Biochemical Engineering Journal (v.99, #C)


Oxidoreductase enzymes have the potential to be used to catalyze the oxidation of aromatic trace contaminants in wastewaters. The feasibility of this approach can be explored by modeling transient kinetics, particularly in the low substrate concentration range, where the model may be used to predict the quantity of enzyme and time required to achieve a particular level of conversion of a target compound. Laccase from Trametes versicolor was selected as a candidate enzyme due to its wide substrate specificity and its use of molecular oxygen as an oxidant. Phenol was selected as a target substrate. A four-parameter kinetic model was developed based on the known reactions of laccase. The model was applied to 30 sets of data collected from batch reactions conducted at pH 5 and 25 °C over a three hour period. Initial phenol concentrations ranged from 0.5 to 50 μM and applied enzyme concentrations ranged from 0.12 to 2.5 μM. The model demonstrated its utility for predicting the quantities of enzyme and reaction times required to achieve desired levels of oxidation of phenol for varying initial concentrations. A three-parameter simplified version of the kinetic model was also developed to facilitate calibration and mathematical solution of its equations.
Keywords: Enzymes; Biocatalysis; Kinetic parameters; Modeling; Laccase; Phenol;

Effects of substrate COD/NO2 -N ratio on simultaneous methanogenesis and short-cut denitrification in the treatment of blue mussel using acclimated sludge by Shinichi Akizuki; Teng Ke Wong; Yutaka Aoki; Germán Cuevas- Rodríguez; Chiaki Niwa; Tatsuki Toda (16-23).
Simultaneous methanogenesis and short-cut denitrification processes have attracted attention because it enables the removal of organic matter and nitrogen in a same reactor. In this study, these processes were applied to the treatment of blue mussels, which are discharged worldwide intermittently and in vast quantities as organic solid waste. The effects of substrate COD/NO2 -N ratios on denitrifying and methanogenic performances were evaluated in batch experiments. NO2 acclimated sludge was used as a part of seed sludge to enhance the tolerance to NO2 . Two acclimated sludges were obtained by two-different acclimating conditions which exposed to influent COD/NO2 -N ratios of 25 with high COD loading rate and 5 with low COD loading rate during acclimation periods (referred to as AS25 and AS5). AS25 added reactors showed the relatively high methanogenic and denitrifying performances compared to AS5 added reactors. The results obtained in AS25 added reactors showed comparable results to different studies which used liquid substrates. High COD and NO2 removal efficiencies were achieved under substrate COD/NO2 -N ratio ranging from 8.6 to 123, representing the successful performance for the treatment of blue mussels which contains COD/N ratio of 16.3 in their tissue.
Keywords: Methanogenesis; Short-cut denitrification; Simultaneous process; Sludge acclimation; COD/NO2 -N ratio; Marine biofouling organisms;

Application of a novel respirometric methodology to characterize mass transfer and activity of H2S-oxidizing biofilms in biotrickling filter beds by Wenceslao Bonilla-Blancas; Mabel Mora; Sergio Revah; Juan Antonio Baeza; Javier Lafuente; Xavier Gamisans; David Gabriel; Armando González-Sánchez (24-34).
The elimination capacity of gaseous H2S biofiltration can be limited either by mass transfer or bioreaction in the biofilm. Assessment of the biological activity of immobilized cells (biofilm) usually implies morphological and physiological changes during the adaptation of cells to respirometric devices operated as suspended cultures. In this study, respirometry of heterogeneous media is advised as a valuable technique for characterizing mass transport and biological activity of H2S-oxidizing biofilms attached on two packing materials from operative biotrickling filters. Controlled flows of liquid and H2S-containing air were recirculated through a closed heterogeneous respirometer allowing a more realistic estimation of the biofilm activity by the experimental evaluation of the oxygen uptake rate (OUR). Specific maximum OUR of 23.0 and 38.5 mmol O2 (g biomass min)−1 were obtained for Pall rings and polyurethane foam, respectively. A mathematical model for the determination of kinetic-related parameters such as the maximum H2S elimination capacity and morphological properties of biofilm (i.e., thickness and fraction of wetted area of packing bed) was developed and calibrated. With the set of parameters obtained, the external oxygen mass transport to the wetted biofilm was found to limit the global H2S biofiltration capacity, whereas the non-wetted biofilm was the dominant route for the gaseous O2 and H2S mass transfer to the biofilm. Oxygen diffusion rate was the limiting step in the case of very active biofilms.
Keywords: OUR; Hydrogen sulfide; Heterogeneous respirometry; Wetted/non-wetted biofilm; Mathematical model; Mass transfer;

Modeling and optimization of acetic acid fermentation: A polynomial-based approach by Ines M. Santos-Dueñas; Jorge E. Jimenez- Hornero; Ana M. Cañete-Rodriguez; Isidoro Garcia-Garcia (35-43).
Vinegar production is a typical bioprocess in the scope of the agrifood industry. Its optimization requires careful modeling which has so far been addressed by using mainly unstructured first principles models. Because of the difficulties in obtaining these models, black box models, such as those used here, are becoming more frequently used. The polynomial models developed in this work, accurately reflect the effect of the major and typical operational variables used in industry for this process. Also, response surfaces were used to identify the optimum operating conditions with a view to maximizing the mean fermentation rate and productivity. The followed strategy has a huge industrial interest since yields a tool that does not only allow finding the best operational conditions depending on different criteria but also is useful for process control. As far as we know this is the first time that these variables have been correlated in this way.
Keywords: Bioreactors; Modelling; Optimisation; Acetic acid; Acetobacter; Vinegar;

Biodegradation of quinoline by Streptomyces sp. N01 immobilized on bamboo carbon supported Fe3O4 nanoparticles by Haifeng Zhuang; Hongjun Han; Peng Xu; Baolin Hou; Shengyong Jia; Dexin Wang; Kun Li (44-47).
A novel immobilizing carrier, bamboo-carbon supported Fe3O4 nanoparticles (Fe3O4/BC), was developed and used for immobilization of cells of a quinoline-degrading, Streptomyces sp. N01. The performance of Fe3O4/BC immobilized cells in the degradation of quinoline was evaluated and compared with bamboo-carbon immobilized cells and the free cells. Experiments were performed at various quinoline concentrations (100–400 mg/L), pH (5–10) and temperature (20–45 °C). The results show that Fe3O4/BC immobilized cells could tolerate a higher quinoline concentration and protect the bacteria much more effectively against changes in temperature and pH. Reusability results revealed that quinoline removal was maintained 85.3% in the ninth cycle. These results demonstrated that Fe3O4/BC possesses a good application potential as a carrier in cell immobilization.
Keywords: Biodegradation; Bamboo-carbon; Immobilized cells; Fe3O4 nanoparticles; Batch processing; Waste-water treatment;

Lipid production by eukaryotic microorganisms isolated from palm oil mill effluent by Jatta M. Marjakangas; Aino-Maija Lakaniemi; Perttu E.P. Koskinen; Jo-Shu Chang; Jaakko A. Puhakka (48-54).
Microbial oil production combined with wastewater management is one option for a more sustainable future. Micrographs of microbial cultures enriched from palm oil mill effluent (POME) showed lipid inclusion in the eukaryotic cells, indicating the cells can accumulate lipids. However, enriching the culture did not increase the total lipids. Therefore, eukaryotic microorganisms were isolated from POME to investigate whether these microorganisms are potential lipid producers. Four strains were isolated, and their lipid synthesis capabilities were compared with known oleaginous yeasts in a synthetic oil-free medium. Two strains (identified as Galactomyces geotrichum and Graphium penicillioides) had the potential to accumulate lipid accumulation based on the increase in triacylglycerol content. G. penicillioides was the most promising strain for lipid production as this strain accumulated more lipids than the well-known oleaginous yeast Cryptococcus curvatus (29.1 ± 3.0 wt% vs. 20.2 ± 2.9 wt%). To our knowledge, oil synthesis and accumulation by G. penicillioides have not previously been reported.
Keywords: Filamentous fungi; Lipid accumulation; Microbial growth; Palm oil mill effluent; Physiology; Yeast;

A model for the autotrophic growth of Chlorella vulgaris (C. vulgaris) under photolimitation and photoinhibition in cylindrical photobioreactor was developed by taking into account the interactions between the biomass concentration and light intensity. The growth kinetic model for a batch system was developed by coupling the light distribution under photolimitation and photoinhibition effects with the biomass growth with respect to incident light intensity on the surface of the reactor. The developed model could successfully simulate the specific growth rates, light intensity distribution in the reactor, average light intensities, and biomass concentrations at a given incident light intensity with good accuracy. The prediction of the cell density primarily depended on the determination of the parameters included in the specific growth rate expression, which is a function of the light intensity distribution. Despite the limitation of the semi-empirical specific growth rate expression, the coupled model could predict the biomass growth in different reactor sizes.
Keywords: Microalgae; Bioreactors; Growth Kinetics; Modelling; Light Distribution; Light Intensity;

A hydrodynamic description of the flow behavior in shaken flasks by E. Mancilla; C.A. Palacios-Morales; M.S. Córdova-Aguilar; M.A. Trujillo-Roldán; G. Ascanio; R. Zenit (61-66).
Shaking flasks bioreactors have been employed extensively in biotechnology research for a long time. Despite of their wide application and importance, there is still insufficient knowledge about the hydrodynamic factors that determine the correct performance of growing cultures. The objective of this work is to provide a hydrodynamic description of the parameters which control the effective performance of such bioprocesses. The flow behavior of shaken flasks was examined using the particle image velocimetry technique (PIV) to capture the mixing dynamics for a range of operating conditions. The velocity fields and turbulent intensities were obtained. For all cases, the chaotic-like fluid motion increased with the orbital speed of the shaker. The behavior of conventional, coiled and baffled flasks was analyzed for shaking speeds ranging from 25 to 250 rpm; at high shaking rates the turbulent distribution increased for each flask configuration but the average value differed significantly. The highest turbulent intensity was found for the one-baffle arrangement, which is about 25% larger than the other configurations. We found that the highest turbulent production for all the different geometric conditions occurred at a shaking speed of about 150 rpm, which is in good agreement with the findings reported for the production of bacterial cultures at such shaking rate.
Keywords: Bioreactors; Fluid mechanics; Mixing; Newtonian fluids; Hydromechanical stresses;

Display OmittedIonic liquids as eco-friendly solvent media and catalyst play very important roles in many enzymatic reactions such as enzymatic biotransformation and all kinds of biosynthesis due to their unique and tunable physical properties. The changes of the cations or anions types in ionic liquids have important effects on the activity, stability and structures of enzymes. In this critical review, we systematically shed light on the key factors affecting the activity and stability of enzymes in ILs and discussed the relationships among these factors. Moreover, the strategies for improving the enzymes’ utility in ionic liquids are exhibited. Finally, we address some novel industrial applications of ionic liquids in biochemical engineering such as the pretreatment of lingocellulosic biomass, transesterification in biodiesel synthesis and non-solvents in biotransformations. This review will thus be helpful for the researchers of various biocatalytic applications.
Keywords: Ionic liquid; Enzyme biocatalysis; Enzyme activity; Enzyme stability; Biotransformation; Application;

As the number of reactions requiring biotransformation continues to grow, manipulating the enzyme substrate specificity becomes very important. Complementary to conventional enzyme engineering techniques based on only natural amino acids, we hypothesized that the site-specific incorporation of a non-natural amino acid in vivo into an enzyme can be used to re-design the active site for the altered substrate specificity. To test our hypothesis, we introduced the non-natural amino acid L-2-naphthylalanine (2Nal) into the active site of the model enzyme murine dihydrofolate reductase (mDHFR). We explored whether the substrate specificity of the enzyme could be switched from the good substrate dihydrofolate (DHF) to the poor substrate folate (FOL). We used two protein design programs (RosettaLigand and RosettaDesign) to calculate ligand docking and conformational stability, respectively, for the evaluation of multiples sites in the mDHFR. From the calculations, position 31 was predicted as an optimal 2Nal incorporation site. One mDHFR variant containing 2Nal at position 31 (mDHFR2Nal31) was expressed in the Escherichia coli expression host cells equipped with the engineered yeast phenylalanyl-tRNA and phenylalanyl-tRNA synthetase pair. As expected, the kinetic assays of purified mDHFR variant revealed that mDHFR2Nal31 has the enhanced binding affinity toward FOL and also exhibits 7.6-fold enhanced catalytic efficiency of FOL over DHF compared to mDHFRWT.
Keywords: Amino acid; Artificial enzyme; Biocatalysis; Enzyme technology; Site-specific incorporation in vivo; Substrate-specificity;

Lactobacillus reuteri is a potential strain for the production of 1,3-propanediol and lactic acid due to its good safety. The aim of this work was to study the inhibition of lactic acid and acetic acid during fermentation, and thus, to investigate the effect of their in situ removal on the growth and metabolism of L. reuteri. The analysis of metabolic flux showed that both of lactic acid and acetic acid severely inhibited the fermentation. Only 30.11 mM and 66.85 mM glucose were consumed in 24 h under the initial concentration of 444 mM lactic acid and 415 mM acetic acid, respectively. The activity of the cell was dramatically decreased by the organic acid, furthermore, the production of 1,3-propanediol and lactic acid was impeded. The adsorption properties of anion exchange resin (D301G) were investigated to facilitate the removal of organic acid in situ during fermentation. The resin D301G was added at initial and during fermentation of 12 h, respectively, which showed different function on the metabolism of L. reuteri. Initial addition of resin increased the biomass of fermentation, while the glucose consuming and the 1,3-propanediol producing did not change significantly. The resin added at 12 h promoted the conversion of glycerol to 1,3-propanediol due to the NADH accumulation caused by the metabolic flux migration to acetic acid pathway, while the biomass decreased in small amount.
Keywords: Product inhibition; Process integration; In situ removal; Lactic acid; Acetic acid; Metabolic flux;

Cell disruption of S. cerevisiae by scalable high-intensity ultrasound by Simon Bystryak; Rasa Santockyte; Alexey S. Peshkovsky (99-106).
Ultrasonic disruption of yeast and other microbial cell cultures is commonly used for laboratory-scale protein preparations because it is rapid, efficient and simple to use compared to such methods as high-pressure homogenization (HPH) and bead milling. Lysing by sonication is also more effective than other cell disruption methods for the recovery of periplasmic, membrane-bound, or insoluble recombinant proteins. Until recently, however, due to amplitude limitations of conventional-design pilot and industrial-size sonication equipment, ultrasonic cell disruption was only feasible on the laboratory scale. In this study, we show that Barbell Horn Ultrasonic Technology (BHUT) can be successfully used for the disruption of Sacharomyces cerevisiae (S. cerevisiae) cells on a large scale. In particular, we show that by using pilot-scale BHUT-based equipment, total protein and alkaline phosphatase can be efficiently extracted from S. cerevisiae cells, achieving about an order of magnitude productivity increase factor with respect to laboratory-scale results. Since the size of BHUT-based ultrasonic processors can be increased further, ultrasonic cell disruption now has the ability to develop into a valuable commercial-scale method, potentially superior to HPH and bead milling techniques in this area of application.
Keywords: Cell disruption; Scale-up; Yeast; Production kinetics; Sonication; Barbell Horn Ultrasonic Technology (BHUT);

Bioavailability of essential trace elements and their impact on anaerobic digestion of slaughterhouse waste by Markus Ortner; Michael Rameder; Lydia Rachbauer; Günther Bochmann; Werner Fuchs (107-113).
Slaughterhouse waste is an energy rich feedstock suitable for anaerobic digestion processes. However, chemical characterization showed a deficiency in essential trace elements which are critical for optimal performance of the process. Hence this study investigated the degree of bioavailability of trace elements in four semi-continuous lab-scale AD tests accepting slaughterhouse waste under mesophilic conditions (38 °C) and a moderate organic loading rate of 2.2 kg/m3  d. Parameters, such as volatile fatty acid (VFA) concentration, COD removal rate and specific methane yield were compared to the results of sequential extraction analysis. The highest methane yield (250–275 Nm3/t COD), lowest accumulation of VFA (<500 mg/l) and high COD removal rate (75–80%) was obtained when the total concentration of 11.4 mg/l Ni, 25.4 mg/l Co and 4.8 mg/l Mo was present in the reactor, of which 62% of Ni and Co, and 68% of Mo were bioavailable for microbial uptake. Based on these results it can be recommended that a supply of 2.5 g/t Ni, 3.5 g/t Co, 0.6 g/t Mo and 0.05 g/t Se provide optimal conditions for anaerobic digestion of slaughterhouse waste.
Keywords: Anaerobic processes; Anaerobic digestion; Trace elements; Sequential extraction; Chemical speciation; Bioavailability; Biogas; Waste treatment; Bioprocess monitoring; Slaughterhouse waste;

Continuous long-term hydrolysis of wheat gluten using a principally food-grade enzyme membrane reactor system by Michael Merz; Thomas Eisele; Wolfgang Claaßen; Daniel Appel; Swen Rabe; Timo Stressler; Lutz Fischer (114-123).
The potential of the enzyme membrane reactor technology has been shown in several studies. In our study, we designed a principally food-grade continuous long-term hydrolysis process of wheat gluten in an enzyme membrane reactor with the proteolytic enzyme preparation Flavourzyme®. Among others, ethanol was the most suitable food-grade processing aid for avoiding contamination and showed high potential for application. The Flavourzyme®/wheat gluten process was comprehensively characterized in the enzyme membrane reactor. Critical factors for the enzyme stability were the temperature, pump stress and enzyme leakage through the membrane. The hydrolysis and process conditions were optimized to increase the space–time yield. Respective process parameters were chosen to obtain sufficient microbial, enzyme and process stability for the whole process time. The long-term hydrolysis was carried out in the presence of 8% (v/v) ethanol with a substrate concentration of 100 g L−1 at 37 °C and pH 7.5 for 96 h. The continuous process resulted in stable product quality (degree of hydrolysis) and space–time yield (6.33 g h−1  L−1) over time. However, a discontinuous removal of accumulated dry matter was inevitable for the present enzyme membrane reactor process and was performed for 30 min every 24 h. Due to the fact that the enzymes were reused, the enzyme productivity could be increased by 450% compared to a reference batch process.
Keywords: Wheat gluten hydrolysis; Enzyme membrane reactor; Continuous; Seasoning; Ethanol; Aspergillus oryzae peptidase; Protein hydrolysates;

Enhancing anaerobic digestibility of lignin-rich submerged macrophyte using thermochemical pre-treatment by Mitsuhiko Koyama; Shuichi Yamamoto; Kanako Ishikawa; Syuhei Ban; Tatsuki Toda (124-130).
The present study investigated the effect of alkaline thermochemical pre-treatment on anaerobic digestibility of two submerged macrophyte species which have significantly different lignin content. The highest hydrolysis efficiency was achieved at NaOH loading rate of 0.20 g g-TSsubstrate −1, 80 °C, 3.0 h for both species. Alkaline delignification was much conspicuous in lignin-rich macrophyte (Potamogeton maackianus) as compared with lignin-poor macrophyte (Egeria densa). Ferulic acid, which is cross-linked with lignin polymer and polysaccharides, remarkably declined with increase of NaOH loading rate. It suggests that alkali removed lignin–ferulate complex from the surface of polysaccharides. The CH4 yield of pre-treated P. maackianus was 243 mL g-VS−1, which is 51% higher than the un-treated (161 mL g-VS−1). In contrast, the CH4 yield of pre-treated E. densa was 24% higher than the un-treated. These results indicated that alkaline thermochemical pre-treatment could be an effective method for anaerobic digestion of lignin-rich macrophytes. However, it was also suggested that the high NaOH addition to lignin-rich macrophyte possibly inhibit the methane recovery, due to the increase of solubilized lignin in the digestate.
Keywords: Anaerobic processes; Waste treatment; Biodegradation; Biogas; Lignin; Pre-treatment; Inhibition;

The antifungal effect of essential oils (EOs) of Thymus vulgaris L. (EO T. vulgaris ) and Cinnamomum cassia L. (EO C. cassia ) against Aspergillus flavus spores was evaluated by determining minimum inhibitory concentration, minimum fungicidal concentrations and fungicidal kinetics. Kinetic model of fungicidal activity of individual EO T. vulgaris and EO C. cassia was developed and its parameters were used to make EO mixture with optimal ratio of individual EOs. Synergism and speed of fungicidal effect of EO mixtures against A. flavus spores were evaluated.Kinetic model revealed optimal ratio EO T. vulgaris : EO C. cassia as 14:1 in mixture. Observed result was validated by comparing fungicidal effect of this mixture to commonly used ratio 1:1 and intermediate 7:1 EO mixtures. All three mixtures showed partial fungicidal synergism, but the time point of fungicidal effect was different. The fastest fungicidal effect was observed in 14:1 EO mixture (after 90 min), followed by 7:1 (110 min) and 1:1 (180 min). The difference in the speed of fungicidal effect could not be detected using standard microdilution susceptibility tests, which demonstrated the importance and usefulness of developed kinetic model for further investigations.
Keywords: Antifungal effect; Aspergillus flavus; Cinnamomum cassia; Kinetic model; Synergism;

Effect of fluiddynamic conditions on growth rate and biodesulfurization capacity of Rhodococcus erythropolis IGTS8 by E. Gomez; A. Alcon; S. Escobar; V.E. Santos; F. Garcia-Ochoa (138-146).
The growth rate and desulfurization capacity accumulated by the cells during the growth of Rhodococcus erythropolis IGTS8 under different fluid dynamic conditions in a stirred and sparged tank bioreactor have been studied. Hydrodynamic conditions were changed using different stirrer speeds and gas flow rates. It was observed that the growth was strongly dependent on the stirrer speed employed. Oxygen transfer limitation was observed at low stirrer speeds (from 100 to 250 rpm). In contrast, at higher stirrer speeds, cell damage was caused by hydrodynamic stress in the turbulent bulk of the broth, yielding again a decrease in growth for stirrer speeds higher than 450 rpm. Moreover, increasing the agitation from 100 to 450 rpm has a positive influence on the development of the desulfurization capacity of the cells during growth, yet this capacity shows a dramatic decrease for higher stirrer speeds. Nevertheless, the change of the air flow rate hardly has any influence on the growth rate and no hydrodynamic stress effect has been detected between 1 and 10 L min−1. A regime analysis of the characteristic times for oxygen mass transfer, oxygen uptake and mixing under different agitation conditions has been made. It was found that the minimum stirrer speed necessary for a satisfactory performance of the bioreactor from the point of view of the cells oxygen demand was 350 rpm, while at stirrer speeds over 450 rpm, growth rate and desulfurization capacity are both negatively affected.
Keywords: Oxygen transfer rate; Oxygen uptake rate; Hydrodynamic stress; Stirred tank bioreactor; Desulfurization capacity; Rhodococcus erythropolis IGTS8;

Microbial lipopeptide synthesis is often associated with the co-production of more than one family of isoforms. The selective lipopeptide production is strongly influenced by the processing conditions and the limiting nutrient sources such as oxygen and nitrogen during a fermentation process. In the current study, comprehensive investigations carried out in shake flasks revealed that the oxygen-limiting conditions increased the selective fengycin production. The optimal conditions of shake flasks studies of 340 mL medium volume (in 1 L conical flask), 4.67% v/v inoculum volume and 121 rpm agitation speed resulted in a total lipopeptide concentration of 5.34 ± 0.1 g L−1, with more selectivity toward fengycin of 74.1%. Among the three rational approaches investigated to recreate these optimal conditions in a 3.7 L stirred tank bioreactor, the strategy, submerged aeration followed by surface aeration effectively reproduced the optimal conditions of shaker flasks resulting in a total lipopeptide concentration of 4.94 ± 0.15 g L−1, with fengycin selectivity of 71%. The present study thus offers a process design strategy of potential industrial significance to the challenge of selectively enhancing the target product, while marginalizing other closely related co-products.
Keywords: Process intensification; Lipopeptide selectivity; Fermentation; Scale up; Bioprocess design; Bioreactors;

Kinetic study of butanol production from various sugars by Clostridium acetobutylicum using a dynamic model by Francesca Raganati; Alessandra Procentese; Giuseppe Olivieri; Peter Götz; Piero Salatino; Antonio Marzocchella (156-166).
This paper presents a kinetic dynamic model of acetone–butanol–ethanol production by Clostridium acetobutylicum DSM 792 developed with the biochemical networks simulator COPASI. This model is an evolution of previous models described in the literature, updated by including various mono-, di-, hexose and pentose sugars: glucose, mannose, fructose, sucrose, lactose, xylose and arabinose. The kinetic relationships of uptake of substrate, butanol production, cell growth and cell death are also included.The batch fermentation tests were carried out at an initial sugar concentration ranging from 5 to 100 g/L. The data from the batch tests were used to assess the kinetic parameters of the model. This model gave satisfactory results for each sugar, both in terms of simulation of fermentation – the square correlation coefficient of metabolite concentrations, calculated by comparing experiments and simulations, ranged between 0.87 and 0.925 – and of comparison with the models reported in the literature.The effects of mono-, di-, hexose and pentose sugars on the growth and production of metabolites, including acids and solvents, were reviewed according to the proposed model. The low fermentation performance measured for xylose and lactose were interpreted taking into account the sugar uptake, the acid production and the hydrolysis path.
Keywords: Substrate inhibition; Product inhibition; Clostridium acetobutylicum; Dynamic modelling; Biokinetics; COPASI;

Improvement of glucoamylase production using axial impellers with low power consumption and homogeneous mass transfer by Wenjun Tang; Ao Pan; Hongzhong Lu; Jianye Xia; Yingping Zhuang; Siliang Zhang; Ju Chu; Henk Noorman (167-176).
Significantly influenced by complex cell morphology, glucoamylase fermentation using Aspergillus niger is characterized by high apparent viscosity and shear-thinning rheology. In this study, the influence of liquid flow field patterns on morphology, broth rheology, mass transfer and glucoamylase production was investigated by applying two different configurations with radial (Ruston Turbine, RT) and axial (Wide-blade hydrofoil upward-pumping, WHu) flow impellers. It was found that empirical correlations for averaged quantities, such as the mass transfer coefficient and viscosity, cannot reasonably explain the observations. Therefore, numerical simulation was carried out to study the detailed characteristics of local field in lab-scale bioreactors. The results showed, under similar glucose and oxygen uptake rates, that the WHu configuration formed relatively homogeneous viscosity and mass transfer fields, while the RT configuration was accompanied with significant heterogeneities. Under these conditions, the fraction of active mycelia in pellets could be highly correlated with enzyme production, and a novel parameter (Active Part Percentage, APP) was defined to introduce the effects of flow field on pelletized morphology. The WHu impellers facilitated the formation of pellets and hairy structures, with a higher APP of the pellets. As a result, the culture with the axial flow impeller configuration exhibited a larger glucoamylase production rate (+25%) and product yield on sugar (+23%) and yield on energy (+60%) in comparison to the radial flow impeller. Computational fluid models were proposed to in-depth understand such results based on local mass transfer and viscosity values, since the average values are similar over the entire fermentation processes.
Keywords: Glucoamylase; Impeller type; Aspergillus niger; Morphology analysis; CFD;

Series fermentation production of ornithine and succinic acid from cane molasses by Corynebacterium glutamicum by Sheng Xu; Ning Hao; Lin Xu; Zhaoxing Liu; Ming Yan; Yan Li; Pingkai Ouyang (177-182).
The production of ornithine and succinic acid by Corynebacterium glutamicum CGMCC1006-ΔldhA media was performed using cane molasses as a low-cost material under aerobic and anaerobic conditions, respectively. The use of a series fermentation process would reduce both production costs and excess bacteria. In this work, the ornithine concentration reached 14.93 g L−1 within 72 h when using the optimal medium containing molasses pretreated with sulphuric acid and coupled with polyacrylamide, whereas a succinic acid concentration of 13.11 g L−1 was attained after 12 h using a medium containing molasses pretreated with sulphuric acid. In fed-batch fermentation, the succinic acid concentration reached 35.13 g L−1 within 54 h when using molasses. The present study suggests that the use of molasses as a raw material in series fermentation for the production of ornithine and succinic acid by C. glutamicum can significantly reduce the production costs and the amount of excess biomass.
Keywords: Cane molasses; Amino acids; Aerobic processes; Anaerobic processes; Fermentation; Bioprocess design;

Aging effect and antibody immobilization on ―COOH exposed surfaces designed for dengue virus detection by Samira Hosseini; Fatimah Ibrahim; Hussin A. Rothan; Rohana Yusof; Cees van der Marel; Ivan Djordjevic; Leo H. Koole (183-192).
Display OmittedPolymethylmethacrylate-co-methacrylic acid, poly(MMA-co-MAA) coatings were produced with different initial molar ratios of monomers (MMA and MAA) in free-radical polymerization reaction. Polymeric platforms were specifically designed with controlled concentration of surface-exposed carboxyl (―COOH) groups that can be used as a desirable functionality for protein immobilization. Spin-coated chips were used for antibody (Ab) immobilization in order to investigate the influence of ―COOH surface concentration on dengue virus detection efficiency in enzyme-linked immunosorbent assay (ELISA) experiment. Successful immobilization of Ab was achieved by two different techniques: (1) physical adsorption; and (2) covalent immobilization by carbodiimide coupling between the surface ―COOH groups and amine functionalities of dengue Ab molecules. Produced polymer coatings were characterized with surface spectroscopy techniques (Raman and X-ray photoelectron spectroscopy, XPS) and water-in-air contact angle (WCA) measurements. In particular, this research concentrated on the aging effect on the availability and activity of surface ―COOH groups. For that reason, WCA and Ab immobilization (ELISA) experiments were repeated on coated biochips after 3, 6 and 9 months of storage. Results in this paper describe the robust and sustainable functionalized polymeric platform that can be used effectively for protein activation and development of novel biosensors.
Keywords: Polymer coatings; Surface functional groups; Immobilization; Enzyme activity; Immunoassay; Biosensors;

The anaerobic digestion model No.1 (ADM1) developed by the IWA Task Group for mathematical modelling of anaerobic digestion processes Batstone et al. [1] is a structural model which describes the main biochemical and physicochemical processes. For such purposes, other models have been proposed to describe anaerobic processes with a reduced set of parameters, state variables and processes. Among them, the anaerobic model No. 2 (AM2) proposed by Bernard et al. [2] which describes the degradation of soluble organic compounds, appears as a model well-suited for control and optimization applications.In this work, we aimed at obtaining a model of reduced dimensions on the basis of which to synthesize regulators or observers with guarantees of performance, stability and robustness. Specifically, our contribution is twofold. First, a modified version of the AM2 is proposed while preserving the simplicity of the new model “AM2HN”. Second, we propose a systematic and generic state association procedure in order to obtain such a simplified model from any validated ADM1.Simulations and comparisons with the predictions of the ADM1 for a case study involving the anaerobic digestion of waste sludge are presented along with satisfactory results.
Keywords: Anaerobic processes; Dynamic modelling; Optimization; Control; AM2; AM2HN;