Growth In Batch Culture Research Articles

Estimating Toxic Effect of Copper on Population of Microalgae Through a Three-dimensional Toxic Effect Growth Model

A newly proposed three-dimensional model for the effects of heavy metals on the growth of batch cultures of algae that allows the estimation of the no detected toxic effect concentration (NDEC) is presented. Two batch assays with exposure to copper were investigated in situ (ship-based enclosure). As an endpoint in these studies, the carrying capacity B(f), a parameter of the logistic growth model, possesses higher sensitivity and reliability than routine ecotoxicological endpoints. Using B(f) as the endpoint, the NDEC from the proposed model is compared to the no observed effect concentration (NOEC), Lowest Observed Effect Concentration (LOEC), the 5% effect concentration (EC₀₅) and no effect concentration (NEC) also calculated from field-derived data. The results show the confidence interval for the NDEC was wholly contained within the corresponding interval between the NOEC and LOEC, as well as within the corresponding much wider confidence interval for EC₀₅. Though the width of the confidence interval for NEC was basically equivalent to the corresponding width for NDEC, the NEC was somewhat higher than the corresponding NDEC. The results indicate that the NDEC is a promising possible alternative parameter to the NOEC.

Microbial lipid production by the oleaginous yeast Cryptococcus curvatus O3 grown in fed-batch culture

This study investigates the capability of the oleaginous yeast Cryptococcus curvatus O3 to synthesize microbial lipids using glucose as its sole carbon source. Both glucose concentration and varying nitrogen sources have a significant effect on cell growth and microbial lipid accumulation in batch and fed-batch cultures. When cultivated in a shaking flask at 30 °C with glucose as sole carbon source, the cellular biomass and lipid content reached 51.8 kg m −3 and 651 g kg −1 , respectively. The fed-batch culture in a 30 × 10 −3 m 3 stirred-tank fermentor run for 185 h produced a cellular biomass, lipid content, and lipid productivity rate of up to 104.1 kg m −3 , 827 g kg −1 , and 0.47 kg m −3 h −1 , respectively. These data indicate that C . curvatus O3 can be used as an ideal oleaginous yeast for microbial lipid production. Gas chromatography analysis of the synthesized microbial lipids revealed that the major constituents are long-chain fatty acids, such as palmitic acid, stearic acid, oleic acid, and linoleic acid. The results suggest that the microbial lipids produced by C. curvatus O3 can be used to produce biodiesel. ► In this manuscript, we use the fed-batch ferementation method to improve the lipid production. ► In a 30 × 10 −3 m 3 stirred-tank fermentor run for 185 h produced a cellular biomass, lipid content, and lipid productivity rate of up to 104.1 kg m −3 , 827 g kg −1 , and 0.47 kg m −3 h −1 , respectively. ► These data indicate that C. curvatus O3 can be used as an ideal oleaginous yeast for microbial lipid production.

Fluorescence in situ hybridization for detection of classical propionibacteria with specific 16S rRNA-targeted probes and its application to enumeration in Gruyère cheese

The classical or dairy propionibacteria have well-documented industrial applications and have been proposed for probiotic applications. Given their industrial importance it is necessary to employ fast and reliable techniques to monitor the growth during products elaboration, industrial fermentations or the intestinal transit. Therefore, the aim of this investigation was to design oligonucleotide probes targeting the 16S rRNA of dairy propionibacteria and optimise the fluorescence in situ hybridization (FISH) protocol to detect these bacteria. Two specific probes were in silico designed to detect Propionibacterium freudenreichii and P. jensenii, named Pfr435 and Pj446 respectively. The FISH protocol was optimised for the hybridisation of propionibacteria cells with the universal probe Eub338 and the designed probes . These probes were assayed in situ for their specificity to hybridise species of propionibacteria by observation using fluorescence microscopy and results were compared with the probe Pap446 previously designed for P. acidipropionici. Probes Pap446, Pfr435 and Pj446 were also evaluated by fluorescence spectrophotometry to assess the influence of cells physiological state during growth in batch culture in the fluorescence intensity. The maximum fluorescence intensity was observed at the onset of the stationary phase of growth and was then reduced. However, changes on the cells permeability did not reduce the efficiency of 16S rRNA hybridisation with the fluorescence-labelled probes. Propionibacteria counts obtained by FISH and plate count methods were compared in a commercial Gruyère cheese. The results showed that this method can be used as a rapid technique for the enumeration of these bacteria in cheese samples.

No detected toxic concentrations in in situ algal growth inhibition tests—A convenient approach to aquatic ecotoxicology

A newly proposed three-dimensional model for the effects of heavy metals on the growth of batch cultures of algae that allows the estimation of the no detected toxic effect concentration (NDEC) is presented. Two batch assays with exposure to copper were investigated in situ. As an endpoint in the in situ studies of ecotoxicology, the carrying capacity, a parameter of the logistic growth model, possesses higher sensitivity and reliability than the routine ecotoxicological endpoints in terms of the analysis based on the theoretical arguments and experimental results. Using the carrying capacity (Bf) as the ecotoxicological endpoint, the NDEC from the proposed model is compared to the NOEC and EC05 on the basis of field derived data. The results indicate that the NDEC is a promising possible alternative parameter to the NOEC.

Fusarium oxysporum degradation and detoxification of a new textile-glycoconjugate azo dye (GAD)

Degradation and detoxification of textile dyes are of interest due to the huge environmental impact of such chemicals. An isolate of Fusarium oxysporum was used to degrade and to detoxify a new chemical class of textile dyes called Glycoconjugate Azo Dye (GAD). After 6d of growth in a liquid batch culture, the fungus degraded the dye and the culture medium at the end of incubation period showed a ˜100% detoxification compared to the initial dye solution. Increasing the initial fungal inoculum, the dye was totally decolourized after 24h of incubation. The degradation ability was found to be common among various isolates of F. oxysporum suggesting this as a specific trait of this species. Degrading rate was enhanced in concomitancy to the glucose depletion and the beginning of the stationary phase of growth, suggesting that the shift from the primary to the secondary metabolism may be the trigger of the degradation pathway. The Daphnia magna acute toxicity test demonstrated a strong detoxification of GAD-4 by F. oxysporum, resulting in non-toxic metabolite production. Fusarium oxysporum could, therefore, be taken into consideration to develop new remediation strategies of textile effluents.

CK2 activity is modulated by growth rate in Saccharomyces cerevisiae

CK2 is a highly conserved protein kinase controlling different cellular processes. It shows a higher activity in proliferating mammalian cells, in various types of cancer cell lines and tumors. The findings presented herein provide the first evidence of an in vivo modulation of CK2 activity, dependent on growth rate, in Saccharomyces cerevisiae. In fact, CK2 activity, assayed on nuclear extracts, is shown to increase in exponential growing batch cultures at faster growth rate, while localization of catalytic and regulatory subunits is not nutritionally modulated. Differences in intracellular CK2 activity of glucose- and ethanol-grown cells appear to depend on both increase in molecule number and kcat. Also in chemostat cultures nuclear CK2 activity is higher in faster growing cells providing the first unequivocal demonstration that growth rate itself can affect CK2 activity in a eukaryotic organism.

Morphology, phylogeny and toxin analysis of Pseudo-nitzschia pseudodelicatissima (Bacillariophyceae) isolated from the Thermaikos Gulf, Greece

Moschandreou K.K., Papaefthimiou D., Katikou P., Kalopesa E., Panou A. and Nikolaidis G. 2010. Morphology, phylogeny and toxin analysis of Pseudo-nitschia pseudodelicatissima (Bacillariophyceae) isolated from the Thermaikos Gulf, Greece. Phycologia 49: 260–273. DOI: 10.2216/09-42.1Pseudo-nitzschia pseudodelicatissima is a diatom species that has been associated with the production of the neurotoxin domoic acid (DA), causative agent of amnesic shellfish poisoning. However, the taxonomic revision of this species complex resulted in uncertain identity and, subsequently, toxicity of the strains tentatively identified under light microscope as P. pseudodelicatissima. Six strains isolated from the Thermaikos Gulf during the period from August to October 2008 were cultured and tested for production of DA. All strains were studied in morphological detail using electron microscopy and were identified as P. pseudodelicatissima. The identification was supported by phylogenetic analyses of the large subunit (LSU) and internal transcribed spacer (ITS) ribosomal rDNA regions, along with the secondary structure of ITS-2 rDNA region. DA was detected in all cultures and in three of the strains in quantifiable amounts up to 3.08 ng ml−1. Two of the strains were studied over the course of a batch culture growth experiment and DA was detected from the mid-exponential until late-stationary phases; the maximum whole-culture DA levels were measured in late-exponential to early-stationary phases. This study confirms the production of DA by P. pseudodelicatissima in culture.

Heterogeneity of Escherichia coli population by respiratory activity and membrane potential of cells during growth and long-term starvation

Assessment of physiological states of individual bacterial cells can be useful in the monitoring of the biotechnological processes. Physiological heterogeneity of Escherichia coli population by respiration activity and membrane potential during growth and starvation in batch cultures was evaluated using 5-cyano-2,3-ditolyl tetrazolium chloride (CTC) and bis-(1,3-dibutylbarbituric acid) trimethine oxo (DiBAC₄)(3) fluorescent probes in combination with flow cytometry. The shares of CTC-reducing cells (CTC(+)-cells) and cells with positively charged outside cytoplasmic membrane which were not stained by DiBAC₄(3) (ΔΨ(+)-cells), were 90% and 95% in the exponential phase of batch culture, respectively. After short-term starvation for 10 h, the shares of CTC(+)-cells and ΔΨ(+)-cells in the samples taken from the exponential phase dropped to 78% and 72%, respectively. After long-term starvation for 40 days, the share of CTC(+)-cells dropped to 5%, whereas the share of ΔΨ(+)-cells was about 50%. The conclusions from this research are as follows: (a) the physiological heterogeneity of bacterial population increased after starvation; (b) the cell respiratory activity is more sensitive to starvation than the cell membrane potential; (c) a probe for the cell membrane potential DiBAC₄(3) is more suitable than a probe for the cell respiratory activity CTC in the detection of viable indicator bacteria in environment.

Kinetic oxygen isotope effects during dissimilatory sulfate reduction: A combined theoretical and experimental approach

Kinetic isotope effects related to the breaking of chemical bonds drive sulfur isotope fractionation during dissimilatory sulfate reduction (DSR), whereas oxygen isotope fractionation during DSR is dominated by exchange between intercellular sulfur intermediates and water. We use a simplified biochemical model for DSR to explore how a kinetic oxygen isotope effect may be expressed. We then explore these relationships in light of evolving sulfur and oxygen isotope compositions (δ 34 S SO4 and δ 18 O SO4 ) during batch culture growth of twelve strains of sulfate-reducing bacteria. Cultured under conditions to optimize growth and with identical δ 18 O H2O and initial δ 18 O SO4 , all strains show 34 S enrichment, whereas only six strains show significant 18 O enrichment. The remaining six show no (or minimal) change in δ 18 O SO4 over the growth of the bacteria. We use these experimental and theoretical results to address three questions: (i) which sulfur intermediates exchange oxygen isotopes with water, (ii) what is the kinetic oxygen isotope effect related to the reduction of adenosine phosphosulfate (APS) to sulfite (SO 3 2− ), (iii) does a kinetic oxygen isotope effect impact the apparent oxygen isotope equilibrium values? We conclude that oxygen isotope exchange between water and a sulfur intermediate likely occurs downstream of APS and that our data constrain the kinetic oxygen isotope fractionation for the reduction of APS to sulfite to be smaller than 4‰. This small oxygen isotope effect impacts the apparent oxygen isotope equilibrium as controlled by the extent to which APS reduction is rate-limiting.

English

More than 80% of the plant diseases are caused by fungi. Usually, fungi not only destroy the plants, but also produce mycotoxins that are harmful to human health. At present, chemical fungicides are mainly used for the prevention of fungi-related plant diseases, however, research and development of biological prevention and controlling are of great importance. In this work, the effects of pH and temperature on cell growth and CF66I formation in batch culture of Burkholderia cepecia CF-66 were studied. The pH value has a marked effect on cell growth and production of CF66I. The lag phase was much longer when pH set lower (e.g.5.0) or higher (e.g.8.0). For earlier phase, optimal pH value was 6.0, because the lag phase can be shortened and the whole fermentation phase can also be shorten and then quickly goes into CF66I production phase. In the late phase, the higher pH is in favor of the production of CF66I. Different temperature have different effect on cell yield, specific growth rate, CF66I yield and specific synthesis rate. In the prophase of fermentation, it is better to set higher temperature to make the cell growth maximizing as soon as possible. However in mid-anaphase, lower temperature shortens the fermentation time, reduce heating energy and the cost. According about results, an optimal control strategy was constructed.

Dynamics of energy charge and adenine nucleotides during uncoupling of catabolism and anabolism in Penicillium ochrochloron

Filamentous fungi are able to spill energy when exposed to energy excess by uncoupling catabolism from anabolism, e.g. via overflow metabolism. In current study we tested the hypothesis that overflow metabolism is regulated via the energetic status of the hyphae (i.e. energy charge, ATP concentration). This hypothesis was studied in Penicillium ochrochloron during the steady state of glucose- or ammonium-limited chemostat cultures as well as during three transient states ((i) glucose pulse to a glucose-limited chemostat, (ii) shift from glucose-limited to ammonium-limited conditions in a chemostat, and (iii) ammonium exhaustion in batch culture). Organic acids were excreted under all conditions, even during exponential growth in batch culture as well as under glucose-limited conditions in a chemostat. Partial uncoupling of catabolism and anabolism via overflow metabolism was thus constitutively present. Under all tested conditions, overflow metabolism was independent of the energy charge or the ATP concentration of the hyphae. There was a reciprocal correlation between glucose uptake rate and intracellular adenine nucleotide content. During all transients states a rapid decrease in energy charge and the concentrations of nucleotides was observed shortly after a change in glycolytic flux (“ATP paradoxon”). A possible connection between the change in adenine nucleotide concentrations and the purine salvage pathway is discussed.

Growth phase-dependent variations in transcript profiles for thioredoxin- and glutathione-dependent redox systems followed by budding and hyphalCandida albicans cultures

We report the absolute transcription profiles of 24 genes coding for putative thioredoxin (Trx)- and glutathione (GSH)-dependent redox system components, accompanying the Candida albicans batch culture growth, under either yeast or hyphal conditions. All mRNAs investigated (plus the housekeeping ACT1) displayed significant alterations in their steady-state copy number. Collectively, these quantifications show that: (1) the most abundant mRNAs during active growth coded for putative thiol peroxidases (TSA1, PRX1, AHP11 and AHP12) and for donor thioredoxin Trx1p, i.e. those five mRNAs represented >74% of all transcript molecules quantified in a late exponential phase; (2) the transcripts under study differed between budding and hyphal cells not only in their abundance but also in their profiles throughout the growth stages; (3) mRNA amounts for four GSH-transferase putative genes (GTT12, GTT13, GTT14 and GST3) increased in the stationary phase in yeast but not under filamentous conditions. Hydrogen peroxide resistance, plus GSH, GSSG and reactive oxygen species contents, throughout yeast and hyphal growth, were also studied, and the differences observed were related to the transcript profiles.

Equalizing growth in high‐throughput small scale cultivations via precultures operated in fed‐batch mode

An often underestimated problem when working with different clones in microtiter plates and shake flask screenings is the non-parallel and non-equal growth of batch cultures. These growth differences are caused by variances of individual clones regarding initial biomass concentration, lag-phase or specific growth rate. Problems arising from unequal growth kinetics are different induction points in expression studies or uneven cultivation periods at the time of harvest. Screening for the best producing clones of a library under comparable conditions is thus often impractical or even impossible. A new approach to circumvent the problem of unequal growth kinetics of main cultures is the application of fed-batch mode in precultures in microtiter plates and shake flasks. Fed-batch operation in precultures is realized through a slow-release system for glucose. After differently growing cultures turn to glucose-limited growth, they all consume the same amount of glucose due to the fixed feed profile of glucose provided by the slow-release system. This leads to equalized growth. Inherent advantages of this method are that it is easy to use and requires no additional equipment like pumps. This new technique for growth equalization in high-throughput cultivations is simulated and verified experimentally. The growth of distinctly inoculated precultures in microtiter plates and shake flasks could be equalized for different microorganisms such as Escherichia coli and Hansenula polymorpha.

Growth and photoregulation dynamics of the picoeukaryote Pelagomonas calceolata in fluctuating light

Growth, photosynthesis, and photoacclimation properties of batch cultures of Pelagomonas calceolata (Pelagophyceae) were compared for 1 week under three different fluctuating light regimes with the same total daily amount of light. Treatments consisted of a sinusoidal diurnal light cycle or a high‐frequency fluctuating light simulating two different regimes of vertical mixing (highly fluctuating light [HFL] or fluctuating light [FL]). Three to five samples were taken every day for analysis of pigments, absorption spectrum, variable fluorescence, nonphotochemical quenching (NPQ), electron transport rate vs. light curves, and cell concentration. Pelagomonas achieved the same growth rate during the exponential growth phase under all three light conditions, revealing a high degree of acclimation to light and also suggesting that the daily light dose is the main factor regulating growth and division. Photophysiological adjustments occurred in the cells in response to the three light regimes. Pelagomonas seems to adopt the n‐type photoacclimation in HFL, whereas the σ;‐type photoacclimation is applied in FL. The cells rapidly trigger photoprotective mechanisms such as the xanthophyll cycle and NPQ, even though these do not appear to be able to fully prevent photoinhibition. The enhanced costs for maintenance and repair associated with HFL may have limited the allocation of energy to growth, thus explaining the shorter duration of the exponential growth phase in this regime with respect to the two others.

An improved model for multistage simulation of glycerol fermentation in batch culture and its parameter identification

The bioconversion of glycerol to 1,3-propanediol is a complex bioprocess. In this study, we aim to explore a novel model for describing the multistage cell growth in batch culture. This is achieved by a modification at the specific rate of cell growth in consideration of its time-dependent changes. The existence, uniqueness and boundedness of solutions to the system and the continuity of solutions with respect to the parameters are discussed. In addition, a parameter identification problem of the system is developed and a feasible optimization algorithm is constructed to solve it. Numerical result shows that the improved model could describe the batch culture well.

Inexpensive fed-batch cultivation for high poly(3-hydroxybutyrate) production by a new isolate of Bacillus megaterium

This research aimed at increasing the cell density and production of the homopolymer polyhydroxybutyrate (PHB) by Bacillus megaterium BA-019, using renewable and inexpensive bioresources as a substrate. A higher cell density and a greater PHB production level were obtained by using sugarcane molasses and urea as carbon and nitrogen sources, respectively. The limitation of nitrogen at a C/N molar ratio of 25 resulted in enhanced cell growth and PHB production in batch cultures. Fed-batch cultivation with the feeding nutrient composed of MSM with sugarcane molasses, urea and trace elements, and controlled by a pH-stat feeding control, lead to a significantly enhanced cell concentration and PHB production. The optimal feeding medium in this system required a higher total sugar concentration (400 g/l) and a C/N molar ratio of 10 mol/mol. Under these conditions the highest attained cell mass (72.6 g/l DW) and PHB content (42% of cell dry wt.) were achieved in a short cultivation time (24 h), leading to improved PHB productivity (1.27 g/l/h). However, dissolved oxygen was limiting and thus the system is likely to be suboptimal and capable of even further improvements to the PHB production rate.

Characterization of a Mesorhizobium loti α-Type Carbonic Anhydrase and Its Role in Symbiotic Nitrogen Fixation

Carbonic anhydrase (CA) (EC 4.2.1.1) is a widespread enzyme catalyzing the reversible hydration of CO(2) to bicarbonate, a reaction that participates in many biochemical and physiological processes. Mesorhizobium loti, the microsymbiont of the model legume Lotus japonicus, possesses on the symbiosis island a gene (msi040) encoding an alpha-type CA homologue, annotated as CAA1. In the present work, the CAA1 open reading frame from M. loti strain R7A was cloned, expressed, and biochemically characterized, and it was proven to be an active alpha-CA. The biochemical and physiological roles of the CAA1 gene in free-living and symbiotic rhizobia were examined by using an M. loti R7A disruption mutant strain. Our analysis revealed that CAA1 is expressed in both nitrogen-fixing bacteroids and free-living bacteria during growth in batch cultures, where gene expression was induced by increased medium pH. L. japonicus plants inoculated with the CAA1 mutant strain showed no differences in top-plant traits and nutritional status but consistently formed a higher number of nodules exhibiting higher fresh weight, N content, nitrogenase activity, and delta(13)C abundance. Based on these results, we propose that although CAA1 is not essential for nodule development and symbiotic nitrogen fixation, it may participate in an auxiliary mechanism that buffers the bacteroid periplasm, creating an environment favorable for NH(3) protonation, thus facilitating its diffusion and transport to the plant. In addition, changes in the nodule delta(13)C abundance suggest the recycling of at least part of the HCO(3)(-) produced by CAA1.

Comparing the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways in arabinose and xylose fermenting Saccharomyces cerevisiae strains

BackgroundEthanolic fermentation of lignocellulosic biomass is a sustainable option for the production of bioethanol. This process would greatly benefit from recombinant Saccharomyces cerevisiae strains also able to ferment, besides the hexose sugar fraction, the pentose sugars, arabinose and xylose. Different pathways can be introduced in S. cerevisiae to provide arabinose and xylose utilisation. In this study, the bacterial arabinose isomerase pathway was combined with two different xylose utilisation pathways: the xylose reductase/xylitol dehydrogenase and xylose isomerase pathways, respectively, in genetically identical strains. The strains were compared with respect to aerobic growth in arabinose and xylose batch culture and in anaerobic batch fermentation of a mixture of glucose, arabinose and xylose.ResultsThe specific aerobic arabinose growth rate was identical, 0.03 h-1, for the xylose reductase/xylitol dehydrogenase and xylose isomerase strain. The xylose reductase/xylitol dehydrogenase strain displayed higher aerobic growth rate on xylose, 0.14 h-1, and higher specific xylose consumption rate in anaerobic batch fermentation, 0.09 g (g cells)-1 h-1 than the xylose isomerase strain, which only reached 0.03 h-1 and 0.02 g (g cells)-1h-1, respectively. Whereas the xylose reductase/xylitol dehydrogenase strain produced higher ethanol yield on total sugars, 0.23 g g-1 compared with 0.18 g g-1 for the xylose isomerase strain, the xylose isomerase strain achieved higher ethanol yield on consumed sugars, 0.41 g g-1 compared with 0.32 g g-1 for the xylose reductase/xylitol dehydrogenase strain. Anaerobic fermentation of a mixture of glucose, arabinose and xylose resulted in higher final ethanol concentration, 14.7 g l-1 for the xylose reductase/xylitol dehydrogenase strain compared with 11.8 g l-1 for the xylose isomerase strain, and in higher specific ethanol productivity, 0.024 g (g cells)-1 h-1 compared with 0.01 g (g cells)-1 h-1 for the xylose reductase/xylitol dehydrogenase strain and the xylose isomerase strain, respectively.ConclusionThe combination of the xylose reductase/xylitol dehydrogenase pathway and the bacterial arabinose isomerase pathway resulted in both higher pentose sugar uptake and higher overall ethanol production than the combination of the xylose isomerase pathway and the bacterial arabinose isomerase pathway. Moreover, the flux through the bacterial arabinose pathway did not increase when combined with the xylose isomerase pathway. This suggests that the low activity of the bacterial arabinose pathway cannot be ascribed to arabitol formation via the xylose reductase enzyme.

Application of comparative proteome analysis to reveal influence of cultivation conditions on asymmetric bioreduction of β-keto ester by Saccharomyces cerevisiae

Industrial bakers' yeast strain Saccharomyces cerevisiae LH1 was selected for asymmetric reduction of ethyl benzoylacetate to (S)-ethyl 3-hydroxy-3-phenylpropionate. Higher reductive efficiency and higher cofactor availability were obtained with the alternation of cultivation condition (mainly growth medium). Compared to the bioreduction by yeast cells grown in malt extract (ME) medium, the concentration of substrate was increased 25-fold (up to 15.6 g/l) in the yeast peptone dextrose (YPD)-grown cells mediated bioreduction with 97.5% of enantioselective excess of (S)-product. The proteomic responses of S. cerevisiae LH1 cells to growth in aerobic batch cultures fed with either YPD or ME medium were examined and compared. Among the relative quantities of 550 protein spots in each gel, changes were shown in the expression level of 102 intracellular proteins when comparing YPD gel to ME gel. Most of the identified proteins were involved in energy metabolism and several cellular molecular biosynthetic pathway and catabolism. For YPD-grown yeast cells, not only enzymes involved in nicotinamide adenine dinucleotide phosphate regeneration, especially 6-phosphogluconate dehydrogenase, but also alcohol dehydrogenase 1 and D: -arabinose 1-dehydrogenase which had been demonstrated activity toward ethyl benzoylacetate to (S)-hydroxy ester were significantly upregulated. These changes provided us insight in the way the yeast cells adapted to a change in cultivation medium and regulated its catalytic efficiency in the bioreduction.

Genome‐scale model for Clostridium acetobutylicum: Part II. Development of specific proton flux states and numerically determined sub‐systems

A regulated genome-scale model for Clostridium acetobutylicum ATCC 824 was developed based on its metabolic network reconstruction. To aid model convergence and limit the number of flux-vector possible solutions (the size of the phenotypic solution space), modeling strategies were developed to impose a new type of constraint at the endo-exo-metabolome interface. This constraint is termed the specific proton flux state, and its use enabled accurate prediction of the extracellular medium pH during vegetative growth of batch cultures. The specific proton flux refers to the influx or efflux of free protons (per unit biomass) across the cell membrane. A specific proton flux state encompasses a defined range of specific proton fluxes and includes all metabolic flux distributions resulting in a specific proton flux within this range. Effective simulation of time-course batch fermentation required the use of independent flux balance solutions from an optimum set of specific proton flux states. Using a real-coded genetic algorithm to optimize temporal bounds of specific proton flux states, we show that six separate specific proton flux states are required to model vegetative-growth metabolism and accurately predict the extracellular medium pH. Further, we define the apparent proton flux stoichiometry per weak acids efflux and show that this value decreases from approximately 3.5 mol of protons secreted per mole of weak acids at the start of the culture to approximately 0 at the end of vegetative growth. Calculations revealed that when specific weak acids production is maximized in vegetative growth, the net proton exchange between the cell and environment occurs primarily through weak acids efflux (apparent proton flux stoichiometry is 1). However, proton efflux through cation channels during the early stages of acidogenesis was found to be significant. We have also developed the concept of numerically determined sub-systems of genome-scale metabolic networks here as a sub-network with a one-dimensional null space basis set. A numerically determined sub-system was constructed in the genome-scale metabolic network to study the flux magnitudes and directions of acetylornithine transaminase, alanine racemase, and D-alanine transaminase. These results were then used to establish additional constraints for the genome-scale model.