Degradation of malic acid by Issatchenkia orientalis KMBL 5774, an acidophilic yeast strain isolated from Korean grape wine pomace

Several yeast strains degrading malic acid as a sole carbon and energy source were isolated from Korean wine pomace after enrichment culture in the presence of malic acid. Among them, the strain designated as KMBL 5774 showed the highest malic acid degrading ability. It was identified as Issatchenkia orientalis based on its morphological and physiological characteristics as well as the nucleotide sequences of the internal transcribed spacer (ITS) I-5.8S rDNA-ITS II region. Phylogenetic analysis of the ITS I-5.8S rDNAITS II sequences showed that the KMBL 5774 is the closest to I. orientalis zhuan 192. Identity of the sequences of the KMBL 5774 was 99.5% with those of I. orientalis zhuan 192. The optimal pH of the media for the growth and malic acid degradation by the yeast was between 2.0 and 3.0, suggesting that the strain is an acidophile. Under the optimized conditions, the yeast could degrade 95.5% of the malic acid after 24 h of incubation at 30 degrees in YNB media containing 2% malic acid as a sole carbon and energy source.     

Co-fermentation of grape must by Issatchenkia orientalis and Saccharomyces cerevisiae reduces the malic acid content in wine

Grape must was fermented by a mixed culture of Saccharomyces cerevisiae W-3 (a wine yeast) and Issatchenkia orientalis KMBL 5774 (a malic acid-degrading yeast). Co-fermentation with 1:1 (v/v) inoculum ratio of W-3 and KMBL 5774 decreased malic acid to 0.33 mg/ml from 1.1 mg ml with W-3 alone. Ethanol production was the same in both cases (7.8%, v/v). Acetaldehyde, 1-propanol, 2-butanol and isoamyl alcohol all decreased, with an increase in methanol, in the co-fermented wine. Sensory evaluation showed a higher score in the wine fermented with 1:1 (v/v) inoculum ratio than those obtained by 4:1 (v/v) inoculum ratio or W-3 alone.     

Malolactic fermentation in wine with high densities of non-proliferating Oenococcus oeni

Five strains of Oenococcus oeni (syn. Leuconostoc oenos) under non-proliferating conditions were assessed for the performance of the malolactic fermentation in wine at various initial pH values, malic acid concentration and densities of cells. We succeeded in inducing the malolactic fermentation after inoculation of high densities of O. oeni G6 even in recalcitrant wines where the traditional malolactic fermentation was inhibited by adverse environmental conditions (low pH and high concentration of malic acid). Optimal degrading conditions in wine, under different physico-chemical environments, were determined in order to achieve rapid depletion of malic acid in red wine. Off-odour compounds were not formed under these conditions, suggesting an attractive alternative for wine production. This revised version was published online in November 2006 with corrections to the Cover Date.     

Reduction of volatile acidity of acidic wines by immobilized Saccharomyces cerevisiae cells

Excessive volatile acidity in wines is a major problem and is still prevalent because available solutions are nevertheless unsatisfactory, namely, blending the filter-sterilized acidic wine with other wines of lower volatile acidity or using reverse osmosis. We have previously explored the use of an empirical biological deacidification procedure to lower the acetic acid content of wines. This winemaker’s enological practice, which consists in refermentation associated with acetic acid consumption by yeasts, is performed by mixing the acidic wine with freshly crushed grapes, musts, or marc from a finished wine fermentation. We have shown that the commercial strain Saccharomyces cerevisiae S26 is able to decrease the volatile acidity of acidic wines with a volatile acidity higher than 1.44 g?L^?1 acetic acid, with no detrimental impact on wine aroma. In this study, we aimed to optimize the immobilization of S26 cells in alginate beads for the bioreduction of volatile acidity of acidic wines. We found that S26 cells immobilized in double-layer alginate–chitosan beads could reduce the volatile acidity of an acidic wine (1.1 g?L^?1 acetic acid, 12.5 % (v/v) ethanol, pH 3.12) by 28 and 62 % within 72 and 168 h, respectively, associated with a slight decrease in ethanol concentration (0.7 %). Similar volatile acidity removal efficiencies were obtained in medium with high glucose concentration (20 % w/v), indicating that this process may also be useful in the deacidification of grape musts. We, therefore, show that immobilized S. cerevisiae S26 cells in double-layer beads are an efficient alternative to improve the quality of wines with excessive volatile acidity.     

Effect of air-blast drying and the presence of protectants on the viability of yeast entrapped in calcium alginate beads with an aim to improve the survival rate

Five yeast strains, Saccharomyces cerevisiae D8, M12, and S13; Hanseniaspora uvarum S6; and Issatchenkia orientalis KMBL5774, isolated from Korean grapes, were entrapped in Ca-alginate beads, which are non-toxic, simple to use, and economical. Ca-alginate beads containing yeast cells were soaked in protective solutions, such as skim milk, saccharides, polyols, and nitrogen compounds, before air-blast drying to improve the yeast survival rate and storage ability. The results showed that both entrapment in Ca-alginate beads and soaking in protective agents favorably affected the survival of all strains. The microenvironment formed by the beads and protective agents can protect the yeast cells from harsh environmental conditions, such as low water (below 10 %). All the yeast strains entrapped in Ca-alginate beads showed greater than 80 % survival and less than 11 % water content after air-blast drying at 37 °C for 5 h. In addition, air-blast dried cells of S. cerevisiae D8, M12, S13; H. uvarum S6; and I. orientalis KMBL5774 entrapped in 2 % Ca-alginate beads and soaked in protective agents (10 % skim milk containing 10 % sucrose, 10 % raffinose, 10 % trehalose, 10 % trehalose, and 10 % glucose, respectively) after air-blast drying at 37 °C for 5 h showed 90, 87, 92, 90, and 87 % viability, respectively. All dried entrapped yeast cells showed survival rates of at least 51 % after storage at 4 °C for 3 months.     

CONTINUOUS ALCOHOL/MALOLACTIC FERMENTATION OF GRAPE MUST IN A BIOREACTOR SYSTEM USING IMMOBILIZED CELLS

Three cultures immobilized by entrapping within alginate gel beads and packed in near-horizontal acrylic columns (15.0° angle) were used for alcohol/malolactic fermentation of grape must. Immobilized cells of Saccharomyces cerevisiae spp. chablis were placed in the 1st column, S. cerevisiae cells (an alcohol-sucrose-tolerant yeast) in the 2nd and the Lactobacillus delbrueckii cells in the 3rd column. Grape must with different levels of sugar(s), were each fed to the bioreactor columns at dilution rate of 0.74 h⁻¹ and recycled at 37.0C. The percent fermentation efficiency and yield using the 1st and 2nd columns for grape must containing 33.3% sugar(s) were 92.9 and 91.5%, respectively, and the wine had 15.5% alcohol after 23 cycles (∼ 50 h fermentation). The viability of the immobilized yeast cells in the alginate gel-bead was 84%± 4.0. Immobilized Lactobacillus delbrueckii cells were then added to the 3rd column (in series 37.0C) and the three cultures resulted in alcohol/malolactic fermentation of the grape must, evidenced by the high level of alcohol formed and simultaneous transformation of malic to lactic acid. Sensory evaluation of the wine scored high (7.8 ± 2.0 based on a value of 10.0) and indicated the potential of using multiple immobilized cells of two specific yeast cultures and a malolactic Lactobacillus for wine production.     

Degradation of chloroform by immobilized cells of <Emphasis Type="Italic">Bacillus</Emphasis> sp. in calcium alginate beads

A Bacillus sp., capable of degrading chloroform, was immobilized in calcium alginate. The beads in 20 g alginate l⁻¹ (about 2 × 10⁸ cells/bead) could be re-used nine times for degradation of chloroform at 40 μM. The immobilized cells had a higher range of tolerance (pH 6.5–9 and 20–41°C) than free cells (pH 7–8.5 and 28–32°C). At 5 g alginate l⁻¹, leakage of the cells from the beads was 0.51 mg dry wt ml⁻¹. This species is the first reported Bacillus that can degrade chloroform as the sole carbon source.     

Oenococcus oeni cells immobilized on delignified cellulosic material for malolactic fermentation of wine

Oenococcus oeni ATCC 23279 cells immobilized on delignified cellulosic material (DCM) were used for malolactic fermentation (MLF). In first, eleven repeated alcoholic fermentation batches of white must of 11-12 degrees Be initial density were performed by Saccharomyces cerevisiae cells immobilized on delignified cellulosic material at 20 degrees C. Subsequently, the induction of MLF in the eleven taken wine batches by O. oeni cells immobilized on DCM took place at 27 degrees C. From the 3rd MLF batch up to 10th, the malic acid degradation was 53.1 up to 67.4% and the cfu of the immobilized cells/g of biocatalyst remained stable. The produced lactic acid was less than the stoichiometric yield and acetic acid content was significantly reduced after MLF not contributing in an important increase of the volatile acidity of wine. Ethanol, higher alcohols acetaldehyde and diacetyl contents in wines after MLF were in acceptable levels.     

Immobilization of Oenococcus oeni in lentikats® to develop malolactic fermentation in wines

Entrapment of Oenococcus oeni into a polymeric matrix based on polyvinyl alcohol (PVA) (Lentikats®) was successfully used to get a better development of malolactic fermentation (MLF) in wine. The incubation of immobilized cells in a nutrient medium before starting the MLF, did not improve the degradation of malic acid. In only one day, 100% of conversion of malic acid was achieved using a high concentration of immobilized cells (0.35 g gel/ml of wine with a cell‐loading of 0.25 mg cells/mg of gel). While a low concentration of 0.21 g gel/ml of wine (cell‐loading of 0.25 mg cells/mg of gel) needed 3 days to get a reduction of 40%. The entrapped cells could be reused through six cycles (runs of 3 days), retaining 75% of efficacy for the conversion of malic acid into lactic acid. The immobilized cells in PVA hydrogels gave better performance than free cells because of the increase of the alcohol toleration. Consequently, the inhibitory effect of ethanol for developing MLF could be reduced using immobilized cells into PVA hydrogels. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013     

Determination of free sulphite in wine using a microbial sensor

Summary A microbial sensor of immobilized Thiobacillus thiooxidans S3 cells was assembled to determine free sulphite in wine. Sulphite oxidation activity of the immobilized cells was sufficiently high for use even after 3 months storage at 4° C. The sensitivity of this sensor was 116 nA·1·mg^-1 for sulphur dioxide. The relationship between the current decrease and the sulphur dioxide concentration was linear up to 17 mg·1^-1. The sampling rate achieved was 10 min per sample including washing time. This sensor method needed no pretreatment of wine samples, and wines diluted with 5 mM sulphuric acid solution could be directly introduced in the computer-aided analysis system. The pigments in red wine did not disturbed the analysis.     

Integrated continuous winemaking process involving sequential alcoholic and malolactic fermentations with immobilized cells

An integrated winemaking process – including sequential alcoholic and malolactic fermentations operated continuously – was developed. For the continuous alcoholic fermentation, yeast cells (Saccharomyces cerevisiae) were immobilized either on grape stems or on grape skins, while bacterial cells (Oenococcus oeni) used for conducting continuous malolactic fermentation were immobilized on grape skins only. The produced wines were subjected to chemical analysis by HPLC (ethanol, glycerol, sugars and organic acids) and by gas chromatography (major and minor volatile compounds). The final proposed integrated continuous process permitted the production of 960 mL/d of a dry white wine, with an alcoholic strength of about 13 vol%, by using two 1.5 L tower bed reactors packed with 260 g of grape skins. The produced wines revealed a good physicochemical quality. Moreover, 67% of the malic acid concentration could be reduced in the second reactor. Both fermentative processes proved to be much more efficient than those conducted traditionally with free cells or even with immobilized cells, but in the batch mode of operation.     

Accumulation of cadmium by immobilizedZoogloea ramigera 115

Summary Zoogloea ramigera 115 was immobilized into beads of calcium-alginate and placed into batch air-bubbled column reactors. In the absence of any added nutrients the immobilized bacterium adsorbed Cd from solutions containing levels of 2 and 20 g ml^–1 per day, over a period of 21 and 20 days, respectively. Adsorption of Cd from solutions containing 20 g ml^–1 Cd was better than 90% for 16 days. Beads treated with Cd at 2 g ml^–1 never adsorbed less than 95% of the metal. Alginate adsorbed Cd as well, but inclusion of cells changed the effectiveness of adsorption. Of a 250 g ml^–1 Cd solution, alginate adsorbed 70.4% Cd in 60 min whereas alginate plus cells adsorbed 90.5% in the same time span. Temperature had no effect on adsorption by immobilized cells at levels of 2 and 10 g ml^–1 Cd. However at higher concentrations, binding was enhanced as temperature increased.Z. ramigera beads were stable during all treatments and for prolonged periods of time (21 days).     

Production of xylanase by immobilized Trichoderma reesei SAF3 in Ca-alginate beads

In the present study, the optimum conditions for the production of xylanase by immobilized spores of Trichoderma reesei SAF3 in calcium alginate beads were determined. The operational stability of the beads during xylanase production under semi-continuous fermentation was also studied. The influence of alginate concentration (1, 2, 3, and 4%) and initial cell loading (100, 200, 300, 400, and 500 beads per flask) on xylanase production was considered. The production of xylanase was found to increase significantly with increasing concentration of alginate and reached a maximum yield of 3.12 ± 0.18 U ml⁻¹ at 2% (w/v). The immobilized cells produced xylanase consistently up to 10 cycles and reached a maximum level at the forth cycle (3.36 ± 0.2 U ml⁻¹).     

Degradation of cationic surfactants using Pseudomonas putida A ATCC 12633 immobilized in calcium alginate beads

In this study, the degradation of tetradecyltrimethylammonium bromide (TTAB) by freely suspended and alginate-entrapped cells from the bacteria Pseudomonas putida (P. putida) A ATCC 12633 was investigated in batch cultures. The optimal conditions to prepare beads for achieving a higher TTAB degradation rate were investigated by changing the concentration of sodium alginate, pH, temperature, agitation rate and initial concentration of TTAB. The results show that the optimal embedding conditions of calcium alginate beads are 4 % w/v of sodium alginate content and 2 × 10⁸ cfu ml^?1 of P. putida A ATCC 12633 cells that had been previously grown in rich medium. The optimal degradation process was carried out in pH 7.4 buffered medium at 30 °C on a rotary shaker at 100 rpm. After 48 h of incubation, the free cells degraded 26 mg l^?1 of TTAB from an initial concentration of 50 mg l^?1 TTAB. When the initial TTAB concentration was increased to 100 mg l^?1, the free cells lost their degrading activity and were no longer viable. In contrast, when the cells were immobilized on alginate, they degraded 75 % of the TTAB after 24 h of incubation from an initial concentration of 330 mg l^?1 of TTAB. The immobilized cells can be stored at 4 °C for 25 days without loss of viability and can be reused without losing degrading capacity for three cycles.     

Production of thermostable and neutral glucoamylase using immobilized <Emphasis Type="Italic">Thermomucor indicae-seudaticae</Emphasis>

Thermomucor indicae-seudaticae was immobilized in alginate, κ-carrageenan, agarose, agar, polyacrylamide and loofah (Luffa cylindrica) sponge (as such or coated with alginate/starch/Emerson YpSs agar), and used for the production of glucoamylase in submerged fermentation. The mycelium developed from alginate-immobilized sporangiospores secreted higher glucoamylase titres (22.7 U ml⁻¹) than those immobilized in other gel matrices and the freely growing mycelial pellets (18.5 U ml⁻¹). Loofah network provided a good support for mycelial growth, but the enzyme production was lower than that attained with alginate beads. Glucoamylase production increased with inoculum density and the optimum levels were achieved when 40 calcium alginate beads (∼5 × 10⁶ immobilized spores) were used to inoculate 50 ml production medium. The alginate bead inoculum displayed high storage stability at 4°C and produced comparable enzyme titres up to 120 days. The glucoamylase production by hyphae emerged from the immobilized sporangiospores was almost stable over eight batches of repeated fermentation. Scanning electron micrographs of alginate beads, after batch fermentation, revealed extensive mycelial growth inside and around the beads.     

Must deacidification with an induced flocculant yeast strain of Schizosaccharomyces pombe

The use of flocculant cells of the yeast strain Schizosaccharomyces pombe for the deacidification of grape musts in continuous culture was developed. An external loop reactor was used to induce flocculation. The flocs obtained were stable in the pH range 3.0–6.0 and in the presence of several sugars. Some inhibition was observed for high (above 6.0) and low (below 3.0) pH values. Once induced, flocculation could no longer be completely inhibited. Vinho Verde, a typical Portuguese wine, has a relatively low ethanol content and a high acid concentration. The external loop reactor loaded with the flocculant cells was used to deacidify a synthetic medium with sugar and malic acid concentrations similar to the ones found in Vinho Verde grape must. A desirable malic acid decrease with moderate glucose consumption was obtained at a dilution rate of 0.7 H^–1. Improved results were obtained when the synthetic medium was replaced by Vinho Verde grape must. Correspondence to: M. Mota     

Biodegradation of Carbofuran phenol by free and immobilized cells of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> ATCC13883T

The Klebsiella sp. strain ATCC13883T capable of degrading carbofuran phenol (2,3-dihydro-2,2-dimethylbenzofuran-7-ol) has been separated from the soil by enrichment culture technique and immobilized in various, namely polyurethane foam (PUF), polyacrylamide, alginate, agar and alginate-bentonite clay-powdered activated charcoal (PAC). The degradation rates of 20 and 30 mM carbofuran phenol by free and immobilized cells in batch and semi-continuous shaken cultures were compared. The PUF-immobilized cells achieved higher degradation rates in a shorter time than freely suspended cells and the cells immobilized in polyacrylamide, alginate and agar. The PUF- and alginate-bentonite clay-PAC-immobilized cells could be reused for more than 36 cycles, polyacrylamide-entrapped cells for 20 cycles and alginate-bentonite-PAC 28 cycles, without losing any degradation capacity and showed better tolerance to pH, temperature and concentration changes than free cells. These results showed that cells immobilized in modified alginate-bentonite-PAC immobilizers tolerated and completely degraded carbofuran phenol at initial concentrations of 20 and 30 mM and also higher. Such a bacterial strain could be used for bioremediation of environments contaminated with phenolic compounds.     

Immobilization of glucosyltransferase fromAureobasidium

Summary Glucosyltransferase fromAureobasidium, which produces panose and isomaltose from maltose, was immobilized by alginate gel or DEAE-cellulose at high efficiency (71 and 41% respectively). Alkylamine porous silica was less efficient as a support. The enzymatic profiles of immobilized enzymes were almost identical to the native one except that their stabilities to extreme pH, metal ions and inhibitors were improved. Both immobilization procedures successfully produced high amounts of panose, 125 mg ml^–1 (alginate gel) or 141 mg ml^–1 (DEAE-cellulose), from 300 mg ml^–1 of maltose.     

Mercury removal by immobilized algae in batch culture systems

The accumulation and volatilization of mercury by non-immobilized and immobilizedChlorella emersonii have been studied in batch culture systems. Reduction in the mercury concentration in the growth medium by non-immobilized cells was highly dependent on inoculum density, whilst reduction in mercury concentration by immobilized cells was rapid at all inoculum densities. Mercury accumulation by immobilized cell biomass was significantly greater than by non-immobilized cells with 10⁶ and 10⁵ cells bead^–1 or ml^–1. Volatilization of mercury by non-immobilized cell systems was greatest at higher inoculum densities, whereas more mercury was volatilized from immobilized cell systems at lower inoculum densities, and was greatest with unstocked alginate beads. Thus, in immobilized systems, mercury removal from solution is complex and involves mercury accumulation by the cells and volatilization by the matrix and cells. Further studies of mercury accumulation and volatilization by unstocked immobilization matrices revealed that agarose volatilized much less mercury than alginate or agar. The precise mechanism of mercury volatilization by alginate remains unclear, though it is thought to be a chemical effect.     

Determination of lactic acid bacteria producing biogenic amines in wine by quantitative PCR methods

Aims: To develop rapid methods allowing enumeration of lactic acid bacteria producing biogenic amines in wines and to analyse wine samples by the methods. Methods and Results: Methods based on quantitative PCR targeting bacterial genes involved in histamine, tyramine and putrescine production were developed and applied to detect and quantify the bacteria producing these biogenic amines in wine. Analysis of 102 samples revealed low populations of the targeted bacteria in grape must samples, an increased bacteria biomass in wine samples after alcoholic fermentation, reaching the highest population levels (above 10⁶ cells ml^?1) during spontaneous malolactic fermentation. A minimum of 10³ ml^?1 producing cells was required for production of more than 1 mg l^?1 of biogenic amines. Accumulation of putrescine in wine was correlated with the presence of bacteria carrying an ornithine decarboxylation pathway. Trials of winemaking showed that the use of selected bacteria for inducing malolactic fermentation was efficient to limit the proliferation of undesirable bacteria and the production of biogenic amines. Conclusion: Methods using quantitative PCR are efficient to enumerate biogenic amines‐producing cells in wine. Significance and Impact of the Study: The methods can help to better control and to improve winemaking conditions in order to avoid biogenic amine production.