Effect of agitation and aeration on production of hexokinase by Saccharomyces cerevisiae

A batch culture of Saccharomyces cerevisiae for the production of hexokinase was carried out in a 5-L fermentor containing 3 L of culture medium, which was in oculated with cell suspension (about 0.7 g/L), and left ferm entingat 35°C and pH 4.0. The aeration and agitation were adjusted to attain k _La values of 15, 60, 135, and 230 h⁻¹. The highest hexokinase productivity (754.6 U/[L h]) and substrate-cell conversion yield (0.21 g/g) occurred for a k _La of 60 h⁻¹. Moreover, the formation of hexokinase and cell growth are coupled events, which is in accordance with the constitutive character of this enzyme. Hexokinase formation for k _La>60 h⁻¹ was not enhanced probably owing to saturation of the respiratory pathway by oxygen.     

Continuous production of lactic acid in a cell recycle reactor

The production of lactic acid from glucose has been demonstrated using a CSTR (continuous stirred-tank reactor) with cell recycle. Studies were conducted withLactobacillus delbrueckii at a fermentation temperature of 42°C and a pH of 6.25. A cell density of 140 g dry weight/L and a volumetric productivity of 150 g/L.h, with complete glucose consumption, were obtained. It was not possible to obtain a lactic acid concentration above 60 g/L because of product inhibition. A cell purge was not necessary to maintain high viability bacteria culture or to obtain a steady state. At steady state the net cell growth appeared to be negligible. The specific glucose consumption for cell maintenance was 0.33 g glucose/g cells-h.     

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

As a novel feeding strategy for aptomizing 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.     

A new approach to improving the performance ofZymomonas in continuous ethanol fermentations

Although most fermentation ethanol is currently produced in traditional batch processes with yeast, the ethanologenic bacteriumZymomonas mobilis is recognized as an alternative process organism for fuel alcohol production. Different strategies for improving the productivity of ethanol fermentations are reviewed. In batch and open-type continuous fermentations the advantage of replacing yeast byZymomonas relates principally to the 10% higher fermentation efficiency (product yield), whereas in high cell density, closed-type continuous systems (operating with cell recycle or retention) the superior kinetic properties ofZymomonas can be exploited to affect about a five-fold improvement in volumetric productivity. Unlike yeast, the rate of energy supply (conversion of glucose to ethanol) inZymomonas is not strictly regulated by the energy demand and a nongrowing culture exhibits a maintenance energy coefficient that is at least 25 times higher than yeast. As an alternative to process improvement through genetic engineering of the process organism this investigation has taken a biochemical and physiological approach to increasing the kinetic performance ofZ. mobilis through manipulation and control of the chemical environment. Energetically “uncoupled” phenotypes with markedly increased specific rates of ethanol production were generated under conditions of nutritional limitation (nitrogen, phosphate, or potassium) in steady-state continuous culture. The pH was shown to influence energy coupling inZymomonas affecting the maintenance coefficient (m _e ) rather than the max growth yield coefficient (Y _x ^sάx ). Whereas the pH for optimal growth ofZ. mobilis (ATCC 29191) in a complex medium was 6.0–6.5, the specific rate of ethanol production in continuous fermentations was maximal in the range 4.0–4.5. Fermentation conditions are specified for maximizing the specific productivity of aZymomonas-based continuous ethanol fermentation where the potential exists for improving the volumetric productivity in dense culture fermentations with an associated 35–40% reduction in capital costs of fermentation equipment and an estimated savings of 10–15% on cost of product recovery (distillation), and 3–7% on overall production costs based on the projected use of inexpensive feedstocks.     

A simultaneous sucrose bioconversion into ethanol and levan byZymomonas mobilis

Two biotechnological systems were developed for sucrose conversion into levan and ethanol withZymomonas mobilis, ensuring a 66.7% transfer of substrate carbon in a batch and 61% carbon transfer in a continuous culture. The effect of glucose, ethanol, and medium pH on sucrose conversion byZ. mobilis was studied. The addition of ethanol to the fermentation medium, in the final conc. of 100 g/L, uncoupled levan synthesis from ethanol fermentation. For a continuous culture, the most efficient conversion of substrate carbon into levan was reached at pH 4.8, giving 64.2 g/L levan, with the levan yield of 0.22 g/g and the productivity of 3.2 g/L/h.     

Immobilization of alkaliphilic Bacillus sp. cells for xylanase production using batch and continuous culture

Agar-immobilized alkaliphilic Bacillus sp. AR-009 cells were used for xylanase production using batch and continuous culture. In a batch culture, maximum enzyme production was observed after 48 h and remained high up to 72 h. In repeated batch cultivation, immobilized cells produced an appreciable level of xylanase activity in seven consecutive batches without any significant decline in productivity. For continuous xylanase production, immobilized cells were packed in a jacketed glass column and sterile medium was continuously pumped. A stable continuous production of xylanase was observed over a period of 1 mo. The volumetric productivity of the continuous culture was 17-fold higher than the batch culture using free cells.     

Continuous production of succinic acid by a fumarate-reducing bacterium immobilized in a hollow-fiber bioreactor

Enterococcus faecalis RKY1, a fumarate-reducing bacterium, was immobilized in an asymmetric hollow-fiber bioreactor (HFBR) for the continuous production of succinic acid. The cells were inoculated into the shell side of the HFBR, which was operated in transverse mode. Since the pH values in the HFBR declined during continuous operation to about 5.7, it was necessary to change the feed pH from 7.0 to 8.0 after 24 h of operation in order to enhance production of succinic acid. During continuous operation with a medium containing fumarate and glycerol, the productivity of succinate was 3.0–10.9 g/(L·h) with an initial concentration of 30 g/L of fumarate, 4.9–14.9 g/(L·h) with 50 g/L of fumarate, and 7.2–17.1 g/(L·h) with 80 g/L of fumarate for dilution rates between 0.1 and 0.4 h⁻¹. The maximum productivity of succinate obtained by the HFBR (17.1 g of succinate /[L·h]) was 1.7 times higher than that of the batch bioconversions (9.9 g of succinate /[L·h]) with 80 g/L of fumarate. Furthermore, the long-term stability of the HFBR was demonstrated with a continuously efficient production of succinate for more than 15 d (360 h).     

Continuous production of butanol from starch-based packing peanuts

Acetone, butanol, ethanol (ABE, or solvents) were produced from starch-based packing peanuts in batch and continuous reactors. In a batch reactor, 18.9 g/L of total ABE was produced from 80 g/L packing peanuts in 110 h of fermentation. The initial and final starch concentrations were 69.6 and 11.1 g/L, respectively. In this fermentation, ABE yield and productivity of 0.32 and 0.17 g/(L·h) were obtained, respectively. Compared to the batch fermentation, continuous fermentation of 40 g/L of starch-based packing peanuts in P2 medium resulted in a maximum solvent production of 8.4 g/L at a dilution rate of 0.033 h⁻¹. This resulted in a productivity of 0.27 g/(L·h). However, the reactor was not stable and fermentation deteriorated with time. Continuous fermentation of 35 g/L of starch solution resulted in a similar performance. These studies were performed in a vertical column reactor using Clostridium beijerinckii BA101 and P2 medium. It is anticipated that prolonged exposure of culture to acrylamide, which is formed during boiling/autoclaving of starch, affects the fermentation negatively.     

Aspects of xylitol formation in sugarcane bagasse hydrolysate by Candida guillie rmondii in the presence of tetracycline

The biocon version of xylose intoxylitol using pH values of 4.0, 5.5 and 7.0 and tetracycline concentrations of 20 and 40 mg/L was carried out to verify the influence of these parameters on Candida guilliermondii metabolism for xylitol production. Experiments were performed with sugarcane bagasse hemicellulosi chydrolysate (48.5 g/L of xylose) in 125-mL Erlenmeyer flasks, at 30°C, 200 rpm, during 88 h. The results demostrated that the bioconversion of xylose into xylitol was significantly influenced by the pH. On the other hand, in media containing 20 or 40 mg/L of tetracycline, this bioconversion was not significantly affected. The best results of xylitol production were obtained in hemicellulosic hydrolysate without tetracycline, at pH 7.0 In these conditions, the maxim um specific growth rate was 0.014/h and the yield factor of xylitol and volumetric productivity were 0.85g/g and 0.70g/L/h respectively. Xylitol and cell growth occureed simultaneously.     

Influence of carbon and nitrogen sources and temperature on hyperproduction of a thermotolerant β-glucosidase from synthetic medium by Kluyveromyces marxianus

The effect of carbon source and its concentration, inoculum size, yeast extract concentration, nitrogen source, pH of the fermentation medium, and fermentation temperature on β-glucosidase production by Kluyveromyces marxianus in shake-flask culture was investigated. These were the independent variables that directly regulated the specific growth and β-glucosidase production rate. The highest product yield, specific product yield, and productivity of β-glucosidase occurred in the medium (pH 5.5) inoculated with 10% (v/v) inoculum of the culture. Cellobiose (20 g/L) significantly improved β-glucosidase production measured as product yield (Y _P/S ) and volumetric productivity (Q _P ) followed by sucrose, lactose, and xylose. The highest levels of productivity (144 IU/[L·h]) of β-glucosidase occurred on cellobiose in the presence of CSL at 35°C and are significantly higher than the values reported by other researchers on almost all other organisms. The thermodynamics and kinetics of β-glucosidase production and its deactivation are also reported. The enzyme was substantially stable at 60°C and may find application in some industrial processes.     

α-amylase production by free and immobilized Bacillus subtilis

The effect of glucose on the α-amylase production by Bacillus subtilis ATCC-21556 was studied. Initial glucose concentrations up to 20 g/L were found to be directly proportional to the specific α-amylase production in an immobilized-cell batch system, whereas a free-cell batch system presented an inversely proportional relationship with the initial glucose concentration. This might be owing to the α-amylase repression by the glucose present in the culture medium. Three hundred eighty-five percent of the specific α-amylase production with the free-cell system was produced by the immobilized-cell batch culture.     

Synthesis and characterization of a novel extracellular polysaccharide by Rhodotorula glutinis

The aim of this work was to characterize an exopolysaccharide by Rhodotorula glutinis KCTC 7989 and to investigate the effect of the culture conditions on the production of this polymer. The extracellular polysaccharide (EPS) produced from this strain was a novel acidic heteropolysaccharide composed of neutral sugars (85%) and uronic acid (15%). The neutral sugar composition was identified by gas chromatography as mannose, fucose, glucose, and galactose in a 6.7:0.2:0.1:0.1 ratio. The molecular weight of purified EPS was estimated to be 1.0−3.8×10⁵ Dalton, and the distribution of the molecular weight was very homogeneous (polydispersity index =1.32). The EPS solution showed a characteristic of pseudoplastic non-Newtonian fluid at a concentration >2.0% in distilled water. The maximum EPS production was obtained when the strain was grown on glucose (30 g/L). Ammonium sulfate was the best suitable nitrogen source for EPS production. The highest yield of EPS was obtained at a carbon to nitrogen ratio of 15. The EPS synthesis was activated at the acidic range of pH 3.0–5.0 and increased when the pH of the culture broth decreased naturally to <2.0 during the fermentation. When the yeast was grown on glucose (30 g/L) and ammonium sulfate (2 g/L) at 22°C at an initial pH of 4.0, EPS production was maximized (4.0 g/L), and the glucose-based production yield coefficient and carbon-based production yield coefficient were 0.30 g of EPS/g of glucose and 0.34 g (carbon of EPS)/g (carbon of glucose), respectively.     

Production of cellulase on mixtures of xylose and cellulose

Cellulase production by the RUT-C30 mutant of the fungusTrichoderma reesei was studied on mixtures of xylose and cellulose. In mixed substrates, the lag phase of the growth cycle was shorter and reached the maximum of total productivity in a shorter time compared to growth on the single substrate, cellulose. A diauxic pattern of utilization of the two carbon sources was observed as well: Xylose was utilized first to support growth, followed by cellulose to induce the cellulase enzyme production and provide an additional carbon source for cellular metabolism. Of the various mixtures of xylose and cellulose used in batch enzyme production, a ratio of 30∶30 g/L of xylose to cellulose was optimal. This mixture produced the highest maximal enzyme productivity of 122 IFPU/L h, and its total productivity reached a maximum value of 55 IFPU/L h in less time than others. However, similar total productivities and higher enzyme titers were observed for growth on cellulose alone.     

Optimization of glucoamylase production by Aspergillus niger in solid-state fermentation

Glucoamylase production by Aspergillus niger in solid-state fermentation was optimized using factorial design and response surface techniques. The variables evaluated were pH and bed thickness in tray, having as response enzyme production and productivity. The bed thickness in tray was the most significant variable for both responses. The highest values for glucoamylase production occurred using pH 4.5 and bed thickness in the inferior limits at 2.0–4.2 cm. For productivity, the optimal conditions were at pH 4.5 as well and bed thickness from 4.4 to 7.5 cm. The optimal conditions for glucoamylase production while obtaining high activity without loss of productivity were pH 4.5 and bed thickness in tray from 4.0 to 4.5 cm, which resulted in an enzyme production of 695 U/g and productivity of 5791 U/h.     

Continuous fermentation for the production of citric acid from glucose

Citric acid is finding new areas of use each year and the demand for the acid is constantly increasing. Being a bulk chemical, the continuous production of citric acid would be advantageous. The paper presents the results from ammonia limited batch and continuous fermentations using the yeast strainSaccharomycopsis (Candida) lipolytica (NRRL Y-7576). Mathematical models were developed for growth and glucose utilization in batch and continuous culture. Cell and acid yields appeared to be almost the same in batch and continuous culture. The specific production rates were found to be constant, equal to 0.053 g/g h, in the batch fermentations but varied in the continuous experiments from 0 to 0.11 g/g h depending on the fermentation conditions. Continuous production in a single stage CSTR was studied for over 1,000 hours without shutdown.     

Overproduction of riboflavin by anArthrobacter sp. mutant resistant to 5-fluorouracil

Effects of pH and dissolved oxygen concentration on batchwise riboflavin production by a 5-fluorouracil (5-FU)-resistant mutant ofArthrobacter sp. were investigated. The reaction was carried out in a jar fermentor. The optimal pH of culture medium was around 7.3. Dissolved oxygen concentration was almost constant during fermentation at 600 rpm of agitation rate. Production of riboflavin reached a maximum of 160 mg/L after 70 h fermentation under the agitation rate of 600 rpm, aeration rate of 1.0 L/min, and pH 7.0.     

Synthesis of 3-substituted cyclopenta[b]indoles

When reacting with I₂, 2-(cyclopent-2-enyl)anilines undergo cyclization into 3-iodo-1,2,3,3a,4,8b-hexahydrocyclopenta[b]indoles in high yields. The minor reaction products were 3,5- or 3,7-diiodoindolines. Ammonolysis of 3-iodo-5-methyl-1,2,3,3a,4,8b-hexahydro-cyclopenta[b]indole or itsN-chloroacetyl derivative results in 3-amino-5-methyl-1,2,3,3a,48b-hexahydro- and 5-methyl-1,3a,4,8b-tetrahydrocyclopenta[b]indoles. Published inIzvestiya Akademii Nauk. Seriya Khimischeskaya, No. 10, pp. 1789–1793, October, 2000.     

Denitrification and removal of heavy metals from waste water by immobilized microorganisms

Pseudomonas fluorescens, immobilized on soft polyvinyl chloride granules containing up to 35% softeners as carbon source, was used for simultaneous removal of nitrate and heavy metals. In typical continuous column operation, a 100 mg/L nitrate input solution was reduced to a 20 mg/L output at a feeding rate of 1500 mL/h, with a capacity of 14 kg/day/m³, and with an efficiency of 79%. In the same column, Pb(NO₃)₂ concentration was reduced from 1.0 to 0.05−0.1 mg/L and ZnSO₄ concentration was reduced from 10 to 5 mg/L.Pseudomonas aeruginosa immobilized on an O₂ plasma-treated melt blown polypropylene web was used for removing 95% of a 1.7 nCi PuCl₄ activity from a nuclear plant waste water in a batch operation.     

Optimization of culture parameters for production of podophyllotoxin in suspension culture of Podophyllum hexandrum

The root explants of the germinated seedlings of Podophyllum hexandrum were grown in MS medium supplemented with indole acetic acid (IAA) (2 mg/L) and activated charcoal (0.5%), and healthy callus culture was obtained after incubation for 3 wk at 20°C. The cultivation of plant cells in shake flask was associated with problems such as clumping of cells and browning of media, which were solved by the addition of pectinase and polyvinylpyrrolidone. The effect of major media components and carbon source was studied on the growth and podophyllotoxin production in suspension culture. It was found that glucose was a better carbon source than sucrose and that NH₄ ⁺:NO₃ ⁻ ratio (total nitrogen concentration of 60 mM) and PO₄ ³⁻ did not have much effect on the growth and product formation. The relative effect of culture parameters (inoculum level, pH, IAA, glucose, NH₄ ⁺:NO₃ ⁻ ratio, and PO₄ ³⁻) on the overall growth and product response of the plant cell suspension culture was further investigated by Plackett-Burman design. This indicated that inoculum level, glucose, IAA, and pH had significant effects on growth and production of podophyllotoxin. To identify the exact optimum concentrations of these parameters on culture growth and podophyllotoxin production, central composite design experiments were formulated. The overall response equations with respect to growth and podophyllotoxin production as a function of these culture parameters were developed and used to determine the optimum concentrations of these parameters, which were pH 6.0, 1.25 mg/L of IAA, 72 g/L of glucose, and inoculum level of 8 g/L.     

Kinetic and Stoichiometric Parameters in the Production of Carotenoids by <Emphasis Type="Italic">Sporidiobolus salmonicolor</Emphasis> (CBS 2636) in Synthetic and Agroindustrial Media

With the objective of determining the kinetic behavior (growth, substrate, pH, and carotenoid production) and obtain the stoichiometric parameters of the fermentative process by Sporidiobolus salmonicolor in synthetic and agroindustrial media, fermentations were carried out in shaken flasks at 25°C, 180 rpm, and initial pH of 4.0 for 120 h in the dark, sampling every 6 h. The maximum concentrations of total carotenoids in synthetic (913 μg/L) and agroindustrial (502 μg/L) media were attained approximately 100 h after the start of the fermentative process. Carotenoid bioproduction is associated with cell growth and the ratio between carotenoid production and cell growth (Y _P/X) is 176 and 163 μg/g in the synthetic and agroindustrial media, respectively. The pH of the agroindustrial fermentation medium varied from 4.2 to 8.5 during the fermentation. The specific growth rate (μ _X) for S. salmonicolor in synthetic and agroindustrial media was 0.07 and 0.04 h⁻¹, respectively. The synthetic medium allowed for greater productivity, obtaining maximum cell productivity (P _x) of 0.08 g L⁻¹ h⁻¹ and maximum total carotenoid productivity (P _car) of 14.2 μg L⁻¹ h⁻¹. Knowledge of the kinetics of a fermentative process is of extreme importance when transposing a laboratory experiment to an industrial scale, as well as making a quantitative comparison between different culture conditions.