高密度发酵.ppt

高密度发酵,定义：即高密度发酵技术,一般指在液体培养中的细胞密度超过常规培养10 倍以上的生长状态或培养技术,达到提高菌体发酵密度的目的。用以描述的单位是干细胞重量升(DCWL)。,优点,(1)提高发酵罐内的菌体密度，提高产物的细胞水平量,(2)减少了生物反应器的体积，提高单位体积设备的生产能力,(3)降低生物量的分离费用，缩短生产周期,(4)降低生产成本，提高生产效率,高密度培养技术最早用于酵母细胞的培养提高生物量或生产单细胞蛋白及乙醇的生产。,（1）固态或挥发性底物在液态培养基中溶解限制,（2）底物对细胞生长可能存在限制或抑制作用,（3）底物和产物的稳定性差或易挥发,（4）产物或代谢副产物的积累对细胞生长产生限制,（5）呼吸作用导致C02和热量的急剧积聚,（6）氧气需求量大,高密度发酵主要限制因素,温度的控制：目前主要的控温策略是手动调节冷却水的流量,发酵中重要参数控制,pH的控制：,初始培养基开始,内源性调节：过程中通过补加C、N源,外源性调节：流加酸（H3PO4）碱（氨水）调节。,溶解氧,物理法：增加空气流量，提高搅拌转速；增大罐压；通入纯氧,化学法：加入H2O2,生物法：在菌体中克隆具有提高养传递能力的透明颤藻蛋白,毕赤酵母表面展示磷脂酶D高密度发酵优化刘逸寒,甲醇主要作为碳源和诱导外源蛋白表达的诱导剂,BMMY培养基诱导培养：于4，8000 rmin离心菌体富集培养液，无菌水洗涤菌体，转接入50 mL250 mL的BMMY培养基中，于30，pH 6.0，220 rmin振荡诱导培养144 h，期间每24小时加入终浓度为05的甲醇。甲醇浓度为1.5时，PLD活力最高达到12.4x10-7katg，菌体量到达13.6gL。,当pH为6.5，对PLD在细胞表面的展示表达最为有利，PLD活力达到13.4xl0-7katg，菌体量到达14.8 gL。,菌体生长24h后以不同速度流加对菌体的影响，得出15mL/(hL)最佳,据DO值调节甲醇流加速率的甲醇恒速流加法 效果较好，诱导132h后，PLD活力达到24.010-7kat/g，此时菌体量为59.1g/LDO水平表明培养过程中菌体的代谢水平，DO的变化能反应出碳源浓度的高低,以摇瓶优化结果作为参考，选取初始pH 65，发酵温度28，进行5 L规模发酵罐试验，15 mL(Lh)速率流加甘油补料培养基6 h后，采用溶氧恒定流加法 流加甲醇诱导132h后，菌体量及PLD活力分别达到最高为67.4g/L及27.310-7kat/g，与未优化条件相比（初始pH6.0，发酵温度30），菌体量提高了12.9%，PLD活力提高了14.6%。,The role of oxygen in yeast metabolismduring high cell density brewery fermentations,Appl Microbiol Biotechnol,The study was carried out with an industrial lager brewing strain of Saccharomyces cerevisiae(酿酒酵母）Sterile all-malt hopped wort with an extract content of 15P and with a free amino nitrogen(FAN)content of 291 ppm was made in a pilot brewery.After centrifugation(3,000 rpm,3 min),the wortwas decantated(倾柱洗涤）.This wort had a reduced fatty acid content(0.1 ppm C16:0,2.6 pp m C16:1,0.6 ppm C18:0,2.9 ppm C18:1,1.6 ppm C18:2,and 0.2 ppm C18:3)and was used throughout the study,Yeast strain and medium,Fermentation conditions and sampling,Yeast preoxygenation was performed at 20C in a membrane loop reactor（膜环流反应器）.Yeast slurries were circulated at 750 ml/min during 5 h.Oxygen was delivered to the slurry via the membrane sparger（分布器）to obtain an oxygen concentration of 8 mg/L in the slurry.Maltose(3%)was added to the yeast solution prior to preoxygenation.The concentration and viability of the yeast slurries were determined by flow cytometry（细胞计数法）before the required amount was pitched in the wort.All fermentations were carried out in duplicate,in tall tubes(75 cm tall,8 cm internal diameter),containing 1.8L sterile 15P wort medium.The fermentations were performed at 15C and were monitored frequently by withdrawing samples through a narrow sampling tube(15 cm from the bottom)with the aid of N2 overpressure.The supernatant（上清液）of the tube(beer)was collected and the remaining slurry was resuspended（重悬）in 11 cold sterile water,after which samples were taken to characterise the cropped yeast,Properties of the five different experimetal conditions,The worts were sparged with air,oxygen or nitrogen,for 10 min before pitching.,Decrease of sugar density as a function of time,The time required to reach an ADF(apparent degree of fermentation)of 80%was approximately 270h for the REF with an inoculum size of 20106 viable cells/ml and between 50 and 70h for the HCD(high-cell density)fermentations.,filled circles=A/NP,empty circles=O/NP,filled triangles=N/PR,empty triangles=A/PR,filled squares=REF,Hence,the oxygen conditions had a significant impact on the extent of yeastgrowth in the HCD fermentations.,When the total biomass was characterised at the end of the fermentation,he cell viability was also measured.The HCD fermentations showed similar viabilities at the end of fermentation,but the REF condition showed a slightly decreased percentage of living cells.This indicates that the long duration of fermentation had a negative influence on the yeast viability but that yeast viability remained at an acceptable level in all fermentations.,The total FAN uptake levels were 68%enhanced whenhigher than normal pitching rates were used.Thehighest FAN uptake was found in the O/NP fermentationcondition and was 15%higher than the other HCDfermentations,which were not significantly different,It was expected that the higher uptake levels of FAN in this fermentation condition should enhance the formation of-acetolactate(-乙酰乳酸）,thereby resulting in higher levels of total diacetyl（二乙酰）.,Fig.3 Fatty acid profiles(in mg/g cell dry weight(CDW)as a function of time,A/NP,filled circles=C16:0（十六碳酸）,empty circles=C16:1,filled triangles=C18:0,empty triangles=C18:1（油酸）,and filled squares=C18:2（亚油酸）,O/NP,N/PR,A/PR,REF,The final UFA levels towards the end of fermentation in case of O/NP were significantly higher than in the other conditions,suggesting that in this condition other nutrients(e.g.,fermentable carbohydrates)were limiting at that point.,Fig.4 Glycogen(a)and trehalose contents(b)(in%of wet weight)of the yeast suspensions 4.5h after pitching(white bars)and of thetotal yeast populations after the different conducted fermentations,compared with the reference fermentation(Fig.4a,white bars)and no significant differences were found between the HCD fermentations The lowest glycogen levels were found in case of the REF.,Init ially(4.5 h after pitching),treha lose level s were minimal and no significan t differences were found between the various fermentations(Fig.4 b,white bars),expression at four fermentation points:(1)1h after pitching,(2)4.5h after pitching,(3)when 35%ADFapparent degree of fermentation,and(4)50%ADF(exponential phase)White bars=A/NP;light grey bars=O/NP;dark grey bars=N/PR;black bars=A/PR;striped bars=REF,Higher expression levels of HSP12 were observed in the yeast cells from the HCD conditions,HSP12 has been evaluated for the use as a molecular marker for stress resistance（抗逆性）,CTT1 encodes the cytosolic catalase T，which catalyses the breakdown ofhydrogen peroxide to oxygen and water.,Superoxide dismutase,encoded by SOD1 and SOD2,catalyses the conversion of the superoxide anion（阴离子）to oxygen and hydrogen peroxide and are both associated with the oxidative stress response（氧化应激反应）,The expres sion of OLE1,which enodes the oxygen dependent fatty acid desaturase（脱氢酶）,was very high throughout the whole experiment,ERG1 catalyses the oxygen-dependen t epoxidation（环氧化作用）of squalene（三十碳六烯）to 2,3-oxidosqualene（2,3-氧鲨烯）and plays an essential role in the ergosterol（麦角甾醇）biosynthesis pathway.,ATF1 encodes the alcohol acetyl（乙酰）transferase,an important enzyme in the synthesis of ethyl acetate（乙酸乙酯）and isoamyl acetate（乙酸异戊酯）,the expression of BAP2 was monitoredbecause the branched chain amino acid permease（透性酶）plays an important role in the assimilation of important amino acids,To investigate the role of ILV2(encoding-acetohydroxy（乙酰羟）acid synthase)and ILV5(encoding-acetohydroxy acid reductoisomerase（还原异构酶）)in the metabolism of diacetyl（二乙酰）,their expression levels were monitored,