Modeling of an integrated fermentation/membrane extraction process for the production of 2-phenylethanol and 2-phenylethylacetate

An unstructured model for an integrated fermentation/membrane extraction process for the production of the aroma compounds 2-phenylethanol and 2-phenylethylacetate by Kluyveromyces marxianus CBS 600 was developed. The extent to which this model, based only on data from the conventional fermentation and separation processes, provided an estimation of the integrated process was evaluated. The effect of product inhibition on specific growth rate and on biomass yield by both aroma compounds was approximated by multivariate regression. Simulations of the respective submodels for fermentation and the separation process matched well with experimental results. With respect to the in situ product removal (ISPR) process, the effect of reduced product inhibition due to product removal on specific growth rate and biomass yield was predicted adequately by the model simulations. Overall product yields were increased considerably in this process (4.0 g/L 2-PE+2-PEA vs. 1.4 g/L in conventional fermentation) and were even higher than predicted by the model. To describe the effect of product concentration on product formation itself, the model was extended using results from the conventional and the ISPR process, thus agreement between model and experimental data improved notably. Therefore, this model can be a useful tool for the development and optimization of an efficient integrated bioprocess.     

Efficient polycyclic aromatic hydrocarbons dihydroxylation in direct micellar systems

Optimization of whole-cell bioconversion of the polycyclic aromatic hydrocarbons (PAHs) anthracene, phenanthrene, and naphthalene to the enantiomerically pure corresponding cis-dihydroxydihydro derivatives by the Escherichia coli JM109 (pPS1778) recombinant strain, carrying the naphthalene dioxygenase and corresponding regulatory genes cloned from Pseudomonas fluorescens N3, in micellar systems, is presented. We show that direct microemulsion systems, where a nonionic surfactant such as 1.5% (v/v) Triton X-100 plus 0.6% to 1.0% (v/v) selected oils are able to solubilize the PAHs tested at relatively high concentrations (initial concentrations in the reaction medium > or =10 mM for naphthalene and phenanthrene and > or =2 mM for anthracene), and allow for more efficient substrate bioconversion. These media, while not affecting bacteria viability and performance, provide increased efficiency and final product yields (100% for naphthalene, >30% for anthracene, >60% for phenanthrene). The phase behavior of the direct microemulsion systems for the different substrates and oils utilized was monitored as a function of their volume fraction by light scattering experiments, and related to the bioconversion results. For anthracene and phenanthrene, the dihydroxylated products have an inhibitory effect on the conversion reactions, thus hindering complete turnover of the substrates. We ascertain that such inhibition is reversible because removal of the products formed allowed the process to start over at rates comparable to initial rates. To allow for complete conversion of the PAHs tested a stepwise or continuous separation of the product formed from the micellar reaction environment is being developed.     

A kinetic model for lutein production by the green microalga Chlorella protothecoides in heterotrophic culture

Production of lutein by the green microalga Chlorella protothecoides grown heterotrophically in a fermentor using glucose as the carbon source and urea as the nitrogen source was investigated. An unstructured kinetic model was proposed to describe the microalgal culture system including cell growth, lutein formation, as well as glucose and nitrogen consumption. The inhibition potentials of biomass, product and substrates on growth and lutein formation were examined and incorporated into the kinetic model. Values of the kinetic model parameters were estimated. The resulting model predictions were in good agreement with the experimental results. The model can be helpful in scale-up, optimization and control of the C. protothecoides culture process, and can also be used as a guideline for similar microalgal cultivation systems. Received 28 January 1999/ Accepted in revised form 27 August 1999     

Application of an extended kalman filter method for monitoring high density cultivation of Escherichia coli

A real-time, on-line extended Kalman filter was used to describe and monitor the growth of Escherichia coli on glycerol. The growth of E. coli showed an inhibition kinetics with μ_max=0.806/h, K_S=0.68 g/l and K_i=87.4 g/l. As a feeding strategy, the conventional DO-stat with a DDC-PID control method, in which the dissolved oxygen concentration is maintained at a desired level by varying the substrate feedrate, was employed. The Kalman filter was based on an unstructured mathematical model and on-line measured data. The mathematical model comprised of mass balances of the biomass and substrate as well as kinetic and stoichiometric data which were measured prior to the process. For biomass concentration up to 50 g dry weight/l, the estimation of the process was rather accurate. At higher biomass concentration, product formation, indicated by an intense brown coloring of the fermentation broth, occured. Since the effect of this product on biomass production was not included in the mathematical model, the estimated data diverged from the experimental data at biomass concentrations greater than 50 g dry weight/l.     

Multisubstrate biodegradation kinetics of naphthalene, phenanthrene, and pyrene mixtures.

Biodegradation kinetics of naphthalene, phenanthrene and pyrene were studied in sole-substrate systems, and in binary and ternary mixtures to examine substrate interactions. The experiments were conducted in aerobic batch aqueous systems inoculated with a mixed culture that had been isolated from soils contaminated with polycyclic aromatic hydrocarbons (PAHs). Monod kinetic parameters and yield coefficients for the individual compounds were estimated from substrate depletion and CO(2) evolution rate data in sole-substrate experiments. In all three binary mixture experiments, biodegradation kinetics were comparable to the sole-substrate kinetics. In the ternary mixture, biodegradation of naphthalene was inhibited and the biodegradation rates of phenanthrene and pyrene were enhanced. A multisubstrate form of the Monod kinetic model was found to adequately predict substrate interactions in the binary and ternary mixtures using only the parameters derived from sole-substrate experiments. Numerical simulations of biomass growth kinetics explain the observed range of behaviors in PAH mixtures. In general, the biodegradation rates of the more degradable and abundant compounds are reduced due to competitive inhibition, but enhanced biodegradation of the more recalcitrant PAHs occurs due to simultaneous biomass growth on multiple substrates. In PAH-contaminated environments, substrate interactions may be very large due to additive effects from the large number of compounds present.     

Microscale process evaluation of recombinant biocatalyst libraries: application to Baeyer–Villiger monooxygenase catalysed lactone synthesis

Microscale processing techniques are rapidly emerging as a cost- effective means for parallel experimentation and hence the evaluation of large libraries of recombinant biocatalysts. In this work, the potential of an automated microscale process is demonstrated in a linked sequence of operations comprising fermentation, enzyme induction and bioconversion using three whole-cell biocatalysts each expressing cyclohexanone monoxygenase (CHMO). The biocatalysts, Escherichia coli TOP 10 [pQR239], E. coli JM107 and Acinetobacter calcoaceticus NCIMB 9871, were first produced in 96-deep square well fermentations at various carbon source concentrations (10 and 20 g L⁻¹ glycerol). Following induction of CHMO activity biomass concentrations of up to 6 g_DCW L⁻¹ were obtained. Cells from each fermentation were subsequently used for the Baeyer–Villiger oxidation of bicyclo[3.2.0]hept-2-en-6-one, cyclohexanone and cyclopentanone. Each bioconversion was performed at two initial substrate concentrations (0.5 and 1.0 g L⁻¹) in order to simultaneously explore both substrate specificity and inhibition. The microscale process sequences yielded quantitative and reproducible data for each biocatalyst on maximum growth rate, biomass yield, initial rate of lactone formation, specific biocatalyst activity and bioconversion yield. E. coli TOP 10 [pQR239] was demonstrated to be an efficient biocatalyst showing substrate specificities and substrate inhibition effects in line with previous studies. Finally, in order to show that the data obtained with E. coli TOP 10 [pQR239] at microwell scale (1,000 μL) could be related to larger scales of operation, the process was performed in a 2-L stirred-tank bioreactor. Using conditions designed to enable microwell kinetic measurements under none oxygen-limited conditions, the fermentation and bioconversion data obtained at the two scales showed good quantitative agreement. This study therefore confirms the potential of automated microscale experimentation for the whole-process evaluation of recombinant biocatalyst libraries and the specification of pilot and process scale operating conditions.     

Comparative study on the action of S-(+)-carvone, in situ, on the potato storage fungi Fusarium solani var. coeruleum and F. sulphureum

Growth of Fusarium sulphureum was inhibited by S-(+)-carvone administered via the gas phase. Under the same conditions, the related fungus F. solani var. coeruleum was not inhibited. In liquid medium, both fungi were found to convert S-(+)-carvone with the same rate, mainly into isodihydrocarvone, isodihydrocarveol and neoisodihydrocarveol. Only a slight difference in the relative amounts of the bioconversion products was observed. Since the bioconversion products did not inhibit the growth of the fungi to the same extent as S-(+)-carvone, the process can be considered as a detoxification mechanism. The bioconversion as such cannot account for the observed difference in growth inhibition.     

Kinetic analysis of 11α-hydroxylation of steroids by Rhizopus nigricans

Optimization of bioconversion of 16,17α-epoxyprogesterone by Rhizopus nigricans TJ 108 was investigated by means of uniform design. Batch cell growth and bioconversion kinetics were simulated under optimal conditions. Contois equation was used in the kinetics study of fungal growth on glucose. Simulation of bioconversion process was done at a constant value of substrate concentration according to the reaction mechanism. It was demonstrated that the above model satisfactorily described the kinetic behaviors of cell growth and bioconversion of the filamentous fungi.     

A model‐driven approach towards rational microbial bioprocess optimization

Due to sustainability concerns, bio‐based production capitalizing on microbes as cell factories is in demand to synthesize valuable products. Nevertheless, the nonhomogenous variations of the extracellular environment in bioprocesses often challenge the biomass growth and the bioproduction yield. To enable a more rational bioprocess optimization, we have established a model‐driven approach that systematically integrates experiments with modeling, executed from flask to bioreactor scale, and using ferulic acid to vanillin bioconversion as a case study. The impacts of mass transfer and aeration on the biomass growth and bioproduction performances were examined using minimal small‐scale experiments. An integrated model coupling the cell factory kinetics with the three‐dimensional computational hydrodynamics of bioreactor was developed to better capture the spatiotemporal distributions of bioproduction. Full‐factorial predictions were then performed to identify the desired operating conditions. A bioconversion yield of 94% was achieved, which is one of the highest for recombinant Escherichia coli using ferulic acid as the precursor.     

Growth dynamics of a methylotroph (Methylomonas L3) in continuous cultures. II. Growth inhibition and comparison against an unstructured model

High methanol concentrations have a negative effect on the growth rate and the biomass yield of growth transients induced by methanol pulses in continuous cultures of Methylomonas L3. The physiological basis of this effect is investigated by measuring the effect of the methanol pulse on the cell energy charge (EC) and ATP, ADP, and AMP concentrations, and by comparing the results of the pulse transients against an unstructured model. The methanol pulse is shown to lead to increased values of the cell EC and ATP concentration, and thus, inhibition and reduced availability of biosynthetic energy are excluded as causes of inhibition. When the biomass and methanol profiles of the transient experiments are compared in phase-plane diagrams against computer simulations based on the model, satisfactory agreement between experimental data and model predictions is found in single-substrate, high-dilution-rate experiments. Conversely, poor agreement between experimental data and simulation results indicates a more severe growth inhibition than the model predicts at low dilution rates and a less severe one in mixed-substrate experiments. Based on these findings and other relevant physiological information, we propose that the large variations in the negative effect of methanol on growth result from the fact that cells accumulate methanol to widely different concentrations depending on their physiological state. In their effort to detoxify from the high intracellular methanol and formaldehyde concentrations, cells oxidize considerably more methanol than they can incorporate into biomass. This leads to a useless ATP surplus, which the cells must hydrolyze without doing any useful biosynthetic work, and this results in lower biomass yields.     

Real-time estimation of biomass and specific growth rate in physiologically variable recombinant fed-batch processes

The real-time measurement of biomass has been addressed since many years. The quantification of biomass in the induction phase of a recombinant bioprocess is not straight forward, since biological burden, caused by protein expression, can have a significant impact on the cell morphology and physiology. This variability potentially leads to poor generalization of the biomass estimation, hence is a very important issue in the dynamic field of process development with frequently changing processes and producer lines. We want to present a method to quantify “biomass” in real-time which avoids off-line sampling and the need for representative training data sets. This generally applicable soft-sensor, based on first principles, was used for the quantification of biomass in induced recombinant fed-batch processes. Results were compared with “state of the art” methods to estimate the biomass concentration and the specific growth rate µ. Gross errors such as wrong stoichiometric assumptions or sensor failure were detected automatically. This method allows for variable model coefficients such as yields in contrast to other process models, hence does not require prior experiments. It can be easily adapted to a different growth stoichiometry; hence the method provides good generalization, also for induced culture mode. This approach estimates the biomass (or anabolic bioconversion) in induced fed-batch cultures in real-time and provides this key variable for process development for control purposes.     

Asymmetric reduction of alpha, beta-unsaturated ketone to (R) allylic alcohol by Candida chilensis

A pilot scale whole cell process was developed for the enantioselective 1,2-reduction of prochiral alpha,beta-unsaturated ketone to (R) allylic alcohol using Candida chilensis. Initial development showed high enantiomeric excess (EE > 95%) but low product yield (10%). Process development, using a combination of statistically designed screening and optimization experiments, improved the desired alcohol yield to 90%. The fermentation growth stage, particularly medium composition and growth pH, had a significant impact on the bioconversion while process characterization identified diverse challenges including the presence of multiple enzymes, substrate/product toxicity, and biphasic cellular morphology. Manipulating the fermentation media allowed control of the whole cell morphology to a predominantly unicellular broth, away from the viscous pseudohyphae, which were detrimental to the bioconversion. The activity of a competing enzyme, which produced the undesired saturated ketone and (R) saturated alcohol, was minimized to < or =5% by controlling the reaction pH, temperature, substrate concentration, and biomass level. Despite the toxicity effects limiting the volumetric productivity, a reproducible and scaleable process was demonstrated at pilot scale with high enantioselectivity (EE > 95%) and overall yield greater than 80%. This was the preferred route compared to a partially purified process using ultra centrifugation, which led to improved volumetric productivity but reduced yield (g/day). The whole cell approach proved to be a valuable alternative to chemical reduction routes, as an intermediate step for the asymmetric synthesis of an integrin receptor antagonist for the inhibition of bone resorption and treatment of osteoporosis.     

Development of a fluorescence-based method for monitoring glucose catabolism and its potential use in a biomass hydrolysis assay

Background  

The availability and low cost of lignocellulosic biomass has caused tremendous interest in the bioconversion of this feedstock into liquid fuels. One measure of the economic viability of the bioconversion process is the ease with which a particular feedstock is hydrolyzed and fermented. Because monitoring the analytes in hydrolysis and fermentation experiments is time consuming, the objective of this study was to develop a rapid fluorescence-based method to monitor sugar production during biomass hydrolysis, and to demonstrate its application in monitoring corn stover hydrolysis.     

Conversion of solvent evaporation residues from the AB- (acetone-butanol) bioprocess into bacterial cells accumulating thermoplastic polyesters

In a bioconversion study based on utilisation of by-products from the AB- (acetone-butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents. The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp. G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate- (PHA-)accumulation up to 40% per dry weight. A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs. Concentrations of 1-butanol, which is known for its membrane damaging properties in micro-organisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth. No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate. Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process. The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.     

Conversion of solvent evaporation residues from the AB- (acetone - butanol) bioprocess into bacterial cells accumulating thermoplastic polyesters

In a bioconversion study based on utilisation of by-products from the AB- (acetone - butanol) bioprocess a new isolated gram-negative solvent tolerant bacterium was used to convert the AB process residue after removal of the major part of the solvents. The bacterium identified as a representative of the genus Alcaligenes (designated as Alcaligenes sp. G) was capable of growth up to optical densities ranging from 8 to 20 and simultaneously of polyhydroxyalkanoate-(PHA-)accumulation up to 40% per dry weight. A standardised medium based on AB by-products containing 7 g/l of butyrate and 5 g/l of acetate at pH 7.5 was used in our studies for bioconversion into PHAs. Concentrations of 1-butanol, which is known for its membrane damaging properties in microorganisms, were tolerated in the AB by-products medium up to 4 g/l without significant inhibition of cellular growth. No inhibition of growth was observed, when the medium was adjusted to 40 g/l butyrate. Due to the toxicity of the remaining 1-butanol maintenance of sterility is of no high priority during the process. The use of acetate and butyrate from an AB process is expected to provide a higher return-on-investment than the combustion of biogas to help meet energy demands.     

Biodegradation Kinetics of Phenanthrene by a Fusant Strain

The fusant strain (F14), which produced by protoplast fusion between Sphingomonas sp. GY2B (GenBank DQ139343) and Pseudomonas sp. GP3A (GenBank EU233280), was tested for phenanthrene biodegradation at 30 °C and pH of 7.0. The kinetics of phenanthrene biodegradation by F14 was investigated over a wide range of initial concentration (15-1,000 mg l(-1)). The rate and the extent of phenanthrene degradation increased with the increase of concentration up to 230 mg l(-1), which indicated negligible inhibition effect at low concentrations. The non-competitive inhibition model was found to be fit for the process. GC-MS analysis showed that biodegradation of phenanthrene by F14 was via dioxygenation at both 1,2- and 3,4-positions and followed by 2-hydroxy-1-naphthoic acid and 1-hydroxy-2-naphthoic acid. The relative intensity of 2-hydroxy-1-naphthoic acid was approximately 3-4 times higher than that of 1-hydroxy-2-naphthoic acid, indicating the 2-hydroxy-1-naphthoic acid was the predominant product in the phenanthrene degradation by fusant strain F14.     

Evaluation of Various Unstructured Kinetic Models for the Production of Protease by Bacillus sphaericus MTTC511

Bacillus sphaericus MTCC511 was used for the production of protease in submerged batch fermentation. Maximum protease activity of 1010 U/L was obtained during a fermentation period of 24 h under optimized conditions of 30 °C in a medium with an initial pH of 7 and at a shaking rate of 120 rpm. The maximum biomass obtained in the batch fermentation was 2.55 g/L after 16 h. Various unstructured models were analyzed to simulate the experimental values of microbial growth, protease activity and substrate concentration. The unstructured models, i.e. the Monod model for microbial growth, the Monod incorporated Luedeking‐Piret model for the production of protease and the Monod‐incorporated modified Luedeking‐Piret model for the utilization of substrate were capable of predicting the fermentation profile with high coefficient of determination (R²) values of 0.9967, 0.9402 and 0.9729, respectively. The results indicated that the unstructured models were able to describe the fermentation kinetics more effectively.     

Enhancement of fruit byproducts through bioconversion by Hermetia illucens (Diptera: Stratiomyidae)

Bioconversion is a biological process by which organic materials are converted into products with higher biological and commercial value. During its larval stage the black soldier fly Hermetia illucens is extremely voracious and can feed on a wide variety of organic materials. To study the impact of different fruit byproducts on the insect's growth, final larval biomass, substrate reduction, bioconversion parameters, and larval nutritional composition, 10 000 black soldier fly larvae (BSFL) were reared on 7.0 kg of one of three substrates (strawberry, tangerine, or orange) or on a standard diet as a control. The results highlight that BSFL can successfully feed and grow on each of these diets, though their development time, growth rate, and final biomass were differently impacted by the substrates, with strawberry being the most suitable. The lipid and protein contents of BSFL were similar among larvae fed on different substrates; however, major differences were detected in ash, micronutrient, fiber, fatty acid, and amino acid contents. Overall, the results indicate that fruit waste management through the BSFL bioconversion process represents a commercially promising resource for regional and national agrifood companies. Our study offers new perspectives for sustainable and environmentally friendly industrial development by which fruit byproducts or waste might be disposed of or unconventionally enhanced to create secondary products of high biological and economic value, including BSFL biomass as animal feed or, in perspective, as alternative protein source for human nutrition.     

Determination of monod kinetic coefficients for volatile hydrophobic organic compounds

A new procedure is presented to determine Monod kinetic coefficients and the microbial yield coefficient for volatile hydrophobic compounds such as phenanthrene. Batch experiments were conducted with a mixed culture capable of degrading phenanthrene. The phenanthrene disappearance and carbon dioxide production were monitored with time. A maximum likelihood estimator was formulated to fit the set of equations that describe the system to the measured data. The model takes into account a number of processes such as partition onto the apparatus, volatilization, and partition onto the biomass. The parameters required to describe these processes were obtained by independent experiments. The yield coefficient could be determined within a small range. However, the specific growth rate and the half-saturation constant were found to vary widely, with pairs of them describing the system adequately. It was shown that partition and volatilization processes can significantly affect the determination of the yield and Monod kinetic coefficients and need to be taken into account. (c) 1996 John Wiley & Sons, Inc.