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Biochemical Engineering Journal (v.46, #1)

BEJ Keywords (pp. ii).

Editorial Board (pp. co2).

Surface modification of the Corynebacterium glutamicum biomass to increase carboxyl binding site for basic dye molecules by Juan Mao; Sung Wook Won; Sun Beom Choi; Min Woo Lee; Yeoung-Sang Yun (pp. 1-6).

The objective of this study was to develop a surface-modified biosorbent with enhanced sorption capacity of cationic dye Basic Blue 3 (BB 3). The biomass of Corynebacterium glutamicum was modified using citric acid (CA). The optimal modification conditions were the mixing of the biomass with 0.8M citric acid solution at a 1:10 ratio, and heating at 120°C for 3.5h. Hydroxyl groups on the biomass surface underwent an esterification with citric acid, forming carboxyl groups which were available for cationic dye binding. From the Fourier transform infrared analysis, it was confirmed that the CA-modified biomass possessed a large amount of surface carboxyl functional groups comparing the raw biomass. The sorption capacity of BB 3 onto CA-modified biomass was 2.02 times higher than that onto the raw biomass. Furthermore, BB 3 sorbed on the CA-modified biomass was easily eluted by shifting the solution pH, making repeated sorption/desorption cycle (up to 4 times) possible without significant performance decrease. Therefore, the method developed for amplifying the carboxyl sites on the biomass surface may be a useful modification tool for the creation of a high-performance and regenerable biosorbent for the cationic dyes.

Keywords: Biosorption; Corynebacterium glutamicum; Citric acid; Modification; Basic dye

The role of disulfide bond formation in the conformational folding kinetics of denatured/reduced lysozyme by Guo-Zhen Wang; Xiao-Yan Dong; Yan Sun (pp. 7-11).

Oxidative folding is of vital importance for producing therapeutic proteins in bacteria via recombinant DNA technology since disulfide bonds exist in most pharmaceutical proteins. Although oxidative protein folding has been extensively investigated in vitro, little is explored concerning the role of disulfide formation to protein conformational folding rate. The effects of oxidized (GSSG)/reduced (GSH) glutathione and pH on the conformational folding kinetics of denatured/reduced lysozyme have been studied herein by fluorescence and circular dichroism. It is found that 83% tryptophan residue burial requires disulfide formation, and increasing GSSG concentration greatly accelerates the tertiary structure formation. The fast phase folding rate constant ( k₁) is linearly related to GSSG concentration, indicating the rate-limiting role of mixed-disulfide formation. Moreover, k₁=0.006(±0.001)s⁻¹ is likely to be a critical value for judging the determinant of the slow phase folding rate ( k₂), namely, k₂ is controlled by disulfide formation rate only at k₁<0.006s⁻¹. These findings have elucidated the determinants of different folding stages and thus may be beneficial for more efficient control of the oxidative folding of proteins.

Keywords: Bioseparations; Protein; Refolding; Kinetic Parameters; Oxidative folding; Disulfide

Simulation of biodegradation process in a fluidized bed bioreactor using genetic algorithm trained feedforward neural network by A. Venu Vinod; K. Arun Kumar; G. Venkat Reddy (pp. 12-20).

The biodegradation process of phenol in a fluidized bed bioreactor (FBR) has been simulated using genetic algorithm trained feedforward neural network. Experiments were carried out using the microorganism Pseudomonas sp. on synthetic wastewater. The steady state model equations describing the biodegradation process have been solved using feedforward artificial neural network (FFANN) and genetic algorithm (GA). The mathematical model has been directly mapped onto the network architecture and the network has been used to find an error function (mean squared error criterion). The minimization of the error function with respect to network parameters (weights and biases) has been considered as training of the network. Real-coded genetic algorithm has been used for training the network in an unsupervised manner. The diffusivities of phenol and oxygen in biofilm obtained from the simulation have been compared with the literature values.

Keywords: Biofilm; Immobilized; Waste treatment; Biodegradation; Neural network; Genetic algorithm

Decolourization and detoxification of pulp and paper mill effluent by Cryptococcus sp. by Anjali Singhal; Indu Shekhar Thakur (pp. 21-27).

Three fungal strains were isolated from sediments of pulp and paper mill in which PF7 reduced colour (27%) and lignin content (24%) of the effluent on 5th day. PF7 was identified as Cryptococcus sp. isolate FIST3 on the basis of ITS1 and ITS2 region sequences. The process of decolourization optimized, in shake flask experiments, by Taguchi approach indicated optimum conditions: temperature (30–35°C); shaking condition (125rpm); dextrose (1.0% w/v); tryptone (0.1% w/v); inoculum size (7.5% w/v); pH (5) and duration (24h). Overall evaluation criterion (OEC) value before optimization was 32.3. There was 38% improvement in the process with final OEC value, 44.6±2.02, at optimum conditions. The colour content of the effluent reduced by 50–53% and lignin content 35–40% after treatment at optimum conditions. Variation in pH from 5 to 6 had most significant effect on decolourization (72%) while variation in temperature from 30°C to 35°C had no effect on the process. Treated effluent was further evaluated for toxicity by alkaline single cell (comet) gel electrophoresis (SCGE) assay using Saccharomyces cerevisiae MTCC 36 as model organism, indicated 45% reduction. The results showed significant reduction in colour, lignin and toxicity of the effluent and this process can be scale up to industrial level.

Keywords: Bioremediation; Comet assay; Lignin; Optimization; Taguchi approach; Waste-water treatment

Increased production of riboflavin by metabolic engineering of the purine pathway in Bacillus subtilis by Shuobo Shi; Zhuo Shen; Xun Chen; Tao Chen; Xueming Zhao (pp. 28-33).

Purine nucleotides are precursors for riboflavin production, and one of its intermediates, GTP, is the immediate precursor for riboflavin biosynthesis. Therefore, activation of the purine pathway would improve the supply of the precursors, which may in turn lead to an increase of riboflavin production. Here we reported the characterization and manipulation of the purine pathway in a riboflavin overproducer Bacillus subtilis PK. Due to the tight regulation of the purine pathway, it was not sufficient to obtain an enhanced production of purine nucleotides or riboflavin by simple overexpression of the purF gene, which is the key gene of the purine pathway. However, co-overexpression of purF, purM, purN, purH, purD genes gave an engineered strain B. subtilis PK-P with much better performance. Metabolites analysis revealed that the precursors, purine nucleotides, were significantly improved in B. subtilis PK-P. Moreover, as much as a 25% increase in the riboflavin yield and a 31% increase in riboflavin titer were obtained in the recombinant B. subtilis PK-P. It was assumed that the co-overexpression of the genes caused higher supply of the purine nucleotides in B. subtilis PK-P, which in turn facilitated riboflavin production.

Keywords: Bacillus subtilis; Metabolic capacity; Purf; gene; Purine pathway; Riboflavin production; Co-overexpression

Gas–solid packed and fluidized bed models for bioremediation of volatile organic compounds in air by Kyla Clarke; Todd Pugsley; Gordon A. Hill (pp. 34-43).

A steady state, one-dimensional model is developed to describe biodegradation of a volatile organic compound (VOC) in a gas–solid fluidized bioreactor. For comparison purposes, biodegradation in a packed bed bioreactor is also modelled. The models are used to predict outlet concentrations of a VOC substrate (ethanol) and the estimates are compared to experimental data. The fluidized bioreactor model includes properties of the bubbles such as rise velocity, bubble size and gas exchange rate with the emulsion phase surrounding the bubbles. The overall rate of biodegradation is modelled with the two limiting cases of zero and first order kinetics with respect to the substrate, which depend on whether the overall resistance is limited by microbial growth or mass transport. The models highlight how maximum elimination capacity and removal efficiency are influenced by bubble properties, residence time, maximum specific growth rate of the microorganisms, and microbial population.

Keywords: Bioreactor systems; Bioremediation; Fluidization; Modelling; Kinetics; Mass transfer; Hydrodynamics

Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis by Rogelio Valadez-Blanco; Andrew Guy Livingston (pp. 44-53).

This work explores product-formation kinetics of the reduction of acetophenone to ( S)-phenylethanol by Rhodotorula glutinis in aqueous media. Different feeding strategies were investigated to improve biotransformation productivity. An empirical kinetic model was formulated considering two different reaction periods: Phase-1, a very fast chemical-shock reaction in the first 5min; Phase-2, a subsequent slower reaction which, depending on the product level, could be subinhibitory or inhibitory. The kinetics of the biotransformation metabolism were modulated by the levels of glucose and acetic acid in the bioreactor. In addition, two substrate feeding regimes were compared: intermittent and continuous. Although similar overall productivities were found, the intermittent method clearly holds greater potential for enhanced space-time yields as it takes advantage of the Phase-1 high reaction rates – additions of substrate pulses produce reaction rates of the same order of magnitude as those achieved in Phase-1. However, product accumulation to a “toxic limit” leads to the total cessation of biocatalytic activity, thus highlighting the importance of in-situ product removal during the biotransformation. Strategies for in-situ product removal are addressed in Part II of this study.

Keywords: Abbreviations; Aph; acetophenone; FB-Int; fed-batch bioreactor with intermittent substrate addition; FB-Cont; fed-batch bioreactor with continuous substrate addition; FB-Cont-Glu; fed-batch bioreactor with continuous substrate and glucose addition; Grow-FB; growing-cells fed-batch bioreactor; Phe; phenylethanol; rac; -Phe; racemic phenylethanol; R; -Phe; phenylethanol; R; enantiomer; S; -Phe; phenylethanol; S; enantiomerMicrobial biocatalysis; Fed-batch bioreactor; Product formation kinetics; Aqueous phase biotransformation; Product inhibition; Asymmetric synthesis

Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis. Part II. Aqueous–organic systems: Emulsion and membrane bioreactors by Rogelio Valadez-Blanco; Andrew Guy Livingston (pp. 54-60).

This paper reports the development and performance of a membrane bioreactor for biotransformations (MBB) using organic solvent nanofiltration membranes for the bioreduction of acetophenone to S-phenylethanol by whole cells of Rhodotorula glutinis. It follows a previous paper dealing with the kinetics of this reaction in aqueous media [R. Valadez-Blanco, A.G. Livingston, Enantioselective whole-cell biotransformation of acetophenone to S-phenylethanol by Rhodotorula glutinis. Part I. Product formation kinetics and feeding strategies in aqueous media, Biochem. Eng. J. 46 (2009) 44–53]. A number of organic solvents were tested, with dibutyl ether found to be the best solvent in terms of toxicity and organic/aqueous partition coefficients. Despite the fact that most of the MBBs were mass-transfer limited, their reaction rates were comparable to those of the aqueous systems, with prolonged biocatalytic activity due to in situ product extraction. The use of direct injection of substrate and glucose to the aqueous phase, together with high biomass concentrations, resulted in the highest overall product formation and acceptable reaction rates. This strategy also avoided the need for additional cumbersome downstream processing to recover the product from the aqueous phase. In contrast, direct-contact biphasic reactors proved to be unsuitable for this biotransformation due to poor product partition or high volatility of the solvents.

Keywords: Abbreviations; Aph; acetophenone; DBE; dibutyl ether; DCB; direct-contact biphasic; ee; enantiomeric excess; MBB; membrane bioreactor for biotransformations; MBB-FB; continuous aqueous substrate addition fed-batch MBB; MBB-FB-Glu; continuous aqueous substrate and glucose addition fed-batch MBB; OSN; organic solvent nanofiltration; Phe; phenylethanol; S; -Phe; phenylethanol; S; enantiomerOrganic-solvent-nanofiltration membrane; Aqueous–organic two-phase biotransformation; Microbial asymmetric synthesis; Reductive biocatalysis; In situ product recovery; Product inhibition

Solid state bioconversion of oil palm empty fruit bunches for cellulase enzyme production using a rotary drum bioreactor by Md. Zahangir Alam; Abdullah A. Mamun; Isam Y. Qudsieh; Suleyman A. Muyibi; Hamzah M. Salleh; Nor Marina Omar (pp. 61-64).

Bioconversion of oil palm lignocellulosic biomass in the form of empty fruit bunches (EFB) as the major substrate into cellulase enzyme was studied in the laboratory. A fungal strain, Trichoderma harzianum T2008 was used to evaluate the solid state bioconversion of EFB for cellulase production. The study was conducted in two systems: an Erlenmeyer flask (EF, 500mL) and a horizontal rotary drum bioreactor (RDB, 50L), designed and fabricated locally. The highest cellulase activity on the fourth day of fermentation in the EF was 8.2filter paper activity (FPA)/gram dry solids (gds) of EFB, while its activity from the RDB was 10.1FPA/gds on the second day of fermentation. Glucosamine, reducing sugar and pH were also determined to evaluate the substrate uptake and growth conditions during the entire fermentation period.

Keywords: Solid state rotary drum bioreactor; Erlenmeyer flask; Oil palm biomass; Cellulase enzyme; Trichoderma harzianum; Solid waste treatment

Immobilization of Escherichia coli cells using polyethyleneimine-coated porous support particles forl-aspartic acid production by Juan Huang; Na Jin; Tomohisa Katsuda; Hideki Fukuda; Hideki Yamaji (pp. 65-68).

Escherichia coli cells were efficiently immobilized in reticulated polyvinyl formal resin biomass support particles (BSPs) that had been simply autoclaved with a solution of a cationic polymer such as polyethyleneimine. When the immobilized E. coli cells containing aspartase were used as whole cell biocatalyst forl-aspartic acid production, they showed specific aspartase activity comparable to that of non-immobilized cells.

Keywords: Immobilized cell; Escherichia coli; Biomass support particles; Polyethyleneimine; Biocatalysis; Whole cell biocatalyst; l; -Aspartic acid

Quantification of DNA damage by the comet assay in radish sprouts exposed to excess light irradiation by Yoshihiro Ojima; Motomu Nishioka; Masahiro Matsumoto; Masahito Taya (pp. 69-72).

The comet assay was adapted for quantifying the degree of photo-induced DNA damage by using radish sprouts exposed to varied light conditions. An index, IN_D, was defined to express the DNA intactness, based on image-analyzing of nuclei in protoplasts prepared from the plant leaves. The IN_D value gradually decreased with increasing light intensity (22–430Wm⁻²) and exposure time (0–6h), and ultimately fell to 21% at 6h under a light intensity of 430Wm⁻², as compared to a reference level in the plants virgin of the exposure. Furthermore, the DNA damage was found to be restored to an appreciable extent when the plants were fed with antioxidants such as ascorbic acid and green tea extract, suggesting that DNA damage from strong light can be caused by photo-oxidative stress generated by the excess energy over a scavenging capacity of antioxidative defense mechanisms in the plant cells.

Keywords: Plant cells; DNA damage; Photo-induced stress; Comet assay; Antioxidant

Persistence and functions of a decolorizing fungal consortium in a non-sterile biofilm reactor by Zhiyuan Lu; Xiao Sun; Qingxiang Yang; Huijun Li; Chunmao Li (pp. 73-78).

In this study, a continuous biofilm decolorizing system with high efficiencies of dye degradation and textile wastewater treatment was established using selected fungal consortium. Function and persistence of this fungal consortium were assessed using traditional and molecular biological methods along with near 4 months running process under non-sterile conditions. The microbial cultivation results indicated that the colony forming units (CFUs) ratio of fungi to bacteria stabilized between 51.8:1 and 6.8:1 under the influent conditions of various simulated and real textile wastewater. Clone libraries and phylogenetic trees of bacteria and fungi were respectively constructed based on their full length 16S and partial 26S rRNA gene sequences retrieved from the biofilm at the end of the system running. The results indicated that the dominant population preserved in the system was yeasts belonging to genus Candida. While most of the filamentous fungi isolated from the enrichment consortium could not be preserved on the biofilm. Among the three fungal isolates with high efficiency of dye decolorization, only Candida tropicalis was retrieved and occupied near 70% in the fungal clone library. 26.5% of the retrieved fungal sequences were grouped with some species of Trichoderma which did not exhibit dye decolorization abilities. Compared to the fungal phylogenetic tree, diversity of bacteria was far higher. 67.9% of the retrieved bacterial sequences were clustered into Alphaproteobacteria.

Keywords: Fungi consortium; Decolorization; Bioreactor; Textile wastewater treatment; Phylogenetic tree

Characterization of benzoate degradation by newly isolated bacterium Pseudomonas sp. XP-M2 by Nengzhong Xie; Hongzhi Tang; Jinhui Feng; Fei Tao; Cuiqing Ma; Ping Xu (pp. 79-82).

An effective benzoate-degradation strain XP-M2 belonging to the genus Pseudomonas, was newly isolated from aromatic compounds contaminated soils. The growing cells of strain XP-M2 completely degraded 10g/L benzoate within 12h, and the degradation efficiency was found to be higher than other reports. Catechol and cis,cis-muconic acid as the metabolic intermediates were identified by gas chromatography–high-resolution mass spectrometry, and the catA gene encoding catechol 1,2-dioxygenase in this strain was cloned and expressed in Escherichia coli, which indicated that benzoate degradation by Pseudomonas sp. XP-M2 was via the ortho-cleavage pathway. In addition, the Andrews model was used to describe the functional relationship between specific growth rate and benzoate concentrations with kinetics parameters: K_S=2.47g/L, K_I=3.81g/L.

Keywords: Benzoate; Kinetics; Andrews model; Microbial; Bioremediation; Waste treatment

Photochemical preparation of magnetic chitosan beads for immobilization of pullulanase by Lianying Zhang; Xinjun Zhu; Shiying Zheng; Hanwen Sun (pp. 83-87).

Magnetic chitosan beads were prepared via photochemical polymerization in Fe₃O₄ magnetite aqueous suspension, and characterized by Fourier transform infrared spectroscopy (FT-IR), scanning electron microscopy (SEM), photocorrelation spectroscopy (PCS) and vibrating sample magnetometer (VSM). The FT-IR result indicated that the magnetic chitosan beads were synthesized successfully under UV irradiation in aqueous system. The magnetic chitosan beads were of regularly spherical shape, had a mean diameter of 86nm, exhibited superparamagnetic property and the saturation magnetization was 52.6emu/g. Pullulanase was covalently immobilized on magnetic chitosan beads by cross-linking with glutaraldehyde. The maximum quantity of pullulanase immobilized onto 1.0mg magnetic chitosan beads was 180μg. The optimum temperature of immobilized pullulanase was 50°C, which was identical to that of free enzyme, and the immobilized pullulanase exhibited a higher relative activity than that of free pullulanase over 50°C. The optimal pH for immobilized pullulanase was 5.0, which was lower than that of the free pullulanase (pH 5.5), and the immobilization resulted in stabilization of enzyme over a broader pH range. The kinetic constant value ( Km) of immobilized pullulanase was three times higher than that of free pullulanase. However, the thermal and operational stabilities of immobilized pullulanase were improved greatly.

Keywords: Magnetic chitosan beads; Photochemical preparation; Immobilization; Pullulanase

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Biochemical Engineering Journal (v.46, #1)