Biochemical Engineering Journal (v.41, #2)

BEJ Keywords (II).

Design and in vivo evaluation of solid-in-oil suspension for oral delivery of human growth hormone by Hiromu Yoshiura; Yoshiro Tahara; Masakazu Hashida; Noriho Kamiya; Akihiko Hirata; Takeru Fujii; Masahiro Goto (106-110).
Solid-in-oil (S/O) suspension containing human growth hormone (hGH), in which hGH was complexed with an edible surfactant, was designed and validated for oral administration of hGH. To optimize the formulation procedures, protein release behavior from the S/O suspensions under physiological conditions was first investigated with horseradish peroxidase (HRP) as a model protein. Evaluation of functional integrity of HRP released from the HRP–surfactant complex suggested that the solubilization process partly impaired the specific activity of HRP, however, the addition of trehalose in the formulation process regained up to about 50% of the biological activity. On the basis of the data collected with HRP, a surfactant–hGH complex prepared under optimized conditions was suspended in soybean oil to formulate S/O suspensions. After oral administration of the S/O suspension to male New Zealand rabbits, we detected hGH in the serum with 3.3% bioavailability, suggesting that hGH can be orally delivered to the systemic circulation by the present formulation.
Keywords: Oral protein delivery; Drug delivery system; Biomedical; Microcarriers; Protein; Amphiphilic materials;

Utilizing whole cell biocatalyst for biodiesel production has some advantages since it can avoid the complex procedures of isolation, purification and immobilization of extracellular lipase. However, during repeated use of Rhizopus oryzae (R. oryzae) IFO4697 whole cell for biodiesel production in solvent-free system, the whole cell exhibited very poor stability; while the whole cell stability has been found to be significantly improved in tert-butanol system compared to that in solvent-free system. The difference in whole cell stability was found to be due to the difference of product accumulation between solvent-free and tert-butanol system. After 144 h reaction, glycerol and methyl ester accumulated in the cell in solvent-free system came up to about 1000 mg/g and 350 mg/g dry biomass, respectively, while in tert-butanol system, glycerol and methyl ester accumulation was kept at a relatively low level, approximately 100 mg/g and 2 mg/g dry biomass, respectively. The accumulated glycerol influenced whole cell stability through mass transfer limitation only, while the accumulated methyl ester influenced whole cell stability through both mass transfer limitation and product inhibition. Further study showed that a slight loss in enzymatic activity in tert-butanol system was caused by protein leakage.
Keywords: Biodiesel; Immobilized cells; Stability; Bioconversion; Enzyme biocatalysis; Product inhibition;

Both vasoactive intestinal peptide (VIP) and humanin (HN) can provide neuroprotection against β-amyloid toxicity and are believed to be beneficial in the treatment of Alzheimer's disease. For the sake of tandem co-expression of recombinant human VIP and HN(rhVIP-HN) fusion protein, new VIP-HN gene is constructed and cloned into the plasmid pET28a(+) and is expressed in Escherichia coli BL21(DE3). Since the fusion protein was present as inclusion bodies, three different preparation approaches are employed to obtain the hexa-histidine tagged rhVIP-HN (His6-rhVIP-HN) fusion protein by Ni2+ chelating resin chromatography. The comparison of the results obtained from the three approaches reveals that renaturation during the separation process (Approach III) is the most efficient for large-scale preparation of His6-rhVIP-HN. Ultimately, 46 mg of the target protein was obtained from one gram of the inclusion body with purity up to 90%. Since there is not separated step in renaturation procedure, Approach III could be more time-saving, buffer-saving and easy to operate than the other two approaches. These results can be useful for preparation of rhVIP-HN in large scale.
Keywords: Bioseparations; Protein denaturation; Refolding; Protein recovery; Recombinant DNA; Vasoactive intestinal peptide;

Biosurfactants have gained attention because they exhibit some advantages such as biodegradability, low toxicity, ecological acceptability and ability to be produced from renewable and cheaper substrates. They are widely used for environmental applications for bioremediation and also in biomedical field. However, the high cost of production is the limiting factor for widespread industrial applications. Thus, optimization of the growth medium for biosurfactant-lichenysin production by Bacillus licheniformis R2 was carried out using response-surface methodology. A preliminary screening phase based on a two-level fractional factorial design led to the identification of NH4NO3, glucose, Na2HPO4 and MnSO4·4H2O concentrations as the most significant variables affecting the fermentation process. The 24 full-factorial central composite design was then applied to further optimize the biosurfactant production. The optimal levels of the aforementioned variables were (g/l): NH4NO3, 1.0; glucose, 34.0; KH2PO4, 6.0; Na2HPO4, 2.7; MgSO4·7H2O, 0.1; CaCl2, 1.2 × 10−3; FeSO4·7H2O, 1.65 × 10−3; MnSO4·4H2O, 1.5 × 10−3 and Na–EDTA, 2.2 × 10−3. With the optimization procedure, the relative lichenysin yield expressed as the critical micelle dilution (CMD) was fourfold higher than that obtained in the non-optimized reference medium.
Keywords: Bioremediation; Optimization; Bacillus licheniformis; Fermentation; Lichenysin;

Substrate and product inhibition kinetics in succinic acid production by Actinobacillus succinogenes by Sze Ki Carol Lin; Chenyu Du; Apostolis Koutinas; Ruohang Wang; Colin Webb (128-135).
The inhibition of substrate and products on the growth of Actinobacillus succinogenes in fermentation using glucose as the major carbon source was studied. A. succinogenes tolerated up to 143 g/L glucose and cell growth was completely inhibited with glucose concentration over 158 g/L. Significant decrease in succinic acid yield and prolonged lag phase were observed with glucose concentration above 100 g/L. Among the end-products investigated, formate was found to have the most inhibitory effect on succinic acid fermentation. The critical concentrations of acetate, ethanol, formate, pyruvate and succinate were 46, 42, 16, 74, 104 g/L, respectively. A growth kinetic model considering both substrate and product inhibition is proposed, which adequately simulates batch fermentation kinetics using both semi-defined and wheat-derived media. The model accurately describes the inhibitory kinetics caused by both externally added chemicals and the same chemicals produced during fermentation. This paper provides key insights into the improvement of succinic acid production and the modelling of inhibition kinetics.
Keywords: Fermentation; Product inhibition; Substrate inhibition; Growth kinetic; Succinic acid; Actinobacillus succinogenes;

Influence of biogas-induced mixing on granulation in UASB reactors by Puspendu Bhunia; M.M. Ghangrekar (136-141).
Studies have been carried out to correlate biogas-induced mixing and granulation in upflow anaerobic sludge blanket (UASB) reactors, treating low-strength as well as high-strength biodegradable wastewaters. A dimensionless granulation index (GI) has been framed taking into account the mixing in sludge bed due to produced biogas. Analysis of full-scale, pilot-scale and lab-scale UASB reactors treating actual wastewaters reveals the significance of biogas-induced mixing, represented by GI, on granulation of biomass in the reactors. For obtaining proper granulation in UASB reactors (percentage granules greater than 50%, w/w), resulting in higher chemical oxygen demand (COD) removal efficiency, it is recommended to maintain GI values in the range of 15,000–57,000.
Keywords: Granulation; Granulation index (GI); Mixing; UASB; Velocity gradient;

The carboxyl groups of the bifunctional cellulase–chitosanase (CCBE), purified from a commercial cellulase prepared from Trichoderma viride were modified using the water-soluble carbodiimide 1-ethyl-3-(3-dimethyl-aminopropyl) carbodiimide (EDC). The EDC modified CCBE lost 80–90% of its chitosnase activity and 20% of its carboxylmethyl cellulase (CMCase) activity; meanwhile, its conformation changed slightly, which altered the substrate binding affinity to chitosan, without affecting its binding to CMC. However, the modification did not alter the structure integrity. The dynamic analysis of modification indicated that the CCBE possessed two carboxylates essential for its chitosanase activity and one carboxyl group for its CMCase activity. One of the two carboxylates involved in chitosanase activity was deduced to be the proton donator, and the other may function for substrate recognition, while the only catalytic carboxyl group for CMCase activity probably also acted as a proton donator.
Keywords: Chemical modification; Chitosanase; Cellulase; Active site; Lysozyme; EDC;

Improvement of olive oil phenolics content by means of enzyme formulations: Effect of different enzyme activities and levels by Danilo De Faveri; Bahar Aliakbarian; Milvio Avogadro; Patrizia Perego; Attilio Converti (149-156).
The present work deals with the development of an enzymatic treatment aiming at producing high-quality olive oil with increased phenolics content and antioxidant activity. Three different enzyme formulations, specifically pectinase, hemicellulase and cellulase (A), pectinase and hemicellulase (B), and only pectinase (C), were used either at three different level of each or in ternary and binary mixtures at a constant level. All of them were added to the olive paste (Italian cultivar Coratina) at the beginning of the malaxation step. Results demonstrated that an increased enzyme level led to higher phenolics content in the oils, and such an effect was found to be enzyme dependent, being greater when using formulation A, followed by formulations B and C. Two significant correlations were obtained between total polyphenols and o-diphenols contents versus antiradical power (R 2  = 0.8743 and R 2  = 0.8635, respectively), which pointed out the effectiveness of the proposed enzymatic treatment to produce olive oils characterized by low susceptibility to oxidation. A synergistic effect between the different enzymatic activities contained in the single formulations was observed by combining enzymes A and B. The ternary mixture was selected as the most efficient enzymatic system ensuring the highest phenolics content increase (40 and 37% for total polyphenols and o-diphenols contents, respectively) compared to the other enzymes when used at the same level.
Keywords: Nutraceutical products; Olive oil; Malaxation; Enzyme; Phenolics; Antioxidant activity;

This study was undertaken to find the optimum conditions of a new enzymatic process to remove plant residues from wool. Commercial enzymatic preparations of Celluclast 1.5 L and Pectinex Ultra SP-L were selected in order to hydrolyze the polysaccharides in primary plant cell walls and middle lamella, resulting into more fragile residues easier to be removed. Since it was intended to define the optimal conditions for enzyme application, a four-factor central composite design was selected to study the effects of pH, temperature, enzyme concentration and wetting agent concentration, on the two selected responses, i.e., soluble reducing sugars (RS) and alkali solubility (AS) of wool to detect plant degradation and to evaluate wool quality, respectively. Results demonstrated that enzyme concentration was the most significant effect in plant residues degradation. A total enzyme concentration loading of 20 mL of both diluted enzymatic preparations in equal parts per 1 L of incubation solution (42.970 U/L of Celluclast preparation and PG 29.3 nkat/L + PME 2.537 nkat/L of Pectinex preparation), yielded an equivalent amount of 240.127 mg of glucose per 1.0 g of plant residue, at the optimal conditions: 40.56 °C, pH 4.0 and 1 mL Plurafac/L. SEM analysis has indicated an identical and important degradation of the plant residues, when compared to the conventional carbonization process, and wool quality has been preserved.
Keywords: Biodegradation; Cell-wall-polysaccharides; Enzyme technology; Response surface methodology; Modeling; Optimization;

Biodegradation of carbazole in oil/water biphasic system by a newly isolated bacterium Klebsiella sp. LSSE-H2 by Yu-Guang Li; Wang-Liang Li; Jie-Xun Huang; Xiao-Chao Xiong; Hong-Shuai Gao; Jian-Min Xing; Hui-Zhou Liu (166-170).
A novel Klebsiella sp. strain LSSE-H2 (CGMCC No. 1624) was isolated from dye-contaminated soil based on its ability to metabolize carbazole as a sole source of carbon and nitrogen. This strain efficiently degraded carbazole from either aqueous and biphasic aqueous–organic media, displaying a high denitrogenation activity and a high level of solvent tolerance. LSSE-H2 could completely degrade 12 mmol/L carbazole after 56 h of cultivation. The co-culture of LSSE-H2 and Pseudomonas delafieldii R-8 strains can degrade approximately 92% of carbazole (10 mmol/L) and 94% of dibenzothiophene (3 mmol/L) from model diesel in 12 h.
Keywords: Biodegradation; Biocatalysis; Carbazole; Microbial growth; Oil/water biphasic system; Biodesulfurization;

The potential of carbonaceous Hibiscus cannabinus L. plant for use in the treatment of oil- and heavy metal-contaminated water is explored. The results from this work demonstrated that the material from this source was capable of sequestering oil and metal ions from the aqueous solutions. The maximum sorption to saturation capacities for diesel and cooking oil were 35 and 30 g g−1, respectively, well above that of the commercial adsorbent. The carbonaceous material was also effective for sequestering Mn2+, Cu2+ and Fe2+. The equilibrium of metal ions adsorption was attained after 30 min for Mn2+ and Cu2+, and 60 min for Fe2+ solutions. The sorption of the metal ions was in the order of Mn2+  > Cu2+  > Fe2+, increased with increase in the dosage in the range between 60% and 92% removal, depending on the dosage amount. The quantitative removal of Mn2+, Cu2+, and Fe2+ at pH 4.5, 50 mg L−1 initial concentration after 150 min equilibration time was 91.2%, 86.0% and 81.0%, respectively.
Keywords: Adsorption; Affinity; Absorption; Kinetic parameters; Separation; Waste-water treatment;

An indigenous phenol-degrading bacterial isolate Cupriavidus taiwanensis R186 was used to degrade phenol from an aqueous solution under fed-batch operation. An exponential feeding strategy combined with dissolved oxygen control was applied based on kinetic characteristics of cell growth and phenol degradation to meet sufficient metabolic needs for cellular growth and achieve the best phenol removal efficiency. Without the stress of phenol inhibition, the optimal set point of specific growth rate of exponential feeding for fed-batch phenol degradation was found to be 0.50–0.55μ max (μ max denotes the maximum specific growth rate from Monod model). Meanwhile, the sufficient set point of dissolved oxygen for maximal phenol degradation efficiency was approximately at 10–55% air saturation. With the optimal operation conditions, the best phenol degradation rate was 0.213 g phenol h−1, while a shortest treatment time of 15 h was achieved for complete degradation of 11.35 mM (ca. 3.20 g) of phenol.
Keywords: Biodegradation; Bioreactors; Cupriavidus taiwanensis; Exponential feeding; Fed-batch culture; Fermentation; Phenol;

Different induction strategies for fed-batch recombinant protein production under the control of the strong T5 promoter in Escherichia coli have been investigated. Since the production of recombinant rhamnulose 1-phosphate aldolase is growth-related, the productivity of the process can be strongly reduced due to the negative effect of protein expression on cell growth. IPTG pulse induction as well as inducer dosage have been applied and their advantages and drawbacks highlighted. Both strategies led to high levels of the recombinant protein, 1000 AU g DCW−1. Inducer concentration and inducer to biomass ratio were identified as the parameters influencing the rate of protein production and final enzymatic activity per gram of biomass. In pulse induction, the maximum enzymatic activity was found at inducer concentration of 70 μM. In continuous induction experiments, inducer concentrations between 4 and 12 μM were identified as the working range in which cell growth and recombinant protein accumulation occurred simultaneously. On the other hand, the amount of IPTG per gram of biomass required was 1.6 μmol IPTG gDCW−1 in pulse induction and between 0.3 and 0.5 μmol IPTG g DCW−1 in continuous induction.
Keywords: Induction strategies; Recombinant protein; E. coli; Fed-batch cultures; Continuous inducer dosage; Aldolase;

Neutralization of acidified water by use of Rhizopus delemar immobilized with a cellulose membrane by Risako Nabeshima; Noriko Kusumoto; Shigeo Katoh; Naofumi Shiomi (188-192).
To solve serious environmental problems caused by the acidification of pond and lake water by acid rain, remediation methods must be used to keep water pH values neutral. In this study, a microbial method to neutralize acidified water was developed. The neutralization activities of 30 strains of bacteria, yeasts and fungi were measured with a medium adjusted to pH 3.0. Because fungi showed high neutralization properties, the Rhizopus delemar fungus was used to study the characteristics of acidified water neutralization. When R. delemar cells were cultured in a media acidified with nitric, sulfuric and hydrochloric acids, the cells neutralized acids by secreting basic compounds including ammonia. The cells also assimilated nitric acid. R. delemar was used to neutralize pond water adjusted to pH 4.0 with nitric acid. R. delemar cells increased the pH value of pond water from 4.0 to around 7.0 within 2 days, although indigenous microorganisms had not been able to neutralize the same pond water. In this study, R. delemar immobilized in a cellulose tube neutralized acidified water repeatedly by the draw-fill method.
Keywords: Rhizopus delemar; Acid rain; Neutralization;

Preparation of vanillin by bioconversion in a silicon rubber membrane bioreactor by Yun-Tao Wu; Ming Feng; Wen-Wu Ding; Xiao-Yu Tang; Yue-Hua Zhong; Ze-Yi Xiao (193-197).
In this study, the bioconversion of clove oil into vanillin using soybean lipoxygenase (SBLOX) as biocatalyst was investigated in a silicon rubber membrane bioreactor (SRMBR) and shaking flasks. High performance liquid chromatography (HPLC) analysis indicated that the vanillin concentration was 8.14 mg/L after 36 h of conversion in a shaking flask. It reached up to 121.53 mg/L in the receiving solution after 36 h of conversion in the SRMBR. The conversion rate of clove oil was 0.033% in the shaking flask. It was 1.01% in the SRMBR. The peak area ratio of vanillin in the receiving solution of the SRMBR was 70.08%. By adding activated carbon into the conversion broth of the SRMBR, the vanillin concentration in the receiving solution reached 140.27 mg/L, the conversion rate of clove oil increased to 1.14%, and the peak area ratio of vanillin in the receiving solution reached 93.53%.
Keywords: Bioreactor; Vanillin; Clove oil; Bioconversion; Product inhibition; Separation;

Recirculation of the leachate in the acidogenic reactor was proposed to enhance anaerobic digestion of food waste in the hybrid anaerobic solid–liquid (HASL) system. Recirculation of the leachate in the acidogenic reactor provided better conditions for extraction of organic matter from the treated food waste and buffering capacity to prevent excessive acidification in the acidogenic reactor. It ensured faster supply of nutrients in the methanogenic reactor in experiment. The highest dissolved COD and VFA concentrations in the leachate from the acidogenic reactor were reached for shorter time and were 16,670 mg/l and 9450 mg/l in control and 18,614 mg/l and 11,094 mg/l in experiment, respectively. Recycling of the leachate in the acidogenic reactor intensified anaerobic digestion of food waste and diminished time needed to produce the same quantity of methane by 40% in comparison with anaerobic digestion of food waste without recirculation.
Keywords: Anaerobic digestion; Food waste; Hybrid anaerobic solid–liquid (HASL) system; Recirculation; Acidogenic reactor;