Within the first 5 min after a sudden relief from glucose limitation, Saccharomyces cerevisiae exhibited fast changes of intracellular metabolite levels and a major transcriptional reprogramming. Integration of transcriptome and metabolome data revealed tight relationships between the changes at these two levels. Transcriptome as well as metabolite changes reflected a major investment in two processes: adaptation from fully respiratory to respiro-fermentative metabolism and preparation for growth acceleration. At the metabolite level, a severe drop of the AXP pools directly after glucose addition was not accompanied by any of the other three NXP. To counterbalance this loss, purine biosynthesis and salvage pathways were transcriptionally upregulated in a concerted manner, reflecting a sudden increase of the purine demand. The short-term dynamics of the transcriptome revealed a remarkably fast decrease in the average halflife of downregulated genes. This acceleration of mRNA decay can be interpreted both as an additional nucleotide salvage pathway and an additional level of glucose-induced regulation of gene expression.

The in vivo kinetics in Saccharomyces cerevisiae CEN.PK 113-7D was evaluated during a 300-second transient period after applying a glucose pulse to an aerobic, carbon-limited chemostat culture. We quantified the responses of extracellular metabolites, intracellular intermediates in primary metabolism, intracellular free amino acids, and in vivo rates of O 2 uptake and CO 2 evolution. With these measurements, dynamic carbon, electron, and ATP balances were set up to identify major carbon, electron, and energy sinks during the postpulse period. There were three distinct metabolic phases during this time. In phase I (0 to 50 seconds after the pulse), the carbon/electron balances closed up to 85%. The accumulation of glycolytic and storage compounds accounted for 60% of the consumed glucose, caused an energy depletion, and may have led to a temporary decrease in the anabolic flux. In phase II (50 to 150 seconds), the fermentative metabolism gradually became the most important carbon/electron sink. In phase III (150 to 300 seconds), 29% of the carbon uptake was not identified in the measurements, and the ATP balance had a large surplus. These results indicate an increase in the anabolic flux, which is consistent with macroscopic balances of extracellular fluxes and the observed increase in CO 2 evolution associated with nonfermentative metabolism. The identified metabolic processes involving major carbon, electron, and energy sinks must be taken into account in in vivo kinetic models based on short-term dynamic metabolome responses.Mathematical models of in vivo enzyme kinetics in microorganisms are important for understanding metabolic control mechanisms operating on the level of the metabolome and can be used to assist the rational redesign of metabolic pathways to enhance desired functionalities of microbes (54). Kinetic parameters in this kind of model can be obtained by stimulusresponse experiments, in which cells grown in a (quasi-)steady state are perturbed by an external stimulus and the dynamic responses of intra-and extracellular metabolites are monitored. The time window of observation is usually within tens to a few hundred seconds after the application of the stimulus, and the responses are usually attributed to rapid (allosteric) enzyme-metabolite interactions (38). Kinetic parameters can be estimated from the measured responses, based on a set of (dynamic) material balances (7, 30, 49), as follows: dx/dt ϭ Sv (equation 1), where x is a vector of the metabolite concentrations, S is the stoichiometry matrix, and v is a vector of the reaction rates as a function of (yet) unknown kinetic parameters. The stimulus-response methodology is an ideal tool for obtaining kinetic information and has been applied to various microorganisms under different growth conditions (7,26,32,38,52).For Saccharomyces cerevisiae, a frequently applied perturbation is the addition of a concentrated glucose solution, i.e., a glucose pulse, to a glucose-limited chemostat culture, thereby inducing a short-term Crabtree effect. Theoba...

With an increase in the organic acid requirement, the publication of organic acids recovery has been increased over the years.

Empty fruit bunches (EFB) of oil palm are the lignocellulosic waste from crude palm oil production. The current practice to deal with the waste is either to burn EFB for energy production or to spread them back on the field as a fertilizer. Both options offer a limited additional value to the industry compared to the use of EFB as a renewable resource for chemicals production. To be used as the raw materials for chemicals production, EFB needs to be hydrolysed first to its sugar-monomer content.This manuscript presents the study of enzymatic hydrolysis of EFB by xylanolytic enzyme. The study covers the evaluation of commercial xylanolytic enzyme in hydrolysing EFB, the effect of temperature, pH, substrate concentration and potential inhibitors in the EFB hydrolysis process, and the influences of thermal pretreatment for enhancing the yield of hydrolysis. The results of this study lead to an increase in the enzymatic hydrolysis process of EFB. The maximum hydrolysis yield was obtained at temperature of 60 ℃ and pH of 5. Both inhibitors, xylose and glucose, affected the hydrolysis process. The results showed that the thermal pretreatment of EFB enhanced the enzymatic hydrolysis yield. Enzymatic hydrolysis was shown to follow Michaelis Menten kinetic model, and the kinetic parameters were obtained to be K m = 22.16 g/L and Vm = 0.17 g/L/min.

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The production of ethanol via syngas fermentation obtained from lignocellulose gasification provides a method for completely utilizing all of the carbon content from lignocellulosic feedstock. The low mass transfer rate of less soluble CO and H2 gas to liquid has been considered a major bottleneck in the overall process; however, microporous membrane has been proposed as a gas diffuser to improve gas-to-liquid mass transfer. In this study, a liquid batch of syngas fermentation employing Clostridium ljungdahlii with continuous gas supply was obtained using the configuration of a bioreactor connected to microporous hydrophobic polypropylene hollow fiber membrane (HFM) as a gas diffuser. Liquid recirculation between the fermentation vessel and membrane module was applied to enhance the gas-liquid contact as well as cell-recycle. The fermentation performance with and without HFM was compared and evaluated by cell growth, CO utilization, ethanol yield, and productivity. A higher ethanol yield, 0.22 mol/mol, was achieved using the system of an HFM-supported bioreactor with a higher ethanol titer of 1.09 g/L and an ethanol-acetate molar ratio of 1.43 mol/mol. The obtained result demonstrates that an HFM-supported bioreactor is the best fermentation system compared to stirred tank reactor (STR) without a membrane.

pH affects many processes on cell metabolism, such as enzyme kinetics. To enhance the understanding of the living cells, it is therefore indispensable to have a method to monitor the pH in living cells. To accomplish this, a dynamic intracellular pH measurement method applying low concentration benzoic acid pulse was developed. The method was thoroughly validated and successfully implemented for measuring fast dynamic intracellular pH of Saccharomyces cerevisiae in response to a glucose pulse perturbation performed in the BioSCOPE set-up. Fast drop in intracellular pH followed by partial alkalinization was observed following the pulse. The low concentration benzoic acid pulse which was implemented in the method avoids the undesirable effects that may be introduced by benzoic acid to cell metabolism.

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