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Biochemical Engineering Journal (v.22, #2)
Biocatalyst preparation from Pseudomonas putida SM-6 for conversion ofdl-lactate to pyruvate by Jingsong Gu; Yuanxiu Wang; Qiang Jiao (pp. 89-96).
The production of pyruvate using enzymatic methods is a valuable process for its cheap substrate, high conversion ratio, simple composition of reaction, and convenience of recovery. Cell-free extracts from Pseudomonas putida SM-6 that contain both lactate oxidase (LOD) and catalase were employed as catalysts for biocatalysis. In order to obtain an efficient and cost-effective biocatalyst, the cultivation of P. putida SM-6 both in shake flask and at bioreactor level was investigated. On enzyme production, the suitability of glucose instead of lactate as a carbon source was studied. Maximum LOD production occurred when a mixture of lactate and glucose was used as a carbon source. Lactate was assimilated first and the increase of nitrogen concentration had a positive effect on cell growth and glucose assimilation. The optimum cultivation temperature was 29–31°C, and the neutral or basic initial medium pH was in favor of both cell growth and LOD production. The interrelation between various process parameters was evaluated for the cultivation of P. putida SM-6 in submerged culture. Using a fed-batch cultivation strategy in a 2-l bioreactor, an increase in cell mass was achieved but without a concomitant increase in LOD production compared with cultivation in shake flask. Bioconversion of lactate to pyruvate was carried out using cell-free extract acquired by cell disruption of P. putida SM-6. Ethylenediaminetetraacetic acid (EDTA) was added into the biocatalytic system to prohibit the degradation of pyruvate by other enzymes in the cell-free extract. As a result, on a preparative scale, cell-free extract which contained 3.04mg/ml crude enzyme (10.0U/ml LOD, 877U/ml catalase) produced pyruvate of 423mM fromdl-lactate of 660mM in a reaction period of 22h. The molar conversion yield of pyruvate on the basis of the amount of lactate consumed was 0.80g/g.
Keywords: Pyruvate; Lactate oxidase; Bioconversion; Enzyme production; Lactate; Glucose
Kinetics for aerobic biological treatment of o-cresol containing wastewaters in a slurry bioreactor: biodegradation by utilizing waste activated sludge by Masahiro Maeda; Asuka Itoh; Yoshinori Kawase (pp. 97-103).
A toxic volatile organic compound (VOC), o-cresol, was biologically degraded by utilizing waste activated sludge in a gas–liquid–solid three-phase slurry bioreactor. The biodegradation kinetics of o-cresol was examined in batch experiments at varying initial o-cresol concentrations (from 30 to 600mg/L), waste activated sludge concentrations (from 1000 to 11,500mg/L) and aeration rates (from 0.05 to 1.0L/min). The kinetic parameters of o-cresol aerobic biodegradation were estimated using Haldane substrate inhibition equation with the correlation factors of approximately 0.95. The oxygen consumption during the biodegradation process was also examined. The oxygen consumption rate was adequately described by the Haldane type model with the correlation factor of 0.965. The biodegradation of o-cresol by waste activated sludge and the change of dissolved oxygen concentration in the slurry bioreactor were successfully simulated.
Keywords: Biodegradation; o; -Cresol; Slurry bioreactor; Haldane kinetics; Oxygen consumption
Gas hold-up and oxygen transfer in a draft-tube airlift bioreactor with petroleum-based liquids by Mohammad Reza Mehrnia; Jafar Towfighi; Babak Bonakdarpour; Mohammad Mehdi Akbarnejad (pp. 105-110).
Gas hold-up ( ɛg) and volumetric gas–liquid oxygen transfer coefficient ( kL a) in a draft-tube airlift bioreactor (24×10−3m3 in volume) were studied using pure kerosene and diesel and their water-in-oil microemulsions, as the model solutions for petroleum biodesulfurization. For comparison, experiments were also done with distilled water. The ɛg and the kL a values for kerosene and diesel systems were in most cases significantly higher than the water system. Increase in water-to-oil phase volume ratio ( φ) of the microemulsion systems resulted in decrease in the values of ɛg, which was attributed to a decrease in the coalescence-inhibiting tendency of the petroleum liquids. Increase in the viscosity (v) of the microemulsion systems to around 32×10−6m2/s resulted in the occurrence of the churn turbulent regime with an associated decrease in the value of ɛg, which could not be solely accounted for by the increase in φ. Furthermore, the kL a values decreased with increase in the viscosity of the petroleum-based liquids. However, when churn turbulent conditions prevailed, increase in viscosity alone could not account for the decrease observed in the kL a values. Empirical correlations were developed that related ɛg and kL a to φ and ν, respectively, under the bubbly flow regime inside the bioreactor.
Keywords: Airlift bioreactors; Gas hold-up; Oxygen transfer; Petroleum; Microemulsions; Viscosity
A highly cost-effective simplification in the design of fast-paced vermireactors based on epigeic earthworms by S. Gajalakshmi; P. Sankar Ganesh; S.A. Abbasi (pp. 111-116).
Epigeics, such as Eudrilus eugeniae, Eisenia fetida and Perionyx excavatus are humus-feeder earthworms which, in natural environments, mostly dwell in the humus and leaf litter lying over the top soil and have very shallow borrows. In order to make the most of this characteristic of the epigeics, we have simplified the design of epigeic-based vermireactors by doing away with layers of gravel, sand and soil, normally used for maintaining the other types (anecic and endogeic) of earthworms in the vermireactors. Instead, we have laid a moistened cotton cloth at the bottom of the circular vermireaction tank and have laid the feed over it. This arrangement provides the epigeics with a thin, moisture-rich, layer of solids beneath the humus-like compost used in the reactors as feed. The bottom layer mimics the moist soil below the mulch and humus which the epigeics use for their shallow burrows in natural environments. By doing away with the conventionally used elaborate vermibed, which used to take up ∼5% of the vermireactor volume in conventional systems, we have effectively increased the utilizable fraction of the reactor volume, thereby enabling a much higher feed throughput per litre of the reactor. Six month long operation of the conventional and the modified vermireactors in two concurrently run batches – one operating at low density of E. eugeniae and the other in high-rate mode – revealed that vermicast production per litre of reactor volume in both batches of modified reactors was dramatically higher than the output in the corresponding conventional reactors.
Keywords: Epigeics; Eudrilus eugeniae; Vermicomposting; Optimization; Design; Municipal solid waste
Aerobic purification of dairy wastewater in continuous regime by F. Carta-Escobar; J. Pereda-Marín; P. Álvarez-Mateos; F. Romero-Guzmán; M.M. Durán Barrantes (pp. 117-124).
Biokinetic equations are needed to determine net microbial growth rate or its reciprocal mean cell residence time, effluent substrate concentrations, observed yield coefficients, decay rate constants… In this paper an attempt is made to establish the kinetic parameters that describe the organic matter biodegradation process in dairy wastewaters. Two different experimental devices in continuous regime were used: a first system with a working volume of 80L, and a second system that was a series three-reactor system. Mass balances to describe the substrate utilisation rate and the microbial growth together with the kinetic models of Michaelis–Menten and Chen–Hashimoto were used to describe the process. While the first system was overloaded under the loading conditions tested, the performance of the second system could be described by the below cited kinetic models at hydraulic residence times higher than 2.3–2.4 days.
Keywords: Aerobic processes; Biodegradation; Biokinetics; Kinetic parameters; Purification; Wastewater treatment
COD, ammonia and dissolved oxygen time profiles in the simultaneous nitrification/denitrification process by J.B. Holman; D.G. Wareham (pp. 125-133).
Research was undertaken to examine the time courses of COD, NH3N and DO during the simultaneous nitrification/denitrification (SND) process, with particular reference to the question of whether SND is a physical or microbiological phenomena. Two lab-scale sequencing batch reactors were operated under controlled conditions using seed material from a local wastewater treatment plant and a synthetic wastewater. The reactors were operated for a period of 9 months while intensive track studies and online monitoring were used to observe the influence of changes to influent conditions on chemical time profiles. Typically, over 75% of nitrogen removal was achieved through the SND process. Two distinct dissolved oxygen plateaus before ultimate DO breakthrough corresponding to COD and NH3N depletion were observed. However, the time courses of the chemical parameters monitored could reasonably support both physical and microbiological explanations for nitrogen removal via SND.
Keywords: Nutrient removal; Simultaneous nitrification and denitrification; Aerobic denitrification
Classification of two-dimensional fluorescence spectra using self-organizing maps by Jong Il Rhee; Kum-Il Lee; Chun-Kwang Kim; Yong-Sik Yim; Sang-Wook Chung; Jiaqiu Wei; Karl-Heinz Bellgardt (pp. 135-144).
A two-dimensional (2D) spectrofluorometer is often used to monitor various fermentation processes. The change in fluorescence intensities resulting from various combinations of excitation and emission wavelengths is investigated by using a spectra subtraction technique. But it has a limited capacity to classify the entire fluorescence spectra gathered during fermentations and to extract some useful information from the data. This study shows that the self-organizing map (SOM) is a useful and interpretative method for classification of the entire gamut of fluorescence spectral data and selection of some combinations of excitation and emission wavelengths, which have useful fluorometric information. Some results such as normalized weights and variances indicate that the SOM network is capable of interpreting the fermentation processes of S. cerevisiae and recombinant Escherichia coli monitored by a 2D spectrofluorometer.
Keywords: Bioprocess monitoring; Fermentation; Self-organizing map; Sensors; 2D spectrofluorometer
Continuous synthesis of 6- O-linoleoyl hexose using a packed-bed reactor system with immobilized lipase by Jie Chen; Yukitaka Kimura; Shuji Adachi (pp. 145-149).
6- O-Linoleoyl mannose, glucose and galactose were continuously synthesized in acetone using a packed-bed reactor system with immobilized-lipase from Candida antarctica. The high solubility of mannose in acetone resulted in the highest product concentration among the hexoses tested. The factors affecting the product concentration such as the linoleic acid concentration in the feed, the residence time in the reactor and the reaction temperature were examined. Although the product concentration was higher at the higher linoleic acid concentration, a concentration four times the solubility of hexose at a given temperature seemed to be proper. The product concentration became almost constant at a residence time in the reactor longer than 20min. Linoleoyl mannose was continuously synthesized at 60 and 70°C, and the operation at 60°C was indicated to be preferable to that at 70°C for long-term steady synthesis.
Keywords: Lipase; Linoleic acid; Linoleoyl hexose; Packed-bed reactor
Biodegradation kinetics of phenol and catechol using Pseudomonas putida MTCC 1194 by Arinjay Kumar; Shashi Kumar; Surendra Kumar (pp. 151-159).
Biological degradation of phenol and catechol by a bacterial strain of Pseudomonas putida (MTCC 1194) in basal salt medium (BSM) was investigated in shake-flask experiments at 29.9±0.3°C and pH of approximately 7.1. The lyophilized cultures of P. putida (MTCC 1194) were revived and exposed to increasing concentrations of phenol, and catechol in shake-flasks. This bacterial strain could be acclimatized to the concentrations of 1000 and 500mg/l for phenol and catechol, respectively, over a period of three months. The higher the concentration of phenol or catechol, the longer was the lag period. The well-acclimatized culture of P. putida (MTCC 1194) degraded the initial phenol concentration of 1000mg/l and initial catechol concentration of 500mg/l completely in 162 and 94h, respectively. Both the phenol and catechol were observed to be the inhibitory compounds. Monod's and linearized-Haldane's model could not represent the growth kinetics over the studied concentration range. However, Haldane's growth kinetics model could be fitted to the growth kinetics data well for the entire concentration range. Further, the decay coefficients have been found to be 0.0056 and 0.0067h−1 for the growth on phenol and catechol, respectively. Besides, the yield coefficient for the growth on phenol and catechol were found to be 0.65 and 0.50mg/mg, respectively. It is our view that the above information would be useful for modeling and designing the units treating phenol- and catechol-containing wastewaters.
Keywords: Biodegradation; Phenol; Catechol; Growth kinetics; P. putida; Substrate inhibition
Activity of glucose oxidase entrapped in mesoporous gels by Kicheol Han; Zhijian Wu; Joun Lee; Ik-Sung Ahn; Jin Won Park; Byoung Ryul Min; Kangtaek Lee (pp. 161-166).
We have immobilized a glucose oxidase (GOD) in mesoporous gels derived from tetraethoxysilane (TEOS) and organosilanes (i.e., methyltriethoxysilane (MTES), vinyltriethoxysilane (VTES), propyltriethoxysilane (PTES), and phenyltriethoxysilane (PhTES)) through a two-step sol–gel process without any templating agent. To find an optimal condition that maximizes the activity of the entrapped GOD, we investigate the effects of the organosilane precursor and the aging and drying temperature on the activity of the entrapped GOD. Our experimental results demonstrate that the activity of the entrapped GOD is highest when the gel is aged and dried at 4°C (except for the PhTES/TEOS gels), while it is lowest when the gel is aged and dried at room temperature. We find that the use of organosilane does not significantly improve the activity of the entrapped GOD. These findings suggest a sol–gel route that can maximize the activity of the entrapped enzyme.
Keywords: Glucose; Glucose oxidase; Enzyme activity; Immobilization; Immobilized enzyme; Mesoporous gel
Metabolic flux analysis of recombinant protein overproduction in Escherichia coli by Pınar Özkan; Berna Sariyar; F. Özde Ütkür; Uğur Akman; Amable Hortaçsu (pp. 167-195).
A metabolic model for cell growth and recombinant protein overproduction in Escherichia coli, which includes expression vector properties such as plasmid copy number and promoter strength as input parameters, was developed. Linear programming was used to obtain pathway fluxes for selected systems.The experimentally observed slow growth of recombinant E. coli XL1 in minimal medium supplemented with glucose accompanied with significant acetic acid secretion was successfully simulated by metabolic flux analysis (MFA) computations. The expression of the fusion protein GI-malE induced by IPTG caused a severe retardation in cell growth rate from μ=0.066 to 0.006h−1 and increased acetic acid secretion to the medium. The metabolic flux analysis of this transition in growth condition show that cells shift the usage of the available substrate from anabolic to catabolic pathways to increase their ability to produce energy for plasmid-encoded protein synthesis, stress protein synthesis and maintenance requirements. The increase in catabolic fluxes led to increased acetic acid secretion rate. MFA analysis of EcoRI endonuclease expression in E. coli 294 at high growth rate and accompanied by low growth rate depression showed that the flux distribution was very similar for both host and induced cells. The pathway utilization simulate exponential growth condition with preference for the PP pathway and activation of the glyoxalate shunt to meet the high demand for NADPH, NADH and biosynthetic intermediates at high growth rate.For both of the systems studied the pathway utilization and relative flux distributions obtained from MFA are in good agreement with reported E. coli gene expression profiles at different stages of growth and during recombinant protein overexpression.
Keywords: Abbreviations; AC; acetate; aceAB; isocitrate lyase and malate synthase; ACCOA; acetyl-coenzymeA; acnB; aconitate hydrase; AKG; α-ketoglutarate; ASP; aspartate; ADP; adenosine diphosphate; ATP; adenosine triphosphate; CTP; cytidine triphosphate; D6PGC; d; -6-phosphoglucono-δ-lactone; DATP; deoxyadenosine triphosphate; DCTP; deoxycytidine triphosphate; DGTP; deoxyguanosine triphosphate; fba; fructose 1,6-biphosphatase; F6P; fructose 6-phosphate; F16P; fructose 1,6-diphosphate; FUM; fumarate; GI; glucose isomerase; GI-malE; glucose isomerase–maltose binding protein fusion; glk; glucose kinase; gltA; citrate synthase; GLU; glutamate; gnd; 6-phosphogluconate dehydrogenase; GTP; guanosine triphosphate; ilvC; acetohydroxy acid isomeroreductase; IPTG; isopropyl-β-; d; -thiogalactopyranoside; leuA; isopropylmalate synthase; lysC; diaminopimelate decarboxylase; mdh; malate dehydrogenase; metE; methionine synthase; MFA; metabolic flux analysis; NADH; nicotinamide adenine dinucleotide; NAPDH; nicotinamide adenine dinucleotide phosphate; NH; 3; ammonia; O; 2; oxygen; OA; oxaloacetate; pckA; phosphoenolpyruvate carboxykinase; Pe; plasmid copy number; Pn; plasmid expression efficiency; PEP; phosphoenolpyruvate; PI; phosphate; P/O; total ATP to oxygen ratio; PP; phosphate pentose pathway; ppc; PEP carboxylase; ppGpp; guanosine 3′-diphosphate 5′-diphosphate; PYR; pyruvate; RecP; recombinant protein; RL5P; d; -ribulose-5-phosphate; rpi; R5P isomerase; RQ; carbon dioxide to oxygen ratio; sdhC; succinate dehydrogenase; TCA; tricarboxylic cycle; TTP; thymidine triphosphate; UTP; uridine triphosphate; X5P; xylulose-5-phosphateAcetic acid; Batch processing; Escherichia coli; Metabolic flux analysis; Microbial growth; Recombinant DNA