Biochemical Engineering Journal (v.25, #1)

IFC (CO2).

BEJ Keywords (II).

The production of dextran and fructose from carob pod extract (CPE) and cheese whey (CW) as carbon source by the bacterium Leuconostoc mesenteroides was investigated. The influence of secondary carbon sources (maltose, lactose and galactose) on dextran molecular weight and fermented broth viscosity were also studied.Significant changes were not observed in broth viscosity during dextran production at initial sucrose concentration of 20 and 120 g/l. Complementary sugars maltose, lactose and galactose together with sucrose promote production of dextran with fewer glucose units. Dextran molecular weight decreases from the range 1,890,000–10,000,000 to 240,000–400,000 Da when complementary sugars are present. Polydispersity was improved when complementary sugars were used.Fermentation using mixtures of carob pod extract and cheese whey confirm these results obtained for production of dextran. Final concentrations of dextran and fructose indicate that reaction yields were not affected. Carob pod and cheese whey can be successfully used as raw material in the fermentation system described.The maximum concentrations of dextran and fructose obtained using carob pod extract resulted in 8.56 and 7.78 g/l, respectively. Combined carob pod extract and cheese whey resulted in dextran and fructose concentrations of 7.23 and 6.98 g/l, respectively. The corresponding dextran mean molecular weight was 1,653,723 and 325,829.
Keywords: Dextran; Fructose; Leuconostoc mesenteroides; Viscosity; Molecular weight; Carob pod; Cheese whey;

We investigated the use of chitosan for removing phenol oxide in the aqueous phase as a water-treatment process. We used p-quinone for the phenol oxide model material and investigated the capabilities of chitosan based on molecular weight (MW) and deacetylation degree (DD). We also examined reaction conditions (temperature and pH). The chitosan specifically removed p-quinone, rather than other phenols. The overall reaction rate constant was sufficiently high in the experimental range of 3.89 × 105 to 1.24 × 106  MW g mol−1 and from 83.1 to 88.6 DD%. Chitosan was suitable for separating p-quinone in the aqueous phase at basic pH levels. Activation energy was found to be 45 kJ mol−1.
Keywords: Chitosan; Molecular weight; Deacetylation degree; Quinone; Separation; Mass transfer; Wastewater treatment;

Immobilization of Pycnoporus sanguineus laccase on magnetic chitosan microspheres by De-Sheng Jiang; Sheng-Ya Long; Jun Huang; Hai-Yan Xiao; Ju-Ying Zhou (15-23).
Magnetic chitosan microspheres were prepared with reversed-phase suspension methodology using glutaraldehyde as cross-linking reagent for the enzyme immobilization. The microspheres had well-shaped spherical form with smooth surface, and its mean particle size was 5.0 μm with a narrow size distribution. Laccase was immobilized on magnetic chitosan microspheres by adsorption and cross-linking with glutaraldehyde. The immobilization conditions and characterization of the immobilized enzyme were investigated. The immobilized laccase exhibited the maximal enzyme activity at pH 3.0. The optimal temperature for immobilized enzyme was 10 °C and 55 °C. The kinetic parameters, K m and V max, for immobilized enzyme were estimated to be 171.1 μM and 5.9 mmol/(l min), respectively. The thermal, operational, and storage stabilities of the enzyme were improved greatly after they were immobilized on the surface of the magnetic chitosan microspheres.
Keywords: Magnetic chitosan microspheres; Immobilization; Adsorption; Laccase; Enzyme activity; Kinetic parameters;

Xylitol production from sugarcane bagasse hydrolysate by W. Carvalho; J.C. Santos; L. Canilha; S.S. Silva; P. Perego; A. Converti (25-31).
Sugarcane bagasse hydrolysate was used for batch xylitol production in stirred tank reactor with Candida guilliermondii cells entrapped in Ca-alginate beads. Experiments were carried out using five-fold concentrated hydrolysate, agitation speed of 300 rpm, air flowrate of 1.3 l min−1, initial cell concentration of 1.4 gDM  l−1, and starting pH 6.0. Xylitol production reached 47.5 g l−1 within 120 h of fermentation, resulting in a bioconversion yield of 0.81 g g−1 and a productivity of 0.40 g l−1  h−1. The metabolic behaviour of C. guilliermondii was then investigated through material balances using the concentrations of consumed substrates and formed products and assuming that xylose was simultaneously assimilated by the yeast for anaerobic and semi-aerobic xylitol productions, complete oxidation by the TCA cycle and cell growth. Data collected at different times were finally used to estimate the overall ATP requirements for biomass growth and maintenance. The energy expenditure increased from 2.1 to 6.6 mo l ATP C mo l DM − 1 throughout the fermentation, highlighting a progressive difficulty of the microbial system in facing the energy needs of its semi-aerobic metabolism.
Keywords: Xylitol; Immobilised cells; Aeration; Bioconversion; Kinetics; Material and bioenergetic balances;

Nitrifying treatment of wastewater from fertilizer production in a multiple airlift loop bioreactor by Wen Jianping; Jia Xiaoqiang; Pan Lei; Wang Changlin; Mao Guozhu (33-37).
An 80 m3 gas–liquid–solid three-phase flow multiple airlift loop bioreactor (ALR) with a low ratio of height to diameter s, in which biofilm replaced the activated sludge, was used in the nitrifying treatment of the effluent from Tengfei Chemical Fertilizer Plant, Tianjin, PR China. The influences of the ratio of bed (settled) volume to bioreactor volume V b/V R, superficial air velocity U g, hydraulic residence time HRT and pH value on ammonia nitrogen reduction were investigated and discussed. The optimum operating conditions were obtained as pH value of 7.0–8.0, V b/V R of 0.65, U g of 0.01 m s−1 and HRT of 6 h were found. Under the optimum operating conditions, the effluent COD and NH4 ―N were less than 50 and 10 mg l−1, which was far below the primary discharge standard for chemical fertilizer industry of PR China: COD < 100 mg l−1 and NH4–N < 40 mg l−1 (GWPB4-1999).
Keywords: Biofilms; Wastewater treatment; Airlift loop bioreactor; Nitrification; Ammonia nitrogen;

Hydrolysis and glycerolysis of l,3-palimitin-2-olein (POP) was studied and mechanism of glycerolysis was discussed. The results showed that the glycerolysis is a complicated process including hydrolysis, esterification and isomerization of monoglycerides (MG) and diacylglycerols (DG). At first 5 h of glycerolysis, hydrolysis dominates the process and then esterification and isomerization will take place. A new mechanism and kinetic model based on hydrolysis, esterification and isomerization were established to correct all reactions in glycerolysis. The average relative error between experimental data and simulated results is 8.37%. The kinetic model can be used to predicate the results of glycerolysis at different molar ratios of POP to glycerol. The relative errors between the results predicted and experimental data are lower that 10%, which means that the kinetic model can be used to describe for glycerolysis of triglycerides.
Keywords: Lipase; 1,3-Palmitin-2-olein; Glycerolysis; Mechanism; Kinetic model;

Under microaerobic condition, key enzymes (glycerol dehydrogenase GDH, 1,3-propanediol oxidoreductase PDOR, glycerol dehydratase GDHt) accumulation of 1,3-propanediol production by Klebsiella pneumoniae was studied. Several carbon sources, organic nutrients, inorganic nitrogen sources and salts were examined for their effects on key enzymes formation. Uniform design and genetic algorithms (GAs) coupling artificial neural networks (ANNs) were developed for the medium optimization. Meanwhile, the optimization of fermentation conditions (initial pH, rotor speed, temperature and inoculum) was conducted. When the strain grew in the optimized medium under optimal fermentation condition in a 5 L stirred tank bioreactor for batch production, GDH, PDOR and GDHt activities were 3700, 3840 and 8.70 U referred to 1 L of fermentation broth after 20 h cultivation and the productivities of GDH, PDOR and GDHt (U/L h) were 185, 192, 0.435 and the maximun concentration of 1,3-PD was 10.5 g/L. Result show that productions of GDH, PDOR and GDHt with K. pneumoniae can be carried out under microaerobic condition and the increment of key enzyme activities is essential to 1,3-PD formation.
Keywords: 1,3-Propanediol; Glycerol dehydrogenase; 1,3-Propanediol oxidoreductase; Glycerol dehydratase; Fermentation; Optimization;

A statistical approach called response surface methodology (RSM) is used for the prediction of the kinetic constants of glucose oxidase (GOx) as a function of reaction temperature and pH. Lineweaver–Burk transformation of the Michaelis–Menten equation was utilized as the integral part of the RSM algorithm. The effects of variables, namely reciprocal of substrate concentration (0.033–0.5 mM−1), reaction temperature (14.9–40.1 °C) and reaction pH (pH 4.4–8.5) on the reciprocal of initial reaction rate were evaluated and a second order polynomial model was fitted by a central composite circumscribed design (CCCD). It was observed that optimum reaction temperature and pH for the GOx reaction depended on the substrate concentration and varied between 27.8 °C and 6.4 pH and 32.7 °C and 6.1 pH in the investigated range of substrate concentration. The maximum reaction rate (V max) and Michaelis–Menten constant (K m) of GOx were obtained for each reaction parameter by using the model equation. The maximum reaction rate varied between 3.5 μmol/min mg enzyme and 29.8 μmol/min mg enzyme. Michaelis–Menten constant was determined between 1.9 mM and 16.8 mM in the tested reaction parameters. The kinetic constants of GOx were also determined with the conventional method at six reaction parameters and compared with the results of the proposed method. The correlation coefficients (R 2) between the results of two methods were determined as 0.940 and 0.869 for V max and K m, respectively.
Keywords: Enzymes; Kinetic constants; Glucose; Glucose oxidase; Response surface methodology;

A novel method for the measurement of oxygen mass transfer rates in small-scale vessels by Kenny Ortiz-Ochoa; Steven D. Doig; John M. Ward; Frank Baganz (63-68).
A novel method for the measurement of the volumetric oxygen transfer coefficient, kLa, using the catechol-2,3-dioxygenase (XylE) bio-oxidation of catechol yielding 2-hydroxymuconic semialdehyde (2-HS), has been developed for small-scale systems. This method was kinetically characterized and validated by comparison to other established techniques. The bio-oxidation rate was found to be zero order over a catechol concentration from 2 to 7 mM l−1. When the enzyme concentration was in excess and the bio-oxidation rate was mass transfer limited, indicated by a percent DOT of zero, the oxygen transfer rate was determined from the linear increase in product concentration. The method was validated in a 2 l stirred tank vessel equipped with a DOT probe and connected to a gas mass spectrometer. The novel method yielded similar kLa values when compared to the dynamic gassing out method under the same conditions. The applicability of this novel method for small-scale devices was demonstrated by measuring kLa values of up to 150 h−1 in shaken microplates with a working volume of only 200 μl.
Keywords: Enzyme kinetics; Bio-oxidation; Mass transfer; Small-scale;

Lipase from Candida rugosa was immobilised onto polypropylene powder by physical adsorption. The immobilised catalyst (CR/PP) was used in the enzymatic synthesis of ethyl oleate in solvent-free medium. The influence of the initial water content, acidity of the aqueous media added, mass of catalyst, reaction temperature, substrate ratio, etc., on enzymatic activity, has been studied. Comparison of specific enzymatic activities achieved using the prepared catalyst and the crude lipase demonstrated that C. rugosa lipase was interfacially activated upon its adsorption on polypropylene. Besides, immobilisation of the lipase led to enhanced thermal stability.Experimental data reported in this manuscript do not belong to equilibrium data but to enzymatic activity attained in the first 2 h of reaction. After this period, it was shown that, in the current synthesis, deactivation/agglomeration/inhibition of the catalyst prevented further CRL activity. However, 2 h measurements allowed fulfilling the aim of this work: the determination of the best conditions for ethyl oleate production in short periods of time, using an immobilised derivative of a relatively cheap lipase as it is C. rugosa lipase. Best results were achieved in the reaction performed at 45 °C and 350 rpm, with an initial stoichiometric ratio of substrates, 20% of aqueous content, and mediated by 50 mg of CR/PP (0.0585 mmol/mg of CR-h). The deleterious effect of ethanol excess and agglomeration of the native and immobilised catalyst have been analysed.
Keywords: Ethyl oleate synthesis; Lipase; Reaction parameters; Aggregation; Inhibiting effect;

We systematically investigated, under stringent control environments, the effects of surfactants on enzymes in dilute solutions. We elucidated how anionic surfactants of various hydrophilicities affected the activities of three metabolically important enzymes –l-glutamate dehydrogenase (GDH), l-lactate dehydrogenase (LDH), and l-malate dehydrogenase (MDH) – of different molecular masses at a pH range important to body functions (6.5–7.4). We also investigated the time course of the surfactant concentration-dependent effects on LDH. As “standards” for comparison with results obtained using the anionic surfactants, we conducted similar studies with the well characterized non-ionic surfactant, Triton X-100. Activity of enzyme protein of larger molecular mass (GDH) in solution showed less variation compared to those with smaller molecular masses (LDH and MDH), with changes in pH, hydrophilicity, and surfactant concentration. For LDH and MDH, relative activities could change tremendously with 1 ppm difference in surfactant concentrations. All three enzymes were more active in hydrophilic than in hydrophobic surfactants. LDH activity also showed time-dependent decreases with different surfactant concentrations. These results suggest that, for data to be comprehensive, surfactant effects should be investigated with a wide range of concentrations and with time as a variable.
Keywords: Anionic surfactants; Enzyme activity; Dehydrogenases; Enzyme kinetics; Enzyme deactivation; Enzyme technology;

This study has been carried out in order to assess the impact of CODbiod. in an UASB effluent applied to a single stage, a two stage RBC system and an anoxic up-flow submerged bio-filter followed by a segmental two stage aerobic RBC on the removal efficiency of different COD fractions, Escherichia coli, ammonia and partially nitrate removal.The two (single stage) RBC's were operated at a constant HRT of 2.5 h and temperature of 21 °C but at different OLR's, viz. of 10 and 14 g CODbiod./m2  day due to the highly different UASB effluent qualities. The results clearly show that the residual values of CODtotal, ammonia and E. coli in the final effluent are significantly lower at the lower imposed OLR of 10 g COD biod./m2  day. In view of the results obtained we recommend to use a single stage RBC system at OLR of 10 g CODbiod./m2  day and at HRT of 2.5 h for post-treatment of the effluent of UASB reactor operated at high temperature of 30 °C as generally prevails in tropical countries.The performance of a single stage versus two stage RBC system for post-treatment of the effluent of an UASB reactor operated at a low temperature of 12 °C has been evaluated. The single stage and a two stage RBC system were operated at the same OLR of 18 g CODbiod./m2  day and at HRT of 2.5 h. The results show that the COD fractions, ammonia and E. coli content in the final effluent of a two stage RBC system were significantly lower than the effluent of the single stage RBC system. Accordingly, we recommend a two stage RBC system at an HRT of 2.5 h and OLR of 18 g CODbiod./m2  day for post-treatment of the effluent of a conventional UASB reactor operating at a low temperature of 12 °C.The nitrogen removal from the nitrified effluent was investigated using a biofilm system consisting of three stages, viz. an anoxic up-flow submerged bio-filter followed by a segmental two stage aerobic RBC. The nitrified effluent of the second stage RBC was recycled to the anoxic up-flow submerged bio-filter reactor. The results obtained reveal that the introduction of an anoxic reactor as a first stage combined with recirculation of the nitrified effluent of the second stage RBC is accompanied with a conversion of nitrate into ammonia, at least in case the content of CODbiod. in the UASB effluent is low. In such a situation the ammonia needs to be nitrified two times, which obviously should be avoided. Therefore in such situations of a too high quality anaerobic effluent in terms of biodegradable COD content, the introduction of a separate anoxic reactor for denitrification as final post-treatment step can not be recommended.
Keywords: UASB; Post-treatment; RBC; COD biodegradable; Nitrification; Denitrification; E. coli;