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Biochemical Engineering Journal (v.56, #3)

Editorial Board (pp. co2).

BEJ Keywords (pp. ii).

Kinetic modeling of lipase-catalyzed glycerolysis of olive oil by Fernando Voll; Roberta L. Krüger; Fernanda de Castilhos; Lúcio Cardozo Filho; Vladimir Cabral; Jorge Ninow; Marcos L. Corazza (pp. 107-115).

▶ We model the lipase-catalyzed glycerolysis of olive oil. ▶ The ordered-sequential Bi Bi mechanism was proposed. ▶ An empirical deactivation term was efficiently introduced. ▶ DAG yields are favored at low initial amounts of glycerol.This work reports experimental data and the kinetic modeling of monoacylglycerols (MAG) and diacylglycerols (DAG) production from olive oil using a commercial immobilized lipase (Novozym 435) and tert-butanol as solvent. The kinetic modeling presented is based on the ordered-sequential Bi Bi mechanism considering glycerolysis and hydrolysis/esterification steps. Moreover, an empirical enzyme deactivation term, due to an excess of glycerol in the system, is proposed. The experiments were performed in batch mode evaluating the effects of temperature (40–70°C), enzyme concentration (2.5–15wt%) and glycerol to oil (G:O) molar ratio (0.5:1.5–6:1). Good conversions of MAG (∼65wt%) and DAG (∼57wt%) were obtained with relatively low enzyme concentrations (10wt %) in relatively short reaction times (720min) for different initial G:O molar ratios. A very satisfactory agreement between the experimental data and modeling results was obtained under various conditions of enzyme concentration, glycerol to olive oil molar ratio and temperature, thus allowing a better understanding of the reaction kinetics.

Keywords: Glycerolysis; Kinetic modeling; Lipase-catalyzed; Olive oil; Monoacylglycerols; Diacylglycerols

Electrochemical immunosensor for detecting carcinoembryonic antigen using hollow Pt nanospheres-labeled multiple enzyme-linked antibodies as labels for signal amplification by Hongchuan Yang; Ruo Yuan; Yaqin Chai; Li Mao; Huilan Su; Wen Jiang; Min Liang (pp. 116-124).

► A new immunosensor for detecting CEA was constructed based on Au–TiO₂ and HRP–Ab₂–HPtNPs. ► The 3D Au–TiO₂ with large surface area and good biocompatibility could efficiently carry proteins. ► HRP–Ab₂ were labeling by HPtNPs, which could load numerous HRP–Ab₂ and result in a synergy with HRP. ► The immunosensor showed a high sensitivity and a wide linear range from 0.02 to 120ng/mL toward CEA.Herein, a sensitive and specific electrochemical immunosensor is constructed for the detection of carcinoembryonic antigen (CEA) using Au–TiO₂ nanoparticles as the sensor platform and multiple horseradish peroxidase (HRP)-labeled antibodies (HRP-Ab₂) functionalized hollow Pt nanospheres (HPtNPs) (abbreviated as HRP-Ab₂–HPtNPs) as labels. Three-dimensional (3D) Au–TiO₂ nanoparticles were uniformly assembled on a glass carbon electrode (GCE) for the immobilization of primary antibodies (Ab₁), which provided a biocompatible immobilization surface and a facile pathway for the electron transfer. After capturing the target CEA, the HRP-Ab₂–HPtNPs bioconjugates were bound to the electrode surface via the formation of a sandwich complex. The electrochemical signals were amplified by HPtNPs and the carried HRP toward the reduction of H₂O₂ using hydroquinone (H₂Q) as the electron mediator. Under optimized conditions, the proposed immunosensor showed a high sensitivity and a wide linear range from 0.02 to 120ng/mL with a low detection limit of 12pg/mL.

Keywords: Electrochemical immunosensor; Au–TiO; ₂; nanoparticles; Hollow Pt nanospheres; Enzyme amplification; Horseradish peroxidase; Carcinoembryonic antigen

Improvement of cellulose conversion caused by the protection of Tween-80 on the adsorbed cellulase by Maohua Yang; Aimin Zhang; Binbin Liu; Wangliang Li; Jianmin Xing (pp. 125-129).

► Tween-80 can increase the conversion of cellulose at high shaking speed. ► Tween-80 cannot protect free cellulase. ► Tween-80 can decrease adsorption of cellulase proteins. ► Tween-80 can protect the adsorbed cellulase.For the hydrolysis of pure cellulose, the positive effect of Tween-80 was controversial. In this study, it was found that Tween-80 had little effect on pure cellulose conversion under the shaking speed of 0 and 100rpm. However, when the shaking speed increased to 180rpm, Tween-80 resulted in an obvious improvement of cellulose conversion. The mechanism of Tween-80 effect was investigated from three aspects: protecting free cellulase from deactivation, decreasing cellulase proteins adsorption and reducing the cellulase deactivation during the interaction between enzyme and substrate. Tween-80 did not show more positive effects at high speed in protecting free cellulase against deactivation and decreasing cellulase proteins adsorption. From the studies of endoglucanase and exoglucanase relative activities in the supernatant of hydrolysis, it was found that the improved conversion of cellulose with Tween-80 at high shaking speed was mainly due to the reduction of the adsorbed cellulase deactivation resulting from enzyme–substrate interaction.

Keywords: Tween-80; Cellulase; Enzymatic hydrolysis; Shaking speed; Deactivation; Mechanism

Integrated utilization of the main components of Hesperaloe funifera by R. Sánchez; A. Rodríguez; E. Navarro; A. Requejo; L. Jiménez (pp. 130-136).

► H. funifera provides an effective alternative to obtain pulps. ► For paper pulp production, hydrothermal treatment is not appropriate. ► pH 6 is the optimal value to obtain solid fractions. ► Pyrolysis and gasification of the solid fraction is an energy alternative.This work aims at the characterization and the biorefinery of Hesperaloe funifera by means of the use of its three main components: separating hemicellulose by hydrothermal treatments; cellulose pulp by various pulping processes (soda, soda–anthraquinone, ethanolamine, ethyleneglycol, diethanolamine and diethyleneglycol); and exploitation of pulping liquor, rich in lignin, by pyrolysis and gasification processes.The contents in lignin, α-cellulose, holocellulose, hemicellulose, ethanol–benzene extractives, hot water solubles, 1% NaOH solubles and ash of H. funifera were found to be 7.3%, 40.9%, 76.5%, 35.6%, 4.0%, 13.5%, 29.5% and 5.9%, respectively. The mean fibre length, 4.19mm, exceeds those for some non-wood materials.By using sulfuric acid in the hydrothermal treatment (170°C, 0, 20min after reaching operating temperature, 8 liquid/solid ratio, and 0.3% sulfuric acid), gives a liquid fraction containing 4.62% of glucose, 10.56% of xylose, 1.28% of arabinose, and a solid fraction with a solid yield of 57.0%.The best pulp of Hesperaloe pulp was obtained by cooking with 10% NaOH and 1% anthraquinone at 155°C for 30min, exhibited good values of yield (48.3%), viscosity (737mL/g), Kappa number (15.2), tensile index (83.6Nm/g), stretch (3.8%), burst index (7.34kN/g) and tear index (3.20mNm²/g). Moreover, the soda–anthraquinone pulps of raw material are better than the pulps from solid fraction of hydrothermal treatments.Finally, acidification (pH 6) of soda–anthraquinone pulping liquor was carried out to separate lignin-rich solids, by which pyrolysis gave a gas containing 1.13% H₂, 31.79% CO and 1.86% CH₄ by weight. Gasification of the same sample provided a gas containing 0.18% H₂, 24.50% CO and 17.75% CH₄.

Keywords: Hesperaloe funifera; Biorefinery; Pulp; Paper; Hemicellulose; Lignin; Pyrolysis gas; Fuel gas

Genetic expression of bacterial merC fused with plant SNARE in Saccharomyces cerevisiae increased mercury accumulation by Masako Kiyono; Yuka Sone; Kiyomi Miyahara; Yumiko Oka; Masumi Nakamura; Ryosuke Nakamura; Masa H. Sato; Hidemitsu Pan-Hou; Kou Sakabe; Ken-ichiro Inoue (pp. 137-141).

► We demonstrate Arabidopsis SNAREs can be used as organelle-targeting markers to direct bacterial heavy metal transporter, MerC, to specific membranes in the model eukaryotic organism, yeast. ► We show yeast genetically engineered to express MerC in its plasma membrane accumulated significantly more mercury than the control cells. ► The use of plant SNARE to direct metal transporter MerC to particular membrane may open new avenues for designing and improving biomass to be more suitable for use in the environmental remediation of toxic metals.MerC encoded by merC in the Tn 21 mer operon, is a heavy metal transporter that may be a useful molecular tool for the bioremediation of cadmium and mercury. To regulate subcellular localization, the Arabidopsis SNARE proteins SYP111 and AtVAM3 were fused to the C-terminal end of MerC. In Saccharomyces cerevisiae, green fluorescent protein (GFP)-MerC-SYP111 fusion proteins located primarily to the plasma membrane, whereas GFP-MerC-AtVAM3 was detected in the vacuolar membranes. These results suggest that SYP111 and AtVAM3 direct MerC fusion proteins to the plasma and vacuolar membranes, respectively. Yeast cells expressing MerC-SYP111 accumulated significantly more mercury than control cells or cells expressing MerC-AtVAM3. Thus, it is possible that localizing MerC to the plasma membrane of plants may represent a similarly promising strategy for improving phytoaccumulation and sequestration of mercury at polluted sites.

Keywords: Bioremediation; Bacterial heavy metal transport; MerC; Plant SNARE; Mercury

Preparation of carriers based on magnetic nanoparticles grafted polymer and immobilization for lipase by Xiao Liu; Lin Lei; Yanfeng Li; Hao Zhu; Yanjun Cui; Huaiyuan Hu (pp. 142-149).

► Super paramagnetic nanoparticles were prepared by a surface-initiated radical polymerization. ► This nanoparticle was provided with positive charge and active epoxy groups simultaneously. ► The candida rugosa lipase (CRL) was immobilized via electrostatic adsorption and covalent binding. ► The lipase loading amount was 68.3±0.5mg CRL/g support and the activity recovery reached to 60.4% (±1.6%).The super paramagnetic nanoparticles Fe₃O₄/(2-hydroxyethty methacrylate-co-dimethyl dially ammonium chloride) copolymer, i.e. Fe₃O₄/P(HEMA–DMDAAC), with positive charge and active epoxy groups simultaneously, were synthesized by a surface-initiated radical polymerization and activation reaction with epoxy chloropropane. The production particles were characterized by Fourier transform infrared spectroscopy (FT-IR), thermogravimetric analysis (TG), transmission electron microscopy (TEM), and vibrating sample magnetometry (VSM). It was verified that the magnetic microspheres held small diameters of 80–100nm and displayed super paramagnetic property with saturation magnetization of 38.9emu/g. The candida rugosa lipase (CRL), meanwhile, was immobilized onto the magnetic microspheres via electrostatic adsorption and covalent binding, the loading amount of lipase was 68.3±0.5mg CRL/g support and the activity recovery of the obtained immobilized lipase reached to 60.4% (±1.6%).

Keywords: Super paramagnetism; Electrostatic adsorption; Covalent binding; Immobilized CRL; Enzyme technology; Reusability

Integrated bioprocess for high-efficiency production of succinic acid in an expanded-bed adsorption system by Qiang Li; Dan Wang; Guangyu Hu; Jianmin Xing; Zhiguo Su (pp. 150-157).

► Expanded-bed adsorption was integrated with in the fermentation process viable for in situ extracellular product removal. ► The coupled fermentation–separation system could overcome product inhibition. ► The final titer of 145.2gl⁻¹ succinic acid was obtained after 126h fermentation.An integrated fermentation process for the production of succinic acid by Actinobacillus succinogenes at high titer, yield and productivity was developed by applying in situ product removal (ISPR) strategy. The ISPR process was conducted by a coupled expanded-bed adsorption (EBA) system. The novel approach during the product inhibitory period of microbial fermentation enhanced the cell growth of A. succinogenes from 48h to 126h, and significantly increased succinic acid production up to the final titer of 145.2gl⁻¹ with an average yield of 0.52gg⁻¹, and productivity of 1.3gl⁻¹h⁻¹. The maximum yield and productivity reached 0.76gg⁻¹ and 2.58gl⁻¹h⁻¹, respectively after the first ISPR operation cycle. ISPR process with EBA leads to the production of succinct acid via fermentation economically feasible.

Keywords: Succinic acid; Expanded-bed adsorption; Bioreactors; Process integration; Product inhibition; Bioconversion

The enhancement of pretreatment and enzymatic hydrolysis of corn stover by FeSO₄ pretreatment by Jing Zhao; Hongman Zhang; Rongping Zheng; Zengxiang Lin; He Huang (pp. 158-164).

► FeSO₄ can improve the removal of hemicellulose and the activity of cellulose. ► This study discussed the role of Fe²⁺ on the hydrolysis mechanism of cellulose. ► FeSO₄ pretreatment is appropriate acidity for enzymatic hydrolysis processing.FeSO₄ was investigated as a catalyst for the pretreatment of corn stover. It was observed that FeSO₄ significantly increased the hemicellulose degradation in aqueous solutions heated between 140 and 200°C with high xylose recovery and low cellulose removal. Optimum pretreatment conditions were 0.1mol/L FeSO₄ at 180°C for 20min. The cellulose conversion was 36.4% higher than that pretreated by hot water under the same enzymatic hydrolysis conditions. The hydrolysis efficiencies of treated materials were characterized by reaction rate constants, Km and the activity of cellulase. The results showed that FeSO₄ pretreatment apparently removed the hemicellulose, promoted the activity of cellulase and enhanced the accessibility of cellulase to cellulose, thus significantly increased the enzymatic digestibility. FeSO₄ pretreatment could disrupt almost all ester linkages between cellulose and hemicellulose. The results obtained indicated that FeSO₄ pretreatment as a special pretreatment method could enhance enzymatic hydrolysis of lignocellulosic biomass by destructing chemical composition and altering structural features.

Keywords: Key words; Hemicellulose; Cellulose; Ferrous sulfate; Pretreatment; Enzymatic hydrolysis; Corn stover

High cell-density cultivation in batch mode for plasmid DNA production by a metabolically engineered E. coli strain with minimized overflow metabolism by René Soto; Luis Caspeta; Blanca Barrón; Guillermo Gosset; Octavio T. Ramírez; Alvaro R. Lara (pp. 165-171).

• A PTS⁻ GalP⁺ E. coli strain was tested for pDNA production and compared to its parental strain. • Both strains were tested in batch mode using 100g/L of initial glucose concentration. • The modified strain produced more than the double of plasmid DNA than its parent. • The modified strain accumulated 2g/L of acetate, while the parental strain produced 5.3g/L.Progress on plasmid-based (pDNA) vaccines requires simpler and efficient cultivation techniques for their production. A prevalent problem in the cultivation of Escherichia coli (the main host for pDNA vaccines production) is the aerobic production of acetate. In this work, a metabolically engineered Escherichia coli strain with strongly reduced acetate formation was tested for the production of a plasmid vaccine at high cell-densities. The wild type (W3110) and engineered (VH33) strains were cultivated in batch mode using 100g/L of initial glucose concentration. This elevated amount of glucose allowed attaining high cell-densities of strain VH33 without external substrate feeding, simplifying the cultivation process. While W3110 produced 17mg/L of pDNA and 5.3g/L of acetate, VH33 reached 40mg/L of pDNA and only 2g/L of acetate. While the plasmid supercoiling degree progressively decreased in W3110 cultivations, it remained nearly constant for VH33. These results show the successful application of cell engineering concepts for improving DNA vaccine production processes.

Keywords: pDNA vaccines; Acetate; Metabolic engineering; High cell-density; Batch; Fed-batch

Enzymatic synthesis of biodiesel from fatty acids. Kinetics of the reaction measured by fluorescent response of Nile Red by Sergey N. Fedosov; Xuebing Xu (pp. 172-183).

• Enzymatic conversion of fatty acids to biodiesel was characterized. • The rate constants were used to model behavior of the system. • It is possible to reduce fatty acids in biodiesel below the specification level. • Different amounts of fatty acids require 1–3 steps of conversion. • The scheme is a part of a general model for enzymatic production of biodiesel.Production of biodiesel (B) from free fatty acids (F) was investigated. Different amounts of F dissolved in B were esterified by methanol or ethanol with help of lipase Novozym 435. The kinetic model included (i) steady-state scheme; (ii) reversible inhibition of the enzyme by alcohols; and (iii) aggregation of water W+W↔WW. The aggregated form WW imitated large water droplets with low chemical activity. Forward and backward reactions were recorded using the calibrated fluorescent signal from Nile Red. The relevant rate constants were calculated and used in computer simulations. The model demonstrated that content of F could be decreased below the specification level of 0.25% by means of the enzymatic conversion exclusively. It was found that the reaction could be accomplished in one step starting from content of F≤1%, water=100ppm and MeOH=6% or EtOH=13%. The higher levels of F (≥4%) would require three cycles where MeOH (≥4%) or EtOH (≥7%) are added at the beginning of each step, and water is dried to 100ppm between the steps. The designed kinetic scheme is a part of the general biodiesel reaction model, which is currently under construction.

Keywords: Abbreviations; B; biodiesel; B; ₁; methanol based B; B; ₂; ethanol based B; CALB; Candida antarctica lipase B; C; alcohol; C; ₁; MeOH, methanol; C; ₂; EtOH, ethanol; E; enzyme; EX; enzyme with attached fatty acid; FAEE; fatty acid ethyl ester; FAME; fatty acid methyl ester; F; fatty acid (free); m/m; mass per mass; m/v; mass per volume; FR; fluorescent ratio; OA; oleic acid; T/D/M; tri-/di-/mono-glyceride; TLC-FID; thin layer chromatography assisted by flame ionization detector; v/v; volume per volume; W; waterBiodiesel; Fatty acids; Enzyme biocatalysis; Lipase; Kinetic parameters; Modeling

Decolorization of a tannery dye: From fungal screening to bioreactor application by R. Baccar; P. Blánquez; J. Bouzid; M. Feki; H. Attiya; M. Sarrà (pp. 184-189).

► Trametes versicolor displayed the greatest decolorization ability of a tannery dye. ► The fungus continue to remove the dye during the three batches. ► The overall color removal by T. versicolor is due to two mechanisms: adsorption and biodegradation. ► Laccase is involved in the decolorization process but mediator is required. ► The appreciable color removal observed by dead cells suggested their use as an effective biosorbent.In the present work, the potential of three white-rot fungi (WRF) ( Trametes versicolor, Ganoderma lucidum and Irpex lacteus) to decolorize the commercial tannery dye – Black Dycem – was investigated. The decolorization ability of the three strains was studied in primary and secondary screenings. The results indicated that T. versicolor displayed the greatest decolorization ability, both in terms of extent and rapidity. To assess the potential of T. versicolor, decolorization tests were performed in single and repeated batches in an air-pulsed bioreactor with biomass reuse. Although low enzyme activity was detected during the repeated batches, the decolorization capability of the fungus did not decrease, and T. versicolor was able to remove 86–89% of the dye. Further experiments were conducted in order to elucidate the enzymatic activities involved in the dye biodegradation process. The results proved that the biodegradation mechanism plays a noticeable role in the decolorization process of the dye, in addition to adsorption phenomenon occurring on the fungal surface. Moreover, it was shown that laccase is involved in the decolorization process, although a mediator is required. Killed fungus presented an appreciable color removal even in repeated batches, suggesting that dead cells are an effective biosorbent.

Keywords: White-rot fungi; Tannery dye; Sequence batch reactor; Enzymatic system

Reversible immobilization of rhamnulose-1-phosphate aldolase for biocatalysis: Enzyme loading optimization and aldol addition kinetic modeling by Inés Ardao; Gregorio Alvaro; M. Dolors Benaiges (pp. 190-197).

Display Omitted• A metal-chelate affinity cobalt support is used for enzyme immobilization. • Immobilized rhamnulose-1-P aldolase exhibits high retained activity and stability. • Optimal enzyme loading was determined for the natural reaction and a synthetic one. • Kinetic modeling of the synthetic aldol addition and secondary reaction was performed. • The model was validated for both soluble and immobilized RhuA catalysis.Reusability of the usually expensive immobilization supports by means of reversible immobilization techniques helps reduce the overall cost of the biocatalytic process. One of these techniques, the reversible binding of the histidine residues of proteins with the metal chelated in affinity supports, has been extensively exploited in purification of His-tagged proteins in one-step. The use of these supports for immobilization of His-tagged recombinant enzymes allows performing simultaneously the enzyme purification and immobilization, thus further reducing the number of process steps, enzyme losses and costs. In this work, His-tagged rhamnulose-1-phosphate aldolase (RhuA) was immobilized onto iminodiacetic acid-functionalized agarose charged with cobalt (Co-IDA) for the catalysis of aldol addition reactions of industrial interest. Enzyme loading was optimized to maximize the immobilized activity avoiding diffusion limitations for both the RhuA natural reaction and the synthetic aldol addition between dihydroxyacetone phosphate (DHAP) and (S)-Cbz-alaninal (10AU/mL of support and 25AU/mL of support, respectively). RhuA:Co-IDA biocatalysts with high retained activity (92%) and enhanced stability (37 days of half life time) were obtained. Kinetic modeling of the synthetic aldol addition between DHAP and (S)-Cbz-alaninal and the unwanted secondary reaction catalyzed by soluble RhuA was carried out. RhuA:Co-IDA catalysis of the same reactions was found to follow the same kinetic model.

Keywords: Abbreviations; RhuA; rhamnulose-1-phosphate aldolase; FucA; fuculose-1-phosphate aldolase; Co-IDA; iminodiacetic acid-functionalized agarose charged with cobalt; DHAP; dihydroxyacetone phosphateEnzyme biocatalysis; Immobilized enzymes; Affinity; Modeling; Rhamnulose-1-phosphate aldolase; Aldol addition biocatalysis

Simulation of equilibrium system and release behaviors of both oxytetracycline and copper on aerobic granules in a sequencing batch reactor by Rui-Hong Song; Xin-Hua Wang; Guo-Hui Zhang; Lei Zhao; Xue-Fei Sun; Shu-Guang Wang; Bao-Yu Gao (pp. 198-204).

► The saturated and equilibrated system was simulated by adsorption test. ► The bonding amounts of oxytetracycline (OTC) and Cu(II) increase in the coexisting system. ► Release of OTC and Cu(II) was conducted to study the influence of concentration, pH, ionic strength, and temperature. ► Relying on concentration, release of OTC and Cu(II) increased with decrease in the coexisting ion, respectively.Accumulation of hard-to-degrade or nondegradable chemicals may be created in a long-time granule sludge treatment process. The excess accumulation of target chemicals on aerobic granules (AG) might induce inevitable release in sequencing batch reactors. In order to investigate the combined extent on AG with oxytetracycline (OTC) and copper as well as the better understanding of the environmental fate of chemicals, the saturated and equilibrated system was simulated by an adsorption test, and then a release experiment was conducted to study the influence of concentration, pH, ionic strength, and temperature. The results showed that isotherms of OTC and Cu(II) with high correlation coefficients ( R²=0.991–0.996) and ( R²=0.992–0.996) fit well with the Redlich–Peterson model (RPM). The bonding amounts of both OTC and Cu(II) increase in the coexisting system, indicating that the OTC–Cu(II) complex had stronger binary ability with AG. Relying on different initial concentrations, release of OTC from AG was 0.799–10.758% with decrease in coexisting Cu(II) ion concentration from 40mg/L to 0mg/L and 0.08–6.21% for Cu(II) with decrease in coexisting OTC concentration from 10mg/L to 0mg/L, indicating that decrease in coexisting ion concentration has adverse effect on their combined ability with AG.

Keywords: Aerobic granules; Adsorption; Release; Oxytetracycline (OTC); Copper; Equilibrium system

A new purification process for goose immunoglobulin IgY(ΔFc) with hydrophobic charge-induction chromatography by Hong-Fei Tong; Dong-Qiang Lin; Yue Pan; Shan-Jing Yao (pp. 205-211).

Display Omitted► Novel bioseparation technology, hydrophobic charge-induction chromatography, was applied to separate IgY(ΔFc). ► The adsorption isotherms of IgY(ΔFc) onto new HCIC adsorbent (MMI-Bestarose) were investigated. ► The HCIC separation conditions were optimized and high separation efficiency was obtained. ► New process with HCIC can be a promising technology for the cost-effective separation of IgY(ΔFc) from plasma.Hydrophobic charge-induction chromatography (HCIC) is a novel bioseparation technique, especially for antibody purification. In this study, HCIC was used for the purification of goose immunoglobulin IgY(ΔFc). IgY(ΔFc) is a kind of unique immunoglobulin existing in waterfowl, which natively lacks C_H3 and C_H4 domains of heavy chains and has some particular therapeutic applications. A new HCIC ligand, 2-mercapto-1-methyl-imidazole (MMI), was coupled to the divinyl-sulfone-activated agarose matrix to prepare the HCIC gel. The adsorption isotherms of IgY(ΔFc) were investigated at different pHs. The saturated capacity of IgY(ΔFc) at pH 5.0 could reach 187.5mg/ml gel, and the pH-dependent adsorption behaviors were found. After the goose plasma was pre-treated with caprylic acid to precipitate some impurities, the supernatant could be directly loaded onto the HCIC column for IgY(ΔFc) separation. The operation conditions were optimized, including the loading pH, elution pH and the loading volume. High separation efficiency was obtained with final purity of 98.6% and the yield of 85.0%. The results indicate that the new process with HCIC can be a promising technology for the cost-effective separation of IgY(ΔFc) from plasma.

Keywords: Hydrophobic charge-induction chromatography; Immunoglobulin; Goose plasma; Purification

Development of a kinetic model for anaerobic dry digestion processes: Focus on acetate degradation and moisture content by J. Bollon; R. Le-hyaric; H. Benbelkacem; P. Buffiere (pp. 212-218).

► An anaerobic digestion model has been established for the digestion of organic waste in dry mode (moisture content below 85%). ► The model was validated for acetate uptake and methane production. ► The moisture content is the key parameter for the biological kinetics: it affects specifically the half-saturation constant K_s. ► The maximum uptake rate is affected only for the lowest value of the moisture content (near the unsaturated water conditions).The objective of this work is to develop a kinetic model to specifically assess the degradation of the organic fraction of municipal solid waste (OFMSW) in dry anaerobic digestion processes. The model description was designed to include several aspects of dry digestion such as the presence of particulate matter and high solid content.The global structure of the kinetic model proposed here is based on the ADM1 (Anaerobic Digestion Model n° 1) which has been modified in order to include specific characteristics of dry media.Batch experimental data of methane production with acetate as organic substrate under dry mesophilic conditions have been obtained and used to calibrate the kinetic parameters associated to acetate uptake.The model was shown to simulate well batch degradation of acetate and methane production in dry mode. The biochemical parameters associated to acetate degradation kinetics were the maximal acetate uptake rate ( k_m X_ac) and the half-saturation constantKSac. After calibration, the value of k_m X_ac was 440mg_CODkg⁻¹d⁻¹ andKSac was 290mg_CODkg⁻¹ for a moisture content (MC) of 82%. The value of K_s was found to be higher in dry digestion than the recommended value in wet digestion. It increased from 290mg_CODkg⁻¹ at 82% MC up to 2000mg_CODkg⁻¹ at 65% MC. This result was attributed to diffusion limitations due to the singular “pasty” aspect of the digesting medium.

Keywords: Modelling; Dry anaerobic digestion; Kinetic parameter; Acetic acid; Moisture content; Biokinetics

Baker's yeast mediated biotransformation of geraniol into citronellol using a continuous-closed-gas-loop bioreactor (CCGLB) system by Aimi Aishah Arifin; Mashitah Mat Don; Mohamad Hekarl Uzir (pp. 219-224).

► The CCGLB system gives an advantage of in situ product removal. ► Gas phase biotransformation promotes better mass transfer between gas and liquid phases. ► High substrate flow rate gives a positive effect to the rate of reaction. ► High agitation leads to an excessive foaming inside the CCGLB.The kinetics of the biotransformation of geraniol into citronellol by the resting cells of Saccharomyces cerevisiae (baker's yeast type-II) was investigated in a continuous-closed-gas-loop bioreactor (CCGLB). Geraniol, which has high vapour pressure, high volatility and low solubility in aqueous medium, is highly suitable to be used in this system. Various operating parameters which affect the reduction reaction were investigated. The optimal conditions were; pH 7, agitation rate of 350rpm, substrate flow rate of 8L/min and glucose concentration of 50g/L. The gas loop led to a maximum citronellol concentration of 2.38g/L with the specific activity of 7.90U/g_cell, which were apparently higher than that of the values obtained using conventional systems. The CCGLB system also has the advantage of in situ product removal and can be readily utilized in large-scale production.

Keywords: Yeast; Biotransformations; In-situ product removal; Continuous-closed-gas-loop bioreactor (CCGLB); Production kinetics; Heterogeneous biocatalysis

Comparative study of natural and synthetic phenolic compounds as efficient laccase mediators for the transformation of cationic dye by Ismat Bibi; Haq Nawaz Bhatti; Muhammad Asgher (pp. 225-231).

• We used commercial laccase for the biotransformation of malachite green. • The effect of natural and synthetic redox mediators was investigated. • The addition of extract of groundnut shell enhanced the decolorization of the dye. • Phenolic compounds could be used as potential redox mediators for decolorization of dyes.Laccase mediator systems are important bioremediation agents as the rate of reactions could be enhanced in the presence of the mediators. In the present study commercial laccase was used for the biotransformation of malachite green (MG), a cationic dye in the presence of phenolic compounds of groundnut shells and lignin-related synthetic redox mediators. The addition of ethanolic phenolic extract of groundnut shell enhanced the decolorization of MG from 38.73 to 87.32%. All the selected natural phenolic compounds enhanced the decolorization of MG like synthetic mediator 1-hydroxybenzotriazole. Characterization of MG transformation products by UV–vis and HPLC analyses revealed that N-demethylation was the key mechanism of decolorization of MG by laccase alone as well as in the presence some of redox mediators. But in case of MG decolorization with laccase mediated system by groundnut shell extract and other natural compounds, leuco-malachite green was the major transformed product. Growth inhibition test based on mycelial growth inhibition of white rot fungus Phanerochaete chrysosporium (ATCC 1556) revealed that treatment with laccase plus natural and synthetic mediators effectively reduced the growth inhibitory levels of MG than that of control. Thus, the study revealed that the natural phenolic compounds could be used as potential redox mediators for enhanced laccase-mediated decolorization of malachite-green dye.

Keywords: Malachite green; Biotransformation; Laccase; Redox mediators; Decolorization

Efficient Agaricus bisporus tyrosinase immobilization on cellulose-based carriers by Karolina Labus; Anna Turek; Jolanta Liesiene; Jolanta Bryjak (pp. 232-240).

► We investigated tyrosinase immobilization on synthesized cellulose-based carriers. ► Covalently bound enzyme was 22 times more stable than native tyrosinase. ► Operational stability was tested with 6 monophenolic and 3 diphenolic substrates. ► Repeated batch tests confirmed suicide inactivation caused by diphenolic substrates.A systematic study of the covalent immobilization of Agaricus bisporus tyrosinase on man-tailored cellulose-based carriers with different pore structure and functionalization showed that DEAE-Granocel with the largest mean pore diameter and activated with divinyl sulphone can offer highly active preparations with a monophenolase (L-Tyrosine) and diphenolase (L-DOPA) activity immobilization efficiency of over 34% and 24%, respectively. A thermal stability test carried out at 55°C indicated monophasic activity depletion with the inactivation rate of immobilized enzyme of 0.021min⁻¹ and 22 times higher (0.454min⁻¹) for the native enzyme. The susceptibility of native and immobilized tyrosinase to suicide inactivation in the presence of monophenolic (L-Tyrosine, phenol, p-chlorophenol, p-cresol, p-aminophenol, o-aminophenol) and diphenolic (L-DOPA, catechol, p- tert-butylcatechol) substrates was tested in repeated batch tests and was found to be critical for obtaining stable biocatalysts in reactions with diphenolic substrates.

Keywords: Tyrosinase; Immobilization; Cellulose carrier; Stability; Batch reactor; Operational stability

Surface activity of heat–alkaline treated excess sludge by Dan Zhao; Kazuhiko Miyanaga; Yasunori Tanji (pp. 241-246).

• We obtained surface activity from sludge treated by heat and alkaline. • Alkaline treatment contributed more to the surface activity. • The specific surface area of n-hexadecane droplets were increased 2.1 folds after emulsified by the treated sludge.Biosurfactants are produced by fermenting pure strains but not treating excess sludge. For reducing the excess sludge, heat and alkaline treatment was reported as a feasible process. To develop a biosurfactant from treated sludge, the surface activity of heat–alkaline treated sludge was investigated in this study. Sludge, Escherichia coli, and extracted E. coli lipids were treated under pH 7, pH 11 and pH 13 conditions that were then combined with or without heat treatment. When the sludge was treated at pH 13 and then heat-treated for 18h, the surface tension of the supernatant of the treated sludge was reduced by 26.6% from 72.5mN/m to 53.0mN/m. As a whole, the alkaline treatment more effectively reduced surface tension than heat treatment because the surface tension of the supernatant was lower than 55.4mN/m after the sludge was treated at pH 13. N-hexadecane droplets were emulsified by the supernatant of treated sludge, and then collected using a designed sampling device. The droplet size distribution and cumulative frequency curve of n-hexadecane droplets showed that over half of the droplets were smaller than 80μm after they were emulsified by supernatant from alkaline-treated sludge at pH 13 treatment condition no matter with or without heat treatment. These findings indicate that heat–alkaline treated sludge has both surface activity and emulsifying capability.

Keywords: Activated sludge; Heat–alkaline treatment; Emulsifying capability; Surface activity; Surface tension reduction

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Biochemical Engineering Journal (v.56, #3)

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Biochemical Engineering Journal (v.56, #3)