Kinetics and metabolic behavior of a composite culture of <Emphasis Type="Italic">Kloeckera apiculata</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> wine related strains

The kinetics and metabolic behavior of Kloeckera apiculata mc1 and Saccharomyces cerevisiae mc2 in composite culture was investigated. K. apiculata showed a higher viability through the fermentation; however the maximum cell density of both yeasts decreased. This behavior was not due to ethanol concentration, killer toxins production or competition for assimilable nitrogenous compounds between both yeasts. Despite the consistent production of secondary products by single culture of K. apiculata, an increase of these compounds was not observed in mixed culture. These results contribute to a better understanding of the behavior of non-Saccharomyces yeasts and their potential application in the wine industry.     

Ethanol and acetate production by <Emphasis Type="Italic">Clostridium ljungdahlii</Emphasis> and <Emphasis Type="Italic">Clostridium autoethanogenum</Emphasis> using resting cells

Combined gasification and fermentation technologies can potentially produce biofuels from renewable biomass. Gasification generates synthesis gas consisting primarily of CO, CO₂, H₂, N₂, with smaller amounts of CH₄, NO_x, O₂, C₂ compounds, ash and tars. Several anaerobic bacteria species can ferment bottled mixtures of pure synthesis gas constituents. However, there are challenges to maintaining culture viability of synthesis gas exposed cells. This study was designed to enhance culture stability and improve ethanol-to-acetate ratios using resting (non-growing) cells in synthesis gas fermentation. Resting cell states were induced in autotrophic Clostridium ljungdahlii cultures with minimal ethanol and acetate production due to low metabolic activity compared to growing cell production levels of 5.2 and 40.1 mM of ethanol and acetate. Clostridium autoethanogenum cultures were not induced into true resting states but did show improvement in total ethanol production (from 5.1 mM in growing cultures to 9.4 in one nitrogen-limited medium) as well as increased shifts in ethanol-to-acetate production ratios.     

Biological acidification during grape must fermentation using mixed cultures of <Emphasis Type="Italic">Kluyveromyces thermotolerans</Emphasis> and <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Four mixed culture fermentations of grape must were carried out with Kluyveromyces thermotolerans strain TH941 and Saccharomyces cerevisiae strain SCM952. In the first culture, both yeasts were added together, whereas in the remaining three cultures S. cerevisiae was added 1, 2, and 3 days after the inoculation of K. thermotolerans. The growth and survival of the K. thermotolerans strain and the amount of the produced l-lactic acid depend on the time of inoculation of the S. cerevisiae strain and provided an effective acidification during alcoholic fermentation. The four cultures contained, respectively, at the end of fermentation 0.18, 1.80, 4.28, and 5.13 g l-lactic acid l⁻¹. The grape must with an initial pH of 3.50 was effectively acidified (70% increase in titratable acidity, 0.30 pH unit decrease) by the production of 5.13 g l-lactic acid l⁻¹.     

The effect of Debina grapevine indigenous yeast strains of <Emphasis Type="Italic">Metschnikowia</Emphasis> and <Emphasis Type="Italic">Saccharomyces</Emphasis> on wine flavour

The spontaneous alcoholic fermentation of grape must is a complex microbiological process involving a large number of various yeast species, to which the flavour of every traditional wine is largely attributed. Whilst Saccharomyces cerevisiae is primarily responsible for the conversion of sugar to alcohol, the activities of various non-Saccharomyces species enhance wine flavour. In this study, indigenous yeast strains belonging to Metschnikowia pulcherrima var. zitsae as well as Saccharomyces cerevisiae were isolated and characterized from Debina must (Zitsa, Epirus, Greece). In addition, these strains were examined for their effect on the outcome of the wine fermentation process when used sequentially as starter cultures. The resulting wine, as analyzed over three consecutive years, was observed to possess a richer, more aromatic bouquet than wine from a commercial starter culture. These results emphasize the potential of employing indigenous yeast strains for the production of traditional wines with improved flavour.     

Effect of cultivation conditions on invertase production by hyperproducing <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> isolates

Invertase (β-D-fructofuranoside fructohydrolase, EC 3.2.1.26) finds major uses in confectionery and in the production of invert syrup. In the present study, we report on invertase production by wild cultures of Saccharomyces cerevisiae. The yeast strains were isolated from dates available in a local market. Five hyperproducing yeast strains (>100- fold higher invertase activity) were kinetically analysed for invertase production. Saccharomyces cerevisiae strain GCA-II was found to be a better invertase-yielding strain than all the other isolates. The values of Q_p and Y_p/s for GCA-II were economical as compared to other Saccharomyces cultures. The effect of sucrose concentration, rate of invertase synthesis, initial pH of fermentation medium and different organic nitrogen sources on the production of invertase under submerged culture conditions was investigated. Optimum concentrations of sucrose, urea and pH were 3, 0.2 (w/v), and 6 respectively. The increase in the enzyme yield obtained after optimization of the cultural conditions was 47.7%.     

The molecular genetic differentiation of cultured <Emphasis Type="Italic">Saccharomyces</Emphasis> strains

A comparative molecular genetic study of cultured Saccharomyces strains isolated from the surface of berries and various fermentation processes showed that bakers yeast and black-currant isolates contain not only Saccharomyces cerevisiae but also S. cerevisiae × S. bayanus var. uvarum hybrids. The molecular karyotyping of bakers, brewers, and wine yeasts showed their polyploidy. The restriction enzyme analysis of noncoding rDNA regions (5.8S-ITS and IGS2) makes it possible to differentiate species of the genus Saccharomyces and to identify interspecies hybrids. The microsatellite primer (GTG)₅ can be used to study the populations of cultured S. cerevisiae strains.__________Translated from Mikrobiologiya, Vol. 74, No. 2, 2005, pp. 215–223.Original Russian Text Copyright © 2005 by Naumova, Zholudeva, Martynenko, Naumov.     

Production of <Emphasis Type="SmallCaps">l</Emphasis>-lactic acid by <Emphasis Type="Italic">Rhizopus oryzae</Emphasis> using semicontinuous fermentation in bioreactor

Semicontinuous fermentation using pellets of Rhizopus oryzae has been recognized as a promising technology for l-lactic acid production. In this work, semicontinuous fermentation of R. oryzae AS 3.819 for l-lactic acid production has been developed with high l-lactic acid yield and volumetric productivity. The effects of factors such as inoculations, CaCO₃ addition time, and temperature on l-lactic acid yield and R. oryzae morphology were researched in detail. The results showed that optimal fermentation conditions for the first cycle were: inoculation with 4% spore suspension, CaCO₃ added to the culture medium at the beginning of culture, and culture temperature of 32–34°C. In orthogonal experiments, high l-lactic acid yield was achieved when the feeding medium was (g/l): glucose, 100; (NH₄)₂SO₄, 2; KH₂PO₄, 0.1; ZnSO₄·7H₂O, 0.33; MgSO₄·7H₂O, 0.15; CaCO₃, 50. Twenty cycles of semicontinuous fermentation were carried out in flask culture. l-lactic acid yield was 78.75% for the first cycle and 80–90% for the repeated cycles; the activities of lactate dehydrogenases (LDH) were 7.2–9.2 U/mg; fermentation was completed in 24 h for each repeated cycle. In a 7-l magnetically stirred fermentor, semicontinuous fermentation lasted for 25 cycles using pellets of R. oryzae AS 3.819 under the optimal conditions determined from flask cultures. The final l-lactic acid concentration (LLAC) reached 103.7 g/l, and the volumetric productivity was 2.16 g/(l·h) for the first cycle; in the following 19 repeated cycles, the final LLAC reached 81–95 g/l, and the volumetric productivities were 3.40–3.85 g/(l·h).     

Evaluation of the acetaldehyde production and degradation potential of 26 enological <Emphasis Type="Italic">Saccharomyces</Emphasis> and non-<Emphasis Type="Italic">Saccharomyces</Emphasis> yeast strains in a resting cell model system

Acetaldehyde is relevant for wine aroma, wine color, and microbiological stability. Yeast are known to play a crucial role in production and utilization of acetaldehyde during fermentations but comparative quantitative data are scarce. This research evaluated the acetaldehyde metabolism of 26 yeast strains, including commercial Saccharomyces and non-Saccharomyces, in a reproducible resting cell model system. Acetaldehyde kinetics and peak values were highly genus, species, and strain dependent. Peak acetaldehyde values varied from 2.2 to 189.4 mg l⁻¹ and correlated well (r ² = 0.92) with the acetaldehyde production yield coefficients that ranged from 0.4 to 42 mg acetaldehyde per g of glucose in absence of SO₂. S. pombe showed the highest acetaldehyde production yield coefficients and peak values. All other non-Saccharomyces species produced significantly less acetaldehyde than the S. cerevisiae strains and were less affected by SO₂ additions. All yeast strains could degrade acetaldehyde as sole substrate, but the acetaldehyde degradation rates did not correlate with acetaldehyde peak values or acetaldehyde production yield coefficients in incubations with glucose as sole substrate.     

In Vitro Susceptibility of <Emphasis Type="Italic">C. albicans</Emphasis> and <Emphasis Type="Italic">C. neoformens</Emphasis> to Potential Metabolites from <Emphasis Type="Italic">Streptomycetes</Emphasis>

Forty two Streptomycetes isolates from soils of Kodachadri region in Western ghats were recovered by soil dilution technique. Cross streak method was followed for primary screening of antifungal activity. Positive isolates were subjected to secondary screening by cold extraction of fermentation broth in butanol solvent. Six isolates exhibited broad spectrum antifungal activity against all the tested yeast pathogens like Candida albicans, Candida lipolytica, Cryptococcus neoformens and Saccharomyces cerevisiae. One isolate showed excellent antifungal activity against all test organisms with maximum zone of inhibition 60 mm each incase of C. neoformens and C. albicans. Partial characterization of antifungal metabolite by TLC resulted in a purple spot with an R_f value 0.50. The UV absorption spectra at 218 nm indicated possible chemical nature of the active metabolite as polyene group and purity was assessed by analytical HPLC.     

Optimizing emulsan production of <Emphasis Type="Italic">A. venetianus</Emphasis> RAG-1 using response surface methodology

Statistical experimental design was used to optimize medium constituents for emulsan production by Acinetobacter venetianus RAG-1 in batch cultivation. The factors affecting emulsan production were screened by a two-level factorial design, and the optimal concentration of medium constituents for emulsan production were determined by the method of steepest path ascent and central composite experimental design. Experimental results showed that the optimal medium constituents were 9.16 g/L ethanol, 8.2 g/L KH₂PO₄, 23.32 g/L K₂HPO₄, 5.77 g/L (NH₄)₂SO₄ and 0.354 g/L MgSO₄•7H₂O. Under this optimal composition, the predicted emulsan production was 72.198 mg/L, and experimental value was 73.312 mg/L for 80 h culture in the shake flasks, and the emulsan yield by A. venetianus RAG-1 was enhanced nearly 1.48-fold (from 49.5 to 73.312 mg/L). Based on the results, we identify the optimal medium constituents for emulsan production and could take advantage of strategy for scale up the fermentation of emulsan production.     

Substrate inhibition kinetics of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> in fed-batch cultures operated at constant glucose and maltose concentration levels

Fed-batch culture is the mode of operation of choice in industrial baker’s yeast fermentation. The particular mode of culture, operated at stable glucose and maltose concentration levels, was employed in this work in order to estimate important kinetic parameters in a process mostly described in the literature as batch or continuous culture. This way, the effects of a continuously falling sugar level during a batch process were avoided and therefore the effects of various (stable) sugar levels on growth kinetics were evaluated. Comparing the kinetics of growth and the inhibition by the substrate in cultures grown on glucose, which is the preferential sugar source for Saccharomyces cerevisiae, and maltose, the most common sugar source in industrial media for baker’s yeast production, a milder inhibition effect by the substrate in maltose-grown cells was observed, as well as a higher yield coefficient. The observed sugar inhibition effect in glucostat cultures was taken into account in modeling substrate inhibition kinetics. The inhibition coefficient K _i increased with increasing sugar concentration levels, but it appeared to be unaffected by the type of substrate and almost equal for both substrates at elevated concentration levels.     

Microbiological and physicochemical factors affecting <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> incidence in Cavendish banana (<Emphasis Type="Italic">Musa cavendishii</Emphasis>) chips production in Southern Philippines

Microbiological and physicochemical factors affecting the incidence of Aspergillus section Flavi in dried Cavendish banana (Musa cavendishii) chips production in Southern Philippines were examined. The average counts of Aspergillus section Flavi (AFC) in fresh and dried Cavendish bananas from 10 production batches of the Philippine Agro-Industrial Development Cooperative in Davao del Norte, Southern Philippines were 1.2 × 10² and 1.6 × 10² cfu/g, respectively. Isolates from both samples were identified to be Aspergillus flavus based on spore type and conidial structure of isolates. An increasing trend in the AFC of Cavendish bananas was observed during dried banana chips processing. Variability in the AFC between production batches was attributed to differences in aerobic and fungal populations and physicochemical characteristics of the fruits, peel damage of the raw materials, concentration of AFC in the air and food-contact surfaces of the production area, and temperature and relative humidity (RH) conditions of the environment during production and storage. Physicochemical characteristics of Cavendish bananas from the receipt of raw materials up to the first day of drying were within the reported range of values allowing growth and toxin production by aflatoxigenic fungi. Air-borne AFC varied depending on the section of the production area examined. The close proximity of the waste disposal area from the production operation to the preparation, drying and storage areas suggests that cross-contamination, probably air-borne or insect-borne was a likely occurrence. The hands of workers were also identified as AFC sources. Results of this study highlight the need for the development of strategies to control aflatoxigenic fungi and aflatoxin contamination in Philippine dried Cavendish bananas.     

Purification and properties of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">S</Emphasis>-adenosylmethionine synthetase expressed in recombinant <Emphasis Type="Italic">Pichia pastoris</Emphasis>

An intracellular S-adenosylmethionine synthetase (SAM-s) was purified from the fermentation broth of Pichia pastoris GS115 by a sequence chromatography column. It was purified to apparent homogeneity by (NH₄)₂SO₄ fractionation (30–60%), anion exchange, hydrophobic interaction, anion exchange and gel filtration chromatography. HPLC showed the purity of purified SAM-s was 91.2%. The enzyme was purified up to 49.5-fold with a final yield of 20.3%. The molecular weight of the homogeneous enzyme was 43.6 KDa, as determined by electro-spray ionization mass spectrometry (ESI-MS). Its isoelectric point was approximately 4.7, indicating an acidic character. The optimum pH and temperature for the enzyme reaction were 8.5 and 35 °C, respectively. The enzyme was stable at pH 7.0–9.0 and was easy to inactivate in acid solution (pH ≤ 5.0). The temperature stability was up to 45 °C. Metal ions, such as, Mn²⁺ and K⁺ at the concentration of 5 mM had a slight activation effect on the enzyme activity and the Mg²⁺ activated the enzyme significantly. The enzyme activity was strongly inhibited by heavy metal ions (Cu²⁺ and Ag²⁺) and EDTA. The purified enzyme from the transformed Pichia pastoris synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K _m of 120 and 330 μM and V _max of 8.1 and 23.2 μmol/mg/min for l-methionine and ATP, respectively.     

Overexpression of <Emphasis Type="Italic">ADH1</Emphasis> and <Emphasis Type="Italic">HXT1</Emphasis> genes in the yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> improves the fermentative efficiency during tequila elaboration

This work assessed the effect of the overexpression of ADH1 and HXT1 genes in the Saccharomyces cerevisiae AR5 strain during fermentation of Agave tequilana Weber blue variety must. Both genes were cloned individually and simultaneously into a yeast centromere plasmid. Two transformant strains overexpressing ADH1 and HXT1 individually and one strain overexpressing both genes were randomly selected and named A1, A3 and A5 respectively. Overexpression effect on growth and ethanol production of the A1, A3 and A5 strains was evaluated in fermentative conditions in A. tequilana Weber blue variety must and YPD medium. During growth in YPD and Agave media, all the recombinant strains showed lower cell mass formation than the wild type AR5 strain. Adh enzymatic activity in the recombinant strains A1 and A5 cultivated in A. tequilana and YPD medium was higher than in the wild type. The overexpression of both genes individually and simultaneously had no significant effect on ethanol formation; however, the fermentative efficiency of the A5 strain increased from 80.33% to 84.57% and 89.40% to 94.29% in YPD and Agave medium respectively.     

Enhanced production of lactic acid by an adapted strain of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">delbrueckii</Emphasis> subsp. <Emphasis Type="Italic">lactis</Emphasis>

Lactobacillus delbrueckii subsp. lactis strains were developed having increased activity, by gradually acclimatizing the bacteria to acidic conditions over repeated batch culture. Cells from one batch culture were used as the inoculum for the subsequent batch culture and thereby an adapted strain of Lactobacillus was obtained showing improved lactic acid productivity, cell growth and total glucose utilization. Furthermore, the acclimatized cells used significantly less nitrogen for a given level of lactic acid production, which is significant from an industrial point of view. The developed procedure decreases fermentation time and nutrient use, leading to reduced operation costs, while providing a lactic acid yield superior to previously reported methods.     

Enhanced 1,3-propanediol production in recombinant <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> carrying the gene <Emphasis Type="Italic">yqh</Emphasis>D encoding 1,3-propanediol oxidoreductase isoenzyme

The yqhD gene from Escherichia coli encoding 1,3-propanediol oxidoreductase isoenzyme (PDORI) and the tetracycline resistant gene (tetR) from plasmid pHY300PLK were amplified by PCR. They were inserted into vector pUC18, yielding the recombinant expression vector pUC18-yqhD-tetR. The recombinant vector was then cloned into Klebsiella pneumoniae ME-308. The overexpression of PDORI in K. pneumoniae surprisingly led to higher 1,3-propanediol production. The final 1,3-propanediol concentration of recombinant K. pneumoniae reached 67.6 g/l, which was 125.33% of that of the original strain. The maximum activity of recombinant PDORI converting 3-HPA to 1,3-PD reached 110 IU/mg after induction by IPTG at 31°C during the fermentation, while it was only 11 IU/mg under the same conditions for the wild type strain. The K _m values of the purified PDORI for 1,3-propanediol and NADP were 12.1 mM and 0.15 mM, respectively. Compared with the original strains, the concentration of the toxic intermediate 3-hydroxypropionaldehyde during the fermentation was also reduced by 22.4%. Both the increased production of 1,3-propanediol and the reduction of toxic intermediate confirmed the significant role of 1,3-propanediol oxidoreductase isoenzyme from E. coli in converting 3-hydroxypropionaldehyde to 1,3-propanediol for 1,3-PD production.     

Winemaking from gaglioppo grapes with hybrid strains of<Emphasis Type="Italic">Saccharomyces</Emphasis>

The fermentative behavior of two hybrid wine yeast strains (first-generation hybrid-strain 12 233×6167—obtained by hybridization of the cryotolerant strainS. bayanus 12 233 with the mesophilic strainsS. cerevisiae 6167, and TT254×6392 arising by hybridization of the thermotolerant strainS. cerevisiae TT254 with the mesophilic strainS. cerevisiae 6392) was compared with that of a commercial wine yeast strainS. cerevisiae K1 in must from black grapes of the Calabrian variety Gaglioppo. The goal was to obtain wines with a high content of ‘polyphenols’ from a grape must with a limited phenolic content such as the Gaglioppo must. The progress of the winemaking was estimated according to residual sugars; at the end of fermentation, the wines were decanted, bottled and principal physico-chemical characteristics determined. Our results point to the possibility to select wine yeasts (significantly differing in the above parameters) by their ability to interact with phenolic compounds.     

Determination of Toxigenic Potentials of <Emphasis Type="Italic">Bacillus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Okpehe</Emphasis>, a Nigerian Fermented Condiment

The toxigenic potential of Bacillus species isolated from the traditional fermented condiment okpehe was determined; this is aimed at selection of non-toxigenic bacilli as starter cultures to bring about production of safe product. B. subtilis and B. cereus strains isolated from okpehe were evaluated for their possible possession of virulence characteristics. Fifty isolates were screened for their ability to produce diarrhoea enterotoxin by reversed passive latex agglutination (BCET-RPLA) test kit; the result showed that 40% of the B. cereus strains were toxigenic. The ability of the selected isolates to compete in situ and in vitro toxin production during the fermentation was also determined. The enterotoxin was not detected using BCET-RPLA kit in the spontaneously fermented samples of okpehe, but the toxin was detected in the okpehe samples fermented using B. cereus enterotoxin producer in mixed starter culture fermentation. The PCR amplification of virulence genes revealed that Bacillus cereus and B. licheniformis, a strain from the B. subtilis group, contained DNA sequences encoding the haemolysin BL (hblD) enterotoxin complex. The growth ability of B. cereus strains to high population during the fermentation and the presence of detectable diarroheagenic genes in B. cereus and B. licheniformis showed that strains carrying virulence characteristics cannot be totally ruled out in traditionally fermented okpehe.     

Elicitation of <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> with dead cells of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> and <Emphasis Type="Italic">Staphylococcus aureus</Emphasis> in a bioreactor increases production of undecylprodigiosin

Since microorganisms normally co-exist with other species in nature, they have developed complex metabolic and physiological responses as a result of such interspecies interactions. We utilized some of these interactions by introducing heat-killed cells of Bacillus subtilis and Staphylococcus aureus to Streptomyces coelicolor cultures and, as a result, stimulated undecylprodigiosin production. Undecylprodigiosin is not only an antibiotic; it has also been attributed with antitumor activities, but, in a defined medium, pure cultures of S. coelicolor produced only low concentrations. Elicitation with B. subtilis increased the maximum undecylprodigiosin concentration by threefold and S. aureus by fivefold compared with the pure culture of S. coelicolor. Growth and glucose consumption of elicited S. coelicolor, however, remained similar to those observed in the pure culture. Furthermore, another positive outcome of the elicitation with both B. subtilis and S. aureus was the earlier onset of undecylprodigiosin production by 24 h compared with the pure culture of S. coelicolor. This is the first time that such a phenomenon has been seen in 2L bioreactors. Our work supports the use of biotic elicitation in order to enhance the production of secondary metabolites for industrial-scale applications.