Modeling and simulation of cephalosporin C production in a fed-batch tower-type bioreactor

Immobilized cell utilization in tower-type bioreactor is one of the main alternatives being studied to improve the industrial bioprocess. Other alternatives for the production of β-lactam antibiotics, such as a cephalosporin C fed-batch process in an aerated stirred-tank bioreactor with free cells of Cephalosporium acremonium, or a tower-type bioreactor with immobilized cells of this fungus, have proven to be more efficient than the batch process. In the fed-batch process, it is possible to minimize the catabolite repression exerted by the rapidly utilization of carbon sources (such as glucose) in the synthesis of antibiotics by utilizing a suitable flow rate of supplementary medium. In this study, several runs for cephalosporin C production, each lasting 200 h, were conducted in a fed-batch tower-type bioreactor using different hydrolyzed sucrose concentrations. For this study's model, modifications were introduced to take intoaccount the influence of supplementary medium flow rate. The balance equations considered the effect of oxygen limitation inside the bioparticles. In the Monod-type rate equations, cell concentrations, substrate concentrations, and dissolved oxygen were included as reactants affecting the bioreaction rate. The set of differential equations was solved by the numerical method, and the values of the parameters were estimated by the classic nonlinear regression method following Marquardt's procedure with a 95% confidence interval. The simulation results showed that the proposed model fit well with the experimental data, and based on the experimental data and the mathematical model, an optimal mass flow rate to maximize the bioprocess productivity could be proposed.

     

Characterization of bioconversion of fumarate to succinate by alginate immobilized Enterococcus faecalis RKY1

In this study, the immobilization characteristics of Enterococcus faecalis RKY1 for succinate production were examined. At first, three natural polymers—agar, κ-carrageenan, and sodium alginate—were tried as immobilizing matrices. Among these, sodium alginate was selected as the best gel for immobilization of E. faecalis RKY1. Efficient conditions for immobilization were established to be with a 2% (w/v) sodium alginate solution and 2-mm-diameter bead. The bioconversion characteristics of the immobilized cellsat various pH values and temperatures were examined and compared with those of free cells. The optimum pH and temperature of the immobilized cells were the same as for free cells, 7.0 and 38°C respectively, but the conversion ratio was higher by immobilization for all the other pH and temperature conditions tested. When the seed volume of the immobilized cells was adjusted to 10% (v/v), 30 g/L of fumarate was completely converted tosuccinate (0.973 g/g conversion ratio) after 12 h. In addition, the immobilized cells maintained a conversion ratio of >0.95 g/g during 4wk of storageat 4°C in a 2% (w/v) CaCl₂ solution. In repetitive bioconversion experiments, the activity of the immobilized cells decreased linearly according to the number of times of reuse.     

Production of ceramide with Saccharomyces cerevisiae

The possibility of producing the biologically active material of the skin, ceramide, was studied using yeasts. The yeast strain that produced the most ceramide, Saccharomyces cerevisiae (KCCM 50515), was selected, and the optimal conditions for ceramide production were determined using shakeflask culture and batch fermentation. By measuring the production rate of ceramide at various pH values and temperatures, the optimal conditions for ceramide production were found to be pH 6.0 and 30°C. When heat shock was applied to the cells for 1 h by increasing the culture temperature from 30 to 40°C after cell growth, the amount of ceramide produced was increased 5.9-fold. A cell growth and ceramide production model was developed with Monod kinetics and the Leudecking-Piret model. It showed that ceramide production was increased when the cells were in the stationary phase.     

Semibatch production of fructo-oligosaccharides from sucrose by immobilized cells ofAureobasidium pullulans

Aureobasidium pullulans cells with fructosyltransferase activity were immobilized in 2% calcium alginate beads, and the production of fructo-oligosaccharides from sucrose was studied in a stirred tank bioreactor. It was found that cells ofA. pullulans were entrapped evenly on the alginate matrix of 2.2 mm in diameter, and an effectiveness factor of the beads was determined to be 0.3. By comparison with the system of free cells in batch operation, the total amount of fructo-oligosaccharides produced by immobilized cells was similar although the composition of fructo-oligosaccharides was found to be different. In semibatch operation with immobilized cells, reproducible results up to 60 cycles were obtained at 50 °C and this operation resulted in no loss of activity of immobilized cells.

     

Production of 2-O-α-glucopyranosyl l-ascorbic acid from ascorbic acid and β-cyclodextrin using immobilized cyclodextrin glycosyltransferase

Cyclodextrin glycosyltransferase (CGTase) isolated and purified from Paenibacillus sp. A11 was immobilized on various carriers by covalent linkage using bifunctional agent glutaraldehyde. Among tested carriers, alumina proved to be the best carrier for immobilization. The effects of several parameters on the activation of the support and on the immobilization of enzyme were optimized. The best preparation of immobilized CGTase retained 31.2% of its original activity. After immobilization, the enzymatic properties were investigated and compared with those of the free enzyme. The optimum pH of the immobilized CGTase was shifted from 6.0 to 7.0 whereas optimum temperature remained unaltered (60°C). Free and immobilized CGTase showed similar pH stability profile but the thermal stability of the immobilized CGTase was 20% higher. Kinetic data (K _M and V _max) for the free and immobilized enzymes were determined from the rate of β-CD formation and it was found that the immobilized form had higher K _M and lower V _max. The immobilized CGTase also exhibited higher stability when stored at both 4°C and 25°C for 2 months. The enzyme immobilized on alumina was further used in a batch production of 2-O-α-glucopyranosyl-l-ascorbic acid (AA-2G) from ascorbic acid and β-cyclodextrin. The yield of AA-2G was 2.92% and the immobilized CGTase retained its activity up to 74.4% of the initial catalytic activity after being used for 3 cycles. The immobilized CGTase would have a promising application in the production of various transglycosylated compounds and in the production of cyclodextrin by the hydrolysis of starch.     

Immobilization of Xylanase from <Emphasis Type="Italic">Bacillus pumilus</Emphasis> Strain MK001 and its Application in Production of Xylo-oligosaccharides

Xylanase from Bacillus pumilus strain MK001 was immobilized on different matrices following varied immobilization methods. Entrapment using gelatin (GE) (40.0%), physical adsorption on chitin (CH) (35.0%), ionic binding with Q-sepharose (Q-S) (45.0%), and covalent binding with HP-20 beads (42.0%) showed the maximum xylanase immobilization efficiency. The optimum pH of immobilized xylanase shifted up to 1.0 unit (pH 7.0) as compared to free enzyme (pH 6.0). The immobilized xylanase exhibited higher pH stability (up to 28.0%) in the alkaline pH range (7.0–10.0) as compared to free enzyme. Optimum temperature of immobilized xylanase was observed to be 8 °C higher (68.0 °C) than free enzyme (60.0 °C). The free xylanase retained 50.0% activity, whereas xylanase immobilized on HP-20, Q-S, CH, and GE retained 68.0, 64.0, 58.0, and 57.0% residual activity, respectively, after 3 h of incubation at 80.0 °C. The immobilized xylanase registered marginal increase and decrease in K _m and V _max values, respectively, as compared to free enzyme. The immobilized xylanase retained up to 70.0% of its initial hydrolysis activity after seven enzyme reaction cycles. The immobilized xylanase was found to produce higher levels of high-quality xylo-oligosaccharides from birchwood xylan, indicating its potential in the nutraceutical industry.     

Production of Lactic Acid from Raw Sweet Potato Powders by Rhizopus Oryzae Immobilized in Sodium Alginate Capsules

Rhizopus oryzae immobilized in calcium alginate was applied in lactic acid fermentation with unhydrolyzed raw sweet potato powders as the sole carbon source. The effects of sodium alginate concentration, calcium chloride concentration, and the immobilized bead diameter on lactic acid production were investigated. Increase in sodium alginate concentration during the gelation process would harden the immobilized capsule, which led to a decrease in lactic acid production. The increase in calcium chloride would increase the thickness of the immobilized capsule, which would increase the mass transfer resistance. Nevertheless, while the calcium chloride was lower than 15%, it would not have obvious effects on lactic acid production. A larger bead could have more space for cell growth, which led to the maximum lactic acid production observed at the 5-mm bead diameter. Moreover, results of repeated-batch operation suggested that immobilized cells could have higher stability in lactic acid production than free cells. The total cumulative lactic acid in immobilized-cell operation could increase by 55% as compared with free-cell operation after 216 h (seven repeated-batches), and no loss of amylolytic activity was observed. The results indicated that immobilized R. oryzae by Ca-alginate could be suitable for lactic acid production from unhydrolyzed raw potato powders.     

Comparative study of amidase production by free and immobilized Escherichia coli cells

Escherichia coli NCIM 2569 was evaluated for its potential for amidase production under submerged fermentation. Among the various amide compounds screened, maximum substrate specificity and enzyme yield (8.1 U/mL) were obtained by using 1% acetamide. Fermentation was carried out at 30°C in shake-flask culture under optimized process conditions. A maximum of 0.52 U/mL of intracellular amidase activity was also obtained from cells incubated for 24 h. Studies were also performed to elucidate the optimal conditions (gel concentration, initial biomass, curing period of beads, and calcium ion concentration in the production medium) for immobilization of whole cells. By using E. coli cells entrapped in alginate, a maximum of 6.2 U/mL of enzyme activity was obtained after 12 h of incubation under optimized conditions. Using the immobilized cells, three repeated batches were carried out successfully, and 85% of the initial enzyme activity was retained in the second and third batches. The study indicated that the immobilized E. coli cells offered certain advantages such as less time for maximum enzyme production, more stability in the enzyme production rate, and repeated use of the biocatalyst.     

Production of fumaric acid with immobilized biocatalysts

Immobilization ofRhizopus arrhizus mycelium improved fumaric acid production. The optimum conditions for fumaric acid production with immobilized cells were investigated using a statistical experimental design. Substrate concentration, carbon:nitrogen ratio, and residence time were chosen as independent variables. In the repeated batch shake flask fermentation, the fumaric acid yield from xylose was as much as 3.5 times higher with immobilized mycelium than with free mycelium. Polyurethane foam cubes, in this case, gave better results than nylon net cubes as a carrier.     

Cephalexin synthesis using immobilizedXanthomonas citri cells

For continuous production of cephalexin, whole cells ofXanthomonas citri were immobilized by entrapment in polyacrylamide gel and kappa-carrageenan gel. It wasfound that cells immobilized with kappa-carrageenan showed better thermal stability compared to those immobilized by polyacrylamide gel. The cells immobilized with kappa-carrageenan were treated with glutaraldehyde and hexamethylenediamine to prevent gel destruction during prolonged operation. By immobilizing intact cells, the optimal temperature for the synthetic enzyme reaction shifted higher by 8°C and the optimal pH became broader around 6.2 In continuous operation, the immobilized cells retained better operational stability at 25 than at 37°C, and also showed maximal conversion up to 83% at 25°C.     

Immobilization on chitosan of a thermophilic βglycosidase expressed inSaccharomyces cerevisiae

ASulfolobus solfataricus β-glycosidase expressed inSaccharomyces cerevisiae (Sβgly) was immobilized on chitosan activated with glutaraldehyde. The yield of immobilization was evaluated as 80%. Compared to the free β-glycosidase, the immobilized enzyme showed a similar pH optimum (pH = 7.0), the same increasing activity up to 80°C, improved thermostability, and no inhibition by glucose. Functional studies pointed out that the kinetic constant values for both enzymes were comparable. A bioreactor, assembled with the immobilized Sβgly, was used for glucose production. The values of cellobiose conversion increased on increasing residence time in the bioreactor, following a nonlinear trend. However, the highest glucose production/ min was obtained at a flow of 0.5 mL/min.     

Pigeonpea (Cajanus cajan L.) urease immobilized on glutaraldehyde-activated chitosan beads and its analytical applications

Urease from pigeonpea (Cajanus cajan L.) was covalently linked to crab shell chitosan beads using glutaraldehyde. The optimum immobilization (64% activity) was observed at 4°C, with a protein concentration of 0.24 mg/bead and 3% glutaraldehyde. The immobilized enzyme stored in 0.05 M Trisacetate buffer, pH 7.3, at 4°C had a t _1/2 of 110 d. There was practically no leaching of enzyme (<3%) from the immobilized beads in 30 d. The immobilized urease was used 10 times at an interval of 24 h between each use with 80% residual activity at the end of the period. The chitosan-immobilized urease showed a significantly higher Michaelis constant (8.3 mM) compared to that of the soluble urease (3.0 mM). Its apparent optimum pH also shifted from 7.3 to 8.5. Immobilized urease showed an optimal temperature of 77°C, compared with 47°C for the soluble urease. Time-dependent kinetics of the thermal denaturation of immobilized urease was studied and found to be monophasic in nature compared to biphasic in nature for soluble enzyme. This immobilized urease was used to analyze blood urea of some of the clinical samples from the clinical pathology laboratories. The results compared favorably with those obtained by the various chemical/biochemical methods employed in the clinical pathology laboratories. A column packed with immobilized urease beads was also prepared in a syringe for the regular and continuous monitoring of serum urea concentrations.     

Screening and Immobilization <Emphasis Type="Italic">Burkholderia</Emphasis> sp. GXU56 Lipase for Enantioselective Resolution of (<Emphasis Type="Italic">R</Emphasis>,<Emphasis Type="Italic">S</Emphasis>)-Methyl Mandelate

Microorganisms producing lipase were isolated from soil and sewage samples and screened for enantioselective resolution of (R,S)-methyl mandelate to (R)-mandelic acid. A strain designated as GXU56 was obtained and identified as Burkholderia sp. Preparing immobilized GXU56 lipase by simple adsorption on octyl sepharose CL-4B, the optimum temperature was shifted from 40 °C (free lipase) to 50 °C (immobilized lipase), and the optimum pH was shifted from 8.0 (free lipase) to 7.2 (immobilized lipase). The immobilized enzyme displayed excellent stability in the pH range of 5.0–8.0, at the temperatures below 50 °C and in organic solvents compared with free enzyme. Enantioselectivity ratio for (R)-mandelic acid (E) was dramatically improved from 29.2 to more than 300 by applying immobilized lipase in the resolution of (R,S)-methyl mandelate. After five cycles of use of immobilized lipase, conversion and enantiomeric excess of (R)-mandelic acid were 34.5% and 98.5%, respectively, with enantioselectivity ratio for (R)-mandelic acid (E) of 230. Thus, octyl-sepharose-immobilized GXU56 lipase can be used as a bio-resolution reagent for producing (R)-mandelic acid.     

Screening thermotolerant white-rot fungi for decolorization of wastewaters

To select a thermotolerant fungal strain for decolorization of wastewaters, ligninolytic enzyme production (lignin peroxidase, manganese peroxidase [MnP], and laccase), decolorization, and removal of total phenol and chemical oxygen demand (COD) were detected. Thirty-eight fungal strains were studied for enzyme production at 35 and 43°C on modified Kirk agar medium including 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) and MnCl₂. Thirteen strains grew on manganese-containing agar and provided green color on ABTS-containing agar plates under culture at 43°C. Decolorization of wastewater from alcohol distillery (WAD) by these strains was compared under static culture at 43°C, and Pycnoporus coccineus FPF 97091303 showed the highest potential. Thereafter, immobilized mycelia were compared with free mycelia for WAD decolorization under culture conditions of 43°C and 100 rpm. The immobilized mycelia on polyurethane foam enhanced the ligninolytic enzyme production as well as total phenol and color removal. At about the same COD removal, MnP and laccase produced by immobilized mycelia were 2 and 19 times higher than by free mycelia; the simultaneous total phenol and color removal were 3.1 and 1.5 times higher than the latter. Moreover, decolorization of synthesis dye wastewater was carried out at 43°C and 100 rpm. More than 80% of 300 mg/L of reactive blue-5 was decolorized by the immobilized mycelia within 1 to 2 d for four cycles.     

Functionalized Alginate as Immobilization Matrix in Enantioselective <Emphasis Type="SmallCaps">l</Emphasis> (+) Lactic Acid Production by <Emphasis Type="Italic">Lactobacillus delbrucekii</Emphasis>

The imperative role of functionalized natural alginate in immobilization of Lactobacillus delbrucekii (NCIM 2365) cells in production of optically pure L (+) lactic acid was studied. L. delbrucekii cells were immobilized in alginate, succinylated alginate and carrageenan to evaluate the bead stability and selectivity towards production of optically pure L (+) lactic acid. The scanning electron microscopic studies of free and immobilized cells show little morphological changes. The present study highlights the use of functionalized alginate-immobilized L. delbrucekii cells in production of L (+) lactic acid in higher yields (0.93 Yp/s in grams) with an improved enantioselectivity (99%). In addition, they further revealed decreased by-product formation (acetic and propionic acid) when compared to free and other immobilized cell fermentation.     

Statistical Optimization of Alpha-Amylase Production with Immobilized Cells of <Emphasis Type="Italic">Streptomyces erumpens</Emphasis> MTCC 7317 in <Emphasis Type="Italic">Luffa cylindrica</Emphasis> L. Sponge Discs

The purpose of this investigation was to study the effect of Streptomyces erumpens cells immobilized in various matrices, i.e., agar–agar, polyacrylamide, and luffa (Luffa cylindrica L.) sponge for production of α-amylase. Luffa sponge was found to be 21% and 51% more effective in enzyme yield than agar–agar and polyacrylamide, respectively. Response surface methodology was used to evaluate the effect of three main variables, i.e., incubation period, pH, and temperature on enzyme production with immobilized luffa cells. The experimental results showed that the optimum incubation period, pH, and temperature were 36h, 6.0, and 50 °C, respectively. The repeated batch fermentation of immobilized cells in shake flasks showed that S. erumpens cells were more or less equally physiologically active on the support even after three cycles of fermentation (3,830–3,575 units). The application of S. erumpens crude enzyme in liquefying cassava starch was studied. The maximum hydrolysis of cassava starch (85%) was obtained with the application of 4ml (15,200 units) of crude enzyme after 5 h of incubation.     

Screening of a growing cell immobilization procedure for the biosynthesis of thermostable α-amylases

Studies were carried out on α-amylase production with immobilized cells of twoBacillus strains. High yields of thermostable αamylases were obtained byBacillus licheniformis 44MB82-G, resistant to glucose catabolite repression and a thermophileBacillus brevis 174, after repeated batch cultivation (270–600 h) of the immobilized biocatalysts. Various cell immobilization techniques were compared, including entrapment in gel matrices (Ca-alginate,x-carrageenan, agar, and their combinations with polyethylene oxide), adsorption on cut disks of polymerized polyethylene oxide, and fixation on formaldehyde activated acrylonitrile-acrylamide membranes. The optimal immobilization parameters (gel and biocatalyst concentration, initial cell quantity) were determined. Among the gels and supports tested, agar,x-carrageenan, agar/polyethylene oxide gels, and the membranes were found to be suitable for immobilization and biocatalysts with high operational stabilities were obtained. An enzyme yield of 2750 U/mL culture medium was reached in the fifth repeated batch run with membrane-immobilizedBacillus licheniformis cells. This activity represented 176% of the corresponding yield obtained in batch fermentation with free cells. Higher amylase yields than the activity of the control were reached in all experiments and repeated batch runs with immobilizedBacillus brevis cells.     

Entrapment of living microbial cells in covalent polymeric networks

The kinetics of oxidative phenol degradation with microbial cellsCandida tropicalis, immobilized in a polyacrylamide and polymethacrylamide matrix, were mathematically simulated assuming zero-order and Michaelis-Menten rate equations. For zero-order kinetics an expanded equation for catalytic effectiveness as a function of the Thiele modulus, Biot number, and partition coefficients was derived and compared with numerical solutions for Michaelis-Menten kinetics. Errors with regard to the zero-order approximation become negligible ifc _o/K _M >2. Experimentally determined catalyst activities as a function of particle size and cell concentration were compared to calculated ones. Additional experiments to determine the diffusion and oxygen consumption ratios have been carried out in an effort to resolve the physical parameters to be used in the above mentioned calculations. Furthermore, experiments on cell growth during reincubation with nutrients and oxygen are reported; an increase in activity up to a factor of ten was observed. These experiments demonstrate that the microbial cells are entrapped in the polymer matrix in the living state.     

A kinetic study on the bioremediation of sodium cyanide and acetonitrile by free and immobilized cells ofPseudomonas putida

Pseudomonas putida capable of utilizing organic nitrile (acetonitrile) and inorganic cyanide (sodium cyanide) as the sole source of carbon and nitrogen was isolated from contaminated industrial sites and waste water. The bacterium possesses nitrile aminohydrolase (EC 3.5.5.1) and amidase (EC 3.5.1.4), which are involved in the transformation of cyanides and nitriles into ammonia and CO₂ through the formation of amide as an intermediate. Both of the enzymes have a high selectivity and affinity toward the⁻Cn group. The rate of degradation of aceotnitrile and sodium cyanide to ammonia and CO₂ by the calcium-alginate immobilized cells ofP. putida was studied. The rate of reaction during the biodegradation of acetonitrile and sodium cyanide, and the substrate- and product-dependent kinetics of these toxic compounds were studied using free and immobilized cells ofP. putida and modeled using a simple Michaelis-Menten equation.     

Enhanced glucose production from cellulose using coimmobilized cellulase and β-glucosidase

β-Glucosidase was covalently immobilized alone and coimmobilized with cellulase using a hydrophilic polyurethane foam (Hypol®FHP 2002). Immobilization improved the functional properties of the enzymes. When immobilized alone, the K_m for cellobiose of β-glucosidase was decreased by 33% and the pH optimum shifted to a slightly more basic value, compared to the free enzyme. Immobilized β-glucosidase was extremely stable (95% of activity remained after 1000 h of continuous use). Coimmobilization of cellulase and β-glucosidase produced a cellulose-hydrolyzing complex with a 2.5-fold greater rate of glucose production for soluble cellulose and a four-fold greater increase for insoluble cellulose, compared to immobilized cellulase alone. The immobilized enzymes showed a broader acceptance of various types of insoluble cellulose substrates than did the free enzymes and showed a long-term (at least 24 h) linear rate of glucose production from microcrystalline cellulose. The pH optimum for the coimmobilized enzymes was 6.0. This method for enzyme immobilization is fast, irreversible, and does not require harsh conditions. The enhanced glucose yields obtained indicate that this method may prove useful for commercial cellulose hydrolysis.