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Biochemical Engineering Journal (v.32, #1)

Editorial Board (pp. co2).

BEJ Keywords (pp. ii).

Optimization of medium composition for biomass production of recombinant Escherichia coli cells using response surface methodology by İ. Emrah Nikerel; EbruToksoy Öner; Betül Kirdar; Ramazan Yildirim (pp. 1-6).

Factorial designs and second order response surface methodology (RSM) for medium optimization were employed for the growth of recombinant Escherichia coli cells carrying a plasmid encoding TaqI endonuclease as a part of the fermentation strategy for general recombinant protein production. The method used was effective in screening for nutritional requirements using limited number of experiments. The concentrations of carbon source (glucose), inorganic nitrogen ((NH₄)₂HPO₄), potassium (KH₂PO₄) and magnesium (MgSO₄·7H₂O) sources in medium were changed according to the central composite rotatable design consisting of 29 experiments, and the biomass yield was calculated. The optimum medium composition was found to be 15gL⁻¹ glucose, 6.6gL⁻¹ (NH₄)₂HPO₄, 20.1gL⁻¹ KH₂PO₄ and 1.7gL⁻¹ MgSO₄·7H₂O. The model prediction of 2.72gDCWL⁻¹ biomass at optimum conditions was verified experimentally as 2.68gDCWL⁻¹ which is much higher than any value obtained in initial experiments as well as in studies carried out previously. The correlation between biomass growth and TaqI endonuclease enzyme yield obtained under the same medium compositions was also analyzed.

Keywords: Medium design; Recombinant DNA; Enzyme production; Response surface methodology optimization

Downstream process engineering evaluation of transgenic soybean seeds as host for recombinant protein production by Goran Robić; Cristiane S. Farinas; Elíbio L. Rech; Sônia M.A. Bueno; Everson A. Miranda (pp. 7-12).

The advantages of using seeds for the production of recombinant proteins with plant-based expression system has been demonstrated by several researchers. The high productivity makes soybean a potential system for large-scale recombinant protein production. However, there is a lack of detailed engineering studies of the downstream process (DSP) of recombinant proteins produced in transgenic soybean. In this work, we evaluated the use of transgenic soybean seeds as hosts for the production of recombinant proteins from a downstream process (DSP) engineering standpoint. Recombinant β-glucuronidase (rGUS), was used as a model for extraction and purification studies. This study showed, that even a protein with acidic p I (rGUS) can be successfully separated from native soybean proteins, which also have acidic p I. Maximum GUS specific activity (9.5×10³U/mg) with high total activity recovery (8.9×10⁴U/mL) was obtained using a simple extraction solution composed of 50mmol/L citrate buffer at pH 5.25. Purification of rGUS was evaluated by a two-step chromatographic procedure – anion-exchange followed by hydrophobic interaction chromatography – which was compared to the purification of rGUS from transgenic corn and canola. Overall purification factor and activity recovery obtained were 97.3 and 110% (a value higher than 100% probably due to removal of an inhibitor). Comparison of this study with similar ones made with corn and canola seeds indicates that in terms of DSP soybean seeds can be considered a potentially viable plant system for the production of recombinant proteins.

Keywords: Soybean seeds; Extraction; Transgenic plants; Protein purification; Glucuronidase

Cultivation of Spirulina platensis in a combined airlift-tubular reactor system by Attilio Converti; Alessandra Lodi; Adriana Del Borghi; Carlo Solisio (pp. 13-18).

This preliminary study aims at evaluating the efficiency of a bench-scale tubular photobioreactor by means of batch cultivations of Spirulina platensis under light-limited conditions. The most interesting feature of this plant configuration is the use of an airlift system for biomass re-cycling instead of traditional pumps to avoid the well-known problems of trichome damage owing to mechanical stress. A maximum cell concentration of 10.6gl⁻¹ was attained after 15 days of cultivation using a photosynthetic active radiation of 120μmolphotonsm⁻²s⁻¹. Although the system operated in laminar flow under all the conditions investigated in this study, excess thricome stress was prevented only at relatively low air flow rates (<4.5lmin⁻¹), corresponding to culture speeds lower than 0.21ms⁻¹.

Keywords: Tubular bioreactor; Spirulina platensis; Light intensity; Biomass growth; Culture speed

Kinetic study on hydrolysis of oils by lipase with ultrasonic emulsification by K.B. Ramachandran; Sulaiman Al-Zuhair; C.S. Fong; C.W. Gak (pp. 19-24).

The hydrolysis of oil by lipase takes place at the interface between the oil and the aqueous solution containing the enzyme. For such systems, interfacial area between the oil phase and the aqueous phase influences the rate of hydrolysis. In this study, to enhance the hydrolysis rates of lipids, ultrasonication instead of mechanical agitation was used for interfacial area generation. By ultrasonication, larger interfacial area with much narrower distribution of oil droplet diameter could be produced as compared to by mechanical agitation. Experiments carried out to assess the hydrolysis rate of lipids showed that it was significantly enhanced by ultrasonic emulsification and the interfacial area saturation with enzyme was also not observed due to large interfacial area generated.

Keywords: Lipase; Ultrasonication; Mixing; Interfacial area; Enzyme catalysis; Kinetic parameters

Effect of metals on biodegradation kinetics for methyl tert-butyl ether by Chi-Wen Lin; Shin-Yuan Chen; Ya-Wen Cheng (pp. 25-32).

Many ground waters are polluted with a complex mixture of organic and metal contaminants. Especially, the presence of heavy metals may be of particular concern, since they may be toxic for microorganisms and may interfere with the biodegradation process. A study of methyl tert-butyl ether (MTBE) biodegradation in the presence of heavy metals with the purpose of determining the effects of type and concentration of metals on reaction kinetics was undertaken. The experiments were performed as batch biodegradation experiments, and the kinetic parameters were determined by a nonlinear regression technique. Experimental results showed that the presence of Mn²⁺ offers a slight stimulating effect on MTBE degradation; however, Cr³⁺ and Zn²⁺ resulted in less biodegradation at metal concentrations of 1mgl⁻¹. The inhibitory effect of adding metals was obvious, with constantly decreasing value of MTBE degradation rate in the following order Cu²⁺>Cr³⁺>Zn²⁺>Mn²⁺ at metal concentrations 10mgl⁻¹ and 50mgl⁻¹. The kinetic parameters of μ_m were found to be virtually unaffected until a threshold concentration (10mgl⁻¹) of metal was reached. However, a significant inhibitory effect on bacterial growth, as manifested by a decrease in both K_i values for Cu²⁺ (at 1 and 10mgl⁻¹), and both Cr³⁺ and Zn²⁺ (at 10mgl⁻¹).

Keywords: Inhibition; Kinetic parameters; Metal ions; MTBE

Hydrogen production by indigenous photosynthetic bacterium Rhodopseudomonas palustris WP3–5 using optical fiber-illuminating photobioreactors by Chun-Yen Chen; Chi-Mei Lee; Jo-Shu Chang (pp. 33-42).

A novel optical fiber-based photobioreactor was utilized to produce H₂ by indigenous purple nonsulfur bacterium Rhodopseudomonas palustris WP3–5 using acetate as the sole carbon source. Plastic cladding of conventional end-light optical fibers was removed to obtain side-light optical fibers (SLOF), which was inserted into photobioreactors as the internal light source. External irradiation by conventional lamps may also be provided for the bioreactor as supplemental light sources. The H₂ production performance and light conversion efficiency of the photobioreactor were assessed when various illumination systems were used. The light sources examined included SLOF excited by halogen lamp (OF-HL), SLOF excited by metal–halide lamp (OF-MH), tungsten filament lamp (TL), halogen lamp (HL), and binary combinations of the above. Compared with bioreactors illuminated by external lamps, the OF-HL system produced more H₂ (625ml), had higher light conversion efficiency (1.80%), and achieved higher H₂ yield (1.19molH₂/mol acetic acid). However, among the single light sources examined, HL gave the highest overall (νH2) and specific (νs,H2) H₂ production rate of 8.68ml/lh and 3.01ml/hg cell, respectively, primarily due to enabling better cell growth. Using OF-MH system resulted in poor H₂ production, indicating that emission spectrum of light sources was critical to photo-H₂ production. Combination of two different light sources appeared to further enhance photo-H₂ production, especially when optical fibers and external lamps were combined. Combination of OF-HL and TL exhibited the highest H₂ yield,νH2, andνs,H2 of 2.64molH₂/mol acetic acid, 17.06ml/hl, and 9.47ml/hg cell, respectively. However, the highest total H₂ production (944ml) and light conversion efficiency (1.42%) were attained when two types of optical fibers were incorporated (i.e., the OF-HL/OF-MH system).

Keywords: Photohydrogen production; Photobioreactor; Optical fiber; Rhodopseudomonas palustris

Influence of immobilization parameters on endopolygalacturonase productivity by hybrid Aspergillus sp. HL entrapped in calcium alginate by N. Reyes; I. Rivas-Ruiz; R. Domínguez-Espinosa; S. Solís (pp. 43-48).

Culture cells of the hybrid Aspergillus HL were immobilized in calcium alginate beads. Different spore concentrations and bead sizes produced surfaces with different mechanical resistances. Bead size of 2.2mm made with 2% alginate and inoculum of 4×10⁷spores/mlgel was determined to be adequate to reduce bead fracture by 32%. The production of endopolygalacturonases (endoPG) was strongly dependent on the temperature alone or in interaction ( P<0.05) with others factors, particularly with the pH of the medium. High temperature (39°C) increased the production of endoPG 10-fold in comparison with low temperature (27°C) without modifying the growth of HL (2.2g/l). Acid pH (2.8) facilitated the production of the enzyme (235U/(lh⁻¹)), achieving a reduction in growth of 71% in relation to the maximum obtained (3.2g/l) at pH 4.5. The best endoPG efficiency was found at 39°C, pH 2.8 using 5ml of bead volume. Under these conditions, the immobilized cells doubled the production of endoPG in comparison with free cells, and minimum support cell detachment.

Keywords: Biosynthesis; Endopolygalacturonase; Enzyme production; Filamentous fungy; Immobilized cells; Mechanical strength

Monitoring of monolayer and multilayer growth for epithelial sheet formation by Masahiro Kino-Oka; Natsuki Ogawa; Koji Murai; Masafumi Manabe; Masahito Taya (pp. 49-55).

The profiles of monolayer and multilayer growth of epithelial cells were kinetically analyzed based on the rates of cell proliferation and glucose consumption. The cells were classified into two populations in proliferative and non-proliferative states, and in the monolayer growth most of the cells were regarded as being proliferative. In the multilayer growth, the proliferative cells decreased with elapsed time, causing an increase in the non-proliferative cells. The kinetic analysis indicated that the proliferative cells consumed glucose for cell mass production (anabolism) and metabolic maintenance (catabolism), while the non-proliferative cells utilized glucose exclusively for the latter. Employing the kinetic equations with the determined parameters, the population balance of proliferative and non-proliferative cells could be estimated throughout the monolayer and the multilayer growth periods. The bioreactor system which permits to determine glucose concentration automatically was constructed, and the monitoring in the culture of epithelial cells was performed in a non-invasive manner by considering glucose consumption of the two populations in the epithelial sheets. It was found that the kinetic analysis was valid for monitoring the growth profiles of cells in the bioreactor, giving fair agreement between calculated and experimental values.

Keywords: Human cell culture; Anchorage-dependent cells; Epithelial sheet formation; Glucose consumption

Extraction penicillin G acylase from Alcaligenes faecalis in recombinant Escherichia coli with cetyl-trimethylammoniumbromide by Shiwei Cheng; Dongzhi Wei; Qingxun Song (pp. 56-60).

Cells were permeabilized with cetyl-trimethylammoniumbromide (CTAB) to extract penicillin G acylase (sp. Alcaligenes faecalis) in recombinant Escherichia coli. The optimum conditions were 0.5% (w/v) CTAB, 0.5M ionic strength, pH 8.0 and at 4°C for 28h. The maximum relative enzyme activity obtained was 93.4%, and the specific activity increased about 1.6 times than that by ultrasonic fragmentation. Furthermore, CTAB-extracted enzyme solution had fine stability, and almost no activity decreased as the enzyme was preserved at 4°C for 30 days. From these results, the method can be used as a high-performance system about the extraction of penicillin G acylase from A. faecalis that resides mainly in the outer periplasmic space of E. coli.

Keywords: Alcaligenes faecalis; Cell disruption; CTAB; Enzyme activity; Enzyme extraction; Penicillin G acylase

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Biochemical Engineering Journal (v.32, #1)