高密度发酵

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 .
Fig. 3 Fatty acid profiles(in mg/g cell dry weight(CDW)) as a function of time 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）
(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。
高密度发酵主要限制因素
（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
empty circles = O/NP
empty triangles = A/PR
filled circles = A/NP
filled triangles = N/PR
Hence, the oxygen conditions had a significant impact on the extent of yeast growth in the HCD fermentations .
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(DCW／L)。 (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
优 点
The role of oxygen in yeast metabolism during high cell density brewery fermentations
Appl Microbiol Biotechnol
Yeast strain and medium
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 15°P 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 wort was 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
The total FAN uptake levels were 68% enhanced when higher than normal pitching rates were used. The highest FAN uptake was found in the O/NP fermentation condition and was 15% higher than the other HCD fermentations, which were not significantly different It was expected that the higher uptake levels of FAN in this fermentation condition should enhance the formation of α-acetolactate(α-乙酰乳酸）, thereby resulting in higher levels of total diacetyl（二乙酰）.
《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 r／min离心菌体富集培养液，无菌水洗涤菌体， 转接入50 mL／250 mL的BMMY培养基中， 于30℃，pH 6.0，220 r／min振荡诱导培 养144 h，期间每24小时加入终浓度为0．5％的甲醇。 甲醇浓度为1.5％时，PLD活力最高达到12.4x10-7kat／g，菌体量到达13.6g／L。
温度的控制：目前主要的控温策略是手动调节冷却水Baidu Nhomakorabea流量
初始培养基开始 发 酵 中 重 要 参 数 控 制 pH的控制： 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。 物理法：增加空气流量，提高搅拌转速；增大罐压；通入纯氧
溶解氧
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
REF
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.
以摇瓶优化结果作为参考，选取初始pH 6．5，发酵温度28℃，进行5 L规模 发酵罐试验，15 mL／(L· h)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27.3×10-7kat/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%。
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
Fermentation conditions and sampling
Yeast preoxygenation was performed at 20°C 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 15°P wort medium. The fermentations were performed at 15°C 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
The time required to reach an ADF(apparent degree of fermentation) of 80% was approximately 270h for the REF with an inoculum size of 20×106 viable cells/ml and between 50 and 70h for the HCD(high-cell density) fermentations .
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)
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13.4xl0-7kat／g，菌体量到达14.8 g／L。
菌体生长24h后以不同 速度流加对菌体的影响， 得出15mL/(h∙L)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 24.0×10 -7kat/g，此时菌体量为 59.1g/L DO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
A/NP
O/NP
N/PR
A/PR 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.