Biochemical Engineering Journal (v.33, #2)

BEJ Keywords (IV).

New cancer diagnosis modeling using boosting and projective adaptive resonance theory with improved reliable index by Hiro Takahashi; Yasuyuki Murase; Takeshi Kobayashi; Hiroyuki Honda (100-109).
An optimal and individualized treatment protocol based on accurate diagnosis is urgently required for the adequate treatment of patients. For this purpose, it is important to develop a sophisticated algorithm that can manage large amount of data, such as gene expression data from DNA microarray, for optimal and individualized diagnosis. Especially, marker gene selection is essential in the analysis of gene expression data.In the present study, we developed the combination method of projective adaptive resonance theory and boosted fuzzy classifier with SWEEP operator method for model construction and marker selection. And we applied this method to microarray data of acute leukemia and brain tumor. The method enabled the selection of 14 important genes related to the prognosis of the tumor. In addition, we proposed improved reliability index for cancer diagnostic prediction of blinded subjects. Based on the index, the discriminated group with over 90% prediction accuracy was separated from the others.PART-BFCS with improved RIBFCS method does not only show high performance, but also has the feature of reliable prediction further. This result suggests that PART-BFCS with improved RIBFCS method has the potential to function as a new method of class prediction for diagnosis of patients.
Keywords: Cancer diagnosis; Fuzzy classifier; Projective adaptive resonance theory; Marker gene selection; Reliability index;

Influence of pH and salt concentration on the cross-flow microfiltration of BSA through a ceramic membrane by Emilio J. de la Casa; Antonio Guadix; Rubén Ibáñez; Emilia M. Guadix (110-115).
In this paper, the influence of pH in the 4–8 interval and NaCl concentration up to 25 mM on the cross-flow microfiltration of BSA was investigated. A tubular ceramic membrane with a pore size of 0.14 μm was employed and its point of zero charge was calculated. The evolution of permeate flow and BSA transmission with time was determined at 45 °C, a cross-flow velocity of 3.5 m/s and a transmembrane pressure of 100 kPa. The curves of permeate flow were explained according to the resistances in series model. Maximum protein transmission was obtained at the isoelectric point of BSA (4.9), with significant transmission also at the point of zero charge of the membrane and null transmission at pH 4 and 8. The highest permeate flow was observed at pH 7 and the lowest at 4.9. Finally, the addition of salt resulted to some extent in an improvement of both protein transmission and permeate flow.
Keywords: Adsorption; Bioseparations; Microporous membranes; Protein; Protein recovery; Separation;

Analysis of the transient response of a CSTR containing immobilized enzyme particles by Arturo Horta; José R. Álvarez; Susana Luque (116-125).
A minimum existence criterion in the transient response of the bulk substrate concentration in a CSTR containing immobilized enzyme (IMEs) in porous solid supports has been obtained from simulation results using several kinetic expressions for the main reaction and the enzyme deactivation reaction. A simple method for the determination of the substrate effective diffusivity and the reaction rate constant is also presented, and applied to the decomposition of hydrogen peroxide, that reacts in a CSTR that contains silica–alumina porous catalyst particles, in which horseradish peroxidase enzyme had been previously immobilized.
Keywords: Transient response; Simulation; Bioreactors; Multiphase reactors; Parameter identification; Immobilized enzyme particles;

A modified biomass of baker's yeast was prepared by grafting poly(amic acid), which was obtained via reaction of pyromellitic dianhydride (PMDA) and thiourea, onto the biomass surface at 50 °C for 4 h. This method was simpler than other reported chemical grafting methods. The presence of poly(amic acid) on the biomass surface was verified by FTIR, X-ray photoelectron spectroscopy (XPS) and microscope analyses, and the amount of carboxylate and amide groups in the biomass surface were found to be 1.36 and 0.7 mmol g−1 through potentiometric titration. Compared with the pristine biomass, the adsorption capacity of the modified biomass increased 15- and 11-fold for Cd2+ and Pb2+, respectively. According to the Langmuir equation, the maximum uptake capacities (q m) for lead and cadmium were 210.5 and 95.2 mg g−1, respectively. The kinetics for cadmium and lead adsorption followed the pseudo-second-order kinetics. FTIR and XPS demonstrated that carboxyl, amide, and hydroxyl groups were involved in the adsorption of lead and cadmium, and the adsorption mechanism for the two metal ions included ion exchange, electrostatic interaction and complexation.
Keywords: Poly(amic acid); Baker's yeast; Grafted; Biosorption;

The effect of glucose concentration in the growth medium on the relationship between glycolysis, glycogen accumulation and vancomycin production of Amycolatopsis orientalis was investigated depending on the incubation time. After a lag phase, bacterial growth of A. orientalis began and biomass concentration increased continuously up to 36th or 48th hours while glucose concentration in the culture medium was consumed rapidly in the same time of incubation. In addition, increase in glucose concentrations of the growth medium lead to increase intracellular glucose as well as glycerol levels. Intracellular pyruvate levels increased significantly up to 15 g/L while extracellular pyruvate levels with respect to increases in glucose concentration. A positive correlation between glucose kinase activities and glucose concentration was determined during the incubation period. Pyruvate kinase activity increased up to 15 g/L glucose and 48th hour of incubation. As a glycopeptide antibiotic, vancomycin production increased with the increases in glucose concentrations up to 15 g/L. These results indicated that glycogen accumulation with respect to glucose concentration of the growth medium was concomitant with the sporulation of A. orientalis. When the initial glucose concentration exceeded 15 g/L, pyruvate excretions as well as intracellular glycogen and glycerol productions were supported in spite of repression in vancomycin production of A. orientalis.
Keywords: Glycolysis; Glycogen; Vancomycin; Pyruvate; Glucose kinase; Pyruvate kinase;

New polyoxometalate–laccase integrated system for kraft pulp delignification by J.A.F. Gamelas; A.S.N. Pontes; D.V. Evtuguin; A.M.R.B. Xavier; A.P. Esculcas (141-147).
New polyoxometalate–laccase integrated system (PLIDS) employing polyoxometalate [SiW11VVO40]5− and laccase of Trametes versicolor for the continuous delignification of eucalypt kraft pulp has been developed. Pulp was delignified in a batch reactor containing catalytic amounts of [SiW11VVO40]5− at about 90 °C under atmospheric pressure. Re-oxidation of reduced polyoxometalate (POM) with laccase was carried out at 45 °C in a separate aerated bioreactor coupled with an ultrafiltration tubular ceramic membrane. This allowed the separation of laccase from re-oxidized POM, which was supplied in turn continuously to the delignification reactor.Proposed PLIDS allowed sustainable pulp delignification with minimal degradation of polysaccharides. The implementation of PLIDS, instead the fist chlorine dioxide stage (D) in conventional DEDED bleaching sequence, showed almost 60% of chlorine dioxide savings with strength properties of the bleached pulp (90% ISO) similar to those obtained after the conventional bleaching.
Keywords: Enzyme biocatalysis; Integrated processing; Lignin; Polyoxometalate; Laccase; Ultrafiltration;

Response surface methodology was used to model and optimise the esterification of ethanol with butyric acid in n-hexane, catalysed by Candida rugosa lipase immobilised in two hydrophilic polyurethane foams (“FHP 2002™” and “FHP 5000™”). Experiments were carried out following central composite rotatable designs (CCRD), as a function of the initial water activity of the biocatalyst ( a w ), initial butyric acid concentration (A) and ethanol:acid molar ratio (MR) in the organic medium. Ester production increased with increasing a w of the biocatalysts, probably due to the hydrophilicity of both substrates in contrast with the hydrophobicity of the product, which is released to the bulk medium. Thus, for each biocatalyst ( a w = 0.98 ) another CCRD was performed as a function of A and MR. With both preparations, higher conversions (>95%) were observed for low A values. For the “FHP 2002™” system, a maximum ester production of 0.23 M is expected, after 18-h reaction, at initial 0.35 M A and 1.51 MR, corresponding to a w of 0.95 and 0.84 M A and 1.65 M ethanol in lipase microenvironment. With “FHP 5000™” system, predicted initial conditions of 0.54 M A and 0.75 MR (0.32 M A; 0.75 M ethanol in microenvironment; a w of 0.95), will lead to the maximum ester production of 0.27 M. These maxima were experimentally confirmed.
Keywords: Ethyl butyrate; Immobilised enzymes; Lipase; Modelling; Optimisation; Polyurethane foam;

Source of isopentenyl diphosphate for taxol and baccatin III biosynthesis in cell cultures of Taxus baccata by Rosa M. Cusidó; Javier Palazón; Mercedes Bonfill; Oscar Expósito; Elisabet Moyano; M. Teresa Piñol (159-167).
To achieve a better understanding of the metabolism and accumulation of taxol and baccatin III in cell cultures of Taxus, three cell lines (I, II and III) of T. baccata were treated (on day 7) with several concentrations of fosmidomycin (100, 200 and 300 μM), an inhibitor of the non-mevalonate branch of the terpenoid pathway, or mevinolin (1, 3 and 5 μM), an inhibitor of the mevalonate branch, in both cases in presence and absence of 100 μM methyl jasmonate (MeJ). They were compared with lines treated only with the elicitor MeJ as well as an untreated control with respect to growth, viability and production of taxol and baccatin III. The results show that the cell line type was an important variable, mainly for taxane accumulation. The blocking effect of fosmidomycin on taxane production was significantly greater than that of mevinolin in all the cell lines, clearly suggesting that the isopentenyl diphosphate (IPP) used for the taxane ring formation was mainly formed via the non-mevalonate pathway. However, the significant reduction in the content of taxol (on average 3.8-fold) and baccatin III (on average 4.3-fold) in line I when treated with the elicitor together with mevinolin concentrations of 5 and 1 μM, respectively, also suggests that both non-mevalonate and mevalonate pathways are involved in the biosynthesis of the two taxanes as a result of cytosolic IPP and/or other prenyl diphosphate transport to the plastids. The observation that the inhibitory effect of fosmidomycin or mevilonin on taxol and baccatinn III yield does not interfere with methyl jasmonate elicitation is discussed.
Keywords: Taxus baccata; Cell lines; Taxol; Baccatin III; Isopentenyl diphosphate; Fosmidomycin; Mevinolin; Methyl jasmonate;

Effect of sulfate and iron on physico-chemical characteristics of anaerobic granular sludge by Eric D. van Hullebusch; Jarno Gieteling; Wim Van Daele; Jacques Defrancq; Piet N.L. Lens (168-177).
This research investigated the effect of the substrate composition (no substrate, glucose, glucose + sulfate or glucose + sulfate + iron) on the physico-chemical characteristics of two different anaerobic granular sludges as a function of time. The sludges were fed batch wise (pH 7, 30 °C) at an organic loading rate of 1.2 g COD l−1  d−1 (0.04 g COD g VSS−1  day−1) for 30 days. The presence of sulfate (COD/sulfate ratio = 1) in the feed of glucose fed anaerobic sludges did not change the physico-chemical characteristics throughout the incubation. In contrast, the presence of iron in the feed (in addition to glucose and sulfate, COD/iron ratio = 1) reduced the protein and carbohydrate content in the SMP and EPS with about 50% after 30 days incubation compared to the other feeding conditions. The sludge grown on glucose + sulfate + iron contained much more iron (+300–500%) and sulfur (+200–350%) than the other incubated sludges both after 14 and 30 days. The higher mineral content (lower VSS content) and the decrease of the EPS content contributed to the disintegration of iron fed granules, as shown by their lower size particles. However, the iron fed sludge displayed a higher granule strength than the other incubated sludges. Although an appreciable variation in the granule strength was noticed between the sludges investigated, it was not possible to relate these differences to their inorganic composition, the chemical composition of the extracted polymers or to the physical characteristics investigated.
Keywords: Anaerobic granular sludge; Substrates; Glucose; Sulfate; Iron; ESEM/EDX; Physico-chemical characteristics;

The effective diffusivity of natural medicinal components is an important property for a detailed pharmaceutical extraction study and a proper design of the extraction process and unit. A mass transfer mathematical model for solvent extraction of andrographolide was developed and numerically solved taking into account particle geometries, solvent concentration, mass fraction of two different geometries of plant materials, external mass transfer resistance and solid–liquid equilibrium concentration. Using inverse simulation approach with genetic algorithm, unknown four key parameters in the developed model, effective diffusivity, partition coefficient, average particle size and mass fraction of leaf particles were determined at different extraction temperatures and solvent concentrations. The simulated data through the genetic algorithm-numerical model (GA-NM) and experimental data showed a good agreement (r 2  > 0.97 and MSE < 0.0016) indicating that the scheme with genetic algorithm can be effectively used for determination of the effective diffusivity of andrographolide in plant materials.
Keywords: Andrographolide; Effective diffusivity; Genetic algorithm; Inverse simulation;

Production of laccase by membrane-surface liquid culture of Trametes versicolor using a poly(l-lactic acid) membrane by Takaaki Tanaka; Satoko Eguchi; Takashi Aoki; Toshiyuki Tamura; Hiroshi Saitoh; Masayuki Taniguchi; Hitomi Ohara; Kazuhiro Nakanishi; Douglas R. Lloyd (188-191).
Membrane-surface liquid culture (MSLC) is a promising method for the bioproduction of highly aerobic filamentous fungi [A. Ogawa, A. Yasuhara, T. Tanaka, T. Sakiyama, K. Nakanishi, Production of neutral protease by membrane-surface liquid culture of Aspergillus oryzae IAM2704, J. Ferment. Bioeng. 80 (1995) 35–40]. This paper reports on the production of laccase by Trametes versicolor on a microporous membrane of poly(l-lactic acid) (PLLA), which can be biodegraded via composting after use. The membrane was stable as a support for 24 days at 30 °C. During the first 9 days in MSLC, the fungus produced half as much laccase as it did in liquid-surface culture (LSC); however, the mycelium on the membrane was able to be re-used five times for laccase production. The laccase production was accelerated in the repeated use of the culture while the mycelium in LSC ceased to produce the enzyme. This study shows that compostable PLLA microporous membranes can be used for enzyme production by MSLC of filamentous fungi.
Keywords: Membrane bioreactors; Filamentous fungi; Microporous membranes; Poly(l-lactic acid); Enzyme production; Laccase;