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Biochemical Engineering Journal (v.60, #)
Exploring the effect of mild hypothermia on CHO cell productivity by Silvana Becerra; Julio Berrios; Nelson Osses; Claudia Altamirano (pp. 1-8).
► We analyzed the framework on the effect of mild hypothermia on CHO cells productivity. ► Cellular processes implied in culture at mild hypothermia case are better understood. ► We propose a different experimental approach. ► Our idea allows estimating the net effect of temperature on the r-protein productivity.The CHO cell line is mainly used to synthesize recombinant proteins for human therapeutic use. Recent efforts have focused on improving the productivity of this system by mainly using two tools: genetic manipulation of the cells and/or changing the operating conditions of the cultures. In most cases, these techniques have resulted in significant improvements to the productivity of the system. For example, a slight decrease in culture temperature has been used over the past ten years to increase culture longevity, resulting in an increased productivity of r-proteins. However, despite large numbers of diverse studies, there are currently no unified hypotheses to explain the increased productivity of r-proteins. This is mainly because the magnitude of this effect is dependent on the cell line and the r-protein, and the effects of temperature on cellular processes of protein synthesis and cell growth are difficult to decouple and quantify experimentally.In this review, we assembled the most relevant backgrounds to address this problem, and we explore a different experimental approach that allowed us to decouple specific cell growth from culture temperature, in order to estimate the net effect of temperature on the productivity of r-proteins and to better understand the molecular and cellular processes involved. The information provided in this study can be used to propose new strategies at an industrial scale for implementing fed-batch or perfusion cultures to improve the production of several important r-proteins.
Keywords: CHO cells culture; Mild hypothermia; Recombinant protein productivity
Decolorization of some synthetic dyes using optimized culture broth of laccase producing ascomycete Paraconiothyrium variabile by M. Aghaie-Khouzani; H. Forootanfar; M. Moshfegh; M.R. Khoshayand; M.A. Faramarzi (pp. 9-15).
Display Omitted► Medium for submerge culture of Paraconiothyrium variabile was optimized for laccase production. ► Optimization experiments increased laccase production from 970U/L to 16,678U/L. ► The optimized medium decolorized five tested dyes during 180min incubation.Optimization of the medium components for production of laccase by Paraconiothyrium variabile using response surface methodology was investigated. Initial screening by Plackett–Burman design was performed to select major variables out of eleven medium components, among which peptone, CuSO4, and xylidine were found to have significant effects on laccase production. After application of the steepest ascent to approach the proximity of the optimum point, a central composite design was employed to optimize the level of the selected variables. In optimum concentrations of the most effective parameters, including peptone, 2.2g/L, CuSO4, 0.03g/L, and xylidine 1.29mM, extracellular laccase activity was enhanced from 970U/L (in basal medium) to 16,678U/L, which means a 17-fold increase in laccase production in the optimized medium. Supernatant of the optimized medium was used for decolorization of five synthetic dyes, among which 93% of Remazol brilliant blue R (with initial concentration of 600mg/L) disappeared after 3h treatment in the presence of 5mM hydroxybenzotriazole.
Keywords: Paraconiothyrium variabile; Laccase; Enzyme production; Submerged culture; Enzyme biocatalysis; Waste treatment
Immobilization of Candida antarctica lipase B by covalent attachment on chitosan-based hydrogels using different support activation strategies by J.A. Silva; G.P. Macedo; D.S. Rodrigues; R.L.C. Giordano; L.R.B. Gonçalves (pp. 16-24).
► Immobilization of Candida antarctica lipase B on chitosan and on chitosan–alginate complex. ► Supports were activated with glycidol, ethylenediamine and glutaraldehyde. ► High activities were achieved in comparison to a commercial immobilized lipase. ► During butyl oleate synthesis, high operational stability was achieved. ► Conversion of 100% was obtained in a 12-h reaction of butyl oleate synthesis. Candida antarctica lipase B immobilization by covalent attachment on chitosan and on chitosan–alginate complex previously activated by different strategies was studied. Hydroxyl and amine groups of support were activated using glycidol and glutaraldehyde. Ethylenediamine (EDA) was also used in the activation process. FT-IR analysis confirmed the reaction of these activating agents with the supports. Several activation–immobilization strategies were performed and the best derivatives showed activities of 422.44±50.4 and 378.30±34.70U/g-support for chitosan and chitosan–alginate complex, respectively, slightly less in comparison to the commercial immobilized lipase Novozym 435 (529.78±11.7U/g-support). Best results of thermal stability (incubation at 60°C) and operational stability (repeated cycles of synthesis of butyl oleate) were obtained for enzyme immobilized on chitosan–alginate, activated with 2% glutaraldehyde. This derivative was 33 times more thermally stable than the soluble enzyme, and it did not lose its initial activity after 8 cycles of a 12-h synthesis of butyl oleate. Chitosan, activated with 72% glycidol, EDA and 5% glutaraldehyde, showed less operational (loss of 16.7% of its initial activity) and thermal stabilities (only 12.5 times more thermally stable than soluble enzyme). Conversion of 100% was obtained in a 12-h reaction of butyl oleate synthesis, using the best derivatives (lipase immobilized on chitosan–Gly72%–EDA–Glu5% and on chitosan–alginate–Glu2%).
Keywords: Chitosan; Chitosan–alginate complex; Immobilized enzymes; Lipase; Biocatalysis
Effect of steam explosion pretreatment with ultra-high temperature and pressure on effective utilization of softwood biomass by Chikako Asada; Chizuru Sasaki; Yoshihiro Uto; Jun Sakafuji; Yoshitoshi Nakamura (pp. 25-29).
► Ultra-high temperature and pressure steam explosion is very effective for softwood. ► Steam explosion at 45atm and 3min provides the highest sugar yield from softwood. ► We present total conversion system of softwood into useful fuels and materials.Effective utilization process of not only cellulose but also lignin contained in softwood biomass into useful fuel and materials was developed using steam explosion with ultra-high temperature and pressure steam, extraction and various conversion methods. The conversion of softwood biomass into useful materials was studied for the effective utilization of its components such as cellulose, water soluble material, methanol soluble lignin, and Klason lignin. The cellulose, water soluble material, methanol soluble lignin, and Klason lignin were converted into useful fuel and materials, i.e. ethanol, antioxidant material, lignin epoxy resin, and activated carbon, respectively. 49.6g of glucose or 17.4g of ethanol was obtained from 100g of steam-exploded product treated at a steam pressure of 45atm and 3min. Water soluble material had a comparatively high antioxidant activity and methanol soluble lignin was converted into a cured epoxy resin with heat-resisting property for solder demanded in the electronic material field.
Keywords: Steam explosion; Ultra-high temperature and pressure; Antioxidant material; Lignin epoxy resin; Bioethanol; Softwood
Enhancement of cordycepin production in submerged cultures of Cordyceps militaris by addition of ferrous sulfate by Dan-dan Fan; Wei Wang; Jian-Jiang Zhong (pp. 30-35).
► Ferrous sulfate addition was found useful to enhance the cordycepin production. ► The related gene expression was studied for better manipulation of the cordycepin biosynthesis. ► The relationship between IMP content and cordycepin production was studied. ► purA gene expression was found important in cordycepin synthesis.The effect of ferrous sulfate addition on production of cordycepin (3′-deoxyadenosine), a useful bioactive product with some pharmacological activities, was investigated in submerged cultures of Cordyceps militaris in shake flasks. The results showed that the optimal addition condition was on day 0 with 1g/L of ferrous sulfate, and the maximal amount of cordycepin reached 596.59±85.5mg/L, about 70% higher than the control without ferrous sulfate addition. Meanwhile, the consumption of inosine 5′-monophosphate (IMP), a potential precursor of cordycepin, was decreased rapidly. Transcription levels of important genes encoding adenylosuccinate synthetase ( purA), IMP cyclohydrolase ( purH) and IMP dehydrogenase ( guaB) in the purine nucleotide biosynthetic pathway were also studied. Compared to the control, the transcription level of purA was significantly up-regulated in ferrous sulfate supplemented cultures, while purH and guaB were slightly down-regulated. This work indicated that ferrous sulfate addition was a simple and useful strategy for improving cordycepin production, and the related information might be helpful for further manipulation and understanding of the cordycepin biosynthesis.
Keywords: Metabolite production; Submerged culture; Physiology; Medicinal mushroom; Cordyceps militaris; Gene transcription
A straightforward logistic method for feeding a fed-batch baker's yeast culture by Daniel Borowiak; Tadeusz Miśkiewicz; Witold Miszczak; Edmund Cibis; Małgorzata Krzywonos (pp. 36-43).
The aim of this work was to find a straightforward, low-cost method for nutrient medium delivery during fed-batch cultivation of baker's yeast; a method that provides a high specific growth rate, a high biomass yield, and a high dough-leavening ability. The time profile for the inflow of the nutrient medium, g( t), is a logistic model which takes the form of g( t)= a/(1+ be− ct), with a, b and c standing for its parameters. The initial values of a, b and c were calculated considering the results of a baker's yeast process where dissolved oxygen tension was used as a control parameter of medium inflow. A method was developed for modifying the parameters of the initial model so as to obtain a formula that would cause the inflow of the nutrient medium to maximize the criterion K( K= Yμ), where Y stands for yeast biomass yield and μ denotes specific growth rate. The parameters of the modified model depended on initial biomass concentration. The relation was described by a logarithmic function and incorporated into the control algorithm. This algorithm produced a biomass yield exceeding 0.55gg−1 and a high dough-leavening ability. The method proposed is recommendable for small and medium-sized firms producing baker's yeast.
Keywords: Baker's yeast cultivation; Dough leavening ability; Control algorithm; Fed-batch culture; Logistic model; Method for nutrient delivery
Toluene mass transfer characterization in a biotrickling filter by Raquel Lebrero; José M. Estrada; Raúl Muñoz; Guillermo Quijano (pp. 44-49).
► A general and simple model to characterize the VOC mass transfer in biotrickling filters by means of the overall mass transfer coefficient ( KL a) was developed. ► The resulting KL a values ranged from 35 to 113h−1. A critical gas-to-liquid flow rate ratio of 200, above which KL a was poorly increased, was observed. ► The estimation of the individual film coefficients revealed that the main resistance for mass transfer was in the liquid film. ► The calculated mass transfer capacity confirmed the high mass transfer potential of biotrickling filters.Biotrickling filters (BTFs) devoted to air pollution control often present mass transfer limitations for hydrophobic volatile organic compounds (VOCs). Under such limiting conditions, BTF design and scale-up should be based on mass transfer data. A general and simple model was developed to characterize the VOC transfer by means of the overall mass transfer coefficient ( KL a). The KL a values were obtained by fitting the model to experimental data of toluene absorption obtained at empty bed residence times (EBRT) from 7 to 50s. The model fitted well the experimental data ( r2=0.97) and the resulting KL a values ranged from 35 to 113h−1. These values are similar to those reported in the literature for BTF despite the lower liquid recycling velocity here used (0.6mh−1). A critical gas-to-liquid flow rate ratio ( QG/ QL) of 200, above which KL a was poorly increased, was observed. In addition, the individual film coefficients were estimated from the Van Krevelen and Hoftijzer correlations, which revealed that the main resistance for toluene mass transfer was in the liquid film regardless of the EBRT used. Finally, the high mass transfer potential of BTFs was confirmed by estimating the mass transfer capacity under varied operating conditions.
Keywords: Biological off-gas treatment; Biotrickling filter; Mass transfer modeling; Toluene; Volatile organic compounds
Design of substrate-type ACE inhibitory pentapeptides with an antepenultimate C-terminal proline for efficient release of inhibitory activity by Sheng-qi Rao; Song Liu; Tao Ju; Wen-qi Xu; Guang-ming Mei; Yan-shun Xu; Yan-jun Yang (pp. 50-55).
► Design of substrate-type ACE inhibitory pentapeptides. ► Stability of the pentapeptides to ACE. ► Pro-Val and Pro-Phe peptide bond were susceptible to hydrolysis by ACE. ► Stability of the pentapeptides to gastrointestinal enzymes. ► IKPVA and IKPFR have the potential antihypertensive activity in vivo.Much research has verified that tripeptides initiated with a branched-chain aliphatic amino acid residue and terminated with a proline have a strong antihypertensive activity in vivo. However, it is difficult to release from their precursor proteins that are orally administered. Based on the selectivity of angiotensin I-converting enzyme (ACE) on C-terminal dipeptides in its substrate, six pentapeptides with the same tripeptide IKP (as a model) at N-terminus including IKPVQ, IKPVA, IKPVK, IKPVR, IKPFR, and IKPHL were designed and chemically synthesized. It was shown that all the pentapeptides released IKP after ACE incubation. The release rate ranged from 23% (IKPHL) to 84.6% (IKPVR) as compared to the peptide sample before incubation. The in vitro digestion experiment demonstrated that all of the pentapeptides except IKPVA with a retention rate of 80.5% were resistant to pepsin hydrolysis but not to pancreatic hydrolysis. It should be noted that IKP could be released from IKPFR by pancreatin digestion. These results suggest that IKPVA and IKPFR potentially have a great antihypertensive effect in vivo. Furthermore, the dipeptides VA and FR described here may be widely used as linkers to help the release of the active peptides with proline at C-terminus from their protein precursors by ACE or gastrointestinal enzymes in human body.
Keywords: Biomedical; Enzyme activity; Substrate inhibition; Food engineering; Angiotensin I-converting enzyme inhibitory peptide; Design
Evolutionary engineering of yeast for closed-circulating ethanol fermentation in PDMS membrane bioreactor by Wenwu Ding; Zeyi Xiao; Xiaoyu Tang; Kewang Deng; Shengwei Fu; Yandong Jiang; Lin Yuan (pp. 56-61).
► A regimen of step transfer cultivation was designed based on the evolutionary engineering. ► The superior yeast strains were obtained in the closed-circulating system. ► The screened strains had an increased fermentation performance compared to the parent strain. ► The fermentation kinetic parameters behaved the same tendency in varied tests. ► It was an effective method to breed yeast strain by the step transfer regimen.Based on evolutionary engineering, a regimen of step transfer cultivation was designed to breed a yeast strain that adapted the process of closed-circulating ethanol fermentation in PDMS membrane bioreactor. The kinetic parameters of the fermentation such as ethanol concentration in the broth, cell concentration and death ratio, ethanol productivity and specific ethanol production rate behaved the same tendency in varied tests. During the fermentation with the screened strain S232, the glucose utilization, ethanol productivity, ethanol yield on glucose, ethanol yield on biomass and specific ethanol production rate were 3.81g/L/h, 1.63g/L/h, 0.427g/g, 194.3g/g and 0.385g/g/h, and had elevation of 7.32%, 10.13%, 2.64%, 14.97% and 19.57% over those of fermentation with the parent strain S3, respectively. These results indicated the screened yeast strain had mutated during the fermentation. The yeast strains bred by the regimen not only adapted to the closed-circulating fermentation process but also could be used for general fermentation process.
Keywords: Membrane; Bioreactor system; Yeast; Evolutionary engineering; Biofuel; Bioseparation
Improving root growth and cichoric acid derivatives production in hairy root culture of Echinacea purpurea by ultrasound treatment by Rui Liu; Wei Li; Li-Yang Sun; Chun-Zhao Liu (pp. 62-66).
► An efficient ultrasound-intensified strategy has been developed for improving CADs biosynthesis in E. purpurea hairy roots. ► The ultrasound-stimulated root growth was related to the increase of rolB gene expression for endogenous IAA biosynthesis. ► The ultrasound-stimulated CADs accumulation in the hairy roots was due to increasing phenylalanine ammonium lyase activity. ► The results presented new opportunities for understanding the hairy root culture process by ultrasound stimulation.An efficient ultrasound-stimulation strategy was developed for improving the hairy root growth and caffeic acid derivatives (CADs) biosynthesis in the hairy root cultures of Echinacea purpurea L. The 15-day-old hairy roots stimulated every 5 days by ultrasound for 6min produced the highest amount of CADs after 30 days of culture among all ultrasound treatment experiments. The obvious increase of CADs production in E. purpurea hairy roots stimulated by ultrasound was related to the increase of both rolB-regulated endogenous indole-3-acetic acid biosynthesis and phenylalanine ammonium lyase (PAL) activity. These results provided a basis for understanding of improving growth and secondary metabolism in the process of hairy root culture stimulated by ultrasound.
Keywords: Ultrasound; Hairy root; Caffeic acid derivatives; Indole-3-acetic acid; Phenylalanine ammonium lyase
Enhanced liver functions in mouse hepatoma cells by induced overexpression of liver-enriched transcription factors by Hideaki Yamamoto; Yoshinori Kawabe; Akira Ito; Masamichi Kamihira (pp. 67-73).
► Overexpression of eight liver-enriched transcription factors enabled the induction of liver functions in hepatoma cells. ► Switching between proliferation and expression of differentiated functions can be controlled in the genetically engineered hepatoma cells. ► This genetic modification approach for hepatoma cells can provide a means for generating cell sources for bioartificial liver support systems.Hepatoma cells, which are derived from liver carcinoma, are able to proliferate infinitely under culture conditions. However, the liver functions of hepatoma cells are generally low compared with those of hepatocytes in a liver. Here, we attempted to create genetically engineered hepatoma cells with enhanced liver functions by overexpression of liver-enriched transcription factors (LETFs), which are associated with the transcription of liver-specific genes and hepatic differentiation. For this purpose, genes for eight LETFs, hepatocyte nuclear factor (HNF)-1α, HNF-1β, HNF-3β, HNF-4α, HNF-6, CCAAT/enhancer binding protein (C/EBP)-α, C/EBP-β and C/EBP-γ, were obtained from the mouse liver. Mouse hepatoma Hepa1-6 cells were transduced with retroviral vectors, in which inducible expression cassettes for the LETF genes were introduced. Cell clones with inducible expression of high liver functions were established. Upon overexpression of the LETF genes, cell proliferation ceased and the cells exhibited an epithelial morphology, indicating hepatic maturation of hepatoma cells. This approach for genetic modification of hepatoma cells may be promising for the construction of cells for use in bioartificial liver support systems.
Keywords: Animal cell culture; Biomedical; Recombinant DNA; Hepatoma cell; Liver-enriched transcription factor; Liver function
Primary recovery of lipase derived from Burkholderia cenocepacia strain ST8 and recycling of phase components in an aqueous two-phase system by Pau Loke Show; Chin Ping Tan; Mohd Shamsul Anuar; Arbakariya Ariff; Yus Aniza Yusof; Soo Kien Chen; Tau Chuan Ling (pp. 74-80).
► Recovery of lipase using an ATPS composed of EOPO/K2H2PO4 with recycling of the phase components. ► EOPO 3900 with a TLL of 48.5% (w/w) was selected as the optimum system. ► Optimum recovery was obtained at a V R of 2.3 and a crude load of 20% (w/w) at pH 7. ► The purification factor and yield using recycled components was 14 and 99%, respectively.The recovery of Burkholderia cenocepacia ( B. cenocepacia) strain ST8 lipase has been investigated using a recycling aqueous two-phase system (ATPS) consisting of the thermoseparating random copolymer ethylene oxide–propylene oxide (EOPO) and potassium phosphate. In the primary ATPS, lipase was satisfactorily partitioned to the polymer-rich top phase in the system composed of EOPO 3900 with a tie-line length (TLL) of 48.5% (w/w), a volume ratio ( V R) of 2.3 and a crude load of 20% (w/w) at pH 7. In the secondary system, the polymer-rich top phase from the primary ATPS was isolated and subjected to thermo-induced phase separation. After thermoseparating, a concentrated solution of EOPO copolymer was formed in the bottom phase, and a water solution containing lipase was formed in the top phase of this secondary two-phase system. Recycling of the components was accomplished by mixing the concentrated polymer recovered from the secondary system and the phosphate solution recovered from the primary system. The average purification factor of lipase and the yield obtained from the four successive purifications were 14 and 99%, respectively. There was no significant difference in the purification factor, yield or partitioning efficiency of purified lipase obtained using fresh or recycled chemicals in the ATPS.
Keywords: Lipase; Aqueous two-phase; Purification; Protein recovery; Bioseparations; Enzymes
Alteration of anaerobic metabolism in Escherichia coli for enhanced hydrogen production by heterologous expression of hydrogenase genes originating from Synechocystis sp by Hao Zheng; Chong Zhang; Yuan Lu; Pei-Xia Jiang; Xin-Hui Xing (pp. 81-86).
► We constructed a vector with hydrogenase genes from Synechocystis sp. PCC 6803, and introduced it into Escherichia coli DH5α to alter the hydrogen metabolism with glucose. ► The recombinant strain reached a yield of 1.89mol/mol glucose, 95% of the theoretical hydrogen yield of E. coli. ► The expression of hoxEFUYH suppressed the transcription of native dehydrogenase, while it had no influence on the hydrogenase 3. ► The expressed HoxEFUYH expanded the substrate specificity of the hydrogen-evolving hydrogenase in E. coli.Metabolic engineering is recognized as one of the most important technologies for improving fermentative hydrogen yield. A vector with hydrogenase genes ( hoxEFUYH) from Synechocystis sp. PCC 6803 under an alkB promoter was constructed, and introduced into Escherichia coli DH5α to alter the hydrogen metabolism with glucose as the sole carbon source. The recombinant strain reached a highest hydrogen yield of 1.89mol/mol glucose, which was 95% of the theoretical hydrogen yield of E. coli. Hydrogenase activities for hydrogen evolution were increased and formic acid assimilation was accelerated with the expression of hoxEFUYH. The expression of hoxEFUYH suppressed the transcription of native hydrogenase 1 and hydrogenase 2, which were responsible for hydrogen uptake activity, while it had no influence on the transcription of the hydrogenase 3. Moreover, as the electron donor of HoxEFUYH is NADH, the expressed HoxEFUYH expanded the substrate specificity of the hydrogen-evolving hydrogenase in E. coli.
Keywords: Biogas; Metabolite over production; Recombinant DNA; Synechocystis; sp.; Hydrogenase; Metabolic engineering
Growth of fungal strains on coffee industry residues with removal of polyphenolic compounds by Ercília M.S. Machado; Rosa M. Rodriguez-Jasso; José A. Teixeira; Solange I. Mussatto (pp. 87-90).
Display Omitted► Fungal strains were cultivated in coffee silverskin (CS) and spent coffee grounds (SCGs). ► Cultivations were performed under solid-state fermentation conditions. ► All the evaluated strains grown and released phenolic compounds from these residues. ► Penicillium purpurogenum, Neurospora crassa and Mucor gave the best results. ► Phenolic compounds extraction is a potential alternative for CS and SCG valorization.Coffee silverskin (CS) and spent coffee grounds (SCG) are abundant residues generated by the coffee industry, but up till now they are practically unutilized being discharged to the environment or burned for elimination. In the present study, the ability of seven different fungal strains from the genera Aspergillus, Mucor, Penicillium, and Neurospora, to grow and release phenolic compounds from these residues under solid-state cultivation conditions was evaluated. All the strains were able to growth on CS and SCG, but Penicillium purpurogenum, Neurospora crassa and Mucor were able to release the highest amounts of phenolic compounds from both residues. Phenolic compounds have wide-range applications in the food and pharmaceutical industries, and thus, the extraction of these compounds could be a valuable alternative for the exploitation of CS and SCG. Additionally, the use of solid-state fermentation to obtain these compounds has a positive environmental impact because the use of chemical solvents is avoided.
Keywords: Coffee silverskin; Spent coffee grounds; Solid-state fermentation; Biodegradation; Microbial growth; Phenolic compounds
The influence of aeration on activity and operational stability of two snake venom amino acid oxidases by Zvjezdana Findrik; Ana Vrsalović Presečki; Đurđa Vasić-Rački (pp. 91-98).
► Two snake venom amino acid oxidases were used as catalysts. ► The influence of oxygen on enzyme activity was measured. ► The influence of aeration rate on enzyme operational stability was examined. ► Aeration increases reaction rate, but has a negative effect on enzyme operational stability.Twol-amino acid oxidases from snake venom Crotalus adamanteus and Crotalus atrox were used as catalysts in the biotransformation ofl-methionine to 2-oxo-4-methylthiobutyric acid. Catalase was present in all reactions to remove the hydrogen peroxide that evolves in the reaction, to prevent its reaction with an α-keto acid, and potential negative influence on amino acid oxidases. Batch reactor experiments without and with aeration were carried out with the two enzymes. Michaelis constants for oxygen were estimated from the initial reaction rate experiments and were found to be 0.030 and 0.053mM forl-AAO from C. adamanteus and C. atrox, respectively. It was found that aeration increases the reaction rate and decreases the time necessary to achieve 100%l-methionine conversion due to the increase of oxygen concentration in the solution. It was also found that the increase of the air flow rate causes faster enzyme operational stability decay rate. The operational stability decay rate constant for C. adamanteus enzyme was estimated to be 6.29 x 10−4min−1 in the experiment without aeration. It increased in the experiment with aeration at 10 Lh−1 to 3.75×10−4min−1, and was practically the same at higher air flow rate. Experiments withoutl-methionine presence proved that oxygen concentration was the reason for the decrease in enzyme activity for this enzyme and not the gas bubbles itself. In these experiments operational stability decay rate constants were one order of magnitude higher than whenl-methionine was oxidized. It was found forl-amino acid oxidase from C. atrox that the operational stability decay rate constant linearly depends on the air flow rate. This dependence can be described by the following equation: kd=3.1×10−4×qv+3.4×10−4 min−1. The operational stability decay rate constants values estimated from the experiments carried out at different air flow rates in the batch reactor were found to be similar to the ones estimated forl-AAO from C. adamanteus.
Keywords: Abbreviations; l; -AAO; l; -amino acid oxidase; l; -met; l; -methionine; 2-oxo; 2-oxo-4-methylthiobutyric acidAmino acid oxidase; Oxygen; Enzyme stability; Enzyme activity
Medium engineering to enhance mushroom tyrosinase stability by Elham Jahangiri; Roshanak Agharafeie; Hermann-Josef Kaiser; Younes Tahmasbi; Raymond L. Legge; Kamahldin Haghbeen (pp. 99-105).
► Development of an entrapping method for mushroom tyrosinase. ► Studying the combination of one-phase-binary solution and cross-linked polyacrylamide for entrapping enzymes. ► Changes in the functional and structural properties of mushroom tyrosinase under the conditions selected.Tyrosinase could have many applications including biotransformation, bioremediation, and in biosensors. However, its application, especially at industrial scale, is seriously limited by its rapid inactivation. To increase mushroom tyrosinase (MT) stability, the enzyme was entrapped in different matrixes. Then, the structural and operational stability of the entrapped mushroom tyrosinase (EMT) were examined in one-phase-binary solutions (OPBS) obtained by mixing different amounts (0–100%) of water-miscible solvents such as acetonitrile and 2-propanol with PBS. Experiments showed MT lost all of its activity upon entrapment in the classical sol–gels and hybrid silica sol–gels, but could retain up to 25% of its activity in cross-linked polyacrylamide (CLP). These studies revealed while EMT in CLP was losing about 0.06% of its structural stability per hour in PBS, OPBS containing 2-propanol (50 or 75%) did not affect the EMT structural stability and increased its cresolase activity up to 1.5 fold during 50 days storage at 4°C. The aquaphilicity of CLP helps EMT to function normally in OPBS and using 2-propanol (50% or more) in such media not only stopped EMT loss from the sieve structure of the CLP, but it was also beneficial to the more hydrophobic substrates. Using tyrosine as a substrate in OPBS containing 2-propanol (75%),l-DOPA production reached 4.21±0.23μM/min−1.
Keywords: Abbreviations; Ac; acrylamide; APS; ammonium persulfate; bAc; bis-acrylamide; CLP; cross-linked polyacrylamide; DC; denaturation capacity; EMT; entrapped mushroom tyrosinase; MePAPh; 4-[(4-methylphenyl)azo]-phenol; MeTPS; methyltripropoxysilane; MT; mushroom tyrosinase; OPBS; one-phase binary solutions; PBS; phosphate buffer solution; PEG; poly ethylene glycol; Pr-TMS; propyltrimethoxysilane; TEOS; tetraethoxyorthosilane; TMOS; tetramethoxyorthosilaneImmobilized; Mushroom tyrosinase; Stability; Kinetic; Sol–gels; Cross-linked polyacrylamide
Continuous production of biodiesel from fat extracted from lamb meat in supercritical CO2 media by Sulaiman Al-Zuhair; Ahmedin Hussein; Ali H. Al-Marzouqi; Isameldin Hashim (pp. 106-110).
► A system of meat fat extraction and biodiesel production in SC-CO2 was developed. ► Two valuable products are simultaneously produced: biodiesel and low-fat lean meat. ► Effects of methanol:fat molar ratio and enzyme stability were investigated. ► Highest biodiesel production rate was achieved when methanol:fat ratio was 10:1. ► The enzyme best maintained its activity at methanol:fat ratio of 5:1.Waste animal fat is considered a promising cheap alternative feedstock for biodiesel production that does not compete with food stock. In addition, using waste animal fat as a feedstock is considered a waste management process. In this work, an integrated process for a continuous fat extraction from lamb meat followed by enzymatic production of biodiesel in supercritical CO2 has been developed and tested. The system simultaneously produces two valuable products, namely biodiesel and healthy low-fat lean lamb meat (HLFLM). For the enzymatic process to be feasible, lipase is preferred to be used in immobilized form, which allows easy reuse. The continuous system was operated at 200bar and a SC-CO2 flow of 0.5mlmin−1, with extraction and transesterification temperatures of 45°C and 50°C, respectively. The effects of methanol:fat (M:F) molar ratio and enzyme stability were investigated. It was found that with fresh enzyme, a M:F molar ratio of 10:1 gave the highest biodiesel production rate of 0.37mgmin−1g-enzyme−1 compared to only 0.09mgmin−1g-enzyme−1 using a M:F molar ratio of 5:1. However, when a M:F molar ratio of 10:1 was used, the activity of the enzyme in the third meat replacement cycle drastically dropped to 18% of its original value, compared to 79% using a M:F molar ratio of 5:1.
Keywords: Continuous operation; Waste animal fat; Biodiesel; Lipase; Supercritical CO; 2
Friction factors, convective heat transfer coefficients and the Colburn analogy for industrial sugarcane juices by Zailer Astolfi-Filho; Eduardo Basílio de Oliveira; Jane Sélia dos Reis Coimbra; Javier Telis-Romero (pp. 111-118).
► Friction factors and Nusselt numbers were determined for sugarcane juices sampled from an industrial processing line. ► These experimentally determined data showed a good correlation with those calculated by means of empirical correlations. ► The Colburn analogy was successfully applied to obtain heat transfer coefficients from friction factor values in turbulent flow. ► New data outlined in this work will be useful to improve the processing of sugarcane juice for ethanol fuel production.Sugarcane ( Saccharum sp .) is a relatively low-cost agricultural resource, produced mainly in tropical and subtropical regions of the planet. One of the most important applications of sugarcane juice is in the production of ethanol biofuel. A correct design of unit operations during the sugarcane juice processing requires an accurate characterization of the flow properties (friction factors, rheological behaviour, thermophysical properties and heat transfer coefficients) of this raw material. In this work, friction factors ( f) and Nusselt number (Nu) were determined for sugarcane juice in different steps of its processing (untreated sugarcane juice, clarified sugarcane juice and mixed sugarcane juice). For both laminar and turbulent flows, the empirical models for f fitted well ( r2≥0.9) with the experimental values obtained by pressure drop measurements. Also, Nu values calculated by applying the empirical correlation of Hausen (for laminar flow) and that of Sieder–Tate (for turbulent flow) presented good adjustment ( r2≥0.9) to the experimentally determined values. Thus, the f and Nu values obtained for turbulent flow were taken to verify the Colburn analogy, which furnished results with small deviations (≤8.9%) from the expected ideal value. This finding indicates the suitability of such analogy in this case, allowing good estimations of heat transfer coefficients from frictions factors, or vice versa, for sugarcane juices in turbulent flow conditions.
Keywords: Biofuel; Colburn analogy; Ethanol; Friction factor; Heat transfer; Sugarcane
Relationship between light intensity and morphology of the moss Physcomitrella patens in a draft tube photo bioreactor by Martin Cerff; Clemens Posten (pp. 119-126).
Display Omitted► Choice of light intensity affects morphology of the moss Physcomitrella patens. ► Pellet formation in a draft tube photo bioreactor at high light intensities. ► Characterization of pellet formation by image- and particle size analysis. ► Low initial light intensity and stepwise increase enables high bio dry mass concentrations (>4gL−1) and maintains isolated filaments. ► Adjustment of illumination conditions as non-invasive process strategy.The moss Physcomitrella patens is a promising expression system for the production of recombinant human biopharmaceuticals. Highly standardized cultivation conditions are required to maintain the desired chloronemal level of cell differentiation during cultivation. Although the life cycle of P. patens is well known and described in literature only little is known about morphology development in closed photo bioreactors (PBR). In the current work we describe the relationship between light intensity and development of moss-morphology in a stirred draft tube PBR during cultivation: pellet formation occurred for high initial photon flux densities (PFD). The application of a low initial PFD followed by subsequent increase could prevent the moss culture from forming dense pellets and the filamentous morphology could be preserved. A high bio dry mass (BDM) of 3–4.5gL−1 could be achieved as well as high specific chlorophyll content of the filaments. Pellet formation is discussed on basis of particle size distributions and data obtained from image analysis. Adjusting the level of illumination is a non-invasive method to maintain the filamentous morphology of P. patens and thus homogeneity of the medium because cells do not need to be disrupted mechanically.
Keywords: Abbreviations; PBR; photo bioreactor; CUR; carbon uptake rate; OPR; oxygen production rate; BDM; bio dry mass; PFD; photon flux density; LED; light emitting diodeLED; Growth kinetics; Photo bioreactors; Image analysis; Plant cell cultures; Morphology
Frothability and surface behavior of a rhamnolipid biosurfactant produced by Pseudomonas aeruginosa MA01 by Hamid Khoshdast; Habib Abbasi; Abbas Sam; Kambiz Akbari Noghabi (pp. 127-134).
Display Omitted► A rhamnolipid biosurfactant produced by a Pseudomonas aeruginosa MA01 was characterized. ► The rhamnolipid product contained R1C10C10 and R2C10C10 in equal molar ratio. ► Rhamnolipid was found to be more surface active than frothers commonly used in mineral flotation. ► Film elasticity and frothability of rhamnolipids were studied. ► Rhamnolipid had more frothing capacity compared to those from commercial flotation frothers.In this work, surface activity and frothability of rhamnolipid biosurfactant produced by a Pseudomonas aeruginosa MA01 were studied and compared with conventional flotation frothers, i.e. methyl isobutyl carbinol (MIBC), pine oil, Dowfroth-250 (DF-250), and Aerofroth-65 (A-65). FTIR and ES-MS analysis indicated that the product contained two types of commonly found rhamnolipids:l-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL-1) andl-rhamnosyll-rhamnosyl-β-hydroxydecanoyl-β-hydroxydecanoate (RL-2). Surface tension measurements showed that the rhamnolipid product reduces surface tension more effectively than frothers due to higher molecular weight and the presence of multiple oxygenated groups in its structure, producing more viscous liquid film, which was confirmed with film elasticity calculations. Both surface tension and elasticity values followed the order: rhamnolipid>A-65>DF-250>pine oil>MIBC. Frothability of the tested surfactants gave the order: rhamnolipid>A-65>DF-250>MIBC>pine oil.
Keywords: Rhamnolipid biosurfactant; Pseudomonas aeruginosa; Frothability; Surface activity; Film elasticity
Influence of mixed liquid suspended solids and hydraulic retention time on oxygen transfer efficiency and viscosity in a submerged membrane bioreactor using pure oxygen to supply aerobic conditions by Francisco A. Rodríguez; Patricia Reboleiro-Rivas; Francisco Osorio; María V. Martínez-Toledo; Ernesto Hontoria; José M. Poyatos (pp. 135-141).
► Alpha-factor significantly decreases with MLSS concentration. ► Changes in the behaviour of the biological system happen with different MLSS and HRT. ► Kinetic coefficients have been changed with different MLSS and HRT in MBR system. ► The efficiency of oxygen transfers is highly influenced by MLSS and HRT.The performance of a wastewater bench-scale membrane bioreactor (MBR) treatment plant using pure oxygen conditions was monitored daily to provide aerobic conditions. The results showed the capacity of the MBR system to remove organic material over at hydraulic retention time (HRT) of 12h and 18h. The alpha-factors of aeration were determined at three different mixed liquid suspended solids (MLSS) concentrations, in order to understand the system performance when pure oxygen was used to provide the aerobic conditions of the MBR system. Under these working conditions, an alpha-factor in the range 0.90–0.12 was obtained when the HRT of 18h was performed, and the MLSS concentration increased from 4300 to 10,275mg/L. Additionally, an alpha-factor in the range of 0.28–0.02 was obtained at HRT of 12h and the MLSS increased from 4071 to 11,192mg/L. Our study suggested significant changes in the behaviour of the biological system (viscosity, aeration efficiency, organic matter removal) when the concentration of MLSS was increased and the HRT decreased in the bioreactor, decreasing the aeration efficiency in both cases. Furthermore, the efficiency of aeration seems to be more affected by MLSS concentration than by HRT under our operating conditions over the range studied.
Keywords: Activated sludge; Pure oxygen; MBR; Wastewater; Alpha-factor
Characteristics of invertase partitioned in poly(ethylene glycol)/magnesium sulfate aqueous two-phase system by Taylan Karkaş; Seçil Önal (pp. 142-150).
► Invertases have many biotechnological applications especially in food processing. ► ATPSs are powerful and attractive for separating of various biomolecules. ► Characterisation of invertase partitioned with an ATPS was searched.The goal of this study was to determine some characteristics of Baker's yeast invertase partitioned with poly(ethylene glycol)/MgSO4 aqueous two-phase system (ATPS). Under optimized conditions [PEG-3000 (15%, w/w) and MgSO4 (23%, w/w) with 5% (w/w) MnCl2 at pH 5.0] yeast invertase was partitioned by using an ATPS with purification factor of 6.2-fold and activity recovery of 217.7%, respectively. The yeast invertase was characterised with respect to its activity and stability at various pH and temperature ranges. Optimum pH and temperature were determined at pH 5.5 and 60°C, respectively. The enzyme was very stable in the range of pH 4.0–7.0 and more than 95% of its initial activity was recovered. The yeast invertase was also stable at the temperature range of 4–50°C and retained nearly about 98% of its initial activity at 50°C. Kinetic parameters, K m and Vmax using sucrose as substrate were measured as 24.1mM and 35.5U, respectively. MnSO4 and MnCl2 efficiently enhanced the activity and also showed an activator effect for invertase. Relative activities were found as 151% and 156% for MnSO4 and MnCl2, respectively. The biochemical properties of the yeast invertase partitioned in an ATPS make the enzyme good candidates for several industrial applications.
Keywords: Baker's yeast; Invertase; Aqueous-two phase system; Bioseparation; Partitioning; Characterisation
Anode modification by electrochemical oxidation: A new practical method to improve the performance of microbial fuel cells by Minghua Zhou; Meiling Chi; Hongyu Wang; Tao Jin (pp. 151-155).
► Anode electrochemical modification was convenient and effective to improve MFCs performance. ► Three modifications posed positive effects, and CM-N was the best with power density increase by 43%. ► Electrochemical oxidation resulted in the change of anode properties and surface characteristics. ► Increasing surface area and current response might contribute to the performance improvement.Electrochemical oxidation as a convenient and effective method was established for anode modification to improve the performance of microbial fuel cells (MFCs). The anode modification was realized by one-step electrochemical treatment in one of the three electrolytes (nitric acid, ammonium nitrate, ammonium persulfate) at ambient temperature. The performances of MFCs before and after anode modification were compared, confirming that all these anode modifications posed positive effects. The maximum power density of the MFC with the anode modified by nitric acid was 792mW/m2, which was 43% larger than the unmodified control (552mW/m2). Furthermore, the Coulombic efficiency (CE) significantly promoted about 71% from 14% (the unmodified MFC) to 24%. It revealed that the electrochemical oxidation resulted in the change of the anode properties, such as surface morphology, internal resistance and anode potential, and thus benefited to the microbial attachment and electron transfer on the anode surface, which might contribute to the performance improvement of the MFCs.
Keywords: Microbial fuel cell; Electrochemical oxidation; Carbon mesh; Power generation; Cyclic voltammetry; Electrode modification
Ionic liquid assisted enzymatic delignification of wood biomass: A new ‘green’ and efficient approach for isolating of cellulose fibers by Muhammad Moniruzzaman; Tsutomu Ono (pp. 156-160).
► Development a new green and efficient process for isolating celluloses. ► Combination of enzymes with ILs enhanced wood delignification. ► Obtained high strength wood fibers to be suitable for biomaterials.The objective of this study was to provide a new environmentally friendly and efficient approach for isolating cellulose fibers with minimum structural alteration from wood biomass. The method comprised enzymatic delignification of ionic liquid (IL) swollen wood biomass in ILs–aqueous systems with the aim of overcoming low delignification efficiency associated with the difficulties in enzyme accessibility to the solid substrate and the poor substrate and products solubility in aqueous system. It was found that the cellulose rich wood fibers obtained from biological pretreatment in IL–aqueous systems contained significantly lower amounts of lignin as compared to those found in conventional methods. The treated wood fibers were characterized using Fourier-transform infrared spectroscopy (FTIR), scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffractometry (XRD) and compared those with untreated wood fibers.
Keywords: Enzymatic delignification; Ionic liquids; Wood biomass; Laccase; Green process; Cellulose rich materials