Independent exponential feeding of glycerol and methanol for fed-batch culture of recombinant Hansenula polymorpha DL-1

As a novel feeding strategy for optimizing human epidermal growth factor (hEGF) production with a recombinant Hansenula polymorpha DL-1 using the methanol oxidase (MOX) promoter in H. polymorpha DL-1, independent exponential feeding of two substrates was used. A simple kinetic model considering the cell growth on two substrates was established and used to calculate the respective feeding rates of glycerol and methanol. In the fedbatch culture with methanol-only feeding, the optimal set point of specific growth rate on methanol was found to be 0.10 h-1. When the fed-batch cultures were conducted by the independent feeding of glycerol and methanol, the actual specific growth rate on glycerol and methanol was slightly lower than the set point of specific growth rate. By the uncoupled feeding of glycerol and methanol the volumetric productivity of hEGF increased from 6.4 to 8.0 mg/(L.h), compared with methanol-only feeding.     

Effect of Constant Glucose Feeding on the Production of Exopolysaccharides by <Emphasis Type="Italic">Tremella fuciformis</Emphasis> Spores

A fed-batch culture system with constant feeding (glucose 80 g L⁻¹, 0.25 ml min⁻¹) was used to study the influence of glucose on cell dry weight and exopolysaccharides production from submerged Tremella fuciformis spores in a 5-L stirred-tank bioreactor. The results showed that high levels of cell mass (9.80 g L⁻¹) and exopolysaccharides production (3.12 g L⁻¹) in fed-batch fermentation were obtained after 1 h of feeding, where the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.267 d⁻¹ and 0.14 g g⁻¹. Unlike batch fermentation, maximal cell mass and exopolysaccharides production merely reached 7.11 and 2.08 g L⁻¹; the specific growth rate (μ) and exopolysaccharides yield on substrate consumed (YP/S) were 0.194 d⁻¹ and 0.093 g g⁻¹, respectively. It is concluded that the synthesis of exopolysaccharides can be promoted effectively when feeding glucose at a late exponential phase.     

Effects of glycerol concentration and pH on growth of recombinantPichia pastoris yeast

Batch fermentations were used to study the effect of different glycerol concentrations and pH conditions on growth of recombinantPichia pastoris. Two strains ofP. pastoris were used: a wild-type in methanol utilization (Mut⁺) and a mutant defective in methanol utilization (Mut^-). Under constant pH conditions of 5.0, glycerol concentrations up to 12% were efficiently utilized. Cell yield (Y_x/s) of about 0.8 and a final cell density of about 95 g/L (dry cell) were achieved. However, there were significant differences (probability [Pr]> F 0.0351) in specific growth rates between the initial glycerol concentrations of 2, 7, and 12%. When fermentations were conducted without pH control, growth continued until the pH had decreased to about 2.5. Growth stopped at pH 2.2 with uncontrolled pH, and residual glycerol concentrations were greater than 2%. As a result, Y_x/s decreased to about 0.3. There were no differences between Mut⁺ and Mut^- strains during cell growth on glycerol. This article is published as paper No. 11607, Journal Series, Nebraska Agricultural Experiment Station, Lincoln, NE 68583-0704.     

Bildung von α-Mannosidase durch Arthrobacter

The production of yeast cell wall mannan degrading -mannosidase was studied in shake flask experiments as well as in a highly instrumented, computer-coupled bioreactor. The enzyme is predominantly excreted into the culture liquid upon submerged cultivation on yeast mannan. Only low activities were detected with mannose or glucose as carbon source whereas the enzyme formation was totally repressed by glycerol. The amount of enzyme produced is proportional to the microbial biomass formed.Carbon-unlimited cultivation on mannose, the primary product of enzymic digestion, resulted in a specific growth rate of 0.10h^–1, a specific oxygen uptake rate ·h and a respiratory quotient ofRQ=1.0. Addition of yeast mannan (0.5%) to nutrient-depleted bacterial cells resulted in an almost complete utilization of this substrate, with 55% of substrate carbon being converted to biomass and 37% to carbon dioxide. The yield coefficient on mannan wasY _x/s =0.51 (g/g). Enzyme formation started with a delay of 30–40 min and stopped with termination of growth. Due to the increased production of mannose by the action of the enzyme the specific growth rate increased from 0.05 to 0.10 h^–1, thus enabling computations of maintenance and yield coefficients for oxygen and carbon dioxide metabolism.

     

Growth ofschizosaccharomyces pombe on glucose-malte mixtures in continuous cell-recycle cultures kinetics of substrate utilization

The aerobic growth ofSchizosaccharomyces pombe on mixtures of glucose and malate was investigated during continuous high cell density cultures with partial cell-recycle using a membrane bioreactor. Determination of the specific metabolic rates relative to substrates and products allowed the capacity of the yeast to metabolize malic acid under both oxidative metabolism (carbon limited cultures) and oxidofermentative metabolism (carbon sufficient cultures) situations to be characterized. Under carbon limiting conditions, the specific rate of malate utilization was dependent on the residual concentration and a limit for a purely oxidative breakdown without ethanol formation was observed for a characteristic ratio between the rates of substrate consumption qm/qg of 1.63 g.g^-1. In addition, the mass balance analysis revealed the incorporation of malic acid into biomass. In carbon excess environments, the specific rate of malate utilization was dependent on both the residual malate and the specific rate of glucose consumption indicating that in addition to its conversion into ethanol malate can be respiratively metabolized for qm/qg ratios higher than 0.4 g.g^-1.     

Enhancing pIFN-α Production and Process Stability in Fed-Batch Culture of Pichia pastoris by Controlling the Methanol Concentration and Monitoring the Responses of OUR/DO Levels

Effective expression of porcine interferon-α (pIFN-α) with recombinant Pichia pastoris was conducted in a bench-scale fermentor using an in situ methanol electrode-based feeding process with the control level of methanol concentration linearly increased to 10 g l^?1 for the first 20 h and maintained at 10 g l^?1 for the rest of expression phase. With this two-stage control process, the highest pIFN-α concentration reached a level of 1.81 g l^?1, which was 1.5-fold of that in the previous constant 10 g l^?1 induction experiments. There is an improvement of the pIFN-α productivity from more distribution of carbon flux to protein expression. The pIFN-α expression stability could be further enhanced by a simple on-line fault diagnosis method for methanol overfeeding based on oxygen uptake rate changing patterns. By implementing corrective action of feeding glycerol after fault detection, the production yield increased to twice the amount it would have been without the diagnosis.     

Expression of Recombinant Human Epidermal Growth Factor in Escherichia coli and Characterization of its Biological Activity

Recombinant human epidermal growth factor (EGF) was successfully expressed as a fusion protein in Escherichia coli system. This system was used OmpA signal sequence to produce soluble protein into the periplasm of E. coli. Human EGF (hEGF) synthesized in bacterial cell was found to be similar in size with the original protein and molecular weight approximately at 6.8 kDa. Cell proliferation assay was conducted to characterize the biological activity of hEGF on human dermal fibroblasts. The synthesized hEGF was found to be functional as compared with authentic hEGF in stimulating cell proliferation and promoting growth of cell. In comparison of biological activity between synthesized and commercial hEGF on cell proliferation, the results showed there was no significant different. This finding indicates the synthesized hEGF in E. coli system is fully bioactive in vitro.     

Production of antitumoral retamycin during fed-batch fermentations of Streptomyces olindensis

Fed-batch runs were performed in order to correlate the production of retamycin, an anthracycline antibiotic produced by Streptomyces olindensis in submerged cultures, with the specific growth rate. Maximum retamycin production was achieved with an exponential feed rate, controlling the specific growth rate at a low value (0.03h⁻¹, about 10% of the maximum specific growth rate). Control of the specific growth rate at higher values (0.10 and 0.17 h⁻¹) caused a decrease in antibiotic production. Morphology, assessed by image analysis, was shown to be highly relevant in this process. Cell growth mainly in the form of clumps (90% clumps and 10% free filaments) led to better results than growth as clumps (75%) and free filaments (25%).     

Biodiesel Residual Glycerol Metabolism by Klebsiella pneumoniae: Pool of Metabolites Under Anaerobiosis and Oxygen Limitation as a Function of Feeding Rates

The metabolism of residual glycerol from biodiesel synthesis by Klebsiella pneumoniae BLh-1 was investigated in this study. Batch and fed-batch cultivations were performed in bioreactors under anaerobic and oxygen limitation conditions. Results of batch cultivations showed that the main product was 1,3-propanediol (1,3-PD) in both conditions, although the higher yields and productivities (0.46 mol mol^?1 glycerol and 1.22 g?L^?1?h^?1, respectively) were obtained under anaerobic condition. Large amounts of ethanol were also produced under batch anaerobic condition, peaking at 12.30 g?L^?1. Batch cultivations under oxygen limitation were characterized by faster growth kinetics, with higher biomass production but lower conversions of glycerol into 1,3-PD, with yields and productivities of 0.33 mol mol^?1 glycerol and 0.99 g?L^?1?h^?1, respectively. The fed-batch cultivations were carried out in order to investigate the effects of feeding of raw glycerol on cells. Fed-batch under anaerobiosis showed that 1,3-PD and ethanol concentrations increased with the feeding rate, with maximal productions of 26.12 and 19.2 g?L^?1, respectively. The oxygen limitation conditions diverted the bacterium metabolism to an elevated lactic acid formation, reaching 59 g?L^?1 in higher feeding rates of glycerol, but lowering the production of ethanol.     

原位水解沉积制备高效氮化钽微球太阳能分解水光阳极

利用一种新的原位水解沉积方法,以在高湿度空气中老化的甲醇中作为溶剂,通过乙醇钽水解而成前驱体微球颗粒沉积,制备出了高效的Ta₃N₅微球光电极,其1.6 V（vs RHE）电极电位下的光电流值达到了6.6 mA·cm^-2。相反地,在新鲜的甲醇溶液中没有钽前驱体微球颗粒沉积。这表明甲醇中水的含量对Ta₃N₅微球光电极的形成十分重要。另外,本制备方法也能方便地在其他透明导电衬底上制备出Ta₃N₅。     

Effects of glucose,glutamine, and malate on the metabolism of spodoptera frugiperda clone 9 (sf9) cells

Optimal design and operation of bioreactors for insect cell culture is facilitated by functional relations providing quantitative information on cellular metabolite consumption kinetics, as well as on the specific cell growth rates (μ_G). Initial specific consumption rates of glucose, malate, and oxygen, and associated changes in μ_G, were measured forSpodoptera frugiperda clone 9 (Sf9) cells grown in batch suspension culture in medium containing 7–35 mM glucose, 0–16 mM malate, and 4–16 mM glutamine. The initial specific glucose consumption rate (q _G ) could be described by a modified Michaelis-Menten equation treating malate as a “competitive” inhibitorK ₁ = 6.5 mM) and glutamine as a “noncompetitive” inhibitorK _I = 14 mM) ofq _G , with aK _m of 7.1 mM for glucose. All three carbon sources were found to increase μ_G in a saturable manner, and a modified Monod equation was employed to describe this relationship (μ_Gmax = 0.047 h^-1). The initial specific oxygen consumption rate (q_O2) in Sf9 cells could be related to μ_G by the maintenance energy model, and it was calculated that, under typical culture conditions, about 15–20% of the cellular energy demand comes from functions not related to growth. Fitted parameters in mathematical expression for μg: K₄, Monod constant for glucose (mM); K₅, modified Monod constant for malate (mM); K₆, Monod constant for glutamine (mM); m_o2, specific consumption rate of oxygen by the cells under zero-growth conditions (nmol/cell/h); q_F, initial specific fumarate production rate (nmol/cell/ h);q _G , initial specific glucose consumption rate (nmol/cell/h); q_Gmax, maximum initial specific glucose consumption rate (nmol/cell/h);q _M , initial specific malate consumption rate (nmol/cell/h); q_o2, initial specific oxygen consumption rate (nmol/cell/h); Y_o2, cell yield on oxygen (cells/nmol); μ, initial specific cell growth rate (h^-1); μ_g, initial specific cell growth rate (h^-1); μG_max, maximum initial specific cell growth rate (h^-1).     

Studies on the overproduction of indole-containing metabolites by a methanol-utilizing yeast

Production of indole-containing metabolites (“indoles”) from methanol has been studied using a mutant ofHansenula polymorpha resistant to 5-fluorotryptophan. Whereas the wild-type culture produces only a small amount of indoles, the mutant is partially deregulated and overproduces indoles. Indoles production was studied in batch and continuous culture and in a washed-cell system. When the pH was above 4.0, indoles production was growth-associated, in both minimal and complex media, and batch or continuous culture. When the pH was below or equal to 4.0, a low phosphate concentration was found to improve production. In a phosphate-deficient washed-cell suspension system, the addition of an amino acid such as methionine at 5 mM increased specific productivity by more than 60%. Addition of cycloheximide at 50 mg/L decreased residual growth and increased maximum productivity of indoles by more than 60%. When the antibiotic was added at 1000 mg/L, growth was completely inhibited and indoles production continued for about 35 h.     

Kinetics of malachite green fading in alcohol–water binary mixtures

The rate constant of alkaline fading of malachite green (MG⁺) was studied in alcohol–water binary mixtures. This reaction was studied under pseudo‐first‐order conditions at 283–303 K. It was observed that the reaction rate constants were increased in the presence of different weight percentages of methanol, ethanol, 1‐propanol, 2‐propanol, ethylene glycol, 1,2‐propanediol, and glycerol (up to 19.3%). In aqueous glycerol solutions higher than 19.3%, the rate constant of reaction slightly decreases, which is due to high viscosity values of solvent mixtures. The fundamental rate constants of MG⁺ fading in these solutions were obtained by using the SESMORTAC model. Owing to the charged character of activated complex, with an increase in the weight percentage of the used cosolvents or temperature, k₂ values change according to the trend of hydroxide ion nucleophilic parameter values. Also, using MG⁺ solvatochromism, a simple test, called MAGUS, is introduced for measuring the glycerol concentration in its aqueous solutions. © 2010 Wiley Periodicals, Inc. Int J Chem Kinet 42: 508–518, 2010     

Kinetics and bioenergetics of Spirulina platensis cultivation by fed-batch addition of urea as nitrogen source

The cyanobacterium Spirulina platensis was cultivated in bench-scale miniponds on bicarbonate/carbonate solutions using urea as nitrogen source. To minimize limitation and inhibition phenomena, urea was supplied semicontinuously using exponentially increasing feeding rates. The average growth rates obtained alternately varying the total mass of urea added per unit reactor volume (275<m _T<725 mg/L) and the total feeding time (9<t _T<15 d) clearly evidenced nitrogen limitation for m _T<500 mg/L and excess nitrogen inhibition above this threshold. The time behavior of the specific growth rate at variable urea feeding patterns allowed estimation of the time-dependent Gibbsenergy dissipation for cell growth under the actual depletion conditions of fed-batch cultivations. Comparison of the yield of growth on Gibbs energy obtained using either urea or KNO₃ pointed to the preference of S. platensis for the former nitrogen source, likely owing to more favorable bioenergetic conditions.     

Simultaneous production of citric acid and erythritol from crude glycerol by Yarrowia lipolytica Wratislavia K1

This study shows a possible microbial process for utilization of crude glycerol generated by the biodiesel industry for citric acid and erythritol production. Simultaneous production of citric acid and erythritol under nitrogen-limited conditions with glycerol as the carbon source was achieved with an acetate negative mutant of Y. lipolytica Wratislavia K1 in fed-batch cultivations. The effect of the initial glycerol concentration (from 30–180 g dm⁻³) on the citrate and erythritol production was investigated. As a result of the experiments, maximum citric acid production (110 g dm⁻³) and a very high amount of erythritol (81 g dm⁻³) were determined after 168 h of fed-batch cultivation with the initial glycerol concentration of 150 g dm⁻³ and the total glycerol concentration of 250 g dm⁻³. In addition, the citric acid to isocitric acid ratio of the products from this strain was 35.5:1. Presented at the 34th International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 21–25 May 2007.     

Anaerobic baffled reactor treatment of biodiesel-processing wastewater with high strength of methanol and glycerol: reactor performance and biogas production

Biodiesel-processing factories employing the alkali-catalyzed transesterification process generate a large amount of wastewater containing high amount of methanol, glycerol, and oil. As such, wastewater has high potential to produce biogas using anaerobic treatment. The aim of this research was to investigate the performance of an anaerobic baffled reactor for organic removal and biogas production from biodiesel wastewater. The effect of different organic loading rates, varying from 0.5 kg m⁻³ d⁻¹ to 3.0 kg m⁻³ d⁻¹ of chemical oxygen demand, was determined using three 22 L reactors, each comprising five separate compartments. Wastewater was pretreated with chemical coagulants to partially remove oil prior to experimentation. Results show that the anaerobic baffled reactor operated at 1.5 kg m⁻³ d⁻¹ of chemical oxygen demand and ten days of hydraulic retention time provided the best removal efficiencies of 99 % of chemical oxygen demand, 100 % of methanol, and 100 % of glycerol. Increasing the organic loading rate over 1.5 kg m⁻³ d⁻¹ of chemical oxygen demand led to excessive accumulation of volatile fatty acids thereby making the pH drop to a value unfavorable for methanogenesis. The biogas production rate was 12 L d⁻¹ and the methane composition accounted for 64–74 %. Phase-separated characteristics revealed that the highest chemical oxygen demand removal percentage was achieved in the first compartment and the removal efficiency gradually decreased longitudinally. A scanning electron microscopic study indicated that the most predominant group of microorganisms residing on the external surface of the granular sludge was Methanosarcina.     

Diminished Respirative Growth and Enhanced Assimilative Sugar Uptake Result in Higher Specific Fermentation Rates by the MutantPichia stipitis FPL-061

A mutant strain ofPichia stipitis, FPL-061, was obtained by selecting for growth on L-xylose in the presence of respiratory inhibitors. The specific fermentation rate of FPL-061, was higher than that of the parent,Pichia stipitis CBS 6054, because of its lower cell yield and growth rate and higher specific substrate uptake rate. With a mixture of glucose and xylose, the mutant strain FPL-061 produced 29.4 g ethanol/L with a yield of 0.42 g ethanol/g sugar consumed. By comparison, CBS 6054 produced 25.7 g ethanol/L with a yield of 0.35 gJg. The fermentation was most efficient at an aeration rate of 9.2 mmoles O₂ L^-1 h^-1. At high aeration rates (22 mmoles O₂ L^-1 h^-1), the mutant cell yield was less than that of the parent. At low aeration rates, (1.1 to 2.5 O₂ L^-1 h^-1), cell yields were similar, the ethanol formation rates were low, and xylitol accumulation was observed in both the strains. Both strains respired the ethanol once sugar was exhausted. We infer from the results that the mutant, P.stipitis FPL-061, diverts a larger fraction of its metabolic energy from cell growth into ethanol production.     

Evaluation of Cashew Apple Juice for the Production of Fuel Ethanol

A commercial strain of Saccharomyces cerevisiae was used for the production of ethanol by fermentation of cashew apple juice. Growth kinetics and ethanol productivity were calculated for batch fermentation with different initial sugar (glucose + fructose) concentrations. Maximal ethanol, cell, and glycerol concentrations were obtained when 103.1 g L⁻¹ of initial sugar concentration was used. Cell yield (Y _X/S) was calculated as 0.24 (g microorganism)/(g glucose + fructose) using cashew apple juice medium with 41.3 g L⁻¹ of initial sugar concentration. Glucose was exhausted first, followed by fructose. Furthermore, the initial concentration of sugars did not influence ethanol selectivity. These results indicate that cashew apple juice is a suitable substrate for yeast growth and ethanol production.     

Electron spin resonance studies of the excited triplet states of DL-5-hydroxytryptophan, 5-hydroxyindole, 6-hydroxynicotinic acid, indole and hippuric acid

Abstract— The electron spin resonances of the metastable triplet states of DL-5-hydroxy-tryptophan, 6-hydroxynicotinic acid, and other related compounds have been observed in various glasses at 77°K. These compounds are biologically interesting in connection with photochemical processes. The Δm= 2 transitions were observed and the spin-spin interaction parameter (D²+ 3E²1/2 was determined. However, for indole, it was also able to observe the Δm= 1 transitions from which D and E were determined separately. From the values of (D²+ 3E²)1/2, D and E whenever possible and from the decay times of the triplet states, it was possible to characterize the lowest triplets as π—π* states in all cases. Studies of the pH dependence of DL-5-hydroxytryptophan, 5-hydroxyindole, and 6-hydroxynicotinic acid indicate that the unionized molecule exhibits a resonance different from that of the related ion with a proton removed from the phenolic hydroxyl group. The zero-field splitting parameters D* and the lifetime of the triplet states have also been measured in various glasses and in Lucite. Both temperature and the specific nature of the host media affected the values of these parameters.     

Citric acid production from raw glycerol by acetate mutants of Yarrowia lipolytica

Three acetate mutants of the yeast species Yarrowia lipolytica were screened using batch cultivation. The strain Y. lipolytica 1.31 was found to be the most suitable for citric acid production from raw glycerol, a by-product of biodiesel production from rapeseed oil. At the initial concentration of glycerol of 200 g dm⁻³, the citric acid production of 124.5 g dm⁻³, yield of 0.62 g g⁻¹, and productivity of 0.88 g dm⁻³ h⁻¹ were achieved. Presented at the 33rd International Conference of the Slovak Society of Chemical Engineering, Tatranské Matliare, 22–26 May 2006.