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Biochemical Engineering Journal (v.37, #3)
Purification of phenylalanine ammonia-lyase in PEG1000/Na2SO4 aqueous two-phase system by a two-step extraction by Haiyan Yue; Qipeng Yuan; Wenchuan Wang (pp. 231-237).
The effects of molecular weight and concentration of PEG and various salts on the partitioning of PAL from Rhodotorula glutinis were studied in PEG/salts aqueous two-phase systems at pH 8.8 and 25°C. With the application of 11.0%PEG1000/14.0%Na2SO4, and 11.0%PEG1000/14.0%Na2SO4/5.3%Na2CO3, purification of PAL by a two-step extraction was proposed in this work. Compared to the conventional salting-out step, 3.7 times of the purification factor (9.3-fold) and a comparable recovery yield (80.6%) were obtained in this method. Therefore, the work offers a useful method for PAL purification and the two-step extraction could replace the conventional salting-out step in the purification.
Keywords: Aqueous two-phase system; Phenylalanine ammonia-lyase; Partition; PEG; Salt
Optimisation of support medium for particle-based biofilm reactors by Dimitar Dimitrov; Dimiter Hadjiev; Iordan Nikov (pp. 238-245).
The aim of this study was to propose a method to improve the biofilm growth on different polymer materials by modifying their surface properties. The ability of two aerobic bacteria strains: Pseudomonas aeruginosa O1 and Bacillus subtilis CIP 5265 to grow on various non-coated and coated polymer materials were investigated. A layer of polymethylmethacrylate and powdered activated carbon (PMMA/PAC) was used to improve the microbial adhesion dynamics. The substratum and cell surface properties were characterized using contact angle measurements. Fluorescent microscopy and SEM were used to observe the support and the biofilm growth. It was determined that better results can be obtained increasing the difference between the surface free energies of the support and the bacteria. It was found that supports with modified surface show higher biofilm development rate and better surface colonization. The influence of the surface free energy on the detachment force and correspondingly on the biofilm formation was demonstrated.
Keywords: Cell immobilization; Surface properties; Thermodynamic approach; Solid surface conditioning
Improved activity and thermostability of ( S)-aminotransferase by error-prone polymerase chain reaction for the production of a chiral amine by Abraham R. Martin; Rocco DiSanto; Irina Plotnikov; Sanjay Kamat; David Shonnard; Sachin Pannuri (pp. 246-255).
The evolution of a mesophilic aminotransferase, isolated from Athrobacter citreus, to a thermostable aminotransferase was accomplished via error-prone PCR. After three rounds of mutagenesis, a mutant was generated that decreased the biocatalyst loading 3-fold. This improved biocatalyst was engineered further and a new mutant was isolated that was capable of the same performance with 5-fold reduction in biocatalyst loading. Overall, the best mutant (#6) enabled a 3-fold reduction in biocatalyst loading, almost a 5-fold increase in product concentration, and a 5-fold reduction in process cycle time. Through these rounds of mutagenesis enzyme specific activity improved from 5.9 to 1582.8IU/g with an overall improvement in product yield due to reduced biocatalyst loading. The new mutants were also able to operate at temperatures greater than 50°C for an extended period of time. A simple cost model was developed to describe the impact of enzyme improvement on product cost.
Keywords: Biocatalysis; Biotransformations; Chiral systems; Enzyme biocatalysis; Enzyme technology; Enzyme production
Fast conversion of glycerol to 1,3-propanediol by a new strain of Klebsiella pneumoniae by GenLin Zhang; BinBin Ma; XiaoLin Xu; Chun Li; Liwei Wang (pp. 256-260).
Five bacterial strains screened from a batch of 39 samples could convert glycerol anaerobically to 1,3-propanediol (1,3-PD). One of the strains, XJ-Li, which could synthesize 1,3-PD with a higher concentration, was identified and characterized. Phylogenetic analysis of the strain XJ-Li included the study of morphology, physiological and biochemical characteristics. In addition, 16SrDNA sequences were created. The results indicated that this strain is a member of Klebsiella pneumoniae. The optimal cultivation parameters for pH and temperature were determined as 8.0 and 40°C, respectively. The optimized nitrogen source and carbon source were 6.0g/L of (NH4)2SO4 and 20g/L of glycerol, respectively. After 8h in batch fermentation, both the 1,3-PD concentration and glycerol consumption reached the maximum, with 12.2g/L of 1,3-PD and 1.53g/Lh of productivity, and a molar yield of 1,3-PD to glycerol of 0.75. Fed-batch fermentation also indicated a higher molar yield of 0.70, and the concentration of 1,3-PD reached 38.1g/L after 66.4g/L of glycerol consumption. The results of batch and fed-batch fermentations demonstrated that K. pneumoniae XJ-Li would be an excellent 1,3-PD producer.
Keywords: Bioconversion; Glycerol; 1,3-Propanediol; Fermentation; Klebsiella pneumoniae; Optimization
Treatment of wet process hardboard plant VOC emissions by a pilot scale biological system by S. Santos; K. Jones; R. Abdul; J. Boswell; J. Paca (pp. 261-270).
A pilot scale biological treatment system for air emissions was installed and tested at a forest products plant in western Oregon, USA, which collected and treated gaseous emissions from the hardboard steam press vents on the top of the plant building. This system was installed mainly to demonstrate the effectiveness of biological treatment technologies in removing volatile organic compounds (VOC) and hazardous air pollutants (HAP) from the wet-process hardboard press emissions, and to test the efficiency of the system on fine particles and condensable organics with the presence of a pre-treatment wet dust collector. The bio-oxidation system was comprised of a particle pre-treatment unit Type W Rotoclone (wet hydrocyclone), a biotrickling filter and a biofilter with airflow capacity of up to 4.72m3/s. This unit operated at approximately 0.71m3/s, which is the optimal flow required for the Rotoclone's throughput, and provided an EBCT (empty bed contact time) of 45s. Analysis of total VOC measurements from the system indicated removals down to less than 5ppm in the effluent emissions. Evaluations of opacity reductions also met project objectives with routine outlet measurements of 0–5%, which are in compliance with state regulatory guidelines. Emissions air samples were collected at different locations in the biological system for GC–MS analysis and characterization to identify specific VOCs and their removals.
Keywords: Biofiltration; VOC removal; Air pollution control; Forest products emissions
Design of optimal solvent for extraction of bio-active ingredients from mulberry leaves by Jong-Min Kim; Sang-Mok Chang; In-Ho Kim; Young-Eun Kim; Ji-Hwan Hwang; Kyo-Seon Kim; Woo-Sik Kim (pp. 271-278).
A method of designing solvents for the optimal extraction of bio-active ingredients from natural resources was developed using an alcohol–water binary solvent. The target bio-active ingredient of polyphenols, anti-oxidation and anti-tyrosinase ingredients exhibited different dependency of extraction efficiency on the alcohol species (methanol, ethanol, n-propanol and i-propanol) and composition of binary solvent. Using the solubility parameter, the extraction efficiency of the bio-active ingredients was correlated with the solvent polarity. As a result, the optimal solvent polarities for the extraction of polyphenols, anti-oxidation and anti-tyrosinase ingredients were predicted as 38.5, 37.33, and 33.0 [MPa1/2], respectively. These predictions also agreed well with the optimal solvent conditions of the water–alcohol mixtures depending on the alcohol species and composition. Plus, the correlation was confirmed with model solvents designed using other solvent species, including acetone and ethylene glycol.
Keywords: Extraction; Alcohol–water binary solvent; Bio-active ingredients; Solvent polarity
Synthesis of 2-ethylhexyl-2-ethylhexanoate catalyzed by immobilized lipase in n-hexane: A kinetic study by A. Daneshfar; H.S. Ghaziaskar; L. Shiri; M.H. Manafi; M. Nikorazm; S. Abassi (pp. 279-284).
A direct esterification reaction for the synthesis of 2-ethylhexyl-2-ethylhexanoate from 2-ethylhexanoic acid and 2-ethyl-1-hexanol in n-hexane as solvent, and Novozym 435, as catalyst, has been carried out. The effect of concentrations of 2-ethylhexyl-2-ethylhexanoate, water, 2-ethylhexanoic acid and 2-ethyl-1-hexanol on ester yield, the initial forward esterification reaction and the reverse reaction rates have been investigated. The experimental results show that the esterification reaction proceeds with a Ping-Pong Bi-Bi mechanism and that 2-ethylhexanoic acid inhibits the reaction.Kinetic parameters were calculated based on this model as follows: Rmax=37.59mmolh−1g−1, KmAl=1.51M, KmAc=0.78M, KiAc=1.55M. The calculated parameters were used to estimate the theoretical initial reaction rate. The calculated initial reaction rates from kinetic parameters for concentrations of less than 0.1M 2-ethylhexanoic acid show good agreements with the experimental data.
Keywords: Esterification; Novozym 435; Ping-Pong mechanism; Bi-Bi mechanism; Kinetic; 2-Ethylhexanoic acid
Modelling diffusivity in porous polymeric membranes with an intermediate layer containing microbial cells by Manuel Mota; Alexander Yelshin; Marcello Fidaleo; Michael C. Flickinger (pp. 285-293).
Three-layer systems (membrane – composite layer (cells+polymer) – membrane) are important in different biochemical applications. Models of latex layered-membranes were evaluated and compared with experimental data in order to predict the diffusivity of substrates in the composite layer containing living E.coli microbial cells. Diffusivity predictions are dependent on the presence or the absence of a ‘skin’ layer, on the degree of polymer particle coalescence and on the thickness of each layer. Simulations with layered models were made to identify the dominant mechanisms in the three-layer system. It was found that the layered system is sensitive to the latex coatings porosity when the composite layer occupies less than 50% of the total membrane system thickness. Whenever the control of polymer particle coalescence and of the layers (coating/composite layer) thickness may be achieved, multi-layer systems presenting a wide range of relative diffusion conductivities may be built for different types of living cells and for a wide variety of practical applications. The diffusivity of the latex layer is proportional to the square of latex porosity.
Keywords: Diffusion; Microporous membrane; Immobilised cells; Modelling; Packing; Mass transfer
Production of bacterial α-amylase by B. amyloliquefaciens under solid substrate fermentation by M. Saban Tanyildizi; Dursun Özer; Murat Elibol (pp. 294-297).
Production of α-amylase by Bacillus amyloliquefaciens, under solid substrate fermentation (SSF) was investigated in shaken-culture. The maximum α-amylase activity was obtained under the following optimized conditions: corn gluten meal (CGM) 30g/l, yeast extract (YE) 10g/l, agitation rate 150rpm and fermentation temperature 33°C. The results showed that α-amylase production in a medium with CGM was five times higher than that in the medium contained starch and other components. The temperature of fermentation was found to be most crucial factor in α-amylase production.
Keywords: Solid substrate fermentation; Corn gluten meal; B. amyloliquefaciens; α-amylase
Structured parallel diffusion model for intraparticle mass transport of proteins to porous adsorbent by Kun Yang; Yan Sun (pp. 298-310).
A structured parallel diffusion model (SParD model) has been developed to describe the intraparticle mass transport of proteins in porous chromatographic matrices. This model takes into account surface diffusion, hindered pore diffusion, pore size distribution of porous supports, and the shrinking of effective pore radius by protein adsorption. Moreover, the Maxwell–Stefan approach was adopted to express the surface diffusion. The uptake kinetics of proteins, bovine serum albumin and γ-globulin, to anion exchanger, DEAE Spherodex M, is studied by batch adsorption. The dynamic adsorption data thus obtained are analyzed by the SParD model. The surface diffusivities of the two proteins are acquired by matching the model with experimental data. The hindrance effect on pore diffusion in different-sized pores was illuminated by the calculated intraparticle concentration profiles of adsorbed protein and the ratios of surface diffusion to pore diffusion rates in different pores. Thus, the validity and usefulness of the SParD model have been confirmed.
Keywords: Mass transport; Structured parallel diffusion model; Protein adsorption; Pore size distribution; Hindered pore diffusion; Surface diffusion
Phase equilibrium and protein partitioning in aqueous two-phase systems containing ammonium carbamate and block copolymers PEO–PPO–PEO by Mayra Codo de Oliveira; Manoel Américo Nogueira de Abreu Filho; Pedro de Alcântara Pessôa Filho (pp. 311-318).
The liquid–liquid equilibrium of aqueous two-phase systems formed by the PEO–PPO–PEO block copolymers F38 or F68 and ammonium carbamate and the partition of the model proteins bovine serum albmin, lysozyme, α-lactalbumin, β-lactoglobulin and γ-globulin in these systems are presented in this paper. Phase diagrams were determined at 4°C (F38) and 25°C (F38 and F68); systems containing F68 present a narrow two-phase region, while systems containing F38 have a suitable working region. Partition coefficients, defined as the ratio of protein concentration in the polymer-rich phase to that in the salt-rich phase, were found to vary in the range of 0.1 to 0.8 for bovine serum albumin, 0.5 to 2.0 for lysozyme, 1.0 to 2.5 for α-lactalbumin, 0.1 to 1.0 for β-lactoglobulin and 0.3 to 1.0 for γ-globulin, depending on the polymer chain-size, temperature and tie-line length. The different trends of protein partition coefficients show that high degrees of separation can be achieved, establishing the system F38 and ammonium carbamate as an alternative to be considered when planning the downstream processing of proteins.
Keywords: Protein; Downstream processing; Aqueous two-phase systems; Salt; Polymer
Sugar production from cellulosic biosludges generated in a water treatment plant of a Kraft pulp mill by Aloia Romaní; Remedios Yáñez; Gil Garrote; José Luis Alonso; Juan Carlos Parajó (pp. 319-327).
A study on the enzymatic hydrolysis of cellulosic biosludges generated in a water treatment plant of a Kraft pulp mill was carried out. The effect of the operational conditions (cellulase to solid ratio (CSR), liquid to solid ratio (LSR), surfactant concentration (SC) and reaction time), on the hydrolysate composition was evaluated and a set of mathematical models able to predict the glucose and xylose concentrations in the reaction media was proposed.Using low cellulase charges (8FPU/g) and high liquid to solid ratios (28–30g/g), a quantitative conversion of the glucan fraction can be reached in 48h, although diluted solutions are produced.However, operating at a cellulase to solid ratio of 12.5FPU/g, a liquid to solid ratio of 12g/g and a surfactant concentration of 0g/L, 74% of the glucan fraction and 67% of the xylan fraction can be saccharified in 34h, leading to solutions containing up to 27.8g/L of glucose and 5.4g/L of xylose.The results demonstrated that this solid residue shows high enzymatic digestibility and that no pretreatments are needed to enhance the saccharification step. These advantages, along with its negative price, make this solid a valuable raw material for lactic acid production.
Keywords: Abbreviations; CSR; Cellulase to solid ratio (FPU/g); FPU; Filter paper unit; G; Glucose concentration (g/L); GM; maximum glucose concentration (g/L); IU; International unit on enzymatic activity; LSR; Liquor to solid ratio (g/g); o.d.b.; Oven-dry basis; PNPG; P; -nitrophenyl β-; d; -glucopyranoside; r; 0G; Initial rate of glucose production (g/Lh); r; 0TS; Initial rate of total sugar production (g/Lh); SC; Surfactant concentration (g/L); SSF; Simultaneous saccharification and fermentation; t; Reaction time (h); TS; Total sugar concentration (g/L); TSm; Maximum total sugar concentration (g/L); X; Xylose concentration (g/L)Biosludges; Cellulose; Waste treatment; Enzymatic hydrolysis; Glucose; Modelling
Improvement of epothilone B production by in situ removal of ammonium using cation exchange resin in Sorangium cellulosum culture by Sang Woo Park; Se Jong Han; Dong-Shik Kim; Sang Jun Sim (pp. 328-331).
A cation exchange resin was added at different stages in the growth of Sorangium cellulosum cultures to remove excess ammonium, which has a negative influence on the production of the anticancer agent, epothilone. When 8gL−1 of cation exchange resin were added at the mid-death phase, the ammonium removal reached 83% and the maximum production of epothilone was 14.3mgL−1, which was 2.6 times greater than that of the control culture.
Keywords: Epothilone; Sorangium cellulosum; Ammonium; Cation resin; Feeding time
Fabrication of superporous cellulose beads with grafted anion-exchange polymer chains for protein chromatography by Dong-Mei Wang; Yan Sun (pp. 332-337).
Glycidyl methacrylate was grafted onto the pore surface in superporous cellulose beads using ceric ammonium nitrate as initiator. By modifying the grafted and ungrafted matrices with diethylaminoethyl (DEAE) groups, anion exchangers were produced. Under a proper grafting reaction condition, the adsorption capacity of bovine serum albumin (BSA) to the DEAE-G-SC was over two times increased as compared to the ungrafted DEAE-SC and no nonspecific adsorption of BSA was observed. Moreover, the intraparticle diffusive mass transfer was not affected by this grafted polymer chains. However, the flow hydrodynamic experiments revealed that the grafted polymer chains led to the decrease of flowthrough pores. So the convective mass transport in the particles was significantly reduced, leading to the decrease of column efficiency with flow velocity. Despite this unfavorable effect, however, due to the high adsorption capacity of the grafted beads, the DEAE-G-SC column could still be operated at a DBC of 20–35% higher than the normal DEAE-SC column in the range of 150–600cm/h.
Keywords: Grafting polymerization; Superporous cellulose bead; Anion exchanger; Protein adsorption; High capacity; Chromatography