Biochemical Engineering Journal (v.39, #2)

BEJ Keywords (II).

The hydrolysis of casein in the presence of proteolytic enzymes: thermolysin and subtilisin, is discussed. For both biocatalysts an appropriate process temperature was chosen and the kind and values of constants in the kinetic equation were determined. Thermolysin was selected for further studies as the more appropriate catalyst because of a strong inhibition of subtilisin.A model of the process was proposed for enzymatic reaction which takes place in a membrane bioreactor with a complete retention of the enzyme and a partial retention of the substrate in the reaction zone. Having selected a proper membrane, the model was successfully verified using the data from the considered casein hydrolysis. It was proved that at a relatively high substrate retention (i.e. ca. 50%), the substrate conversion degree could increase by ca. 50%. Owing to a high cost of biocatalysts such process intensification is significant. It is all the more promising, as separation of the enzyme on a membrane allows us to use the biocatalyst many times without applying any surface immobilisation techniques.
Keywords: Casein hydrolysates; Membrane bioreactor; Membrane separation; Native enzyme immobilisation; Integrated system;

Organosolv ethanolamine pulping of olive wood by L. Jiménez; A. Rodríguez; L. Serrano; A. Moral (230-235).
This paper reports on the influence of independent variables in the pulping of olive wood trimmings [viz. cooking temperature (165–195 °C) and time (30–90 min), ethanolamine concentration (5–15%), soda concentration (2.5–7.5%) and liquid/solid ratio (4–6)], on the yield and kappa number of the pulps and breaking length, burst index and tear index of the resulting paper sheets.By using a central composite factorial design, equations that relate each dependent variable to the different independent variables were obtained that reproduced the experimental results for the dependent variables with errors less than 15%. These equations could be used to find the suitable operations conditions, so that operating with not too high values of operating variables (with the consequent minor costs of capital and of operation) pulps could be obtained by acceptable strength properties.Optimizing pulp yield, the kappa number and the strength properties of the resulting paper sheets entails using rather different conditions in each case.Obtaining acceptably strong paper sheets while saving on immobilized capital through the use of smaller facilities and less chemical reagents entails using a medium soda concentration and low values of all other independent variables. In this way, the yield is 22.2% lower, the kappa number 42.5% higher, and the breaking length, burst index and tear index 6.2, 29.1 and 29.6%, respectively, lower than their optimum values; in any case, the strength properties increase although the pulp was suitably refining.
Keywords: Organosolv; Ethanolamine; Soda; Pulping; Paper; Strength properties; Olive wood trimmings;

Response surface methodology study of laccase production in Panus tigrinus liquid cultures by Daniele Quaratino; Mario Ciaffi; Ermanno Federici; Alessandro D’annibale (236-245).
The growth medium for Panus tigrinus laccase production was studied in shaken culture through response surface methodology. The impact of five crucial variables on enzyme production, including glucose and nitrogen concentrations and three distinct putative inducers, namely, copper sulphate, 2,5-xylidine and olive-mill wastewater was thoroughly investigated. Among them, 2,5-xylidine remarkably stimulated enzyme production while Cu2+ ions within the tested concentration range (0–0.25 mM) had an opposite effect. The maximal laccase activity (about 2 IU/mL on day 11) was observed in the medium containing glucose and nitrogen at concentrations of 12.5 g/L and 20 mM, respectively, and 0.25 mM 2,5-xylidine as the sole inducer. A 35-fold increase in laccase activity was obtained with respect to the same medium where 2,5-xylidine had been omitted. With the medium thus optimised, further experiments were performed in a 3-L stirred-tank reactor (STR) leading to a laccase activity of 2200 IU/L on day 9 with a productivity of 10.2 IU/(L h) and a specific activity of 26.7 IU/mg protein. RT-PCR analyses of total RNA from both non-induced and putatively induced cultures conducted in STR showed that the transcript levels of only one out of two constitutively expressed laccase genes were markedly increased by the presence of 0.25 mM 2,5-xylidine in the optimised medium.
Keywords: Laccase production; Panus tigrinus; Response surface methodology; Process optimisation; Bioreactor; Gene expression;

The paper discusses various aspects of biosorption of microelements by marine seaweeds to be used as mineral feed supplement for livestock. In the study, biosorptive properties of a single marine species Cladophora rupestris and mixed species of algae from the Baltic Sea (a mixture of Cladophora sp. and Enteromorpha prolifera) were investigated. The mechanism of biosorption was identified as cation-exchange with light metals. Cation-exchange capacity was determined and compared with biosorption capacity. Biosorption was carried out at different pH and temperature, to choose the best operation conditions. Biosorption was performed in single- and multi-metal system to select the best production mode. The experiments showed that the biomass can bind high quantities of microelements. The maximum biosorption capacity, a model parameter of Langmuir equation for C. rupestris was 6.46 meq g−1 for Cr(III) ions, 1.37 meq g−1 for Co(II), 1.24 meq g−1 for Cu(II), 1.16 meq g−1 for Zn(II), 0.984 meq g−1 for Mn(II) and for mixed species of seaweeds from the Baltic Sea was as follows: Cr(III) 5.20 meq g−1, Co(II) 1.68 meq g−1, Mn(II) 1.28 meq g−1, Zn(II) 1.16 meq g−1, Cu(II) 0.988 meq g−1. It was found that in single-metal system, the biomass bound more metal ions than in multi-metal system. The recommended level of supplementation to feed for laying hens and swine was discussed. Also, multielemental analysis of the biomass was carried out and compared with the composition of other representatives of Cladophora and Enteromorpha.
Keywords: Biological feed supplement for livestock; Biosorption; Microelements; Macroalgae; Cladophora rupestris; Seaweeds;

Confocal laser scanning microscopy (CLSM) has been introduced in the research of chromatographic adsorption and the technique provides a direct and noninvasive way to measure and characterize intraparticle concentration distributions of proteins on a single particle level. In this work, an optics-intrinsic double-circle phenomenon that occurs in protein adsorption visualized by CLSM is exhibited and its optical mechanism is reasonably explained by an evolutive version of the attenuation equation. The time course of intraparticle fluorescence intensity profiles obtained from CLSM (even the double-circle phenomenon) in this research is vividly reproduced by model simulation, which takes into account the light attenuation by combining the attenuation equation with an effective pore diffusion model.
Keywords: Protein adsorption; Confocal laser scanning microscopy; Attenuation equation; Pore diffusion model;

A new flow-through voltammetric immunoassay protocol based on a novel transparent immunoaffinity reactor for the detection of carcinoembryonic antigen (CEA) was proposed. To construct such an affinity reactor, the reactor was initially fabricated by means of immobilization HRP-labeled anti-CEA antibody (HRP-anti-CEA)-functionalized magnetic CoFe2O4 nanoparticles (bionanoparticles) on a home-made carbon paste electrode (CPE) with an external magnet. With a non-competitive immunoassay format, the as-prepared bionanoparticles were incubated with the analyte CEA, and the formed antigen–antibody complex inhibited partly the active center of the immobilized HRP, which decreased the HRP towards H2O2 reduction. Under optimized conditions, the decrease in current response was proportional to the CEA concentration in the range of 1.5–60 ng/ml with a detection limit of 0.5 ng/ml (at 3δ). The flow-injection immunoassay system could automatically control the incubation, washing and measurement steps with acceptable reproducibility and good stability. Moreover, the proposed immunosensors were used to analyze CEA in human serum specimens. Analytical results of clinical samples show the developed immunosensor has a promising alternative approach for detecting CEA in the clinical diagnosis.
Keywords: Carcinoembryonic antigen; Magnetic nanoparticles; Voltammetric immunosensor;

Using the logistic model as a starting point, a set of reparameterised equations were established which permit the easy calculation of the relevant parameters of microbial kinetics, together with their confidence limits, with the aim of establishing rigorous comparison between cultures under differing conditions. When the resource was used to evaluate the aptitude of peptones from diverse sources for the culture of lactic acid bacteria (LAB), a great variability was found, even among the results of commercial formulations with the same denomination. None of the peptones was able to maximise the growth – very active in fish peptones – and the production of the characteristic metabolites at the same time. Under these conditions, the application of the cluster analysis to kinetic parameters of proven descriptive capability becomes a useful exploratory method, which allows to decide combinations of protein sources apt to make compatible different potential purposes of LAB cultures. This way, it is possible to design factorial experiments to search optimum values for these purposes on the basis of hypothesis which avoid the selection of superfluous variables and inadequate domains.
Keywords: Logistic equation; Lactic acid bacteria; MRS media; Commercial peptones; Fish viscera peptones; Lactic acid; Nisin; Pediocin;

Changes of catabolic genes and microbial community structures during biodegradation of nonylphenol ethoxylates and nonylphenol in natural water microcosms by Yu Zhang; Kazunari Sei; Tadashi Toyama; Michihiko Ike; Jing Zhang; Min Yang; Yoichi Kamagata (288-296).
Changes of possible key catabolic genes and microbial community structures during the degradation of NPEOs and NP in natural water microcosms were investigated using the most-probable-number-polymerase chain reaction (MPN-PCR) and terminal restriction fragment length polymorphism (T-RFLP). The copy number of catechol 2,3-dioxygenase (C23O) DNA increased significantly during NPEO and NP degradation, suggesting that meta-cleavage of the aromatic rings of NPEOs and NP might have happened. Catechol 1,2-dioxygenase (C12O) DNA, alkane-catabolic genes (alk), and 16S rDNA, on the other hand, did not change notably, suggesting that the two genes might not be the relevant genes for NPEOs and NP degradation. The 16S rRNA gene-based T-RFLP analysis results indicated that specific and different dominant (or degrading) bacteria should be selected, depending on the substances. A strain with a DNA length of 78 bp, which might be affiliated with the beta subclass of Proteobacteria, became the dominant species for NPEO degradation, while strains at 88 and 198 bp were dominant in the NP microcosm. Diversity of microbial community structure tended to be simplified after NPEO degradation, while that in the NP microcosm remained relatively stable. Five clusters were obtained according to the similarity in community structures of different microcosms by cluster analysis, which were consistent with the biodegradation behaviors of different microcosms. This is the first report on genetic evidence of a possible aromatic ring meta-cleaving pathway of NPEOs and NP in an aquatic environment.
Keywords: Functional genes; Microbial community; Nonylphenol ethoxylates; Nonylphenol; Biodegradation;

The tissue plasminogen activator analogue (pamiteplase) gene was introduced into CHO cells, and several methotorexate (MTX) resistant clones were isolated. In a medium turnover culture of the recombinant CHO cells under varied serum concentrations, the specific growth rate of cells and the specific production rate of pamiteplase showed positive dependencies on serum concentrations; however, the relationship between them was not linear. To investigate the relationship between cell growth and pamiteplase production during the cell cycle, synchronous cultures of MTX-resistant clones were grown and mitotic selection was applied using various concentrations of growth factor (Daigo's GF21). The production rate of pamiteplase showed an immediate response to the differences of GF21 concentrations during their cell cycle transition. The production rate peaked at G2/M phase at a lower level with clone PM200 and at a higher level with its derivative clone PM400, which are resistant to 200 and 400 nM MTX concentration, respectively. Clone PM400 had two peaks of pamiteplase production, a smaller one at early S phase and a larger one at G2/M phase, and the heights of these peaks were proportional to the concentration of GF21. Pamiteplase production varied more dramatically than cell cycle time depending on the medium conditions, and each parameter was affected independently. The cell cycle time became saturated at a lower concentration of GF21; however, the pamiteplase production rate kept increasing even at higher concentrations.
Keywords: Cell cycle; CHO; Recombinant protein production; Specific production rate; Cell cycle time; Mitosis; t-PA; Modelling;

Effect of culture conditions on 3-hydroxypropionaldehyde detoxification in 1,3-propanediol fermentation by Klebsiella pneumoniae by Zong-Ming Zheng; Ke-Ke Cheng; Qiu-Long Hu; Hong-Juan Liu; Ni-Ni Guo; De-Hua Liu (305-310).
1,3-Propanediol (1,3-PD) production is favored by high amount of glycerol which also contributes to the lethal 3-hydroxypropionaldehyde (3-HPA) accumulation. The influences of three culture conditions including initial glycerol concentration, stirring rate, and oxidoreduction potential (ORP) regulation on 3-HPA accumulation and 1,3-PD production were investigated. 3-HPA detoxification was available at initial glycerol concentration, aeration rate and stirring rate of up to 40 g l−1, 0.5 vvm air flow and 250 rpm, respectively. Alternatively, the stirring rate of 300 rpm was sufficient to fulfill the fermentation with initial 50 g glycerol l−1. In addition, 3-HPA accumulation could be avoided at the initial 50 g glycerol l−1 under the regulated reducing conditions (−200 and −400 mV). However, ORP regulation was not convenient and feasible for the industrial production in that more carbon was distributed to weak organic acids. Consequently, two-stage fed-batch strategy was presented to achieve 3-HPA detoxification and maintain the expected high productivity of 1,3-PD, in which the batch culture stage was conducted at initial glycerol concentration and stirring rate of 40 g l−1 and 250 rpm, respectively, and the feeding culture stage was performed at stirring rate of 300 rpm. 74.07 g 1,3-PD l−1 was produced in 24 h with the yield and productivity of 0.62 mol mol−1 and 3.08 g l−1  h−1.
Keywords: Detoxification; Fermentation; 3-Hydroxypropionaldehyde; Klebsiella pneumoniae; 1,3-Propanediol;

The kinetic characteristics of the reduction of phenylglyoxylic acid (PGA) to R-(−)-mandelic acid (R-MA) by the immobilized yeast Saccharomyces cerevisiae FD11b were studied. Compared with the free cells, the immobilized cells showed a higher enantioselectivity for the production of R-MA. However the specific production rate (q p) of R-MA with the immobilized cells was lower than that with free cells due to the effects of the external and internal diffusion. When the agitation rate was above 200 rpm, the effect of the external diffusion could be eliminated. The effects of the granule diameter and the cell density immobilized in the granules on the internal diffusion rate of the substrate and the specific production rate of R-MA (q p) were investigated. A mathematical model considering the internal diffusion and reduction kinetics was developed to analyze the effect of the internal mass transfer. The experimental data and the simulative results showed that the effect of the internal diffusion on q p could be eliminated when the granule diameter was less than 2.2 mm and the cell density immobilized was lower than 66.7 gdw/L-granules. After excluding the effect of the external and internal diffusion, q p reached 0.355 mmol gdw−1  h−1, close to that with free cells (0.360 mmol gdw−1  h−1) in the same reaction system. The optimal pH for the reduction of PGA with immobilized FD11b was 7.5, and the optimal temperature was 32 °C.
Keywords: Asymmetric production; R-(−)-Mandelic acid; Immobilization; Numerical simulation;

In this paper l-methionine oxidation catalyzed by l-phenylalanine dehydrogenase from Rhodococcus sp. M4 was studied. It was found that the reaction equilibrium is shifted to the side of reduction, and it was therefore necessary to regenerate NAD+ to increase l-methionine conversion. NADH oxidase from Lactobacillus brevis was used for that purpose. The enzyme was kinetically characterized. It was found that the enzyme is inhibited by NAD+. Hence, NADH oxidation catalyzed by NADH oxidase was described by the Michaelis–Menten equation which included anticompetitive NAD+ inhibition. l-Methionine oxidation was described by formal double-substrate Michaelis–Menten model which included competitive product inhibition by NADH. 2-Oxo-4-methylthiobutyric acid reduction was described by formal three-substrate Michaelis–Menten kinetics which included competitive inhibition by NAD+. Experiments were carried out in the batch and in the continuously operated enzyme membrane reactor. 100% l-methionine conversion was achieved in the batch reactor. The conversion was lower in the continuously operated enzyme membrane reactor where enzyme deactivation occurred.
Keywords: Amino acid; Coenzyme regeneration; NADH oxidase; l-Phenylalanine dehydrogenase; Enzymes; Enzyme biocatalysis;

Amino acids yields during proteolysis catalyzed by carboxypeptidase A are strongly dependent on substrate pre-hydrolysis by Gilson A. Pinto; Paulo W. Tardioli; Rebeca Y. Cabrera-Padilla; Célia M.A. Galvão; Roberto C. Giordano; Raquel L.C. Giordano (328-337).
The hydrolysis of polypeptides (45 °C, pH 7.0), catalyzed by bovine pancreas carboxypeptidase A (CPA) immobilized on 6%-agarose beads (weight basis), activated with linear aliphatic aldehyde groups (glyoxyl-agarose) is studied here. Reaction substrates (polypeptides) were the product of previous sequential hydrolyses of cheese whey proteins by trypsin and/or chymotrypsin. Yields of individual amino acids were correlated with respect to the overall rate of hydrolysis, thus allowing the prediction of the rates of production of each amino acid during the reaction course. Two kinetic models were compared, resulting respectively from a classic Briggs–Haldane formulation and from the relaxation of the pseudo-steady-state assumption. This approach grants lumped-parameter models, since it considers the number of hydrolysable peptide bounds as a pseudo-component, and in the presence of mass transport effects within the biocatalyst pores. Yet, our methodology was able to provide a detailed composition of a tailor-made pool of amino acids and peptides, allowing the optimization of the enzymatic reactor operation. It was observed that different conditions of substrate pre-hydrolysis caused significant changes on these yields. CPA hydrolyzed up to 11 amino acids, but its activity was higher for Phe, followed by Leu, Tyr and Trp.
Keywords: Whey proteolysis; Carboxypeptidase A; Enzyme catalysis; Phenylalanine removal;

In this paper, durian peel (DP), an agricultural waste, was evaluated for its ability to remove acid dye from aqueous solutions. Adsorption equilibrium and kinetics of acid green 25 (AG25) from aqueous solutions at various initial dye concentrations (50–500 mg/L), pH (2–10), and temperature (30–50 °C) on DP were studied in a batch mode operation. Equilibrium isotherms were analyzed by Langmuir and Freundlich isotherm models. The equilibrium data were best represented by Langmuir isotherm model with maximum monolayer adsorption capacity of 63.29 mg/g at 30 °C. Kinetics analyses were conducted using pseudo-first-order, pseudo-second-order and intraparticle diffusion models. It was found that the adsorption kinetics of AG25 on DP obeyed pseudo-second-order sorption kinetics. The results indicate the potential of DP as sorbent for the removal of acid dye from aqueous solution.
Keywords: Durian peel; Acid green 25; Adsorption isotherm; Equilibrium; Kinetics;

Aiming for the reduction of excess sludge and the simultaneous removal of organic carbon and nitrogen, a new two-stage bioreactor system constructed by combining three-phase fluidized-bed (TFB) and sludge-reduction fixed-bed bioreactor (SFB) processes with different structured porous carriers was proposed. In this system, wastewater was first treated in the TFB bioreactor, in which the organic and nitrogenous compounds were simultaneously removed, and then the excess sludge produced in the TFB and residual organic substances flowed into the SFB for further degradation of suspended solids (SS) and nutrient removal. During a 470-day continuous operation, the average COD removal ratio reached about 95% at the influent COD of 500–600 mg/L. The removal ratio of T-N could reach up to 28–55% and was carried out through both microbial growth and simultaneous nitrification and denitrification (SND). The SS from TFB could be reduced in the following fixed-bed from 160 mg/L to 28 mg/L with two SFBs without the settling tank. Therefore, the simultaneous removal of organic carbon and nitrogen with an on-site reduction of excess sludge could be achieved in this two-stage bioreactor.
Keywords: Fluidized-bed bioreactor; Fixed-bed bioreactor; Porous carriers; Excess sludge reduction; Wastewater treatment; Nitrogen removal;

Among the different agro-industrial waste products and kitchen waste materials, viz. mustered oil cake, wheat bran, rice bran, Imperata cylindrica grass, banana leaves, potato peels and used tea leaves screened as substrates/solid supports for the production of alkaline protease by a thermophilic strain of Bacillus subtilis DM-04 under solid-state fermentation, potato peel followed by I. cylindrica grass supported maximum protease production. Further, potato peel and I. cylindrica grass mixed in a ratio of 1:1 (w/w) significantly (P  < 0.05) enhanced the protease production by B. subtilis DM-04 compared to individual substrate. Among the tested nitrogen compounds, 0.1% (w/w) beef extract followed by yeast extract served as the best co-nitrogen sources for protease production by B. subtilis DM-04 strain on I. cyindrica grass. Distilled water adjusted to pH 8.0 was most efficient moistening agent for protease production. The crude protease displayed optimum activity at 37–45 °C temperature range and showed characteristics pH optima at pH 8.0–9.0. The protease from B. subtilis DM-04 retained 67% of its original activity post-heating at 60 °C for 15 min. Among the tested protein substrates, casein served as the most preferred substrate for the enzyme. Crude protease exhibited a significant stability and compatibility with most of the tested commercial laundry detergents, demonstrating its feasibility for inclusion in laundry detergent formulation.
Keywords: Alkaline protease; Bacillus subtilis; Imperata cylindrica; Laundry detergent formulation; Potato peel; Solid-state fermentation; Ulukher;

A course of simple enzymatic reactions in a batch reactor in the presence of the whole cells of microorganisms has been analyzed. A possibility of analytical determination of the optimal temperature profiles has been pointed out. An influence of the effectiveness factor on the said profiles and the reaction time of the batch process have been considered by accounting for the mass transfer across the cell membrane.
Keywords: Bioreactors; Biocatalysis; Enzyme deactivation; Optimization; Optimal temperature profiles; Microorganism cells;

The behavior of S. platensis was investigated in this study through fed-batch pulse-feeding cultures performed at different carbon dioxide feeding rates (F  = 0.44–1.03 g L−1  d−1) and photosynthetic photon flux density (PPFD = 80–250 μmol photons m−2  s−1) in a bench-scale helical photobioreactor. To achieve this purpose, an inorganic medium lacking the carbon source was enriched by gaseous carbon dioxide from a cylinder. The maximum cell concentration achieved was 12.8 g L−1 at PPFD = 166 μmol photons m−2  s−1 and F  = 0.44 g L−1  d−1 of CO2. At PPFD = 80 and 125 μmol photons m−2  s−1, the carbon utilization efficiency (CUE) reached maximum values of 50 and 69%, respectively, after about 20 days, and then it decreased, thus highlighting a photolimitation effect. At PPFD = 166 μmol photons m−2  s−1, CUE was ≥90% between 20 and 50 days. The photosynthetic efficiency reached its maximum value (9.4%) at PPFD = 125 μmol photons m−2  s−1. The photoinhibition threshold appeared to strongly depend on the feeding rate: at high PPFD, an increase in the amount of fed CO2 delayed the inhibitory effect on biomass growth, whereas at low PPFD, excess CO2 addition caused the microalga to stop growing.
Keywords: Helical photobioreactor; Spirulina platensis; Photosynthetic photon flux density; Carbon dioxide; Fed-batch process; Pulse feeding;

Characterization of Bacillus kaustophilus leucine aminopeptidase immobilized in Ca-alginate/k-carrageenan beads by Meng-Chun Chi; Rui-Cin Lyu; Long-Liu Lin; Hsien-Bin Huang (376-382).
Properties of Bacillus kaustophilus leucine aminopeptidase (BkLAP) immobilized by entrapment in Ca-alginate/k-carrageenan hydrogel were examined. For the free and immobilized BkLAP, optimum pH was found to be 8.0 and 7.5, respectively. The optimum temperature of the free and immobilized enzymes was also observed to be 55 and 60 °C, respectively. Thermal stability of BkLAP was increased as a result of immobilization. Maximum reaction rate (V max) and Michalis–Menten constant (K m) of BkLAP were changed significantly upon immobilization. The immobilized enzyme could be reused at least up to 10 cycles without any further loss of activity. After incubating at 4 °C for 30 days, the stability values for the immobilized enzyme system were found to be 90%. In the presence of 50 mM H2O2, the immobilized BkLAP was more stable respective to the free enzyme, indicating that the oxidative stability of the enzyme can be improved by immobilization.
Keywords: Leucine aminopeptidase; Bacillus kaustophilus; Immobilization; Alginate; k-Carrageenan; Stability;

The dimorphic filamentous fungus Mucor indicus was used as a biofilter and fermenting microorganism in a bioreactor for continuous cultivation of dilute-acid lignocellulosic hydrolyzates up to dilution rate (D) 0.3 h−1. The hydrolyzate was so toxic that the fungus could not grow in traditional stirred-tank bioreactors even at D 0.1 h−1. On the other hand, the fungus could be manipulated to grow as a mixture of short dispersed filaments and yeast-like cells. This morphology was suitable to create a biological filter, when attached to a specific screen located across the outlet stream. This filter was fully permeable to the liquid, but not the biomass inside the bioreactor. While the biomass concentration in the outflow was between 0.51 and 3.43 g/l at dilution rates 0.3–0.1 h−1, the biomass concentration inside the bioreactor was held at 8.34–9.91 g/l. The cells consumed 95.7%, 94.3%, and 75.3% of hexoses and 64.5%, 25.9%, and 7.6% of xylose present in the hydrolyzate at D 0.1, 0.2, and 0.3 h−1, respectively. The ethanol yields at these dilution rates were 0.37, 0.45, and 0.46 g/g consumed sugars, respectively. The only important by-products were glycerol and xylitol, which at D 0.2 h−1 yielded 0.036 and 0.022 g/g consumed sugars, respectively.
Keywords: Ethanol; Mucor indicus; Filamentous fungi; Dilute-acid lignocellulosic hydrolyzates; Continuous cultivation; Biological filter;

Many microbial processes exhibit sustained oscillations under practical conditions. Often these oscillations are corrupted by noise from the environment. It is then important to filter out the noise and recover ‘true’ oscillations for subsequent studies. Previous studies have used algorithmic or neural or hybrid filters in conjunction with mathematical equations for the kinetics. However, this approach is limited because in real situations it is difficult to measure and model some of the variables. Therefore, a hybrid neural simulator (HNS) has been developed here and tested with the continuous fermentation with Saccharomyces cerevisiae. The HNS combines a hybrid neural filter (HNF) for the noise, a hybrid description of the fermentation kinetics and macroscopic balance equations for the bioreactor. The HNS achieved 96% recovery of noise-free oscillations, compared to 91% with an HNF and lower efficiencies with pure neural and algorithmic filters. The commonly employed extended Kalman filter was ineffective as a stand-alone device but contributed to good filtering by the HNF and the HNS, thus indicating that a proper distribution of variables between the mathematical and neural components can significantly improve the performances of both.
Keywords: Saccharomyces cerevisiae; Continuous fermentation; Sustained oscillations; Noise inflow; Hybrid neural simulator;

The problem of parameters identification in multiparameter models of the complicated kinetics processes is analyzed. In this case the minimization of the least square function is very difficult because it is multiextremal. The problem solution needs to be obtained very well initial parameters values. A polynomial approximation of the experimental data permits to propose a hierarchical approach for obtaining initial parameters values in the global minimum area, using a consecutive approximations method. The method for parameter identification of multiparameter models is tested for modeling of fermentation systems. The model parameter values are obtained on the bases of real experimental data. The results obtained show a decrease of the model error variance on every next hierarchical level and a good agreement with the experimental data on the last level.
Keywords: Multiparameter model; Model parameter identification; Hierarchical approach; Fermentation systems;

Keywords: Shear stress; Mass transfer; Chitinase production;