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Biochemical Engineering Journal (v.55, #3)
Mass production of the entomopathogenic nematode, Steinernema carpocapsae CABA01, through the submerged monoxenic culture in two internal-loop airlift bioreactors with some geometric differences by Norberto Chavarría-Hernández; Gabriela Maciel-Vergara; Juan-Carlos Chavarría-Hernández; Javier Castro-Rosas; Blanca-Rosa Rodríguez-Pastrana; Mayra de la Torre-Martínez; Adriana-Inés Rodríguez-Hernández (pp. 145-153).
► The culture of the nematode Steinernema carpocapsae CABA01 was carried out in two airlift reactors with some geometric differences. ► Global changes in both hydrodynamics (as Reynolds number: 170–10,060) and oxygen transfer conditions (as kL a: 1.9–3.5×10−2 s−1) during the processes were estimated, involving final concentrations up to 250,000 infective juvenile nematodes per mL. ► Results suggest that higher inertial forces would promote the mating among nematodes due to better mixing conditions.The submerged monoxenic culture of the entomopathogenic nematode, Steinernema carpocapsae CABA01, was carried out in two internal-loop airlift reactors (R1 and R2) with some geometric differences. Two runs each reactor were carried out at operating conditions VL=4.22L, Q=1–1.5vvm, T=22°C. The culture broth apparent viscosity changed from 2 to 12mPas determining global changes in both hydrodynamics (expressed as the dimensionless Reynolds number, Re) and oxygen transfer conditions (expressed as the kL a coefficient). Such changes were 170kL a (10−2s−1)<3.5 for R1 experiments, and 400kL a (10−2s−1)<3.5, for R2 experiments. Infective juvenile stage (IJ) concentrations of 250,000/mL were achieved in R2 experiments whereas notably lower ones were obtained in R1 experiments (110,000IJ/mL, approximately). The hydrodynamic conditions in both riser and downcomer sections of R2 experiments involved higher inertial forces than the corresponding ones to R1 runs, implying values of (ReR2/ReR1)riser from 2.3 to 3.3 and (ReR2/ReR1)downcomer from 1.8 to 2.5. These results suggest that higher inertial forces would promote the mating process among the vigorous adult nematodes of the first generation, probably due to encourage female–male encounters in response to a better mixing of the culture broth. Further, the nematode reproduction appeared to be greatly influenced by the reactor geometry.
Keywords: Bio-insecticides; Bio-reactors; Nematode mass production; Hydrodynamics; Oxygen transfer conditions
Optimization of in vitro erythropoiesis from CD34+ cord blood cells using design of experiments (DOE) by Mayasari Lim; Nicki Panoskaltsis; Hua Ye; Athanasios Mantalaris (pp. 154-161).
► Effective and efficient use of DOE for analysis, characterisation, and optimization. ► In vitro erythropoiesis to the stage of enucleation. ► Systematic platform for stem cell bioprocessing. In vitro culture studies of hematopoietic cells (HCs) have traditionally been limited to dose-response, empirical methods which can be uninformative, misleading and suboptimal. Herein, we use design of experiments methodology to quantitatively define and optimize in vitro erythropoiesis from human cord blood CD34+ cells to enucleation. DOE was used to theoretically define the effects of 7 cytokines known to affect red cell maturation in humans. Of these, stem cell factor (SCF) and erythropoietin (EPO) were found to be the most significant factors in red cell production ( p<0.01). DOE defined process characteristics and interactions of SCF and EPO on total cell expansion and maturation of cord blood HCs towards the erythroid lineage and delineated optimal in vitro concentrations of each cytokine (75ng/mL and 4.5IU/mL, respectively). The DOE-optimized cytokine cocktail achieved an average of 26,460-fold total cell expansion and accelerated erythroid maturation yielding over 90% glycophorin-A-expressing cells by day 7. Maturation of red cells was confirmed by morphology, immunophenotype, clonogenic capacity, gene and protein expression of adult and neonatal hemoglobins (α, β, and γ) and by progressive expression of GATA-1 with maturation. The use of DOE was effective and efficient for the analysis, characterization and optimization of in vitro erythropoiesis to the stage of enucleation and provides a systematic platform for the use of other growth factors in vitro for the study of hematopoiesis and expansion of cell products.
Keywords: Erythropoiesis; Erythrocytes; Cord blood stem cells; Design of experiments
PEGylated liposomes encapsulating human hemoglobin enhance oxygen transfer and cell proliferation while decreasing cell hypoxia in fibrin by Valérie Centis; Pierre Proulx; Patrick Vermette (pp. 162-168).
► Liposomes encapsulating hemoglobin enhance oxygen transfer. ► Liposomes encapsulating hemoglobin enhance cell proliferation in fibrin. ► Liposomes encapsulating hemoglobin reduce HIF-1α (hypoxia) expression in cells grown in fibrin.Oxygen supply to cells in three-dimensional cultures occurs mainly by diffusion, with insufficient oxygen concentration reaching the center of cell aggregates, thus creating hypoxic areas and leading eventually to cell death. The aims of this study were, firstly to measure and characterize the oxygen transfer from liposomes encapsulating human hemoglobin (LEH) to an aqueous environment and, secondly, to study the effects of LEH to cells grown in a three-dimensional environment. LEH were loaded with pure oxygen and the oxygen transfer from the liposomes to an aqueous phase was monitored. Human umbilical vein endothelial cells (HUVEC) were cultured in a fibrin gel mimicking a diffusion-limited environment. After 24h, HUVEC seeded in fibrin covered with culture medium supplemented with oxygen-loaded LEH showed a decrease in HIF-1α expression and an increase in proliferation compared to those in fibrin cultured either with medium containing liposomes with no hemoglobin or non-modified medium.
Keywords: Hypoxia; Oxygen carriers; Liposomes encapsulating hemoglobin; Fibrin; Endothelial cells; Oxygen transfer
Peptide disulfides CGC and RKCGC facilitate oxidative protein refolding by Guo-Zhen Wang; Xiao-Yan Dong; Yan Sun (pp. 169-175).
► A new oxidant, RKCGC disulfide, was designed to catalyze oxidative protein refolding. ► RKCGC facilitated oxidative protein refolding more effectively than CGC disulfide. ► RKCGC was effective not only in neutral pH but also in weak alkaline solutions. ► The design reveals that adding nearby positive charges to peptide disulfide is effective to improve its catalytic activity by lowering the p Ka of the corresponding thiol.Oxidative protein refolding is a big challenge for the recovery of disulfide bonds containing recombinant proteins expressed as inclusion bodies. In this study, the disulfide form of a small peptide mimic of protein disulfide isomerase, CGC, was explored to facilitate oxidative protein refolding with residual dithiothreitol (DTT) as the reductant. Working with DTT, CGC gave higher yield and folding rate than oxidized glutathione. Then, a new pentapeptide, RKCGC disulfide, was designed. It was found that RKCGC was more effective than CGC in increasing the folding rate and final yield, as demonstrated in the oxidative refolding of lysozyme and ribonuclease A. It was because the pentapeptide has lower p Ka and higher reduction potential than CGC. Moreover, it efficiently facilitated protein refolding not only at neutral pH but also in a weak alkaline buffer. With CGC or RKCGC as the oxidant, the DTT removal step and the addition of other thiol reductants were not needed. This would benefit in decreasing the processing time and cost in protein refolding processes. This design has thus provided more efficient oxidant, and it may pave the way for the design of more effective thiol-disulfide redox agents.
Keywords: Bioseparation; Protein; Refolding; Kinetic parameters; Peptide disulfide; Redox system
Development of a reversible binding process for in situ removal of 3-hydroxypropionaldehyde during biotechnological conversion of glycerol by D.P. Rütti; C. Lacroix; T. Jeremiç; M. Mathis; A. Díe; S. Vollenweider (pp. 176-184).
► 3-HPA can be selectively removed during its biotechnological production. ► 3-HPA can be selectively recovered from the support. ► The support was a resin in the bisulfite form.3-Hydroxypropionaldehyde (3-HPA) has a great potential in the pharmaceutical-, food-, and chemical industry as a potent antimicrobial compound and chemical precursor. Until now it is not commercially available, occurring only as intermediate in production of petro-derived 1,3-propanediol. In this study, we developed a new method for 3-HPA isolation, biotechnologically produced by Lactobacillus reuteri. A composition of an Amberlite anion exchange resin and hydrogensulfite (IRA-SO3H) was generated to extract 3-HPA from the complex mixture of the production medium. 3-HPA was successfully recovered and concentrated in a pure form. Using this approach, IRA-SO3H was evaluated to bind 3-HPA throughout the bioconversion. The IRA-SO3H successfully bound already produced 3-HPA without decreasing L. reuteri viability. However, new 3-HPA formation was repressed. To conclude, our data showed for the first time the selective extraction of 3-HPA from the bioconversion medium and its subsequent recovery. Future work is needed to adapt this system to an in situ product removal process not interfering with the bioconversion.
Keywords: Reuterin; Biotransformation; Bisulfite; ISPR; Antimicrobial; Platform chemical
Electrochemical DNA sensor for simultaneous detection of genes encoding two functional enzymes involved in lignin degradation by Zhen Li; Guangming Zeng; Lin Tang; Yi Zhang; Yuanping Li; Ya Pang; Jie Luo; Yuanyuan Liu (pp. 185-192).
► Simultaneous detection of two functional genes in the same system was studied. ► PCR and restriction endonuclease digestion enhanced the sensitivity of sensor. ► Horseradish peroxidase and laccase labels were used for signal amplification. ► Application for simultaneous detection of MnP and CDH genes from white rot fungi. ► The results of DNA detection were consistent with UV spectrometry.An electrochemical DNA sensor for simultaneous detection of functional genes encoding manganese peroxidase (MnP) and cellobiose dehydrogenase (CDH) on a gold electrode was developed. After two thiolated capture probes assembled on the electrode surface, the electrode was exposed to a monolayer of 6-mercapto-1-hexanol (MCH) solution to prevent nonspecific adsorption of target DNA and detection probes. Horseradish peroxidase–streptavidin (HRP–SA) conjugate and laccase–streptavidin (LAC–SA) conjugate were applied for enzyme-amplified amperometric measurement. The two target genes were simultaneously quantified in the same system. The DNA conformation and surface coverage on electrode were characterized by impedance spectroscopy and cyclic voltammetry. The amperometric current responses to HRP and LAC-catalyzed reactions were linearly related to the common logarithm of two target nucleic acids concentrations, ranging from 1×10−11M to 4×10−8M and 1×10−10M to 4×10−8M. The correlation coefficients were 0.9884 and 0.9881, and the detection limits were 6.2×10−12M and 3.0×10−11M, respectively. The effectiveness of this DNA sensor was confirmed by simultaneous detection of two gene fragments extracted from Phanerochaete chrysosporium using polymerase chain reaction (PCR) and restriction endonuclease digestion. The DNA biosensor exhibited good selectivity, precision, stability and reproducibility.
Keywords: DNA biosensor; Simultaneous detection; Encoding gene; Functional enzyme; Lignin degradation; Polymerase chain reaction
Optimization on thermophilic aerobic treatment of anaerobically digested palm oil mill effluent (POME) by Yi Jing Chan; Mei Fong Chong; Chung Lim Law (pp. 193-198).
► The current research is aimed to improve thermophilic aerobic treatment system of anaerobically digested palm oil mill effluent (POME) through optimization of organic loading rate (OLR), mixed liquor suspended solids (MLSS), dissolved oxygen (DO) concentrations and settling time. ► High MLSS concentration range which has yet to be reported was successfully applied in this study ► The optimum operating conditions to achieve the highest COD, BOD and TSS removal efficiencies for the thermophilic aerobic treatment of anaerobically digested POME were successfully attained from the present study. ► In comparison with other thermophilic treatment system at equivalent OLR, greater TCOD removal efficiencies were achieved in the present investigation.This research is aimed at improving the thermophilic aerobic treatment system of anaerobically digested palm oil mill effluent (POME) through optimization of mixed liquor suspended solids (MLSS) at high concentration range for the first time besides other parameters of organic loading rate (OLR), dissolved oxygen (DO) concentrations and settling time. Chemical oxygen demand (COD), biochemical oxygen demand (BOD) and total suspended solids (TSS) removal ratios were measured as indicators of the system performance. Experimental results clearly indicate that MLSS and OLR are the crucial parameters in thermophilic aerobic treatment. Optimal COD, BOD and TSS removals up to 86%, 87% and 89%, respectively, were achieved with MLSS concentration and OLR of 27,000mg/L and 2.5gCOD/(Lday), respectively. More significantly, high MLSS concentration range which is yet to be reported was successfully applied and this has resulted in superior performance as compared to other thermophilic treatment studies. No significant improvement of the treatment efficiency was attained with DO concentration exceeding 5mg/L and settling time up to 24h. Hence, operation at DO concentration of 2mg/L and settling time of 2h gives a good compromise between effluent quality and system operating costs.
Keywords: Palm oil mill effluent (POME); Optimization; Sequencing batch reactor (SBR); Aerobic; Organic loading rate (OLR); Mixed liquor suspended solids (MLSS)
Preparation and modification of chitosan particles for Rhizomucor miehei lipase immobilization by Camila A. Palla; Consuelo Pacheco; María E. Carrín (pp. 199-207).
► Chitosan particles, suitable as immobilization support, were prepared. ► Carbonated chains were incorporated to chitosan structure by reductive amination. ► Aldehyde concentration and reaction time change the hydrophobicity of the particles. ► Lipases from Rhizomucor miehei were immobilized onto modified chitosan microspheres. ► During acidolysis reaction, biocatalysts showed higher activity than free lipase.Chitosan particles, suitable as immobilization support, were prepared by precipitation and modified by reductive amination in order to graft linear aliphatic chains of 12 carbon atoms to their native amine groups. Their physical characterization was performed by different techniques: differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), air–water contact angle analysis, among others. Lipases from Rhizomucor miehei (RM) were immobilized by adsorption at low ionic strength onto different modified chitosan microspheres. Their ability to catalyze the acidolysis reaction between sunflower oil and palmitic and stearic free fatty acids was evaluated in a solvent medium. Effects of modification conditions on the particles hydrophobic character, lipase adsorption and acidolysis activity were investigated. Modified particles were bigger and more hydrophobic than unmodified ones. The most active biocatalyst achieved a change in the composition of palmitic and stearic acid from a value of 9.6% in the original oil to 49.1% in the final structured lipids, representing an almost 3-fold enzyme hyperactivation. This high conversion was maintained during 7 reuse cycles (168 total hours). The results demonstrated that the chitosan modification was effective in order to adsorb and hyperactivate RM lipases.
Keywords: Chitosan; Reductive amination; Adsorption; Rhizomucor miehei; lipase; Acidolysis; Structured lipids
Vacuum fermentation integrated with separation process for ethanol production by Viet D. Nguyen; Joseph Auresenia; Hitoshi Kosuge; Raymond R. Tan; Yolanda Brondial (pp. 208-214).
► Fermentation reaction and separation of ethanol in the packed column. ► Kinetic parameters for the fermentation under the vacuum are predicted. ► A combination of CSTR mode and plug flow reaction mode for ethanol production.In this study, continuous fermentation integrated with separation process at atmospheric and vacuum pressures are investigated. An initial glucose concentration of 200gL−1 was used to produce yeast cells under batch operating mode. After a period of 18–20h, a medium of 350gL−1 glucose concentration was fed continuously to the fermentation-separation column. During the continuous experiments, fermented broth was recirculated back to the fermentation-separation column. At atmospheric pressure, the dry mass concentration decreased rapidly and remained at 2.4gL−1 after long period, while glucose concentration still remained at 240gL−1. On the other hand, at vacuum pressure, a quasi-steady state condition was reached after 96h, corresponding to 4.2gL−1 of cell dry mass, 165gL−1 of glucose and 44.2gL−1 of ethanol levels in the fermentation broth and 33.2wt% of ethanol concentration at the outlet. These results indicate reduced product and substrate inhibition, suggesting that the integrated process could be used to replace the first bioreactor in a fermentation cascade system. A mathematical model was then developed and used to predict the kinetic parameters. The simulation results agreed well with experimental data.
Keywords: High substrate fermentation; Ethanol inhibition; Vacuum separation; Continuous column fermentation; Kinetic model
Long-term partial nitrification in an intermittently aerated sequencing batch reactor (SBR) treating ammonium-rich wastewater under controlled oxygen-limited conditions by Jianping Li; David Elliott; Michael Nielsen; Mark G. Healy; Xinmin Zhan (pp. 215-222).
► Development of a novel technology for partial nitrification at ambient temperature. ► First time to use online NH4−–N measurement as a control parameter for bioreactors. ► RT-PCR analysis shows the population size of AOB was up to 10% of total bacteria. ► Intermittent aeration and oxygen-limited conditions sustained partial nitrification. ► The bioreactor recovered quickly after 47 days non-operation.In this study, a novel technology was developed to achieve efficient partial nitrification at moderately low temperature, which would save the aeration cost and have the capacity to treat a wide range of ammonium-rich wastewaters with low chemical oxygen demand-to-nitrogen (COD:N) ratios. At pH of 7.1–7.4 and a sludge retention time (SRT) of greater than 100 days, a laboratory-scale intermittently aerated sequencing batch reactor (IA-SBR) was operated at about 20°C to treat synthetic wastewater containing readily biodegradable COD of 100–300mgL−1 and ammonium–nitrogen (NH4+–N) of 300mgL−1. During the aeration periods, DO concentrations were controlled at less than 0.2mgL−1, and the aeration was terminated when on-line NH4+–N measurement reached 20mgL−1. During 180 days of operation, the mass ratio of nitrite–N (NO2−–N) to the total oxidized nitrogen was over 90% in the effluent. Molecular analyses show that ammonium oxidizing bacteria (AOB) represented up to 10% of the total bacterial population in the sludge biomass, and the ratio of AOB to nitrite oxidizing bacteria (NOB) was generally over 100. The IA-SBR quickly recovered after non-operation for 47 days. The three main factors enabling long-term stable partial nitrification in this study were: enrichment of AOB in the start-up period; controlled oxygen-limited conditions and the intermittent aeration strategy.
Keywords: Aeration control; Intermittent aeration; Nitrification; Nitrite accumulation; Molecular analysis; Online monitoring
Biocalorimetric and respirometric studies on production of Penicillin G acylase from Bacillus badius pac in E. coli DH5α by Karthikeyan Rajendran; Surianarayanan Mahadevan; Sudharshan Sekar; Gunasekaran Paramasamy; Asit Baran Mandal (pp. 223-229).
► The growth of E. coli DH5α containing Bacillus badius pac gene can be calorimetrically monitored. ► Reliable information on growth dynamics and production of Penicillin G acylase obtained. ► A maximum PAC activity of 43U/ml was achieved for the first time at 0.66vvm oxygen supply with fructose as carbon source. ► Change in metabolic heat flow rate profiles and heat yields indicated that the recombinant construct is an obligate aerobic. ► The heat yields due to biomass, 15.26kJ/g, substrate, 16.52kJ/g and OUR, 413.2kJ/mol reflects physiological activity of the organism.An isothermal heat flux biological reaction calorimeter is employed to monitor the production of Penicillin G acylase (PAC) from E. coli DH5α harboring recombinant construct pPROPAC (pET-30b carrying pac gene from Bacillus badius with its own promoter) cultivated in fermentation medium earlier with different flow rates of oxygen. Three oxygen flow rates were studied. At 0.66vvm (volume of gas per volume of liquid per minute [ll−1min−1]) a maximum enzyme yield of 43U/ml was achieved with fructose as carbon source. Metabolic heat flow rate profile and heat yields at different levels helped to conclude that the recombinant construct was an obligate aerobic. Oxygen uptake rate correlated well with enzyme activity, biomass growth and heat production rate at exponential and stationary growth phases in optimized conditions. It was observed that metabolic heat, enzyme activity, substrate utilization rate and oxygen uptake rate matched well. The heat yields due to biomass growth, 15.26kJ/g, substrate consumption, 16.52kJ/g and oxygen uptake, 413.2kJ/mol led to understanding the process behavior and physiological activity of the organism under study. The results can contribute to scale up studies, possibly.
Keywords: Biocalorimetry; Respirometry; Heat flow; Enzymes; pac; gene; Penicillin G acylase (PAC)
Selective partition of plasmid DNA and RNA from crude Escherichia coli cell lysate by aqueous two-phase systems by Frank Luechau; Tau Chuan Ling; Andrew Lyddiatt (pp. 230-232).
► The application of PEG-phosphate ATPS could be extended to the selective removal of RNA from pDNA in crude cell lysate. ► It is demonstrated that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342mM (kg ATPS)−1 NaCl were required to partition pDNA to the bottom phase.In this paper, the partition of plasmid DNA (pDNA) and RNA in polyethylene glycol (PEG) and di-potassium phosphate aqueous two-phase systems (ATPS) by adding up of NaCl salt was studied with crude Escherichia coli ( E. coli) cell lysate. The partition of nucleic acid was studied in an ATPS composed of 17% (w/w) PEG300, 14% (w/w) di-potassium phosphate (designated as 17/14) and 40% (w/w) lysate. It is demonstrated that in a 17/14 system with 40% (w/w) lysate, at least 2% (w/w) or 342mM (kg ATPS)−1 NaCl were able to selective partition the pDNA to the bottom phase.
Keywords: Bioseparations; Aqueous two-phase system; Purification; Integrated processing; RNA; DNA