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Biochemical Engineering Journal (v.30, #3)

Editorial Board (pp. co2).

BEJ Keywords (pp. ii).

Superfine grinding of steam-exploded rice straw and its enzymatic hydrolysis by Shengying Jin; Hongzhang Chen (pp. 225-230).

The combination of low severity steam explosion and superfine grinding has been studied with respect to side products generation and enzymatic hydrolysis efficiency. Chemical compositions, fiber characteristics and composed cells contents in the superfine ground product and the ground residue particles produced by superfine grinding were also studied. At the determined parameters using FJM-200 fluidized bed opposed jet mill, 78% superfine ground steam-exploded rice straw (SERS) products with the mean fiber length of 60μm were obtained, the particles yield was 179% higher than that from the native rice straw (RS). Enzymatic hydrolysis, chemical composition, fiber characteristics and composed cells proportion of the superfine ground SERS product and the ground residue all show great differences. The difference in enzymatic hydrolysis and structural properties indicates that superfine grinding is a good way to fractionate SERS into easily bio-converted part and difficultly hydrolysed part.

Keywords: Superfine grinding; Steam explosion; Enzymatic hydrolysis; Fiber characteristics; Fluidized bed opposed jet mill; Rice straw

Antimicrobial activity of steady-state cultures of Bacillus sp. CCMI 1051 against wood contaminant fungi by Ana Teresa Caldeira; Sonia Savluchinske Feio; J.M. Santos Arteiro; J. Carlos Roseiro (pp. 231-236).

The effect of changing dilution rate ( D) on Bacillus sp. CCMI 1051 at dilution rates between 0.1 and 0.55h⁻¹ in a glucose-limited medium was studied. Biomass values varied between 0.88 and 1.1gL⁻¹ at D values of 0.15–0.35h⁻¹. Maximal biomass productivity was found to be 0.39gL⁻¹h⁻¹, obtained at D=0.35h⁻¹ and corresponding to a 54.4% conversion of the carbon into cell mass. The highest rate of glucose consumption was 4.45mmolg⁻¹h⁻¹ occurring at D=0.4h⁻¹. The glucose concentration inside the chemostat was below the detection level starting to accumulate around 0.4h⁻¹. Growth inhibition of fifteen strains of fungi by the broth of the steady-state cell-free supernatants was assessed. Results showed that the relative inhibition differ among the target species but was not influenced by the dilution rate changing.

Keywords: Bacillus; sp.; Chemostat; Fermentation; Aerobic processes; Antibiotic; Physiology

Effect of aprotic solvents on the enzymatic activity of lipase in AOT reverse micelles by Muhammad Moniruzzaman; Yoshishige Hayashi; Md. Mahabubur Rahman Talukder; Eiji Saito; Takuya Kawanishi (pp. 237-244).

The modification of reverse micellar systems composed of AOT, isooctane, water by the addition of aprotic solvents has been performed. The impact of this change on the activity, stability and kinetics of solubilized Chromobacterium viscosum lipase (glycerol-ester hydrolase, EC 3.1.1.3) was investigated. Of seven aprotic solvents tested, dimethyl sulfoxide (DMSO) was found to be most effective. It was found that lipase activity was enhanced by optimizing some relevant parameters, such as water–AOT molar ratio ( W₀), buffer pH and surfactant concentration. A kinetic model that considers the free substrate in equilibrium with the substrate adsorbed on the micellar surface was successfully used to deduce some kinetic parameters ( V_max, K_m and K_ad), and the values of K_m and K_ad were significantly reduced by the presence of DMSO. Higher lipase stability was found in AOT reverse micelles with DMSO compared with that in simple AOT systems with half-life of 125 and 33 days, respectively. Fluorescence spectroscopy and Fourier transform infrared spectroscopy (FT-IR) were used to elucidate the effects of DMSO on the properties of AOT reverse micelles.

Keywords: Reverse micelles; Lipase; Hydrolysis; DMSO; Enzyme stability and kinetics

Effect of organic solvents on oxygen mass transfer in multiphase systems: Application to bioreactors in environmental protection by E. Dumont; Y. Andrès; P. Le Cloirec (pp. 245-252).

The absorption of oxygen in aqueous–organic solvent emulsions was studied in a laboratory-scale bubble reactor at a constant gas flow rate. The organic and the gas phases were dispersed in the continuous aqueous phase. Volumetric mass transfer coefficients ( k_L a) of oxygen between air and water were measured experimentally using a dynamic method. It was assumed that the gas phase contacts preferentially the water phase. It was found that addition of silicone oils hinders oxygen mass transfer compared to air–water systems whereas the addition of decane, hexadecane and perfluorocarbon PFC40 has no significant influence. By and large, the results show that, for experimental conditions (organic liquid hold-up ≤10% and solubility ratio ≤10), the k_L a values of oxygen determined in binary air–water systems can be used for multiphase (gas–liquid–liquid) reactor design with applications in environmental protection (water and air treatment processes).

Keywords: Absorption; Mass transfer; Multiphase reactors; Silicone oil; Oxygen; Bioprocesses

Enrichment in axial direction of aqueous foam in continuous foam separation by Hideo Maruyama; Akira Suzuki; Hideshi Seki; Norio Inoue (pp. 253-259).

Estimation of overhead production enrichment in continuous foam separation was conducted with a surfactant: sodium n-dodecylbenzenesulfonate (SDBS) and soluble proteins: ovalbumin (OA) and hemoglobin (HB). Axial profiles of the volumetric flow rate and the concentration of the collapsed foam liquid within the column were measured, and the enrichment ratio and the liquid holdup in axial direction were determined experimentally. The proposed model was fitted to the experimental results obtained with various experimental conditions (superficial gas velocity, feed concentration and pH) and was in reasonable agreement with the experimental data by using the least square regression. The present model makes it possible to estimate the foamate concentration at a desired foam height.

Keywords: Adsorption; Bioseparations; Bubble columns; Separation; Foam separation; Protein

Recovery of medium-chain-length polyhydroxyalkanoates (PHAs) through enzymatic digestion treatments and ultrafiltration by K. Yasotha; M.K. Aroua; K.B. Ramachandran; I.K.P. Tan (pp. 260-268).

Medium-chain-length (mcl) polyhydroxyalkanoates (PHAs) are biodegradable polyesters accumulated intracellularly as energy resources by bacterial species such as Pseudomonas putida. The most popular method for PHA recovery in the downstream processing is solvent extraction using chloroform and methanol. An alternate method is bioseparation using enzymatic digestion process which eliminates the need for hazardous solvents. This research focuses on an attempt to optimize the recovery of PHAs by solubilisation of non-PHA granules through enzymatic treatments such as; Alcalase (to digest the denatured proteins), sodium dodecyl sulfate (SDS) to assist solubilisation, ethylene diamine tetra acetic acid (EDTA) to complex divalent cations and lysozyme to digest the peptidoglycan wall enveloping the cell. The experiment was designed through Taguchi's design of experiment (DOE) using Qualitek-4 software. The results show that Alcalase enzyme used had the most significant effect on the treatment process and contributed to about 71.5% in terms of process factor importance among the different factors on treatment performance for PHA recovery. It is desired to recover the PHA granules in water suspension after the enzymatic treatment by removing the solubilised non-PHA cell material through crossflow ultrafiltration system and purified through continuous diafiltration process. Final purity of PHA in water suspension obtained using GC analysis is 92.6%, with a nearly 90% recovery, thus concluding that this method is indeed a suitable alternative.

Keywords: mcl-Polyhydroxyalkanoate; Downstream processing; Bioseparation; Enzymatic digestion; Taguchi's method; Ultrafiltration and continuous diafiltration

Immobilization of lipase from Candida rugosa on Eupergit^® C supports by covalent attachment by Zorica Knezevic; Nenad Milosavic; Dejan Bezbradica; Zivana Jakovljevic; Radivoje Prodanovic (pp. 269-278).

The present study compares the results of three different covalent immobilization methods employed for immobilization of lipase from Candida rugosa on Eupergit^® C supports with respect to enzyme loadings, activities and coupling yields. It seems that method yielding the highest activity retention of 43.3% is based on coupling lipase via its carbohydrate moiety previously modified by periodate oxidation. Study of thermal deactivation kinetics at three temperatures (37, 50 and 75°C) revealed that the immobilization method also produces an appreciable stabilization of the biocatalyst, changing its thermal deactivation profile. By comparison of the t_1/2 values obtained at 75°C, it can be concluded that the lipase immobilized via carbohydrate moiety was almost 2-fold more stable than conventionally immobilized one and 18-fold than free lipase. The immobilization procedure developed is quite simple, and easily reproduced, and provides a promising solution for application of lipase in aqueous and microaqueous reaction system.

Keywords: Immobilized enzymes; Candida rugosa; lipase; Eupergit; Enzyme deactivation; Kinetic parameters; Microemulsions

Run-to-run fed-batch optimization for protein production using recombinant Escherichia coli by Chih-Lung Ko; Feng-Sheng Wang (pp. 279-285).

A two-phase design approach is introduced to determine the optimal feed rate, fed glucose concentration and fermentation time to maximize protein productivity using recombinant Escherichia coli BL21 (pBAW2) strain. The first phase is applied to determine a primary S-system kinetic model using batch time-series data. Two runs were carried out in the second phase to achieve the maximum protein productivity for the fed-batch fermentation process. The computational results using the S-system kinetic model obtained from the second run are in better agreement with the experiments than those using the kinetic model obtained from batch time-series data. For cross-validation, two extra fed-batch experiments with different feed strategies were carried out for comparison with the optimal fed-batch result. From the experimental results, this approach could improve productivity by at least 3%.

Keywords: S-system; Optimization; Hybrid differential evolution; Fed-batch culture; Fermentation; Bioreactor

Effect of sucA or sucC gene knockout on the metabolism in Escherichia coli based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic fluxes by¹³C-labeling experiments by Mai Li; Pei Yee Ho; Shanjing Yao; Kazuyuki Shimizu (pp. 286-296).

The effect of sucA or sucC gene knockout on the metabolism in Escherichia coli was investigated for the aerobic cell growth in batch and continuous cultivations based on gene expressions, enzyme activities, intracellular metabolite concentrations and metabolic flux analysis. In the batch cultivation, the cell growth rate and the glucose uptake rate were lower for sucA mutant as compared with the parent strain, while it was not the case for sucC mutant. A significantly higher amount of acetate was produced, and it was not utilized in s ucC mutant, while a little less acetate was produced in sucA mutant as compared with the parent strain. Unlike the parent strain and sucC mutant, sucA mutant excreted a little amount ofl-glutamate. Enzyme activity results show that some of the glycolytic enzymes such as Tpi and Pgk were up-regulated, while Pfk, Fba and Pyk activities were down-regulated for sucA mutant as compared with the parent strain. For sucC mutant, the activities of Pfk, Fba, Tpi, GAPDH, Pgk and Pyk activities were down-regulated. As for the TCA cycle enzymes, the activities of CS and ICDH were down-regulated, while those of Icl, MS, Fum and MDH were up-regulated for sucA mutant. The activities of the oxidative pentose phosphate (PP) pathway enzymes such as G6PDH and 6PGDH and the gluconeogenic pathway enzyme such as Mez were up-regulated in sucA mutant. The Ack activity was down-regulated for sucA mutant, but not for sucC mutant. In continuous cultivation, the gene expression results indicate that the global regulatory genes such as fadR and iclR were slightly down-regulated in sucA mutant, which enhanced the expression of aceA gene and caused the up-regulation of the isocitrate lyase activity in sucA mutant, while fadR and iclR of sucC mutant changed little and no isocitrate lyase activation was observed for sucC mutant. Some other global regulatory genes such as arcA and fnr genes were down-regulated in both mutants, which caused some of the TCA cycle genes to be up-regulated. The effect of the sucA gene knockout on the metabolic flux distributions was investigated based on¹H–¹³C NMR spectra and GC–MS signals obtained from¹³C-labeling experiments. Flux analysis results indicate that the knockout of sucA gene caused the activation of PP pathway and the glyoxylate shunt. The fluxes through glycolysis and the TCA cycle were down-regulated in the sucA mutant. On the other hand, the fluxes through PP pathway and the anaplerotic reactions of Ppc-Pck and Mez increased.

Keywords: sucA; gene knockout; sucC; gene knockout; RT-PCR; Enzyme activity; Intracellular metabolite; ¹³; C-Labeling experiment; Metabolic flux analysis

Performance of a three-stage aerobic RBC reactor in food canning wastewater treatment by G.D. Najafpour; A.A.L. Zinatizadeh; L.K. Lee (pp. 297-302).

Biological treatment using attached growth in a three-stage lab-scale rotating biological contactor (RBC) was implemented for wastewater from food cannery industries. The wastewater contained high level of organic compounds due to fish and fruit cleaning, cooking and filling processes. Nutrients available in the wastewater enhanced the growth of microorganisms and allowed the biological treatment to be effective. The RBC consisted of 54 parallel discs rotating in a reservoir and was arranged in three stages, i.e. 18 discs oriented in each stage. Effect of major operating and physical variables such as hydraulic retention time (HRT), disc submergence and disc rotational speed were examined in COD removal. For duration of 5 days, 96.4% BOD removal was achieved in batch experiment. BOD constant rate ( k) and ultimate BOD were determined respectively, 0.8198day⁻¹ and 6349mg/l by Thomas graphical method. COD removal efficiency was increased from 85.3 to 97.4% while the HRT was increased from 24 to 48h. The COD removal efficiency increased from 74.9 to 87.5% as the disc submergence was increased from 31 to 36%. At submergence level of 23.7%, removal efficiency was increased due to activation of second and third compartments. When the rotational speed was increased from 3 to 11rpm, the COD removal efficiency was also increased from 62.7 to 93.7%, respectively. The stage COD removal efficiency was gradually decreased with an increase number of stage and about 88% of organic compounds were removed in the first stage of aerobic RBC, indicating that the single stage reactor may be sufficient in practical application.

Keywords: Three-stage RBC; Food canning wastewater; Disc submergence; Rotational speed; Ultimate BOD

Kinetics of molecular weight reduction of poly(glutamic acid) by in situ depolymerization in cell-free broth of Bacillus subtilis by Andrew Richard; Argyrios Margaritis (pp. 303-307).

Poly(glutamic acid) (PGA) is a water soluble, biodegradable biopolymer that is produced by microbial fermentation. Recent research has shown that poly(glutamic acid) can be used in drug delivery applications for the controlled release of paclitaxel (Taxol) in cancer treatment. The molecular weight of microbial poly(glutamic acid) is generally larger than what is required for drug delivery. As such, molecular weight reduction is a necessary step in producing poly(glutamic acid) for this application. Poly(glutamic acid) produced by Bacillus subtilis IFO 3335 was subjected to in situ depolymerization in the cell-free fermentation broth. Molecular weight reduction was measured, and an empirical kinetic model was used to correlate the experimental data. The kinetic rate constant, k, was found to be 6.92×10⁻⁶h⁻¹ at pH 7.0 and 37°C, which were the optimum depolymerization conditions.

Keywords: Poly(glutamic acid); In situ depolymerization; Optimization; Modeling; Kinetic parameters; Polypeptides

Mathematical modelling of the aerobic degradation of two-phase olive mill effluents in a batch reactor by Marco Pelillo; Bárbara Rincón; Francisco Raposo; Antonio Martín; Rafael Borja (pp. 308-315).

A laboratory-scale study was conducted on the aerobic degradation of two-phase olive mill effluents (TPOME) made up of the mixture of the washwaters derived from the initial cleansing of the olives and those obtained in the washing and purification of virgin olive oil. The process was carried out in a 1-l working volume stirred tank reactor operating in batch mode at room temperature (25°C). The reactor was operated at influent substrate concentrations of 2.80g COD/l (TPOME 25%), 5.45g COD/l (TPOME 50%), 8.18g COD/l (TPOME 75%) and 10.90g COD/l (TPOME 100%). After five days of operation time, total and soluble COD removal efficiencies of 64.3% and 66.6% were achieved respectively for the most concentrated influent used (TPOME 100%). A simplified kinetic model for studying the hydrolysis of insoluble organic matter, oxidation of soluble substrate and biomass production was proposed on the basis of the experimental results obtained. The following kinetic constants with their standard deviations were obtained for the above stages in the case of the most concentrated influent used (TPOME 100%): k₁ (kinetic constant for hydrolysis of suspended organic matter): 0.11±0.01l/(g VSS day); k₂ (kinetic constant for total consumption of soluble substrate): 0.30±0.02l/(g VSS day); k₃ (endogenous metabolism constant): 0.07±0.01 per day). Finally, the biomass yield coefficient was found to be 0.30g VSS/g COD_removed. The values of non-biodegradable total and soluble CODs obtained from the model were found to be 3 and 2g/l, respectively. The kinetic constants obtained and the proposed equations were used to simulate the aerobic degradation process of TPOME and to obtain the theoretical values of non-soluble and soluble CODs and biomass concentration. The small deviations obtained (equal or lower than 10%) between the theoretical and experimental values suggest that the parameters obtained represent and predict the activity of the microorganisms involved in the overall aerobic degradation process of this wastewater.

Keywords: Mathematical modelling; Aerobic degradation; Two-phase olive mill effluents (TPOME); Batch reactor

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Biochemical Engineering Journal (v.30, #3)