一株法夫酵母虾青素高产菌株的生产性能

为了评价虾青素高产菌株-法夫酵母JMU-MVP14的生产性能及建立虾青素高产发酵技术,通过测定糖、生物量、虾青素产量、总类胡萝卜素产量等发酵参数,用摇瓶试验对比了法夫酵母JMU-MVP14和出发菌株的差异,用7 L罐试验对比了pH值调控方式及补料培养基成分对发酵的影响,用1 m3罐试验评估了法夫酵母JMU-MVP14高密度发酵虾青素的产量水平。摇瓶发酵结果表明,法夫酵母JMU-MVP14虾青素及总类胡萝卜素的细胞产率分别达到6.01 mg/g及10.38 mg/g;7 L罐分批发酵试验结果表明,自动流加调     

Fed-batch Cultivation of Mucor indicus in Dilute-acid Lignocellulosic Hydrolyzate for Ethanol Production

Mucor indicus fermented dilute-acid lignocellulosic hydrolyzates to ethanol in fed-batch cultivation with complete hexose utilization and partial uptake of xylose. The fungus was tolerant to the inhibitors present in the hydrolyzates. It grew in media containing furfural (1 g/l), hydroxymethylfurfural (1 g/l), vanillin (1 g/l), or acetic acid (7 g/l), but did not germinate directly in the hydrolyzate. However, with fed-batch methodology, after initial growth of M. indicus in 500 ml enzymatic wheat hydrolyzate, lignocellulosic hydrolyzate was fermented with feeding rates 55 and 100 ml/h. The fungus consumed more than 46% of the initial xylose, while less than half of this xylose was excreted in the form of xylitol. The ethanol yield was 0.43 g/g total consumed sugar, and reached the maximum concentration of 19.6 g ethanol/l at the end of feeding phase. Filamentous growth, which is regarded as the main obstacle to large-scale cultivation of M. indicus, was avoided in the fed-batch experiments.     

Modeling of Xanthophyllomyces dendrorhous growth on glucose and overflow metabolism in batch and fed-batch cultures for astaxanthin production

An astaxanthin-producing yeast Xanthophyllomyces dendrorhous ENM5 was cultivated in a liquid medium containing 50 g/L glucose as the major carbon source in stirred fermentors (1.5-L working volume) in fully aerobic conditions. Ethanol was produced during the exponential growth phase as a result of overflow metabolism or fermentative catabolism of glucose by yeast cells. After accumulating to a peak of 3.5 g/L, the ethanol was consumed by yeast cells as a carbon source when glucose in the culture was nearly exhausted. High initial glucose concentrations and ethanol accumulation in the culture had inhibitory effects on cell growth. Astaxanthin production was partially associated with cell growth. Based on these culture characteristics, we constructed a modified Monod kinetic model incorporating substrate (glucose) and product (ethanol) inhibition to describe the relationship of cell growth rate with glucose and ethanol concentrations. This kinetic model, coupled with the Luedeking-Piret equation for the astaxanthin production, gave satisfactory prediction of the biomass production, glucose consumption, ethanol formation and consumption, and astaxanthin production in batch cultures over 25-75 g/L glucose concentration ranges. The model was also applied to fed-batch cultures to predict the optimum feeding scheme (feeding glucose and corn steep liquor) for astaxanthin production, leading to a high volumetric yield (28.6 mg/L) and a high productivity (5.36 mg/L/day).     

Use of dynamic step response for control of fed-batch conversion of lignocellulosic hydrolyzates to ethanol

Optimization of fed-batch conversion of lignocellulosic hydrolyzates by the yeast Saccharomyces cerevisiae was studied. The feed rate was controlled using a step response strategy, in which the carbon dioxide evolution rate was used as input variable. The performance of the control strategy was examined using both an untreated and a detoxified dilute acid hydrolyzate, and the performance was compared to that obtained with a synthetic medium. In batch cultivation of the untreated hydrolyzate, only 23% of the hexose sugars were assimilated. However, by using the feed-back controlled fed-batch technique, it was possible to obtain complete conversion of the hexose sugars. Furthermore, the maximal specific ethanol productivity (q(E,max)) increased more than 10-fold, from 0.06 to 0.70 g g(-1) h(-1). In addition, the viability of the yeast cells decreased by more than 99% in batch cultivation, whereas a viability of more than 40% could be maintained during fed-batch cultivation. In contrast to untreated hydrolyzate, it was possible to convert the sugars in the detoxified hydrolyzate also in batch cultivation. However, a 50% higher specific ethanol productivity was obtained using fed-batch cultivation. During batch cultivation of both untreated and detoxified hydrolyzate a gradual decrease in specific ethanol productivity was observed. This decrease could largely be avoided in fed-batch cultivations.     

Fed-batch cultivation of Wautersia eutropha

Batch kinetics of polyhydroxybutyrate (PHB) synthesis in a bioreactor under controlled conditions of pH and dissolved oxygen gave a biomass of 14 g l(-1) with a PHB concentration of 6.1 g l(-1) in 60 h. The data of the batch kinetics was used to develop a mathematical model, which was then extrapolated to fed-batch by incorporating the dilution due to substrate feeding. Offline computer simulation of the fed-batch model was done to develop the nutrient feeding strategies in the fed-batch cultivation. Fed-batch strategies with constant feeding of only nitrogen and constant feeding of both nitrogen and fructose were tried. Constant feeding strategy for nitrogen and fructose gave a better PHB production rate of 0.56 g h(-1) over the value obtained in batch cultivation (PHB production rate - 0.4 g h(-1)).     

Perfusion culture process plus H2O2 stimulation for efficient astaxanthin production by Xanthophyllomyces dendrorhous

A semicontinuous perfusion culture process (repeated medium renewal with cell retention) was evaluated together with batch and repeated fed-batch processes for astaxanthin production in shake-flask cultures of Xanthophyllomyces dendrorhous. The perfusion process with 25% medium renewal every 12 h for 10 days achieved a biomass density of 65.6 g/L, a volumetric astaxanthin yield of 52.5 mg/L, and an astaxanthin productivity of 4.38 mg/L-d, which were 8.4-fold, 5.6-fold, and 2.3-fold of those in the batch process, 7.8 g/L, 9.4 mg/L, and 1.88 mg/L-d, respectively. The incorporation of hydrogen peroxide (H(2)O(2)) stimulation of astaxanthin biosynthesis into the perfusion process further increased the astaxanthin yield to 58.3 mg/L and the productivity to 4.86 mg/L-d. The repeated fed-batch process with 8 g/L glucose and 4 g/L corn steep liquor fed every 12 h achieved 42.2 g/L biomass density, 36.5 mg/L astaxanthin yield, and 3.04 mg/L-d astaxanthin productivity. The lower biomass and astaxanthin productivity in the repeated fed-batch than in the perfusion process may be mostly attributed to the accumulation of inhibitory metabolites such as ethanol and acetic acid in the culture. The study shows that perfusion process plus H(2)O(2) stimulation is an effective strategy for enhanced astaxanthin production in X. dendrorhous cultures.     

Effect of sugar-feeding strategies on astaxanthin production by <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis>

Summary Astaxanthin, a main carotenoid pigment, has a strong antioxidant activity. The effects of sugar-feeding strategies on astaxanthin production by Xanthophyllomyces dendrorhous fed-batch fermentation were studied. Sugar was added instantaneously to a 30 l fermentor at various discrete time instants (pulse-feeding), or continuously to the fermentor to maintain the constant sugar concentration. Pulse-feeding experiments were initiated with sugar concentrations from 30 to 50 g/l, and then one, two, three and four feedings were made. The results showed that optimal sugar feeding depended on feeding time and pulse sugar feeding was the best among all experiments, in which a significant increase (54.9%) in production of astaxanthin was achieved at 132 h in a pulse fed-batch process compared with a batch process.     

Effect of feeding strategy on Zymomonas mobilis CP4 fed-batch fermentations and mathematical modeling of the system

In this work, the effect of the feeding strategy in Zymomonas mobilis CP4 fed-batch fermentations on the final biomass and ethanol concentrations was studied. Highest glucose yields to biomass (0.018 g/g) and to ethanol (0.188 g/g) were obtained in fed-batch fermentations carried out using different feeding rates with a glucose concentration in the feed equal to 100 g/l. Lower values (0.0102 g biomass/g glucose and 0.085 g ethanol/g glucose) were obtained when glucose accumulated to levels higher than 60 g/l. On the other hand, the highest biomass (5 g/l) and ethanol (39 g/l) concentrations were obtained using a glucose concentration in the feed equal to 220 g/l and exponentially varied feeding rates. Experimental data were used to validate the mathematical model of the system. The prediction errors of the model are 0.39, 14.36 and 3.24 g/l for the biomass, glucose and ethanol concentrations, respectively. Due to the complex relationship for describing the specific growth rate, a fed-batch culture in which glucose concentration is constant would not optimize the process. Received: 30 November 1999 / Received revision: 24 March 2000 / Accepted: 7 April 2000     

Calorimetric control of fed-batch cultures of Saccharomyces cerevisiae

Calorimetry has been used to control the glucose feeding in fed-batch cultures of S. cerevisiae in order to avoid ethanol formation and maintain a fully respiratory metabolism. Comparisons between batch and fed-batch cultivations showed that the former had a much lower growth yield. The growth yields for fed-batch cultivations were more than 30% higher than for batch cultures. However, energy balance calculations showed that a large part of the increase could be explained by the evaporation of ethanol during batch cultivations. When the growth yields obtained from the batch cultures were corrected for the evaporation of ethanol, the increase in growth yield for fed-batch cultures was about 10%.     

Astaxanthin from Jerusalem artichoke: Production by fed-batch fermentation using Phaffia rhodozyma and application in cosmetics

Jerusalem artichoke extract or powder was used for astaxanthin production using Phaffia rhodozyma without acidic or enzymatic inulin hydrolysis. The culture medium containing Jerusalem artichoke as carbon source was optimized, and feeding strategies, including constant, exponential, pH-stat, and substrate feedback fed-batch fermentations, were also compared for enhancing the cell biomass and astaxanthin synthesis by P. rhodozyma. Substrate-feedback fed-batch fermentation resulted in the highest dry cell weight of 83.60 g/L, with a carotenoid concentration and yield of 982.50 mg/L and 13.30 mg/g, respectively, under optimized medium components using Jerusalem artichoke extract as carbon source in a 3-L stirred-tank bioreactor. Moreover, 482.50 mg/L of carotenoids and 253.10 mg/L of astaxanthin were obtained by continuous feeding of Jerusalem artichoke powder, which was used as carbon source. Astaxanthin essence with high DPPH-scavenging activity was obtained from the extracted astaxanthin, and the DPPH free radical scavenging rate of 40 ppm astaxanthin essence reached 76.29%. When stored at 4 °C, astaxanthin essence showed the highest stability, with a minimum k value of 0.0099 week⁻¹ and maximum half-life (t_1/2) value of 70 weeks.     

Effect of galactose feeding on the improved production of hirudin in fed-batch cultures of recombinant Saccharomyces cerevisiae

Different feeding strategies of galactose were employed to improve the production of anticoagulant, hirudin, by fed-batch mode of cultivation from recombinant Saccharomyces cerevisiae. The structural gene coding for hirudin was harboured with GAL10 promoter for controlled expression of hirudin and the MFα 1 signal sequence for secretion into the growth medium. A step-wise feeding of galactose was found as more suitable feeding strategy of galactose which resulted in the final hirudin volumetric productivity of 6,840?μg/l?·?h, than intermittent, continuous and ethanol controlled feeding of galactose. The final volumetric productivity of hirudin obtained by step-wise feeding of galactose was 3.88 fold higher compared with simple batch fermentation.     

Constant-volume fed-batch operation for high density cultivation of hyperthermophilic aerobes

When hyperthermophilic archaebacteria are cultivated under aerobic conditions, the loss of the culture volume becomes significant due to vaporization of water with aeration. To eliminate the problems caused by evaporation of the medium, we have devised a constant-volume fed-batch system, where the loss of water due to evaporation is compensated by feeding additional water into the fermentor. The feeding strategy for maintaining a constant volume during fed-batch cultivation was developed from the water balance and the cell growth and substrate consumption kinetics, and the developed method was applied to high density cultivation of Sulfolobus solfataricus DSM 1617. The maximum cell density obtained was 22.6 g/L, which is the highest value reported for hyperthermophiles in fed-batch operations.     

基于动力学模型的法夫酵母发酵生产虾青素的补料策略优化

对法夫酵母的不同补料发酵方式进行了研究.基于底物抑制模型,提出了一种优化的两阶段补料策略,用于法夫酵母产虾青素的高密度发酵.在发酵的延迟期和对数生长期早期,糖浓度控制在25 g/L左右,在此条件下,生物量可以达到最大,且时间缩短.在对数生长期后期及稳定期,糖浓度控制在5 g/L,虾青素的合成时间可以有效延长.与传统的补料方式相比,采用此补料策略取得了较好的发酵效果.发酵终点细胞干重达到23.8g/L,虾青素产量达到29.05 mg/L,分别比分批发酵提高了52.8%和109%.     

Exploring low-cost carbon sources for microbial lipids production by fed-batch cultivation of <Emphasis Type="Italic">Cryptococcus albidus</Emphasis>

Volatile fatty acids (VFAs), acetic acid, acetates, and ethanol were used as carbon sources for the production of microbial lipids using Cryptococcus albidus in batch cultures. C. albidus utilized organic acids less than glucose in the production of lipids, resulting in a lipid yield coefficient on VFAs of 0.125 g/g. In a two-stage batch culture, the lipid content increased to 43.8% (w/w) when VFAs were used as the sole carbon source in the second stage, which was two times higher than that of the batch culture. Furthermore, a 192 h, two-stage fed-batch cultivation of C. albidus produced a dry cell weight, lipid concentration, and lipid content of 26.4 g/L, 14.5 g/L, and 55.1% (w/w), respectively. The fed-batch culture model used in this study featured pure VFA solutions, with intermittent feeding, under oxygen-enriched air supply conditions. This study investigated several alternative carbon sources to reduce the cost of microbial lipids production and proved the feasibility of using VFAs as the carbon source for the provision of a high lipid content and productivity.     

Utilization of cane molasses towards cost-saving astaxanthin production by a Chlorella zofingiensis mutant

The aim of the present study was to survey the growth and astaxanthin production of E17, an astaxanthin-rich mutant of Chlorella zofingiensis, through feeding the low-cost carbon source cane molasses. In heterotrophic batch cultivation, E17 fed with pretreated molasses achieved biomass (1.79 g L^?1 day^?1) and astaxanthin (1.99 mg L^?1 day^?1) productivities comparable to those with glucose, which were about 2- and 2.8-fold of those fed with untreated molasses, respectively. Molasses-induced astaxanthin accumulation may be attributed to the elicited expression of carotenogenic genes, in particular the genes specifically responsible for the ketolation and hydroxylation of β-carotene to form astaxanthin. A two-stage fed-batch strategy was employed to grow E17 and induce astaxathin accumulation, resulting in 45.6 g L^?1 biomass and 56.1 mg L^?1 astaxanthin, the highest volumetric astaxanthin yield ever reported for this alga. In addition, the astaxanthin production by E17 was tested with a semi-continuous culture method, where the directly diluted raw molasses (giving 5 g L^?1 sugar) was used as the carbon source. Little growth inhibition of E17 was observed in the semi-continuous culture with a biomass productivity of 1.33 g L^?1 day^?1 and an astaxanthin productivity of 0.83 mg L^?1 day^?1. The mixotrophic semi-continuous cultures enhanced the biomass and astaxanthin productivities by 29.3 % and 42.2 %, respectively. This study highlights the potential of using the industrially cheap cane molasses towards large-scale cost-saving production of the high-value ketocarotenoid astaxanthin.     

Integration of the production and the purification processes of cutinase secreted by a recombinant Saccharomyces cerevisiae SU50 strain

By expanded bed adsorption (EBA) it was possible to simultaneously recover and purify the heterologous cutinase directly from the crude feedstock. However, it was observed that in a highly condensed and consequently economically advantageous purification process as EBA, the cultivation step highly influences the following purification step. Thus, the yeast cultivation and cutinase purification by EBA cannot be considered as independent entities, and the understanding of the interactions between them are crucial for the development of a highly cost effective overall cutinase production process. From the cultivation strategies studied, one batch, one continuous and two fed-batch cultivations, the strategy that resulted in a more economical cutinase overall production process was a fed-batch mode with a feeding in galactose. This last cultivation strategy, exhibited the highest culture cutinase activity and bioreactor productivity, being obtained 3.8-fold higher cutinase activity and 3.0-fold higher productivity that could compensate the 40% higher cultivation medium costs when compared with a fed-batch culture with a feeding on glucose and galactose. Moreover, a 3.8-fold higher effective cutinase dynamic adsorption capacity and 3.8-fold higher effective purification productivity were obtained in relation to the fed-batch culture with the feeding on glucose and galactose. The cultivation strategy with a feeding on galactose, that presented 5.6-fold higher effective purification productivity, could also compensate the 32% effective adsorption capacity obtained with a continuous cultivation broth. Furthermore, a 205-fold higher cutinase activity, 24-fold higher bioreactor productivity and 6% of the cultivation medium costs were obtained in relation to the continuous culture.     

Growth kinetics ofSaccharomyces cerevisiae in batch and fedbatch cultivation using sugarcane molasses and glucose syrup from cassava starch

Growth kinetics ofSaccharomyces cerevisiae in glucose syrup from cassava starch and sugarcane molasses were studied using batch and fed-batch cultivation. The optimum temperature and pH required for growth were 30°C and pH 5.5, respectively. In batch culture the productivity and overall cell yield were 0.31 g L^–1 h^–1 and 0.23 g cells g^–1 sugar, respectively, on glucose syrup and 0.22 g L^–1 h^–1 and 0.18 g cells g^–1 sugar, respectively, on molasses. In fed-batch cultivation, a productivity of 3.12 g L^–1 h^–1 and an overall cell yield of 0.52 g cells g^–1 sugar in glucose syrup cultivation and a productivity of 2.33 g L^–1 h^–1 and an overall cell yield of 0.46 g cells g^–1 sugar were achieved in molasses cultivation by controlling the reducing sugar concentration at its optimum level obtained from the fermentation model. By using an on-line ethanol sensor combined with a porous Teflon^® tubing method in automating the feeding of substrate in the fed-batch culture, a productivity of 2.15 g L^–1 h^–1 with a yield of 0.47 g cells g^–1 sugar was achieved using glucose syrup as substrate when ethanol concentration was kept at a constant level by automatic control.     

Controlled pilot development unit-scale fed-batch cultivation of yeast on spruce hydrolysates

Yeast production on hydrolysate is a likely process solution in large-scale ethanol production from lignocellulose. The hydrolysate will be available on site, and the yeast has furthermore been shown to acquire an increased inhibitor tolerance when cultivated on hydrolysate. However, due to over-flow metabolism and inhibition, efficient yeast production on hydrolysate can only be achieved by well-controlled substrate addition. In the present work, a method was developed for controlled addition of hydrolysate to PDU (process development unit)-scale aerobic fed-batch cultivations of Saccharomyces cerevisiae TMB 3000. A feed rate control strategy, which maintains the ethanol concentration at a low constant level, was adapted to process-like conditions. The ethanol concentration was obtained from on-line measurements of the ethanol mole fraction in the exhaust gas. A computer model of the system was developed to optimize control performance. Productivities, biomass yields, and byproduct formation were evaluated. The feed rate control worked satisfactorily and maintained the ethanol concentration close to the setpoint during the cultivations. Biomass yields of 0.45 g/g were obtained on added hexoses during cultivation on hydrolysate and of 0.49 g/g during cultivation on a synthetic medium with glucose as the carbon source. Exponential growth was achieved with a specific growth rate of 0.18 h-1 during cultivation on hydrolysate and 0.22 h-1 during cultivation on glucose.     

Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture

Production of astaxanthin by sequential heterotrophic-photoautotrophiccultivation of a green alga, Haematococcus pluvialis was investigated.This involved cultivating the cells heterotrophically to high cellconcentration, followed by illumination of the culture for astaxanthinaccumulation. The optimum pH and temperature for heterotrophic biomassproduction were 8 and 25 ^°C, respectively. There was no significantdifference in the specific growth rate of the cells when acetateconcentration was varied between 10 mM and 30 mM. However, cellgrowth was inhibited at higher acetate concentrations. A pH stat methodwas then used for fed-batch heterotrophic culture, using acetate as theorganic carbon source. A cell concentration of 7 g L^-1 wasobtained. Higher cell concentration could not be obtained because the cellschanged from vegetative to cyst forms during the heterotrophic cultivation.However, by using repeated fed-batch processes, the cells could bemaintained in the vegetative form, leading to more than two times increasein cell number output rate. When the vegetative cells were transferred tophotoautotrophic phase, there was a sharp decrease in the cell number andonly very few cells encysted and accumulated astaxanthin. On the otherhand, when the shift from heterotrophic to photoautotrophic condition wasdone when most of the cells had encysted, there was still a decrease in cellnumber but astaxanthin accumulation was very high. The astaxanthinconcentration (114 mg L^-1) and productivity (4.4 mg L^-1d^-1) obtained by this sequential heterotrophic-photoautotrophiccultivation method are very high compared to the data in the literature.     

Simple fed-batch cultivation strategy for the enhanced production of a single-sugar glucuronic acid-based oligosaccharides by a cellulose-producing <Emphasis Type="Italic">Gluconacetobacter hansenii</Emphasis> strain

The production of water-soluble single-sugar glucuronic acid-based oligosaccharides (WSOS) by a cellulose producing strain Gluconacetobacter hansenii PJK was studied in a periodically recycled and fed-batch cultivations using glucose/ethanol or glucose only. Fermentations were carried out in a 2 L jar fermenter equipped with a turbine impeller with 6 flat blades. WSOS were produced constantly but the bacterial cellulose (BC) production stopped at 48 h of cultivation in a periodically recycled culture using the exhausted medium supplemented with glucose and ethanol. Tremendous quantities of WSOS were obtained in fed-batch cultivations using glucose/ethanol (35.6 g/L at 132 h of cultivation) or glucose only (86 g/L after 240 h of cultivation) as the nutritional source. However, the BC production yield under these nutritional conditions decreased significantly in comparison to previous studies about the BC production by the same strain. The overall results revealed that G. hansenii is capable of producing enormous quantities of WSOS compared to those reported previously for compounds of a related chemical nature. Moreover, the WSOS production was found to be dependent on the pH of the culture broth.