Aquacultural characteristics of <Emphasis Type="Italic">Rhizoclonium</Emphasis><Emphasis Type="Italic">riparium</Emphasis> and an evaluation of its biomass growth potential

A study was made of environmental factors affecting the growth of Rhizoclonium riparium in order to evaluate its suitability for large-scale culturing. The results indicate that under the natural conditions prevailing at Taishi, Taiwan, this species can grow year-round, with a monthly biomass production (oven-dried) of 945–1540 kg ha⁻¹ pond surface (assuming a pond depth of 1 m). The specific growth rate ranged from –2.1 to 10.4% per day. Salinity and temperature, both influenced the rate significantly, with optimal values at 20% and 25 °C, respectively. Short (2-mm) lengths of filaments had a higher specific growth rate than longer (20 mm) filaments. Under rotational culturing conditions, the specific growth rate was reduced when flow was increased.     

Improving intracellular production of recombinant protein in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using an optimized preinduction glycerol-feeding scheme

High-cell-density production of recombinant growth hormone of Lateolabrax japonicus (rljGH) expressed intracellularly in Pichia pastoris was investigated. In the regular strategy of induction at a cell density of 160 g l⁻¹, short duration of intracellular rljGH accumulation (17 h) resulted in a low final cell density of 226 g l⁻¹. Thus, a novel strategy of induction at a cell density of 320 g l⁻¹ was investigated. In this strategy, the preinduction glycerol-feeding scheme had a significant effect on the post-induction production. Constant glycerol feeding led to a decrease of the specific rljGH production and specific production rate because of low preinduction specific growth rate. This decrease was avoided by exponential glycerol feeding to maintain a preinduction specific growth rate of 0.16 h⁻¹. The results from exponential glycerol feeding indicated that the rljGH production depended on the preinduction specific growth rate. Moreover, mixed feeding of methanol and glycerol during induction improved the specific production rate to 0.07 mg g⁻¹ h⁻¹ from 0.043 mg g⁻¹ h⁻¹. Consequently, both high cell density (428 g l⁻¹) and high rljGH production could be achieved by the novel strategy: growing the cells at the specific growth rate of 0.16 h⁻¹ to the cell density of 320 g l⁻¹ and inducing the expression by mixed feeding.     

Studies on bacteriocin (thermophilin T) production by <Emphasis Type="Italic">Streptococcus thermophilus</Emphasis> ACA-DC 0040 in batch and fed-batch fermentation modes

Growth conditions that support bacteriocin (thermophilin T) production by Streptococcus thermophilus ACA-DC 0040 were identified. Synthesis of thermophilin T occurred during primary metabolic growth, while its specific rate of synthesis seemed to be optimal at T = 30°C. Thermophilin T activity rapidly decreased in the stationary phase, especially at high growth temperature (i.e. T = 42°C). In media with high content of complex nitrogen sources, high amounts of bacteriocin were detected in the growth environment, while about an 8-fold increase of thermophilin T titer and a 2-fold increase of specific synthesis rate was achieved when a fed-batch fermentation mode was applied.     

Temperature management strategy for efficient gene expression in a thermally inducible <Emphasis Type="Italic">Escherichia coli</Emphasis>/bacteriophage system

In a two-phase operation, E. coli containing λSNNU1 (Q ⁻ S ⁻) in the chromosome is typically cultured at 33°C and cloned gene expression is induced by elevating the temperature. At least 40°C is necessary for complete induction of cloned gene expression; however, temperatures above 40°C have been shown to inhibit cloned gene expression. This suggests that a three-phase operation, which has an induction phase between the growth and production phases, may result in higher gene expression. In this study, optimal temperature management strategies were investigated for the three-phase operation of cloned gene expression in thermally inducible E. coli/bacteriophage systems. The optimal temperature for the induction phase was determined to be 40°C. When the temperature of the production stage was 33°C, the optimal time period for the induction phase at 40°C was determined to be 60 min. In contrast, when the temperature of the production phase was 37°C, the optimal period for the induction phase at 40°C was 20∼30 min. When the three-phase temperature and temporal profile were set at a growth phase of 33°C, an induction phase at 40°C for 30 min, and a production phase at 37°C, the highest level of cloned gene expression was achieved.     

Influence of culture conditions on glutathione production by <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

A strategy of experimental design using a fractional factorial design (FFD) and a central composite rotatable design (CCRD) were carried out with the aim to obtain the best conditions of temperature (20–30°C), agitation rate (100–300 rpm), initial pH (5.0–7.0), inoculum concentration (5–15%), and glucose concentration (30–70 g/l) for glutathione (GSH) production in shake-flask culture by Saccharomyces cerevisiae ATCC 7754. By a FFD (2^5–2), the agitation rate, temperature, and pH were found to be significant factors for GSH production. In CCRD (2²) was obtained a second-order model equation, and the percent of variation explained by the model was 95%. The results showed that the optimal culture conditions were agitation rate, 300 rpm; temperature, 20°C; initial pH, 5; glucose, 54 g/l; and inoculum concentration, 5%. The highest GSH concentration (154.5 mg/l) was obtained after 72 h of fermentation.     

A phenomenological model to represent the kinetics of growth by <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis> for lysine production

Corynebacterium glutamicum is commonly used for lysine production. In the last decade, several metabolic engineering approaches have been successfully applied to C. glutamicum. However, only few studies have been focused on the kinetics of growth and lysine production. Here, we present a phenomenological model that captures the growth and lysine production during different phases of fermentation at various initial dextrose concentrations. The model invokes control coefficients to capture the dynamics of lysine and trehalose synthesis. The analysis indicated that maximum lysine productivity can be obtained using 72 g/L of initial dextrose concentration in the media, while growth was optimum at 27 g/L of dextrose concentration. The predictive capability was demonstrated through a two-stage fermentation strategy to enhance the productivity of lysine by 1.5 times of the maximum obtained in the batch fermentation. Two-stage fermentation indicated that the kinetic model could be further extended to predict the optimal feeding strategy for fed-batch fermentation.     

Modeling and optimization of alpha-amylase production in a recombinant yeast fed-batch culture taking account of the cell cycle population distribution.

A simple mathematical model describing the cell cycle dependency of rice alpha-amylase production by a recombinant yeast was constructed to investigate the efficiency of cell cycle population control. First, the effects of the glucose concentration and cultivation temperature on the specific growth rate, the specific production rate of rice alpha-amylase, and the distribution of the cell cycle population were studied under balanced growth conditions. On the basis of the results, parameter values for the mathematical model were then estimated. The proposed model was shown to be applicable for unbalanced as well as balanced growth phases. The optimal control strategy in respect of temperature and glucose concentration for maximum rice alpha-amylase production, taking into account the cell cycle population, was determined and the result was compared with that obtained by a simple mathematical model in which cell cycle distribution was not considered. Finally, the effect of the initial population of each cell cycle phase on the final amount of the product under optimal operational conditions was investigated. The simulation and experimental data coincided well with each other, and the model was used to optimize the control strategy for maximum alpha-amylase production.     

Alcoholic fermentation of xylose and mixed sugars using recombinant <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> engineered for xylose utilization

Previously, a Saccharomyces cerevisiae strain was engineered for xylose assimilation by the constitutive overexpression of the Orpinomyces xylose isomerase, the S. cerevisiae xylulokinase, and the Pichia stipitis SUT1 sugar transporter genes. The recombinant strain exhibited growth on xylose, under aerobic conditions, with a specific growth rate of 0.025 h⁻¹, while ethanol production from xylose was achieved anaerobically. In the present study, the developed recombinant yeast was adapted for enhanced growth on xylose by serial transfer in xylose-containing minimal medium under aerobic conditions. After repeated batch cultivations, a strain was isolated which grew with a specific growth rate of 0.133 h⁻¹. The adapted strain could ferment 20 g l⁻¹ of xylose to ethanol with a yield of 0.37 g g⁻¹ and production rate of 0.026 g l⁻¹ h⁻¹. Raising the fermentation temperature from 30°C to 35°C resulted in a substantial increase in the ethanol yield (0.43 g g⁻¹) and production rate (0.07 g l⁻¹ h⁻¹) as well as a significant reduction in the xylitol yield. By the addition of a sugar complexing agent, such as sodium tetraborate, significant improvement in ethanol production and reduction in xylitol accumulation was achieved. Furthermore, ethanol production from xylose and a mixture of glucose and xylose was also demonstrated in complex medium containing yeast extract, peptone, and borate with a considerably high yield of 0.48 g g⁻¹.     

Overproduction of glutathione by <Emphasis Type="SmallCaps">l</Emphasis>-cysteine addition and a temperature-shift strategy

The present study investigated the effects of three constituent amino acids on glutathione production in flask culture of Candida utilis. Although l-glutamic acid and glycine had little impact on cell growth and glutathione biosynthesis, l-cysteine positively influenced glutathione production, despite inhibiting cell growth when it was added prior to stationary phase. Adding 8 mmol/L of l-cysteine to the culture broth at 16 h boosted glutathione production by 91%, increasing the intracellular glutathione content by 106% compared to untreated controls. A temperature-shift strategy, in which we shifted batch and fed-batch cultures of C. utilis from 30 to 26°C, also significantly enhanced glutathione production. Applying both strategies (i.e. adding 20 mmol/L l-cysteine and shifting the temperature from 30 to 26°C) at 33 h enhanced the glutathione concentration and the intracellular glutathione content to 1,312 mg/L and 3.75%, respectively, during fed-batch cultivation (glucose feeding at a constant rate of 18.3 g/h). The average specific glutathione production rate under this condition was 129% higher than that of the control without strategy.     

Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.     

Improving arachidonic acid fermentation by Mortierella alpina through multistage temperature and aeration rate control in bioreactor

Effective production of arachidonic acid (ARA) using Mortierella alpina was conducted in a 30-L airlift bioreactor. Varying the aeration rate and temperature significantly influenced cell morphology, cell growth, and ARA production, while the optimal aeration rate and temperature for cell growth and product formation were quite different. As a result, a two-stage aeration rate control strategy was constructed based on monitoring of cell morphology and ARA production under various aeration rate control levels (0.6–1.8 vvm). Using this strategy, ARA yield reached 4.7 g/L, an increase of 38.2% compared with the control (constant aeration rate control at 1.0 vvm). Dynamic temperature-control strategy was implemented based on the fermentation performance at various temperatures (13–28°C), with ARA level in total cellular lipid increased by 37.1% comparing to a constant-temperature control (25°C). On that basis, the combinatorial fermentation strategy of two-stage aeration rate control and dynamic temperature control was applied and ARA production achieved the highest level of 5.8 g/L.     

Contrasting ‘Top-Down’ Effects of Crustacean Zooplankton Grazing on Bacteria and Phytoflagellates

The combined effects of grazing and nutrient regeneration by Daphnia and Eudiaptomus on the growth of Rhodomonas and heterotrophic bacteria was assessed experimentally. The responses of Rhodomonas and bacteria to the grazers were measured as net specific growth rate over the entire experimental periods, as well as production and specific production at the end of the experiments. Both zooplankton species had a negative effect on Rhodomonas net specific growth rate due to grazing and a positive effect on specific primary production due to nutrient regeneration. Daphnia had no effect on bacterial net specific growth rate, bacterial production or specific bacterial production in one of two experiments. In the other experiment, however, both bacterial growth rate and production decreased as a result of grazing. Furthermore, Daphnia had a negative effect on specific bacterial production, but Eudiaptomus had a positive effect on all bacterial parameters due to nutrient regeneration, probably of phosphorus. Positive effects of copepods on bacterial growth has previously been attributed to trophic cascades via protozoa. However, the present experiments show that regeneration of nutrients, especially phosphorus, may account for a large part of the stimulation of bacterial growth.     

Modelling faecal <Emphasis Type="Italic">streptococci</Emphasis> mortality in constructed wetlands implanted with <Emphasis Type="Italic">Eichhornia crassipes</Emphasis>

Faecal streptococci mortality was investigated in a water hyacinth (Eichhornia crassipes) constructed wetland pond. The wetland was 7.5 m long, 1.5 m wide and 1.0 m deep, and was implanted with E. crassipes. In order to assess the performance of the system towards bacterial mortality, a mathematical model, based on plug flow philosophy was developed. The model incorporated the role of factors, namely solar intensity, pH, dissolved oxygen, temperature, sedimentation, and root attached growth. Model analysis strongly suggests that bacterial mortality rate constant was largely influenced by two factors, namely solar intensity and root biofilm attachment, with both contributing approximately 70.5% of removal. The contribution of other factors like temperature, dissolved oxygen, pH and sedimentation on bacterial mortality rate were less significant. For example, dissolved oxygen, pH and sedimentation contributed 5%, 8% and 0.82%, respectively. Thus, the sedimentation factor was omitted from the model because of its insignificant contribution. The same was done for temperature, due to low ambient temperature range (3.1°C) in the study area. The overall model bacterial removal efficiency was 83%.     

Effects of temperature,photosynthetic photon flux density,photoperiod and O<Subscript>2</Subscript> and CO<Subscript>2</Subscript> concentrations on growth rates of the symbiotic dinoflagellate, <Emphasis Type="Italic">Amphidinium</Emphasis> sp.

Symbiotic dinoflagellates of the species Amphidinium are expected to be pharmaceutically useful microalgae because they produce antitumor macrolides. A microalgae production system with a large number of cells at a high density has been developed for the efficient production of macrolide compounds. In the present study, the effects of culture conditions on the cellular growth rate of dinoflagellates were investigated to determine the optimum culture conditions for obtaining high yields of microalgae. Amphidinium species was cultured under conditions with six temperature levels (21–35°C), six levels of photosynthetic photon flux density (15–70 μmol photons m⁻² s⁻¹), three levels of CO₂ concentration (0.02–0.1%), and three levels of O₂ concentration (0.2–21%). The number of cells cultured in a certain volume of solution was monitored microscopically and the cellular growth rate was expressed as the specific growth rate. The maximum specific growth rate was 0.022 h⁻¹ at a temperature of 26°C and O₂ concentration of 5%, and the specific growth rate was saturated at a CO₂ concentration of 0.05%, a photosynthetic photon flux density of 35 μmol photons m⁻² s⁻¹ and a photoperiod of 12 h day⁻¹ upon increasing each environmental parameter. The results demonstrate that Amphidinium species can multiply efficiently under conditions of relatively low light intensity and low O₂ concentration.     

Temperature profiles of cellular growth and exopolysaccharide synthesis by Botryococus braunii Kütz. UC 58

Temperature profiles (range 20–33 °C) were obtained for growth and exopolysaccharide (EPS) biosynthesis of the microalga Botryococcus braunii strain UC 58 under photoautotrophic conditions. The maximum temperature for growth was 32 °C and the temperature dependence of the specific growth rate was described by the Hinshelwood equation based on the Arrhenius relationship. The optimal range of temperatures for growth and extracellular EPS synthesis (25–30 °C) concurred and production of 4.5–5 g l⁻¹ of EPS was obtained routinely, leading to high broth viscosities. Below 23 °C EPS biosynthesis was negligible, although the specific growth rate maintained high values. At supraoptimal temperatures EPS biosynthesis decreased, accompanying the increase in doubling time. The polymers formed at temperatures within the optimal range for production, when dissolved in water, produced solutions (2 gl⁻¹) with the highest viscosity, suggesting that their molecular weight showed the highest values. The degree of polymerization of the EPS synthesized at suboptimal and supraoptimal temperatures was significantly below the values within the optimal range.     

Optimal pH control of batch processes for production of curdlan by Agrobacterium species

We sought an optimal pH profile to maximize curdlan production in a batch fermentation of Agrobacterium species. The optimal pH profile was calculated using a gradient iteration algorithm based on the minimum principle of Pontryagin. The model equations describing cell growth and curdlan production were developed as functions of pH, sucrose concentration, and ammonium concentration, since the specific rates of cell growth and curdlan production were highly influenced by those parameters. The pH profile provided the strategy to shift the culture pH from the optimal growth condition (pH 7.0) to the optimal production one (pH 5.5) at the time of ammonium exhaustion. By applying the optimal pH profile in the batch process, we obtained significant improvement in curdlan production (64 g L⁻¹) compared to that of constant pH operation (36 g L⁻¹). Received 24 November 1998/ Accepted in revised form 17 June 1999     

A maximum production strategy of lysine based on a simplified model derived from a metabolic reaction network

The aim of this study is to develop a strategy for maximum production of a target product with a simplified model derived from a metabolic reaction network through an example of lysine production. Based on the model, a search for the optimal specific growth rate profile was conducted among the available conditions of batch fermentation based on the derived model, when the total fermentation time was fixed. The optimal specific growth rate was obtained as a boundary control: initially, the specific growth rate was maintained at a maximum value and was subsequently switched to a critical value giving the maximum specific production rate. To make the specific growth rate follow this optimal profile as accurately as possible in batch mode, first, an appropriate initial concentration of leucine was employed in the experiment. Second, the feeding strategy of leucine was further studied. The specific growth rate profile with feeding was closer to the optimal one and the amount of lysine produced at the final stage of fermentation was increased about twofold, compared to that in the batch fermentation. Finally, the strategy was summarized as an algorithm for general use of this method.     

Scale-up fermentation of recombinant <Emphasis Type="Italic">Candida rugosa</Emphasis> lipase expressed in <Emphasis Type="Italic">Pichia pastoris</Emphasis> using the GAP promoter

The high-cell-density fermentation of Candida rugosa lipase in the constitutive Pichia pastoris expression system was scaled up from 5 to 800 l in series by optimizing the fermentation conditions at both lab scale and pilot scale. The exponential feeding combined with pH-stat strategy succeeded in small scale studies, while a two-stage fermentation strategy, which shifted at 48 h by fine tuning the culture temperature and pH, was assessed effective in pilot-scale fermentation. The two-stage strategy made an excellent balance between the expression of heterogeneous protein and the growth of host cells, controlling the fermentation at a relatively low cell growth rate for the constitutive yeast expression system to accumulate high-level product. A stable lipase activity of approximately 14,000 IU ml⁻¹ and a cell wet weight of ca. 500 g l⁻¹ at the 800-l scale were obtained. The efficient and convenient techniques suggested in this study might facilitate further scale-up for industrial lipase production.     

Cultural characteristics and extraction of the fungal pigment phleichrome from the phytopathogenic fungus<Emphasis Type="Italic">Cladosporium phlei</Emphasis>

The cultural characteristics of the fungusCladosporium phlei were assessed in order to develop an improved method for the production of the fungal pigment, phleichrome, which is an intermediate in the production of a photodynamic therapeutic agent. The growth ofC. phlei, as measured by the hyphal growth rate and increase in biomass, varies significantly depending on the culture media utilized (V8 juice-based medium proved optimal for both growth rate and biomass increase). How-ever, even on a V8 juice plate, the growth ofC. phlei occurred slowly and in a limited fashion, in that the colony covered only 75% of the agar surface after more than 4 weeks of cultivation at 20°C. Supplementations of glucose, fructose, galactose, and sucrose increased both hyphal expansion and mass production, whereas supplementations of other carbon sources, including glycerol and sorbitol, exerted no detectable effects. The effect of inorganic nitrogen supplementation was negligible, whereas organic nitrogen evidenced significant effects, with enhanced growth with malt extract and growth inhibition with yeast extract and tryptone. Sporulation was enhanced under conditions of continuous light, and a minimum of 10³ spores per mL of liquid media was found to be necessary for the optimal mass increase. A simple extraction procedure was established in order to isolate the deep red pigment which was subsequently identified as phleichrome via NMR analysis. WhenC. phlei was cultured on V8 medium containing 5% glucose and 2% malt extract, the quantity of mycelial mass was estimated as 20.6 g (dry weight) per liter of culture. The expected phleichrome yields from the mycelia and culture filtrates were estimated to be 43 and 2 mg/L, repectively. There was an equal contribution of the reported research by the first two authors.     

Modeling and optimization of hairy root growth in fed‐batch process

This article proposes a feeding strategy based on a kinetic model to enhance hairy roots growth. A new approach for modeling hairy root growth is used, considering that there is no nutrient limitation thanks to an appropriate feeding, and the intracellular pools are supposed to be always saturated. Thus, the model describes the specific growth rate from extracellular concentration of the major nutrients and nutrient uptakes depend on biomass growth. An optimized feeding strategy was determined thanks to the model to maintain the major nutrient levels at their optimum assuming optimal initial concentrations. The optimal feed rate is computed in open loop using kinetic model prediction or in closed loop using conductivity measurements to estimate biomass growth. Datura innoxia was chosen as the model culture system. Shake flask cultures were used to calibrate the model. Finally, cultures in bioreactor were performed to validate the model and the control laws. © 2010 American Institute of Chemical Engineers Biotechnol. Prog., 2010