Biochemical Engineering Journal (v.34, #3)

BEJ Keywords (II).

Optimization of biological treatment of paper mill effluent in a sequencing batch reactor by Y.F. Tsang; F.L. Hua; H. Chua; S.N. Sin; Y.J. Wang (193-199).
Laboratory scale research on the effects of operating parameters, including mixed liquor suspended solid (MLSS) concentration, volumetric exchange rate (VER), aeration time, temperature and daily operation cycle on biological treatment of the pulp and paper mill effluent was studied using four 4 l sequencing batch reactors (SBR). The results revealed that chemical oxygen demand (COD) removal efficiency was up to 93.1 ± 0.3% and the volumetric loading reached 1.9 kg BOD m−3  day−1 under optimal operating conditions. The treatment performance of organic removal by the SBR system remained stable during the operation. The effluent quality was satisfied with the discharge standard set by the local authority and the filamentous bulking problem was solved. At the same time, the sludge settleability, in terms of sludge volume index (SVI), was improved to the healthy level (SVI = 52.7 ± 1.3 ml g−1).
Keywords: Sequencing batch reactor; Activated sludge; Paper mill wastewater; Wastewater treatment; Optimization;

Calculating liquid distribution in shake flasks on rotary shakers at waterlike viscosities by Jochen Büchs; Ulrike Maier; Stefan Lotter; Cyril P. Peter (200-208).
Screening projects in biotechnological industry performed in shake flasks risk unwanted development if not failure, when operating conditions are not suitable. Limited knowledge, however, is available for the mechanistical design of operating conditions in this type of bioreactor. The fundamental engineering variables are influenced by the geometry of the rotating bulk liquid: for momentum transfer, the contact area between the liquid and the flask inner wall is the friction area, and for mass transfer, the wetted wall exposed to the surrounding air is the mass exchange area. To assess the geometry of the rotating bulk liquid moving inside a shaken Erlenmeyer flask, with respect to the mentioned important engineering variables mentioned, a mechanistical model for the liquid distribution in shake flasks is described in this work. The model is based on a superposition of two individual movements: a circular translatoric movement and a rotation of the flask counteracting the first motion to keep the shake flasks’ spatial orientation. If the effect of viscosity is neglected, the liquid distribution results in an exactly symmetrical paraboloid. A comparison of the calculated liquid distribution with photographs shows very good qualitative agreement of the real liquid distributions by the model equations. Quantitative agreement has been demonstrated by comparison of the liquid height. Furthermore, model equations are presented for the calculation of the contact area between the liquid and the flask wall. This may eventually lead to a prediction of the volumetric power consumption. Similarly, the calculation of the mass transfer area (i.e. liquid surface area and wetted flask wall) is presented.
Keywords: Fermentation; Shake flask; Power consumption; Mass transfer; Modelling; Scale-down;

Substrate specificity, regioselectivity and hydrolytic activity of lipases activated from Geotrichum sp. by Karel Stránský; Marie Zarevúcka; Zdeněk Kejík; Zdeněk Wimmer; Martina Macková; Kateřina Demnerová (209-216).
Substrate specificity (typoselectivity), regioselectivity and hydrolytic activity of induced lipases from three strains (4012, 4013, 4166) of Geotrichum candidum and that of Geotrichum ludwigii (48) were investigated. The lipases were induced in two types of culture media, of which the medium containing peptone as nitrogen source was proved to give better results. Olive oil was employed as inductor for the lipase activity. Activated lipases represented mostly extracelullar lipases, which penetrated through cellular membrane into medium. The activity of cell-bound lipase was also determined. Most of lipases belong to the group of specific lipases able to hydrolyse ester bonds in the positions sn-1 and sn-3 ester of triacylglycerols (1,3-selective lipases) and display specificity to saturated fatty acids. All activated lipases from Geotrichum sp., extracellular and cell-bound, were used as biocatalyst in the blackcurrant oil hydrolysis.
Keywords: Biocatalysis; Enzyme activity; Gas chromatography; Lipase; Blackcurrant oil; Geotrichum sp;

Neutral lipase from aqueous solutions on chitosan nano-particles by Zhen-Xing Tang; Lu-E Shi; Jun-Qing Qian (217-223).
The size of chitosan nano-particles had been prepared by ionic cross-linking of chitosan and tri-polyphosphate (CS-TPP), which were well dispersed and stable in aqueous solution. The physicochemical properties of nano-particles were characterized by IR spectra, SEM. Its sorption kinetics and sorption mechanism for neutral lipase were studied. The effect factors of adsorption kinetics were investigated, such as neutral lipase concentration, chitosan nano-particles solution concentration, adsorption temperature, size of chitosan nano-particles, stirring rate, solution pH, etc. Adsorption of neutral lipase on chitosan nano-particles was fitted into Lagergren first-order equation at initial neutral lipase of 3.0 mg/ml (pH 7.0). The first-order constant for neutral lipase was 23.34 h−1. When neutral lipase concentration was controlled under certain region, it was fitted into Freundlich isothermal linear equation. Mechanism of adsorption for neutral lipase was presumed by analyzing IR spectra. The hydrogen in the carboxyl group was connected with electronegative oxygen. Electron pair was attractive to oxygen, so hydrogen atom became cation and proton. When electronegative neutral lipase was closed to chitosan nano-particle, the hydrogen bond might be formed. It was the main force between hydroxyl group, NH2 group and neutral lipase.
Keywords: Chitosan; Nano-particles; Neutral lipase; Sorption kinetics; Sorption mechanism;

Disposable bioreactors are increasingly used in bioprocess development of animal cell cultures. However, the engineering parameters of the present disposable bioreactors are very difficult to characterise. Characterisation of any bioreactor is important to evaluate the suitability of operating conditions and absolutely necessary for successful scale-up. In this research work, an attempt is made to validate a characterisation method which is simple and can be used for any given volume of disposable bioreactors larger than 2 L. Two evaluation methods, namely the temperature method and the well established torque method were used for determination of power consumption in 2 L and 20 L disposable shaken bioreactors. The trend of the values of power consumption was shown with respect to shaking frequency and filling volume. Results indicate that quite reasonable values of power consumption can be obtained by the temperature method and the torque method, which are in good agreement with each other (error <30%).
Keywords: Bioprocess development; Disposable; Shaken bioreactor; Characterisation; Power consumption; Torque; Temperature; Validation;

Hydrolysis of palm and olive oils by immobilised lipase using hollow fibre reactor by Marwan M. Shamel; K.B. Ramachandran; Masitah Hasan; Sulaiman Al-Zuhair (228-235).
Lipase from Mucor miehei immobilised by adsorption on microporous, asymmetric hollow fibre membrane reactors was used to hydrolyse two different oils, namely, palm and olive oils. The hydrolysis reaction was carried out at a temperature of 40 °C, an average transmembrane pressure (TMP) of 115 mmHg and oil and aqueous flow rates of 2.5 and 3.0 ml min−1, respectively. It was experimentally proven that adsorption of lipase increased with temperature and was higher on hydrophobic membranes than hydrophilic ones. The effluent concentrations of fatty acid products were measured using gas chromatograph with FID detector. Hydrolysis experimental results were fitted to a multisubstrate kinetic model derived from the Ping Pong Bi Bi mechanism. The final model expression is useful for predicting the free fatty acid profile of the enzymatic hydrolysis of palm and olive oils for different substrate flow rates and enzyme loading.
Keywords: Lipase; Adsorption; Hydrolysis; Hollow fibre reactor; Immobilised enzyme; Kinetic parameters;

Continuous counter-current purification of glucose-6-phosphate dehydrogenase using liquid–liquid extraction by reverse micelles by Francislene A. Hasmann; Daniela V. Cortez; Daniela B. Gurpilhares; Valéria C. Santos; Inês C. Roberto; Adalberto Pessoa-Júnior (236-241).
This work details the continuous counter-current purification of glucose-6-phosphate dehydrogenase from Saccharomyces cerevisiae cells by liquid–liquid extraction using reverse micelles. A biocompatible reverse micellar system consisting of 0.05 M soybean lecithin (zwiterionic surfactants) in isooctane with hexanol was employed to study the influence of different flow-rates on G6PD purification. The results showed that the reverse micellar system was able to remove proteins (impurities) from the cell-free. The enzyme recovery yield varied from 32% to 115% and G6PD purification factor from 1.0 to 2.2, under flow-rates ranging from 1.6:1.6 mL min−1 to 6.0:6.0 mL min−1 for both phases (micellar and aqueous phases). The steady hold-up values demonstrated that the mass transfer capability of this extraction apparatus was practically constant. The continuous counter-current purification system employed proved to be useful not only for purifying G6PD, but also for maintaining the enzyme activity.
Keywords: Purification; Continuous system; Reverse micellar system; Downstream processing; Glucose-6-phosphate dehydrogenase; Liquid–liquid extraction;

Elimination of 4-chlorophenol by soybean peroxidase and hydrogen peroxide: Kinetic model and intrinsic parameters by Antonio Bódalo; José L. Gómez; Elisa Gómez; Asunción M. Hidalgo; María Gómez; Ana M. Yelo (242-247).
A kinetic model for the initial rate reaction, based on the Ping-Pong Bi-Bi mechanism, has been assumed for the removal of 4-chlorophenol with soybean peroxidase and hydrogen peroxide. Several sets of experimental data, obtained in a batch reactor at different concentrations of hydrogen peroxide and 4-chorophenol, were used to estimate the kinetic parameters. A new method for determining the intrinsic parameters of the reaction in processes involving a high reaction rate was developed. The method consists of determining several sets of apparent parameters at different times and then extrapolating these to zero time.
Keywords: SBP/4-chlorophenol; Enzyme bioreactors; Kinetic parameters; Modelling; Intrinsic parameters; Waste-water treatment;

Selection of an easy-to-measure, well-correlated surrogate parameter is critical for ensuring the effective operation of a nitrification/denitrification process on a real-time basis. The study aimed at investigating alkalinity as a reliable indicator for effluent nitrogen concentration under a series of operational conditions (chemical oxygen demand (COD), NH4 +, hydraulic retention time) and under allylthiourea (ATU, a chemical inhibited nitrification) shock. The accuracy of alkalinity indication was compared with redox potential (ORP) in a sequencing batch reactor (SBR). Although both ORP and alkalinity exhibited clear variations in a SBR cycle, alkalinity presented a better indication than ORP, especially when there was a progressive decease in nitrification/denitrification efficiency. Effluent alkalinity exhibited a linear reverse correlation with nitrogen concentration (Alk = −4.26[N] + 180, R 2  = 0.92), with alkalinity lower than 100 mg/L indicating insufficient denitrification, while alkalinity higher than 200–250 mg/L indicating insufficient nitrification. Furthermore, alkalinity difference between influent and effluent (ΔAlk), which reflected an overall result of alkalinity consumption in nitrification and alkalinity production in denitrification, was studied as another indicator. ΔAlkinf.-eff. decreased with better denitrification (ΔAlk = 6.99[N] + 22, R 2  = 0.82) but increased with better nitrification (ΔAlk = −5.54[N] + 126, R 2  = 0.76). The strong correlations of alkalinity with effluent nitrogen concentration, and ΔAlkinf.-eff. with nitrification/denitrification efficiency demonstrate that both alkalinity and ΔAlkinf.-eff. can be used as indicators in nitrification/denitrification processes. In addition, the benefits and problems of alkalinity, ORP and pH indications for nitrogen removal were comprehensively compared in the paper.
Keywords: Alkalinity; Redox potential (ORP); Nitrification; Denitrification; Chemical oxygen demand (COD); Oxygen; Hydraulic retention time (HRT); Allylthiourea (ATU) shock;

Thermodynamic equilibrium model in anaerobic digestion process by Sung T. Oh; Alastair D. Martin (256-266).
Catabolic reactions provide the chemical energy necessary for the maintenance of living microorganisms. The catabolic reactions in anaerobic digestion process may progress close to the equilibrium state (ΔG  = 0) depending strongly on the microorganisms in the digester. The thermodynamic equilibrium of catabolic reactions in the anaerobic digestion process was modelled under isothermal and isobaric conditions. Three thermodynamic models were considered; the ideal, the Debye-Hückel–Praunitz, and the Pitzer–Praunitz. The models in this paper concentrate on the methanogenic equilibrium of the anaerobic digestion process. The thermodynamic equilibrium model shows that the methanogenesis step requires thermal energy and electrons, so that anaerobic digestion may achieve high substrate degradation and high conversion to methane. Some thermodynamic recommendations are suggested for the future development of the methanogenic phase of anaerobic digestion.
Keywords: Anaerobic digestion; Methanogenesis; Thermodynamics;

A long-term hepatocyte model in vitro is preferable for chronic hepatotoxicity research because hepatocytes in this model of culture can preserve liver-specific functions for long period. Micro-hollow fiber reactors (MHFR), composed of polysulphone (PS) hollow fibers with a molecular weight cut-off 100 kDa, were applied to test the hepatotoxicity of acetaminophen, isoniazid and rifampicin, respectively. Monolayer culture was used as a control model for hepatocyte culture. It was found that hepatocytes within MHFR were more sensitive to toxicity of acetaminophen (0.38–1.51 g/L) than those in monolayer cultures. Furthermore, significant hepatotoxicity of isoniazid (15 mg/L) and rifampicin (10 mg/L) were detected in hepatocytes cultured in MHFR but not detected in hepatocyte monolayer, which could be due to well-preserved drug metabolizing enzymes in MHFR. These results indicate that the MHFR may be an effective model for long-term hepatotoxicity research in vitro.
Keywords: Micro-hollow fiber reactor; Hepatocyte monolayer; Rat hepatocytes;

Biodiesel-fuel production in a packed-bed reactor using lipase-producing Rhizopus oryzae cells immobilized within biomass support particles by Shinji Hama; Hideki Yamaji; Takahiro Fukumizu; Takao Numata; Sriappareddy Tamalampudi; Akihiko Kondo; Hideo Noda; Hideki Fukuda (273-278).
A packed-bed reactor (PBR) system using fungus whole-cell biocatalyst was developed for biodiesel fuel production by plant oil methanolysis. Lipase-producing Rhizopus oryzae cells were immobilized within 6 mm × 6 mm × 3 mm cuboidal polyurethane foam biomass support particles (BSPs) during batch cultivation in a 20-l air-lift bioreactor. Emulsification of the reaction mixture containing soybean oils and water improved the methanolysis reaction rate. Using a high flow rate for the reaction mixture in the PBR caused exfoliation of the immobilized cells from the BSPs, while the inefficient mixing of the reaction mixture at low flow rates allowed the BSPs to be covered with a hydrophilic layer of high methanol concentration, leading to a significant decrease in lipase activity. A high methyl ester content of over 90% was achieved at a flow rate of 25 l/h in the first cycle of repeated batch methanolysis and a high value of around 80% was maintained even after the tenth cycle. Comparison with methanolysis reaction in a shaken bottle suggested that the PBR enhances repeated batch methanolysis by protecting immobilized cells from physical damage and excess amounts of methanol. The process presented here is therefore considered to be promising for industrial biodiesel-fuel production.
Keywords: Biodiesel fuel; Methanolysis; Packed-bed reactor; Whole-cell biocatalyst; Filamentous fungi; Rhizopus oryzae; Biomass support particles; Immobilized cells;

Effects of geometrical design on hydrodynamic and mass transfer characteristics of a rectangular-column airlift bioreactor by Peter M. Kilonzo; Argyrios Margaritis; M.A. Bergougnou; Juntang Yu; Qin Ye (279-288).
Gas holdup and gas–liquid mass transfer coefficients were measured in a 21-L rectangular-column airlift bioreactor with aspect ratio of 10 and working volumes ranging from 10 to 16 L. The effect of the bottom and top clearances was investigated using water and mineralized CMC solutions and covering a range of effective viscosity from 0.02 to 0.5 Pa s and surface tension from 0.065 to 0.085 N m−1. The gas holdup and mass transfer results were successfully correlated using expressions derived via dimensional analysis. The separator gas holdup was found to be similar to the total gas holdup in the airlift bioreactor. The downcomer gas holdup (ɛ d) increased two-fold when the bottom clearance (h b) was increased from 0.014 to 0.094 m while the top clearance (h t) had no effect. Increasing h b decreased the mass transfer by 50% compared to 31% when the top clearance (h t/D hr) was increased. It was found that the gas–liquid separator diameter ratio (D hs/D hc) exerted the maximal influence of over 65% on mass transfer as compared to both clearances.
Keywords: Airlift bioreactor; Clearance; Gas holdup; Liquid velocity; Pressure drop; Mass transfer;