Fermentation, Volume 2, Issue 1 (March 2016) – 7 articles

Malvar grape juice offers relatively little in the way of a sensory experience. Our interest lies in the use of locally-selected yeast strains in experimental fermentations to improve the sensory characteristics of Malvar wines. Two locally-selected strains of Saccharomyces cerevisiae were used as starter cultures in vinifications and compared with spontaneous fermentations of the same cultivar musts. Wine quality was investigated by their principal oenological parameters, analysis of the volatile aroma components, and corroborated by an experienced taster panel. The most salient chemical attributes were its high concentrations of isoamyl acetate and hexyl acetate and the high acidity, which have been detected to be key constituents in setting the fruity and fresh character of Malvar wines. Winemakers of winegrowing areas where this grape variety is cultivated will have improved options to elaborate new white wines styles, using selected yeast strains that enhance its aromatic properties. Full article

Despite the rise of ‘omics techniques for the study of biological systems, the quantitative description of phenotypes still rests to a large extent on quantitative data produced on chromatography platforms. Here, we describe an improved liquid chromatography method for the determination of sugars, carboxylates, alcohols and aldehydes in microbial fermentation samples and cell extracts. Specific emphasis is given to substrates and products currently pursued in industrial microbiology. The present method allows quantification of 21 compounds in a single run with limits of quantification between 10⁻⁷ and 10⁻¹⁰ mol and limits of detection between 10⁻⁹ and 10⁻¹¹ mol. Full article

Aerobically cultivated cells of Corynebacterium glutamicum produce mixed organic acids, including succinic acid (SA), from glucose when the cells are transferred to oxygen-deprived conditions. Genetic modification, including inactivation of lactate dehydrogenase and overexpression of pyruvate carboxylase, allows this microbe to be an efficient SA producer under the conditions of oxygen deprivation. High productivity and high titers can be achieved in the production process by using the genetically engineered strain of C. glutamicum under the given conditions. However, glucose consumption for cell preparation decreases process yield (defined as the quantity of SA produced divided by the total quantity of glucose used in cell preparation and SA production). In this study, we investigated cell recycle fed-batch fermentation for SA production to improve the process yield by reducing the effect of glucose consumption for cell preparation on the process yield. A genetically stable and markerless strain, harboring nine genomic copies of the pyruvate carboxylase gene, was newly constructed and used for cell recycle fermentation. During 26 reaction cycles, only 0.7% decrease in specific productivity per reaction was observed. Overall, the process yield was improved by 79% compared to that in a single fed-batch reaction without cell recycling. Full article

Production of small molecule drugs in a recombinant host is becoming an increasingly popular alternative to chemical synthesis or production in natural hosts such as plants due to the ease of growing microorganisms with higher titers and less cost. While there are a wide variety of well-developed cloning techniques to produce small molecule drugs in a heterologous host, there are still many challenges towards efficient production. Therefore, this paper reviews some of these recently developed tools for metabolic engineering and categorizes them according to a chronological series of steps for a generalized method of drug production in a heterologous host, including 1) pathway discovery from a natural host, 2) pathway assembly in the recombinant host, and 3) pathway optimization to increase titers and yield. Full article

The phenotypic responses of E. coli cells exposed to 1.2% (v/v) of 1-butanol, 2-butanol, isobutanol, tert-butanol, and 1,4-butanediol were studied in near real-time using Raman spectroscopy. A method of “chemometric fingerprinting” was employed that uses multivariate statistics (principal component analysis and linear discriminant analysis) to identify E. coli phenotypic changes over a 180 min post-treatment time-course. A toxicity study showed extreme variability among the reduction in culture growth, with 1-butanol showing the greatest toxicity and 1,4-butanediol showing relatively no toxicity. Chemometric fingerprinting showed distinct phenotype clusters according to the type of alcohol: (i) 1-butanol and 2-butanol (straight chain alcohols); (ii) isobutanol and tert-butanol (branched chain alcohols); and (iii) control and 1,4-butanediol (no terminal alkyl end) treated cells. While the isobutanol and tert-butanol treated cells led to similar phenotypic responses, isobutanol was significantly more toxic. In addition, the phenotypic response was found to take place largely within 60 min of culture treatment; however, significant responses (especially for 1,4-butanediol) were still occurring at 180 min post-treatment. The methodology presented here identified different phenotypic responses to seemingly similar 4-carbon alcohols and can be used to study phenotypic responses of virtually any cell type under any set of environmental conditions or genetic manipulations. Full article

Koji, rice fermented with Aspergillus, is used for saccharification of starch contained in crops during the manufacturing of many of Japanese traditional foods and drinks. Japanese people have long eaten koji, and many beneficial substances have been reported to be contained in koji. However, there has been no report on the existence or content of galactosylceramide in koji. To address this issue, we analyzed the chemical composition of the sugar moiety of monohexosylceramide contained in koji, and elucidate that 30.3% of yellow koji is galactosylceramide, 69.7% of that is glucosylceramide, 19.2% of white koji is galactosylceramide, and 80.8% of that is glucosylceramide. This is the first report of the existence and content of galactosylceramide in koji. Full article

Some non-Saccharomyces yeasts, including Hanseniaspora, participate in the first stages of wine fermentation. Besides their importance in the wine production process some of these yeasts have been described to be potential producers of hydrolytic enzymes to industrial level. In this work, we pretend to evaluate the technological abilities of the Hanseniaspora strains deposited in the Spanish Type Culture Collection (CECT). First of all, we considered verification of the correct identification of the strains using several miniaturized biochemical systems and molecular techniques (PCR, RFLP and sequencing of the ribosomal D1/D2 region). The results allowed us to verify the correct adscription of the 26 strains included in this study, which exhibited concordant profiles of restriction with one of the three species described in previous studies (H. occidentalis, H. osmophila and H. valbyensis). Some other strains assigned to the species (H. uvarum, H. vineae and H. guilliermondii) showed at least two different profiles. The other objective of this study was to perform an initial screening to characterize both at quantitative and qualitative levels, the ability of these yeasts to produce valuable enzymes for wine fermentation (increase of aroma) and other applications. The more important enzymatic activities detected were β-glucosidase, β-xylosidase, and protease. The HU7, HU8, HV1, HV3, HO2 and HOC1 strains showed high levels of β-glucosidase and β-xylosidase activity, whereas some strains (HG1, HG3, HVA1, HOC 3 and HOC4 were useful for protease production. Full article