Biochemical Engineering Journal (v.40, #2)

BEJ Keywords (II).

Canonical correlation analysis for multivariate regression and its application to metabolic fingerprinting by Hiroyuki Yamamoto; Hideki Yamaji; Eiichiro Fukusaki; Hiromu Ohno; Hideki Fukuda (199-204).
Multivariate regression analysis is one of the most important tools in metabolomics studies. For regression of high-dimensional data, partial least squares (PLS) has been widely used. Canonical correlation analysis (CCA) is a classic method of multivariate analysis; it has however rarely been applied to multivariate regression. In the present study, we applied PLS and regularized CCA (RCCA) to high-dimensional data where the number of variables (p) exceeds the number of observations (N), N  ≪  p. Using kernel CCA with linear kernel can drastically reduce the calculation time of RCCA. We applied these methods to gas chromatography–mass spectrometry (GC–MS) data, which were analyzed to resolve the problem of Japanese green tea ranking. To construct a quality-predictive model, the optimal number of latent variables in RCCA determined by leave-one-out cross-validation (LOOCV) was significantly fewer than in PLS. For metabolic fingerprinting, we successfully identified important metabolites for green tea grade classification using PLS and RCCA.
Keywords: Canonical correlation analysis; Partial least squares; Kernel method; Multivariate analysis; Metabolic fingerprinting; Metabolomics;

Bilirubin and tryptophan adsorption in albumin-containing solutions by Maria Cristina Annesini; Chiara Di Carlo; Vincenzo Piemonte; Luca Turchetti (205-210).
Albumin-bound toxins are involved in the aetiology of many liver failure associated pathologies and specific processes aimed to their removal have been developed. In general, adsorption plays an important role in all of these processes. In this paper, two albumin-bound toxins (bilirubin and tryptophan) were considered and their adsorption equilibrium on activated carbon in albumin-containing solutions was investigated. The experimental results clearly show that the higher the albumin concentration in the liquid phase, the lower the toxin adsorbed amount. This effect was quantitatively analysed by means of a mathematical model that accounts for chemical and non-chemical interactions between albumin and toxin adsorption.
Keywords: Adsorption; Amino acid; Artificial liver; Bilirubin; Modelling; Artificial liver;

Viscoelasticity and wearability of hyaluronate solutions by Syang-Peng Rwei; Saint-Wei Chen; Ching-Feng Mao; Hsu-Wei Fang (211-217).
This work systematically studied the viscoelastic properties of hyaluronic acid (HA) solution, a major component of synovial fluid, under various testing conditions. The optimum relaxation time of HA solution is around 4.5 s at pH 6.8, in the absence of salt at room temperature, indicating that synovial fluid is viscous when the shear rate is less than 1/4.5 s−1 but elastic when the shear rate exceeds this critical value. HA viscosity declines markedly as the following factors are increased in their order, salt concentration > pH level > temperature, demonstrating that these factors weaken the intermolecular attraction among HA molecules. The non-thixotropic behavior of the HA solution suggests that the breakdown and recovery of the HA structure proceed through the same intermediate states, reconfirming the strong performance of HA as a main component of synovial fluid. The wear results reveal that when the shear rate exceeds a critical value of around 20 s−1, the drop in viscosity leveled out independent of any further increase in shear rate. However, in a broad range of wear rates (20–300 s−1), the HA viscosity sustains for wearing time less than 20 min but declines without leveling off as the wear duration increases thereafter. Finally, experimental results verify that bovine albumin (BA), in HA solution, acts as both hydrodynamic and boundary lubricant, substantially improving the wearability of HA.
Keywords: Hyaluronic acid (HA); Relaxation time; Viscoelasticity; Wear result; Bovine albumin (BA); Lubrication;

Suspension culture of Azadirachta indica produces the biopesticide azadirachtin. Some elicitors (salicylic acid, chitosan, jasmonic acid, methyl jasmonate, yeast extract and yeast extract carbohydrate fraction) at different concentrations were added in shake flask suspension culture of A. indica. Chitosan, salicylic acid and jasmonic acid stimulated the highest increase in azadirachtin content, which ranged from 2 to 3-fold greater than the control. The combined effect of these elicitor(s) on azadirachtin content was then studied by Response Surface Methodology. A synergistic effect of these elicitor(s) on azadirachtin production resulted in 5-fold higher azadirachtin production (15.9 mg/g DCW versus 3.2 mg/g in control cultures). Exposure time studies with elicitor(s) addition on 8th day revealed that highest azadirachtin accumulation reached after 48 h of combined addition of elicitor(s) (17.4 mg/g). Cultivation of A. indica cells was also carried out with combined (statistically optimized) elicitors addition on 8th day in Stirred Tank Bioreactor. This led to more than 3-fold greater azadirachtin accumulation (161.1 mg/l) as opposed to control bioreactor with no elicitor addition (50 mg/l) in 10 days of cultivation period. The present study not only identifies the elicitor(s) and their respective concentrations for enhanced azadirachtin synthesis but also establishes the role of combined elicitors to improve secondary metabolite production of plant cell cultures more efficiently.
Keywords: Azadirachtin; Bioreactor; Growth kinetics; Elicitor; Statistical design;

A simpler phosphate removal process has been proposed involving only aerobic cultivation of Acinetobacter calcoaceticus in a fed-batch mode of treatment. Batch cultivation of A. calcoaceticus was conducted under statistically optimized culture conditions in order to study the kinetics of growth and phosphate uptake. Studies with respect to carbon, nitrogen and phosphate were performed to quantify their inhibitory effect on specific growth rate (if any). Using kinetics/inhibition data, a mathematical model was proposed. The batch model was extrapolated for fed-batch cultivation by incorporating the dilution terms in the model equations so as to predict, suitable nutrient feeding strategy for better phosphate removal. According to the developed feed-strategy, the bioreactor was operated in batch mode for 4 h, after which model based predetermined fresh nutrient feeding (sodium acetate 5.0 g/l) at 2.25 ml/min was started. Model predictions indicated that after 7 h of cultivation process phosphate would be totally consumed. Upon implementation of this model based fed-batch cultivation strategy a biomass accumulation of 4.02 g/l was obtained experimentally against 3.88 g/l concentration predicted by model after 7 h with a residual phosphate concentration of 0.01 g/l. An improvement to 94% phosphate removal efficiency (initial loading 0.175 g/l) in only 7 h was observed in fed-batch cultivation as opposed to 78% (initial loading 0.175 g/l) in 11 h for batch cultivation.
Keywords: Acinetobacter calcoaceticus; Phosphate removal; Aerobic conditions; Substrate inhibition; Mathematical model; Fed-batch;

Monitoring of aromatic pollutants biodegradation by Zlatka Alexieva; Maria Gerginova; Plamena Zlateva; Jordan Manasiev; Danka Ivanova; Nely Dimova (233-240).
Trichosporon cutaneum R57 is known as effective biodegradant able to utilize and thus remove a number of toxic aromatic compounds from the environment. In the present study, the dynamics of the processes of degradation of monohydoxyl derivatives of phenol (resorcinol, catechol and hydroquinone) in concentrations up to 1.6 g/l were investigated and inhibitory constants (k i) of these compounds were determined 0.58, 0.55, and 0.6 g/l correspondingly. The biodegradation of significantly more toxic aromatic compounds 2,6-dinitrophenol (k i  = 0.13 g/l), α-methylstyrene (k i  = 0.25 g/l) and acetophenone (k i  = 0.15 g/l) was also described. Based on summarized qualitative and quantitative data and the “if-then” rules developed, a fuzzy model was designed to describe the processes despite the degree of toxicity and concentration of the aromatic compounds. The output linguistic variables “normal”, “check” and “attention” were defined, which determine further measures for improvement of process quality. The model suggested provides opportunity for early estimation of the biodegradation process quality.
Keywords: Yeast; Biodegradation; Fuzzy logic; Aromatic compounds; Growth kinetics;

High-pressure gas-assisted absorption of protein within biopolymeric micro-patterned membrane by Keti Vezzù; Valentina Betto; Nicola Elvassore (241-248).
The loading of bio-molecules within polymeric matrices, such as particles, sponges or thin membranes, by completely solvent-free processes has an enormous interest in biomedical engineering applications, in particular, for the production of drug carriers and biomimetic scaffolds. In this research, we developed a process based on the use of high-pressure carbon dioxide (CO2) in order to absorb bioactive molecules within poly(lactic-co-glycolic acid) (PLGA). A dye, methylene blue, was used to optimize process conditions, whereas lysozyme was used to evaluate the protein-loading efficiency. The in situ UV–vis measurements of the gas-assisted absorption of methylene blue showed that the dye concentration within the polymer increased tenfold with respect to the control sample, after 35 min at a relatively low pressure of 2.0 MPa. Experimental measurements in the range of 0.1–6.7 MPa, and at 308 and 313 K, showed that the protein loading was highly enhanced by CO2 assisted absorption, whereas no detectable amount of protein was observed in the control. This gas-assisted technique is a completely solvent-free process, operates at mild temperature conditions (<313 K), and is particularly promising for the absorption of thermo-degradable molecules within PLGA membranes without affecting their morphology at macro- and micro-scale.
Keywords: Absorption; Protein; PLGA; High pressure; Biomimetic scaffold; In situ UV–vis measurement; Diffusion coefficient;

Combined treatment of 2,2′,5,5′-tetrachlorobenzidine (TCB) industrial wastewater by Xia Shibin; Tang bin; Xia Shuichun; Zhu Changqing (249-252).
2,2′,5,5′-Tetrachlorobenzidine (TCB), an intermediate in the industrial manufacture of dyes and pigments, is classified as a human carcinogen or a probable putative human carcinogen in USA. During the production of TCB, substantial quantities of industrial effluents are generated. A lab-scale combination process, composed by micro-electrochemical oxidation, air-stripping and aerobic biological processes, was carried out for the treatment of TCB wastewater from the two-staged neutralization dissolved by methanol. Performances of the processes were mainly investigated in this study. On a condition that hydraulic retention time (HRT) 1 h and pH 2.9 in iron-chipping filtration (ICF); HRT 24 h and the gas–liquid ratio 15 in air-stripping reactor (ASR); mixed liquor suspended solid (MLSS) 2.2–3.2 g/l, HRT 24 h, SRT 30 d, F/M 0.37–0.42 kg COD/kg MLSS d in aerobic reactor, when the influent COD was 12,910 mg/l, TCB 83.5 mg/l and NH4 +-N 1144 mg/l, the removals of COD, TCB and NH4 +-N were 15.4%, 28% and 0.3%, respectively, in ICF; 90.2%, 71.7% and 61.9%, respectively, in ASR; 86.3%, 70.6% and 90%, respectively, in aerobic reactor. The total removal of COD, TCB and NH4 +-N, respectively, was 98.9%, 98% and 88.8%, respectively. Concerning about the toxicity of TCB contained in industrial discharge, more mineralization need to be done with the treated TCB wastewater in current study.
Keywords: Aerobic; Air-stripping; Biological treatment; Electrolytic oxidation; 2,2′,5,5′-Tetrachlorobenzidine (TCB); Wastewater;

A study of the effect of organic loading rate (OLR) and hydraulic retention time (HRT) on the performance, stability and microbial communities of a laboratory-scale completely stirred tank anaerobic reactor treating two-phase olive mill solid residue (OMSR) was carried out at mesophilic temperature (35 °C). The reactor operated at a fixed influent substrate concentration of 162 g total chemical oxygen demand (COD)/L and 126 g volatile solids (VS)/L. The OLR and HRT varied in the ranges of 0.8–11.0 g COD/L day and 108–15 days, respectively. COD removal efficiencies in the range of 97–77% were achieved for OLRs and HRTs in the ranges of 1.5–9.2 g COD/L day and 108–17 days, respectively. The maximum methane production rate was found to be 1.7 L CH4 STP/L day and it was achieved for an OLR of 9.2 g COD/L day and HRT of 17 days. The methane yield coefficient was 0.244 ± 0.005 L methane at STP conditions/g COD removed. The results obtained demonstrated that an OLR of 11.0 g COD/L day and a HRT of 15 days brought about a decrease in the pH and total volatile fatty acids (TVFA)/alkalinity ratios up to values of 5.3 and 1.5 (mequiv. acetic acid/mequiv. CaCO3), respectively, causing the destabilization of the reactor and process failure. Microbial communities, both Bacteria and Archaea, were studied by molecular fingerprinting methods, cloning and sequencing. Molecular fingerprints of bacterial communities showed higher number of major bands at increasing OLR. Firmicutes, mostly represented by the genus Clostridium, were the predominant bacteria at low OLR. Other bacterial communities such as Gammaproteobacteria, Actinobacteria, Bacteroidetes and Deferribacteres were the most abundant at high OLR. The Archaea were mainly represented by four phylotypes belonging to the genus Methanosaeta independently of the OLR. This study remarks the interest of relating OMSR decomposing bioreactor performance with the microbial communities carrying out the process in order to better understand and monitor this anaerobic digestion.
Keywords: Two-phases olive mill solid residue; Anaerobic digestion; Performance; Stability; Microbial communities;

Preparation of nanofibrous polymer grafted magnetic poly(GMA-MMA)-g-MAA beads for immobilization of trypsin via adsorption by Gülay Bayramoğlu; Meltem Yılmaz; Ayşegül Ülkü Şenel; M. Yakup Arıca (262-274).
Poly(glycidylmethacrylate-methylmethacrylate), poly(GMA-MMA) beads were prepared via suspension polymerization in the presence of ferric ions. The epoxy groups of the poly(GMA-MMA) beads were converted into amino groups during magnetization reaction, and then were grafted with methacrylic acid (MAA) via graft copolymerization. The magnetic beads were characterized by surface area measurement, swelling test, scanning electron microscope (SEM), electron spin resonance (ESR) and Mössbauer spectroscopy. The enzyme “trypsin” was immobilized on the magnetic beads via adsorption. The maximum adsorption was obtained at pH 7.0. At 2.0 mg/mL initial trypsin concentration, the maximum immobilization capacity was 123.2 mg trypsin/g beads and retained about 84.2% of its initial activity. The immobilized trypsin could not be desorbed by enzyme reaction solution in the pH range of 5.0–9.0, and could be desorbed by 1.0 M formic acid solution containing 1 M NaCl.
Keywords: Magnetic beads; Adsorption; Enzyme technology; Protease; Protein; Peptide maps;

Linseed oil was used as the carbon source for the production of polyunsaturated polyhydroxyalkanoates in cultures of Pseudomonas aeruginosa 42A2. The polymer obtained, PHA-linseed (PHA-L) contents 36.5% of unsaturated side chains. 16% of the monomers were polyunsaturated, with two or three unsaturations (C12:2Δ6,9, C14:2Δ5,8 and C14:3Δ5,8,11), whereas the saturated part was mainly poly(3-hydroxyoctanoate) and poly(3-hydroxydecanoate). Up to 13 different monomers, ranging from C6 to C14, were identified. We found that the stability of the PHA-linseed side chain olefinic groups was affected by time-dependent autoxidation, which induced a crosslinking reaction with the other polymer chains at room temperature. To accelerate this process, the polymer was UV-treated (λ  = 300 nm) and changes were monitored at different reaction times (0, 3, 6, 15 and 24 h). Among other techniques, curing reaction and polymer composition were also followed by FTIR, GC/MS and NMR. Thermal properties of the PHA-L and the UV-irradiated polyhydroxyalkanoates were studied, showing an increase in the glass transition temperature from −51 to −32 °C due to the crosslinking reaction. The accelerated curing of irradiated PHA-L films was compared to the natural curing of the films at room conditions.
Keywords: Pseudomonas; PHA; Linseed oil; Crosslinking;

Isolation, identification and characterization of a Hypocrea tawa strain with high Cr(VI) reduction potential by Liliana Morales-Barrera; Flor de María Guillén-Jiménez; Alicia Ortiz-Moreno; Thelma Lilia Villegas-Garrido; Antonio Sandoval-Cabrera; César Hugo Hernández-Rodríguez; Eliseo Cristiani-Urbina (284-292).
A fungal strain that exhibits high Cr(VI) reduction potential was isolated from surface water by enrichment culture techniques. The isolated fungus was identified as Hypocrea tawa by the D1/D2 domain sequence of its 26S rRNA gene. The Cr(VI) reduction process catalyzed by H. tawa was characterized in batch cultures conducted at initial Cr(VI) concentrations ranging from 0.59 to 4.13 mM. The fungus showed a remarkable capacity to completely reduce very high concentrations of Cr(VI) (4.13 mM) under aerobic conditions. Higher volumetric (1.75 mg Cr(VI)/L h) and specific rates (0.67 mg Cr(VI)/g biomass h) as well as a greater capacity (77 mg Cr(VI)/g biomass) to reduce Cr(VI) were obtained with higher initial Cr(VI) concentrations (4.13 mM), which suggests that the fungal strain could be potentially useful for detoxification of Cr(VI)-laden wastewaters.
Keywords: Bioremediation; Fungi; Hexavalent chromium (Cr(VI)); Hypocrea tawa; Kinetics; Cr(VI) reduction;

Biodegradation kinetic studies for the removal of p-cresol from wastewater using Gliomastix indicus MTCC 3869 by Ravi Kant Singh; Shashi Kumar; Surendra Kumar; Arinjay Kumar (293-303).
Biodegradation of p-cresol by a pure culture of new filamentous fungal strain Gliomastix indicus was studied. The fungus strain was acclimatized to the higher concentration of 700 mg/l of p-cresol in modified czapeck medium over a period of two months. The lag period was 24 h. The shake flask batch experiments were carried out at temperature of 28 °C and pH of 6 over a wide range of initial p-cresol concentration (10–700 mg/l) keeping initial biomass concentration constant at 18.72 mg/l. Experimentally it was observed that p-cresol was inhibitory type substrate and the inhibition effect of p-cresol became predominant above a concentration of 50 mg/l. The kinetics of cell growth and p-cresol degradation were investigated. Six kinetic models (Haldane, Andrews, Webb, Yano, Aiba, and Teissier) were fitted to the experimental growth kinetic data. Models by Haldane, Andrews, Webb and Yano (correlation coefficient R 2  > 0.99) were found to be most suitable models. In addition, the variation of observed yield coefficient Y X with specific growth rate μ g was investigated. The maintenance energy coefficient and true growth yield coefficient, were found to be 0.0227 h−1 and 1.0708 (mg/mg), respectively. The specific substrate utilization rate was represented in terms of specific biomass growth rate. The decay coefficient was found to be 0.0055 h−1. The effect of pH and nitrogen source of medium on biodegradation of p-cresol was investigated as well.
Keywords: Gliomastix indicus; p-Cresol; Wastewater treatment; Biodegradation; Kinetic parameters; Substrate inhibition;

The food and drink industry is one of the largest industrial sectors worldwide. Milk is one of the most important raw materials employed due to both its unique nutritional value and important functional properties. However, the functionality of the various components in milk could be employed more effectively if they were available separately. The aim of this work is the evaluation of the performance of an electro membrane technology, electrodialysis with bipolar membranes (EDBM), in the separation of casein from milk. The influence of the main operation variables, i.e., milk concentration (2.5–10 wt.%) and current density (100–400 A m−2), has been analysed. Several process parameters have been recorded while the energy consumption calculations have also been performed in order to compare this membrane technology with the conventional process.Obtained results demonstrate the technical viability of EDBM and the high influence of selected variables in the separation of casein from milk. As a result, high purity casein of 95% purity was achieved by EDBM technology instead of the 85% purity casein obtained by the conventional process. The use of clean technologies, as EDBM, will contribute towards the sustainable development of this sector.
Keywords: Protein; Separation; Food engineering; Electrodialysis; Bipolar membrane; Casein;

Metabolic engineering of Escherichia coli for the production of malic acid by Soo Yun Moon; Soon Ho Hong; Tae Yong Kim; Sang Yup Lee (312-320).
Malic acid is a C4-dicarboxylic acid and an intermediate of tricarboxylic acid (TCA) cycle. It has been widely used in the polymer, food and pharmaceutical industries. Metabolic flux analysis was performed to find a strategy for enhanced malic acid production in Escherichia coli. The simulation results suggested that the amplification of phosphoenolpyruvate (PEP) carboxylation flux allowed increased malic acid production. Since the PEP carboxylase of E. coli converts PEP to oxaloacetate without generating ATP, thus losing the high-energy phosphate bond of PEP, the PEP carboxykinase, which generates ATP during this conversion, was chosen. However, the E. coli PEP carboxykinase catalyzes the reaction that converts oxaloacetate to PEP rather than the desirable opposite reaction. Thus, we cloned the PEP carboxykinase (enconded by the pckA gene) of Mannheimia succiniciproducens, which converts PEP to oxaloacetate as a favorable reaction. The pta mutant E. coli strain WGS-10 harboring the plasmid p104ManPck containing the M. succiniciproducens pckA gene was constructed and cultured at 37 °C. The final malic acid concentration of 9.25 g/L could be obtained after 12 h of aerobic cultivation.
Keywords: Malic acid; Mannheimia succiniciproducens; Recombinant DNA; Fermentation; Glucose; Protein;

The aim of the study was to optimize nutrient inflow in baker's yeast cultivation where the nutrient was dosed in portions, according to the cyclic changes in dissolved oxygen concentration; the optimization criterion being a simultaneous maximization of biomass yield and specific growth rate. The maximization of such a criterion required that the glucose concentration in the culture medium should be higher than the glucose critical level. Furthermore, the size of the nutrient portion had to be limited, the limiting parameter being the set value of the respiratory quotient. Two factors were found to be of significance to the optimization process: the set value of the respiratory quotient and the dynamics of changes in the portion size. Portions for a real respiratory quotient value below the set value (which was 1.1) and for portion value changes of ±10% were regarded as optimal. The biomass yield and specific growth rate of the culture where the set value of the respiratory quotient totaled 1.1 and the dynamics of changes in the portion size were within the range of ±10% amounted to 0.55 g g−1 and 0.14 h−1, respectively. Initial biomass concentration was also a parameter of major importance to the optimization of nutrient inflow. The increase in the initial biomass concentration value stimulates aerobic fermentation, brought about a decrease in biomass yield and specific growth rate.
Keywords: Baker's yeast cultivation; Crabtree effect; Fuzzy logic controller; Optimization of nutrient inflow; Pulsed feeding; Saccharomyces cerevisiae;

In this paper, the performance of immobilized packed bed glucose isomerase enzyme was mathematically modeled. A modified Michaelis–Menten type relation was used to describe the enzyme kinetics. Mass transfer inside the biocatalyst particle and through the bed column was analyzed simultaneously. Using measured data, physicochemical properties including diffusivity, viscosity and density of sugar solutions were correlated with its concentrations and were used to provide precision in solving the set of model equations. Model equations were solved using the Runge–Kutta and Gauss–Seidel algorithms and finite difference numerical method in MATLAB environment. Model output was used to demonstrate the effect of parameters such as velocity and bulk substrate concentration on the concentration profile within the biocatalyst particle, effectiveness factor and bulk substrate concentration along the bed. Model predictions were further validated against experimental data collected from a lab scale isomerization bioreactor. Measurements of the overall bioreactor conversion at various substrate concentrations were shown to lie within 5% of the values as put forth by the model. Using experimental data, a criteria was proposed to maximize the isomerization conversion from the immobilized bioreactor.
Keywords: Glucose; Fructose; Glucose isomerase; Packed bed; Bioreactor; Immobilized enzyme; HFS;

Acetic acid is an important industrial feedstock which can be produced from renewable resources such as carbon monoxide (CO) for an infinite supply for future usage. The biological route of acetic acid production from CO through fermentation by acetogenic bacteria, Clostridium aceticum appeared to be potential substitution for current process technology. The fermentation medium which is responsible for sustaining and maintaining the biocatalyst growth usually accounts for 60–70% of the production cost. Therefore, efforts in creating cost effective process have been carried out by optimizing the nutrient requirements of NH4Cl and yeast extract compositions which can still attain satisfactory responses during acetic acid fermentation. In this study, medium composition of NH4Cl (studied ranges: 0.0–1.50 g/L) was optimized in the early stage and then followed by optimizing the yeast extract component (studied ranges: 0.0–2.00 g/L). The high-end concentration selected for each medium composition was based on the concentration consisted in the DSMZ growth medium 1496 as a guideline. Response surface methodology (RSM) which consisted of 2-D contour plot and 3-D surface plot was used to determine the optimum concentrations of NH4Cl and yeast extract that enabled the maximum acetic acid production with high CO conversion in an actively growing culture. Fermentation when launched in medium of 0.2 g/L of NH4Cl coupled with 1.5 g/L of yeast extract and operated for 72 h cultivation time resulted in 1.01 g/L of acetic acid production and 100% CO conversion. Optimum fermentation medium was defined as 0.2 and 1.5 g/L of NH4Cl and yeast extract respectively in this experiment based on the 67% of response desirability achieved through RSM.
Keywords: Anaerobic processes; Acetic acid; Clostridium aceticum; Fermentation; Medium optimization; Response surface methodology (RSM);

Optimal xylanase production using Penicilium janthinellum NCIM 1169: A model based approach by Mukesh Meshram; Abhijit Kulkarni; V.K. Jayaraman; B.D. Kulkarni; S.S. Lele (348-356).
Xylanases are an industrially important class of hydrolytic enzymes that degrade xylans. Production of xylanase from a fungal culture by submerged fermentation and optimization of the operating conditions for maximum activity are the two aims of the present study. Penicillium janthinellum NCIM 1169 with Mandels–Weber medium, sugarcane bagassse (40#) as a carbon source and beef extract as a nitrogen source were used in the experiments. We did 41 experiments to see the effect of variations in carbon, nitrogen source, pH, and inoculum on xylanase activity. This data was then used to build an input/output model using multiple linear regression, back propagation neural network and lazy learning algorithm. It was found that lazy learning model correlated well in mapping input/output data. This model was then utilized as an objective function in genetic algorithm to find the optimal combination of the operating conditions to get the maximum xylanase activity. It was observed that with carbon source, 1.63%, nitrogen source, 0.16%, pH, 4.1, and inoculum, 5.5%, maximum xylanase activity of 28.98 ± 1.73 U/ml was achieved.
Keywords: Xylanase; Fermentation; Enzyme activity; Artificial intelligence; Modeling; Optimization;

Ponkan peel: A potential biosorbent for removal of Pb(II) ions from aqueous solution by Flávio A. Pavan; Ana C. Mazzocato; Rosângela A. Jacques; Silvio L.P. Dias (357-362).
The ability of ponkan peel, a natural biosorbent, to remove Pb(II) ions from aqueous solution by adsorption was studied. The experiments were carried out by batch method at 25 °C. Important parameters such as pH, initial Pb(II) concentration and contact time on the biosorption were investigated. The optimal pH value for adsorption capacity was 5.0. The biosorption was relatively quick, about 60 min. Based on the Langmuir-type isotherms the maximum uptake capacity of lead ions on ponkan peel was 112.1 mg g−1. The result shows that ponkan peel is a good biosorbent for removing lead ions from aqueous solution and does not need any chemical or physical pre-treatment. The ponkan peel biosorbent is a low cost material that shows potential to be applied in wastewater technology for remediation of toxic metal.
Keywords: Biosorption; Ponkan peel; Toxic metal; Wastewater; Batch method;

Yeast cells of Saccharomyces cerevisiae were found to accumulate 152,154Eu radioisotopes selectively from a synthetic mixture of 152,154Eu, 137Cs, and 60Co radiotracers at neutral pH in both trace and macro level. The extent of Eu uptake increased with time. The uptake of Co was observed only when concentration of cobalt was more than 10 mg kg−1 at pH 6.5. None of the strains selected in our study showed any accumulation for Cs, even at trace level. Transmission electron microscopic (TEM) images of thin sections of the Eu accumulated yeast strains showed that it was accumulated inside the cell. The growth of four different strains of Saccharomyces cerevisiae was monitored at different concentrations of these metal salts. The yeast cells can grow upto 4.6 mM (700 mg kg−1) Eu concentration and 7.3 mM (1 g kg−1) Cs concentration irrespective of their histidine auxotrophy. In agreement with earlier reports it was observed that at 1.2 mM Co concentration, His+ could grow at a pH range 5–6.5, whereas His strains could not grow at any pH. Thus, the mechanism of uptake of the rare earth element, Eu, was found to follow a different pathway than the transition element Co, which causes toxicity to yeast cells in the absence of histidine in the biosynthetic pathway. The result may encourage for pilot experiments in preconcentration of rare earths using bioreagents in commercial scale.
Keywords: Saccharomyces cerevisiae. Bioaccumulation; Histidine auxotrophy; 152,154Eu; 60Co; 137Cs; Fission products;

Enzymatic synthesis of conjugated linoleoyl ascorbate in acetone by Yoshiyuki Watanabe; Yuki Sawahara; Hideyuki Nosaka; Kazuhiro Yamanaka; Shuji Adachi (368-372).
Conjugated linoleoyl ascorbates were synthesized through the condensation of cis-9,trans-11-conjugated linoleic acid (c9,t11CLA) or trans-10,cis-12-conjugated linoleic acid (t10,c12CLA) with l-ascorbic acid (AsA) using immobilized lipase in a water-soluble organic solvent at 50 °C. The conversion was higher at higher polarity of the solvent and a higher ratio of CLA to AsA. The conversion for the synthesis of t10,c12CLA-AsA was higher than those for c9,t11CLA-AsA and linoleoyl ascorbate (LA-AsA), when Chirazyme® L-2 C2 from Candida antarctica type B, which has been commonly used for enzymatic reaction in organic solvent, was used as a catalyst. The unmodified CLA isomers were almost completely oxidized within 4 h at 65 °C and 75% relative humidity, whereas all the CLA-AsAs were significantly resistant to oxidation. The oxidation kinetics was empirically expressed by the Weibull equation, and the rate constant, k, was estimated. The k values for CLA-AsAs and LA-AsA were about 1/30 and 1/60, respectively, of that for the corresponding unmodified CLA isomers. The enzymatic modification of CLA with AsA was an effective technique for improvement in the oxidative stability of CLA.
Keywords: Conjugated linoleic acid; Immobilized lipase; Linoleoyl ascorbate; Oxidative stability;

Nitrification was investigated in two laboratory-scale sequencing batch reactors (SBRs) at 11 °C, one with glucose addition (G-Reactor) and the other without (N-Reactor). The characteristics of nitrification and the distribution of ammonia oxidizing bacteria (AOB) within activated sludge flocs in the two reactors were examined. A high specific nitrification rate existed in the N-Reactor, while a high volumetric nitrification rate existed in the G-Reactor. The proportion of AOB enriched in the N-Reactor was about three times that in the G-Reactor. The activated sludge flocs in the N-Reactor had a larger floc size and a higher biomass density than in the G-Reactor. AOB were mainly in a form of clusters in the activated sludge flocs in the N-Reactor, but were well dispersed throughout the flocs in the G-Reactor.
Keywords: Ammonia oxidizing bacteria; Fluorescence in situ hybridization; Glucose; Heterotrophs; Nitrification;

Mathematical model for change in diameter distribution of baculovirus-infected Sf-9 insect cells by Takeshi Gotoh; Masamichi Fukuhara; Ken-Ichi Kikuchi (379-386).
Sf-9 insect cells were examined for diameter before and after baculovirus infection. The diameter distribution of uninfected Sf-9 insect cells was described by the normal distribution function with a mean cell diameter and standard deviation of 18.5 ± 1.5 μm. After synchronous infection at a high MOI and high cell density, the infected cells grew in diameter almost linearly with time for about 48 h to have a steady mean diameter 1.45 times larger than that of uninfected cells, though still normally distributed. The distribution of the infected cells began to broaden at around 6 h-post-infection and eventually had standard deviation twice as large as that of uninfected cells. A mathematical model was developed to describe the change in the diameter distribution of virus-infected insect cells, integrating the distributions of infected cell subsets that were generated according to the Poisson distribution function between time t and t  + dt and individually characterized with a post-infection time. The growth of uninfected cells and post-infection events such as progeny virus replication and secondary infection of uninfected remained cells were simulated according to a previous report. The model calculation predicts the change in diameter distribution of Sf-9 insect cells that were infected under various conditions, giving an indication to assess the degree of virus infection.
Keywords: Baculovirus; Insect cell; Sf-9; Modelling; Diameter distribution;

The aim of this study was to evaluate the expression of albumin production and drug metabolism activities of primary rat hepatocytes under a two-dimensional monolayer culture condition formed on a RGD-immobilized culture dish. Albumin production activity on the RGD-immobilized dish (2500 ng cm−2 Pronectin F-coated dish) was 1.6 (p  < 0.05) and 1.2 (p  < 0.05) times higher than that on the collagen-coated dish and that of the spheroid culture, respectively. p-Acetamidophenol (APAP) metabolism rate of the collagen-monolayer at 5 days of culture was decreased to 16% of the activity at 1 day of culture. On the other hand, APAP metabolism activities of the RGD-monolayer and spheroid cultures were well maintained during 7 days of culture. However, we could not detect the effectiveness of the RGD-monolayer on lidocaine metabolism of hepatocytes. This is the first report suggesting that a hepatocyte monolayer on a RGD-immobilized culture substratum expresses albumin production and APAP metabolism activities equal to those of a hepatocyte spheroid culture.
Keywords: Arg-Gly-Asp (RGD); Hepatocyte; Amino acid; Animal cell culture; Tissue cell culture; Bioconversion;

A zero-dimensional (0D) biofilm model for dynamic simulation of biological wastewater treatment systems is proposed and applied to a pilot-scale moving bed bioreactor (MBBR). The model, written in Peterson matrix format, does not describe biofilm structure in any form. Biochemical conversions in the model are based on the activated sludge model no. 1 (ASM1). Diffusional mass transport limitations are taken into account implicitly in the model using adapted half-saturation coefficients in the Monod terms of the model. In addition to biochemical conversions, the model contains attachment of particulates from the bulk of the liquid onto the biofilm and detachment of particulates from the biofilm into the bulk of the liquid. Model performance is evaluated using data originating from two intensive measurement campaigns covering 9 days of wastewater treatment with the pilot-scale MBBR. The process dynamics of the MBBR were well reproduced by the model, since a good fit between measured effluent ammonia and nitrate concentrations to the simulation results was obtained. Biofilm related parameters, like biofilm age, attachment rate and detachment rate, were extracted from the model.
Keywords: Biofilms; Biofilm age; Dynamic simulation; Moving bed bioreactors; Zero-dimensional model; 0D; Modelling;