Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

On-line analysis of avermectin fermentation cell growth kinetics in an industrial pilot plant

Experimental data are presented which show that on-line calculation of oxygen uptake rate can be used to estimate the cell concentration of Streptomyces avermitilis during the active growth phase of this fermentation. Moreover, by dividing the oxygen uptake rate by the total oxygen consumed, an on-line estimate of specific growth rate of this culture can be generated. A theoretical basis is provided for this model. Use of a mass spectrometer for vent gas analysis coupled with computer data acquisition has made this information both very accurate and readily available. Examples are given which illustrate the kinetics of the avermectin fermentation as well as the effect of a temperature shift on the specific growth rate.     

Effect of oxygen transfer on lipase production by Acinetobacter radioresistens

The influence of oxygen on alkaline lipase production by Acinetobacter radioresistens was studied under two operating modes: controlled dissolved oxygen (DO) concentration and controlled aeration rate. Compared with cell growth, the lipase production depended more extensively on oxygen. The intrinsic factor determining cell growth and lipase production was oxygen transfer rate (OTR) rather than DO concentration. Improvements in OTR, either by aeration or agitation, resulted in an increase in lipase yield and/or a reduction in fermentation time. The formation of A. radioresistens lipase could be described by a mixed-growth-associated model, and the enzyme was mainly a growth-associated product. The overall productivity for the lipase, which depended more strongly on agitation than aeration, could be related with kLa. DO concentration could not be employed in this correlation, though it has been useful as a criterion for ensuring no oxygen limitation in an aerobic fermentation.     

Kinetics of <Emphasis Type="SmallCaps">l</Emphasis>-lysine fermentation: a continuous culture model incorporating oxygen uptake rate

For process design and optimization, it is essential to have a mathematical model that represents the system well. Many past studies do not go beyond empirically fitting experimental data. In the present study, an unstructured model incorporating oxygen uptake and dissolved oxygen concentration was developed for a continuous culture of L-lysine. Specific rate expressions of cell growth, substrate consumption, product formation, and oxygen uptake were developed and incorporated in the model. The model predicts very well the effects of operational parameters, such as the dilution rate and the feed substrate concentration. It is also able to predict the unsteady-state dynamics of continuous L-lysine fermentation.     

Oxygen consumption by anaerobic Saccharomyces cerevisiae under enological conditions: effect on fermentation kinetics

The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.     

重组大肠杆菌高密度发酵生产RGD-TRAIL的条件优化

本文以一株产RGD-TRAIL的重组大肠杆菌为研究对象,在10L发酵罐中考查了诱导温度、pH值、溶氧、流加葡萄糖对重组大肠杆菌生长和RGD-TRAIL蛋白表达的影响。结果表明:诱导温度25℃,pH值控制7.0,溶氧控制30%,以5g·L~(-1)·h~(-1)流速流加葡萄糖最有利于菌体生长和蛋白表达,菌体收率和RGD-TRAIL产量分别达到45.99g·L~(-1)和160.2mg·L~(-1)。     

表达血管生长抑制素的重组毕赤酵母在诱导阶段混合碳源的流加

为进行高密度发酵并实现外源基因的高表达,在表型为Mut^S的重组毕赤酵母（Pichia pastoris）表达人血管生长抑制素的诱导阶段,采用了甘油甲醇混合补料的培养方式。以溶氧水平作为甘油代谢指针来控制甘油限制性流加既可维持一定菌体生长,又不会发生发酵液中残余甘油及有害代谢产物（乙醇）阻遏蛋白表达。当表达阶段的菌体平均比生长速率控制于0.012h^-1,菌体浓度达150 g/L,血管生长抑制素浓度最高达到108 mg/L,血管生长抑制素的平均比生产速率为0.02 mg/(g·h),菌体关于甘油的表观得率为0.69 g/g,菌体关于甲醇的表观得率为0.93g/g,较没有采用甘油限制性流加时都有所提高。     

Improved poly-ε-lysine biosynthesis using Streptomyces noursei NRRL 5126 by controlling dissolved oxygen during fermentation

The growth kinetics of Streptomyces noursei NRRL 5126 was investigated under different aeration and agitation combinations in a 5.0 l stirred tank fermenter. Poly-epsilon-lysine biosynthesis, cell mass formation, and glycerol utilization rates were affected markedly by both aeration and agitation. An agitation speed of 300 rpm and aeration rate at 2.0 vvm supported better yields of 1,622.81 mg/l with highest specific productivity of 15 mg/l.h. Fermentation kinetics performed under different aeration and agitation conditions showed poly- epsilon-lysine fermentation to be a growth-associated production. A constant DO at 40% in the growth phase and 20% in the production phase increased the poly-epsilon-lysine yield as well as cell mass to their maximum values of 1,992.35 mg/l and 20.73 g/l, respectively. The oxygen transfer rate (OTR), oxygen utilization rate (OUR), and specific oxygen uptake rates (qO2) in the fermentation broth increased in the growth phase and remained unchanged in the stationary phase.     

Oxygen Consumption by Anaerobic Saccharomyces cerevisiae under Enological Conditions: Effect on Fermentation Kinetics

The anaerobic growth of the yeast Saccharomyces cerevisiae normally requires the addition of molecular oxygen, which is used to synthesize sterols and unsaturated fatty acids (UFAs). A single oxygen pulse can stimulate enological fermentation, but the biochemical pathways involved in this phenomenon remain to be elucidated. We showed that the addition of oxygen (0.3 to 1.5 mg/g [dry mass] of yeast) to a lipid-depleted medium mainly resulted in the synthesis of the sterols and UFAs required for cell growth. However, the addition of oxygen during the stationary phase in a medium containing excess ergosterol and oleic acid increased the specific fermentation rate, increased cell viability, and shortened the fermentation period. Neither the respiratory chain nor de novo protein synthesis was required for these medium- and long-term effects. As de novo lipid synthesis may be involved in ethanol tolerance, we studied the effect of oxygen addition on sterol and UFA auxotrophs (erg1 and ole1 mutants, respectively). Both mutants exhibited normal anaerobic fermentation kinetics. However, only the ole1 mutant strain responded to the oxygen pulse during the stationary phase, suggesting that de novo sterol synthesis is required for the oxygen-induced increase of the specific fermentation rate. In conclusion, the sterol pathway appears to contribute significantly to the oxygen consumption capacities of cells under anaerobic conditions. Nevertheless, we demonstrated the existence of alternative oxygen consumption pathways that are neither linked to the respiratory chain nor linked to heme, sterol, or UFA synthesis. These pathways dissipate the oxygen added during the stationary phase, without affecting the fermentation kinetics.     

Kinetics of Lactococcus lactis growth and metabolite formation under aerobic and anaerobic conditions in the presence or absence of hemin

The study of batch kinetics of Lactococcus lactis cell growth and product formation reveals three distinct metabolic behaviors depending upon the availability of oxygen to the culture and the presence of hemin in the medium. These three cultivation modes, anerobic homolactic fermentation, aerobic heterolactic fermentation, and hemin-stimulated respiration have been studied at pH 6.0 and 30 degrees C with a medium containing a high concentration of glucose (60 g/L). A maximum cell density of 5.78 g/L was obtained in the batch culture under hemin-stimulated respiration conditions, about three times as much as that achieved with anerobic homolactic fermentation (1.87 g/L) and aerobic heterolactic fermentation (1.80 g/L). The maximum specific growth rate was 0.60/h in hemin-stimulated respiration, slightly higher than that achieved in homolactic fermentation (0.56/h) and substantially higher than that in heterolactic fermentation (0.40/h). Alteration of metabolism caused by the supplementation of oxygen and hemin is evidenced by changes in both cell growth kinetics and metabolite formation kinetics, which are characterized by a unique pseudo-diauxic growth of L. lactis. We hypothesise that Lactococcus lactis generates bioenergy (ATP) through simultaneous lactate formation and hemin-stimulated respiration in the primary exponential phase, when glucose is abundant, and utilizes lactate for cell growth and cell maintenance in the stationary phase, after glucose is exhausted. We also examined the applicability of a modified logistic model and the Luedeking-Piret model for cell growth kinetics and metabolite formation kinetics, respectively.     

The role of oxygen in yeast metabolism during high cell density brewery fermentations

The volumetric productivity of the beer fermentation process can be increased by using a higher pitching rate (i.e., higher inoculum size). However, the decreased yeast net growth observed in these high cell density fermentations can have a negative impact on the physiological stability throughout subsequent yeast generations. The use of different oxygen conditions (wort aeration, wort oxygenation, yeast preoxygenation) was investigated to improve the growth yield during high cell density fermentations and yeast metabolic and physiological parameters were assessed systematically. Together with a higher extent of growth (dependent on the applied oxygen conditions), the fermentation power and the formation of unsaturated fatty acids were also affected. Wort oxygenation had a significant decreasing effect on the formation of esters, which was caused by a decreased expression of the alcohol acetyl transferase gene ATF1, compared with the other conditions. Lower glycogen and trehalose levels at the end of fermentation were observed in case of the high cell density fermentations with oxygenated wort and the reference fermentation. The expression levels of BAP2 (encoding the branched chain amino acid permease), ERG1 (encoding squalene epoxidase), and the stress responsive gene HSP12 were predominantly influenced by the high cell concentrations, while OLE1 (encoding the fatty acid desaturase) and the oxidative stress responsive genes SOD1 and CTT1 were mainly affected by the oxygen availability per cell. These results demonstrate that optimisation of high cell density fermentations could be achieved by improving the oxygen conditions, without drastically affecting the physiological condition of the yeast and beer quality.     

表达透明颤菌血红蛋白基因对酿酒酵母生长及细胞内氧化状态的影响*

透明颤菌血红蛋白基因vgb在多种研究和工业发酵菌中异源表达很好的解决了高密度发酵中的溶氧率问题。酿酒酵母是经典的真核模型,且在发酵工业中具有重要的应用价值,但vgb在酿酒酵母中异源表达对细胞生长的影响并不清楚。以ADH1为启动子构建了含透明颤菌(Vistreoscilla)血红蛋白基因vgb的异源表达质粒YEplac195-ADH1pr-vgb,并转化至酿酒酵母BY4741。通过生长敏感性实验,发现在发酵碳源和非发酵碳源中,vgb的异源表达均抑制了菌株生长。接着,通过2',7'-二氯荧光黄双乙酸盐和PI染色和脂质过氧化产物检测分析,发现过表达vgb的酿酒酵母细胞中活性氧(ROS)的积累、细胞膜通透性改变以及脂质过氧化。结果表明,酿酒酵母中过表达vgb改变细胞的氧化状态促进活性氧的累积,氧化应激导致菌株的生长抑制。     

毕赤酵母高密度发酵工艺的研究

高密度发酵是毕赤酵母提高蛋白表达量的一种重要策略,发酵工艺是高密度发酵的一个重要因素。采用下列措施均可以有效地提高表达水平：调节基础培养基,采用变pH和变温发酵,提高DO,选择最适的诱导前菌体密度和比生长速率并降低甘油初始浓度和采用分段式指数流加进行调控。选择合适的甲醇补料策略：甲醇限制补料（MLFB）、氧气限制补料（OLFB）、甲醇不限制补料（MNLFB）和温度限制补料（TLFB）。采用两种方式调控补料：诱导阶段菌体生长时,甲醇比消耗速率(qMeOH)为0.02-0.03gg-1h-1,而菌体不生长时,qMeOH采用较高值。     

Engineering a Saccharomyces cerevisiae wine yeast that exhibits reduced ethanol production during fermentation under controlled microoxygenation conditions

We recently showed that expressing an H(2)O-NADH oxidase in Saccharomyces cerevisiae drastically reduces the intracellular NADH concentration and substantially alters the distribution of metabolic fluxes in the cell. Although the engineered strain produces a reduced amount of ethanol, a high level of acetaldehyde accumulates early in the process (1 g/liter), impairing growth and fermentation performance. To overcome these undesirable effects, we carried out a comprehensive analysis of the impact of oxygen on the metabolic network of the same NADH oxidase-expressing strain. While reducing the oxygen transfer rate led to a gradual recovery of the growth and fermentation performance, its impact on the ethanol yield was negligible. In contrast, supplying oxygen only during the stationary phase resulted in a 7% reduction in the ethanol yield, but without affecting growth and fermentation. This approach thus represents an effective strategy for producing wine with reduced levels of alcohol. Importantly, our data also point to a significant role for NAD(+) reoxidation in controlling the glycolytic flux, indicating that engineered yeast strains expressing an NADH oxidase can be used as a powerful tool for gaining insight into redox metabolism in yeast.     

Computer-aided baker's yeast fermentations.

The economics of yeast production depend heavily upon the cellular yield coefficient on the carbon source and the volumetric productivity of the process. The application of an on-line computer to maximize these two terms during the fermentation requires a continuous method of measuring cell density and growth rate. Unfortunately, a direct sensor for biomass concentration suitable for use in industrial fermentations is not available. Material balancing, with the aid of on-line computer monitoring, offers an indirect method of measurement. Laboratory results from baker's yeast production in a 14-liter fermentor (with a PDP-11/10 computer for on-line analyses) show this indirect measurement technique to be a viable alternative. From the oxygen uptake and carbon dioxide production data, gas flow rate, and ammonia addition rate, the cell density during the fermentation has been estimated and found to compare well with actual fermentation data.     

Origin of the phyla and cancer

Multicelled animals with specialized cells (metazoans) emerged shortly after rising oxygen levels in the seas permitted formation of collagen-family molecules. Certain unicells then formed 3-D clusters, some with disc- or ball-like shapes that happened to resemble blastulas. These became unstable beyond a certain size due to contrasting metabolic styles among their component cells. For whereas cells near their exteriors could employ oxygen respiration, cells closer to the oxygen-deprived interiors were obliged to rely on anaerobic metabolism (fermentation), a process that produces waste molecules that, if retained within cells, cause disproportionate cell growth. Unstable blastula-like forms would either disintegrate or reorganize along surfaces of relative weakness in a process that may be likened to gastrulation. Initial cell-differentiation depended on the quantity and diversity of retained fermentation products and on the pumping of molecules from cell to cell by the consequent electro-chemical gradients. In subsequent contexts, oxygen deprivation, fermentation, excess cell growth, and disintegration or reorganization of tissues produce cancer.     

Modeling cell concentration in complex media

A model that continuously predicts the concentration of microorganisms in complex medium fermentations is suggested. The model uses carbon dioxide evolution as its primary input and assumes that respiration activity can be differentiated into growth-related and maintenance-related functions. This model can be programmed on computer-coupled vessels and used to standardize on a physiological fermentation inoculum transfer time. The cell concentration estimate can also be used to calculate specific growth rate and can be combined with additional monitored information to calculate other important fermentation parameters such as specific oxygen uptake.     

Influence of oxygen transfer rate on vinegar production by Acetobacter aceti in submerged fermentation

Summary A study was carried out on the influence of oxygen transfer rate on bacterial growth and acid production in submerged vinegar fermentation by an industrial culture of Acetobacter aceti. The production rate grew as did growth rate even if no increment in cellular mass was noticed when oxygen transfer rate was increased. The relationship between growth and production followed a mixed growth associated model.     

Growth, death, and oxygen uptake kinetics of Pichia stipitis on xylose

Pichia stipitis NRRL Y-7124 has potential application in the fermentation of xylose-rich waste streams, produced by wood hydrolysis. Kinetic models of cell growth, death, and oxygen uptake were investigated in batch and oxygen-limited continuous cultures fed a rich synthetic medium. Variables included rates of dilution (D) and oxygen transfer (K(1)a) and concentrations of xylose (X), ethanol (E), and dissolved oxygen (C(ox)). Sustained cell growth required the presence of oxygen. Given excess xylose, specific growth rate (micro) was a Monod function of C(ox). Specific oxygen uptake rate was proportional to mu by a yield coefficient relating biomass production to oxygen consumption; but oxygen uptake for maintenance was negligible. Thus steady-state C(OX) depended only on D, while steady-state biomass concentration was controlled by both D and K(1)a. Given excess oxygen, cells grew subject to Monod limitation by xylose, which became inhibitory above 40 g/L. Ethanol inhibition was consistent with Luong's model, and 64. 3 g/L was the maximum ethanol concentration allowing growth. Actively growing cells died at a rate that was 20% of micro. The dying portion increased with E and X.