostreococcus tauri and the most abundant member of its phycosphere, Marinobacter algicola. The prevalence of M. algicola in O. tauri cultures raises questions about how this bacterium maintains itself under laboratory conditions in the microalga culture. The results showed that M. algicola did not promote O. tauri growth in the absence of vitamin B12 while M. algicola depended on O. tauri to grow in synthetic medium, most likely to obtain organic carbon sources provided by the microalgae. M. algicola grew on a range of lipids, including triacylglycerols that are known to be produced by O. tauri in culture during abiotic stress. Genomic screening revealed the absence of genes of two particular modes of quorum-sensing in Marinobacter genomes which refutes the idea that these bacterial communication systems operate in this genus. To date, the ‘opportunistic’ behaviour of M. algicola in the laboratory is limited to several phytoplanktonic species including Chlorophyta such as O. tauri. This would indicate a preferential occurrence of M. algicola in association with these specific microalgae under optimum laboratory conditions.