Glutathione production coupled with an ATP regeneration system

Summary Escherichia coli cells possessing glutathione synthetase and acetate kinase activities were immobilized with carrageenan gel. To enhance the operational stability, immobilized cells were treated with hardening agent, glutaraldehyde in the presence of hexamethylenediamine. The continuous production of glutathione was investigated using the column packed with immobilized Escherichia coli cell preparations. Glutathione was continuously produced by this column in the presence of acetyl phosphate and the half-life of this column was calculated to be 8 days at the flow rate of S.V.=0.1 h^–1 at 37°C.     

Glycolytic pathway as an ATP generation system and its application to the production of glutathione and NADP

Efficient ATP generation is required to produce glutathione and NADP. Hence, the generation of ATP was investigated using the glycolytic pathway of yeast. Saccharomyces cerevisiae cells immobilized using polyacrylamide gel generated ATP from adenosine, consuming glucose and converting it to ethanol and carbon dioxide. Under optimal conditions, the ATP-generating activity of immobilized yeast cells was 7.0 μmol h^?1 ml^?1 gel. A column packed with these immobilized yeast cells was used for continuous ATP generation. The half-life of the column was 19 days at a space velocity of (SV) 0.3 h^?1 at 30°C. The properties of glutathione- and NADP-producing reactions coupled with the ATP-generating reaction were investigated. Escherichia coli cells with glutathione synthesizing activity and Brevibacterium ammoniagenes cells with NAD kinase activity were immobilized in a polyacrylamide gel lattice. Under optimal conditions, the immobilized E. coli cells and immobilized B. ammoniagenes cells produced glutathione and NADP at the rates of 2.1 and 0.65 μmol h^?1 ml^?1 gel, respectively, adding ATP to the reaction mixture. In order to produce glutathione and NADP economically and efficiently, the glutathione- and NADP-producing reactions were finally coupled with the ATP-generating reaction catalysed by immobilized S. cerevisiae cells. To compare the productivities of glutathione and NADP, and to compare the efficiency of ATP utilization for the production of these two compounds, the two reactor systems, co-immobilized cell system and mixed immobilized cell system, were designed. As a result, these two compounds were also found to be produced by these two kinds of reactor systems. Using the data obtained, the feasibility and properties of ATP generation by immobilized yeast cells are discussed in terms of the production of glutathione and NADP.     

Contributions of immobilized and free cells of salt-tolerantZygosaccharomyces rouxii andCandida versatilis to the production of ethanol and 4-ethylguaiacol

Summary The contribution of immobilized cells and free cells released from gel beads to ethanol production by the salt-tolerant yeastsZygosaccharomyces rouxii andCandida versatilis, and 4-ethylguaiacol (4-EG) production byC. versatilis were investigated using an airlift reactor. The amounts of ethanol produced by free cells were about 65% and about 90% of total ethanol in the reactor forZ. rouxii andC. versatilis, respectively. It was found that immobilized cells gave a much lower specific productivity of ethanol (ethanol production per hour per cell) than free cells of both yeasts, especially ofC. versatilis. 4-EG was produced mainly by immobilized cells ofC. versatilis; the amount of 4-EG produced by free cells was about 20% of the total 4-EG, in contrast to the results of ethanol production. However, the specific productivity of 4-EG (4-EG production per hour per cell) by immobilized and free cells was fairly similar.     

Enhancement of penicillin acylase activity by cultivating immobilized Kluyvera citrophila

Summary Whole cells of Kluyvera citrophila were immobilized in polyacrylamide gel. The penicillin acylase activity of immobilized whole cells was 60%–70% of native cells. When the immobilized cells were continuously cultivated for 40 h in an aerated fermentor containing peptone medium and were treated with alkali in order to remove -lactamase activity, the immobilized cells produced ampicillin up to 4.4 times faster than noncultivated cells.Ampicillin production was investigated in a column system using these cultivated immobilized whole cells. The cultivated immobilized cells showed excellent performance in continuous ampicillin production.     

Semicontinuous cultivation of immobilized Claviceps purpurea

Summary Mycelia of Claviceps purpurea CBS 164.59 were immobilized in 2%, 4%, and 8% calcium alginate. Alkaloid production by free cells declined after 60 days, while immobilized cells retained their activity for 200 days. The cumulative alkaloid production for all fermentation cycles using 8% calcium alginate immobilized mycelia was 25 times higher than that from free cells. The best yields of the ergopeptide ergometrine were reached with 4% gel immobilized mycelia, while higher gel concentrations caused a shift in the alkaloid biosynthesis towards high clavine alkaloid production.Beginning with the third cycle of reincubation the immobilized mycelia showed a marked tendency to fragmentize into vacuolated arthrosporoid-like structures and produced violet-black pigments so that the beads recalled sclerotial structures of parasitically living Claviceps.Dedicated to Prof. Dr. K. Esser to his 60th birthday     

Production of L-lysine by free and PVA-cryogel immobilizedCorynebacterium glutamicum cells

Summary Free and immobilized cells ofC. glutamicum were analyzed for production of L-lysine. Non-growing free cells as well as immobilized ones produced higher levels of lysine than growing free cells, when cultured for 24 h in a medium containing 80 g/L glucose as a carbon source. The effect of initial biomass and yeast extract concentrations was investigated in order to improve lysine production.     

Hydrogen production from glucose by immobilized growing cells ofClostridium butyricum

Summary Clostridium butyricum was immobilized in a porous carrier (acetylcellulose filter) with agar. Addition of peptone to the reaction mixture increased the hydrogen productivity from glucose. The number of cells in the agaracetylcellulose increased during incubation in the medium containing glucose and peptone, and the immobilized growing cells converted 45% of the glucose to hydrogen. Riboflavin enhanced the hydrogen productivity and the lactate produced by the native cells decreased remarkably. Therefore, the immobilized whole cells incubated with riboflavin were employed for repeated hydrogen production in the medium containing glucose and peptone. The hydrogen productivity of the immobilized cells increased markedly after repeated use, and the immobilized cells produced hydrogen in stoichiometric amounts from glucose.     

Enhanced production of alkaline thermostable keratinolytic protease from calcium alginate immobilized cells of thermoalkalophilic Bacillus halodurans JB 99 exhibiting dehairing activity

The thermoalkalophilic Bacillus halodurans JB 99 cells known for production of novel thermostable alkaline keratinolytic protease were immobilized in calcium alginate matrix. Batch and repeated batch cultivation using calcium alginate immobilized cells were studied for alkaline protease production in submerged fermentation. Immobilized cells with 2.5% alginate and 350 beads/flask of initial cell loading showed enhanced production of alkaline protease by 23.2% (5,275 ± 39.4 U/ml) as compared to free cells (4,280 ± 35.4 U/ml) after 24 h. In the semicontinuous mode of cultivation, immobilized cells under optimized conditions produced an appreciable level of alkaline protease in up to nine cycles and reached a maximal value of 5,975 U/ml after the seventh cycle. The enzyme produced from immobilized cells efficiently degraded chicken feathers in the presence of a reducing agent which can help the poultry industry in the management of keratin-rich waste and obtaining value-added products.     

Physical Factors and Reuse Affect the Production of Cholesterol Oxidase in Reactors Containing Calcium Alginate-Immobilized Rhodococcus equi No. 23

Summary Some physical factors including initial pH of medium, cultivation temperature and shaking speed as well as reuse affecting the production of cholesterol oxidase (CholOx) in reactors containing calcium alginate-immobilized cells of Rhodococcus equi No. 23 were investigated. Results revealed that the free cells showed the maximum CholOx in the culture with an initial pH of 5.0, while culture inoculated with immobilized cells exhibited a broad pH range, 6.0–9.0, for maximum CholOx production. The immobilized and free cells produced the maximum CholOx in the culture incubated at 30 and 25°C, respectively. The CholOx production decreased upon increasing the cultivation temperature. Increasing CholOx activity was also noted for both immobilized and free cells of R. equi No. 23 in the culture with increasing shaking speed. Under the optimal culture conditions, that were established, a higher maximum CholOx production of 0.94 unit/ml was found for immobilized R. equi No. 23 compared to that of 0.84 unit/ml for free cells after 48 h of cultivation. Furthermore, no gel leakage was noted after re-use of the calcium alginate-immobilized R. equi No. 23 for seven consecutive 48 h batch culture. The CholOx production in the seventh cycle was about 60.4% of that obtained in the first cycle.     

Production of acrylamide using immobilized cells of<Emphasis Type="Italic">Rhodococcus rhodochrous</Emphasis> M33

The cellsof Rhodococcus rhodochrous M33, which produce a nitrile hydratase enzyme, were immobilized in acrylamide-based polymer gels. The optimum pH and temperature for the activity of nitrile hydratase in both the free and immobilized cells were 7.4 and 45°C, respectively, yet the optinum temperature for acrylamide production by the immobilized cells was 20°C. The nitrile hydratase of the immobilized cells was more stable with acrylamide than that of the free cells. Under optimal conditions, the final acrylamide concentration reached about 400 g/L with a conversion yield of almost 100% after 8 h of reaction when using 150 g/L of immobilized cells corresponding to a 1.91 g-dry cell weight/L. The enzyme activity of the immobilized cells rapidly decreased with repeated use. However, the quality of the acrylamide produced by the immobilized cells was much better than that produced by the free cells in terms of color, salt content, turbidity, and foam formation. The quality of the aqueous acrylamide solution obtained was found to be of commercial use without further purification.     

Semicontinuous production of red pigment by immobilized cells of<Emphasis Type="Italic">Bacillus</Emphasis> sp. BH-99 using column bioreactor

The semicontinuous production of red pigment by immobilized cells ofBacillus sp. BH-99 was investigated in comparison with free cells. The red pigment produced highest productivity under the conditions of aeration of 0.2 mL/min and 2 mm diameter of gel beads by using 3.0% sodium alginate. Semicontinuous production by immobilized cells showed the highest productivity with replacement of fresh production medium in every 72 h for fourth fermentation cycle following the conditions of red pigment productivity.     

Galactose oxidase production by immobilized cells ofDactylium dendroides

Summary Dactylium dendroides cells were immobilized with calcium alginate, calcium pectate and k-carrageenan. Alginate immobilized cells produced relatively small amounts of (D-galactose: O₂ oxidoreductase, EC 1.1.3.9, GOase). Pectate immobilized cells gave the best yield of GOase, which was comparable with that obtained with free cells, and productivity could be extended up to 28 days (7 cycles). Controlled dosage of phosphate to the medium markedly improved GOase production with higher yields per cycle than with free cells.     

Cellulase production by free and immobilized <Emphasis Type="Italic">Aspergillus terreus</Emphasis>

Aspergillus terreus, isolated from rotting bagasse, showed comparable cellulolytic activities when grown either in the free or immobilized states with cellulose as the sole carbon source. The cultural and nutritional requirements for maximum cellulase production by the organism either in the free or immobilized states were similar, except an increase in the temperature optimum from 30 to 40°C, occurred upon immobilization. In the free state, the maximum filter paper hydrolase, carboxymethylcellulase and β-glucosidase activities produced were 2.1, 13.6, and 3.2 U/ml, respectively, while in the immobilized state, the levels were 1.8, 12.0, and 2.4 U/ml. Production of cellulolytic enzymes by immobilized cells was influenced by the surface area of the support material. In addition, cells in the immobilized state sustained enzyme production for a much longer period with a 4.5-fold increase in productivity during repeated batch when compared to free cells.     

Glucoamylase and α-amylase production by immobilized Aspergillus niger

Summary Aspergillus niger mycelia or spores were immobilized in calcium alginate gel beads and employed for production of glucoamylase and -amylase by repeated batch process. The immobilized mycelium produced lower enzyme activities than immobilized spores germinated in a growth medium and subsequently cultured in an enzyme production medium. In repeated batch experiments, free cells could be used for only 4 4-day batches, whereas with immobilized spores at least 11 4-day batches with a gradual increase in enzyme activities in each successive batch were possible. The activity ratio of glucoamylase and -amylase produced was altered by immobilization.     

Continuous production ofL-malic acid by immobilizedBrevibacterium ammoniagenes cells

Summary Continuous production ofL-malic acid from fumaric acid using immobilized microbial cells was investigated. Several microorganisms having fumarase activity were immobilized into a polyacrylamide gel lattice. Among the microorganisms tested, immobilizedBrevibacterium ammoniagenes IAM 1645 showed the highest enzyme activity, but produced an unwanted by-product, succinic acid. Conditions for suppression of this side reaction were investigated, and bile extract treatment of immobilized cells was found to be effective.The bile extract treatment of immobilized cells also resulted in a marked increase of reaction rate forL-malic acid formation.No difference was observed between the native enzyme and immobilized cells in optimal pH and temperature of the enzyme reaction.The effect of temperature on the reaction rate and the stability of fumarase activity of an immobilized cell column were investigated under conditions of continuous enzyme reaction. The decay of enzyme activity during continuous enzyme reaction was expressed by an exponential relationship. Half-life of the fumarase activity of the immobilized cell column at 37°C was calculated to be 52.5 days.Presented at the Annual Meeting of the Society of Fermentation Technology, Japan, Osaka, Japan, October 30, 1975.     

NADPH production from NADP+ with a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans

Summary A convenient and efficient method of NADPH production from NADP⁺ has been established using a glucose dehydrogenase system involving whole cells and immobilized cells of Gluconobacter suboxydans IFO 3172. Using airdried cells of the bacterium, the optimum conditions for NADPH production were examined, including the cell and glucose concentrations, NADP⁺ concentration, pH, buffer and reaction temperature. Under suitable conditions, the conversion ratio and the amount of NADPH accumulated reached about 100% and 73 mg/ml of the reaction mixture, respectively, after 1-h reaction. Intact cells of the bacterium also showed high NADPH production even in the reaction mixture without a surfactant. The addition of Triton X-100 to the reaction mixture and freeze-thawing treatment of intact cells enhanced the production. The NADPH production method was further improved by using cells of the bacterium immobilized by entrapment in a -carrageenan gel lattice. The immobilized cells had almost the same enzymatic properties as the air-dried cells. The conditions for the continuous production of NADPH with an immobilized cell column were also investigated. NADPH was produced in a good yield (about 95%) with this continuous process.     

Continuous production of glutathione using immobilized microbial cells containing ATP generating system

Acetate kinase reaction in Escherichia coli cells and glycolytic pathway in Saccharomyces cerevisiae cells were utilized as ATP generation systems for glutathione synthetic processes. These two ATP generation systems were well coupled with glutathione synthetase reactions and glutathione was produced by coimmobilized E. coli cells with dextran-bound ATP or by immobilized S. cerevisiae cells. The glycolytic pathway in S. cerevisiae cells was further utilized for the biosynthetic processes of other useful compounds.     

Protease production by immobilized growing cells of Serratia marcescens and Myxococcus xanthus in calcium alginate gel beads

Summary Serratia marcescens and Myxococcus xanthus cells were immobilized in calcium alginate gel beads. Immobilization under various conditions had no effect on the extracellular proteolytic activity of S. marcescens cells. Protease production seemed rather to depend on the free cells in the medium. However, the stability over time of enzyme production was enhanced, as immobilization increased protease production half-life from 5 to 12 days. On the other hand, Myxococcus xanthus produced proteases inside the gel beads which could diffuse into the medium. The proteolytic activity increased as a function of the initial cell content of the beads and of the bead inoculum. Compared to free cells, immobilized cells of Myxococcus xanthus could produce 8 times more proteolytic activity, with a very low free-cell concentration in the medium.     

Process and technoeconomics of ethanol production by immobilized cells

Summary A two-stage fermentation process has been developed for continuous ethanol production by immobilized cells of Zymomonas mobilis. About 90–92 kg/m³ ethanol was produced after 4 h of residence time. Entrapped cells of Zymomonas mobilis have a capability to convert glucose to ethanol at 93% of the theoretical yield. The immobilized cell system has functioned for several weeks, and experience indicates that the carrageenan gel apparently facilitates easy diffusion of glucose and ethanol.The simplicity and the high productivity of the plug-flow reactor employing immobilized cells makes it economically attrative. An evaluation of process economics of an immobilized cell system indicates that at least 4 c/l of ethanol can be saved using the immobilized cell system rather than the conventional batch system. The high productivity achieved in the immobilized cell reactor results in the requirement for only small reactor vessels indicating low capital cost. Consequently, by switching from batch to immobilized processing, the fixed capital investment is substantially reduced, thus increasing the profitability of ethanol production by fermentation.     

The production of ethanol by immobilized yeast cells

Saccharomyces cerevisiae cells were immobilized in calcium alginate beads for use in the continuous production of ethanol. Yeasts were grown in medium supplemented with ethanol to selectively screen for a culture which showed the greatest tolerance to ethanol inhibition. Yeast beads were produced from a yeast slurry containing 1.5% alginate (w/v) which was added as drops to 0.05M CaCl₂ solution. To determine their optimum fermentation parameters, ethanol production using glucose as a substrate was monitored in batch systems at varying physiological conditions (temperature, pH, ethanol concentration), cell densities, and gel concentration. The data obtained were compared to optimum free cell ethanol fermentation parameters. The immobilized yeast cells examined in a packed-bed reactor system operated under optimized parameters derived from batch-immobilized yeast cell experiments. Ethanol production rates, as well as residual sugar concentration were monitored at different feedstock flow rates.