Dewey D. Y. Ryu

Production of L(+)-lactic acid using immobilized rhizopus oryzae reactor performance based on kinetic model and simulation

The production of L(+)-lactic acid using alginate immobilizedRhizopus oryzae in tapered-column fluidized-bed batch reactor was tested and simulated using the kinetic data taken independently in shake-flask cultures. The data show saturation kinetics with substrate and product inhibitions in linear form. Analysis of the kinetic data gave kinetic constants:Vm, 11.04 g lactic acid/(L-bead. h);Km, 20.9 g glucose/L; andKi, 365 g glucose/L for lactic acid production. The product inhibition constant,Kp, was found to be 316 g lactic acid/L. The simulation results showed a good agreement with the experimental results when the initial lag phase was taken into account in the simulation model. Without the adjustment for the initial lag period, the kinetic model showed higher conversion. Starting with a glucose concentration of 150 g/L, it was possible to produce 73 g/L of L(+)-lactic acid in 44.5 h. The lactic acid yield was 64.8% by weight based on the amount of glucose consumed.

Recombinant bioprocess optimization for heterologous protein production using two-stage, cyclic fed-batch culture

Abstract A two-stage, cyclic fed-batch bioprocess was designed, and its performance evaluated to improve rice α-amylase productivity by the yeast Yarrowia lipolytica SMY2 (MatA, ade1, ura3, xpr2), ATCC 201847, containing a replicative plasmid coding for a rice α-amlyase. Transcription of the recombinant gene is controlled by the XPR2 promoter. The first stage (or growth stage) was operated in the fed-batch mode, and the growth medium, designed to maintain a constant high cell density (i.e., 60 g/l), was fed according to a predetermined and preprogrammed optimal feed rate which, in turn, maintained the specific cell growth rate at an optimal value (i.e., 0.1 h−1). Typically, when the volume in the first stage reached a preset value, a portion of culture broth (i.e., 55%) was transferred to the second stage (or production stage). The remaining cells in the growth stage were then fed with fresh growth medium according to the bioprocess control strategy developed, while induction of α-amylase expression and its production was taking place in the second stage. The second stage was also operated in the fed-batch mode, and the production medium designed to maintain a constant high cell density and high productivity of heterologous protein was fed at a predetermined and preprogrammed rate, which maintained the specific cell growth rate at an optimal level. The volumetric α-amylase productivity achieved (1835 units l−1 h−1) from the two-stage, cyclic fed-batch culture process was twofold higher than that of the fed-batch culture process. The genetic stability of the recombinant strain and the design of optimal media for growth and production stages are also critically important to a successful implementation of the two-stage, cyclic fed-batch process for production of heterologous protein.

A biosensor stabilized by polyethylene glycol for the monitoring of hydrogen peroxide in organic solvent media

Abstract Since many chemical/biochemical reactions containing hydrogen peroxide are performed in organic solvent media, the development of a stabilized biosensor in organic solvent media is very crucial. A stable hydrogen peroxide sensor with a wide measurement range and a long life in organic solvent as well as aqueous solution was developed. To maintain the stability of the sensor in the organic solvent system, catalase was mixed with polyethylene glycol (PEG). The treatment could apparently enhance the stability of the enzyme activity. The induction of hydrogen bonding between enzyme and PEG was assumed to be the possible reason for the stabilization, and was also confirmed by infrared spectrophotometry and circular dichroism (CD). The stability of the enzyme depended upon the content and molecular weight of PEG. PEGs (MW 3,350–6,000) with a mixing ratio of 0.2 g PEG to 2.8 × 10 4 catalase activity units showed the highest stability level. The biosensor developed in the present study, therefore, worked well even in 50% (v/v) dioxane solution for 2 days; 90% of the initial activity was maintained. The detection limit of the sensor was about 140 m m and the response time was 40 s in aqueous buffer and 60–90 s in the organic solvent .

Improvement of ethanol productivity and energy efficiency by degradation of inhibitors using recombinant Zymomonas mobilis (pHW20a-fdh)

Toxic compounds, such as formic acid, furfural, and hydroxymethylfurfural (HMF) generated during pretreatment of corn stover (CS) at high temperature and low pH, inhibit growth of Zymomonas mobilis and lower the conversion efficiency of CS to biofuel and other products. The inhibition of toxic compounds is considered as one of the major technical barriers in the lignocellulose bioconversion. In order to detoxify and/or degrade these toxic compounds by the model ethanologenic strain Z. mobilis itself in situ the fermentation medium, we constructed a recombinant Z. mobilis ZM4 (pHW20a‐fdh) strain that is capable of degrading toxic inhibitor, formate. This is accomplished by cloning heterologous formate dehydrogenase gene (fdh) from Saccharomyces cerevisiae and by coupling this reaction of NADH regeneration reaction system with furfural and HMF degradation in the recombinant Z. mobilis strain. The NADH regeneration reaction also improved both the energy efficiency and cell physiological activity of the recombinant organism, which were definitely confirmed by the improved cell growth, ethanol yield, and ethanol productivity during fermentation with CS hydrolysate. Biotechnol. Bioeng. 2013; 110:2395–2404.

Design and Construction of Improved New Vectors for Zymomonas mobilis Recombinants

Zymomonas mobilis is a very important gram‐negative bacterium having a potential application to simultaneous co‐production of biofuel and other high value‐added products through biorefinery process technology development. Up to now, pLOI193 has been used as the plasmid of choice for Z. mobilis strains. However, its application has been limited due to its relatively low transformation efficiency, a large plasmid size (13.4 kb), and limited choice of cloning sites for gene manipulations. Some of these limitations can be overcome by the newly designed and constructed plasmid pHW20a, which provides significantly higher transformation efficiency (about two orders of magnitude greater), better stability (for at least 120 generation times), and an ease of gene manipulations. The pHW20a contains three complete cis‐acting genes (repA, repB, and repC) encoding the Rep proteins for primosome formation. It has the origin of replication (oriV) to ensure replication in gram‐negative bacteria, two mob genes that enhances transformation efficiency, a screening marker (lacZα), expanded multiple cloning sites (MCS) that enables easy gene manipulation, and the tetracycline resistance gene (tcr). The utility of screening marker, lacZα with MCS, was confirmed by the blue‐white screening test. Several examples of applications of gene expression in Z. mobilis ZM4 have been demonstrated in this article by using several new pHW20a‐derived plasmids and expressing the homologous genes (gfo and ppc) and the heterologous genes (bglA, mdh, and fdh1). The results show that pHW20a is a very useful new vector for construction of new Z. mobilis recombinant strains that will enable simultaneous co‐production of biofuel and high value added products. Biotechnol. Bioeng. 2011; 108:1616–1627.

Sorbitol production using recombinant Zymomonas mobilis strain

A recombinant Zymomonas mobilis strain harboring the plasmid pHW20a-gfo for over-expression of glucose-fructose oxidoreductase (GFOR) was constructed. The specific activity of GFOR enzyme in the new recombinant strain was at least two folds greater than that in the wild strain. The maximum GFOR activity achieved in terms of the volumetric, and the cellular were 2.59 U ml(-1), and 0.70 U mg(-1), respectively, in the batch cultures. A significant improvement of the bioconversion process for the production of sorbitol and gluconic acid from glucose and fructose was made using divalent metal ions which drastically reduced the ethanol yield and significantly increased the yield of target product. Among several divalent metal ions evaluated, Zn(2+) was found to be most effective by inhibiting the Entner-Doudoroff pathway enzymes. The yield of the byproduct ethanol was reduced from 16.7 to 1.8 gl(-1) and the sorbitol yield was increased to almost 100% from 89%. The Ca(2+) enhanced the sorbitol yield and the formation of calcium gluconate salt made the separation of gluconate from the reaction system easier.

Effect of oxygen on ethanol fermentation in packed-bed tapered-column reactor

SummaryIn ethanol production with immobilized yeast a major problem is the provision of nutrients to these highly concentrated cells. O2 being one of the nutrients of utmost importance to yeast cells, was fed into a column packed with beads with a cell loading of more than 40 g/l. Since addition of large volume of air or O2 to a cylindrical column reactor would aggravate the problems of pressure build up and channelling caused by the evolving CO2 gas, a tapered-column reactor and pulsed flow of oxygen gas was used. The supplement of O2 gas to the tapered column increased the productivity from 21.1 g ethanol x (l gel x h)-1 to 26.7 g x (l gel x h)-1, when the ethanol concentration at the outlet was about 80 g/l. The yield coefficient of ethanol was also increased from 0.41 g ethanol/g glucose to 0.43 after O2 supplement was started. The effects of frequency and duration of O2 supplement were also determined.

Application of protoplast fusion technique to genetic recombination of Micromonospora rosaria

Abstract The optimum conditions for efficient formation and regeneration of Micromonospora rosaria protoplasts have been determined. The state of inoculum culture and stage of growth in a medium containing partially growth-inhibiting concentrations of glycine had significant effects on protoplasting. A high frequency of regeneration was accomplished with a hypertonic regeneration agar medium. A slight difference was found in the optimum culture age for formation and regeneration of protoplasts. Protoplast fusion was carried out using these optimum conditions. The recombinant frequency varied from 0.7 to 5.9% in the intraspecific crosses employing single and multiple auxotrophic markers. Electron microscopy showed stable and intact protoplasts when they were prepared with a hypertonic buffer. However, many protoplasts were shown to be damaged and many membraneous vesicles were observed when prepared in buffer without sucrose. The fusion process of protoplasts of Micromonospora was observed with the aid of electron microscopy.

New recirculating bioreactor-separator combination system for continuous bioconversion and separation of products

Abstract A new recirculating continuous bioreactor-separator combination system is designed and its practical application demonstrated for continuous production and separation of high-purity product. The process performance data and the production cost were evaluated and compared to those available from the conventional batch or semicontinuous biotransformation processes. The new design concept consists of three main components: a tapered column bioreactor, the crystallizer-separator unit, and the substrate feeding reservoir unit. The operating principles of the new bioreactor system are as follows: (1) the tapered column bioreactor unit could employ immobilized cell or enzyme and could be operated as a fluidized-bed or a packed-bed reactor system; (2) the effluent from the bioreactor is transferred to the crystallizer-separator unit in which the product is crystallized and separated intermittently or continuously; (3) the filtrate from the crystallizer-separator unit containing residual substrate is transferred to the substrate reservoir unit, where an additional amount of substrate is dissolved to make up the amount of substrate converted to the product during the last cycle; (4) the second-time filtered substrate solution from the substrate reservoir unit is fed back into the bioreactor proper unit for further bioconversion, and this completes the recycle loop of the new bioreactor-separator combination system. By this new design, a significant improvement in terms of product purity and production cost was achieved as compared to the conventional batch or semicontinuous bioconversion process systems. Based on our experimental results, we were able to conclude that highly pure amino acid product (>99.95% pure product) could be obtained, and at the same time, by employing this new biotransformation system the estimated production cost can be reduced by 25%, as compared to the conventional batch process system. It is anticipated that the principles involved in the design and operation of this new bioreactor system, as illustrated in this paper with an amino acid bioconversion process, from l -aspartate to l -alanine, as a model system will find many practical applications in the area of process biotechnology for products of pharmaceutical and agricultural importance.

Effect of intercalating dyes on the production of antibiotics by Micromonospora rosaria and micromonospora purpurea

The effect of treatment with various intercalating dyes on the ability to produce antibiotics in Micromonospora rosaria and Micromonospora purpurea was studied. Treatment with acriflavine resulted in a high frequency loss of antibiotic productivity in both species. In M. rosaria, the loss of antibiotic-producing ability appeared to be strain-dependent. In M. purpurea, up to 90% of colonies were found to have lost gentamicin-producing ability when protoplasts were used in the test. These antibiotic-nonproducing strains were further studied. The following observations were made: (1) Unlike the producing ability, the resistance to the antibiotics is a very stable character in both species. (2) Protoplast fusion analysis indicates that rosamicin-nonproducing characteristics of MR 217-AF2 and MR 217-AF3 strains induced by the acriflavine treatment is due to chromosomal mutation or rearrangement but not to loss of a plasmid. (3) Gentamicin-nonproducing strains of M. purpurea responded differently to the supplementation of streptamine or DOS in the culture medium. When supplemented with streptamine or DOS, some of these strains regained the ability to produce antibiotic, showing that the biosynthesis of intermediate was affected in these strains.