Biochemical Engineering Journal (v.73, #C)

BEJ Keywords (II).

Extraction method for increasing antioxidant activity of raw garlic using steam explosion by Yuko Noda; Chikako Asada; Chizuru Sasaki; Saki Hashimoto; Yoshitoshi Nakamura (1-4).
► Steam explosion was used for increasing antioxidant activity of raw garlic. ► 93.7 mg-catechin equiv./g-dry raw garlic was obtained at 45 atm and 5 min of steam explosion. ► Antioxidant activity of steam-exploded raw garlic was higher than that of black garlic.Novel extraction method for increasing the antioxidant activity of raw garlic was proposed using steam explosion. Raw garlic was hydrolyzed by high temperature (183–258 °C) and pressure steam (10–45 atm), and then crushed by the rapid decompression. The antioxidant activity of raw garlic treated by steam explosion was higher than that of black garlic, i.e. aging garlic. The lowest EC50 value, i.e. the highest antioxidant activity, of extract from raw garlic was obtained at a steam pressure of 45 atm for a steaming time of 5 min, but the highest amount of phenolic compounds, i.e. 93.7 mg-catechin equiv./g-dry raw garlic, was obtained at a steam pressure of 30 atm for a steaming time of 5 min.
Keywords: Antioxidant activity; Phenolic compounds; Radical scavenging activity; Raw garlic; Steam explosion;

Fermentations were performed in an external recycle bioreactor using CO2 and d-glucose at feed concentrations of 20 and 40 g L−1. Severe biofilm formation prevented kinetic analysis of suspended cell (‘chemostat’) fermentation, while perlite packing enhanced the volumetric productivity by increasing the amount of immobilised cells. The highest productivity of 6.35 g L−1  h−1 was achieved at a dilution rate of 0.56 h−1. A constant succinic acid yield of 0.69 ± 0.02 g/(g of glucose consumed) was obtained and found to be independent of the dilution rate, transient state and extent of biofilm build-up – approximately 56% of the carbon that formed phosphoenolpyruvate ended up as succinate. Byproduct analysis indicated that pyruvate oxidation proceeded solely via the formate-lyase pathway. Cell growth and corresponding biofilm formation were rapid at dilution rates higher than 0.35 h−1 when the product concentrations were low (succinic acid < 10 g L−1), while minimal growth was observed at succinic acid concentrations above this threshold.
Keywords: Continuous fermentation; Succinic acid; Actinobacillus succinogenes; Bioreactors; Biofilms; Glucose;

Sampling location of the inoculum is crucial in designing anodes for microbial fuel cells by Stephanie F. Ketep; Alain Bergel; Marie Bertrand; Wafa Achouak; Eric Fourest (12-16).
► Microbial anodes formed from pulp and paper effluents. ► Efficiency and microbial composition of the anodes depend on the inoculum location. ► Predominant species Desulfuromonas acetexigens was detected by DGGE.A Kraft pulp mill effluent was used as the inoculum to form microbial bioanodes under controlled potential at +0.4 V/SCE. Samples were collected at the inlet and outlet of the aerated lagoon of the treatment line. The outlet sample allowed efficient bioanodes to be designed (5.1 A/m2), which included Geobacter and Desulfuromonas sp. in their microbial community. In contrast, the bioanodes formed with the inlet sample did not contain directly connecting anode-respiring bacteria and led to lower currents. It was necessary to re-form this bioanode at lower applied potential (−0.2 V/SCE) to select more efficient electroactive species and increase the current density to 5 A/m2.
Keywords: Microbial fuel cell (MFC); Waste-water treatment; Pulp mill effluent; Bioanode; Inoculum;

Kinetic study of the acidolysis of high oleic sunflower oil with stearic–palmitic acid mixtures catalysed by immobilised Rhizopus oryzae lipase by Joydeep Ray; Zoltan K. Nagy; Kevin W. Smith; Krishnadath Bhaggan; Andrew G.F. Stapley (17-28).
Display Omitted► High oleic sunflower oil can be acidolysed with palmitic and stearic acids to produce TAG mixtures for CBE formulations. ► Equilibrium TAG compositions were well predicted by a probability based model. ► Acidolysis reactions were successfully modelled using first order kinetics. ► Fitted rate constants were similar for equivalent reactions involving palmitic and stearic acids. ► Allowing for the formation of DAGs slightly improved kinetic model fits.The acidolysis of high oleic sunflower oil (containing >70% triolein) with 11 different stearic–palmitic acid mixtures at an oil:acid ratio of 1:1.3 (w/w) has been performed using a 1,3 regiospecific lipase (Rhizopus oryzae), to produce specialised fats with a high disaturated triacylglycerol (TAG) content (∼40%). The final conversions corresponded well to predictions from a probability model. The variation of TAG composition with time was also measured to study the reaction kinetics. Initially, a reaction scheme was formulated allowing all possible acidolysis reactions of TAGs with stearic, palmitic and oleic fatty acids at the 1 and 3 TAG positions. It was found that a first order scheme produced good fits to data and that reactions involving stearic and palmitic reactions in equivalent positions produced very similar fitted rate constants. When these rate constants were constrained to be equal, acceptable fits were also obtained. As the acidolysis reactions occur via the formation of diacylglycerols (DAGs) by hydrolysis (7.1–10.9%), a further scheme was tested whereby all possible reactions involving DAGs were included (with equal rate constants for equivalent reactions with palmitic and stearic acid to limit the number of fit parameters). This produced only a small increase in goodness of fit. Assuming a single value of rate constant for all reactions produced poor fits.
Keywords: Triacylglycerol; Diacylglycerol; Stearin; Cocoa butter equivalent; Probability model; Enzyme biocatalysis;

Metabolic studies of γ-polyglutamic acid production in Bacillus licheniformis by small-scale continuous cultivations by Anja Wilming; Jens Begemann; Stefan Kuhne; Lars Regestein; Johannes Bongaerts; Stefan Evers; Karl-Heinz Maurer; Jochen Büchs (29-37).
► Viscosity increase seen in protease production with γ-PGA forming B. licheniformis. ► High viscosity severely affects fermentation, results in undefined conditions. ► Continuous cultivation and pulse experiments were performed. ► Excess carbon and oxygen limitation have similar effect on viscosity. ► Key trigger for γ-PGA production is catabolite controlled overflow.For the cultivation of microorganisms shake flasks are frequently used. The development of the broth viscosity during the cultivation is usually not paid special attention to. However, changes in viscosity may have a severe impact on shake flask cultivations, ranging from effects on mass and heat transfer to the occurrence of out-of-phase conditions. Significant changes in viscosity were observed during batch cultivations with Bacillus licheniformis on defined mineral medium. The formed biopolymer was identified to be γ-polyglutamic acid (γ-PGA), a polymer, Bacillus species are known to excrete as a side-product. γ-PGA leads to increasing viscosity and, thus, has critical influences on shake flask cultivations. Consequently, the oxygen transfer is strongly influenced by viscosity and, therefore, γ-PGA formation. Furthermore, γ-PGA has chelating characteristics, which might affect nutrient and trace element availability, possibly resulting in substrate limitations. In total, these influences lead to undefined conditions in the fermentation. Therefore, the trigger for γ-PGA production was investigated. By performing continuous cultivation experiments as well as pulse experiments, catabolite controlled overflow was identified as one of the key triggers for γ-PGA production. The results suggest, that catabolite controlled overflow leads to inhibition of the 2-oxoglutarate complex, resulting in accumulation of 2-oxoglutarate and a redirection of metabolic flux toward glutamic acid and finally to γ-PGA. Additionally, oxygen limitations were determined to further potentiate γ-PGA production. As a result, fermentation conditions favoring catabolite controlled overflow as well as oxygen limitation need to be strictly avoided in order to assure defined conditions at low viscosity throughout the fermentation.
Keywords: Bioreactors; Viscosity; Fermentation; Protease; Bacillus licheniformis; Polyglutamic acid;

► Created a rapid methodology to determine kinetic parameters for complex enzymatic reactions. ► Used statistical analysis to allow selection of the most appropriate kinetic model. ► Quantified kinetic parameters for the transaminase synthesis of chiral amino alcohols. ► Used microscale methods for rapid and efficient collection of enzyme kinetic data. ► Identified potential dead-end complex formation and bottlenecks for bioprocess design.This work describes the establishment of a full kinetic model, including values of apparent kinetic parameters, for the whole cell E. coli mediated synthesis of the chiral amino-alcohol (2S,3R)-2-amino-1,3,4-butanetriol (ABT), using (S)-(−)-α-methylbenzylamine (MBA) as amino donor. The whole cell biocatalyst expressed the CV2025 ω-transaminase from Chromobacterium violaceum. Establishment of the most suitable reaction mechanism and determination of the complete forward and reverse kinetic parameter values for the reversible bioconversion where obtained using a hybrid methodology. This combined traditional initial rate experiments to identify a solution in the vicinity of the global minimum, with nonlinear regression methods to determine the exact location of the solution. The systematic procedure included selection and statistical evaluation of different kinetic models that best described the measured reaction rates and which ultimately provided new insights into the reaction mechanism; in particular the possible formation of a dead end complex between the amino donor and the cofactor enzyme complex. The hybrid methodology was combined with a microscale experimental platform, to significantly reduce both the number of experiments required as well as the time and material required for full kinetic parameter estimation. The equilibrium constant was determined to be 849, and the forward and reverse rate constants were found to be 97 and 13 min−1, respectively, which greatly favoured the asymmetric synthesis of chiral ABT. Using the established kinetic model, the asymmetric synthesis of ABT was simulated, and excellent agreement was found between the experimental and predicted data over a range of reaction conditions. A sensitivity analysis combined with various simulations suggested the crucial bottleneck of the reaction was the second half reaction of the ping pong bi–bi mechanism, in part due to the low Michaelis constant of substrate l-erythrulose (ERY). The toxicity of MBA towards the transaminase was identified as another major bottleneck. The kinetic model was useful to give early insights into the most appropriate bioconversion conditions, which can improve the rate and yield of ABT formation, as well as minimizing the toxicity and inhibition effects of the substrates and products. The systematic methodology developed here is considered to be generic and useful in regard to speeding up bioconversion process design and optimization.
Keywords: Nonlinear kinetic modelling; Transaminase; Reversible bioconversions; Amino-alcohols; Microscale high-throughput; Reaction mechanism selection;

The use of cloth fabric diffusion layers for scalable microbial fuel cells by Yong Luo; Fang Zhang; Bin Wei; Guangli Liu; Renduo Zhang; Bruce E. Logan (49-52).
► Goretex fabric was tested as a diffusion layer (DL) in microbial fuel cells (MFCs). ► The DL performance was similar to that of liquid-applied polytetrafluoroethylene (PTFE). ► The pre-manufactured DL is a promising approach for building a scalable MFC cathode.A scalable and pre-manufactured cloth material (Goretex® fabric) was used as a diffusion layer (DL) material as a replacement for a liquid-applied polytetrafluoroethylene (PTFE) DL. Cathodes with the Goretex fabric heat-bonded to the air-side of carbon cloth cathode (CC-Goretex) produced a maximum power density of 1330 ± 30 mW/m2, similar to that using a PTFE DL (1390 ± 70 mW/m2, CC-PTFE). This method was also successfully used to produce cathodes made of inexpensive carbon mesh, which resulted in only slightly less power (1180 ± 10 mW/m2) (CM-Goretex). Coulombic efficiencies were a function of current density, with the highest value for CC-PTFE cathodes (63%), similar to CC-Goretex cathodes (61%), and slightly larger than that obtained for the CM-Goretex cathodes (54%). These results show that a commercially available fabric can easily be used as the DL in an MFC, achieving performance similar to that obtained with a more labor-intensive process based on liquid-applied DLs using PTFE.
Keywords: Goretex fabric; Diffusion layer; Microbial fuel cell; Carbon mesh;

Overview on the developments of microbial fuel cells by V.B. Oliveira; M. Simões; L.F. Melo; A.M.F.R. Pinto (53-64).
► We present a recent review concerning the most recent MFC optimization studies. ► The effect of pH, temperature, organic load, feed rate and shear stress are described. ► The current status of the MFC modelling and scale-up studies is provided. ► This work is a helpful tool for discovery new designs and operation regimes. ► Development of low-cost materials and optimization of operating and design parameters is crucial for MFCs.Microbial fuel cells (MFCs) are a promising technology for electricity production from a variety of materials, such as natural organic matter, complex organic waste or renewable biomass, and can be advantageously combined with applications in wastewater treatment. The problem with MFCs is that they are technically still very far from attaining acceptable levels of power output, since the performance of this type of fuel cells is affected by limitations based on irreversible reactions and processes occurring both on the anode and cathode side. However, in the last years, there has been a growing amount of work on MFCs which managed to increase power outputs by an order of magnitude.The present review article discusses a number of biological and engineering aspects related to improvement of MFC performance including the effect of important parameters, such as pH, temperature, feed rate, shear stress and organic load. The recent progresses on scale-up MFC are summarized and the different modelling approaches to describe the different biological and transport phenomena in MFCs are also provided.
Keywords: Microbial fuel cells; Modelling; Operating conditions; Scale-up; Sustainable energy;

► Using airlift reactor with solid draft tube yielded the highest biomass content. ► Aeration rate of 0.1 vvm offered a better environment and increased biomass production. ► A. cinnamomea is sensitive to shear stress and prefers lower k L a. ► Scale up to 500-L airlift bioreactor will increase biomass production and time-saving. ► The ethanol extract of mycelium effectively inhibited proliferation of HepG2 cells.Three 5-L airlift bioreactors including airlift reactor with solid draft tube (ALs), airlift reactor with net draft tube (ALn) and bubble column reactor (BC) were investigated for their suitability for cultivating Antrodia cinnamomea, and a stirred tank reactor (ST) was used for comparison. Results indicated that after 7 days fermentation, ALs yielded the highest mycelium content (313 mg/100 mL) and had the lowest dissolved oxygen in the broth. Among different aeration rates (0.025, 0.05, 0.1, 0.5, 1 vvm) used during cultivation of A. cinnamomea in ALs, the aeration rate 0.1 vvm resulted in a volumetric oxygen transfer coefficient of 10.8 h−1 and produced the highest mycelium content. When the optimal conditions were used for the fermentation of A. cinnamomea in an industrial 500-L ALs, the mycelium content in the broth reached 542 mg/100 mL in 28 days. The IC50 values of the ethanol extracts of A. cinnamomea mycelium cultivated in 5-L and 500-L ALs for 28 days were 23 and 17 μg/mL, respectively, for hepatocellular carcinoma cells HepG2. And after 42 days cultivation in 500-L ALs, the IC50 value of the mycelium ethanol extract was reduced to 10 μg/mL.
Keywords: Antrodia cinnamomea; Airlift bioreactors; Oxygen transfer; Aeration; Bioprocess design; Sacle-up;

► We design a new recyclable biocatalyst system for ethyl isovalerate production. ► System provides a combination of enzyme stability, recovery and reusability. ► 80% esterification yield in 48 h was observed for non-ionic surfactant coated lipase. ► System retained higher catalytic activity (more than 65%) after reusing seven times. ► Enhanced activity due to interfacial activation was attained for this system.Gum arabic coated magnetic Fe3O4 nanoparticles (GAMNP) were prepared by chemical co-precipitation method and their surface morphology, particle size and presence of polymer-coating was confirmed by various measurements, including transmission electron microscopy (TEM), X-ray diffraction (XRD), thermo gravimetric analysis (TGA), and Fourier transform infra red (FTIR) analysis. Magnetic particles were employed for their potential application as a support material for lipase immobilization. Glutaraldehyde was used as a coupling agent for efficient binding of lipase onto the magnetic carrier. For this purpose, the surface of a Candida rugosa lipase was initially coated with various surfactants, to stabilize enzyme in its open form, and then immobilized on to the support. This immobilized system was used as a biocatalyst for ethyl isovalerate, a flavor ester, production. The influence of various factors such as type of surfactant, optimum temperature and pH requirement, organic solvent used, amount of surfactant in coating lipase and effect of enzyme loadings on the esterification reaction were systematically studied. Different surfactants were used amongst which non-ionic surfactant performed better, showing about 80% esterification yield in 48 h as compared to cationic/anionic surfactants. Enhanced activity due to interfacial activation was observed for immobilized non-ionic surfactant–lipase complex. The immobilized surfactant coated lipase activity was retained after reusing seven times.
Keywords: Magnetic nanoparticles; Gum arabic; Lipase; Esterification;

Adding Fe0 powder to enhance the anaerobic conversion of propionate to acetate by Xusheng Meng; Yaobin Zhang; Qi Li; Xie Quan (80-85).
► Adding Fe0 to enhance propionate decomposition in an acidogenic reactor. ► Accelerating homoacetogenesis to reduce the H2 content with the presence of Fe0. ► Fe0 could decrease the Gibbs free energy and improve the enzymes activities.Propionate is an unfavorable substrate for the anaerobic digestion because it is thermodynamically difficult to be decomposed into acetate. An attempt to enhance the decomposition of propionate by adding Fe0 powder (10 g) into an acidogenic reactor (A1) with propionate as the sole carbon source was made in this study. The results showed that the propionate conversion rate (67–89%) in A1 were higher than that in a reference reactor (43–77%) without dosing of Fe0 (A2). The enhanced conversion of propionate caused both chemical oxygen demand removal (COD) (57–79%) and acetate production (178–328 mg/L) in A1 to increase significantly. Although Fe0 contributed the H2 production chemically, the H2 content of A1 was less than that of A2. The reason was ascribed to the enhanced utilization of H2 for the homoacetogenesis. It was calculated that the Gibbs free energy in the decomposition of propionate was decreased by about 8.0–10.2% with the dosing of Fe0. Also, the activities of enzymes related to the acetogenesis were enhanced by 2–34-folds. Fluorescence in situ hybridization (FISH) and denaturing gradient gel electrophoresis (DGGE) analysis indicated that Fe0 increased the abundance of microbial communities, especially propionate-utilizing bacteria and homoacetogenic bacteria.
Keywords: Propionate; Fe0; Acetate; Acetogenesis; Homoacetogenesis; Anaerobic;

Convenient one-step purification and immobilization of lipase using a genetically encoded aldehyde tag by Anming Wang; Fangchuan Du; Fen Wang; Yingqiang Shen; Weifang Gao; Pengfei Zhang (86-92).
► One-step purification and immobilization of recombinant lipase using aldehyde tag. ► The novel immobilized enzyme presented high activity, k cat/K m and thermal stability. ► Bioorthogonal covalent linkage may be appropriate protection of enzyme's active site.To avoid the unwanted and random covalent linkage between the cross-linker and enzyme's active site in covalent immobilization, a genetically encoded “aldehyde tag” was introduced into recombinant lipase and applied for the one-step purification and covalent immobilization of this enzyme. The effects of the immobilization time, temperature and the amount of enzyme were investigated, and the thermo-stability of immobilized lipase was also examined. The specific activity and the k cat/K m of the immobilized lipase using aldehyde tag (IL-AT) were 2.50 and 3.02 fold higher, respectively, than those of the traditionally immobilized lipase using glutaraldehyde (IL-GA). The newly immobilized lipase also presented better thermo-stability than the traditionally immobilized one. The results show that the recombinant enzyme could be conveniently immobilized without glutaraldehyde and that the enzyme's active site was well protected. This is a new immobilization method able to avoid glutaraldehyde or 2,4,6-trichloro-1,3,5-triazine as an activating agent. The greener method without hazardous chemicals for the one-step purification and immobilization of an enzyme using a genetically encoded “aldehyde tag” can be exploited for numerous other enzyme purification and immobilization applications.
Keywords: Aldehyde-tag; Bioseparations; Immobilized enyzme; Enzyme activity; Thermo-stability; Lipase;