Biochemical Engineering Journal (v.41, #3)

BEJ Keywords (IV).

Production of functional antibody Fab fragment by recombinant insect cells by Hideki Yamaji; Toshitaka Manabe; Keizo Watakabe; Masaru Muraoka; Ikuo Fujii; Hideki Fukuda (203-209).
The production of an Fab fragment of the catalytic antibody 6D9 in stably transformed lepidopteran insect cells was investigated. On the basis of an expression vector that utilizes the Bombyx mori cytoplasmic actin promoter, from which foreign gene expression is stimulated with the B. mori nucleopolyhedrovirus (BmNPV) IE-1 transactivator and the BmNPV HR3 enhancer, two plasmid vectors were constructed which contain either a neomycin or a blasticidin resistance gene for use as a selectable marker. The genes encoding the heavy chain (Hc; Fd fragment) and light chain (Lc) of the 6D9 Fab fragment were inserted separately into the expression vectors. After cotransfection with the resulting plasmids to introduce the Hc and Lc genes and the two different antibiotic resistance genes, Trichoplusia ni BTI-TN-5B1-4 (High Five) cells were cultured in the presence of G418 and blasticidin. Colonies of cells resistant to the antibiotics were obtained around 2 weeks after cotransfection. Western blotting and enzyme-linked immunosorbent assay (ELISA) of the cell culture supernatant suggested that the resistant cells stably secrete an Fab fragment which retains an antigen-binding activity. High yields of over 300 μg/ml of Fab fragment were achieved in simple batch shake-flask culture of transfected insect cells. These results indicate that recombinant insect cells may offer a novel approach for efficient production of antibody molecules.
Keywords: Insect cell culture; Recombinant protein production; Cell transformation; Antibody; Fab fragment; High Five cells;

Lactic acid production from recycled paper sludge by simultaneous saccharification and fermentation by Susana Marques; José A.L. Santos; Francisco M. Gírio; J. Carlos Roseiro (210-216).
Concentrated sludge generated in large amounts by the wastewater treatment facilities of recycling paper plants raises a serious disposal problem requiring urgent solution. This recycled paper sludge (RPS) is an industrial waste with high polysaccharide content. As previously demonstrated, cellulosic and hemicellulosic fractions of RPS can be completely converted by enzymatic hydrolysis (using Celluclast®1.5 L with Novozym®188) into the constitutive glucose and xylose. These monosaccharides can be used on fermentation media to obtain a variety of products, such as lactic acid, which has an expanding market as precursor of biodegradable polylactides. Hence the purpose of the present work is to evaluate the performance of RPS as feedstock for fermentative production of lactic acid (LA) with Lactobacillus rhamnosus ATCC 7469, a very efficient lactic acid bacterium. Maximum production of lactic acid from RPS was obtained by performing the hydrolysis and fermentation steps simultaneously on medium supplemented with MRS components and calcium carbonate. L. rhamnosus produced 73 g L−1 of lactic acid, corresponding to a maximum productivity of 2.9 g L−1  h−1, with 0.97 g LA produced per g of carbohydrates on initial substrate. A process simplification was also implemented by minimizing RPS supplementation and suppressing Novozym cellobiase addition.
Keywords: Lactic acid; Recycled paper sludge; Bioconversion; Cellulase; Fermentation; Lactobacillus rhamnosus;

Transport of biomolecules within cells plays a critical role in the maintenance of the cellular functions. Compared to diffusion in aqueous phase, cytoplasmic diffusion is partly retarded by the elevated viscosity of cytosol. In addition, intracellular diffusion is affected by cytoplasmic physical properties such as tortuosity, binding, and molecule crowding. This work develops a two dimensional mathematical model describing the intracellular diffusion and yielding probe concentration distribution within the cell in time course. Initially, cytoplasmic mobility is quantified as apparent diffusion coefficient (ADC), with which all the diffusion resistances encountered are accounted for. Binding, however, as an important diffusion resistance, is then isolated from all others included in ADC. Using the theoretical analysis from the literature, furthermore we quantify the contribution of steric hindrance in retarded cytoplasmic mobility. In all, three major diffusion resistances including molecular binding to cytoarchitecture, molecule detouring caused by cytoskeleton obstacles, and elevated cytosol viscosity are elucidated and quantified in this work. All of these factors play significant roles in mobility retardness in intracellular transport.
Keywords: Diffusion; Modelling; Mass transfer; Viscosity; Cytoplasmic binding; Cytoskeleton hindrance;

This research highlights the possibility of employing a fermentation industry waste (Corynebacterium glutamicum) for the removal of nickel(II) ions from aqueous solution. Furthermore, it necessitates the importance of detailed examinations on the possible differences in the biosorption performance, even for the same biomass, but from different origins. Two types of C. glutamicum, obtained from different industrial sources, were used in this study. With respect to nickel speciation and biosorption performance, pH 6 was identified as an optimal condition. Of the two types of C. glutamicum used, the biomass with excess negatively charged groups performed well in the binding of Ni2+ ions. To enhance the feasibility of using the biomass in column mode, as well as its reuse for multiple cycles, C. glutamicum was immobilized in a polysulfone matrix. Both the free and immobilized biomasses performed relatively well, with maximum experimental uptakes of 111.4 and 102.4 mg g−1, respectively. An up-flow packed column loaded with immobilized biomass was employed for the removal of Ni2+ ions. The column performed well in the biosorption of nickel(II), and exhibited a delayed and favorable breakthrough curve, with Ni2+ uptake and percentage removal of 48.1 mg g−1 biomass and 60.4%, respectively.
Keywords: Biosorption; Immobilization; Waste-water treatment; Packed bed bioreactors; Fermentation waste; Nickel;

Optimization of amino acids addition for efficient production of glutathione in Candida utilis by Guobin Liang; Xianyan Liao; Guocheng Du; Jian Chen (234-240).
Strategy of precursor amino acids addition for glutathione (GSH) synthesis was optimized in this study. The addition of cysteine and glycine showed a significant effect on GSH accumulation. Cysteine increased intracellular GSH content greatly but inhibited cell growth at the same time. A two-step amino acids addition strategy was developed. First, 6 mmol/L glycine was added at 4 h, and a mixture of glutamic acid, glycine and cysteine was added at 12 h at stationary phase of cell growth. The optimum concentrations of three amino acids (glutamic acid, 6 mmol/L; glycine, 5.5 mmol/L; cysteine, 6.5 mmol/L) were obtained by response surface methodology.With the two-step addition strategy, biomass and GSH yield reached 11.65 g/L and 241.4 mg/L in flask experiments, increased by 10.5% and 69.5%, respectively, as compared with the control without amino acids addition.By applying this amino acid addition strategy further to batch fermentation in 7 L fermentor, GSH yield reaches 383.3 mg/L, which is 78.1% higher than the control.
Keywords: Amino acids; Glutathione (GSH); Candida utilis; Optimization; Fermentation; Glucose;

The hydrolytic kinetics of mixtures of cotton gin waste (CGW) and recycled paper sludge (RPS) at various initial enzyme concentrations of Spezyme™ AO3117 was investigated. The experiments showed that the concentrations of reducing sugars and the conversions of the mixtures increased with increasing initial enzyme concentration. The reducing sugar concentration and conversion of the mixture of 75% CGW and 25% RPS were higher than those of the mixture of 80% CGW and 20% RPS. The conversion of the former can reach 73.8% after a 72-h hydrolysis at the initial enzyme loading of 17.4 Filter Paper Unit (FPU)/g substrate. A three-parameter kinetic model based on enzyme deactivation and its analytical expression were derived. Using nonlinear regression, the parameters of the model were determined for the experimental data of hydrolytic kinetics of the mixtures. Based on this kinetic model of hydrolysis, two profit rate models, representing two kinds of operating modes with and without feedstock recycling, were developed. Using the profit rate models, the optimal enzyme loading and hydrolytic time can be predicted for the maximum profit rate in ethanol production according to the costs of enzyme and operation, enzyme loading, and ethanol market price. Simulated results from the models based on the experimental data of hydrolysis of the mixture of 75% CGW and 25% RPS showed that use of a high substrate concentration and an operating mode with feedstock recycle can greatly increase the profit rate in ethanol production. The results also demonstrated that the hydrolysis at a low enzyme loading is economically required for systematic optimization of ethanol production.
Keywords: Enzyme hydrolysis; Kinetic model; Cotton gin waste; Recycled paper sludge; Profit rate; Ethanol;

An intensified degradation of phenanthrene with macroporous alginate–lignin beads immobilized Phanerochaete chrysosporium by Kun Zhang; Yuanyuan Xu; Xiufu Hua; Huilong Han; Jiannan Wang; Jun Wang; Yongmin Liu; Zheng Liu (251-257).
Macroporous Ca-alginate (MCA) beads and macroporous Ca-alginate–lignin (MCAL) beads were prepared to immobilize Phanerochaete chrysosporium for the degradation of phenanthrene. The diameters of MCA and MCAL beads were 3.8 ± 0.2 and 4.1 ± 0.4 mm, respectively, while the diameters of macropores within them were determined to be 10–40 μm. Compared with conventional Ca-alginate (CA) beads, MCA beads prepared with 5% CaCO3 granules of 15 μm as porogenic diluent yielded a 1.5-fold increase in the diffusivity of glucose, an index of mass transfer performance. This difference was magnified to 2.2-fold when CA and MCA beads were applied to the cultivation of P. chrysosporium pre-cultivated for 2 days. Moreover, the net increase in the biomass of P. chrysosporium immobilized in MCAL beads was 1.1-, 1.7- and 3.4-fold than that immobilized in MCA beads, CA beads and in free form, respectively, within 8-day cultivation. The presence of lignin in MCAL beads led to a 28.7-fold increase in the phenanthrene adsorption capacity compared with that by MCA beads. The degradation of phenanthrene was also increased from 28.2% (MCA) to 42.3% (MCAL) after 4-day degradation. The enhanced growth and degradation indicated a high potential of MCAL-immobilized P. chrysosporium as an effective microbial model for an intensified biodegradation of phenanthrene.
Keywords: Ca-alginate bead; Diffusion; Lignin; Adsorption; Biodegradation; Phenanthrene;

In this study, the computer-aided process/solvent design is introduced to find an optimal biocompatible solvent and to maximize the ethanol production rate simultaneously for the single- or double-stage extractive fermentation process with cell recycling. Such a process/solvent design problem is formulated as a mixed-integer nonlinear programming problem that is solved by mixed-integer hybrid differential evolution in order to obtain a global design. The double-stage process can use a smaller amount of fresh solvent to increase ethanol productivity compared with that of the single-stage process, but it will also decrease overall conversion. Comparing the case studies, the simultaneous process/solvent design could yield higher overall ethanol productivity than that of the process design. The maximum ethanol production rate for the double-stage extractive fermentation with cell recycling was about 10-fold higher than that of continuous fermentation and about twofold higher than that of continuous fermentation with cell recycling.
Keywords: Ethanol; Fermentation; Liquid–liquid extraction; Optimization; Solvent design; Hybrid differential evolution;

Response surface methodology (RSM) is the most preferred method for fermentation media optimization so far. In last two decades, artificial neural network-genetic algorithm (ANN-GA) has come up as one of the most efficient method for empirical modeling and optimization, especially for non-linear systems. This paper presents the comparative studies between ANN-GA and RSM in fermentation media optimization. Fermentative production of biopolymer scleroglucan has been chosen as case study. The yield of scleroglucan was modeled and optimized as a function of four independent variables (media components) using ANN-GA and RSM. The optimized media produced 16.22 ± 0.44 g/l scleroglucan as compared to 7.8 ± 0.54 g/l with unoptimized medium.Two methodologies were compared for their modeling, sensitivity analysis and optimization abilities. The predictive and generalization ability of both ANN and RSM were compared using separate dataset of 17 experiments from earlier published work. The average % error for ANN and RSM models were 6.5 and 20 and the CC was 0.89 and 0.99, respectively, indicating the superiority of ANN in capturing the non-linear behavior of the system. The sensitivity analysis performed by both methods has given comparative results. The prediction error in optimum yield by hybrid ANN-GA and RSM were 2% and 8%, respectively.
Keywords: Scleroglucan; Sclerotium rolfsii; Response surface methodology; Artificial neural network; Genetic algorithms; Sensitivity analysis;

To elucidate the underlying signal mechanisms of Taxus cuspidata in response to oleic acid (OA) in two-liquid-phase suspension cultures (TLPSCs), the mitogen-activated protein kinase (MAPK) activation and release of cytochrome c (CytC) from mitochondria in TLPSC T. cuspidata were investigated with Western blot. The results showed that a long-term treatment with 4% (v/v) OA caused apoptosis and release of CytC from mitochondria and inhibited the activation of extracellular signal-regulated kinase-like (ERK-like) MAPK (approximately 46 kDa) in TLPSC of T. cuspidata, but a short-term treatment significantly increased the ERK-like MAPK activity. These findings suggest that the coordinate expression of p-ERK and CytC is fundamentally involved in cell survival or death in TLPSC T. cuspidata. Further investigation confirmed that anthracene-9-carboxylate (an ion-channel blocker) and suramin (a G-protein inhibitor) markedly blocked the ERK-like MAPK activity, inhibited DNA ladder formation and O2 • burst and reduced Taxol production. Although diphenyliodonium (an inhibitor of O2 • formation) successfully blocked DNA ladder formation and decreased Taxol production, it did not significantly affect the ERK-like MAPK activity, indicating that the ERK-like MAPK activation should depend on the OA-stimulated G-protein and ion-channel activity, but not on the oxidative burst during the apoptosis and Taxol production in TLPSC T. cuspidata cells.
Keywords: Two-liquid-phase; Mitogen-activated protein kinase; Cytochrome c; Oleic acid; Taxus cuspidata;

In order to separate ursodeoxycholic acid (UDCA) from its isomeric mixture, the molecular imprinting polymers (MIPs) were synthesized by using core–shell emulsion polymerization. In the porous imprinting polymer, ursodeoxycholic acid was used as imprinting molecule, acrylamide (AM) and α-methacrylic acid (MAA) were functional monomers, and CaCO3 was used for the porogen in the polymerization to obtain large pore. Characterization of the MIP structure with IR spectra demonstrated the expected MIPs. Through adsorption and selectivity assays, AM as the functional monomer showed better separation efficiency than MAA, and nonspecific and specific adsorption capacities of MIP with AM were 43.52 and 13.93 mg/g, respectively. The separation factor of MIP with AM for UDCA was 2.20. Furthermore, MIP with AM could be applied to separate UDCA from the isomeric mixture by column chromatography successfully.
Keywords: Ursodeoxycholic acid; Chenodeoxycholic acid; Core–shell molecular imprinting polymers;

Removal of endocrine disrupter compounds from municipal wastewater using an aerobic granular biomass reactor by Lydia Balest; Antonio Lopez; Giuseppe Mascolo; Claudio Di Iaconi (288-294).
The removal of selected endocrine disrupter compounds (EDCs), namely estrone (E1), 17β-estradiol (E2), 17α-ethynylestradiol (EE2), bisphenol A (BPA) and 4-tert-octylphenol (4t-OP) from municipal wastewater was investigated using a sequencing batch biofilter granular reactor (SBBGR), a new technology for biological treatment based on aerobic granular biomass. This new biological treatment is characterized by high biomass concentration (up to 40 g L−1), high sludge retention times (up to 6 months) and low sludge production (i.e., an order of magnitude lower than commonly reported for conventional biological technologies). The investigation was carried out comparing both a lab-scale SBBGR system and a demonstrative unit with a conventional full-scale activated sludge process. Results showed that the SBBGR, beside its already known benefits (removal efficiencies around 90% COD even at a high organic load (i.e., 5.7 kg COD m−3  d−1) and a sludge production value of around 0.06 kg TSS kg−1 COD removed), performed better than a conventional activated sludge process in removing E1, E2, BPA and 4t-OP. The average removal percentages of the above-mentioned EDCs, obtained during a 4-month operating period, were 60, 69, 93, 81% and 53, 41, 72, 67% for the demonstrative SBBGR system and the conventional activated sludge process of the municipal sewage treatment plant, respectively. In addition, lab-scale and demonstrative SBBGR systems showed similar efficiency for the removal of the investigated EDCs.
Keywords: Endocrine disrupter compounds; Municipal wastewater; Aerobic granular reactor; Demonstrative plant;

Compactin production in solid-state fermentation using orthogonal array method by P. brevicompactum by Nikhil S. Shaligram; Sudheer Kumar Singh; Rekha S. Singhal; George Szakacs; Ashok Pandey (295-300).
In the present study, compactin production by Penicillium brevicompactum WA 2315 was studied using solid-state fermentation. Initially, various agro-industrial residues were screened as a support and carbon source. The effect of various process parameters such as initial moisture content, incubation time, effect of additional carbon and nitrogen supplementation were studied by single factor optimization for improved compactin production. Subsequently, levels of supplement components were studied statistically using Taguchi orthogonal array (L25-orthogonal array). Out of six parameters studied using L25 array, glycerol was the most significant contributor to compactin production. The supplements at optimized levels resulted in a compactin production of 771 μg/gds (microgram/gram of dry substrate). Subsequent studies resulted in further improvement of compactin yield by varying the pH of the supplement solution and an initial pH 7.5 resulted in a compactin yield of 815 μg/gds.
Keywords: Compactin; Penicillium brevicompactum WA 2315; Solid-state fermentation; L25-orthogonal array;