Biochemical Engineering Journal (v.28, #3)

BEJ Keywords (II).

IFC (CO2).

Enhanced plasmid stability and production of hEGF by immobilized recombinant E. coli JM101 by Xin-ai Chen; Zhi-nan Xu; Pei-lin Cen; W.K.R. Wong (215-219).
Recombinant Escherichia coli JM101 was immobilized with porous polyurethane foam (PUF) particle as supporter matrix for human epidermal growth factor (hEGF) production. Flask culture showed that cell immobilization in PUF can improve cell growth and hEGF expression. A bubble column and a three-phase fluidized bed bioreactor by self-design was further applied to produce hEGF, respectively. The results demonstrated that PUF is a feasible immobilized supporter material with good biocompatibility. Immobilization could also decrease the probability for segregational plasmid loss and overgrowth of plasmid-free cells. Cell density, plasmid stability and hEGF productivity were higher than those without the foam matrix, respectively. hEGF productivity was enhanced from 8.73 mg/l h of free-culture to 11.4 mg/l h of immobilized cultivation.
Keywords: hEGF productivity; Immobilized cultivation; Plasmid stability; Polyurethane foam; Recombinant E. coli JM101;

Computational prediction of impact of rerouting the carbon flux in metabolic pathway on cell growth and nisin production by Lactococcus lactis by Agustin Krisna Wardani; Sunao Egawa; Keisuke Nagahisa; Hiroshi Shimizu; Suteaki Shioya (220-230).
The detrimental effect of a low pH due to the accumulation of lactate on cell growth and nisin production by Lactococcus lactis is well known. To avoid such an inhibitory effect of lactate and to enhance nisin production, a new strategy by rerouting the carbon flux was considered. This strategy will be conducted in two ways: (i) rerouting the carbon flux to ethanol, or (ii) rerouting the carbon flux to alanine. To confirm the feasibility of both compounds as targets to reroute the carbon flux, their inhibitory effects on cell growth and nisin production were investigated. For this purpose, a mathematical model was developed to investigate the nisin production during the cultivation of L. lactis subsp. lactis ATCC11454 with addition of the compounds. The constructed model then was applied to estimate the optimum redirection ratio of the carbon flux to ethanol. The model described that nisin production with rerouting the carbon flux to ethanol was 1.7 times higher than that without rerouting the carbon flux. Investigation of inhibitory effect of alanine was also carried out and the result confirmed that alanine gave no significant inhibitory effect on both cell growth and nisin production. Based on the calculation, the optimum of nisin production was estimated twice higher than that without rerouting the carbon flux. The proposed model here provided a reasonable description to estimate and to evaluate quantitatively the feasibility of rerouting the carbon flux to ethanol or alanine to achieve a high nisin production.
Keywords: Carbon flux; Mathematical modeling; Batch culture; Lactate; Nisin; Lactococcus lactis;

Layer-by-layer self-assembly of films of nano-Au and Co(bpy)3 3+ for the determination of Japanese B encephalitis vaccine by Lingyan Zhang; Ruo Yuan; Yaqin Chai; Shihong Chen; Na Wang; Qiang Zhu (231-236).
Nano-Au and Co(bpy)3 3+ in a variety of charge states were assembled layer-by-layer into films on the gold electrode modified by l-cysteine to immobilize anti-Japanese B encephalitis for the assay of Japanese B encephalitis vaccine. The electrode modification process was characterized by electrochemical impedance spectroscopy (EIS). The factors influencing the performance of the potentiometric immunosensor were studied in detail. The immunosensor exhibited fast potentiometric response and the linear range was from 8.1 × 10−8 to 3.0 × 10−6  lgpfu/ml (plaque forming unit) with a detection limit of 3.5 × 10−8  lgpfu/ml. Moreover, the studied immunosensor exhibited high sensitivity and long-term stability. The present work supplied a promising test method for biological products.
Keywords: Potentiometric immunosensor; Layer-by-layer self-assembly; Nano-Au; Japanese B encephalitis vaccine;

The oxygen transfer rate (OTR) is a key parameter in aerobic bioprocesses, and in particular, in hydrocarbon-based bioprocesses where a proportionately higher rate is required due to the oxygen deficient structure of the substrate. The OTR is dependent on the oxygen transfer coefficient (K L a) and the differing oxygen solubilities in the immiscible liquid phases. In this study, K L a was evaluated at six discrete alkane concentrations (0, 2.5, 5, 10, 15 and 20 vol.%) over a range of agitation rates (200–1200 rpm) and aeration rates (0.5–1.5 vvm). The influence of alkane on K L a and hence OTR, was shown to be markedly dependent on the agitation rate. At agitation rates of 800 rpm and above, K L a was enhanced with increasing alkane concentration up to 10%, after which K L a repression became evident. At agitation rates below 800 rpm, K L a was repressed below that in water, at all alkane concentrations. Maximum K L a was obtained at an agitation rate between 1000 and 1200 rpm and an alkane concentration between 5 and 10%. Maximum OTR was similarly attained between 1000 and 1200 rpm although the high oxygen solubility in the alkane shifted the optimum alkane concentration to between 15 and 20%.
Keywords: Agitation; Aeration; Bioreactors; Dissolved oxygen; Gas–liquid mass transfer; Oxygen transfer; Alkane concentration;

Development of new unstructured model for simultaneous saccharification and fermentation of starch to ethanol by recombinant strain by Alexander Dimitrov Kroumov; Aparecido Nivaldo Módenes; Maicon C. de Araujo Tait (243-255).
The development of simultaneous saccharification and fermentation of starch to ethanol (SSFSE) by genetically modified microbial strains has been studied intensively [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445; G. Birol, Z.Ï. Önsan, B. Kirdar, S.G. Oliver, Ethanol production and fermentation characteristics of recombinant Saccharomyces cerevisiae strains grown on starch, Enzyme Microb. Technol. 22 (1998) 672–677; F. Kobayashi, Y. Nakamura, Effect of repressor gene on stability of bioprocess with continuous conversion of starch into ethanol using recombinant yeast, Biochem. Eng. J. 18 (2004) 133–141; F. Kobayashi, Y. Nakamura, Mathematical model of direct ethanol production from starch in immobilized recombinant yeast culture, Biochem. Eng. J. 21 (2004) 93–101; M.M. Altintas, K.Ö. Ülgen, B. Kirdar, Z.Ï. Önsan, S.G. Oliver, Improvement of ethanol production from starch by recombinant yeast through manipulation of environmental factors, Enzyme Microb. Technol. 31 (2002) 640–647; K.Ö. Ülgen, B. Saygili, Z.Ï. Önsan, B. Kirdar, Bioconversion of starch into ethanol by a recombinant Saccharomyces cerevisiae strain YPG-AB, Process Biochem. 37 (2002) 1157–1168]. Saccharomyces cerevisiae YPB-G strain secretes a bifunctional fusion protein containing enzymatic activity of the B. subtilis alpha-amylase and of the Aspergillus awamori glucoamylase [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445], and therefore is distinguished in relation to SSFSE step. In this work we have used the experimental data, presented in the paper [M.M. Altintas, B. Kirdar, Z.Ï. Önsan, K.Ö. Ülgen, Cybernetic modelling of growth and ethanol production in a recombinant Saccharomyces cerevisiae strain secreting a bifunctional fusion protein, Process Biochem. 37 (2002) 1439–1445] to develop two-hierarchic-level unstructured mathematical model describing kinetics of direct bioconversion of starch to ethanol. The first level has modeled enzymatic hydrolysis of starch to glucose by bifunctional protein and the second level includes utilization and bioconversion of glucose to ethanol by yeasts. The second level has unified the enzymatic degradation of starch, and glucose metabolization to ethanol by microorganisms. The response surface analysis was used to develop the rates models. A hybrid genetic algorithm and a decomposition approach were used in the nonlinear parameters identification procedure. The proposed model demonstrated excellent flexibility for different operational conditions of SSFSE process, and can be used successfully to describe microbial physiology of genetically modified strains.
Keywords: Recombinant yeast; Modeling; Kinetics; Ethanol; Simultaneous saccharification and fermentation;

Scale-up is traduced in practice by an increase of the dimensions of the bioreactors, leading to a modification of the time scale and thus of the process dynamics. In the present work, a methodology to study the effect of scale-up on bioreactors hydrodynamics and to put in place scale-down reactors representative of the flow properties encountered in real scales bioreactors is detailed.In order to simplify the analysis, we have proposed the use of a stochastic model which is directly affected by the time scale. Indeed, to run simulations with such models, we have to specify the time taken to achieve a transition Δt. Stochastic models are thus reliable to study scale-up effect on stirred reactors hydrodynamics. In addition, these models permit to have an insight on the internal dynamic of the process.In the case of the circulation process, qualitative aspects have to be taken into account and induce a modification of the flow regions arrangement of the model. The stochastic analysis of large-scale bioreactors permits to propose a translating methodology into a scale-down context. Optimised scale-down reactors can be used further to carry out fermentation tests with the hydrodynamic conditions of the industrial scale. In a general rule, the performances of stochastic model allow to facilitate greatly the analysis of the scale-up effect and the hydrodynamic characteristics of both large-scale and scale-down reactors.
Keywords: Bioreactors; Mixing; Circulation; Modelling; Stochastic; Scale-down;

As a control measure for substrate inhibition phenomenon in biofilters caused by toxic gases with high concentration, an appropriate mathematical model is required to calculate gas concentrations at various positions within and outside biofilms. Thus validation of the Deshussess model, Devinny–Hodge model and Luong Model were carried out for high toluene load conditions, and the results were examined for their veracity. Calculated concentrations using the modified Deshussess model, which considers sorption volume of carriers, approximated to measurements. This appears to mean the contribution of porosity in inorganic ceramic carriers to the biological removal of toluene vapor. Since toluene removal capacities for high liquid concentrations, which were calculated using the Luong model, approximated to the measurements, the generality and usefulness of the Luong model in predicting the substrate elimination capacity in biofilters with substrate inhibition appear to be manifested. Simplified Devinny–Hodge model turned out to be not applicable to predicting gas concentration along longitudinal axis of biofilters with high gas load. Various model parameters, needed for modeling studies, including critical toluene load per unit biomass and time, maximum toluene degradation rate, half velocity toluene concentration, and maximum toluene concentration in liquid, above which biodegradation is inhibited, were experimentally determined.
Keywords: Biofilter; Model verification; Elimination capacity; Toluene vapor; Substrate inhibition; Deshussess model;

Lipase-catalyzed enantioselective acylation of 3-benzyloxypropane-1,2-diol in supercritical carbon dioxide by Ildikó Kmecz; Béla Simándi; László Poppe; Zoltán Juvancz; Katalin Renner; Viktória Bódai; Enikő R. Tőke; Csaba Csajági; János Sawinsky (275-280).
Lipase-catalyzed acylation of 3-benzyloxypropane-1,2-diol with vinyl acetate as acyl donor using different lipases [porcine pancreas lipase (PPL), Lipase AK “Amano”, Lipase PS “Amano”, and crude enzymes from Trichoderma reesei RUT-C30, Thermoascus thermophilus (NRRL5208), Talaromyces emersonii (NRLL3221)] was studied in supercritical carbon dioxide (scCO2). In the reactions catalyzed by different lipases different amounts of monoacetate and diacetate products along with minor amounts of cyclic acetals forming from the diol and acetaldehyde were obtained.Application of Lipase AK led to the highest conversion (84.7%) and the highest enantiomeric excess values (eemonoacetates  = 38%, eediacetate  = 85%). Effect of water content of scCO2 on the productivity and the enantiomer selectivity of the reactions with Lipase AK was also investigated.
Keywords: Supercritical carbon dioxide; Enantioselective; Acylation; Water activity; Lipase; Filamentous fungi;

Sorbitan trioleate was modified with Cibacron Blue F-3GA (CB) to create an affinity surfactant and to form affinity-based reverse micelles in n-hexane. The partitioning equilibria and the extraction kinetics of lysozyme and bovine serum albumin (BSA) were then examined. The solubilization capacity of the reverse micellar system for lysozyme increased linearly with increasing the CB concentration from 0.1 to 0.5 mmol L−1. In contrast, the capacity for BSA at 0.5 mmol L−1 of coupled CB was only about one-fifth that for lysozyme. It indicates a strong steric hindrance effect of the micelles for the high molecular mass protein. The overall volumetric mass transfer coefficient of lysozyme in the forward extraction increased from 0.43 × 10−3 to 1.25 × 10−3  s−1 with increasing CB concentration from 0.1 to 0.5 mmol L−1. Due to the high molecular mass of BSA, its volumetric mass transfer coefficient in the forward extraction was only one-sixth that of lysozyme. The ratio of the coefficient in the back extraction to that in the forward extraction was less than 0.03, much lower than those in other micellar systems. It indicates that the interfacial resistance in this system was severer than in others.
Keywords: Affinity; Reverse micelles; Nonionic surfactant; Protein; Equilibrium; Extraction kinetics;

Production of chitosan oligosaccharides by chitosanase directly immobilized on an agar gel-coated multidisk impeller by Mei Ming; Takashi Kuroiwa; Sosaku Ichikawa; Seigo Sato; Sukekuni Mukataka (289-294).
An immobilized enzyme bioreactor consisting of an agar gel-coated multidisk impeller was developed for the hydrolysis of highly viscous chitosan solutions, and the operating conditions for the production of physiologically active chitosan oligosaccharides (pentamers and hexamers) were investigated. Chitosanase was directly immobilized on the agar gel-coated multidisk impeller by a multipoint attachment method. The high stability of the immobilized enzyme was confirmed by means of five repetitions of a batch hydrolysis reaction. When the enzyme activity at the support surface was relatively high, the yield of the target products was higher at an impeller speed of 2 s−1 than at a speed of 1 s−1. However, no significant increase in yield was observed at impeller speeds higher than 2 s−1 in reactions at either of the two substrate concentrations tested (5 and 20 kg/m3). When the surface enzyme activity was low, the impeller speed did not affect the yield of the target products. The maximum yield of pentamers and hexamers increased as the surface enzyme activity decreased, and high yields (>30%) were obtained at activities below 160 U/m2. From the viewpoint of productivity, the optimal surface-enzyme activity was about 340 U/m2, and at that activity, the yield of target products was 22%. This yield was higher than that reported for conventional acid hydrolysis. To maximize both the productivity and the yield of the target products, the surface area for the immobilized enzyme should be increased. Our results suggest that it may be possible to obtain high yields of pentamers and hexamers of chitosan oligosaccharides from highly viscous chitosan solutions with this reactor.
Keywords: Chitosan hydrolysis; Chitosan oligosaccharides; Enzymes; Bioreactors; Bioconversion; Immobilized enzymes;

A novel treatment system of wastewater contaminated with copper by a moss by Fumihisa Kobayashi; Rumiko Kofuji; Yuya Yamashita; Yoshitoshi Nakamura (295-298).
A novel treatment system of wastewater contaminated with copper was developed by using some mosses that are demonstrably metal tolerant and accumulate heavy metals into the cells. Scopelophila cataractae could remove copper more efficiently than other mosses, i.e. Physcomitrella patens and Polytrichum formosum. One hundred milligram per liter of copper ion was removed completely for 9 d using the suspended cultivation system flowing air coupled with intermittent mechanical disruption of the protonema filaments of S. cataractae by a homogenizer.
Keywords: Bioaccumulation; Heavy metals; Moss; Plant cell bioreactors; Submerged culture; Wastewater treatment;

Microbial enantioselective reduction of ethyl-2-oxo-4-phenyl-butanoate by Paulo S. Bergo de Lacerda; Joyce Benzaquem Ribeiro; Selma G.F. Leite; Ricardo B. Coelho; Edson Luiz da Silva Lima; O.A.C. Antunes (299-302).
Different microorganisms (MOs) were used to carry out the enantioselective reduction of ethyl-2-oxo-4-phenylbutanoate to (S)-(+)-2-hydroxy-4-phenylbutanoate or (R)-(+)-2-hydroxy-4-phenylbutanoate. Commercially available Saccharomyces cerevisiae and Dekera sp. led to over 92% ee of (S)-(+)-2-hydroxy-4-phenylbutanoate. Kluyveromyces marxianus gave the opposite isomer with 32% ee (R). All reactions, except those with Hansenula sp., proceeded to greater than 90% conversion. This the first report on the use of Dekera sp., Hansenula sp. and K. marxianus in the reduction of α-ketoesters.
Keywords: Saccharomyces cerevisiae; Dekera sp.; Hansenula sp.; Kluyveromyces marxianus; Lisinopril; ACE inhibitors;

To investigate the effect of crystalline structures on biocidal activity of TiO2 particles, the photocatalytic deactivation of phage MS2 was conducted using the mixture of anatase- and rutile-type TiO2 particles under irradiation with a black light lamp. Apparent deactivation rate constant, k′, was determined by applying the first-order kinetics to the time profiles of phage deactivation and the k′ value was found to be maximized at anatase ratio of R an  = 70 wt%. Considering the quantity of phage particles adsorbed on TiO2 surfaces, q T, it was also found that the k′/q T value at R an  = 70 wt% was 4.3 and 5.4 times, respectively, as compared with those at R an  = 0 and 100 wt%. The kinetic analysis of phage deactivation supported the consideration that the close contact between both types of TiO2 particles caused the enhancement of quantum yield and thereby the increased generation of reactive oxygen species in TiO2 photoreaction.
Keywords: Titanium dioxide; Bacteriophage MS2; Photocatalytic deactivation; Crystalline structure; Deactivation kinetics; Reactive oxygen species;