Substrate Feeding Strategy Research Articles

A toolbox approach for the rapid evaluation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli expression system with microscale experimentation

This work describes an experimental ‘toolbox’ for the rapid evaluation and optimisation of multi-step enzymatic syntheses comprising a ‘mix and match’ E. coli-based expression system and automated microwell scale experimentation. The approach is illustrated with a de novo designed pathway for the synthesis of optically pure amino alcohols using the enzymes transketolase (TK) and transaminase (TAm) to catalyze asymmetric carbon-carbon bond formation and selective chiral amine group addition respectively. The E. coli expression system, based on two compatible plasmids, enables pairs of enzymes from previously engineered and cloned TK and TAm libraries to be evaluated for the sequential conversion of different initial substrates. This is complemented by the microwell experimentation which enables efficient investigation of different biocatalyst forms, use of different amine donors and substrate feeding strategies. Using this experimental ‘toolbox’, one-pot syntheses of the diastereoisomers (2S,3S)-2-aminopentane-1,3-diol (APD) and (2S,3R)-2-amino-1,3,4-butanetriol (ABT) were designed and performed, which gave final product yields of 90% mol/mol for APD and 87% mol/mol for ABT (relative to the initial TK substrates) within 25 hours. For the synthesis of APD, the E coli TK mutant D469E was paired with the TAm from Chromobacterium violaceum 2025 while for ABT synthesis the wild-type E. coli TK exhibited the highest specific activity and ee( enantiomeric excess) of >95%. For both reactions, whole-cell forms of the TK-TAm biocatalyst performed better than cell lysates while isopropylamine (IPA) was a preferable amine donor than methylbenzylamine (MBA) since side reactions with the initial TK substrates were avoided. The available libraries of TK and TAm enzymes and scalable nature of the microwell data suggest this ‘toolbox’ provides an efficient approach to early stage bioconversion process design in the chemical and pharmaceutical sectors.

Enhancing the production of gamma-linolenic acid in Hansenula polymorpha by fed-batch fermentation using response surface methodology

AbstractGamma-linolenic acid (GLA, C18:3Δ6,9,12) is an n-6 polyunsaturated fatty acid (PUFA) that has been used for the alleviation and treatment of a number of symptoms and diseases. Increasing GLA demand has led to a search for alternative producers and potential strategies for GLA production. Based on the successful performance of Hansenula polymorpha, a methylotrophic yeast, as a “cell factory” for the production of valuable bioproducts, a bioprocess development approach was implemented for GLA production in the recombinant yeast carrying the mutated Δ6-desaturase gene of Mucor rouxii. Using a substrate-feeding strategy under glycerol-limited conditions, the physical-chemical variables during the fed-batch fermentation of the recombinant H. polymorpha were optimised for GLA production through response surface methodology using a Box-Behnken design. The medium composition, including yeast extract and trace elements, and dissolved oxygen tension (DOT) were targeted. We found that DOT was the most effective variable for enhancing GLA yield. These results also suggest that the optimum conditions for GLA production are 28 % saturation of DOT, 1 g L−1 of yeast extract and 3.6 mL L−1 of the Pichia trace metals 1 (PTM1).

Lipid production by Rhodosporidium toruloides Y4 using different substrate feeding strategies

Microbial lipid is a potential alternative feedstock for the biodiesel industry. New culture strategies remain to be developed to improve the economics of microbial lipid technology. This work describes lipid production by the oleaginous yeast Rhodosporidium toruloides Y4 using a 15-l bioreactor with different substrate feeding strategies. Among these strategies, the intermittent feeding mode gave a lipid productivity of 0.36 g l(-1) h(-1), whereas the constant glucose concentration II (CC-II) mode gave the highest lipid productivity of 0.57 g l(-1) h(-1). The repeated fed-batch mode according to the CC-II mode was performed with a duration time of 358 h, and the overall lipid productivity was 0.55 g l(-1) h(-1). Our results suggested that substrate feeding modes had a great impact on lipid productivity and that the repeated fed-batch process was the most appealing method by which to enhance microbial lipid production.

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1(19)) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1(19) in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1(19). The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1(19) in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1(19). It was observed that the higher expression level of PfMSP-1(19) was essentially associated with the generation of higher level of incorrectly folded PfMSP-1(19). A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1(19) from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1(19) suggested that it was highly pure, homogeneous, and correctly folded.

Substrate utilization and feeding strategies of mammals: description and classification

Describing species' ecological strategies enables us to condense ecological information and to express it in evolutionary terms. However, the process of categorizing species is hampered by methodological difficulties and insufficient development of the typology and nomenclature of different strategies. In this article an approach for overcoming these difficulties is proposed. For a precise description of mammalian substrate utilization, it is better to combine two characteristics rather than use only one. The categorization should reflect: (1) the media or substrates primarily used for foraging; and (2) the media or substrates primarily used for sleeping. The numerous substrate utilization strategies of mammals fall into five broad groups: (1) aquatic and semiaquatic; (2) subterranean; (3) terrestrial and subterranean-terrestrial; (4) arboreal and semiarboreal; and (5) aerial and semiaerial. Three main mammalian feeding strategies are proposed: animalivorous, frugivorous, and herbivorous. An example of ecological classification of mammals in terms of substrate utilization and feeding strategy is provided.

Optimization of pH and acetic acid concentration for bioconversion of hemicellulose from corncobs to xylitol by Candida tropicalis

Abstract Hemicellulose hydrolysate from corncobs, separated by diluted sulfuric acid and sequently detoxed by boiling, overliming and solvent extraction, was used for xylitol production by Candida tropicalis W103. The effect of glucose and acetate in hydrolysate on xylitol production was investigated. It was found that glucose in hydrolysate promoted growth of Candida tropicalis while acetate at high concentration was inhibitory. The acetate inhibition can be alleviated by adjusting pH to 6 prior to fermentation and a substrate feeding strategy. Under these optimum conditions, a maximal xylitol concentration of 68.4 g l −1 was obtained after 72 h of fermentation, giving a yield of 0.7 g g −1 xylose and a productivity of 0.95 g l −1 h −1 .

Amidase-catalyzed production of nicotinic acid in batch and continuous stirred membrane reactors

The aim of the present study is to explore the potential use of Microbacterium imperiale CBS 498–74 resting cells as a catalyst for the bioconversion of nicotinamide into nicotinic acid. This strain converts nitrile into the corresponding acid following a two-step reaction catalysed by nitrile hydratase and amidase via an amide as intermediate. The effect of temperature, cell load and substrate feeding strategy were investigated with controlled continuous stirred membrane bioreactors (CSMR) in an attempt to improve reaction conversion as well as reactor performances. The temperature dependence of amidase activity was investigated in both batch reactors and CSMR. An activation energy of about 52.6/53.5 kJ mol −1 was determined, indicating absence of mass-transport phenomena. Amidase operated under mild conditions suitable for the synthesis of labile organic molecules and it was stable up to 50 °C. Nicotinamide (substrate) at concentrations of ≥300 mM partially inhibited the enzyme. In the batch reactor, total conversion was achieved. In CSMR, the residence time was optimized to attain high conversion (up to 88%). These data indicate the potentiality of the continuous bioprocess for industrial application.

Ethanol production from sweet sorghum juice in batch and fed-batch fermentations by Saccharomyces cerevisiae

Sweet sorghum juice supplemented with 0.5% ammonium sulphate was used as a substrate for ethanol production by Saccharomyces cerevisiae TISTR 5048. In batch fermentation, kinetic parameters for ethanol production depended on initial cell and sugar concentrations. The optimum initial cell and sugar concentrations in the batch fermentation were 1 × 108 cells ml−1 and 24 °Bx respectively. At these conditions, ethanol concentration produced (P), yield (Yps) and productivity (Qp) were 100 g l−1, 0.42 g g−1 and 1.67 g l−1 h−1 respectively. In fed-batch fermentation, the optimum substrate feeding strategy for ethanol production at the initial sugar concentration of 24 °Bx was one-time substrate feeding, where P, Yps and Qp were 120 g l−1, 0.48 g g−1 and 1.11 g l−1 h−1 respectively. These findings suggest that fed-batch fermentation improves the efficiency of ethanol production in terms of ethanol concentration and product yield.

Automated feeding strategies for high-cell-density fed-batch cultivation of Pseudomonas putida KT2440

Four automatic substrate feeding strategies were developed and investigated in this study to obtain rapid, repeatable, and reliable high cell densities of Pseudomonas putida KT2440 from glucose. Growth yield data of the key nutrients, Y(X/Glucose), Y(X/NH4), Y(X/PO4), Y(X/Mg), and Y(CO2/Glucose), were determined to be 0.41, 5.44, 13.70, 236, and 0.65 g g(-1), respectively. Although standard exponential feeding strategy worked well when the predetermined mu was set at 0.25 h(-1), an exponential glucose feeding strategy with online mu(max) estimation resulted in a higher average biomass productivity (3.4 vs 2.8 g l(-1) h(-1)). A CO2 production rate based pulse glucose feeding strategy also resulted in good overall productivity (3.0 g l(-1) h(-1)) and can be used as an alternative to pH-stat or DO-stat feeding. A cumulative CO2 production based continuous feed with real-time cumulative glucose consumption estimation resulted in much higher biomass productivity (4.3 g l(-1) h(-1)) and appears to be an excellent and reliable approach to fully automating high-cell-density fed-batch cultivation of P. putida.

Optimization of cell growth and poly(ɛ-lysine) production in batch and fed-batch cultures by Streptomyces albulus IFO 14147

Streptomyces albulus IFO 14147 was cultivated in a stirred tank reactor; the pH and nutrient effects were evaluated in batch and fed-batch fermentation, particularly the control of pH and glucose feeding for enhancing the cell growth and ɛ-PL production were demonstrated. It was concluded that high cell growth was obtained and the cells remained stable at pH above 4.5 in the broth, while high ɛ-PL accumulation was achieved at lower pH; the high rate of cell growth at high pH was accompanied by a high rate of glucose consumption. To enhance the ɛ-PL production, a two-stage pH control and glucose feeding strategy was employed. At first sage the pH was maintained at its initial 6.8 for 48 h for achieving high cell density; the second stage aimed at increasing the ɛ-PL accumulation by maintaining pH at 4.0. In addition, pulsed feeding of glucose was carried out when it was depleted from the culture medium. By using this control and feeding strategy, the increase of ɛ-PL accumulation in 10-days cultivation is 258%. We have demonstrated that pH control and substrate feeding strategy is a feasible method for enhancing ɛ-PL production by S. albulus IFO 14147.

Efficient bioreduction of ethyl 4-chloro-3-oxobutanoate to (S)-4-chloro-3-hydrobutanoate by whole cells ofCandida magnoliae in water/n-butyl acetate two-phase system

The asymmetric biosynthesis of ethyl (S)-4-chloro-3-hydrobutanoate from ethyl 4-chloro-3-oxobutanoate was investigated by using whole cells ofCandida magnoliae JX120-3 without the addition of glucose dehydrogenase or NADP+/NADPH. In a one-phase system, the bioconversion yield was seriously affected on the addition of 12.1 g/L ethyl 4-chloro-3-oxobutanoate. In order to reduce this substrate inhibition, a water/n-butyl acetate two-phase system was developed, and the bioreduction conditions optimized with regard to the yield and product enantiometric excess value. The optimal conditions were as following: water ton-butyl acetate volume ratio of 1∶1, 4.0 g DCW/L active cells, 50 g/L glucose and 35°C. By adopting a dropwise substrate feeding strategy, high concentration of ethyl 4-chloro-3-oxobutanoate (60 g/L) could be asymmetrically reduced to ethyl (S)-4-chloro-3-hydrobutanoate with high yield (93.8%) and high enantiometric excess value (92.7%).

A history of research on yeasts 9: regulation of sugar metabolism1

A history of research on yeasts 9: regulation of sugar metabolism1

Polymer Development for Enhanced Delivery of Phenol in a Solid-Liquid Two-Phase Partitioning Bioreactor

Two-Phase Partitioning Bioreactors (TPPBs) have traditionally been used to partition toxic concentrations of xenobiotics from a cell-containing aqueous phase by means of an immiscible organic solvent and to deliver these substrates back to the cells on the basis of metabolic demand and the maintenance of thermodynamic equilibrium between the phases. A limitation of TPPBs, which use organic liquid solvents, is the possibility that the solvent can be bioavailable, and this has therefore limited organic liquid TPPBs to the use of pure strains of microbes. Solid polymer beads have recently been introduced as a replacement for liquid organic solvents, offering similar absorption properties but with the capability to be used with mixed microbial populations. The present work was aimed at identifying a polymer with a greater capacity for and more rapid uptake and release of phenol for use as the second phase in a mixed culture TPPB. Polarity and hydrogen bonding capabilities between polymer and phenol were considered in the screening and selection process of candidate polymers. Hytrel (a copolymer of poly(butylene terephthalate) and butylene ether glycol terephthalate) polymer beads, offered improved capacity (19 mg phenol/g polymer at a fixed initial phenol concentration of 2000 mg/L) and a greater diffusivity (1.54 x 10(-7) cm2/s) when compared to the capacity and diffusivity of previously used EVA (ethylene vinyl acetate) beads (12.4 mg phenol/g polymer and 3.73 x 10(-9) cm2/s, respectively). Hytrel polymer beads were then used in a TPPB for the investigation of various substrate feeding strategies (fed-batch, bead replacement, and concentrated spikes of phenol), with rapid and complete phenol degradation shown in all cases.

Fed-batch and batch operating mode analysis of a stirred anaerobic sequencing reactor with self-immobilized biomass treating low-strength wastewater

This work presents an analysis of a stirred anaerobic sequencing discontinuous reactor with different substrate feeding strategies resulting in batch, fed-batch/batch and fed-batch operating modes. The reactor, containing granulated biomass, was fed with approximately 2.0 L of synthetic domestic wastewater with Chemical Oxygen Demand of nearly 500 mg/L per cycle and operated at 30 °C and 50 rpm. Three feeding strategies with a total cycle time of 6 h, including 30-min settling, were adopted: batch mode with a fill cycle of 6 min, a fed-batch/batch mode with fill cycles of 60, 120 and 240 min and fed-batch mode with a fill cycle of 320 min. The system attained average non-filtered and filtered substrate removal efficiency of 78 and 84%, respectively, for all operating conditions, presenting good stability, solid retention and no granule break-up. A first order kinetic model with a residual organic matter concentration was proposed to analyze the influence of the feeding strategy on the performance during a cycle and bicarbonate alkalinity and total volatile acids concentration profiles were also quantified in order to verify the transient stability behavior.

Computer coupled substrate feeding strategies for efficient conversion of d-sorbitol to l-sorbose in fed-batch culture

l-sorbose fermentation is of industrial importance since it is an intermediate step in the industrial production of vitamin C. It involves the biological oxidation of d-sorbitol to l-sorbose using Acetobacter suboxydans. Batch sorbose fermentation features substrate inhibition and hence it is impossible to obtain high sorbose productivity in batch bioreactors. Fed-batch fermentations can eliminate substrate inhibition and improve sorbose productivity. Fed-batch sorbose fermentations were conducted using two different nutrient feeding strategies. Fed-batch fermentation conducted by feeding nutrients containing 600 g l −1 of sorbitol at a constant feed rate of 0.6 l h −1 yielded a sorbose productivity of 12.64 g l −1 h −1. While fed-batch fermentation conducted by feeding nutrients containing 600 g l −1 of sorbitol at a linearly decreasing feed rate (0.5–0.04 l h −1) demonstrated a productivity of 17.01 g l −1 h −1. The productivity obtained by feeding nutrients at a linearly decreasing feed rate was higher than that obtained by the constant fed-batch fermentation and, therefore, is a better substrate feeding strategy for improving sorbose productivity.

Fermentation strategies for recombinant protein expression in the methylotrophic yeastPichia pastoris

Fermentation strategies for recombinant protein production inPichia pastoris have been investigated and are reviewed here. Characteristics of the expression system, such as phenotypes and carbon utilization, are summarized. Recently reported results such as growth model establishment, application of a methanol sensor, optimization of substrate feeding strategy, DOstat controller design, mixed feed technology, and perfusion and continuous culture are discussed in detail.

High-cell-density cultivation of yeasts on disaccharides in oxygen-limited batch cultures

Many facultatively fermentative yeast species exhibit a "Kluyver effect": even under oxygen-limited growth conditions, certain disaccharides that support aerobic, respiratory growth are not fermented, even though the component monosaccharides are good fermentation substrates. This article investigates the applicability of this phenomenon for high-cell-density cultivation of yeasts. In glucose-grown batch cultures of Candida utilis CBS 621, the onset of oxygen limitation led to alcoholic fermentation and, consequently, a decrease of the biomass yield on sugar. In maltose-grown cultures, alcoholic fermentation did not occur and oxygen-limited growth resulted in high biomass concentrations (90 g dry weight L(-1) from 200 g L(-1) maltose monohydrate in a simple batch fermentation). It was subsequently investigated whether this principle could also be applied to Kluyveromyces species exhibiting a Kluyver effect for lactose. In oxygen-limited, glucose-grown chemostat cultures of K. wickerhamii CBS 2745, high ethanol concentrations and low biomass yields were observed. Conversely, ethanol was absent and biomass yields on sugar were high in oxygen-limited chemostat cultures grown on lactose. Batch cultures of K. wickerhamii grown on lactose exhibited the same growth characteristics as the maltose-grown C. utilis cultures: absence of ethanol formation and high biomass yields. Within the species K. marxianus, the occurrence of a Kluyver effect for lactose is known to be strain dependent. Thus, K. marxianus CBS 7894 could be grown to high biomass densities in lactose-grown batch cultures, whereas strain CBS 5795 produced ethanol after the onset of oxygen limitation and, consequently, yielded low amounts of biomass. Because the use of yeast strains exhibiting a Kluyver effect obviates the need for controlled substrate-feeding strategies to avoid oxygen limitation, such strains should be excellently suited for the production of biomass and growth-related products from low-cost disaccharide-containing feedstocks. (c) 1996 John Wiley & Sons, Inc.

Bioreactor operating strategy inThalictrum rugosum plant cell culture for the production of berberine

Optimal substrate feeding strategy in bioreactor operation was investigated to increase the production of secondary metabolite in a high density culture of plant cell. It was accomplished by the previously proposed structured kinetic model that describes the cell growth and synthesis of the secondary metabolite, berberine, in a batch suspension culture ofThalictrum rugosum. Four types of operation strategies for sugar feeding intoT. rugosum culture were proposed based on the model, which were the periodic fedbatch operations to maintain the cell activity, the cell viability, and the specific production rate, and the perfusion operation to maintain the specific production rate. From the simulation results of these strategies, it could be found that the periodic fed-batch operation and the perfusion operation could achieve the higher volumetric production of berberine (mg berberine/L) and specific production yield (mg berberine/g dry cell weight) than those of batch cultures. Although the highest productivity (mg berberine/day) of berberine could be achieved by the periodic fed-batch operation to maintain the cell activity compared with the other strategies in the periodic fed-batch operations, the specific production yield was low due to the higher maximum dry cell weight than other cases. The periodic fed-batch operation to maintain cell viability resulted in the highest volumetric production of berberine and specific production yield compared with the other strategies. In the cases of maintaining the specific production rate, the per-formance of the periodic fed-batch operation was better than that of the perfusion operation in the respect of the volumetric production and productivity of berberine. In order to increase the volumetric production of berberine and to get the highest specific production yield, the periodic fed-batch operation to maintain cell viability could be chosen as the optimal operating strategy in high density, culture ofT. rugosum plant cell.

Optimization of feeding profile for baker's yeast production by dynamic programming

An optimal substrate feeding strategy for an industrial scale fed-batch fermenter is determined through iterative dynamic programming in order to maximize cell-mass production and to minimize ethanol formation. The objective function is maximized with respect to the sugar feed rate. The obtained optimal policy indicates considerable increase in yeast production achieved while minimizing the undesired by-product-ethanol.

Application of genetic algorithm to self-organizing fuzzy controller in fed-batch culture ofScutellaria baicalensis G. plant cell

A self-organizing fuzzy controller is constructed for control of substrate concentration in fed-batch operation of a cell culture process. A genetic algorithm is used to generate fuzzy rules of the self-organizing fuzzy controller and to modify the universe of discourse automatically. The fuzzy controller is designed for the application ofScutellaria baicalensis G. plant cell culture process as a model system. A substrate feeding strategy of a two stage culture method to maximize flavone glycoside production in fed-batch culture ofScutellaria baicalensis G. plant cell is proposed based on structured model of growth and product synthesis. As a two stage culture, the feeding strategy consists of the first period at 22 g/L of glucose concentration to promote cell growth and the second period at 10 g/L of glucose concentration to promote flavone glycoside synthesis. The designed self-organizing fuzzy controller is applied to regulate the glucose concentration at a given set-point to increase flavone glycoside synthesis. The simulation results show that the proposed feeding strategy in a fed-batch culture enhances flavone glycoside production and the self-organizing fuzzy logic controller generated by genetic algorithm improves controller performance.