Increased production of laccase by <Emphasis Type="Italic">Cerrena unicolor</Emphasis> in submerged liquid cultures

The wood-degrading basidiomycete Cerrena unicolor C-139 has been suggested as a potential producer of the industrially important enzyme laccase. Basic culture parameters influencing the enzyme synthesis in shaken-flask and aerated bioreactor cultures were evaluated to improve the yields of the process. Production of extracellular laccase was considerably enhanced by the addition of Cu²⁺ in the micromolar range to a carbon-sufficient and nitrogen-sufficient culture medium (C/N = 16.69). When an optimised medium containing glucose (10 g/L) and l-asparagine (1.5 g/L) was used, and enzyme synthesis was stimulated by addition of 10 μM Cu²⁺ to the culture medium on days 3, 6 and 9, maximal laccase productivity obtained after 17 days’ cultivation in shaken flask cultures was above 100,000 nkat/L. In fermenter fungal cultures, the influence of stabilisation of medium pH on laccase activity was additionally studied. The use of a bioreactor with an automatic pH control set at pH 6.5 after 48-h incubation resulted in the enzyme activity of 65,000 nkat/L after 8 days’ cultivation.     

Physiology of the growth and supersynthesis of DNA-polymerase in Escherichia coli during 2-stage continuous cultivation

The physiology of growth under the conditions of batch and continuous cultivation was studied with the recombinant strain of Escherichia coli CM 5199 capable of DNA polymerase I superproduction. The specific growth rate of the strain is 0.8 h-1 under the conditions of continuous cultivation which is almost 2.5 times greater than that in the exponential phase of batch cultivation. When the strain was cultivated at a flow rate above 0.3 h-1, the biomass concentration in the fermenter decreased and the culture was no more limited by the carbon source in the absence of other growth limiting components of the medium. Apparently, the metabolic product ceased to inhibit high growth rates of the culture under the conditions of continuous cultivation. The rate of DNA polymerase synthesis correlated with the specific growth rate and the respiration activity of the culture when the lambda pol A prophage was induced in the cells. The authors discuss the effectiveness of ribosome operation in the cells at a growth rate of 0.05 to 0.3 h-1 and the content of ribosomes at a higher growth rate in relation to DNA polymerase I synthesis.     

High cell density culture of the diatom Nitzschia laevis for eicosapentaenoic acid production: fed-batch development

A fed-batch process was developed for high cell density culture of the diatom Nitzschia laevis for enhanced production of eicosapentaenoic acid (EPA). Firstly, among the various medium components, glucose (Glu) was identified as the limiting substrate while nitrate (NO₃⁻), tryptone (Tr) and yeast extract (Ye) were found to promote cell growth by enhancing specific growth rate. Therefore, these components were considered essential and were included in the feed medium for subsequent fed-batch cultivation. With the optimized ratio of NO₃⁻:Tr:Ye being 1:2.6:1.3 (by weight), the relative proportions of glucose to the nitrogen sources in the feed were investigated. The optimal ratios of Glu:NO₃⁻ for specific growth rate and EPA productivity were both determined to be 32:1 (by weight). Finally, based on the residual glucose concentration in the culture, a continuous medium feeding strategy for fed-batch fermenter cultivation was developed, with which, the maximal cell dry weight and EPA yield obtained were 22.1 g l⁻¹ and 695 mg l⁻¹, respectively, which were great improvements over those of batch cultures.     

Growth ofEscherichia coli B in a continuous culture under limitation by inorganic phosphate

During batch cultivation ofEscherichia coli in a medium deficient in inorganic phosphate, the growth curve after exhaustion of phosphate is linear. Results obtained in batch cultivation were used for deriving expressions for bacterial growth at a constant rate in a single-and multi-stage continuous system. It was found experimentally that the theoretical relations derived are satisfactory.     

Production of carbonyl reductase by Geotrichum candidum in a laboratory scale bioreactor

Fungal fermentation is very complex in nature due to its nonlinear relationship with the time, especially in batch culture. Growth and production of carbonyl reductase by Geotrichum candidum NCIM 980 have been studied in a laboratory scale stirred tank bioreactor at different pH (uncontrolled and controlled), agitation, aeration and dissolved oxygen concentration. The yield of the process has been calculated in terms of glucose consumed. Initial studies showed that fermenter grown cells have more than 15 times higher activity than that of the shake flask grown cells. The medium pH was found to have unspecific but significant influence on the enzyme productivity. However, at controlled pH 5.5 the specific enzyme activity was highest (306U/mg). Higher agitation had detrimental effect on the cell mass production. Dissolved oxygen concentration was maintained by automatic control of the agitation speed at an aeration rate of 0.6 volume per volume per minute (vvm). Optimization of glucose concentration yielded 21g/l cell mass with and 9.77x10(3)U carbonyl reductase activity/g glucose. Adaptation of different strategies for glucose feeding in the fermenter broth was helpful in increasing the process yield. Feeding of glucose at a continuous rate after 3h of cultivation yielded 0.97g cell mass/g glucose corresponding to 29.1g/l cell mass. Volumetric oxygen transfer coefficient (K(L)a) increased with the increasing of agitation rate.     

Investigation of acid proteinase biosynthesis by the fungus Humicola lutea 120-5 in an airlift bioreactor

Acid proteinase production using filamentous fungus Humicola lutea 120-5 was studied under batch and continuous fermentation conditions in an airlift bioreactor. A comparison with proteinase production by fungal cells, cultivated in stirred tank bioreactor was made. The process performance in both fermentation devices was similar with respect to substrate utilization, biomass, and enzyme concentration. Continuous acid proteinase production was achieved for 14 days at an optimal dilution rate of 0.05/h with maximum specific activity of 90 U/mg DW of mycelia and yield of 38 U/mg glucose. The volumetric productivity (50 U/ml. h) was approximately 3 times higher than this of the batch system. All continuous experiments were carried out without any bacterial contamination, due to the low pH (3.0-3.5) during the process. The "pellet" type growth of the fungus in the airlift reactor prevented the system from plugging with filaments.     

A filter fermenter-apparatus and control equipment

Laboratory equipment for cultivation of microorganisms with the removal of cell-free medium is described. Separation is accomplished by filtration through a membrane which is built into a top-driven fermenter. A system is presented which controls the flow rate by automatic cleaning of the filter. Flow schemes for batch and continuous fermentations are shown. The filter fermenter is useful when studying continuous culture with feedback of biomass or processes where growth or product formation rates are influenced by inhibitory metabolites. Results are presented from experiments with Saccharomyces cerevisiae, Pedicoccus pentosaceus, and Zymomonas mobilis.     

Production of Maltase by Wild-Type and a Constitutive Mutant of Saccharomyces italicus

The production of maltase, an inducible and repressible catabolic enzyme in Saccharomyces italicus, was studied and compared in batch, fed-batch, and continuous fermentations. Tight genetic controls on maltase synthesis limited the effect of environmental manipulations such as fed-batch or continuous culture in enhancement of maltase synthesis, and neither approach was able to improve the performance above the batch process for maltase production. S. italicus was mutated, and a constitutive producer of maltase was isolated. The mutant was detected by its ability to grow on sucrose, which is a noninducing substrate that is hydrolyzed by maltase; S. italicus does not possess invertase and will not normally grow on sucrose. Maltase production by this mutant was studied during growth on sucrose in batch and continuous cultures and marked improvement in enzyme productivity was observed. The specific activity of maltase produced by this mutant was more than twice that of the parent wild type: 2,210 and 1,370 U/g of cells for the mutant versus 890 and 510 U/g of cells for the wild type in batch and continuous cultures, respectively. Maltase specific productivity was increased from 74 to 288 U/g of cells per h by switching from batch growth of the wild type to continuous cultivation of the mutant.     

Improved production of live cells of Lactobacillus rhamnosus by continuous cultivation using glucose-yeast extract medium

In this study, the growth kinetics of Lactobacillus rhamnosus and lactic acid production in continuous culture were assessed at a range of dilution rates (0.05 h(-1) to 0.40 h(-1)) using a 2 L stirred tank fermenter with a working volume of 600 ml. Unstructured models, predicated on the Monod and Luedeking-Piret equations, were employed to simulate the growth of the bacterium, glucose consumption, and lactic acid production at different dilution rates in continuous cultures. The maximum specific growth rate of L. rhamnosus, mu-max, was estimated at 0.40 h(-1), and the Monod cell growth saturation constant, Ks, at approximately 0.25 g/L. Maximum cell viability (1.3 x 10(10) CFU/ml) was achieved in the dilution rate range of D = 0.28 h(-1) to 0.35 h(-1). Both maximum viable cell yield and productivity were achieved at D = 0.35 h(-1). The continuous cultivation of L. rhamnosus at D = 0.35 h(-1) resulted in substantial improvements in cell productivity, of 267% (viable cell count) that achieved via batch cultivation.     

Biodegradation of 2-chloroethanol by freely suspended and adsorbed immobilized Pseudomonas putida US2 in soil

The degradation of 2-chloroethanol by Pseudomonas putida US2 was investigated in batch, repeated batch and continuous cultures especially in a packed-bed fermenter with sand. The degradation of 2-chloroethanol was connected with a release of protons, which led to a decrease of the pH in the medium. Higher initial concentration than 25 mM 2-chloroethanol were not degraded completely because they entailed a decrease of the pH to 5.0, which inhibited further growth and degradation. P. putida US2 showed a typical repression of catabolites and diauxic growth with succinate as cosubstrate. The addition of succinate as a second substrate caused a decrease in degradation of 2-chloroethanol. Activated sludge added to adsorbed cultures in a continuous fermentation did not lead to a decrease in metabolic activity. After 2 weeks of continuous cultivation the specialized strain could be retained.     

Enhanced continuous production of lovastatin using pellets and siran supported growth of Aspergillus terreus in an airlift reactor

Lovastatin, a hypocholesterolemic agent, is a secondary metabolite produced by filamentous microorganism Aspergillus terreus in submerged batch cultivation. Lovastatin production by pellets and immobilized siran cells was investigated in an airlift reactor. The process was carried out by submerged cultivation in continuous mode with the objective of increasing productivity using pellet and siran supported growth of A terreus. The continuous mode of fermentation improves the rate of lovastatin production. The effect of dilution rate and aeration rate were studied in continuous culture. The optimum dilution rate for pellet was 0.02 h⁻¹ and for siran carrier was 0.025 h⁻¹. Lovastatin productivity using immobilized siran carrier (0.0255 g/L/h) was found to be greater than pellets (0.022 g/L/h). The productivity by both modes of fermentation was found higher than that of batch process which suggests that continuous cultivation is a promising strategy for lovastatin production.     

The surface growth and activity ofNitrobacter

The effect of surface attachment on oxidation of nitrite to nitrate byNitrobacter was studied in batch culture, on glass coverslips, and in continuous culture on glass beads and anion exchange resin beads in an air-lift column fermenter. In batch culture, the surfaces stimulated specific growth rate, while in continuous culture, activity of attached cells was less than that of freely suspended cells. Nitrate productivity, free cell productivity, and attached cell concentration increased exponentially at the same specific rate, termed the colonization rate, and nitrate productivity was found to be a convenient estimate of biomass concentration. Permanent attachment was mediated by production of slime material. Surface growth resulted in multiple steady states and the ability to respond quickly to changes in dilution rate. The air-lift column fermenter system provided a convenient system for the study of growth and activity of attached cells and was most suitable when using ion exchange resins as a substratum for attachment.     

Low-cost propionate salt as road deicer: evaluation of cheese whey and other media constituents

Propionate and acetate salts are environmentally friendly, effective road deicer substitutes for widely used sodium chloride. A low-cost medium, using raw cheese whey and hydrolyzed whey permeate/whey permeate powder as substrates, and corn-steep liquor as a nutrient supplement, was studied for lactic acid production, replacing synthetic lactose and other high-cost nutrients. A non-sterile stage-I fermentation process for improved lactate productivity using an inexpensive commercial medium was performed at a 20-L fermenter level. A lactate yield of 0.98 g/g lactose and a productivity of 1.1 g/L/h was obtained with complete lactose utilization. When synthetic lactate and glucose were used as substrates in propionate and acetate fermentation, a total acid yield of 0.55 g/g glucose and lactate consumed and a batch productivity of 0.22 g/L/h was obtained. A stage-II fermentation process to produce propionate and acetate salts from cheese whey-derived lactate (stage-I fermentation broth) resulted in 1.6%( w/v) propionate after a total of 161 h (stages I and II).     

The regulating action of oxygen on the nutritional requirements of Neisseria meningitidis

The study has revealed regularities in changing nutritional requirements of Neisseria meningitidis with changes in the degree of the oxygen saturation of the culture medium in a fermenter under the conditions of the controlled cultivation of N. meningitidis in a synthetic culture medium in the process of batch, semicontinuous and continuous flow cultivation. As shown in this study, when oxygen supply is limited, the consumption of carbohydrates prevails, while in the presence of surplus oxygen the prevalence of the consumption of amino nitrogen is observed.     

Repeated-batch production of kojic acid in a cell-retention fermenter using Aspergillus oryzae M3B9

A cell-retention fermenter was used for the pilot-scale production of kojic acid using an improved strain of Aspergillus oryzae in repeated-batch fermentations. Among the various carbon and nitrogen sources used, sucrose and yeast extract promoted pellet morphology of fungi and higher kojic acid production. Repeated-batch culture using a medium replacement ratio of 75% gave a productivity of 5.3 g L^–1 day^–1 after 11.5 days of cultivation. While batch culture in shake-flasks resulted in a productivity of 5.1 g L^–1 day^–1, a productivity of 5 g L^–1 day^–1 was obtained in a pilot-scale fermenter. By converting the batch culture into repeated batches, the non-productive downtime of cleaning, filling and sterilizing the fermenter between each batch were eliminated, thereby increasing the kojic acid productivity.     

Immobilization of fungus Aspergillus sp. by a novel cryogel technique for production of extracellular hydrolytic enzymes

Aerobic cells of a fungus isolate Aspergillus sp. CX-1 have been immobilized in macroporous cryoPAG and in different composite cryoPAGs — fibrous adjunct carriers. The productivity of the extracellular enzymes (exo-1.4-β-glucanase, endo-1.4-β-glucanase, β-glucosidase and xylanase), and the viability, growth and ultrastructure of the immobilized fungus have been studied. The enzyme activities and stability during long-term repeated batch cultivation in the immobilized fungus were higher than in free mycelia when batch cultivated. The fungus immobilized in the composite cryoPAG, containing polypropylene non-woven fabric, possessed the highest exo-1.4-β-glucanase activity, the longest durability of enzyme production (85 days) and the most reliable mechanical strength. The fungus immobilized in porous composite cryogel possessed a variety of advantages including easy control of cryogel porosity, improved mechanical strength and durability, simplicity of construction, high enzyme productivity and high stability.     

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.     

Continuous two-stage ABE-fermentation using Clostridium beijerinckii NRRL B592 operating with a growth rate in the first stage vessel close to its maximal value

A two-stage continuous cultivation experiment with Clostridium beijerinckii NRRL B592 is described. The experiment was designed to mimic the two phases of batch culture growth of the organism in a two-stage continuous process. Thus in the first stage turbidostat the organism was grown acidogenically as rapidly as possible, and transferred to the second stage at the 'acid break point'. The second stage was designed to mimic the solventogenesis of the batch culture when it enters late exponential/early stationary phase. The volume of the second stage vessel was calculated to provide the necessary residence time for complete sugar utilization. It was hoped that the experimental set-up chosen would show whether data obtained from batch fermentation could be transferred directly to continuous culture. The culture maintained its ability to produce acetone, 1-butanol and ethanol at a dilution rate of 0.12 h(-1) for the first stage and 2.2 x 10(-2) h(-1) for the second stage and achieved an average overall solvent concentration of 15 g/l and an overall solvent productivity of 0.27 g/l/h for a period of steady-state operation of more than 1600 hours. The productivity of solventogenesis in the first stage was dependent on the value of the growth rate of the culture which was in turn determined in part by the organism employed but also by the medium composition.     

Production of pullulanase by free and immobilized cells of <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> NRC22 in batch and continuous cultures

The production of extracellular pullulanase by Bacillus licheniformis NRC22 was investigated using different fermentation modes. In batch culture maximal enzyme activity of 18 U/ml was obtained after 24 h of growth. In continuous fermentation by the free cells, maximal reactor productivity (4.15 KU/l/h) with enzyme concentration of 14.8 U/ml and specific productivity of 334.9 U/g wet cells/h was attained at a dilution rate of 0.28/h, over a period of 25 days. B. licheniformis NRC22 cells were immobilized on Ca-alginate. The immobilization conditions with respect to matrix concentration and cell load was optimized for maximal enzyme production. In repeated batch operation, the activity of the immobilized cells was stable during the 10 cycles and the activity remained between 9.8 and 7.7 U/ml. Continuous production of pullulanase by the immobilized cells was investigated in a packed–bed reactor. Maximal reactor productivity (7.0 KU/h) with enzyme concentration of 16.8 U/ml and specific productivity of 131.64 U/g wet cells/h was attained at dilution rate of 0.42/h. The enzyme activity in the effluent started to decline gradually to the level of 8.7 U/ml after 25 days of the operation.     

Thermostable alpha-amylase production by an extreme thermophile Bacillus thermooleovorans

AIMS: alpha-Amylase production by a newly isolated thermophile, Bacillus thermooleovorans, was studied under different cultivation conditions. METHODS AND RESULTS: The influence of various carbon and nitrogen sources on alpha-amylase production was quantified in batch fermentation in shake flasks. Starch and tryptone were observed to be the ideal carbon and nitrogen sources, respectively. Cultivation of the organism in a chemically defined medium consisting of glucose, riboflavin, cysteine, MgSO4, K2HPO4 and NaCl led to a near twofold increase in the production of alpha-amylase in comparison with that in the complex medium. The increase in enzyme production was achieved using vitamins and amino acids. When the organism was grown in a laboratory fermenter in the optimized complex medium, the noticeable effects were the near abolition of the lag phase, a 2.2-fold increase in enzyme production and a reduction in optimal production time from 12 to 4-5 h. CONCLUSION: Enhancement of amylase production was achieved under various cultivation conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacillus thermooleovorans produces a calcium-independent and thermostable amylase which can find use in starch saccharification.