Continuous fermentation of soy sauce by immobilized cells of Zygosaccharomyces rouxii in an airlift reactor

Summary The optimum conditions for continuous alcohol fermentation of soy sauce with immobilized Zygosaccharomyces rouxii cells were investigated using an airlift reactor. The optimum pH and temperature of the fermentation were 4.5–5.5 and 25°–27.5° C, respectively. Ethanol content in the fermented liquid was increased with increasing height to diameter ratio of the reactor and the ratio of air to nitrogen in the supplied gas (total supplied gas: 0.08 vvm). A notable decrease in ethanol content was observed when only nitrogen gas was supplied. The products fermented by supplying air (0.02 vvm) had a higher conent of aroma components than that by supplying only nitrogen gas, and the aroma of the former products was similar to that of conventional soy sauce. This alcohol fermentation using an airlift reactor was continued for about 50 days without problems even if conditions such as residence time and aeration were altered.     

Xylulose fermentation by mutant and wild-type strains of Zygosaccharomyces and Saccharomyces cerevisiae

Anaerobic xylulose fermentation was compared in strains of Zygosaccharomyces and Saccharomyces cerevisiae, mutants and wild-type strains to identify host-strain background and genetic modifications beneficial to xylose fermentation. Overexpression of the gene (XKS1) for the pentose phosphate pathway (PPP) enzyme xylulokinase (XK) increased the ethanol yield by almost 85% and resulted in ethanol yields [0.61 C-mmol (C-mmol consumed xylulose)⁻¹] that were close to the theoretical yield [0.67 C-mmol (C-mmol consumed xylulose)⁻¹]. Likewise, deletion of gluconate 6-phosphate dehydrogenase (gnd1Δ) in the PPP and deletion of trehalose 6-phosphate synthase (tps1Δ) together with trehalose 6-phosphate phosphatase (tps2Δ) increased the ethanol yield by 30% and 20%, respectively. Strains deleted in the promoter of the phosphoglucose isomerase gene (PGI1) – resulting in reduced enzyme activities – increased the ethanol yield by 15%. Deletion of ribulose 5-phosphate (rpe1Δ) in the PPP abolished ethanol formation completely. Among non-transformed and parental strains S. cerevisiae ENY. WA-1A exhibited the highest ethanol yield, 0.47 C-mmol (C-mmol consumed xylulose)⁻¹. Other non-transformed strains produced mainly arabinitol or xylitol from xylulose under anaerobic conditions. Contrary to previous reports S. cerevisiae T23D and CBS 8066 were not isogenic with respect to pentose metabolism. Whereas, CBS 8066 has been reported to have a high ethanol yield on xylulose, 0.46 C-mmol (C-mmol consumed xylulose)⁻¹ (Yu et al. 1995), T23D only formed ethanol with a yield of 0.24 C-mmol (C-mmol consumed xylulose)⁻¹. Strains producing arabinitol did not produce xylitol and vice versa. However, overexpression of XKS1 shifted polyol formation from xylitol to arabinitol. Received: 2 July 1999 / Accepted in revised form: 12 October 1999     

Enhanced anthocyanin production by repeated-batch culture of strawberry cells with medium shift

Repeated-batch cultures of strawberry cells (Fragaria ananassa cv. Shikinari) subjected to four medium-shift procedures (constant LS medium, constant B5 medium, alternation between LS and B5 starting from LS and alternation between LS and B5 starting from B5) were investigated for the enhanced anthocyanin productivity. To determine the optimum period for repeated batch cultures, two medium-shift periods of 9 and 14 days were studied, which represent the end of the exponential growth phase and the stationary phase. By comparison with the corresponding batch cultures, higher anthocyanin productivity was achieved for all the repeated-batch cultures at a 9-day medium-shift period. The average anthocyanin productivity was enhanced 1.7- and 1.76-fold by repeated-batch cultures in constant LS and constant B5 medium at a 9-day shift period for 45 days, respectively. No further improvement was observed when the medium was alternated between LS (the growth medium) and B5 (the production medium). Anthocyanin production was unstable at a 14-day shift period regardless of the medium-shift procedures. The results show that it is feasible to improve anthocyanin production by a repeated-batch culture of strawberry cells.     

Improved production of viable cells of the salt-tolerant yeast Zygosaccharomyces rouxii by continuous culture

Summary A continuous culture system of the salt-tolerant yeast Zygosaccharomyces rouxii (soy yeast) was investigated in order to obtain high production efficiency of viable cells. The optimum pH and C/N ratio of the feed medium for cell production were about 5.0 and 16–20, respectively. About a fivefold increase in viable cell number and cell productivity (viable cell number per litre per hour) were obtained in glucose-limited culture at a dilution rate (D) of 0.06 h^–1 as compared with batch culture. However, the fermentative activity of the cells from glucose-limited culture was significantly lower than those from batch and dissolved-oxygen (DO)-limited cultures, and the former cells showed lower specific activity of glycolytic enzymes. On the other hand, at the boundary conditions between glucose and DO limitation almost the same cell productivity and higher fermentative activity of the cell were obtained as compared with glucose-limited conditions. The cultivation continued for about 60 days without any problems even if the D was altered. It was found that the continuous cultivation method was suitable for industrial production of viable cells of soy yeasts. Offprint requests to: T. Hamada     

Efficient transformation of the osmotolerant yeast Zygosaccharomyces rouxii by electroporation

A rapid and simple electroporation method to transform osmotolerant yeast Zygosaccharomyces rouxii has been developed and conditions for efficient transformation of mutants derived from different Z. rouxii wild-type strains optimized.     

Gamma-linolenic acid production of Mucor rouxii by solid-state fermentation using agricultural by-products

Aims:  This study aims to maximize the yield of gamma-linolenic acid by a filamentous fungus, Mucor rouxii , using low cost production by solid-state fermentation.
Methods and Results:  We optimized substrate types and culture conditions including inoculum size and temperature. The optimal growth of M. rouxii was found in the cultures inoculated with 5 × 10⁵ spores g⁻¹ substrate. The fungal cells grew well on rice bran and soy bean meal, whereas a lower biomass was found in the cultures grown on polished rice, broken rice and spent malt grain. The GLA content was highly accumulated in rice bran ferment and its maximal content of about 6 g kg⁻¹ fermented mass was observed in the 5th-day culture grown at 30°C. However, the GLA content in the rice bran ferment was not enhanced by low temperature.
Conclusions:  The GLA production of M. rouxii could be enhanced by optimizing the agricultural by-product substrates and culture condition.
Significance and Impact of the Study:  Low cost GLA production process was achieved, and fermented product containing GLA can be incorporated into feed additives without further oil extraction to alternate the expensive plant oils.     

Water relations of polyol accumulation by Zygosaccharomyces rouxii in continuous culture

Summary Glycerol and arabitol were the main polyols accumulated by Zygosaccharomyces rouxii in continuous culture but the intracellular and extracellular concentrations of the polyols varied with the dilution rate and osmoticum used to adjust the water activity (a_w) to 0.960. When the a_w was adjusted with NaCl, glycerol was the main polyol accumulated intracellularly whereas glycerol and arabitol were accumulated when polyethylene glycol (PEG) 400 was used. The extracellular glycerol and arabitol concentrations at 0.960 a_w (NaCl or PEG 400) were similar or decreased relative to cultures at 0.998 a_w. Compared to steady-state cultivation at 0.998 a_w, the yeast retained at 0.960 a_w (NaCl or PEG 400) a greater proportion of the total glycerol intracellularly against an increased concentration ratio without significantly greater production of glycerol. Arabitol was only significant in osmoregulation when cultivated at 0.960 a_w (PEG 400). The intracellular glycerol concentration was insufficient to balance the a_w across the membrane, but an equilibrium could be achieved under certain conditions if arabitol was also osmotically active. Offprint requests to: P. J. van Zyl     

Genomic exploration of the hemiascomycetous yeasts: 8. Zygosaccharomyces rouxii

This paper reports the genomic analysis of strain CBS732 of Zygosaccharomyces rouxii, a homothallic diploid yeast. We explored the sequences of 4934 random sequencing tags of about 1 kb in size and compared them to the Saccharomyces cerevisiae gene products. Approximately 2250 nuclear genes, 57 tRNAs, the rDNA locus, the endogenous pSR1 plasmid and 15 mitochondrial genes were identified. According to 18S and 25S rRNA cladograms and to synteny analysis, Z. rouxii could be placed among the S. cerevisiae sensu lato yeasts.     

Synthesis and Secretion of Acid Phosphatase in Halotolerant Zygosaccharomyces rouxii

Changes in the contents of defensive substances against the active oxygen in water-stressed spinach plants were examined. The contents of ascorbate peroxidase (AP; EC 1.11.1.7), glutathione reductase (GR; EC 1.6.4.2) and α-tocopherol increased remarkably in water-stressed spinach leaves, while those of Superoxide dismutase (SOD; EC 1.15.1.1), dehydroascorbate reductase (EC 1.8.5.1), ascorbate and glutathione changed little. The content of α-tocopherol in chloroplast thylakoid membranes isolated from water-stressed leaves was higher than that from normal leaves. It is, therefore, conceivable that GR, AP and α-tocopherol might be related to the tolerance of plants to water deficiency.     

Breeding of flocculent industrial alcohol yeast strains by self-cloning of the flocculation gene FLO1 and repeated-batch fermentation by transformants

A nonflocculent industrial polyploid yeast strain, Saccharomyces cerevisiae 396-9-6V, was converted to a flocculent one by introducing a functional FLO1 gene at the URA3 locus. The flocculent strain FSC27 obtained was a so-called self-cloned strain, having no bacterial DNA. FSC27 cells could be easily recovered for reuse from fermentation mash without any physical energy. The strain produced a concentration of alcohol as high as 396-9-6V, although the fermentation rate of FSC27 was slightly lower than that of 396-9-6V. When uracil was added to the medium or when URA3 was reintroduced into FSC27 (named FSCU-L18), the fermentation rate and the growth rate increased, and the ethanol concentration produced was higher than that produced by the parent strain. The stable flocculation and high ethanol productivity were observed by using FSCU-L18 during 10 cycles of repeated-batch fermentation test.     

Ethanol production from sweet sorghum juice in repeated-batch fermentation by Saccharomyces cerevisiae immobilized on corncob

Ethanol fermentation from sweet sorghum juice containing 240 g/l of total sugar by Saccharomyces cerevisiae TISTR 5048 and S. cerevisiae NP 01 immobilized on low-cost support materials, corncob pieces, was investigated. In batch fermentation, S. cerevisiae TISTR 5048 immobilized on 6 × 6 × 6 mm³ corncobs gave higher ethanol production than those immobilized on 12 × 12 × 12 mm³ corncobs in terms of ethanol concentration (P), yield (Y _p/s ) and productivity (Q _p ) with the values of 102.39 ± 1.11 g/l, 0.48 ± 0.01 and 2.13 ± 0.02 g/l h, respectively. In repeated-batch fermentation, the yeasts immobilized on the 6 × 6 × 6 mm³ corncobs could be used at least eight successive cycles with the average P, Y _p/s and Q _p of 97.19 ± 5.02 g/l, 0.48 ± 0.02 and 2.02 ± 0.11 g/l h, respectively. Under the same immobilization and repeated-batch fermentation conditions, P (90.75 ± 3.05 g/l) and Q _p (1.89 ± 0.06 g/l h) obtained from S. cerevisiae NP 01 were significantly lower than those from S. cerevisiae TISTR 5048 (P < 0.05), while Y _p/s from both strains were not different. S. cerevisiae TISTR 5048 immobilized on the corncobs also gave significantly higher P, Y _p/s and Q _p than those immobilized on calcium alginate beads (P < 0.05).     

Depression of synthesis of linoleyl residue in Zygosaccharomyces rouxii cells by sodium chloride

The syntheses of fatty acyl residues in a salt-tolerant yeast, Zygosaccharomyces rouxii, were compared under 4 growth conditions as follows, transfer from; (A) NaCl-free medium to the same medium, (B) NaCl-free medium to 2 M NaCl medium, (C) 2 M NaCl medium to the same medium, and (D) 2 M NaCl medium to NaCl-free medium. The synthesis of linoleyl residue 18:2) was depressed by the presence of 2 M NaCl while that of oleyl residue (18:1) was not. Under condition D, however, the synthesis of 18:2 recovered in the NaCl-free medium. These phenomena were confirmed through pulse-labeling and chase experiments. It is suggested that desaturation from 18:1 to 18:2 may be depressed by the presence of 2 M NaCl.     

Enantioselective reduction of 3,4-methylene-dioxyphenylacetone using Candida famata and Zygosaccharomyces rouxii

In an effort to prepare 3,4-methylene-dioxyphenyl-(S)-isopropanol from 3,4-methylene-dioxyphenylacetone, an initial screen of microbes indicated that Candida famata could catalyze this reaction efficiently at low substrate concentration. A dilute, large-scale process was developed to provide experimental material for the chemical synthesis to be explored. However, the productivity number of this process [0.134 g product (g␣wet␣weight cells)⁻¹ day⁻¹ was too low to be practical. C.␣famata was also extremely sensitive to concentrations of both the ketone and the alcohol greater than 2 g/l. A more extensive screen of yeast and fungi revealed that Zygosaccharomyces rouxii was more tolerant to higher substrate concentrations and had a higher productivity number [0.8 g (g wet weight cells)⁻¹ day⁻¹]. These characteristics suggested that Z. rouxii could be used in a large-scale process at high substrate concentrations. Received: 8 July 1996 / Received revision: 9 September 1996 / Accepted: 18 September 1996     

酱香型白酒酿造主要功能菌株对拜耳接合酵母的作用

【背景】拜耳接合酵母(Zygosaccharomyces bailii)是酱香型白酒自然酿造过程中的优势菌株,但白酒酿造功能菌株对其酿造特征的影响尚不清晰。【目的】分析酱香型白酒酿造体系中3株主要功能菌株对Z.bailii的作用,揭示其在白酒酿造过程中的发酵特征。【方法】分别构建拜耳接合酵母与酿酒酵母、布氏乳酸杆菌和地衣芽孢杆菌的共培养体系,比较生物量、p H、乙醇及风味物质代谢差异;基于表型差异,从转录组学角度进一步分析拜耳接合酵母与地衣芽孢杆菌共培养的代谢机制。【结果】在共培养体系中,拜耳接合酵母的生长及乙醇代谢受到酿酒酵母的抑制,而不受布氏乳酸杆菌和地衣芽孢杆菌的影响。同时,拜耳接合酵母与酿酒酵母、布氏乳酸杆菌共培养时的风味物质产量下降;但与地衣芽孢杆菌共培养时,其风味代谢却显著提高,其中醇类、酸类、酯类和醛类物质含量较其纯培养时分别上升了41%、36%、44%和73%。转录组数据分析表明,与地衣芽孢杆菌共培养时,拜耳接合酵母中与碳水化合物代谢和氨基酸代谢相关的基因表达显著上调(≥2-fold,P0.05),而碳水化合物和氨基酸是风味物质的主要来源,其相关基因的表达上调有助于拜耳接合酵母的风味代谢。【结论】共培养体系中,地衣芽孢杆菌促进了拜耳接合酵母风味代谢,使之形成更多的醇类、酸类、酯类及醛类物质。研究拜耳接合酵母与主要酿造微生物共培养时的发酵特征,有助于正确认识其在酱香型白酒发酵过程中的功能和应用。     

Isolation and characterization of salt-sensitive mutants of a salt-tolerant yeast Zygosaccharomyces rouxii

Abstract Twenty salt-sensitive (ss) mutants were isolated from the salt-tolerant yeast Zygosaccharomyces rouxii by treatment with N -methyl- N '-nitro- N -nitrosoguanidine. The mutants were divided into five classes on the basis of their ability to grow in media containing various high concentrations of NaCl. The mutant with the greatest sensitivity to NaCl of all the mutants tested was able to grow very slowly with a longer lag phase in medium containing 2 M NaCl, in contrast to the wild strain which had the capacity to grow in medium containing 3.5 M NaCl. Most of the ss mutants exhibited, to some extent, less tolerance to high concentrations of glucose than the wild strain. It appeared from the characterization of the ss mutants that the following factors are necessary for growth of Z. rouxii in high concentrations of NaCl: (a) the ability to produce glycerol under these conditions; (b) the ability to maintain a defined concentration of glycerol within the cells; (c) the ability to take up glycerol that has leaked into the medium, and to assimilate glycerol; and (d) unknown factor(s).     

Visualization of site-specific recombination catalyzed by a recombinase from Zygosaccharomyces rouxii in Arabidopsis thaliana

Excision of a DNA segment can occur in Arabidopsis thaliana by reciprocal recombination between two specific recombination sites (RSs) when the recombinase gene (R) from Zygosaccharomyces rouxii is expressed in the plant. To monitor recombination events, we generated several lines of transgenic Arabidopsis plants that carried a cryptic β-glucuronidase (GUS) reporter gene which was designed in such a way that expression of the reporter gene could be induced by R gene-mediated recombination. We also made several transgenic lines with an R gene linked to the 35S promoter of cauliflower mosaic virus. Each transgenic line carrying the cryptic reporter gene was crossed with each line carrying the R gene. Activity of GUS in F₁ and F₂ progeny was examined histochemically and recombination between two RSs was analyzed by Southern blotting and the polymerase chain reaction. In seedlings and plantlets of F₁ progeny and most of the F₂ progeny, a variety of patterns of activity of GUS, including sectorial chimerism in leaves, was observed. A small percentage of F₂ individuals exhibited GUS activity in the entire plant. This pattern of expression was ascribed to germinal recombination in the F₁ generation on the basis of an analysis of DNA structure by Southern blotting. These results indicate that R gene-mediated recombination can be induced in both somatic and germ cells of A. thaliana by cross-pollination of parental transgenic lines.     

Enhanced lovastatin production by solid state fermentation ofMonascus ruber

The purpose of this study was to optimize the solid state cultivation ofMonascus ruber on sterile rice. A single-level-multiple-factor and a single-factor-multiple-level experimental design were employed to determine the optimal medium constituents and to optimize carbon and nitrogen source concentrations for lovastatin production. Simultaneous quantitative analyses of the β-hydroxyacid form and β-hydroxylactone for of lovastatin were performed by the high performance liquid chromatography (HPLC) method with a UV photodiode-array (PDA) detector. The total lovastatin yield (4≈6 mg/g, average of five repeats) was achieved by adding soybean powder, glycerol, sodium nitrate, and acetic acid at optimal composition of the medium increased by almost 2 times the yield observed prior to optimization. The experimental results also indicated that the β-hydroxylactone form of lovastatin (LFL) and the β-hydroxyacid form of lovastatin (AFL) simultaneously existed in solid state cultures ofMonascus ruber, while the latter was the dominant form in the middle-late stage of continued fermentation. These results indicate that optimized culture conditions can be used for industrial production of lovastatin to obtain high yields.     

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of α-isopropylmalate synthase

Abstract Mutants of the 'miso' yeast, Zygosaccharomyces rouxii , that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro-dl-leucine-resistant mutants, an analogue of l-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of α-isopropylmalate synthase, one of the enzymes used for l-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of l-leucine, a feedback inhibitor.     

Optimization of L-(+)-lactic acid production by ring and disc plastic composite supports through repeated-batch biofilm fermentation.

Four customized bioreactors, three with plastic composite supports (PCS) and one with suspended cells (control), were operated as repeated-batch fermentors for 66 days at pH 5 and 37 degrees C. The working volume of each customized reactor was 600 ml, and each reactor's medium was changed every 2 to 5 days for 17 batches. The performance of PCS bioreactors in long-term biofilm repeated-batch fermentation was compared with that of suspended-cell bioreactors in this research. PCS could stimulate biofilm formation, supply nutrients to attached and free suspended cells, and reduce medium channelling for lactic acid production. Compared with conventional repeated-batch fermentation, PCS bioreactors shortened the lag time by threefold (control, 11 h; PCS, 3.5 h) and sixfold (control, 9 h; PCS, 1.5 h) at yeast extract concentrations of 0.4 and 0.8% (wt/vol), respectively. They also increased the lactic acid productivity of Lactobacillus casei subsp. rhamnosus (ATCC 11443) by 40 to 70% and shortened the total fermentation time by 28 to 61% at all yeast extract concentrations. The fastest productivity of the PCS bioreactors (4.26 g/liter/h) was at a starting glucose concentration of 10% (wt/vol), whereas that of the control (2.78 g/liter/h) was at 8% (wt/vol). PCS biofilm lactic acid fermentation can drastically improve the fermentation rate with reduced complex-nutrient addition.