Continuous ATP production by spinach thylakoid in a stirred tank reactor

Spinach thylakoid was immobilized by two different methods for the purpose of retention within a continuous-flow stirred tank reactor (CSTR). The glutaraldehyde crosslinked albumin polymer method completely inactivated the cyclic ATP photophosphorylation of thylakoid. In contrast, agarose-entrapped thylakoid retained about 17% of the activity of the cyclic photophosphorylation of non-immobilized thylakoid. This activity declined continuously during ATP production in the CSTR. Fifty percent of the initial activity was lost within about 5.5 h. Ascorbate was found to increase the stability of ATP photophosphorylation; about twice as much ATP was produced at the optimal ascorbate concentration of 5 mM. Under the optimal dilution rate of 2.36 h⁻¹, about 60 μmol of ATP per mg chlorophyll were produced in 20 h by agarose-entrapped thylakoid in the CSTR. These results showed that, compared to non-immobilized thylakoid in batch operation, agarose-entrapped thylakoid produced only a low amount of ATP under continuous operation.     

Self-cycling fermentation in a stirred tank reactor

Self-cycling fermentations (SCFs) were conducted in a stirred tank apparatus using Bacillus subtilis and Acinetobacter calcoaceticus. The systems were very stable and the experiments lasted through many cycles. The variation of parameters such as biomass and doubling time from cycle to cycle was small. The stirred tank reactor (STR) allowed a much better control of the working volume in the fermentor from cycle to cycle, compared to the cyclone column, and it was not necessary to make periodic corrections.The production of surfactin from B. subtilis was achieved without extending the cycle time. The harvested broth at the end of each cycle was allowed to remain in a secondary vessel, at ambient temperature, before being collected. It is exhaustion of the limiting nutrient which causes an increase in dissolved oxygen (DO). At this point, the computer, which constantly monitors the DO, triggered the harvesting sequence to end the cycle. Thus, the mature culture in the secondary vessel experienced appropriate conditions for the production of the secondary metabolite. Meanwhile, the next batch of cells was being grown in the primary reactor.The response of a gas analyzer on the effluent paralleled that of the DO measurements in the fermentor. These data for oxygen and carbon dioxide exhibited less noise than the DO readings. Either would be a more reliable parameter for feedback control of the SCF because the problem of fouling of the DO probe after extended runs of many cycles would be eliminated. (c) 1993 John Wiley & Sons, Inc.     

Influence of medium composition on penicillin V production in a stirred tank reactor.

Penicillin production with a high-producing strain Penicillium chrysogenum was investigated under well-controlled conditions in a stirred tank reactor with complex media containing lard oil and lactose on the one hand, and lactose on the other hand. With lard oil, cell growth and product formation rates were higher, and the production time was shorter by 40 h than without lard oil. On account of the longer production time without lard oil, the amount of beta-lactam compounds was higher (29.93 g l-1), but the mole fraction of the decomposed products (penicilloic acid and penilloic acid) was larger (0.282) than the amount of penicillin V (23.25 g l-1) and the decomposed mole fraction (0.0747) with lard oil. The final product concentrations were about the same (20.86 g l-1 or 35,462 IU ml-1 with lard oil, and 20.43 g l-1 or 34510 IU ml-1 without lard oil). The mole fractions of the by-product (p-OH-penicillin V) were 0.0365 and 0.066. The substitution of lard oil with lactose is possible without a considerable reduction of process performance.     

Phosphorus removal in aerated stirred tank reactor

The possibility to obtain biological phosphorus removal in strictly aerobic conditions has been investigated. Experiments, carried out in a continuous stirred tank reactor (CSTR), show the feasibility to obtain phosphorus removal without the anaerobic phase. Reactor performance in terms of phosphorus abatement kept always higher then 65% depending on adopted sludge retention time (SRT). In fact increasing SRT from 5 days to 8 days phosphorus removal and reactor performance increase but overcoming this SRT value a decreasing in reactor efficiency was recorded.     

Azadirachtin production by hairy root cultivation of Azadirachta indica in a modified stirred tank reactor

Present investigation involves hairy root cultivation of Azadirachta indica in a modified stirred tank reactor under optimized culture conditions for maximum volumetric productivity of azadirachtin. The selected hairy root line (Az-35) was induced via Agrobacterium rhizogenes LBA 920-mediated transformation of A. indica leaf explants (Coimbatore variety, India). Liquid culture of the hairy roots was developed in a modified Murashige and Skoog medium (MM2). To further enhance the productivity of azadirachtin, selected growth regulators (1.0?mg/l IAA and 0.025?mg/l GA₃), permeabilizing agent (0.5?% v/v DNBP), a biotic elicitor (1?% v/v Curvularia (culture filtrate)) and an indirectly linked biosynthetic precursor (50?mg/l cholesterol) were added in the growth medium on 15th day of the hairy root cultivation period in shake flask. Highest azadirachtin production (113?mg/l) was obtained on 25th day of the growth cycle with a biomass of 21?g/l DW. Further, batch cultivation of hairy roots was carried out in a novel liquid-phase bioreactor configuration (modified stirred tank reactor with polyurethane foam as root support) to investigate the possible scale-up of the established A. indica hairy root culture. A biomass production of 15.2?g/l with azadirachtin accumulation in the hairy roots of 6.4?mg/g (97.28?mg/l) could be achieved after 25?days of the batch cultivation period, which was ~27 and ~14?% less biomass and azadirachtin concentration obtained respectively, in shake flasks. An overall volumetric productivity of 3.89?mg/(l?day) of azadirachtin was obtained in the bioreactor.     

Batch xylitol production from wheat straw hemicellulosic hydrolysate using Candida guilliermondii in a stirred tank reactor

Batch production of xylitol from the hydrolysate of wheat straw hemicellulose using Candida guilliermondii was carried out in a stirred tank reactor (agitation speed of 300 rpm, aeration rate of 0.6 vvm and initial cell concentration of 0.5 g l^–1). After 54 h, xylitol production from 30.5 g xylose l^–1 reached 27.5 g l^–1, resulting in a xylose-to-xylitol bioconversion yield of 0.9 g g^–1 and a productivity of 0.5 g l^–1 h^–1.     

Factors affecting the production of a single-chain antibody fragment by Aspergillus awamori in a stirred tank reactor

A recombinant strain of Aspergillus awamori expressing anti-lysozyme single chain antibody fragments (scFv), under the control of a xylanase promoter, was studied in order to investigate the impact of medium, induction regime and protease production on the expression of the product. Experiments with the time of induction showed that the optimum results are achieved when induction is started in the late exponential phase (21 h after inoculation) improving the titer of the product from 14.5 mg L(-1), obtained in the early exponential phase (7 h after inoculation), to 16.2 mg L(-1). A 100% increase of the carbon (fructose) and nitrogen (ammonium sulfate) sources in the growth medium resulted in an increase in product concentration from 16.2 to 108.9 mg L(-1) and an increase in maximum dry cell weight from 7.5 to 11.5 g L(-1). A 50% reduction in the concentration of the inducer resulted in an increase in the product yield from 10 mg g(-1) dry cell weight to 12 mg g(-1). Proteolytic enzymes were produced during the fermentation up to concentrations equivalent to 1.4 g L(-1) trypsin, but they had no detrimental effect on the concentration of the antibody fragment.     

Tween 80 influences the production of intracellular lipase by Schizochytrium S31 in a stirred tank reactor

Marine microorganisms are a potential source of enzymes with structural stability, high activity at low temperature and unique substrate selectivity. Thraustochytrids are marine heterotrophic microbes, well known for the production of omega-3 fatty acids. In this study the effect of Tween 80 as a carbon source was investigated with regard to biomass, lipase and lipid productivity in Schizochytrium sp. S31. Tween 80 (1%) and 120 h of incubation were the optimum condition period for biomass, lipid and lipase productivity in a stirred tank reactor. The yields obtained were 0.9 g L⁻¹ of biomass, 300 mg g⁻¹ of lipid and 39 U/g of lipase activity. Sonication was optimised in terms of time and acoustic power to maximise the yield of extracted lipase. The extracted lipase from Schizochytrium S31 was observed to hydrolyse long chain polyunsaturated fatty acids DHA and EPA.     

Study of microencapsulated urease in a continuous feed,stirred tank reactor

Urease, (urea amidohydrolase, EC 3.5.1.5) co-encapsulated with haemoglobin in cellulose nitrate membranes was found to exhibit apparent Michaelis-Menten kinetics; however, a steadily increasing apparent Michaelis-Menten constant over the lifetime of the preparation was observed. The activity of the enzyme in a continuous feed stirred tank reactor (CSTR) was investigated and correlated with a mathematical model derived from basic Michaelis-Menten kinetics. Plots relating substrate conversion to feed substrate concentration and tank reactor capacity were constructed and found to be accurate to less than 15% error under the experimental conditions studied.     

Mechanistic analysis of xanthan gum production in a stirred tank

The overall effect of agitation on xanthan gum production by Xanthomonas campestris ATCC13951 in a stirred vessel was mechanistically analyzed considering local variation of the specific production rate due to variation of shear stress in the vessel. The whole liquid volume in a fermentor was roughly divided into three regions; the micromixing region around the impeller with high shear stress, the macromixing region dominated by a circulating flow and the stagnant region. The value of the shear rate was first ascertained by experiments in order to obtain a picture of shear rate variation in a radial direction from the impeller, and the equivalence between the volumes of the high shear stress region and micromixing region was confirmed. The shear stress obtained using a correlation between the shear rate at the impeller tip and Reynolds number of Wichterle et al. was used as a representative of the shear stress in the micromixing region, and the shear stress estimated by use of an empirical correlation between the average shear rate in a fermentor and agitation speed derived by Metzner et al. was adopted as a representative of the shear stress in the macromixing region. The information about the circulation time distribution was also used to take into account oxygen deficiency during circulation of liquid elements in the macromixing region, considering that oxygen from the gas phase was supplied mainly in the high shear region. The calculated values of xanthan gum concentrations which were obtained by the proposed simulation method agreed well with the experimental data in the time course of xanthan gum production at various agitation speeds. Experimental results of the relationship between the overall specific production rate and ND (N, agitation speed, and D, impeller diameter) was also verified by the proposed method.     

Consecutive immobilized enzymatic reactions in continuous stirred tank reactor systems

The performance characteristics of two-enzyme reaction in a continuous stirred tank reactor (CSTR) are analytical investigated in this work. A model is formulated to describe the substrate concentration variations by taking into account the external and internal diffusion resistances. It is found that the reaction system exhibits the characteristics of reaction control or diffusion control depending on the operating conditions. The single CSTR model is also extended to describe the multiple CSTR system. The latter model enables the prediction of the number of CSTRs in series required to achieve a prescribed substrate conversion.     

Semi-continuous xylose-to-xylitol bioconversion by Ca-alginate entrapped yeast cells in a stirred tank reactor

Candida guilliermondii FTI 20037 cells were entrapped in Ca-alginate beads and used for xylose-to-xylitol bioconversions during five successive batches in a stirred tank reactor. Supplemented sugarcane bagasse hemicellulosic hydrolysate was used as the fermentation medium. The average volume of the Ca-alginate beads was reduced by about 30% after the 600 h taken to perform the five bioconversion cycles, thus demonstrating physical instability under the conditions prevailing in the reactor vessel. In spite of this, almost steady bioconversion rates and yields were observed along the repeated batches. In average values, a production of 51.6 g l(-1), a productivity of 0.43 g l(-1 )h(-1) and a yield of 0.71 g g(-1) were attained in each batch, variation coefficients being smaller than 10%.     

Bioleaching of chalcopyrite concentrate by a moderately thermophilic culture in a stirred tank reactor

A mixed culture of moderately thermophilic microorganisms was enriched from acid mine drainage samples collected from several chalcopyrite mines in China. Such mixed culture can be used to effectively extract copper from chalcopyrite. Furthermore, after being adapted to gradually increased concentration of chalcopyrite concentrate, the tolerance of the mixed culture to chalcopyrite concentrate was brought up to 80 g/L. The effects of several leaching parameters on copper recovery in stirred tank reactor also had been investigated. The results of the investigation show that it was possible to achieve a copper extraction rate of 75% in 44 days at a pulp density of 8%. The leaching rate of chalcopyrite concentrate tended to increase with dissolved total iron concentration. At low pH ranges, more microscopic counts of microorganisms were found in the solution. Furthermore, the analysis of leached residues indicates that the passivation of chalcopyrite concentrate was mainly due to a mass of jarosite and PbSO(4) on the mineral surface, other than the elemental sulphur layer. The bacterial community composition was analyzed by using Amplified Ribosomal DNA Restriction Analysis. Two moderately thermophilic bacteria species were identified as Leptospirillum ferriphilum and Acidithiobacillus caldus with abundance of 67% and 33% in the bio-pulp, respectively.     

Operational stability of immobilized d-glucose isomerase in a continuous feed stirred tank reactor

d-Glucose isomerization has been studied using immobilized cells of Streptomyces phaeochromogenes in a continuous feed stirred tank reactor (CSTR) where the external film diffusion resistance was negligible. Experiments conducted with various sizes of enzyme particles indicated that a strong internal diffusion resistance improved the apparent stability of these particles. The performance equations of the CSTR were constructed by associating the material balances for the inside porous support matrix with the bulk liquid phase, and enzyme deactivation was also taken into consideration. An iterative method together with the orthogonal collocation method is proposed for the evaluation of effectiveness factor and the substrate concentration profile within the enzyme particles. The numerical results offer an alternative analytical proof for the observation that under strong internal diffusion control the apparent operational stability of immobilized enzyme is improved.     

Optimization of operational conditions for adipate ester synthesis in a stirred tank reactor

Esterification of adipic acid and oleyl alcohol in a solvent-free system featuring a stirred tank reactor containing commercially immobilized Candida antarctica lipase B was performed. The process was carried out using an artificial neural network (ANN) trained by the Levenberg-Marquardt (LM) algorithm. The effects of four operative variables, temperature, time, amount of enzyme, and impeller speed, on the reaction yield were studied. By examining different ANN configurations, the best network was found to consist of seven hidden nodes using a hyperbolic tangent sigmoid transfer function. The values of the coefficient of determination (R²) and root mean squared error (RMSE) between the actual and predicted responses were determined to be 1 and 0.0058178 for training and 0.99467 and 0.622540 for the testing datasets, respectively. These results imply that the developed model was capable of predicting the esterification yield. The operative variables affected the yield, and their order of contribution was as follows: time > amount of enzyme > temperature > impeller speed. A high percentage of yield (95.7%) was obtained using a low level of enzyme (2.5% w/w), and the temperature, time, and impeller speed were 66.5°C, 354 min (about 6 h), and 500 rpm, respectively. A simple protocol for efficient substrate conversion in a solvent-free system evidenced by high enzyme stability is indicative of successful ester synthesis.     

A study on xylitol production from sugarcane bagasse hemicellulosic hydrolysate by Ca-alginate entrapped cells in a stirred tank reactor

Candida guilliermondii FTI 20037 cells were entrapped in Ca-alginate beads and used for xylitol production from sugarcane bagasse hemicellulosic hydrolysate in a stirred tank reactor (STR). Screening design and response surface methodologies were used to determine adequate cultivation conditions for this fermentation system. Quadratic models were fitted to the experimental data by regression analysis, considering the yield (Y_P/S) and the productivity (Q_P) of the xylose-to-xylitol bioconversion as dependent variables. Using a five-fold concentrated hydrolysate, air flowrate of 1.30 l/min, agitation speed of 300 rpm, initial cell concentration of 1.4 g/l and value 6.0 for the initial pH of the fermentation medium resulted in a xylitol production of 47.5 g/l after 120 h of fermentation, corresponding to a Y_P/S of 0.81 g/g and to a Q_P of 0.40 g/l h.     

Citrus peel influences the production of an extracellular naringinase by Staphylococcus xylosus MAK2 in a stirred tank reactor

Staphylococcus xylosus MAK2, Gram-positive coccus, a nonpathogenic member of the coagulase-negative Staphylococcus family was isolated from soil and used to produce naringinase in a stirred tank reactor. An initial medium at pH 5.5 and a cultivation temperature of 30°C was found to be optimal for enzyme production. The addition of Ca⁺² caused stimulation of enzyme activity. The effect of various physico-chemical parameters, such as pH, temperature, agitation, and inducer concentration was studied. The enzyme production was enhanced by the addition of citrus peel powder (CPP) in the optimized medium. A twofold increase in naringinase production was achieved using different technological combinations. The process optimization using technological combinations allowed rapid optimization of large number of variables, which significantly improved enzyme production in a 5-l reactor in 34 h. An increase in sugar concentration (15 g l⁻¹) in the fermentation medium further increased naringinase production (8.9 IU ml⁻¹) in the bioreactor. Thus, availability of naringinase renders it attractive for potential biotechnological applications in citrus processing industry.     

A comparative study of immobilized amyloglucosidase in a packed bed reactor and a continuous feed stirred tank reactor

The catalytic activity of amyloglucosidase covalently attached to DEAE-cellulose was studied in a packed bed reactor and a continuous feed stirred tank reactor (CSTR) for the reaction maltose → glucose. At low flow rates mass-transfer limitations in the bed reactor lead to lower conversions for this reactor compared to the CSTR. Simple theoretical expressions for these reactors were compared with the experimental results. There are significant differences between the kinetic parameters and pH profile of the immobilized and free enzyme. The immobilized enzyme also showed greater stability at 50°C than did free amyloglucosidase. The temperature dependence of the reaction rate was the same for immobilized and free enzyme.