培养基pH调控法高密度发酵唾液乳杆菌XH4B研究

本研究以MRS培养基为基础,通过优化碳、氮源及无机盐的配方和用量,再结合补料发酵,最终实现唾液乳杆菌XH4B高密度培养的目的。以乳酸菌的生物量为指标,同时考查发酵液pH值、乳酸含量等,最终确定酵母粉和蔗糖为最佳氮、碳源,同时增加乙酸钠用量至2%、磷酸二氢钠0.6%,可以对发酵液酸化时提供一定的缓冲作用。采用优化的PY-Suc培养基,唾液乳杆菌XH4B的生物量最高能达到6.91 g/L,明显高于MRS培养基的5.01~6.30 g/L(P0.05)。等量补料培养并且采用NaOH中和发酵液pH值时,乳酸最高积累速度可以达到5.958 g/(L·h),但是随着培养时间延长,积累速度迅速下降。发酵酸化较严重时(乳酸含量9~10 g/L),唾液乳杆菌XH4B的生物量积累变缓。结论:优化MRS培养基,并加大乙酸钠、磷酸二氢钠等能够缓冲发酵液的无机盐用量,结合补料发酵,可以实现唾液乳杆菌XH4B的高密度培养。     

Development of a fed-batch cultivation strategy for the enhanced production and secretion of cutinase by a recombinant Saccharomyces cerevisiae SU50 strain

Saccharomyces cerevisiae SU50 strain was cultivated with different concentrations of glucose and galactose with the aim of increasing cutinase activity, cutinase yield on the carbon source, and bioreactor productivity. Cultivations in shake flasks with galactose as the sole carbon source, with sugar concentrations between 10 and 40 g/l, exhibited growth-associated cutinase production and a constant specificity of cutinase secretion. Furthermore, as the galactose concentration increased to values higher than 15 g/l, a progressively higher maximum specific galactose consumption rate and a consequent higher alcoholic fermentation occurred, resulting in progressively lower biomass yields on the carbon source and cutinase yields on biomass. Using high glucose and galactose concentrations in a well-aerated bioreactor resulted in a high biomass productivity (0.5 g dcw/l/h), a high cutinase yield on biomass (21.5 U/mg dew), a final high cutinase secretion efficiency (97%), and plasmid stability (99%). Based on these studies, a two phase fed-batch cultivation strategy was developed. A batch phase with high glucose and galactose concentrations, followed by a fed-batch with a constant feed rate with galactose as the sole carbon source in order to minimize the repression of the GAL 7 promoter, were established. The feed rate was estimated to maintain a pre-determined concentration of galactose (20 g/l) on the culture medium in order to maximize the efficiency of cutinase secretion and minimize the galactose alcoholic fermentation. By this cultivation strategy, enhancements of 3.6-fold in cutinase activity, 1.2-fold in cutinase yield on the carbon source, and 8.7-fold culture productivity were obtained in relation to a batch cultivation performed in shake flasks with 20 g/l of galactose.     

Real-time viable-cell mass monitoring in high-cell-density fed-batch glutathione fermentation by Saccharomyces cerevisiae T65 in industrial complex medium

An on-line monitoring of viable-cell mass in high-cell-density fed-batch cultivations of Saccharomyces cerevisiae grown on an industrial complex medium was performed with an in situ capacitance probe fitted to a 50-l fermentor. Conventional off-line biomass determinations of several parameters, including dry cell weight (DCW), optical density at 600 nm wavelength (OD(600)), packed mycelial volume (PMV) and number of colony forming units (CFU), were performed throughout the bioprocess and then compared with on-line viable-cell concentrations measured using a capacitance probe. Capacitance versus viable biomass and all off-line biomass assay values were compared during glutathione fermentation in industrial complex culture media. As a result, the relationship between the number of colony forming units and capacitance with a correlation coefficient (R) of 0.995 was achieved. Simultaneously, compared with those determined by at-line indirect estimation methods including oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER), the specific growth rates estimated by on-line capacitance measurement could be more reliable during glutathione fermentation. Therefore, it is concluded that a capacitance probe is a practical tool for real-time viable biomass monitoring in high-cell-density fed-batch cultivation in a complex medium.     

Characteristics of alpha-glucosidase production from recombinant Aspergillus oryzae by membrane-surface liquid culture in comparison with various cultivation methods

alpha-Glucosidase was produced using recombinant Aspergillus oryzae by membrane-surface liquid culture (MSLC), a method previously developed by the authors and the results compared with other methods, including shaking flask culture (SFC), agar-plate culture (APC), culture on urethane sponge supports (USC), and liquid surface culture (LSC) to determine possible reasons for the advantageous features of MSLC. When yeast extract was used as a nitrogen source, the amount of enzyme produced by MSLC was 5 or more times higher than those for SFC and LSC, but similar to that using APC. Enzyme production in USC was slightly lower than in MSLC and APC. Cell growth was similar irrespective of the cultivation method used. When NaNO3, a typical inorganic nitrogen source was used, enzyme production in all the cultures was lower than that using yeast extract. However, even using NaNO3, the amount of the enzyme produced by MSLC was 8 to 20 times higher than those by SFC, APC, USC, and LSC. Although cell growth using NaNO3 was similar to that for yeast extract in MSLC, it was markedly decreased in SFC, APC, and LSC. The reason for the difference in enzyme productivity for various cultivation methods using yeast extract and NaNO3 as a nitrogen source is discussed, on the basis of the experimental findings. The role of the oxygen transfer effect and gene expression levels in enzyme production were also examined.     

应用渗透膜高密度培养富硒螺旋藻

应用透析袋(OT法)组装光生物反应器,与鼓泡式生物反应器(AB法)和摇瓶(SB法)进行对比实验,探讨利用渗透膜建立高密度培养富硒螺旋藻(Se-SP)方法的可行性。称质量法监测并推算藻细胞生长率和最大生物量,荧光光度法测定Se-SP及其培养液中硒含量和形态变化,分光光度法检测Se-SP中总蛋白(TP)、藻蓝蛋白(PC)、叶绿素a(Chla)、总胡萝卜素(Caro)、水溶性多糖(SPS)及巯基(-SH)含量等主要营养构成,培养液中无机碳(IC)和有机溶解碳(DOC)的含量采用自动分析仪测定。结果发现：OT法培养Se-SP的最大生物量可达9.6g/L,是AB和SB方法培养的3～5倍,其中有机硒转化率及TP、PC、Chla、SPS和-SH等主要营养成分的含量均有明显提高。相应地,培养基中 IC剩余减少, DOC无明显累积。结果提示,OT法可实现Se-SP高密度培养,并有望获得优质Se-SP产品,其优势体现在高细胞增殖速率、高有机硒转化率、高无机营养源消耗率和低有机碳的积累,便于培养液的再生利用,减少废液污染。     

马克斯克鲁维酵母高密度发酵条件的优化研究

对影响马克斯克鲁维酵母高密度发酵的培养基营养成分和培养条件展开分析研究。单因素实验发现,YPD作为基础培养基有利于马克斯克鲁维酵母的增殖;培养基成分响应面分析和培养条件正交实验结果表明,当培养基成分为蔗糖67.37 g/L,酵母浸粉29.7 g/L,玉米浆15.61 g/L,KH₂PO₄4.13 g/L,MgSO₄0.3 g/L,初始pH为6.0、发酵温度为30℃、搅拌速度为160 r/min时,发酵培养18 h马克斯克鲁维酵母的生物量最大,为（9.34±0.12）g/L。进一步进行乳饮品发酵实验,优化培养的马克斯克鲁维酵母与乳源培养基培养的马克斯克鲁维酵母,在菌种的生物量和乳饮品的口感风味上无明显差异。因此,优化后的高密度发酵培养基配方和工艺条件适宜于马克斯克鲁维酵母菌的高密度发酵。      

氮源对L-色氨酸发酵的影响

以L-色氨酸生产菌E.coli TRTH为供试菌株,研究了氮源对L-色氨酸发酵的影响。利用30 L发酵罐进行分批补料发酵试验,确定了最佳有机氮源和无机氮源分别为酵母粉和硫酸铵,进一步确定酵母粉和硫酸铵的最佳用量为1 g/L和10 g/L,最后采用NaOH和氨水混合补料控制发酵液中NH4+浓度在120 mmol/L以下,发酵38 h,菌体生物量和L-色氨酸产量分别达到53.42 g/L和32.6 g/L,实现了大肠杆菌的高密度培养。     

Yeast extract stimulates production of glycolipid biosurfactants, mannosylerythritol lipids, by Pseudozyma hubeiensis SY62

We improved the culture conditions for a biosurfactant producing yeast, Pseudozyma hubeiensis SY62. We found that yeast extract greatly stimulates MEL production. Furthermore, we demonstrated a highly efficient production of MELs in the improved medium by fed-batch cultivation. The final concentration of MELs reached 129 ± 8.2g/l for one week.     

酵母对无机硒的富集及其影响因素

在蔗糖培养基中添加无机硒,利用生物转化技术,通过酵母的生命活动,将无机硒有效的富集到细胞内,研究不同的碳源、硒离子浓度、通气量、加硒时间与不同方法对酵母的生物量及细胞含硒量的影响。结果表明,硒离子浓度对酵母的生长繁殖能力影响较大,浓度越高,酵母细胞含硒量越大,但达到某一个峰值后又开始下降;在对数生长期加硒,保持适当的通风量有利于酵母的生长和对硒的富集。     

基于麦汁培养基的富硒酵母培养条件优化

以啤酒酵母为材料,对富硒酵母的最适培养条件进行研究。结果显示：以2oBx 的麦汁培养酵母菌,其生长得率最高,过高的麦汁质量浓度会产生克拉勃脱效应而影响酵母生长。在2oBx 的麦汁培养基中适当添加酵母提取物,可显著提高酵母的生物量,而添加硫酸铵和磷酸二氢钾对酵母生物量的提高不明显;添加葡萄糖在一定程度上也能增加酵母的生物量,但这种增加会被硫酸铵抑制。培养基中的初始硒含量能显著影响酵母的生物量和细胞中硒的积累,在初始质量浓度为10mg/L(最终质量浓度为50mg/L)的Na2SeO3 初始质量浓度下培养24h,酵母的生物量可达2.83g/L,细胞中积累的总硒量为266.3mg/kg。     

酵母对无机锌的富集及其影响因素研究

在培养基中添加无机锌，利用生物转化技术，通过酵母的生命活动，将无机锌有效地富集到细胞内，研究锌离子浓度对酵母生长繁殖、细胞含锌量、锌富集率及酵母发酵力的影响，并通过控制锌离子浓度，使酵母细胞既能富集锌，又对其发酵力不产生显著的影响，以便将仍具有发酵活力的富锌酵母用于食品加工之中。研究结果表明，锌离子浓度对酵母的生长繁殖和富集能力影响较大，浓度越高，酵母细胞含锌量越大。但无机锌对酵母细胞有显著的损伤，且这种损伤是非遗传性的，当无机锌去掉后，酵母细胞形态又恢复正常。     

Effect of Culture Variables on Mycelial Arachidonic acid Production by Mortierella alpina

Effect of culture conditions on biomass, lipid, and arachidonic acid production was investigated in the oleaginous fungus Mortierella alpina CBS 528.72 under shake flask conditions. Several factors have been found to affect the biomass buildup and lipogenesis in this fungus, complicated by the fact that different strains demonstrate varying optimization conditions. Growth, lipid accumulation, and arachidonic acid production in the strain investigated were influenced by media, pH, temperature, carbon source, nitrogen source, etc. The results indicated that the most effective medium for growth and arachidonic acid production was glucose yeast extract medium. The optimum pH and temperature were found to be 6.5 and 28°C, respectively. On the same weight basis, glucose was the most efficient carbon source for biomass and lipid production in this fungal strain which yielded 6.8 g/L dry biomass and 40.2% (w/w) total lipid after 7 days of cultivation. Maximum arachidonic acid (ARA) production of 40.41% achieved in rhamnose-containing media was not concomitant with higher biomass and lipid yields. Efficacy of organic carbon sources, viz, yeast extract and peptone over inorganic sources like sodium nitrate, ammonium sulfate, ammonium chloride, etc, was established in the present study. M. alpina CBS 528.72 grown in peptone acquired the highest lipid content (42.0% (w/w)). However, the ARA content (28.74%) proved to be significantly less than that grown in yeast extract (35.28%). Furthermore, it was found that the biomass and ARA production declined drastically in a medium with vegetable oils as the sole carbon source but triggered the lipogenic pathway leading to higher accumulation of total lipids. Under the ideal conditions mentioned above, the maximum biomass, total lipid, and arachidonic acid production were 6.8 g/L, 41.6%, and 35.28% total fatty acid, respectively, in shake flask system.     

Synchronized fresh cell bioreactor system for continuous L-(+)-lactic acid production using Lactococcus lactis IO-1 in hydrolysed sago starch

An efficient bioreactor, termed a 'synchronized fresh cell bioreactor', was developed and consisted of a pH-dependent substrate feed system coupled with cross flow filtration and turbidity control. The effect of high dilution rate and high cell density coupled with high cell viability on the production of l-lactic acid in continuous culture by Lactococcus lactis IO-1 in enzyme-hydrolysed sago starch medium was investigated. For all changes in dilution rate, cells responded in a synchronized way to the addition of glucose by increasing the rate of biomass formation. Consequently, a glucose-free feed solution was required to maintain the cell concentration at a particular pre-set value. This set-up facilitated the maintenance of the cells in a permanent log phase. At a cell concentration of 15 gl(-1) and a feed glucose concentration of 53 gl(-1), volumetric LA productivities of 8.2, 19.3 and 33.1 gl(-1)h(-1) were obtained at dilution rates of 0.21, 0.50 and 1.1 h(-1), respectively. The respective residual glucose concentrations in the spent medium were 1.90, 0.24 and 3.80 gl(-1). By increasing the cell density, the volumetric productivity increased proportionally. At high cell density, higher dilution rates resulted in lower lactate concentrations in the culture medium resulting in higher productivity. This reactor facilitated efficient operation with high cell viability by maintaining the cells in continuous growth phase for long-term fermentation. Therefore, the growth rate (mu) was calculated according to the Monod equation. Using this system, high specific productivities can be obtained which guarantees high commercial productivity at economical cost with only a small investment for setting up the sago industry.     

利用甲醇厂CO_2尾气培养小球藻

利用甲醇厂所排放的高浓度CO2尾气培养小球藻,研究不同的通气方式、培养方式对小球藻生长的影响。实验结果表明,当通气速率为300 mL/min、CO2浓度为10%并以10 min/h的间歇通气方式培养时能够提高小球藻的生物量,生物量和产率分别为0.681 g/L和0.082 g/(L.d)。对氮源和磷源采用补料的方式进行培养时对小球藻生长具有促进作用,最高生物量和产率分别达到0.789 g/L和0.088 g/(L.d)。当培养基的更新率为20%时能够获得较高的生物量。利用醋酸进行兼性培养时,一次性添加50～125μL/L醋酸都可促进小球藻生长,每日添加10～25μL/L的醋酸时,可明显促进小球藻生长,最高生物量和产率分别为0.933 g/L和0.111 g/(L.d),分别是空气对照的1.3倍和1.4倍。说明利用甲醇厂高浓度CO2尾气培养小球藻提高生物量是可行的。     

Stable maintenance of plasmid in continuous culture of yeast under non-selective conditions

A recombinant yeast plasmid containing the gene for beta-galactosidase was tested for stability in a host auxotrophic for leucine. Plasmid loss was studied at different dilution rates in continuous culture under selective as well as non-selective conditions. It was observed that the instability of the culture was higher at low dilution rates in selective medium, while the pattern was reversed when complex non-selective medium was used, with plasmid-containing cells competing effectively with plasmid-free cells at low dilution rates. This was attributed to a low residual yeast extract concentration in the medium at low dilution rates. Since yeast extract was the sole source of leucine, this limited the growth of plasmid-free cells, which were auxotrophic for leucine. Growth rate studies also indicated a competitive advantage of the plasmid-containing cells over the plasmid-free cells at low yeast extract concentrations in semi-defined medium. Using the above data, a modified continuous culture was run using non-selective medium at a low dilution rate of 0.05 h(-1). This resulted in stable coexistence of plasmid-containing and plasmid-free cells and hence sustained expression of beta-galactosidase at approximately 330 OD420l(-1) h(-1) throughout the period of cultivation (134 h).     

碳酸氢铵为氮源对螺旋藻培养的影响

以碳酸氢铵作为氮源,研究在分批培养及流加培养条件下其对螺旋藻生长的影响。结果表明：当培养液中碳酸氢铵浓度小于5mmol/L时,螺旋藻生长正常;碳酸氢铵浓度超过5mmol/L时,螺旋藻生长受到抑制,解体死亡。采用生物量反馈补料的流加策略可以使培养液中螺旋藻生物量达到3.08g/L,产率达到0.26g/(L ·d),藻体中蛋白质及叶绿素含量分别达到65.06%和13.37mg/g,结果证实了碳酸氢铵为氮源高密度培养螺旋藻的可行性。     

Production of Candida utilis biomass as aquaculture feed

Fish offal-peat compost was hydrolysed and the resultant liquid extracts used as substrate in the cultivation of the yeast Candida utilis. The yeast was able to grow in this culture medium, attaining a growth yield of approximately 260 g biomass kg^?1 total carbohydrate consumed. The biomass produced had a good amino acid composition, with a protein content of 520 g kg^?1, and a relatively low lipid content. Dry yeast was used as a feed component in the diet of cultivated rainbow trout (Oncorhynchus mykiss) in which it substituted well for other, traditional sources of protein.     

布拉酵母高密度发酵培养基及发酵工艺优化

为实现布拉酵母高密度培养,对其高密度发酵培养基和发酵工艺进行优化。采用Plackett-Burman试验筛选培养基中的显著因素,并进行中心组合设计。通过人工神经网络（artificial neural network,ANN）和响应面试验建立菌体布拉酵母产量与培养基之间的关系模型,利用遗传算法（genetic algorithm,GA）进行全局寻优。结果表明,ANN模型有较好的数据拟合能力和预测能力,更适合处理复杂的非线性问题。GA优化获得最佳培养基组合：葡萄糖40.52 g/L、蛋白胨36.8 g/L、玉米浆17.32 g/L、硝酸钾14 g/L、酵母营养盐1.5 g/L、磷酸二氢钾0.6 g/L、硫酸镁0.8 g/L。利用该培养基进行摇瓶培养,菌体布拉酵母产量可达到8.21 g/L,比优化前提高1.39 倍。在此基础上利用1 L发酵罐培养确定最佳发酵工艺：温度30 ℃、接种量10%、pH 5.0、溶氧40%。利用50 L发酵罐进行扩大培养,流加葡萄糖和蛋白胨控制发酵液中葡萄糖3 g/L、氨氮0.06 g/L,菌体布拉酵母产量达到51.21 g/L。     

产朊假丝酵母核酸高产株的选育及其发酵性能研究

为了获得一株发酵性能优良的高核酸酵母,以产朊假丝酵母(Candida utilis)CL1501为出发菌株,采用紫外-亚硝基胍复合诱变,筛选获得一株高核酸含量的突变株CL15013,经测定其核酸含量占菌体干质量的16.8%,高于出发菌47.8%。对比研究其流加及分批培养工艺,流加培养细胞收获量为16.9 g/L,比分批培养提高94.3%;正交试验设计优化流加培养条件后,CL15013的收获量达21.3 g/L,比分批培养提高144.8%,具有良好的生产应用潜力。     

产酶培养条件对一株嗜酸乳杆菌亚油酸异构酶活力的影响

本实验用MRS培养基嗜酸乳杆菌,并通过添加亚油酸(LA)诱导其产亚油酸异构酶。单因素试验结果表明,在MRS培养基中添加糖类物质总体上不利于产酶,添加尿素、牛肉膏、酵母膏、酪蛋白等有机氮源后,酶活力比对照组有较大幅度提高,而添加无机氮源酶活力增加幅度不如有机氮源。在培养基中添加一定量的NaCl和卵磷脂也有助于酶活力的提高。由正交试验结果确定的最佳产酶条件为:培养温度37℃,培养时间24h,每100ml培养基中添加10mgLA,接种量3%(V/V)。