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Biochemical Engineering Journal (v.57, #)

Editorial Board (pp. co2).

BEJ Keywords (pp. iv).

Sensitive label-free electrochemical immunoassay based on a redox matrix of gold nanoparticles/Azure І/multi-wall carbon nanotubes composite by Ai-Li Sun; Gai-Rong Chen; Qing-Lin Sheng; Jian-Bin Zheng (pp. 1-6).

► In this work we construct the GNPs/Azure І/MWCNT–Nf matrix for the CEA antibody immobilization. ► The GNPs/Azure І/MWCNT–Nf matrix provides porous nanostructure, good electrochemical redox reversibility and biocompatibility. ► Based on the direct immunoassay format, the label-free immunosenors achieve a broader linear range, high sensitivity and good stability.A sensitive label-free electrochemical immunosensing platform was designed by a redox matrix of gold nanoparticles (GNPs), Azure І and multi-wall carbon nanotubes (MWCNT) self-assemblying nanocomposite. To construct the immunosensor, MWCNT was first dispersed in Nafion (Nf) to obtain a homogeneous solution and then it was dropped on the surface of the gold electrode (Au). Then the positively-charged redox molecule, Azure І, was entrapped into MWCNT–Nf film to form a redox nanostructural membrane. Next, the negatively charged gold nanoparticles (GNPs) were assembled to the interface through the electrostatic force. Finally, carcinoembryonic antibody molecules could be absorbed into the GNPs/Azure І/MWCNT–Nf immobilization matrix. Using carcinoembryonic antigen (CEA) as a model protein, the electrochemical immunosensor exhibited good stability and reproducibility, as well as good selectivity and storage stability. This strategy presented a promising platform for sensitive and facile monitoring of CEA.

Keywords: Label-free immunosensor; Multi-wall carbon nanotubes; Gold nanoparticles; Azure I; Carcinoembryonic antigen

Cultivation of aerobic granular sludge with a mixed wastewater rich in toxic organics by Li Liu; Guo-Ping Sheng; Wen-Wei Li; Zhong-Hua Tong; Raymond J. Zeng; Jun-Xin Liu; Jie Xie; Shu-Chuan Peng; Han-Qing Yu (pp. 7-12).

► Aerobic granules were cultivated in an SBR fed with a mixed industrial wastewater rich in toxic organics. ► The granules exhibited a “core” structure and were beneficial to reactor operation. ► Various approaches were used to characterize the structure of the granules. ► The granule-based reactor had a high removal efficiency and ability to resist wastewater toxicity. ► The results are useful for cultivation of aerobic granules for treatment of toxic wastewaters.Aerobic granular sludge was successfully cultivated in a sequencing batch reactor fed with a mixture of chemical industrial wastewater rich in toxic organics and the effluent from an anaerobic acidogenic reactor. After 30-day operation, stable granules with a size of 1.0–3.0mm were obtained. These granules appeared to have rougher surface than those cultivated with the carbohydrate- or acetate-rich wastewaters. There exhibited a “core” in the internal structure of the granules, which might benefit microorganisms to survive and resist the harsh environment. The formation of granules significantly improved the ability of sludge to withstand the toxic substances. The chemical oxygen demand removal efficiency of the granule-based reactor could reach around 80%, while its ammonia and total nitrogen removal efficiencies reached 90% and 40%, respectively. The aerobic-granule-based reactor showed an ability to resist the wastewater toxicity.

Keywords: Aerobic granule; Chemical industrial wastewater; Sequencing batch reactor (SBR); Sludge; Toxic substances

Characterization of a laccase gene from the white-rot fungi Trametes sp. 5930 isolated from Shennongjia Nature Reserve in China and studying on the capability of decolorization of different synthetic dyes by Yang Yang; Fuying Ma; Hongbo Yu; Fangfang Fan; Xia Wan; Xiaoyu Zhang; Mulan Jiang (pp. 13-22).

► Trametes sp. 5930 was isolated from the virgin forest of Shennongjia Nature Reserve in China. ► The laccase gene lac5930-1 and its corresponding full-length cDNA were cloned and characterized. ► lac5930-1 gene was successfully expressed in the heterologous Pichia pastoris system. ► The capability of decolorizing different dyes was positively related to the laccase activity.Laccase belongs to a family of multi-copper oxidases which is especially useful for biotechnological and industrial applications. A laccase-producing white-rot fungi strain designated as Trametes sp. 5930 was nearly isolated from Shennongjia Nature Reserve in China. Trametes sp. 5930 had the high yield of laccase and was capable of decolorizing different dyes efficiently. Laccase played a very important role in the decolorization of different dyes by this fungus. The laccase gene lac5930-1 and its corresponding full-length cDNA were then cloned and characterized from Trametes sp. 5930. The 1563bp full-length cDNA of lac5930-1 encoded a mature laccase protein consisting of 499 amino acids preceded by a signal peptide of 21 amino acids. lac5930-1 gene was successfully expressed in Pichia pastoris, which verified the function of lac5930-1 encoding active laccase by means of gene expression. The recombinant laccase produced by the yeast transformant in which lac5930-1 was efficiently expressed, conferred the ability to decolorize different dyes. The capability of decolorizing different dyes was positively related to the laccase activity, which provided strong evidence for the important function of laccase used in decolorizing industrial dyes.

Keywords: White-rot fungi; Laccase gene; Decolorization of dyes; Enzymes; DNA; Yeast

Thermal analysis for differentiating between oleaginous and non-oleaginous microorganisms by Bongmun Kang; Kohsuke Honda; Kenji Okano; Tsunehiro Aki; Takeshi Omasa; Hisao Ohtake (pp. 23-29).

► We apply thermal analysis for screening oleaginous microorganisms. ► TG/DTA analysis is performed using a two-step linear temperature program. ► The exothermic heat is related to the total lipid content of microorganisms. ► Heat evolved between 280 and 360°C is useful for screening oleaginous species.The potential of thermal analysis for differentiating between oleaginous and non-oleaginous microorganisms was investigated using thermogravimetry (TG) and differential thermal analysis (DTA). The model oleaginous microorganisms used in the present study were the fungi, Mortierella alpina IFO32281 and Mortierella alliacea YN-15, the unicellular alga, Aurantiochytrium sp. CB 15-5, and the yeast, Rhodosporidium toruloides DMKU3-TK 16. Escherichia coli JM109, Rhodococcus opacus B-4, and Saccharomyces cerevisiae were used as the control non-oleaginous microorganisms. In simultaneous TG and DTA, the furnace temperature was linearly increased from 30 to 280°C, decreased to 30°C, linearly increased from 30 to 360°C, and then isothermally held at 360°C for 30min. This two-step linear temperature program was effective in resolving overlapping exothermic peaks in the DTA curves in the temperature range from 280 to 360°C. Heat evolved from a microbial sample was estimated from the area under the exothermic peak between 280 and 360°C using indium as a standard material. There was a linear relationship between the exothermic heat and total lipid content of the tested microorganisms. Exothermic heat per dry sample mass (kJ/g) in the temperature range from 280 to 360°C is a promising measure for differentiating between oleaginous and non-oleaginous microorganisms.

Keywords: Differential thermal analysis; Oil production; Oleaginous and non-oleaginous microorganisms; Thermogravimetry

The effect of glycerol mixed substrate on the heterologous production of a Rhizopus oryzae lipase in Pichia pastoris system by Carolina Arnau; Carles Casas; Francisco Valero (pp. 30-37).

► Mixed substrates are good strategy to increase protein production in Pichia pastoris. ► Methanol set-point and μ are the key parameters in the bioprocess production. ► Glycerol represses PAOX1 at μ highest than 0.04h⁻¹ in Mut^s phenotype. ► Proteolytic activity was detected only when glycerol was used as co-substrate. ► Sorbitol was better co-substrate than glycerol for ROL production.A recombinant Rhizopus oryzae lipase producing Mut^s Pichia pastoris strain was used as a model organism to study the effect of mixed substrates (glycerol and methanol) on the specific product productivity. Different fed-batch cultivations were performed under three constant specific growth rates (0.02, 0.05 and 0.1h⁻¹), maintaining a constant methanol concentration of 2gl⁻¹.At the lowest μ tested (0.02h⁻¹), the specific productivity was 1.23 and 1.61 fold higher and the specific methanol consumption rate ( q_sMeOH) was 3 and 3.5 fold higher than values obtained when μ was 0.05 and 0.1h⁻¹, respectively. This implies a relation between the q_sMeOH and the specific productivity, yielding higher specific productivities whenever the consumption of methanol is higher. Although glycerol was maintained under limiting conditions in all μ tested, when the relation between the μ_Gly and μ_MeOH was larger than 4, an important decrease on the maximal activity value was observed.Finally, a comparison under the same conditions using glycerol or sorbitol as co-substrates was also performed, obtaining better specific productivity when sorbitol was used. In addition, protease activity was detected when glycerol was used as co-substrate.

Keywords: Pichia pastoris; Mixed substrate; Fed-Batch culture; Glycerol; Rhizopus oryzae; lipase; Bioprocess monitoring

Influence of surface topography on biofilm development: Experiment and modeling by Muatasem Alnnasouri; Cécile Lemaitre; Caroline Gentric; Christophe Dagot; Marie-Noëlle Pons (pp. 38-45).

► Biofilm development was monitored successfully on rough surfaces by image analysis. ► Biofilm sloughs more easily from flat surfaces than from rough ones. ► Wall stress across the rotating rough biofilm substratum was modeled by CFD.The effect of surface topography on the long-term development (≈10 weeks) of biofilms has been investigated using a monitoring technique based on images produced by a flat-bed scanner and initially developed for flat surfaces. The biofilm response to rotation speed changes in lab-scale rotating biological contactors (RBCs) has been studied. Two RBCs, each containing five discs (two with flat surfaces and three with rough surfaces) were run initially at two different rotation speeds: 4rpm for reactor I and 40rpm for reactor II. After 47 days, the rotation speed was increased in reactor I to 40rpm and decreased in reactor II to 4rpm. Prior to the rotation speed change, the biofilm on the flat discs underwent large detachments in both reactors, but the biofilm on rough discs was less extensively damaged. The increase in rotation speed induced large detachments of the biofilm in reactor I on all discs, but the biofilm on the rough discs recovered more effectively with faster regrowth. In reactor II, the decrease in rotation speed favored the development of the biofilm. Wall stress distributions obtained from CFD simulations on flat and rough discs at different rotation speeds were well correlated with experimental observations.

Keywords: Biofilm; CFD; Image analysis; Rotating biological contactor; Rough surface

The investigation of dairy industry wastewater treatment in a biological high performance membrane system by Burhanettin Farizoglu; Suleyman Uzuner (pp. 46-54).

► Dairy industry wastewaters were treated aerobically in a high rate compact system. ► Oxygen transfer rates of the bioreactor were found to be very high (100–285h⁻¹). ► A loading rate of 53kgCODm⁻³d⁻¹ achieved with 97–98% COD removal efficiencies. ► The high MLSS concentrations were retained in the system (up to 38,000mgL⁻¹). ► Cake formation fouling was determined as dominant fouling mechanisms.The dairy industry is generally considered to be the largest source of food processing wastewater in many countries. The highly variable nature of dairy wastewaters in terms of volumes and flowrates and in terms of high organic materials contents such as COD 921–9004mgL⁻¹, BOD 483–6080mgL⁻¹, TN of 8–230mgL⁻¹ and SS of 134–804mgL⁻¹ makes the choice of an effective wastewater treatment regime difficult. A high performance bioreactor, an aerobic jet loop reactor, combined with a ceramic membrane filtration unit, was used to investigate its suitability for the treatment of the dairy processing wastewater. The oxygen transfer rates of the bioreactor were found to be very high (100–285h⁻¹) on the operating conditions. A loading rate of 53kgCODm⁻³d⁻¹ resulted in 97–98% COD removal efficiencies under 3h hydraulic retention time. The high MLSS concentrations could be retained in the system (up to 38,000mgL⁻¹) with the contribution of UF (ultrafiltration) unit. During the filtration of activated sludge, the fluxes decreased with increasing MLSS. Cake formation fouling was determined as dominant fouling mechanisms. The results demonstrate that jet loop membrane bioreactor system was a suitable and effective treatment choice for treating dairy industry wastewater.

Keywords: Jet loop reactor; Dairy wastewater; Industrial wastewater treatment; Ultrafiltration; Membrane bioreactor; Membrane fouling

Fed-batch fermentation of recombinant Citrobacter freundii with expression of a violacein-synthesizing gene cluster for efficient violacein production from glycerol by Cheng Yang; Peixia Jiang; Su Xiao; Chong Zhang; Kai Lou; Xin-Hui Xing (pp. 55-62).

► A violacein productivity of 82.6mgL⁻¹h⁻¹ was obtained by the implementation of a fed-batch fermentation strategy. ► It is the first time that an efficient production of violacein by genetically engineered strains was studied with the highest productivity ever reported. ► Violacein could be produced by recombinant Citrobacter freundii economically and efficiently using glycerol as the sole carbon source and endogenousl-tryptophan as the precursor. ► We found that the production of violacein partly leads to the increase of intracellular pH.The extensive prospects of violacein in the pharmaceutical industry have attracted increasing interest. However, the fermentation levels of violacein are currently inadequate to meet the demands of industrial production. This study was undertaken to develop an efficient process for the production of violacein by recombinant Citrobacter freundii. The effects of dissolved oxygen (DO) and pH on cell growth and violacein production in batch cultures were investigated first. When the DO and pH of the medium were controlled at around 25% and 7.0, respectively, the biomass and concentration of violacein were maximized. Based on the consumption of nutrients in the medium observed during batch culture, a fed-batch fermentation strategy with controlled DO and pH was implemented. By continuously feeding glycerol, NH₄Cl, andl-tryptophan at a constant feeding rate of 16mLh⁻¹, the final concentration of violacein reached 4.13gL⁻¹, which was 4.09-fold higher than the corresponding batch culture, and the maximal dry cell weight (DCW) and average violacein productivity obtained for the fed-batch culture were 3.34g DCWL⁻¹ and 82.6mgL⁻¹h⁻¹, respectively. To date, this is the first report on the efficient production of violacein by genetically engineered strains in a fermentor.

Keywords: Citrobacter freundii; Fed-batch culture; Fermentation; Heterogeneous biocatalysis; Production kinetics; Violacein

Improving Pseudomonas sp. esterase performance by engineering approaches for kinetic resolution of 2-acetoxyphenylacetic acids by Xin Ju; Jiang Pan; Hui-Lei Yu; Chun-Xiu Li; Jian-He Xu (pp. 63-68).

Display Omitted► Repeated resolution of ( R, S)-2-acetoxyphenylacetic acids with a new esterase (PsE) with high enantioselectivity. ► Reaction rate was raised 3.7-folds by using sodium salt of the acidic substrates. ► Spontaneous hydrolysis of substrate is depressed by half in the reformed reaction system. ► Permeabilization and immobilization of whole-cell PsE as provide efficient and stable biocatalysts.The catalytic performance of Pseudomonas sp. ECU1011 esterase (PsE) in the kinetic resolution of ( R, S)-2-acetoxyphenylacetic acid (APA) was significantly improved by substrate modification, biocatalyst permeabilization and immobilization. The reaction system was modified, and the sodium salt of the substrate (APA Na), instead of APA, was hydrolyzed in aqueous phase without buffer. Considering the improved substrate solubility and the decreased biocatalyst inactivation, the reaction rate increased 3.7-folds and the spontaneous hydrolysis of the substrate reduced by 48%. During the cell permeabilization, the hydrolytic activity of the whole-cell biocatalyst was increased by 2.3-fold after 2h of pretreatment with 10% (v/v) toluene. The permeabilized cells were further entrapped in calcium alginate, resulting in 171% activity recovery with a half-life of 123h at 30°C. Using the modified reaction system with high reaction rates and the modified biocatalyst with high activity and stability, this biocatalytic process can be transformed into a practical and environmentally friendly bioprocess for the efficient production of ( S)-mandelic acid and ( S)- o-chloromandelic acid.

Keywords: Biocatalysis; Bioprocess design; Enantioseparation; Pseudomonas; sp. Esterase; Immobilized cells; Mandelic acid; o; -Chloromandelic acid

Comparison of substance supply in static and perfusion cultures based on mass transport phenomena by Shinji Sugiura; Toshiyuki Kanamori (pp. 69-74).

► Mathematical modeling was used to assess substance supply in cell cultures. ► Simulation of static culture conditions resembles actual culture conditions. ► Simulation was used to evaluate previously reported perfusion chambers. ► The mathematical model could be use to design a microfluidic cell culture chip.Mass transport phenomena in cell culture can be formulated by using classical reaction-diffusion equations; however, in practice, it is difficult to solve these equations analytically. Here, we used computer simulation to solve these equations, and compared the time-dependent concentration profile of substances in conventional static culture with that in perfusion culture. The simulated glucose consumption in static culture agreed with that of actual culture conditions used in a general cell culture experiment. The simulation of perfusion culture revealed that the geometry of the chamber and its operating parameters are critical to obtaining a sufficient supply of substances. We also found that the previously reported perfusion chambers are well designed and operate under adequate conditions. Our kinetic model of time-dependent concentration profiles for general substances based on mass transport phenomena could, therefore, be used for the optimal design of a microfluidic perfusion culture chip.

Keywords: Mass transfer; Tissue cell culture; Bioprocess design; Oxygen transfer; Static culture; Microfluidic perfusion culture

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Biochemical Engineering Journal (v.57, #)