High-cell-density fermentation for ergosterol production by Saccharomyces cerevisiae

The direct feedback control of glucose using an on-line ethanol concentration monitor for ergosterol production by high-cell-density fermentation was investigated and the fermentation parameters (e.g., pH, dissolved oxygen, ethanol concentration, oxygen uptake rate, carbon dioxide evolution rate and respiratory quotient) were analyzed. Controlling glucose feeding rate in accordance with ethanol concentration and adjusting pH with ammonia during the fermentation process were effective fed-batch methods for ergosterol production. The fermentation parameters well described the variation of the whole fermentation process. Cultivation in a 5 l fermentor was carried out under the following conditions: culture temperature, 30 degrees C; pH, 5.5; agitation speed, 600 rpm; fermentation time, 60 h; controlling ethanol concentration below 1% and keeping respiratory quotient (RQ) at approximately 1.0. Under these conditions, the yeast dry weight reached 120 g/l and the ergosterol yield reached 1500 mg/l.     

Bioethanol production from sugar beet molasses and thick juice by free and immobilised Saccharomyces cerevisiae

The aim of this study was the investigation and comparison of the potential of sugar beet molasses and thick juice as raw materials for bioethanol production, as renewable and sustainable energy sources. Ethanol fermentation of a wide range of initial sugar concentrations (100–300 g/L) was performed using either free or immobilised Saccharomyces cerevisiae in calcium alginate beads in the absence of any added nutrients. In general, immobilised cells showed better fermentative performance, enhanced ethanol productivity, stability and cell viability compared with free cells, under the same fermentation conditions. The high concentration of non‐sugar components contained in molasses affected yeast fermentation performance and viability. Maximum ethanol concentration in fermented media of 84.6 and 109.5 g/L were obtained by immobilised cells for initial sugar concentrations of 200 and 250 g/L for molasses and thick juice, respectively. However, the highest ethanol yields of 31.7 L per 100 kg of molasses and 37.6 L per 100 kg of thick juice were obtained by immobilised cells at an initial sugar concentration of 175 g/L. In the high gravity fermentation process, thick juice resulted in a higher ethanol yield per mass of raw material compared with molasses. This study shows the advantage of immobilised yeast for the efficient production of high gravity bioethanol from thick juice, which was a more favourable raw material than molasses. © 2018 The Institute of Brewing & Distilling     

ISOLATION AND SELECTION OF ETHANOL-RESISTANT AND OSMOTOLERANT YEASTS FROM REGIONAL AGRICULTURAL SOURCES IN MEXICO

In Mexican alcohol distilleries using sugarcane molasses, one reason for low alcoholic fermentation efficiency is the use of inferior yeast cultures. The objective of the present study was to isolate and select yeast strains from alcoholic fermentations of natural sources (sugarcane molasses, grape juice, cane juice and pineapple) from Veracruz city market and Mexican distilleries, and to evaluate their performance under laboratory conditions in an effort to select superior strains for industrial fermentations. Ethanol production, glucose composition, growth rate, "Killer" activity, ethanol and glucose tolerance of the most promising strains were monitored on synthetic and molasses media. A total of 112 yeast strains were isolated by their capacity to produce ethanol, and from these, only 58 were selected on the basis of best ethanol theoretical yields (88–96%). These strains were exposed several times to high concentrations of glucose and ethanol in order to select ethanol- and glucose-tolerant yeast; 10 were obtained that adapted best to these conditions and that showed "Killer" activity. Of these strains, U3-11, M11, JC10 and U2-10 (obtained from grape juice, sugarcane molasses and cane juice) demonstrated the highest adaptation to both ethanol (5–7% w/v) and glucose (20% w/v). The maximum yield obtained was 0.46 g/g (90% theoretical yield) in a 20-L bioreactor with cane molasses under nonsterile conditions.

PRACTICAL APPLICATIONS

The selected yeasts could be introduced into industrial processes in Mexican distilleries using sugarcane molasses in order to improve productivity and diminish contamination problems.     

以大豆糖蜜为原料高产乙醇酵母的筛选鉴定及其发酵特性研究

从大豆糖蜜中进行高产乙醇酵母的筛选和鉴定,并对其发酵特性进行研究。从大豆糖蜜中通过菌种的富集分离,TTC平板法初筛,耐乙醇能力及乙醇发酵能力的测定,筛选出一株乙醇产量达9.07%(V/V)的菌株P14。通过个体形态、菌落特征、生理生化及26S rDNA D1/D2区序列分析将菌株P14鉴定为酿酒酵母。研究了大豆糖蜜浓度及添加氮源和无机盐对酿酒酵母P14发酵生产乙醇的影响及酿酒酵母P14对大豆糖蜜中低聚糖的利用,结果表明大豆糖蜜浓度、添加氮源和无机盐对乙醇发酵影响显著,最佳的大豆糖蜜浓度为40%,添加氮源为1.2 g/L的蛋白胨;补加的无机盐为0.4 g/L MgSO4。在此培养基中发酵72 h后,糖蜜中90.10%的葡萄糖,91.23%的蔗糖,92.56%的棉籽糖和96.97%的水苏糖被酵母利用。因此大豆糖蜜中筛选出来的酿酒酵母P14具有较强的利用大豆糖蜜中的大豆低聚糖发酵产生乙醇的能力。     

Cultivation of the carotenoid-hyperproducing mutant 2A2N of the red yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) with molasses

The carotenoid-hyperproducing mutant 2A2N of the yeast Xanthophyllomyces dendrorhous (Phaffia rhodozyma) was cultivated using sugar beet blackstrap molasses. This molasses was composed of 70% (w/v) total solid and 50% (w/v) total sugar. Biomass yield (biomass/carbohydrate) significantly decreased at >5% (v/v) molasses. Atomic emission spectrometry revealed that Na and P were the limiting nutrients when molasses was used. Molasses (5%, v/v) containing urea (30 g/l molasses) and sodium phosphate (NaH2PO4, 5 g/l molasses) was formulated for biomass production by the mutant. The optimal pH for carotenoid production was 4.9 during the growth phase and 2.6-3.5 during the stationary phase. The three main sugars in molasses (sucrose, glucose, and fructose) were assimilated by the mutant but fructose was consumed slowly. When the formulated medium with pH 4.5-5.5 was used, the maximal biomass yield was 36 g/l (0.18 g of yeast l(-1)h(-1) and 40 mg of carotenoid l(-1)) in fed-batch pilot-scale 100-l cultivation.     

Structural Complexity of the Nitrogen Source and Influence on Yeast Growth and Fermentation

The structural complexity of the nitrogen source strongly affects both biomass and ethanol production by industrial strains of Saccharomyces cerevisiae, during fermentation in media containing glucose or maltose, and supplemented with a nitrogen source varying from a single ammonium salt (ammonium sulfate) to free amino acids (casamino acids) and peptides (peptone). Diauxie was observed at low glucose and maltose concentrations independent of nitrogen supplementation. At high sugar concentrations diauxie was not easily observed, and growth and ethanol production depended on the nature of the nitrogen source. This was different for baking and brewing ale and lager yeast strains. Sugar concentration had a strong effect on the shift from oxido‐fermentative to oxidative metabolism. At low sugar concentrations, biomass production was similar under both peptone and casamino acid supplementation. Under casamino acid supplementation, the time for metabolic shift increased with the glucose concentration, together with a decrease in the biomass production. This drastic effect on glucose fermentation resulted in the extinction of the second growth phase, probably due to the loss of cell viability. Ammonium salts always induced poor yeast performance. In general, supplementation with a nitrogen source in the peptide form (peptone) was more positive for yeast metabolism, inducing higher biomass and ethanol production, and preserving yeast viability, in both glucose and maltose media, for baking and brewing ale and lager yeast strains. Determination of amino acid utilization showed that most free and peptide amino acids present, in peptone and casamino acids, were utilized by the yeast, suggesting that the results described in this work were not due to a nutritional status induced by nitrogen limitation.     

高浓度甘蔗汁酒精发酵过程的研究

研究高浓度甘蔗汁酒精发酵过程中糖的消耗,酒精生成以及酵母细胞生长的变化.采用液相色谱法测定各种糖的含量、气相色谱法测定酒精的生成量.试验结果表明,发酵12h时葡萄糖消耗速率最大(9.46(g/L)/h),14h时果糖、蔗糖、总糖消耗速率最大,分别为12.61(g/L)/h,12.30 (g/L)/h,20.59 (g/L)/h;发酵16h时酒精生成速率(7.89(g/L)/h)最大,细胞死亡率(22.9％)开始大于出芽率(12.6％),随后细胞活力逐渐下降.试验最终发酵酒精度12.8％vol,残糖22.50g/L,糖利用率90.9％,发酵效率79.69％.     

Significance of Copper in Alcohol Production with Fermentation of Raisin Extracts by the Cell Recycle Process

The ethanol fermentation rate, cell growth and yield were reduced from one batch to the next in presence of 50 ppm copper as compared to 5 ppm. The decreases of these parameters are attributed to the increased uptake of copper causing lessening of the glucose uptake by yeast cells. These changes were studied through the uptake of ¹⁴C-glucose in fermentations of synthetic media containing ¹⁴C-glucose, in presence of various copper concentrations.     

High-temperature ethanol fermentation by immobilized coculture of Kluyveromyces marxianus and Saccharomyces cerevisiae

Suspended and immobilized cocultures of the thermotolerant yeast, Kluyveromyces marxianus DMKU 3-1042 and the mesophilic flocculent yeast, Saccharomyces cerevisiae M30 were studied for their abilities to improve production and stability of ethanol fermentation. Sugarcane juice and blackstrap molasses, at initial sugar concentrations of 220 g/L, were used as carbon sources. The results indicated that the coculture system could improve ethanol production from both sugarcane juice and blackstrap molasses when the operating temperature ranged between 33 °C and 45 °C. High temperature tolerances were achieved when the coculture was immobilized. The immobilized coculture was more effective in high-temperature ethanol fermentation than the suspended cultures. The coculture immobilized on thin-shell silk cocoon and fermented at 37 °C and 40 °C generated maximal ethanol concentrations of 81.4 and 77.3 g/L, respectively, which were 5.9-8.7% and 16.8-39.0% higher than those of the suspended cultures, respectively.     

不同补料方式对酿酒酵母高密度发酵的影响

以酿酒酵母为发酵菌种,使用自主优化筛选配制的糖蜜培养基,分别采用残糖反馈分批补料和连续流加补料方式,优化发酵工艺条件以提高发酵液中菌体密度。残糖反馈分批补料,拟设定3个梯度,分别控制对数期发酵液中残糖含量在0.5%～1.5%,1.0%～2.0%,1.5%～2.5%。三次试验结果分别为,试验1菌体培养32 h时达到最大生物量,为28.88 g/L;试验2菌体培养52 h时达到最大生物量,为27.43 g/L;试验3菌体培养24 h时达到最大生物量,为22.06 g/L。通过连续流加补料培养菌体,结果培养30 h时达到最大生物量,为29.29 g/L。     

红酵母发酵废糖蜜产类胡萝卜素的研究

对红酵母利用糖蜜发酵产类胡萝卜素进行了研究,结果表明:对糖蜜的预处理的最佳酸为硫酸;利用经过酸处理后的糖蜜发酵,菌株的生物量为30.75g/L,单细胞色素含量为104μg/g,色素产量为26mg/L;最佳发酵时间为168h。使用糖蜜代替葡萄糖做廉价碳源对红酵母进行发酵,确定添加量为170g/L,色素产量最大值为葡萄糖发酵的0.97倍,生物量为1.04倍。      

豆腐黄浆水中补加乙醇和葡萄糖发酵虾青素的研究

在豆腐黄浆水中补加乙醇和葡萄糖发酵虾青素进行了研究,结果表明,黄浆水中添加6g/L乙醇,可提高生物量、虾青素含量以及虾青素产量,但发酵周期有所延长;黄浆水中添加10g/L葡萄糖,有利于酵母菌的生长和提高虾青素产量。     

Identification of genes whose expressions are enhanced or reduced in baker's yeast during fed-batch culture process using molasses medium by DNA microarray analysis

Genes whose expression levels are enhanced or reduced during the cultivation process that uses cane molasses in baker's yeast production were identified in this study. The results showed that baker's yeast grown in molasses medium had higher fermentation ability and stress tolerance compared with baker's yeast grown in synthetic medium. Molasses apparently provided not only sugar as a carbon source but also provided functional components that enhanced or reduced expression of genes involved in fermentation ability and stress tolerance. To identify the genes whose expression is enhanced or reduced during cultivation in molasses medium, DNA microarray analysis was then used to compare the gene expression profile of cells grown in molasses with that of cells grown in synthetic medium. To simulate the commercial baker's yeast production process, cells were cultivated using a fed-batch culture system. In molasses medium, genes involved in the synthesis or uptake of vitamins (e.g., biotin, pyridoxine and thiamine) showed enhanced expression, suggesting that vitamin concentrations in molasses medium were lower than those in synthetic medium. Genes involved in formate dehydrogenase and maltose assimilation showed enhanced expression in molasses medium. In contrast, genes involved in iron utilization (e.g., siderophore, iron transporter and ferroxidase) showed enhanced expression in synthetic medium, suggesting that iron starvation occurred. The genes involved in the metabolism of amino acids also showed enhanced expression in synthetic medium. This identification of genes provides information that will help improve the baker's yeast production process.     

高产酸扣囊复膜酵母的筛选与培养基配方优化

该研究对10种不同的米酒曲和黄酒曲中的高产酸酵母菌进行了分离、筛选及鉴定,并以酵母菌的生物量为评价指标,采用单因素试验和响应面法,对菌株的液态发酵培养基进行优化。结果表明,经过分子生物学鉴定,共分离获得6株扣囊复膜酵母（Saccharomycopsis fibuligera）。通过菌株产酸能力和耐受性的比较,筛选到一株产酸率高、耐高温和乙醇能力强的扣囊复膜酵母菌株3-1,其总酸（以乳酸计）产量达5.4 g/L。最佳培养基配方为：糖蜜7.5 g/L,葡萄糖7.7 g/L,大豆蛋白胨1.7 g/L,酵母浸粉1.7 g/L。在此优化条件下,菌株3-1的生物量达2.45×108个/mL,总菌数比对照培养基提高了63.3%。     

Continuous baker's yeast production using orange peel as promoting support in the bioreactor

Improvements in maximum growth rate and productivity in biomass production using orange peel as promoter were observed in batch aerobic fermentation. Daily biomass productivity and biomass yield up to 14.9 g (dry weight) L^?1 and 0.22 g (dry weight) g^?1 utilized sugar respectively in batch aerobic fermentation by the presence of orange peel as promoter were reported. A novel bioreactor for continuous aerobic fermentation of molasses is described and its suitability for baker's yeast production using orange peel as promoter is investigated. The continuous bioreactor was operated for 12 days, stored for 20 days at 10 °C and operated again for another 13 days without any diminution of biomass productivity. Daily biomass productivity, yield and conversion up to 11.2 g (dry weight) L^?1, 0.16 g (dry weight) produced g^?1 utilized sugar and 90.4% respectively were recorded. The possibility of using such a system for industrial continuous baker's yeast production is discussed. Copyright © 2005 Society of Chemical Industry     

金属离子对低糖酵母发酵活性的影响

该研究考察了3种金属离子对低糖酵母发酵活力的影响。首先,采用响应面设计法对酵母发酵培养基进行了优化。通过Plackett-Burman设计试验筛选出3个主要因素：镁离子（Mg2+）、锌离子（Zn2+）、锰离子（Mn2+）。在这个基础上应用最陡爬坡路径法逼近最大响应值区域,然后利用响应面分析法确定最佳培养基配方为酵母抽提物（FM888） 10 g/L、蛋白胨（FM318） 20 g/L、葡萄糖20 g/L、六水合氯化镁 6.95 g/L、氯化锌1.78 mg/L、一水合硫酸锰0.069 mg/L。其次,将优化培养基配方应用于低糖酵母发酵,干酵母活力可达426.86 mL/g。经过3次平行试验的验证,实际的平均发酵活力与预测的发酵活力值相近,比优化前提高了24.8%。此研究对低糖酵母的工业化生产具有一定的指导意义。     

Comparison of Industrial Scale Ethanol Production from a Palmyrah‐Based Carbon Source by Commercial Yeast and a Mixed Culture from Palmyrah Toddy

Palmyrah (Borassus flabellifer) based products were used as an alternative carbon source for industrial scale ethanol production. The fermentation medium was enriched with spent wash obtained from a distillation column. The performance of a commercially available baker's yeast in the media was compared with a ‘palmyrah toddy mixed culture’ where the organisms were obtained from the sedimentation of palmyrah toddy. In a laboratory scale study, the ethanol produced from a palmyrah fruit pulp extract, diluted with distilled water, was 16.5 gL^?1 (36 h) and 13.0 gL^?1 (48 h) with ‘palmyrah toddy mixed culture’ and baker's yeast respectively. The ‘palmyrah toddy mixed culture’ performed better than the baker's yeast with palmyrah fruit pulp extract, diluted either with distilled water or spent wash. Among the different palmyrah based carbon sources, both cultures preferred molasses diluted with spent wash and both performed best in the medium containing the spent wash supplemented with sucrose. In a 5,000 L industrial scale fermentation of 20° Brix molasses supplemented with 10 gL^?1 ammonium sulphate, 72 gL^?1 and 65 gL^?1 ethanol was produced by the ‘palmyrah toddy mixed culture’ (72 h) and the baker's yeast (90 h) respectively. As the performance of the ‘palmyrah toddy mixed culture’ was better than that of the baker's yeast, the former was selected for the industrial scale studies of molasses fermentation media diluted with spent wash. In these studies the temperature reached 42°C by 36 h and resultant cell death was observed. However ethanol production was higher and more rapid in the molasses diluted with spent wash, rather than in the molasses diluted with tap water and supplemented with (NH₄)₂SO₄. Cell recycle operation obviated the interruption in fermentation caused by temperature induced cell death and increased rates and efficiency of ethanol production were observed.     

基于面包酵母中海藻糖提取工艺优化及菌株筛选的高密度培养工艺

确定使用酶-3,5-二硝基水杨酸（3,5-dinitrosalicylic acid,DNS）法作为酵母胞内海藻糖定量测定方法,通过微波-浸提法对活性干酵母胞内海藻糖进行提取,采用单因素试验以酵母中海藻糖含量为参考指标优化该工艺中的微波功率、微波时间、三氯乙酸浓度、三氯乙酸体积、浸提温度、浸提时间,在此基础上对微波功率、微波时间、三氯乙酸浓度、三氯乙酸体积、浸提温度5?个因素进行正交试验,优化后工艺条件为：针对1.5?g活性干酵母,微波功率231?W、微波时间40?s、三氯乙酸浓度0.7?mol/L、三氯乙酸体积40?mL、浸提温度55?℃、浸提时间150?min。使用该工艺得到的活性干酵母中的海藻糖含量为280.15?mg/g,相对于优化前提高了15.2%。同时,利用该优化后工艺在现有面包酵母菌种中筛选出了一株高产海藻糖的菌株。在50?L自吸式发酵罐中将该菌株进行流加发酵,采用糖蜜进行溶氧反馈流加时最大酵母湿质量浓度为198.34?g/L,将流加工艺进行改进,提出称质量补料的流加方法将糖蜜、尿素、磷酸二氢铵作为流加营养源,实验发现发酵后最大酵母湿质量浓度可提高到264.82?g/L,相对于原工艺提高了33.52%,达到了面包酵母高密度培养的要求。发酵过程中将乙醇体积分数控制在0.7%～1.0%之间,将有效提高酵母湿质量浓度且保证其发酵力在650?mL/h以上,且工艺稳定性良好。     

CARBON SOURCES AND THEIR EFFECT ON GROWTH, ACETIC ACID AND ETHANOL PRODUCTION BY BRETTANOMYCES BRUXELLENSIS IN BATCH CULTURE

The influence of available low‐cost carbohydrates as carbon sources on Brettanomyces bruxellensis growth, acetic acid and ethanol production was studied in order to ascertain the viability of this yeast to eventually become an industrial acetic acid producer. Six different raw materials were included as carbon sources (glucose, sugarcane molasses, refined cane sugar, pineapple, sugarcane and beet juices). B. bruxellensis develops in a complex culture medium like plant juices and sugarcane molasses better than in a medium with a simple carbohydrate such as glucose. The maximum acid acetic yield (0.24 g/g) and productivity (0.14 g/L/h) were attained in tests carried out with sugarcane molasses containing 60 g/L sucrose. The strain produced low levels of ethanol in a refined sugarcane medium, but was able to produce a substantial quantity of acetic acid (13 g/L).     

Chitosan from Syncephalastrum racemosum Using Sugar Cane Substrates as Inexpensive Carbon Sources

A strain of Syncephalastrum racemosum isolated from herbivores feces was studied for chitosan production using sugar cane substrates, such as juice or molasses, from Northeast Brazil. S. racemosus was batch grown in shake flasks over 5 days using sugar cane juice or molasses as carbon source. The effect of supplementary nitrogen source was studied by supplying medium with yeast extract or ammonium sulphate. The highest yield of the biopolymer (30 mg chitosan. g^-1 sucrose) was obtained using sugar cane juice (21 g sucrose. L^-1) plus 0.3% yeast extract. Batch fermentation in a 5-L bioreactor using the same medium, increased the yield to 50 mg chitosan. g^-1 sucrose. Chitosan extracted from S. racemosus cultured in sugar cane juice medium showed a low degree of acetylation with a high D-glucosamine content.