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Biochemical Engineering Journal (v.26, #2-3)
Heterologous production of Rhizopus oryzae lipase in Pichia pastoris using the alcohol oxidase and formaldehyde dehydrogenase promoters in batch and fed-batch cultures by Oriol Cos; David Resina; Pau Ferrer; José Luis Montesinos; Francisco Valero (pp. 86-94).
A Rhizopus oryzae lipase (ROL) has been expressed in Pichia pastoris as a reporter gene using two different regulated promoters. Both phenotypes, Muts and Mut+, have been used for the expression under the control of alcohol oxidase 1 promoter ( PAOX1). Moreover, the new formaldehyde dehydrogenase 1 promoter ( PFLD1) has been tested. PFLD1 allows the design of methanol-free culture strategies, being methylamine a less volatile and flammable inducer. The comparison in batch and fed-batch cultures of the two promoters has shown that Muts ( PAOX1) is somewhat more efficient than PFLD1 and Mut+ (PAOX1) systems, with a 1.1- and 1.4-fold higher specific productivity (U lipase/g biomass h), respectively. However, the productivity in all three systems was very similar (2500U lipase/lh).These results demonstrate that the expression of heterologous proteins under the transcriptional control of PFLD1 is a promising alternative to the well-known PAOX1.
Keywords: Fed-batch culture; Bioreactors; Lipase; Pichia pastoris; AOX1; FLD1
Lipolytic enzyme production by Thermus thermophilus HB27 in a stirred tank bioreactor by Alberto Dominguez; Lorenzo Pastrana; Maria A. Longo; Maria L. Rúa; M. Angeles Sanroman (pp. 95-99).
In this study, lipolytic enzyme production by Thermus thermophilus HB27 at bioreactor scale has been investigated. Cultivation was performed in a 5-L stirred tank bioreactor in discontinuous mode, at an agitation speed of 200rpm. Different variables affecting intra- and extra-cellular lipolytic enzyme production such as culture temperature and aeration rate have been analysed. The bacterium was able to grow within the temperature range tested (from 60 to 70°C) with an optimum value of 70°C for intra- and extra-cellular lipolytic enzyme production.On the other hand, various aeration levels (from 0 to 2.5L/min) were employed. A continuous supply of air was necessary, but no significant improvement in biomass or enzyme production was detected when air flow rates were increased above 1L/min. Total lipolytic enzyme production reached a maximum of 167U/L after 3 days, and a relatively high concentration of extra-cellular activity was detected (40% of the total amount). Enzyme yield was around 158U/g cells. Moreover, it is noteworthy that the lipolytic activity obtained operating at optimal conditions (70°C and air flow of 1L/min) was about five-fold higher than that attained in shake flask cultures
Keywords: Aeration; Esterase; Lipase; Thermophilic; Thermus; Batch operation; Bioreactor
Hydrolytic enzyme production by Aspergillus awamori on grape pomace by C. Botella; I. de Ory; C. Webb; D. Cantero; A. Blandino (pp. 100-106).
Grape pomace, the main waste in the wine industry, has been shown to be the sole nutrient source for solid state fermentation to produce hydrolytic enzymes (cellulases, xylanases and pectinases) using Aspergillus awamori. Petri dishes with this natural support inoculated with spores were incubated under static conditions during 7 days and the enzymatic extracts obtained at different time intervals were analysed. The enzymes analyses demonstrated that grape pomace could be competitive with other typical agroindustrial wastes used as substrates in SSF processes.
Keywords: Solid state fermentation; Agroindustrial residues; Grape pomace; Aspergillus awamori; Cellulase; Xylanase; Pectinase
Modelling of growth and accumulation of carotenoids in Haematococcus pluvialis as a function of irradiance and nutrients supply by M.C. García-Malea; C. Brindley; E. Del Río; F.G. Acién; J.M. Fernández; E. Molina (pp. 107-114).
This paper analyzes the feasibility of the autotrophic production of vegetative cells of Haematococcus pluvialis under conditions resembling outdoors. The experimental design simulates in laboratory with artificial light an outdoors circadian cycle similar to solar illumination. The influence of the irradiance and nutrient concentration on the growth rate and carotenoids accumulation in batch cultures is studied. The cultures were not photoinhibited even under the maximum irradiance-level tested (2500μEm−2s−1). Growth was kept nutrient-limited by using nutrients concentration below the standard inorganic medium (10mM nitrate). When no nutrient-limitation occurs, the growth rate and biomass productivity measured 0.57day−1 and 0.28gL−1day−1, respectively, were similar to the maximum values reported, regardless of the nutritional regime: autotrophic, mixotrophic or heterotrophic. On the other hand, carotenogenesis was only observed under nutrient-limiting conditions when the medium strength was reduced to 0.2- or 0.3-fold of the standard medium. On the other hand, carotenogenesis ceased under severe nutrient deprivation (i.e. nutrient strength of 0.1-fold of the standard medium). The growth rate and the carotenoids accumulation rate were demonstrated to be a function of the average irradiance inside the culture, and of the nutrient content of the medium. A mathematical model for the observed behaviour is proposed. This model was adequate to fit all the experimental data obtained. The values determined for the characteristics parameters are in agreement with those found by other authors. Therefore, the proposed model can be a useful tool for the design and management of Haematococcus cultures, and could allow improving the yield of this production process.
Keywords: Haematococcus; Growth rate; Irradiance; Nutrients; Carotenoids; Secondary metabolism
Shear effects on suspended marine sponge cells by F. García Camacho; E.H. Belarbi; M.C. Cerón García; A. Sánchez Mirón; T. Chile; Y. Chisti; E. Molina Grima (pp. 115-121).
Fractions of viable cells, apoptotic and irreversibly damaged cells, dead whole cells and cell fragments were measured by flow cytometry during the production of freely suspended primary cells from explants of the marine sponge Axinella damicornis. The explants were disintegrated using the well-known Müller protocol [W.E.G. Müller, M. Wiens, R. Batel, R. Steffen, R. Borojevic, M.R. Custodio, Establishment of a primary cell culture from a sponge: primmorphs from Suberites domuncula, Mar. Ecol. Progr. Ser. 178 (1999) 205–219]. Supplementation of the standard Ca2+- and Mg2+-free artificial seawater of the Müller protocol, with the shear protectant Pluronic F68 (0.1%, w/v) greatly reduced the cell damage and enhanced the recovery of viable cells at each of the four stages of the protocol. Agitation of cells on an orbital shaker at 75rpm essentially killed all the viable cells within 2.5h, but no loss of viability occurred at a higher agitation speed of 100rpm for up to 6h when the cells were supplemented with Pluronic F68. This time-dependent loss in viability could be significantly reduced by processing at 3°C instead of the normal 17°C. A four-step mechanistic model was shown to describe the kinetics of cell death and fragmentation within ±10% of the measured values. The damage to cells was modeled as a web of first-order processes that did not depend on cell–cell interactions. The forces in the agitated fluid killed the viable cells by impact, which was not accompanied by cell rupture (i.e. the cell was left dead, but intact).
Keywords: Sponge cell culture; Axinella damicornis; Pluronic F68; Shear effects; Cell damage
Kinetic analysis of hybridoma cell culture in a protein-free medium: Substrate and agitation effects by Lorea Legazpi; Jaime Díaz; Adriana Laca; Mario Díaz (pp. 122-130).
A kinetic study of a hybridoma cell line that produces monoclonal antibodies against lactoferrin was carried out. A well defined protein-free culture medium was employed to facilitate the subsequent purification of the monoclonal antibodies. It should be highlighted that most of the existing work has been carried out employing culture media enriched with fetal bovine serum (FBS). Cell growth and monoclonal antibody production were monitored and kinetic parameters were determined. Besides, fundamental nutrients such as glucose and glutamine, inhibitory products such as ammonium and lactate, and several amino acids were followed throughout the culture. Additional experiments were carried out supplementing the medium with glutamine and ammonium, none of them resulting the key compound that halted the cell growth under the tested conditions and an unstructured model can be used to describe the system. Finally, agitation of the culture by a rocker set-up has shown high values of the specific death rate.
Keywords: Abbreviations; Am; ammonium; Asp; aspartic acid; Glc; glucose; Gln; glutamine; Ile; isoleucine; Lac; lactate; Leu; leucine; Lys; lysine; Met; methionine; Phe; phenilalanine; Pro; proline; S.D.; standard deviation; Ser; serine; Tyr; tyrosine; Val; valineHybridoma; Kinetics; Monoclonal antibodies; Protein-free; Agitation
Rheology and processing of gluten based bioplastics by A. Jerez; P. Partal; I. Martínez; C. Gallegos; A. Guerrero (pp. 131-138).
Knowledge of processing effects on the rheological properties of wheat gluten based biomaterials is important for the optimization of the productive process and to obtain biomaterials with suitable mechanical properties. In this paper, the linear viscoelasticity behaviour of glycerol/gluten bioplastics obtained by casting and thermoplastic processing has been studied. The effects of plasticizer content, mixing speed and thermal history were evaluated by oscillatory shear experiments and modulated differential scanning calorimetry in a wide range of temperatures. The mechanical spectrum of these materials was characterized during processing, showing a similar microstructure and, therefore, rheological behaviour at room temperature for both processing procedures. However, bioplastic obtained by mechanical processing showed higher thermal susceptibility, which was related to the denaturation degree and thermosetting and cross-linking potentials of proteins.
Keywords: Wheat gluten; Bioplastics; Rheology; Viscoelasticity; Processing
Effects of pellet morphology on broth rheology in fermentations of Aspergillus terreus by E.M. Rodríguez Porcel; J.L. Casas López; J.A. Sánchez Pérez; J.M. Fernández Sevilla; Y. Chisti (pp. 139-144).
Effects of pellet morphology on broth rheology are reported for pelleted submerged cultures of the lovastatin producing filamentous fungus Aspergillus terreus, growing in fluidized bed and stirred tank bioreactors. The pellet diameter and compactness were affected by the agitation intensity of the broth; however, the total biomass productivity was not affected. In fluidized beds and stirred tanks with agitation intensity of up to 300rpm (impeller tip speed of 1.02ms−1), the fungal pellets were stable at diameters of up to about 2300μm. In more intensely agitated stirred tanks (≥600rpm; impeller tip speed of ≥2.03ms−1), the stable pellet size was only about ≤900μm. The biomass concentration and the pellet diameter were the main factors that influenced the flow index and the consistency index of the power-law broths. Because the biomass productivity was the same in all experiments in a given type of reactor and the oxygen concentration was kept at ∼400% of air saturation, the pellet size and morphology were not influenced by oxygen mass transfer effects. Pellets were always dense in the core region and no necrosis of the biomass occurred.
Keywords: Agitation; Aspergillus terreus; Filamentous fungi; Fluidized bed bioreactors; Pellet morphology; Rheology
Factors affecting the biotransformation of trimethylammonium compounds intol-carnitine by Escherichia coli by M. Cánovas; V. Bernal; M. González; H.P. Kleber; J.L. Iborra (pp. 145-154).
The biotransformation ofd-carnitine and crotonobetaine intol-carnitine with wild and transformed E. coli strains under batch and continuous operation was optimised. In batch, the best conditions for the transformed strain were 30% oxygen saturation, a temperature of 41°C and a minimal medium, whereas anaerobic cultures in either complex or minimal media at 37°C and pH 7.5 were optimal for the wild strain. Studies on the expression of the enzymes involved in trimethylammonium metabolism showed thatl-carnitine dehydratase activity was always higher than that ofd-carnitine racemase. Experiments with the transformed strain in continuous cell-recycle reactors showed that, despite the higher productivity that could be achieved (0.65–1.2g/Lh), plasmid-bearing cells were segregated even when a selective medium was used. This fact was also confirmed by studying the evolution of thed-carnitine racemase level. Immobilization of the transformed strain in κ-carrageenan gels allowed continuous operation forl-carnitine production with no plasmid loss. In continuous processes with cell-retention systems, the wild strain showed higher productivity and stability than the transformed strain. Moreover, crotonobetaine was a better substrate for both strains used. Recycling with hollow-fiber cartridges provided the highest biomass level even though thel-carnitine dehydratase/biomass ratio was lower. However, membrane composition and cut-off had less influence on reactor performance as similar levels of productivity were attained. In spite of this, continuous processes attained al-carnitine production as high as 11.5g/Lh as a result of the high enzyme induction and biomass levels.
Keywords: Crotonobetaine hydration; d; -Carnitine racemization; d; -Carnitine; l; -Carnitine; E. coli; strains
Enzymatic biosynthesis of ricinoleic acid estolides by A. Bódalo-Santoyo; J. Bastida-Rodríguez; M.F. Máximo-Martín; M.C. Montiel-Morte; M.D. Murcia-Almagro (pp. 155-158).
Candida rugosa lipase has been shown to have sufficient activity to catalyse the enzymatic synthesis of ricinoleic acid estolides in a batch reactor. The water requirements of the reactor change during the reaction: at the beginning of the process a minimum amount of water is necessary but, later, the reaction mixture must be dried out to obtain an estolide with a high degree of condensation. The influence on the reaction rate of variables, such as water content, enzyme concentration and mixing devices, was established and optimised. Using an initial water content of 144,000ppm and a lipase concentration of 13.33mgE/g ricin, and maintaining the temperature at 40°C by mean of hot air circulation and using a three-bladed propeller stirrer as mixing device, an estolide of ricinoleic acid with an acid number of 65 was obtained in 48h.
Keywords: Biotransformations; Biosynthesis; Enzyme biocatalysis; Lipase; Ricinoleic acid; Estolide
Agro-industrial oily wastes as substrates for PHA production by the new strain Pseudomonas aeruginosa NCIB 40045: Effect of culture conditions by D. Fernández; E. Rodríguez; M. Bassas; M. Viñas; A.M. Solanas; J. Llorens; A.M. Marqués; A. Manresa (pp. 159-167).
Production of poly(3-hydroxyalkaonates) (PHA) by Pseudomonas aeruginosa 42A2 from agro-industrial oily wastes was studied. PHA accumulation, throughout the cell cycle, was observed as intracellular accumulation associated to polyphosphate granules. A 54.6% PHA accumulation was obtained when technical oleic acid (TOA) was used as carbon source. Molecular weight of the polymer was 54.7 Da. The polymer was amorphous, with glass transition at −47.5°C and thermal degradation at 293°C. PHA production and monomer composition were affected by KLa and temperature. The most relevant characteristic of the polymer produced at low aeration rates ( KLa, 0.06s−1) were the unusual C14:2 (13%) and the increase of C12:1 (42.2%). The highest amount of unsaturated monomers was found in the polymer produced at 18°C (64.4%).PHA accumulation ranged between 66.1% when waste-free fatty acids from soybean oil (WFFA) were used as carbon substrate, 29.4% when waste frying oil (WFO) was used and 16.8% when glucose was used. Depending on the substrate supplied a wide range of components was observed. Major saturated or unsaturated components of the polymer found were C10:0, C12:0 and C8:0 or C12:1 and C14:1, respectively. When glucose was used as carbon substrate C9:0, C11:0 and C16:0 were found.
Keywords: PHA; Waste-free fatty acids; Low aeration rates
Biodesulfurisation of DBT with Pseudomonas putida CECT5279 by resting cells: Influence of cell growth time on reducing equivalent concentration and HpaC activity by Almudena Alcon; Victoria E. Santos; Ana B. Martin; Pedro Yustos; Felix Garcia-Ochoa (pp. 168-175).
Dibenzothiophene (DBT) biodesulfurisation (BDS) route using a genetically modified organism, Pseudomonas putida CECT 5279, is studied. Tests of BDS with whole cells and with homogenized cells are carried out by taking samples of the cells during growth. The influence of the growth phases in the evolution of the intermediates of the 4S DBT desulfurising route is shown.Conversions of the five key compounds of the 4S route (DBT, DBTO, DBTO2, HBPS and HBP) are measured. DBT conversion values are maximal with cells obtained after 30h of growth time. HBP conversion values do not coincide with DBT conversion values, the maximum HBP production is obtained with cells grown for 10h. A greater intermediate DBTO and DBTO2 accumulation in broth is produced with cells obtained at 5 and 10h of growth time. Nevertheless, the accumulation in broth of HBPS, another intermediate, is considerably lower than that observed with cells obtained at 23, 30 and 45h of growth time.Also, the concentration of the reducing equivalents (NADH and FMNH2) and flavin-oxido-reductase activity inside the cells is measured. This showed that the concentration of the reducing equivalents and the activity of the HpaC enzyme in the P. putida cytoplasm do not limit BDS rate.The influence of 4S compound transport across cellular membrane is studied by comparison of results obtained by resting cell assays (whole cells) and with homogenized cells assays (disrupted cells). The results show that there is no accumulation of any compound inside the cells, and that the transport rate across the cellular membrane does not limit the overall biodesulfurisation rate.
Keywords: Biodesulfurisation; Biotransformations; Enzymes activity; Diffusion reactions; Pseudomonas putida; CECT 5279; 4S Intermediates route
Multiple analysis reprogrammable titration analyser for the kinetic characterization of nitrifying and autotrophic denitrifying biomass by Priscila Artiga; Fernando González; Anuska Mosquera-Corral; Jose Luis Campos; Juan Manuel Garrido; Elena Ficara; Ramón Méndez (pp. 176-183).
The system Multiple Analysis Reprogrammable TItratioN Analyser (MARTINA) based on titrimetric techniques has been used to kinetically characterize different types of sludges: nitrifying, enriched ammonia oxidizing and autotrophic denitrifying biomass. The titration system employed, combines the addition of NaOH solution and H2O2 solution in the mixed liquor to keep the pre-established value of pH and the dissolved oxygen concentration, respectively. Results obtained from repeated experiments performed with nitrifying sludge from municipal and industrial origin present slight differences (coefficient of variation lower than 30%) indicating that the method is highly reproducible. Besides, the kinetic parameters of the enriched ammonia oxidizing sludge obtained using the MARTINA system are comparable to those obtained using the respirometry indicating the reliability of this methodology. Changes in the procedure may be easily implemented in order to estimate half saturation constants with high values. On the other hand, experiments in anoxic conditions applied to the estimation of the kinetic parameters of the autotrophic denitrifying biomass have been successfully performed, even if this process involves a reaction characterized by slight pH changes. The titration system MARTINA is a reproducible, reliable, versatile and precise alternative to the traditional respirometric and substrate monitoring tests for the characterization of kinetics for a wide range of sludges in aerobic or anoxic conditions.
Keywords: Autotrophic denitrification; Kinetic parameters; Nitrifying bacteria; Respirometry; Titrimetry
Aerobic phosphorus release linked to acetate uptake: Influence of PAO intracellular storage compounds by M. Pijuan; A. Guisasola; J.A. Baeza; J. Carrera; C. Casas; J. Lafuente (pp. 184-190).
The mechanisms of polyphosphate accumulating organisms (PAO) are not fully established yet under conditions that differ from the classical anaerobic/aerobic conditions. Recently, some research in the field of biological phosphorus removal has been focused on studying systems where the electron donor (substrate) and the electron acceptor (nitrate or oxygen) are present simultaneously. The aim of this study was to assess the influence of the levels of intracellular storage compounds in the net phosphorus removal process occurring under continuous aerobic conditions. An enhanced biological phosphorus removal (EBPR) system was enriched in a sequencing batch reactor working under alternating anaerobic/aerobic conditions and the percentage of “ Candidatus Accumulibacter phosphatis� (the PAO in this study) was 45%. Three different batch experiments were performed to obtain a better insight of this process: one under standard anaerobic–aerobic conditions and two experiments under strictly aerobic conditions. One of the aerobic experiments was developed with the EBPR biomass withdrawn at the end of the anaerobic period and the other one with biomass withdrawn at the end of the aerobic period of the standard process. Although net phosphorus removal was achieved in both aerobic experiments, the ratios of phosphorus released and glycogen degraded versus acetate taken up were higher when the sludge was withdrawn at the end of the aerobic period, where higher levels of polyphosphate and glycogen were present in the biomass.
Keywords: Enhanced biological phosphorus removal; Polyphosphate accumulating organisms; Oxygen uptake rate; Wastewater treatment
Biodegradation kinetics of stored wastewater substrates by a mixed microbial culture by A. de Lucas; L. Rodríguez; J. Villaseñor; F.J. Fernández (pp. 191-197).
The anaerobic accumulation of several organic pollutants from industrial wastewaters, as storage substrates, and their subsequent aerobic biodegradation using a wastewater treatment mixed microbial culture for biological nutrient removal has been studied. The amount and the kinetics of substrate accumulation in the anaerobic stage depended on the characteristics of the wastewater fed to the anaerobic stage. Depending on the substrate used, levels of between 27 and 86% of storage polymers were accumulated with respect to the level obtained on feeding with acetate. The biodegradation kinetics were studied by modelling respirometry results. During the aerobic stage, oxygen-consumption data obtained in the respirometric tests were fitted to a model using a non-linear fitting estimation method. The simulation data obtained correlated well with the experimental oxygen-consumption data. The estimated kinetic parameters obtained indicate that each storage polymer was degraded at a different rate. However, the values obtained for the storage polymer half-saturation coefficient, KS: 16 mgCODl−1, and for the coefficient for endogenous respiration, b: 0.008h−1, were similar in all the experiments. The results indicate that each substrate produces the synthesis of a specific storage polymer that is degraded at a different rate.
Keywords: Biodegradation; Kinetic parameters; Storage polymers; Modelling; Wastewater treatment; Electrolytic respirometry