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Biochemical Engineering Journal (v.43, #2)

BEJ Keywords (pp. ii).

Editorial Board (pp. co2).

Development of novel wheat biorefining: Effect of gluten extraction from wheat on bioethanol production by N. Arifeen; I.K. Kookos; R. Wang; A.A. Koutinas; C. Webb (pp. 113-121).

Wheat has been used in a novel biorefinery as the sole raw material for the production of bioethanol and some co-products (bran-rich pearlings, gluten and pure yeast cells). Minimisation in waste production has been achieved either by generating co-products from major wheat components that are not required for bioethanol fermentation or by re-generating nutrients contained initially in wheat via microbial autolysis. On-site production of enzymes required to hydrolyse wheat macromolecules has been achieved by Aspergillus awamori fermentation of pearled wheat flour. Complete gluten extraction was made feasible by providing the required amount of free amino nitrogen (FAN) for bioethanol fermentations via on-site fungal autolysis. A wheat to ethanol conversion yield of 0.296gg⁻¹, which constitutes 77% of the maximum theoretical conversion (0.385gethanol (gwheat)⁻¹) calculated from the starch content in the wheat used, has been achieved including the starch requirements for enzyme production. A modified unstructured model has been proposed to describe fermentations of Saccharomyces cerevisiae on wheat-derived media for bioethanol production.

Keywords: Biorefinery; Wheat; Gluten; Bioethanol; Modelling

Estimation of the contribution of immobilized biofilm and suspended biomass to the biodegradation of phenol in membrane contactors by Ruey-Shin Juang; Hsiang-Chien Kao (pp. 122-128).

The biodegradation of phenol by Pseudomonas putida BCRC 14365 at 30°C and pH 7 was studied in microporous polypropylene (PP) hollow-fiber membrane contactors. The fibers were pre-wetted by ethanol to make them more hydrophilic. The initial cell concentration was fixed at 25g/m³ (optical density, 0.064). In this system, microbial cells are separated from phenol-containing wastewater to avoid the toxic effect of phenol. Phenol could be completely degraded, even though the initial phenol level was high up to 2150g/m³, with the help of not only suspended biomass but also membrane-attached biofilm. In order to quantitatively estimate the contribution of biofilm to the overall process, a simplified kinetic model was proposed that combines the steady mass transfer equations and dynamic growth kinetics of suspended cells but assumes the absence of membrane-attached biofilm. Under the phenol levels studied (500–2150g/m³), it was shown that the amount of phenol removed by the biofilm was less than 50% during the process. Also, the contribution of biofilm remained not more than 35% when the initial phenol level was increased to 2150g/m³.

Keywords: Biodegradation; Mass transfer; Phenol; Microporous membranes; Biofilms; Dynamic modeling

Ferulic acid and p-coumaric acid solubilization by alkaline hydrolysis of the solid residue obtained after acid prehydrolysis of vine shoot prunings: Effect of the hydroxide and pH by Belén Max; Ana María Torrado; Ana Belén Moldes; Attilio Converti; José Manuel Domínguez (pp. 129-134).

Laboratory-scale experiments were conducted with the goal of releasing ferulic acid (FA) and p-coumaric acids ( p-CA) from the solid residue obtained after prehydrolysis of prunings of vine shoots. Firstly, an alkaline hydrolysis with different hydroxides at mild conditions of temperature (35°C) was performed, followed by acidification of liquors, and liquid–liquid extraction with ethyl acetate. The influence of two operational variables was tested on three levels in a 9 factorial design. The independent variables were the type of hydroxide (3N NH₄OH, KOH or NaOH) and pH in the range 1.5–7.5. Under the optima conditions (3N KOH, pH 2.88), 228.6mg FA/L, 0.41mg FA/g vine shoots, 102.6mg p-CA/L, and 0.147mg p-CA/g vine shoots, were achieved.

Keywords: Trimming wastes; Ferulic acid; p; -coumaric acid; Alkaline hydrolysis

Production of violet pigment by a newly isolated psychrotrophic bacterium from a glacier in Xinjiang, China by Yuan Lu; Liyan Wang; Yuan Xue; Chong Zhang; Xin-Hui Xing; Kai Lou; Zhidong Zhang; Yong Li; Guifeng Zhang; Jingxiu Bi; Zhiguo Su (pp. 135-141).

A psychrotrophic XT1 strain (CGMCC 1194) isolated from a glacier in China's Xinjiang province produced violet pigments at a high content. This strain was identified as Janthinobacterium lividum XT1 by API kit and 16S rDNA analysis. The main fraction of violet pigment was violacein based on the analysis by HPLC-MS and¹H NMR. The pigment production by J. lividum XT1 was not associated with the cell growth, and a cultivation temperature lower than 20°C was required for the cell growth and pigment production. Sucrose was the most suitable carbon source, and casein was the best nitrogen source for cell growth and pigment formation. Mineral components in the medium, including CaNO₃, FeSO₄, riboflavin, MgSO₄ and nicotinic acid, showed different effects on the cell growth and pigment production. The maximum pigment productivity reached 0.8g pigment per dry cell weight. The pigment production pattern by our isolate was different from that by other strains such as J. lividum reported so far.

Keywords: Cell growth; Fermentation; Janthinobacterium; sp.; Pigment production; Violet pigment; Violacein

Aerobic biodegradation kinetics of tannic acid in activated sludge system by W.W. Li; X.D. Li; K.M. Zeng (pp. 142-148).

Biological treatment of simulated tannic acid (TA)-containing wastewater by activated sludge was investigated to find the optimal biodegradation conditions and investigate the aerobic biodegradation kinetics. Activated sludge was acclimatized to simulated wastewater with TA concentration up to 950mg/l over a period of 27 days. Batch experiments on TA biodegradation under different pH, temperature, aeration conditions and with different initial substrate concentrations were conducted. The initial biomass concentration was kept at 2000mg/l. By comparing the degradation rates under different conditions, the optimal ranges of pH, temperature and DO for TA biodegradation in the activated sludge system were determined: pH≈7, temperature=20–35°C, DO>1.0mg/l. A biodegradation kinetic model that takes into consideration substrate inhibition and endogenous decay was established, and its kinetic parameters were determined. The estimated values of μ_max, K_s, K_i, k_d and Y were 0.208h⁻¹, 226mg/l, 522mg/l, 0.0092h⁻¹ and 0.594, respectively.

Keywords: Tannic acid; Activated sludge; Biodegradation; Kinetic parameters; Substrate inhibition; Wastewater treatment

To study the influence of different components of fermentable substrates on induction of extracellular α-amylase synthesis by Bacillus subtilis DM-03 in solid-state fermentation and exploration of feasibility for inclusion of α-amylase in laundry detergent formulations by Ashis K. Mukherjee; Munindra Borah; Sudhir K. Rai (pp. 149-156).

Study on potential of different agro-industrial waste residues for supporting α-amylase production by a thermophilic strain of Bacillus subtilis DM-03 in solid-state fermentation (SSF) was initiated and potato peel was found to be the best substrate for α-amylase production. Linear multiple regression analysis showed that higher starch contents of the fermentable substrates enhanced the α-amylase synthesis while the higher sugar content of the substrates imposed a negative effect presumably due to catabolic repression of the enzyme synthesis. Furthermore, C/N ratio of the substrates also played a critical role in induction of α-amylase synthesis. The result of batch fermentation showed 532±5U of α-amylase production per gram of dry substrate by B. subtilis DM-03 post-72h of incubation under optimized SSF condition. The crude α-amylase enzyme from B. subtilis DM-03 strain demonstrated excellent stability and compatibility with the laundry detergents that favors its inclusion in commercial laundry detergent formulations.

Keywords: Amylase; Bacillus subtilis; Enzyme production; Fermentation; Linear regression; Solid-state fermentation

Pichia pastoris fermentation for phytase production using crude glycerol from biodiesel production as the sole carbon source by Shuiquan Tang; Lindsay Boehme; Howard Lam; Zisheng Zhang (pp. 157-162).

Efficient utilization of crude glycerol, a by-product from biodiesel production, could bring significant economic and environmental benefits. In this work, a low-grade glycerol was used as the sole carbon source in phytase production with recombinant Pichia pastoris possessing a pGAP-based constitutive expression vector. In batch and fed-batch modes, the effects of important cultivation conditions were investigated using both analytical and biodiesel glycerols in batch and fed-batch fermentations. The following factors were analyzed: initial glycerol concentration, dissolved oxygen level, and the effect of feeding strategy on cell growth biomass and protein production. Significant cell growth inhibition was observed in batch fermentation when the initial crude glycerol concentration was as high as 70g/L. However, such inhibition was overcome in fed-batch mode by starting the cultivation with a lower crude glycerol level. Finally, cell densities and phytase activity levels of as high as 146g (dry cell weight)/L broth and 1125U/mL supernatant were achieved in the fed-batch fermentation with crude biodiesel glycerol as the sole carbon source. The study has proven the potential of using crude glycerol from biodiesel production as the carbon source for industrial scale phytase mass production in high cell density fermentations with recombinant P. pastoris.

Keywords: Crude glycerol; Biodiesel; Phytase; Pichia pastoris; High cell density fermentation

Development of a fed-batch process for the production of anticancer drug TAT_m-survivin(T34A) in Escherichia coli by Haiyi Zhang; Yu Zheng; Qinghai Liu; Xinyi Tao; Wenyun Zheng; Xingyuan Ma; Dongzhi Wei (pp. 163-168).

A fed-batch process was developed for intracellular production of recombinant TAT_m-survivin(T34A) in Escherichia coli under the control of T7 promoter. The effects of induction mode and nutritional conditions were investigated. Compared to the one-point addition of inducer, the step-wise addition of isopropyl beta-d-thiogalactopyranoside (IPTG) maintained higher plasmid stability and increased the production level by 52%. Insufficient glucose supply after induction was observed to control acetate accumulation effectively and improved the expression of the target gene evidently. Remarkably, the pre-induction supplement of inorganic nitrogen source had a positive influence on the production of TAT_m-survivin(T34A). High ammonium concentration of 4.8gl⁻¹ was the most efficient in enhancing the production level (as the percentage of total cellular protein) and the specific productivity of TAT_m-survivin(T34A). As a result, the production of TAT_m-survivin(T34A) was optimized from 11.6% to 36.8% of total cellular protein (corresponding to 1.68gl⁽¹). The findings provide valuable information for optimization of recombinant protein expression.

Keywords: TAT; _m; -survivin(T34A); Escherichia coli; Fed-batch culture; Optimization; Acetic acid; Bioprocess design

Mathematical modeling of the biofiltration of ethyl acetate and toluene and their mixture by F. Javier Álvarez-Hornos; Carmen Gabaldón; V. Martínez-Soria; Paula Marzal; Josep-Manuel Penya-roja (pp. 169-177).

A dynamic model for ethyl acetate and toluene removal by biofiltration is presented. Haldane-type kinetic expressions that include the oxygen limitation, the inhibition effect due to high concentration of substrate, and the cross-inhibition between substrates have been considered. A decrease in the biomass density with the bed height was proposed to represent the performance of peat biofilters over a broad range of operating conditions. Experimental yield coefficients have been derived from the experimental CO₂ production data. The unknown kinetic parameters of the model along with the coefficient for axial biomass density distribution were calibrated using experimental data from two peat biofilters treating ethyl acetate and toluene as single pollutants. The biofilters were operated during 2 years, operating in the empty bed residence time range between 22 and 90s and in the inlet load range between 20 and 200gm⁻³h⁻¹. Good agreement between the gaseous concentration profiles and the predicted profiles was found in almost all cases. The model was adequately validated by simulation of the removal of a 1:1 (w/w) ethyl acetate/toluene mixture in a peat biofilter operating under similar operating conditions than in the single pollutant biofiltration. The model proposed herein can be used for design purposes.

Keywords: Biofiltration; Biomass stratification; Ethyl acetate; Mathematical model; Toluene

Use of erythrocyte adhesion assay to predict the risk of diabetic complications by Yayoi Isaji; Mina Okochi; Fumihiko Horio; Hiroyuki Honda (pp. 178-184).

Simple diagnostic tools for the monitoring of an oxidative state are important in the self-monitoring of diabetic complications and other obesity-related disorders closely linked to oxidative stress. Cellular membranes are protected from oxidative damage by antioxidative enzymes and vitamins. A decrease in the level of these protective systems results in oxidative cellular damage by reactive oxygen species (ROS). In the evaluation of oxidative cellular damage that leads to numerous diseases, it is not only necessary to determine the amount of antioxidants or ROS, but also the accumulative cellular damages. In the present study, we developed a method for evaluation of the oxidative state of erythrocytes using less than 5μl of whole blood. The method is based on two observations: (i) a decrease of the surface electrical charge in oxidized erythrocytes and (ii) a change in the oxidized erythrocytes’ adhesion to the positively charged surfaces. The proportion of human erythrocytes, which adhered to the high density amine-coated slides with the surface zeta potential of 18.4mV reflected the cell oxidative state caused by t-butylhydroperoxide in vitro. In addition, the method was used to monitor the peripheral blood of diabetic Akita mice, whose risk of diabetic complications was interminably increased. The erythrocytes’ adhesion of diabetic Akita mice significantly decreased compared with that of control mice. The results of the adhesion assay correlated with the results of serological assays.

Keywords: Oxidative stress; Erythrocytes; Adhesion

Immobilization of high catalytic acid protease on functionalized mesoporous activated carbon particles by A. Ganesh Kumar; S. Swarnalatha; P. Kamatchi; G. Sekaran (pp. 185-190).

The functionalized mesoporous activated carbon (FMAC) was used as support material for immobilization of acid protease (AP). Immobilization of acid protease on functionalized mesoporous activated carbon (AP–FMAC) performs as a suitable enzyme carrier. Under optimized condition pH (6.0) acid protease 150mgg⁻¹ FMAC has been adsorbed. The optimum temperature for both free and immobilized AP activities was 50°C. After incubation at 50°C, the immobilized AP maintained 50% of its initial activity, while the free enzyme was completely inactivated. A significant catalytic efficiency was maintained along for more than five consecutive reaction cycles in AP–FMAC combination immobilized system. The functional groups of the AP, FMAC and AP–FMAC were observed by Fourier transformer infrared spectroscopy (FT-IR). The scanning electron microscopy (SEM) allowed us to observe the morphology of the surface of FMAC and the AP–FMAC.

Keywords: Immobilization; Immobilized enzymes; Enzymes; Protease

Simultaneous nitrification and denitrification coupled with phosphorus removal in an modified anoxic/oxic-membrane bioreactor (A/O-MBR) by Zhimin Fu; Fenglin Yang; Yingyu An; Yuan Xue (pp. 191-196).

In this study, simultaneous carbon, nitrogen and phosphorus (PO₄³⁻-P) removals using an anoxic/oxic-membrane bioreactor (A/O-MBR) system without any circulation was investigated. The total nitrogen (TN) removal efficiency was above 84.6%, and the average removal efficiencies of COD and PO₄³⁻-P were 94.6 and 90.0%, respectively. Ammonium (NH₄⁺-N) removal without nitrite (NO₂⁻-N) and nitrate (NO₃⁻-N) accumulation in the reactor suggested that simultaneous nitrification and denitrification (SND) occurred. The phosphorus-accumulating organisms (PAOs) accumulated in the reactor. AnOx/Oxic test indicated that intracellular organic carbon might be utilized as denitrification electron donor in nitrogen removal process.

Keywords: Biological nitrogen removal; Biological assimilation; Phosphorus-accumulating organisms; Poly-β-hydroxybutyrate; Membrane bioreactor

Optimization of refolding with simultaneous purification of recombinant human granulocyte colony-stimulating factor from Escherichia coli by immobilized metal ion affinity chromatography by Chaozhan Wang; Lili Wang; Xindu Geng (pp. 197-202).

Immobilized metal ion affinity chromatography (IMAC) is a new technique for protein refolding, but it is limited to the refolding of fusion proteins with histidine affinity tags. In the present work, a non-fusion recombinant protein, recombinant human granulocyte colony-stimulating factor (rhG-CSF) expressed in Escheriachia coli ( E. coli) in the form of inclusion bodies was successfully refolded with simultaneous purification by IMAC. rhG-CSF inclusion bodies solubilized in 8.0mol/L urea was injected into a Cu(II)-iminodiacetic acid (IDA)-IMAC column, the soluble and active form of rhG-CSF in aqueous solution was obtained after desorbed from the column by linear increase of imidazole concentration. Several factors in the refolding process, including urea concentration and pH of mobile phases, type of buffer, glycerol concentration and loading sample volume, were investigated, respectively. When 200μL of denatured/reduced rhG-CSF solution at a total protein concentration of 2.8mg/mL was loaded on the IMAC column, rhG-CSF with a specific activity of 2.3×10⁸IU/mg and a mass recovery of 39% was obtained after IMAC refolding, and rhG-CSF was also purified during this chromatographic process, its purity was determined to be 97%.

Keywords: Recombinant human granulocyte colony-stimulating factor; Inclusion bodies; Protein refolding and purification; Immobilized metal ion affinity chromatography; Protein folding liquid chromatography

Optimization of pH and acetic acid concentration for bioconversion of hemicellulose from corncobs to xylitol by Candida tropicalis by Ke-Ke Cheng; Jian-An Zhang; Hong-Zhi Ling; Wen-Xiang Ping; Wei Huang; Jing-Ping Ge; Jing-Ming Xu (pp. 203-207).

Hemicellulose hydrolysate from corncobs, separated by diluted sulfuric acid and sequently detoxed by boiling, overliming and solvent extraction, was used for xylitol production by Candida tropicalis W103. The effect of glucose and acetate in hydrolysate on xylitol production was investigated. It was found that glucose in hydrolysate promoted growth of Candida tropicalis while acetate at high concentration was inhibitory. The acetate inhibition can be alleviated by adjusting pH to 6 prior to fermentation and a substrate feeding strategy. Under these optimum conditions, a maximal xylitol concentration of 68.4gl⁻¹ was obtained after 72h of fermentation, giving a yield of 0.7gg⁻¹ xylose and a productivity of 0.95gl⁻¹h⁻¹.

Keywords: Corncob; Fermentation; Hydrolysate; Pretreatment; Xylitol; Xylose

The acid hydrolysis of potato tuber mash in bioethanol production by Marija B. Tasić; Budimir V. Konstantinović; Miodrag L. Lazić; Vlada B. Veljković (pp. 208-211).

The hydrolysis of starch from fresh potato tubers by HCl and H₂SO₄ at different ratios of plant material to acid solution was investigated. The final reducing sugars concentration in the hydrolyzates depended on the type and concentration of acid and the ratio of plant material to acid solution but not on the type of potato. The highest dextrose equivalent of 94%, the greatest reaction rate constant of 0.089min⁻¹ and the 5-hydroxymethylfurfural (5-HMF) yield of 0.04g/L were achieved using 1M HCl at the ratio of plant material to acid solution of 1:2 (w/v). The ethanol yield of 31g/L was obtained in the fermentation of hydrolyzate prepared under the optimal hydrolysis conditions by commercial bakery yeast at 28°C for about 18h.

Keywords: Acid hydrolysis; Ethanol; Glucose; Production kinetics; Potato tuber; Starch

Isolation and screening of a novel extracellular organic solvent-stable protease producer by Yaowei Fang; Shu Liu; Shujun Wang; Mingsheng Lv (pp. 212-215).

Using enrichment procedures, 68 organic solvent tolerant strains were screened from sea mud samples. Twelve of these strains demonstrated high protease activity on skim-milk agar. Among them, the DS11 isolate was selected based on the stability of its proteolytic enzyme in the presence of various organic solvents and later identified as Bacillus sphaericus by morphological, physiological, biochemical tests and 16S rDNA gene sequence analysis. Strain DS11 was able to sustain and grow in a wide range of organic solvents. The crude protease also exhibited remarkable solvent stability and retained most of the activity at least up to 14 days at 37°C and 200rpm in the presence of various organic solvents at 25% (v/v) concentration. More than 80% activity was recovered when the log P value of organic solvents were from 1.8 to 3.5, whereas in methanol ethanol, and 1-butanol the residual activity was 35%, 36%, and 42%, respectively. However, when n-decane, octane, isooctane, and heptane, of which the log P values are equal to or more than 4.0 were added, the activities of the proteases was enhanced. This organic solvent-stable protease could be used as a biocatalyst for peptide synthesis in organic media.

Keywords: Biocatalysis; Microbial; Enzyme; Biocatalyst reparation; Screening; Organic solvent-stable protease

Expression and in situ processing of human prorenin to active renin in baculovirus-infected Sf-9 insect cell cultures under several infective conditions by Takeshi Gotoh; Hirono Awa; Ken-Ichi Kikuchi; Saori Takahashi (pp. 216-220).

In the baculovirus expression vector system (BEVS), intrinsic proteases concomitantly produced by infected insect cells have been generally regarded as a defect, because they sometimes degrade expressed recombinant proteins and decrease the productivity. The present study successfully used the proteolysis to generate active recombinant human- (rh) renin after the expression of inactive rh-prorenin. Sf-9 insect cells were infected with recombinant baculoviruses having human preprorenin cDNA in the site of polyhedron gene at several MOIs. At any MOIs, rh-prorenin was expressed in a late phase of infective cultures and processed to active rh-renin in a very late phase. The maximum volumetric yield of active rh-renin was obtained at MOIs of 1 and 10pfu/cell. The protease activity was examined with an internally quenched fluorogenic substrate newly designed for the processing. The generation of rh-renin was coincided with a considerable increase in a protease activity that was classified into the cysteine protease family, and significantly suppressed by supplementing the culture medium with leupeptin, a cysteine protease inhibitor. This suggested that the cysteine protease was responsible to the processing of rh-prorenin to rh-renin.

Keywords: Sf-9 insect cell; Baculovirus; Protease; Renin; Prorenin

C-phycocyanin from Spirulina fussiformis exposed to blue light demonstrates higher efficacy of in vitro antioxidant activity by H.K. Madhyastha; S. Sivashankari; T.M. Vatsala (pp. 221-224).

In this study we made the comparative evaluation of antioxidant activities of C-phycocyanins produced from Spirulina fussiformis exposed to blue light (C-pc (B)) and normal light (C-pc (N)), by various antioxidant assays such as 2,2-azino-bis(3)ethylbenzthiazoline-6-sulfonic acid (ABTS), 1,1-diphenyl-2-picrylhydrazyl (DPPH), super oxide anion (O₂⁻) scavenging, hydroxy radical (OH) scavenging, hypochlorous acid (HOCl) scavenging, ferrous oxidation xylenol orange (FOX), oxygen radical absorbance capacity (ORAC), ferric reducing ability of plasma (FRAP) and reducing power assays. The observed antioxidant activities were compared with that of ascorbic acid, a standard antioxidant. C-pc (B) exhibited significantly ( p<0.05) higher radical scavenging effect than in DPPH, OH, HOCl, ORAC and FRAP assay conditions in a dose-dependent manner. Higher reducing power of C-pc (B) demonstrated a dose-dependent pattern and indicated the presence of free radical scavenger moieties. Furthermore, sequencing of α and β chains of both C-pcs revealed marginal changes in α chains of both kinds of C-pcs and realignment of sequences from 76 to 97 amino position in β chain of C-pc (B) indicating modification of the polypeptide with higher cysteine. In conclusion the present study demonstrated that C-pc (B) showed higher antioxidant activity.

Keywords: Abbreviations; C-pc; C-phycocyanin; C-pc (B); C-phycocyanin produced from; Spirulina fussiformis; exposed to blue light; C-pc (N); C-phycocyanin produced from; Spirulina fussiformis; exposed to normal (white) lightC-phycocyanin; Antioxidation; α and β chains; Amino acid sequence; Spirulina fussiformis

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Biochemical Engineering Journal (v.43, #2)