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Biochemical Engineering Journal (v.27, #3)
Simultaneous nitrogen and phosphorus removal using denitrifying phosphate-accumulating organisms in a sequencing batch reactor by Satoshi Tsuneda; Takashi Ohno; Koichi Soejima; Akira Hirata (pp. 191-196).
In this study, an anaerobic/aerobic/anoxic process (referred to as an AOA process) using a sequencing batch reactor (SBR) was proposed for simultaneous phosphorus and nitrogen removal from wastewater. The AOA process was stably operated over more than one year when a certain amount of carbon substrate (40mg-C/L in a reactor) was supplemented to inhibit aerobic phosphate uptake. The average nitrogen and phosphorus removal efficiencies were 83% and 92%, respectively. It was demonstrated that phosphate-accumulating organisms (PAOs) capable of utilizing nitrite as an electron acceptor, the so-called denitrifying phosphate-accumulating organisms (DNPAOs), could exist in the AOA process. Moreover, the ratio of anoxic phosphate uptake rate (PUR) to aerobic PUR (anoxic/aerobic PUR ratio), which indicates the fraction of DNPAOs in total PAOs, was experimentally evaluated. The results indicate that the AOA process has a much larger anoxic/aerobic PUR ratio than the conventional A2O (anaerobic/anoxic/aerobic) and AO (anaerobic/aerobic) processes. In conclusion, the AOA process allows DNPAOs to take an active part in simultaneous nitrogen and phosphorus removal in an SBR when a suitable amount of carbon substrate is supplied at the start of aerobic conditions.
Keywords: Enhanced biological phosphorus removal (EBPR); Anaerobic/aerobic/anoxic (AOA) process; Denitrifying phosphate-accumulating organisms (DNPAOs); Sequencing batch reactor (SBR)
Computer simulated fed-batch cultivation for over production of PHB: A comparison of simultaneous and alternate feeding of carbon and nitrogen by Shilpi Khanna; A.K. Srivastava (pp. 197-203).
Batch kinetics for cultivation of Ralstonia eutropha, NRRL B14690 featured 19.7g/l biomass and 10.89g/l PHB accumulation in 60h under statistically optimized culture conditions. For in-depth understanding of culture dynamics and for suitable reactor design a mathematical model was developed. The model was highly instrumental in guiding the appropriate experiments to optimize the process with minimum efforts. Offline computer simulated fed-batch cultivations allowed selection of two different nutrient (nitrogen (as urea) and fructose) feeding strategies. Fed-batch fermentation, conducted by feeding nitrogen (as urea) (8g/l) at the rate of 70ml/h and fructose (300g/l) at the rate of 80ml/h simultaneously yielded 17.57g/l PHB in 50h with an overall productivity of 0.35g/lh. In another strategy, alternate feeding of the nitrogen (as urea) (15g/l) and fructose (350g/l) at a flow rate of 80ml/h resulted in PHB accumulation of 18.46g/l in 40h resulting in overall productivity of 0.46g/lh. Model-based fed-batch operation was very helpful for over producing PHB by R. eutropha. The increase in PHB productivity in fed-batch cultivation was almost 2.6 times as compared to batch.
Keywords: Modelling; Fructose; Growth kinetics; Fed-batch culture; Ralstonia eutropha; PHB
Kinetic modeling of the biodegradation of the aqueous p-xylene in the immobilized soil bioreactor by A. Kermanshahi pour; D. Karamanev; A. Margaritis (pp. 204-211).
Biodegradation kinetics of p-xylene in aqueous solution was studied in a new type of biofilm reactor—immobilized soil bioreactor. Biofilm modeling was performed in order to determine the concentration gradients within the biofilm based on a coupled diffusion-reaction model for cylindrical geometry. The total amount of biomass in the 0.83L bioreactor changed from 460 to 780mg during continuous operation. The concentration gradient was typical for a shallow biofilm and also predicts low mass transfer resistance within the biofilm. Rate constants for the continuous regime were determined based on the biomass growth and the amount of substrate utilization, resulting in the values of: maximum specific growth rate ( μmax) of 0.0047h−1, half saturation constant ( Ks) of 3.9mgL−1 and yield ( Yx/s) of 0.05mg biomassmg−1 substrate. For batch operation, a similar yield coefficient was assumed and the experimental data was fitted to the Monod equation. μmax of 0.0047h−1 (similar to continuous) and Ks of 10mgL−1 were obtained.
Keywords: Microbial kinetics; p; -Xylene; Biodegradation; Bioremediation; Bioreactor; Biofilm
Pulsed electric field treatment of Saccharomyces cerevisiae suspensions: A mechanistic approach coupling energy transfer, mass transfer and hydrodynamics by L. Schrive; A. Grasmick; S. Moussière; S. Sarrade (pp. 212-224).
Pulsed electric field (PEF) treatment is a promising sterilization technique because of its nonthermal characteristics. It is well known that PEF applied at field values about 30×105Vm−1 promotes cell disruption, internal component leakage, and subsequent death. Classically, lethality is only related to electric and biological parameters. However, it is conceivable that hydrodynamic or process parameters are also involved. The goal of this work was therefore to understand the synergetic effects between electric energy transfer and hydrodynamics, based on trials carried out with a model biomass composed of Saccharomyces cerevisiae yeast treated in an experimental set-up.This work demonstrates that survival rates can be drastically decreased at constant electric energy input, by strong mixing between each pulse. Taking into account the cell permeabilization, leakage phenomena were considered through internal component diffusion and convection from the biologic cell to the surrounding medium. A new approach was therefore proposed in which the survival rate was associated with a modified Sherwood number. For the first time with PEF treatment, this approach integrated all kinds of parameters: biological (mainly through the cell radius), electrical (through the electric field and electrical resistance), and hydraulic (through the Reynolds number and number of recyclings). Finally, this study contributes to optimizing the specific energy consumption, making the PEF sterilization process more efficient.
Keywords: Pulsed electric field (PEF); Cell permeabilization; Sterilization; Energy and mass transfer
Soft-sensor development for fed-batch bioreactors using support vector regression by Kiran Desai; Yogesh Badhe; Sanjeev S. Tambe; Bhaskar D. Kulkarni (pp. 225-239).
In the present paper, a state-of-the-art machine learning based modeling formalism known as “support vector regression (SVR)�, has been introduced for the soft-sensor applications in the fed-batch processes. The SVR method possesses a number of attractive properties such as a strong statistical basis, convergence to the unique global minimum and an improved generalization performance by the approximated function. Also, the structure and parameters of an SVR model can be interpreted in terms of the training data. The efficacy of the SVR formalism for the soft-sensor development task has been demonstrated by considering two simulated bio-processes namely, invertase and streptokinase. Additionally, the performance of the SVR based soft-sensors is rigorously compared with those developed using the multilayer perceptron and radial basis function neural networks. The results presented here clearly indicate that the SVR is an attractive alternative to artificial neural networks for the development of soft-sensors in bioprocesses.
Keywords: Artificial neural networks; Bioreactor; Soft-sensors; Support vector regression; Multilayer perceptron; Radial basis function network
Support material selection for anaerobic fluidized bed reactors by phospholipid analysis by C. Arnaiz; J.C. Gutierrez; J. Lebrato (pp. 240-245).
Many different types of support materials are used in anaerobic fluidized bed in order to obtain high levels of biomass in the reactors. A material is usually considered the most suitable in terms of physical–chemical parameters or reactor performance, but not in terms of microbial adhesion. In this work, two commercial materials were tested as support for anaerobic biomass: perlite and sepiolite. Biofilm accumulation was estimated by two methods, attached volatile solids and lipid phosphate concentration, both determined directly on attached biofilm. The biomass adhesion on sepiolite was higher than the perlite for all the tested initial total organic carbon concentration in the reactors. We have found that biomass measurement method developed directly on attached biofilm is relevant to the amount of living cells. We also discuss the suitability of estimating attached biomass by means of direct measures on biocovered particles rather than on enrichment cultures of free cells detached from the supports.
Keywords: Anaerobic processes; Biofilms; Fed-batch culture; Fluidized bed bioreactors; Phospholipid analysis; Wastewater treatment
Effect of intermittent aeration on the decrease of biological sludge amount by Soo-Jung Jung; Kazuhiko Miyanaga; Yasunori Tanji; Hajime Unno (pp. 246-251).
As a biochemical approach to decrease the amount of biological sludge, the sludge solubilization by repeatedly changing the microbial environments from aerobic to anaerobic was studied. The sludge reduction amount in batch operation was related to the degradation of intracellular materials released through sludge solubilization. The shorter the operation cycle time was, the more effective the sludge amount reduction was. On the other hand, under the cycle time of 3–24h, longer anaerobic period compared to aerobic period was effective in removing nitrogenous compounds. This was partly ascribed to the excretion of enzyme such as protease and lipase.
Keywords: Sludge reduction; Aerobic–anaerobic; Solubilization; Enzyme
Effects of oxygen, carbon dioxide and ethylene on growth and bioactive compound production in bioreactor culture of ginseng adventitious roots by Cheol-Seung Jeong; Debasis Chakrabarty; Eun-Joo Hahn; Hyung-Lae Lee; Kee-Yoeup Paek (pp. 252-263).
The gaseous composition in plant cell and tissue cultures is an important factor affecting the plant physiology. Gaseous composition, especially O2, CO2 and C2H4 has profound effect on cell growth and secondary metabolite production. In this work, we investigated the effects of O2, CO2 and C2H4 on adventitious root growth, ginsenoside and polysaccharide production using bioreactor system. The results showed significant increase of dry weight with the gaseous compositions of 10ppm (57.9g) and 20ppm (55.17g) ethylene as compared to control as well as other treatments at the end of culture but CO2 and C2H4 were unfavorable to the adventitious root culture due to low ginsenoside production. Oxygen concentration of 40% was found optimal for the production of adventitious root mass, ginseng saponin and polysaccharide. The kinetics of changes in ion concentrations, sugar and electrical conductivity (EC) in the media was monitored to understand the relationship between growth, ginsenoside production and nutrient partitioning. Our results indicate that EC, sugar and mineral ions decreased with the progress of the experiment. With the progress of the experiment, sugars and mineral ions were utilized by the adventitious roots for the production of biomass resulting continuous decrease in EC. The interaction among gaseous metabolites, kinetics of changes in ion concentrations, sugar uptake and ginsenoside formation were also elucidated. This information will facilitate the rational operation of bioreactor culture system producing secondary metabolites.
Keywords: Carbon dioxide; Ethylene; Ginsenoside production; Nutrient uptake; Oxygen; Panax ginseng
Evaluation of the optical sulfite oxidation method for the determination of the interfacial mass transfer area in small-scale bioreactors by V. Linek; M. KordaÄ?; T. Moucha (pp. 264-268).
The optical sulfite oxidation method was recently used by Hermann et al. [R. Hermann, M. Lehmann, J. Büchs, Characterization of gas–liquid mass transfer phenomena in microtiter plates, Biotechnol. Bioeng. 81 (2003) 178–186] and Kensy et al. [F. Kensy, H.F. Zimmermann, I. Knabben, T. Anderlei, H. Trauthwein, U. Dingerdissen, J. Büchs, Oxygen transfer phenomena in 48-well microtiter plates: determination by optical monitoring of sulfite oxidation and verification by real-time measurement during microbial growth, Biotechnol. Bioeng. 89 (2005) 698–708.] for determination of interfacial mass transfer area a in a microtiter plate at different shaking intensities. They have determined relative increase of a liquid surface area due to the shaking as a ratio of average specific oxygen absorption rates Nave in 0.5M sulfite solution with 10−4M CoSO4 under shaken and unshaken conditions. In this paper it is shown that the method is based on the assumption that the profiles of instantaneous oxygen flux Φ during the whole experiments are the same for the shaken and nonshaken solutions. Our experiments performed in a stirred cell did not confirm validity of this assumption. Monitoring of pH and sulfite concentration during the oxidation of sulfite solutions revealed lower values of Φ into nonagitated solution compared to agitated one. The value of Nave in the nonagitated (nonshaken) sulfite solution was by 30% lower than the rate in the agitated one. Consequently, the mass transfer areas measured by the optical sulfite oxidation method are overestimated seriously, cca. 1.4 times. The lower oxygen transfer rate in the nonagitated solution is caused by a local drop of the sulfite concentration near the interface which is due to insufficiently fast transport of sulfite ions from the bulk of a liquid phase to interface under nonagitated (nonshaken) conditions. It is shown that the optical method for measuring of a is fundamentally flawed and should not be used in the presented form.
Keywords: Bioreactors; Gas–liquid mass transfer; Oxygen transfer; Microtiter plates; Sulfite oxidation; Optical method
Preparation and biocompatibility of chitosan microcarriers as biomaterial by Xi-Guang Chen; Cheng-Sheng Liu; Chen-Guang Liu; Xiang-Hong Meng; Chong M. Lee; Hyun-Jin Park (pp. 269-274).
Chitosan microcarriers (100–200μm) were prepared by the methods of emulsification and ethanol coagulant. It has smooth surface and was stable in phosphate buffer solution (PBS) of at pH 7.2 in the treatment of temperature 120°C and pressure 150kPa. The chitosan microcarriers showed molecular affinity to the bovine serum proteins at pH 7.2. The adsorptive capacity of the microcarriers to the serum albumin was 6.8mg protein/g chitosan bead. The chitosan microcarriers were found to have good biocompatibility and no cytotoxicity to both human and mouse fibroblasts in tissue cell culture. The fibroblasts well adhered on the smooth surface of the chitosan microcarriers and grew in high cell density. The results suggest a good potential of the chitosan microcarriers as a wound-healing biomaterial.
Keywords: Affinity; Microcarrier; Tissue cell culture; Adsorption
Modelling ofl-DOPA enzymatic oxidation catalyzed byl-amino acid oxidases from Crotalus adamanteus and Rhodococcus opacus by Z. Findrik; B. Geueke; W. Hummel; Ä?. Vasić-RaÄ?ki (pp. 275-286).
l-amino acid oxidases (l-AAO) are well known for their broad substrate specificity.l-amino acid oxidases from Crotalus adamanteus and Rhodococcus opacus were applied for biotransformation of 3,4-dihydroxyphenyl-l-alanine (L-DOPA) as a substrate to its corresponding α-keto acid. In this reaction, hydrogen peroxide formed as a by-product causes chemical decarboxylation of α-keto acids and acts as competitive product inhibitor. Beef liver catalase was used to decompose it.It was shown that both enzymes were able to oxidizel-DOPA to corresponding products.l-AAO from R. opacus was more specific (lowerKml−DOPA value) and more active towardsl-DOPA substrate thanl-AAO from C. adamanteus. Its catalytic constant, k3, estimated by Levenspiel's method, was found to be 10-fold higher than the one forl-AAO from C. adamanteus.l-AAO from R. opacus exhibits slightlyl-DOPA inhibition, which is not the case forl-AAO from C. adamanteus.The biotransformations ofl-DOPA were carried out in batch enzyme membrane reactor (EMR), as well as in the repetitive batch EMR. The reactor and kinetics were modelled. Parameters were estimated by differential and integral method and presented in this article.
Keywords: l; -amino acid oxidase; l; -DOPA; Enzyme membrane reactor; α-Keto acid; Enzyme kinetics; Modelling
Optimization of lipase production in a triple impeller bioreactor by Meenal S. Puthli; Virendra K. Rathod; Aniruddha B. Pandit (pp. 287-294).
The fermentation kinetics for the synthesis of lipase by Candida rugosa has been studied in a batch system in a 2l batch bioreactor. The studies illustrated the influence of gas–liquid mass transfer coefficient on the cell growth and hence the lipase production. In order to maintain sufficient oxygen concentration for the optimum cell growth and lipase activity, fermentation has been carried out using triple impeller system at an operating speed of 600rpm (optimum operating speed as found in the earlier work) and at different aeration rates. Gas-flow rate of 50.34cc/s has been observed to be optimum. Under optimized conditions of the bioreactor, cell production was enhanced and the lipase activity increased by 2.5 folds. The Monod's kinetics was fitted to the data of the operating parameters to understand the cell growth and substrate consumption. Luedking and Piret model was applied to the data to determine the relationship between the cell growth and lipase production. The lipase production was found to be microbial growth associated function.
Keywords: Bioreactor; Gas–liquid mass transfer; Fermentation; Triple impeller; Aerobic process; Growth kinetics
Ethanol production from crude whey by Kluyveromyces marxianus by Salman Zafar; Mohammad Owais (pp. 295-298).
Kluyveromyces marxianus strain MTCC 1288 was employed to study the batch kinetics of ethanol and biomass production from crude whey. The yeast was able to metabolize most of the lactose within 22h to give 2.10gL−1 ethanol and 8.9gL−1 biomass. The growth rate reached the peak value of 0.157h−1 during the exponential phase but decreased significantly after the fermentation time of 12h, presumably due to product inhibition. The specific ethanol formation rate attained the maximum value of 0.046h−1 between 6 and 8h of batch fermentation. The relationship between ethanol concentration and specific growth rate suggested a strong inhibitory effect of ethanol on the specific culture growth rate.
Keywords: Fermentation; Whey; Ethanol; Batch kinetics; Biomass
Thermal deactivation of a commercial α-amylase from Bacillus licheniformis used in detergents by V. Bravo Rodríguez; E. Jurado Alameda; J.F. Martínez Gallegos; A. Reyes Requena; A.I. García López (pp. 299-304).
We have studied experimentally the enzymatic hydrolysis of soluble starch with a commercial α-amylase from Bacillus licheniformis (commercial enzyme Termamyl 300 L Type DX) at pH 7.5 within the temperature range of 60–75°C. To follow the reaction we applied a procedure based on the iodometric method of measuring α-amylase activity. We found for each of the temperatures studied that at the same treatment intensity the different enzyme concentrations and reaction times assayed gave practically the same conversion values, whereas these values were altered according to the length of time the enzyme solutions were previously kept at reaction temperature. Therefore, the thermal denaturation of the enzyme can be fitted to a second-order kinetic. We have established a way of calculating the deactivation constant for each temperature both for the experiments made with different waiting times before the reaction and for those with different treatment intensity. Finally, we fitted the thermal-deactivation constant against temperature according to Arrhenius’ equation, obtaining an activation energy of 172kJ/mol.
Keywords: Amylase; Starch; Kinetic parameters; Enzyme deactivation; Detergents; Termamyl
Biodesulfurization of hydrodesulfurized diesel oil with Pseudomonas delafieldii R-8 from high density culture by Shan Guobin; Zhang Huaiying; Xing Jianmin; Chen Guo; Li Wangliang; Liu Huizhou (pp. 305-309).
Biodesulfurization (BDS) process of hydrodesulfurized diesel oil by Pseudomonas delafieldii R-8 was studied. Twenty-five grams (dry cell)l−1 of cell density was obtained by production of P. delafieldii R-8. The desulfurization activity of cells grown in logarithmic and stationary phases is higher than that of cells grown in other phases. The maximal desulfurization rate can reach 0.33mgg−1h−1 during the first 4h. The metabolites do not affect desufurization activity of diesel oil. Cell suspension grown at stationary phase was directly mixed with diesel oil, and had a high desulfurization activity as 0.32mgg−1h−1 during the first 4h. In a 5-l fermentor, 500ml of diesel oil was added when desulfurized cells grown in a stationary phase and the total sulfur was reduced to 313 from 591mgl−1. Thus, the BDS process can be simplified by directly mixing cell cultivation suspension with diesel oil.
Keywords: High cell density; 2-Hydroxybiphenyl (2-HBP); Pseudomonas delafieldii; Sulfate
Sorption and desorption of Pb2+ ions by dead Sargassum sp. biomass by Bruno L. Martins; Claudio C.V. Cruz; Aderval S. Luna; Cristiane A. Henriques (pp. 310-314).
Dead Sargassum sp. as a biosorbent for Pb2+ ions from aqueous solutions was studied. Kinetics and equilibrium of Pb2+ biosorption were evaluated. The biosorption process used in this work followed a second-order kinetics and was not influenced by temperature, at least within 298–328K. The equilibrium of biosorption was well described by Langmuir isotherm and the maximum Pb2+ uptake capacity (1.26mmolg−1) is higher than those reported in the literature, for different sorbents. More than 95% of sorbed Pb2+cations could be removed by desorption with a 0.10moll−1 Na2EDTA solution at 298K.
Keywords: Biosorption; Equilibrium; Kinetic; Lead; Sargassum; sp.
Viscosity evolution of anaerobic granular sludge by A. Pevere; G. Guibaud; E. van Hullebusch; P. Lens; M. Baudu (pp. 315-322).
The evolution of the apparent viscosity at steady shear rate of sieved anaerobic granular sludge (20–315μm diameter) sampled from different full-scale anaerobic reactors was recorded using rotation tests. The “limit viscosity� of sieved anaerobic granular sludge was determined from the apparent viscosity. The limit viscosity values depended on the applied shear rate, indicating a non-Newtonian behaviour of anaerobic granular sludge. The tests to determine the apparent viscosity values were carried out at a constant shear rate of 500s−1, which was found to be the optimal value to obtain the limit viscosity. The total suspended solids (TSS) content of the sieved granular sludge was shown to strongly influence the limit viscosity value and an exponential relationship was found between the TSS content and this rheological parameter. The limit viscosity value increases with decreasing size and surface charge of the granules. This underlines the importance of the basic physical sludge characteristics (TSS content, size of granule, surface charge) on the quantitative interactions between granules (i.e. limit viscosity value). Significant differences in limit viscosity values were found for granular sludges of different origin. This indicates the ability of the rheological parameter “limit viscosity� to describe different overall characteristics (TSS, granulometry, origin, charge of surface…) of anaerobic granular sludge within the size class investigated.
Keywords: Rheology; Viscosity; Anaerobic granular sludge
Mixing time in bioreactors under aerated conditions by Dimiter Hadjiev; Nour Eddine Sabiri; Adel Zanati (pp. 323-330).
The major difficulty needing a solution in agitated and aerated bioreactors is closely connected to the optimum hydrodynamics regime in the stirred tank. It is evident that even a simple stirred reactor is geometrically complex and it seems that the mixing time can be one useful criterion for estimation of mixing intensity and therefore the estimation of oxygen profiles and mass transfer coefficients. The mixing time measures are relevant to the length of time for which detectable inhomogeneities last in the extremities of the vessel and can identify the extent of possible concentration gradients in a reactor that should be used as a gradientless unit for bio-kinetic studies. The parameter depends mainly on the mixing system characteristics, the physicochemical properties, the aeration and the rate of cell multiplication. To quantify the influence of some of these factors experiments have been performed using a wide spread laboratory scale stirred dual-impeller bioreactor configuration marketed by “Bioflow�. Experimental data have been obtained for various internal geometries of the reactor under aerated and non-aerated conditions administering real wastewater. A multi-regression analysis method was used to find mathematical correlation relating the influence of the rotation speed and the gas flowrate with the reactor's capacity to mix.
Keywords: Bioreactor; Mixing time; Aeration
Characterization of oxygen transfer conditions and their effects on Phaffia rhodozyma growth and carotenoid production in shake-flask cultures by Yuan-Shuai Liu; Jian-Yong Wu; Kwok-ping Ho (pp. 331-335).
This study characterizes the effects of oxygen transfer on the growth and carotenoid (astaxanthin) production of the red yeast Phaffia rhodozyma in liquid cultures. The yeast culture on a liquid medium at 20°C had a critical dissolved oxygen (DO) concentration of 10% air-saturation (0.8mg/L) and a maximum specific oxygen uptake rate of 60mg O2/gcellh. In 250-mL shake-flasks with 50ml liquid medium, poor oxygen transfer resulted in constantly lower DO level than the critical DO in the culture. Both the biomass and carotenoid yields increased with the decrease in liquid volume in flasks and the increase in shaker speed, e.g., 8.0g dw/L biomass and 7.4mg/L carotenoid with 50mL liquid at 200rpm versus 10.7g dw/L biomass and 15.5mg/L carotenoid with 20mL liquid at 250rpm. The oxygen transfer coefficient ( KL a) was correlated to shaker speed ( N) and liquid volume ( VL) by KLa =0.141 N0.88( VL /Vo)−0.80 based on linear regression of experimental data ( R2=0.997, Vo=flask volume). The carotenoid yield showed a strong linear correlation with the oxygen transfer rate ( R2=0.989). These results show that oxygen supply is crucial for carotenoid production in P. rhodozyma liquid cultures.
Keywords: Phaffia rhodozyma; Shake-flask culture; Oxygen transfer; Dissolved oxygen; Yeast growth; Carotenoid production
Dextran-grafted-PNIPAAm as an artificial chaperone for protein refolding by Diannan Lu; Zhixia Liu; Minlian Zhang; Xiaogong Wang; Zheng Liu (pp. 336-343).
A temperature stimuli-responsive polymer, dextran-grafted-PNIPAAm (DGP), was prepared by radical polymerization using cerium nitrate as the initiator. The structure and the grafting ratio of DGP were determined by FT-IR and elemental analysis. The temperature-stimuli responsive behavior of DGP and the size of the DGP self-assemblies at different temperatures were determined by transmittance and dynamic light scattering, respectively. The use of DGP as an artificial chaperone to assist protein refolding in vitro was demonstrated using hen egg white lysozyme (lysozyme) and carbonic anhydrase bovine (CAB) as model proteins. It was shown that the hydrophobic interaction between DGP and the protein being refolded gave a significant reduction in the rate of protein aggregation and a slight reduction in the refolding rate, and consequently gave an improved refolding yield. Moreover, the refolding of lysozyme and CAB at a temperature gradient starting above the LCST of DGP and ending at a lower temperature, which make DGP change from hydrophobic to hydrophilic, gave a significant increase in the refolding yield compared to that carried out at a constant temperature. The process mechanism of DGP assisted protein refolding was presented and the importance of controlling the hydrophobicity of the solution environment for protein refolding was discussed.
Keywords: Protein; Refolding; Protein recovery; Dextran; Dextran-; g; -PNIPAAm; Temperature gradient