Biochemical Engineering Journal (v.52, #1)

BEJ Keywords (II).

In this study, an optimization of xylanase expression by recombinant Pichia pastoris was carried out with a 23 factorial design. The influence of initial cell density, methanol and yeast nitrogen base concentration was evaluated, and the cell density was found to be the most important parameter. Considering with the yield of enzyme activity and the amount of methanol and YNB used for fermentation, the optimal conditions in this study for enzyme production were 0.5% (w/v) methanol, 0.8% (w/v) YNB, and initial cell density of 3 × 108  cells/ml. The enzyme production in 15-l fermenter with the optimized conditions was performed and a twofold higher xylanase activity was obtained. The produced enzyme was practically free of cellulolytic activity. The molecular mass (21 kDa), optimal pH (6.0) and substrate specificity of the enzyme were both identical to native enzyme. However, the thermostability of the recombinant enzyme was better than the native enzyme. The optimal temperature for the recombinant Xyn2 was 5 °C higher than native Xyn2, and remained at least 90% of its activity after 30 min incubation at 55 °C. The high level of fully active recombinant xylanase obtained in P. pastoris makes this expression system attractive for fermenter growth and industrial applications.
Keywords: Xylanase; Expression; Pichia pastoris; Trichoderma reesei; Optimization; Fermentation;

Dynamic modeling of partial nitrification in a rotating disk biofilm reactor: Calibration, validation and simulation by César Huiliñir; Rodrigo Romero; Carlos Muñoz; Christian Bornhardt; Marlene Roeckel; Christian Antileo (7-18).
Partial nitrification to nitrite saves oxygen and organic matter. A dynamic model of a rotating disk biofilm reactor was developed in order to determine optimal operation conditions for partial nitrification. The biofilm model considered the active biomass fraction as a state variable; it was calibrated at steady state using the adjustment of two parameters –μ max,AOB (1.32 d−1) and k L (0.44 m h−1) and validated with long-term experiments. The experimental data were attained running the reactor in continuously and in batch modes for more than 700 days at different pH values, oxygen concentrations and nitrogen load rates (NLRs). A good agreement between the measured and modeled results was obtained with a Theil inequality coefficient lower than 0.3 for both calibration and validation. The model predicts that the active biomass fraction does not reach steady-state before 40 operating days, and the active nitrite-oxidizing bacteria fraction is much more sensitive than the ammonia-oxidizing bacteria fraction to the pH. Results demonstrated that nitrite accumulation (β) is strongly influenced by the NLR and the pH. The optimal operation zone for β  > 70% is reached for NLRs between 8.5 and 11.5 g N m−2  d−1, pH > 8 and dissolved oxygen concentration <3 mg O2  L−1.
Keywords: Partial nitrification; Nitrite accumulation; RBC; Biofilm; Modeling;

The production of a novel thermostable amidase (EC 3.5.1.4) from Geobacillus pallidus RAPc8 using recombinant Escherichia coli BL21 (DE3) was investigated. Volumetric and specific enzyme activities were investigated in relation to inducer concentration in a batch process using a defined medium with glucose as the carbon source. While IPTG is routinely used to induce expression of genes under the control of lac promoter, the impact of high biomass concentration on IPTG induction has not been reported rigorously. In this study, biomass production was unaffected by IPTG concentration across the range 0–1000 μM. Induction of recombinant protein expression by 400 μM IPTG at late lag phase of growth (3rd hour) inhibited cell growth while induction at early exponential phase of growth (5th hour) gave a 3 fold increase in volumetric amidase activity compared to induction at mid exponential phase (8th hour). Protein production increased by a factor of two with IPTG addition, independent of IPTG concentration in the range of 40–1000 μM. Amidase activity, measured on a volumetric basis and relative to protein and biomass concentrations, increased with increasing IPTG concentration up to 400 μM. While inducer concentrations are typically reported on a volumetric basis, their mode of action is consistent with a biomass dependence. Analysis of the data across a range of biomass concentration confirmed that induction was a function of inducer concentration per unit biomass. The amidase enzyme was predominantly soluble and cytoplasmic with less than 3% retained within the cell debris.
Keywords: Thermostable amidase; Iso-propyl-β-d-1-thiogalactopyranoside (IPTG); Volumetric amidase activity; Heterologous protein expression;

Biological modeling of wastewater processes has gained importance for their design, control and operation. However, this requires consistent information on the biological activity. Microcalorimetry has also been developed to investigate biological processes and measure bacterial metabolisms. This measurement is done on the basis of the heat (produced or consumed) and therefore has a wider range of applicability compared to other methods based on the chemical analyses of substrate consumption or product formation. For this purpose, a 2 L Bio-RC1 (Mettler-Toledo) has been modified to reach a resolution of 5–10 mW L−1 and applied to research projects on activated sludge characterization. Several calorimetric applications are presented regarding studies of biomass activity (useful to achieve kinetic and stoichiometric coefficients) and pollutants degradation in aerobic, anoxic and anaerobic conditions.
Keywords: Wastewater treatment; Biological activity; Activated sludge; Microcalorimetry; Inhibition; Adaptation;

The biotechnologically important enzyme laccase (benzenediol: oxygen oxidoreductase; EC 1.10.3.2) is secreted by white rot fungi. However, these organisms produce insufficient amount of laccase for use in various biotechnological areas. The main aim of this study is to enhance the laccase production in the repeated-batch cultures of Funalia trogii ATCC 200800 and Trametes versicolor ATCC 200801 isolated in Turkey. In this study, laccase production in the repeated-batch cultures of F. trogii and T. versicolor pre-grown pellets were investigated under various conditions. Retention time, temperature, agitation, pH and the amount of pellets were found to be important for laccase production during repeated-batch studies. The culture filtrate showed one protein band. Zymogram gel also showed only one band of activity. The enzyme remained fully active when frozen for 300 days. The immobilized fungi were also able to produce high amounts of laccase during reuse. It is possible to obtain high laccase amounts with free and immobilized repeated-batch cultures under the most appropriate culture conditions determined. Considering the various biotechnological applications of laccase, an enhancement in laccase production through the selection of appropriate culture conditions could facilitate the development of more economical and environmentally friendly processes.
Keywords: Immobilization; Laccase; Pellet; Repeated-batch; White rot fungi;

Cationic liposome can interfere mRNA translation in an E. coli cell-free translation system by Huong Thi Bui; Hiroshi Umakoshi; Keishi Suga; Tomoyuki Tanabe; Kien Xuan Ngo; Toshinori Shimanouchi; Ryoichi Kuboi (38-43).
The effect of cationic liposome prepared from 1,2-dioleoyl-3-trimethyl- ammonium-propane (DOTAP) on the gene expression at the mRNA translation level was investigated using an E. coli cell-free translation system. DOTAP liposome at 3 mM inhibited the mRNA translation of green fluorescent protein (GFP), as indicated both by the fluorescence intensity of GFP and by SDS-PAGE analysis. The role of DOTAP liposome on the inhibition of mRNA translation was revealed that the cationic quaternary amine groups on the liposome surface can interact and neutralize the anionic phosphate groups on mRNA by an electrostatic interaction. mRNA molecules still existed without any degradation in the presence of DOTAP liposome although it could not be translated. These results clearly illustrated that the DOTAP liposome could knock down mRNA and silence its activity of translation in an E. coli cell-free system.
Keywords: Membrane stress biotechnology; Liposome; GFP; mRNA; Translation system; Cell free;

Lysis of bacterial cells is considered a key step in biological treatment process of waste activated sludge. Most cells in waste activated sludge are long-cultured cells in stationary- or death-phases, which are proved more difficult to be lysed than actively-growing cells. However, few attentions have been paid to the lysis of long-cultured cells. In this study, synergistic action of different lytic enzymes from two thermophilic bacteria, Geobacillus spp. SY9 and SY14, were discovered to promote the lysis of long-cultured cells. The combinational action of the lytic enzymes from the two thermophilic bacteria were significantly better than their individual action and the cell lysis rates at the optimized combinational ratio were higher than that of their individual action by 2–3 times. The results of selective inhibition test infer that the potential lytic enzymes of SY9 and SY14 are glycosidase and peptidase, respectively. Hence, the combinational action of different lytic enzymes is a potential measure to promote the lysis of long-cultured cells.
Keywords: Geobacillus; Stationary-phase cells; Lytic-enzyme; Sludge solubilization; Thermophilic bacteria; Waste activated sludge;

Rheological property of self-flocculating yeast suspension by L. Yu; H. Wang; L. Wang; F.W. Bai (50-54).
Using the online monitoring technique established for yeast flocs with the focused beam reflectance measurement system, the rheological property of the simulation system with self-flocculating yeast and water was examined. By controlling yeast flocs size relatively stable at the range from 350 to 470 μm, the impact of biomass concentration on the rheological property was first studied. Compared with regular yeast suspension, which is a Newtonian fluid in general, the suspension of yeast flocs exhibited non-Newtonian fluid behavior, from a pseudoplastic fluid with biomass concentration lower than 40 g/L to a yield pseudoplastic fluid as biomass concentration increased, since the higher the biomass concentration, the more resistant the suspension to shear force. On the other hand, it was found that, when yeast flocs size decreased to about 200 μm, a linear correlation between shear rate and shear stress was established, and the rheological property of the suspension was close to a Newton fluid, but non-Newtonian flow behavior developed when the size of yeast flocs increased to above that criterion, indicating its main contribution to the non-Newtonian flow behavior of the suspension. Based on these results and rheological model established with the simulation system, the rheological property of the ethanol fermentation system with the self-flocculating yeast was predicted.
Keywords: Self-flocculating yeast suspension; Rheological property; Ethanol fermentation;

1,1,2,2-Tetrachloroethane aerobic cometabolic biodegradation in slurry and soil-free bioreactors: A kinetic study by Dario Frascari; Martina Cappelletti; Stefano Fedi; Davide Zannoni; Massimo Nocentini; Davide Pinelli (55-64).
In this work the aerobic cometabolic biodegradation of 1,1,2,2-tetrachloroethane (TeCA) by propane-utilizing bacteria was studied in slurry bioreactors containing soil and groundwater from 5 aquifers as well as in soil-free bioreactors. The main goals were: (a) to identify and calibrate a kinetic model of TeCA cometabolism; (b) to select and characterize a TeCA-degrading bacterial consortium; (c) to compare the results obtained in slurry and in soil-free bioreactors. The results showed that 4 of the 5 tested aquifers contain TeCA-degrading bacteria, indicating that aerobic cometabolism is a potentially effective approach for TeCA-contaminated aquifers. In bioaugmentation tests, a TeCA-cometabolizing consortium developed in the slurry bioreactors induced a strong reduction of the lag-time for the onset of TeCA cometabolism. The soil-free tests yielded a satisfactory TeCA degradation performance, indicating that on-site soil-free bioreactors represent an interesting technical solution for the aerobic cometabolic bioremediation of CAH-contaminated groundwaters. The mineralization of the organic Cl was equal to about 97%. The prolonged TeCA biodegradation determined a progressive selection of the bacterial strains more effective in TeCA degradation and less affected by degradation product toxicity. The tested Michaelis–Menten-based kinetic model proved an effective tool to interpret the experimental data of TeCA aerobic cometabolism.
Keywords: Aerobic cometabolism; Tetrachloroethane; Bioremediation; Bioreactor; Kinetic model;

Influence of fermentation on the acid tolerance and freeze drying survival of Lactobacillus rhamnosus GG by Antonis Ampatzoglou; Benjamin Schurr; Gurjot Deepika; Sasitorn Baipong; Dimitris Charalampopoulos (65-70).
The aim of this work was to investigate the effect of fermentation pH and time on the acid tolerance, glycolytic activity, and survival during freeze drying of Lactobacillus rhamnosus GG. Two fermentations were performed, one was pH controlled (pH 6.8), and the other was pH uncontrolled. Cells were collected after 10 h (late exponential phase), 25 h (mid stationary phase) and 37 h (late stationary phase) of fermentation. In the case of the pH controlled fermentation, the acid tolerance of the cells collected at 10 h was remarkably high, as the viability decreased only by 0.2 logs; in the case of the 25 h and 37 h, it decreased 1.5 and 6.0 logs, respectively. In the case of the pH uncontrolled fermentation, for all three time points the cell reduction was high, ranging between 3 and 4 logs. The cells from the pH controlled fermentation, and in particular the cells from the 10 h sample, demonstrated considerably higher glycolytic activity compared to the cells from the uncontrolled fermentation. The type of fermentation had a lesser effect on the ability of the cells to survive during freeze drying, as in all cases the cell reduction was small (<1 log). The cells from the 10 h sample from the pH controlled fermentation survived significantly (P  < 0.05) better than the cells from the 25 and 37 h sample; the opposite was the case for the cells from the pH uncontrolled fermentation. However, in all cases, the differences between the samples were less than 0.5 log. In conclusion, both the fermentation pH and time were found to have a significant impact on the glycolytic activity of L. rhamnosus GG, and on its ability to survive under acidic conditions and during freeze drying.
Keywords: Fermentation; Lactobacillus rhamnosus GG; Freeze drying; Acid tolerance; Survival; Probiotics;

A xylanase purified from Streptomyces rameus L2001 and the biobleaching effect on wheat straw pulp was investigated. The extracellular xylanase was purified 13.3-fold by precipitation with 40–60% (NH4)2SO4, DEAE-52 and CM Sepharose Fast Flow ion exchange chromatography. It appeared as a monomeric protein on SDS-PAGE gel and had a molecular mass of approx. 21.1 kDa, with a specific activity of 3236.6 U/mg. The purified xylanase had an optimum pH of 5.3 and was stable over pH 4.3–6.7. The stable optimal temperature of the enzyme was 70 °C. The xylanase was activated by Co2+ by up to 329% of baseline activity. The xylanase was highly specific towards xylan, but did not exhibit other enzyme activity. Apparent K m values of the xylanase for birchwood and beechwood were 5.8 and 5.3 mg mL−1, respectively. The potential application of the xylanase was further evaluated in wheat straw pulp. The amount of reducing sugars released by the xylanase from wheat straw pulp was significantly greater with increasing time. Enzymatic treatment at a charge of 20 U/g dry pulp for 1 h before hypochlorite (3.8%) treatment revealed an increase in brightness index by 2.8% and increase in residual chlorine by 14.5%.
Keywords: Streptomyces rameus L2001; Characterization; Purification; Xylanase; Wheat straw pulp; Biobleaching;

Bioprocess design and economics of recombinant BM86/BM95 antigen production for anti-tick vaccines by Mario Canales; Juan A. Moreno-Cid; Consuelo Almazán; Margarita Villar; José de la Fuente (79-90).
Cattle ticks, Rhipicephalus (Boophilus) spp., infestations are a major veterinary problem that impacts cattle production worldwide. Recently, vaccination has emerged as an alternative to control tick infestations that has shown the advantage of being cost-effective, avoids environmental contamination and prevents selection of drug-resistant ticks resulting from repeated acaricides applications. However, the success of vaccination is highly dependent on the availability of effective vaccines at affordable prices. Recombinant DNA technology has provided the mean for producing antigens in large quantities making possible the development and commercialization of anti-tick vaccines. In the early 1990s, a technology was developed to produce a commercial anti-tick vaccine based on the recombinant BM86 (rBM86) antigen isolated from R. (Boophilus) microplus and intracellularly expressed in Pichia pastoris. However, the technological process for its production was expensive. Herein we conducted an economical analysis of this technology in comparison with other reported processes. The results allowed the identification of critical steps in the technology and addressed research for bioprocess optimization. As a result, two alternative bioprocesses were developed to produce anti-tick vaccines based on the rBM86 protein secreted in P. pastoris and by displaying antigenic peptides on the Escherichia coli membrane (BM95-MSP1a antigen). Vaccine production costs were reduced from US$0.92/dose for the intracellular rBM86 to US$0.72/dose and US$0.56/dose for the secreted rBM86 and BM95-MSP1a, respectively.
Keywords: Tick; Pichia pastoris; Boophilus microplus; Bm86; Bm95; Bioprocess economics;

Porcine interferon-α (pIFN-α) production by recombinant Pichia pastoris with standard induction strategy at 30 °C often suffers problems such as low antiviral activity, long cells adaptation period, etc. To solve these problems, a combinational induction strategy by simultaneously controlling induction temperature at 20 °C and dissolved oxygen concentration (DO) over 50% was proposed and the relevant fermentation runs were conducted in a 5 l bioreactor. With this control strategy, pIFN-α antiviral activity could be continuously enhanced and eventually reached a level of 3.62 × 107  IU/ml, which was about 16-fold of the maximum obtained when induction was done at 20 °C but without DO control, and more than 100-fold of the maximum obtained with the standard induction strategy at 30 °C. The novel control strategy could enhance alcohol oxidase (AOX) activity and relieve oxygen supply limitation in oxidative phosphorylation reaction to accelerate ATP regeneration simultaneously. As a result, the metabolic flux towards pIFN-α synthesis was enhanced and the adaptation period was shortened, enabling the entire system to be operated in a most efficient way.
Keywords: Induction; Metabolic analysis; On-line fed-batch; Optimization; Pichia pastoris; Recombinant protein production;

Stirring effect in a stirred membrane cell and the type of pumping action in a cross-flow filtration system were assessed from the viewpoint of recovery of polygalacturonase (PG) from Aspergillus carbonarius culture broth. PG and protein loss increased with stirring speed (shear stress) as well as processing time under simulated conditions of a stirred membrane cell. When the stirring conditions did not exceed the critical speed of 400 rpm, corresponding to a shear stress of 2.1 Pa, higher recoveries of PG (88%) and protein (96%) were obtained. Processing culture broth using a gear pump increased PG and protein loss with processing time and resulted in 71% PG and 54% protein loss after 3 h of pumping, while the loss of PG and protein was negligible when a peristaltic pump was used. The results revealed that processing shear sensitive enzymes like PG required determination of critical shear stress and selection of suitable pump to prevent losses in the membrane process.
Keywords: Aspergillus carbonarius; Polygalacturonase; Protein; Shear stress; Process pumps;

We tested the nitrogen removal ability of a dual bag system. The dual bag was constructed with an outer non-woven fabric bag and an inner non-porous polyethylene film bag. Nitrosomonas europaea and Paracoccus pantotrophus were immobilized on the outer bag and 23 mL of 99.5% ethanol was packed into the inner hermetic bag. The dual bag removes ammonia as follows: ammonia in the solution is oxidized to nitrite, and the produced nitrite is reduced to nitrogen gas by cooperation of the N. europaea and P. pantotrophus on the outer bag. Ethanol as an electron donor for denitrification is supplied to the P. pantotrophus from the inner bag. Ammonia solution (20 mg N L−1) flowed into a reactor tank (200 mL volume) with three dual bags was continuously treated for a hydraulic retention time of 2.6 h at 25 °C. Total nitrogen in the outflow of the reactor was kept below 4.0 mg N L−1 for more than 90 days without accumulating organic carbons. The dual bags removed 80.0% of the nitrogen from the solution without additional operations. The inner bag containing 23 mL of ethanol probably releases ethanol for approximately 1000 days without refilling since the inner bag released only 0.022 mL ethanol per day. The dual bag system would effectively simplify the nitrogen removal system since it is easily installed in vacant spaces of various waterways and tanks.
Keywords: Nitrification; Denitrification; Wastewater treatment; Polyethylene film;

Tissular model/sensor seamless system for qualified analysis and its characterization by Chisato Tanoue; Hitoshi Asakawa; Michael Cauchi; Conrad Bessant; Shinya Ikeno; Tetsuya Haruyama (110-115).
Clinical drug discovery in most cases begins with molecular screening in order to select a lead-substance. This process is a key step in successful drug development. Lead-substances are marked based on their ability to affect objective biological properties. In order to judge the molecular efficacy of a lead-substance, both animal experimentation and cell-based bioassay have been employed. However, in high throughput assay, cellular biosensing is one of the smart methods. In this study, an NO sensor device was developed which has two functions: NO sensing in cell culture media, and cell adhesion for mammalian cell cultivation. Endothelium cells with intact functionality were also investigated on the molecular level as a tissular model. Furthermore, we analyzed the response graph of the sensor output from the cellular NO sensor. We conclude that, using the multivariate data analysis method, of principal component analysis (PCA), it is possible to deduce typical biological events from the sensor output.
Keywords: Qualified analysis; Cellular biosensing; Nitric oxide; High throughput analysis (HTA); Electrochemical sensing; Multivariate data analysis;