Biochemical Engineering Journal (v.12, #1)

BEJ Keywords (II).


The forward and backward extraction of bovine serum albumin (BSA) has been studied using mixed reverse micellar systems of cetyl trimethyl ammonium bromide (CTAB) and alkyl halides (including 1-chlorobutane (R4Cl), 1-bromooctane (R8Br) and 1-iodobutane (R4I)). The addition of alkyl halide R8Br to 50 mmol/l CTAB/20% (v/v) hexanol/petroleum ether reverse micellar system could improve the BSA transfer from the aqueous phase to reverse micellar phase, while the addition of R4Cl or R4I had almost no effect on the BSA transfer. The mixed reverse micelles formed with CTAB and alkyl halides exhibit excellent backward extraction behavior for BSA. The mixed reverse micelles formed with CTAB and R4I can realize the recovery of BSA effectively in a wide range of pH up to or higher than the isoelectric point of BSA. The mixed reverse micelles formed with CTAB and R4Cl, R8Br or R4I can obviously enhance the BSA backward transfer at low ionic strength with addition of KBr or KCl as electrolyte. The mixed reverse micellar system indicates that it requires less time to reach the mass transfer equilibrium in comparison with the reverse micellar system with CTAB only. The mechanism of backward extraction proposed that with the addition of alkyl halides to CTAB reverse micelles, the hydrophobic interaction between the reverse micelles decreased.
Keywords: Reverse micelles; Backward extraction; Alkyl halide; BSA; CTAB;

Characterization and control of stimuli-induced membrane fusion of liposomes in the presence of proteins and stimuli responsive polymers by Matundu Menayame Félix; Hiroshi Umakoshi; Toshinori Shimanouchi; Makoto Yoshimoto; Ryoichi Kuboi (7-19).
The process of fusion of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) liposomes in the presence of stimuli responsive copolymers (poly(N-isopropylacrylamide-co-methacrylic acid) and poly(N-isopropylacrylamide-co-methacrylic acid-co-octadecylacrylamide)) or proteins (α-chymotrypsin (α-CT), bovine carbonic anhydrase (CAB), and β-galactosidase (β-gal)) was quantitatively characterized under the stimuli by varying pH and temperature. After the liposomes were exposed to the specific pH and heat stimuli in the presence of the stimuli responsive polymers or proteins, the percentage of the fusion was determined by using two kinds of liposomes entrapping cobalt-calcein and EDTA (cobalt-calcein method). In the presence of stimuli responsive copolymers, the percentage of fusion was increased to 20% above the phase transition condition of the copolymer (above 37 °C and below pH 5.7). The percentage of fusion in the presence of proteins was varied under the stimuli, depending on the type of proteins. In the case of the α-CT, the maximal percentage of fusion was only 8%. The addition of the CAB and β-gal improved the percentage of fusion to 15 and 20%, respectively, by selecting the optimal stimuli conditions. Although some disagreements were observed in the region of strong acidic conditions, it was found that the increase of the percentage of fusion was well corresponding with the increase of the membrane fluidity of the POPC liposome, followed by the increase of the local hydrophobicity of proteins or copolymers under the stimuli. Based on the above results, a possible model for the stimuli-induced fusion of POPC liposome membranes was finally presented.
Keywords: Liposomes; Membrane fusion; Stimuli responsive polymers; Proteins; Membrane fluidity;

Effects of exogenous polyamines on embryogenic carrot cells by Toshiya Takeda; Fumino Hayakawa; Kanako Oe; Hiroshi Matsuoka (21-28).
We investigated the effects of exogenous polyamines on somatic embryo formation in carrot cells. The polyamines, spermidine and spermine, were added to a regeneration culture of carrots. A greater enhancement of somatic embryo formation was achieved with the addition of spermine. This enhancement was not, however, always observed. Additional spermine increased the DNA content regardless of whether somatic embryo formation increased. Based on the analysis of profile in somatic embryo formation, we note that the addition of spermine increased the lag time of somatic embryo formation and suppressed the protein secretion from cells.
Keywords: Somatic embryo; Carrot; Polyamine; Spermine;

Preparation and characteristics of resting cells of bioluminescent Pseudomonas putida BLU by Takakuni Tanaka; Xin-Hui Xing; Kanji Matsumoto; Hajime Unno (29-36).
A bioluminescent strain, Pseudomonas putida BLU, which was created by inserting the lux gene into the chromosome of P. putida mt-2 (TOL), a well-known degrader for benzene, toluene and xylene (BTX), produces luminescence depending on both cell concentration and intracellular metabolic activity, including the intracellular level of reduced riboflavin phosphate (FMNH2). To estimate the cell concentration of a BTX degrader by the rapid detection of bioluminescence in a bioaugmentation system, the effect of metabolic activity on the output of bioluminescence needs to be considered. For this purpose, the preparation of resting cells of P. putida BLU, defined as incapable of growing but having metabolic activity, was tried using different surfactants. Resting cells formed on addition of n-dodecyltrimethylammonium bromide (DTAB, a cationic surfactant) at a concentration below the critical micellar concentration (CMC) to the bacterial culture, and exhibited morphological change. The P. putida BLU showed a decrease in bioluminescence output to a constant level and stopped growing immediately after the addition of DTAB, while maintaining activity for oxygen consumption. When the bacterial culture treated with DTAB was washed with a 0.9% saline solution, the growth and bioluminescence of the resting cells recovered to the normal level. The bioluminescence output (LUX) of the resting cells prepared at different time points from the bacterial culture correlated only with the cell concentration (X), LUX∝X 1.0, suggesting that generating resting cells enables the cell concentration to be simply and rapidly quantified by measuring the luminescence.
Keywords: Bioaugmentation; Biodegradation; Bioluminescence; Microbial growth; Metabolic activity; Pseudomonas putida;

Broth rheology of Beta vulgaris cultures growing in an air lift bioreactor by Marco Juárez Sánchez; Antonio Jiménez-Aparicio; Gustavo Gutiérrez López; Gabriela Trejo Tapia; Mario Rodrı́guez-Monroy (37-41).
Cell cultures of Beta vulgaris were developed in an air lift bioreactor of 10 dm3. Culture broth rheology exhibited non-Newtonian, shear thinning characteristics. The pseudoplasticity of the broth was governed by the presence of the cells as well as by the proteins secreted by the cells in the medium. The accumulation of extracellular proteins produced an increase in the viscosity and a change in the rheological properties of the cell-free medium. This phenomena may be a response of the cells to hydrodynamic stress. The accumulation of extracellular proteins and the change in the rheology of cell-free medium were discussed with respect to those data reported in literature obtained in shake flasks and stirred tank bioreactor.
Keywords: Air lift bioreactor; Rheology; Hydrodynamic stress; Extracellular proteins;

Properties of the biofilm of Thiobacillus ferrooxidans formed in rotating biological contactor by L Nikolov; D Karamanev; V Mamatarkova; D Mehochev; D Dimitrov (43-48).
The physico-chemical properties of the Thiobacillus ferrooxidans biofilm formed on the surface of rotating disks in a rotating biological contactor (RBC) were studied under steady-state conditions. The main independent variable was the input substrate (ferrous iron) concentration in the bioreactor. It has been shown that the biofilm thickness was maximal, the biofilm density was minimal and the specific surface area of pores in the biofilm was maximal when the input ferrous iron concentration was between 1 and 2.1 g/l. The biofilm volume remained nearly the same when input substrate concentrations were between 0.49 and 14.21 g/l, corresponding to oxidation rates between 0.35 and 8.6 g/m2  h.
Keywords: Biofilms; Bioreactors; Kinetic parameters; Thiobacillus ferrooxidans; Iron oxidation;

Growth model and prediction of oxygen transfer rate for xylitol production from d-xylose by C. guilliermondii by Wilson B Aguiar; Luı́s F.F Faria; Maria A.P.G Couto; Ofélia Q.F Araujo; Nei Pereira (49-59).
The K L a values for optimum xylitol productivity using Candida guilliermondii with initial cell concentrations of 1.0 and 4.0 g/l and an initial d-xylose concentration of 50 g/l were predicted as 20 and 100 h−1, respectively, by means of growth, substrate and oxygen uptake simulations. The highest xylitol production rate (1.52 g/l h) was experimentally obtained with an initial cell concentration of 4 g/l and K L a 100 h−1. Kinetic parameters were estimated for Monod, Contois and Tessier growth models using a non-linear method. The Contois model was chosen as the most suitable due to the agreement with data reported in literature for yeasts and statistical confidence of its parameters. The specific oxygen uptake rate (SOUR) was determined by the dynamic oxygen balance. It had a maximum value of 153 mg O2/g cell h after 2 h growth. The choice of the best K L a value was made through dissolved oxygen concentration simulations for fermentations carried out with two different inoculum sizes, resulting in a maximum volumetric productivity of 1.52 g/l h, for an initial cell concentration of 4 g/l, employing a K L a value of 100 h−1.
Keywords: Candida guilliermondii; Growth model; Oxygen transfer; Non-linear estimation; K L a prediction; Xylitol production;

Modeling of mixing in stirred bioreactors by C Oniscu; A.-I Galaction; D Cascaval; F Ungureanu (61-69).
The mixing time is one of the most useful criterion for mixing intensity of fermentation broths and for scale-up of biosynthesis processes. This parameter value depends mainly on the rheological properties of the broths, biomass concentration and morphology, mixing system characteristics and fermentation conditions.For quantifying the influence of these factors on mixing efficiency for stirred bioreactors, these studies were carried out for non-aerated suspensions of bacteria (Propionibacterium shermanii), yeasts (Saccharomyces cerevisiae) and fungus (Penicillium chrysogenum, free mycelia and mycelial aggregates) of different concentrations, using a laboratory bioreactor with double turbine impeller. By means of the experimental data and using a multiregression analysis method, some mathematical correlations for mixing time having the general expression of: t m=α(C x β L δ /N γ ) were established. The proposed equations are adequate for the flow regime of Re<25,000.
Keywords: Stirred bioreactor; Mixing time; Anaerobic culture; Bacteria suspension; Yeasts suspension; Fungus suspension; Free mycelia; Mycelial aggregates;

Extraction techniques using microwave-assisted extraction (MAE), extraction at room temperature (ERT), heat reflux extraction, ultrasonic extraction and Soxhlet extraction were evaluated for the extraction of tanshinones (Cryptotanshinone, Tanshinone I and Tanshinone IIA) from Salvia miltiorrhiza bunge. The extracts were analyzed by high performance liquid chromatography (HPLC) without any treatment. The results showed that the percentage extraction of Cryptotanshinone, Tanshinone I and Tanshinone IIA from S. miltiorrhiza bunge by MAE was equivalent with and in fact higher than that of conventional extraction methods. MAE only needs 2 min, whereas ERT, heat reflux extraction, ultrasonic extraction and Soxhlet extraction need 24 h, 45, 75 and 90 min, respectively. Due to the considerable saving of time and high extraction efficiency, MAE was more effective than the conventional methods.
Keywords: Salvia miltiorrhiza bunge; Tanshinones; Microwave-assisted; Extraction method;

The identification of a psychrotrophic bacterium, which produced a violet pigment, isolated from the organic residue of a water tank keeping rainbow trout and the antibacterial effect of the violet pigment were investigated experimentally. The psychrotrophic bacterium was found to be a new species very closely related to Janthinobacterium lividum. 1 H NMR, 13 C NMR, and FT-MS spectra analyses results showed that the chemical structure of the violet pigment was a mixture of violacein and deoxyviolacein. The antibacterial effect of the violet pigment was confirmed for putrefactive bacteria such as Bacillus subtilis, Bacillus megaterium, Staphylococcus aureus, and Pseudomonas aeruginosa. The growth-inhibitory and lethal effects of the violet pigment on the putrefactive bacteria were evaluated by increasing the concentration of the violet pigment, ranging from 5 to 20 mg dm−3. It was found that higher concentrations of the violet pigment caused not only growth inhibition but also the death of the putrefactive bacteria.
Keywords: Antibiotic; Glucose; Purification; Viability; Violet pigment; Microbial growth;