Substrate Consumption Research Articles

Toward standardized solid medium cultivations: Online microbial monitoring based on respiration activity.

Cultivating microorganisms on solid agar media is a fundamental technique in microbiology and other related disciplines. For the evaluation, most often, a subjective visual examination is performed. Crucial information, such as metabolic activity, is not assessed. Thus, time-resolved monitoring of the respiration activity in agar cultivations is presented to provide additional insightful data on the metabolism. A modified version of the Respiration Activity MOnitoring System (RAMOS) was used to determine area-specific oxygen and carbon dioxide transfer rates and the resulting respiratory quotients of agar cultivations. Therewith, information on growth, substrate consumption, and product formation was obtained. The validity of the presented method was tested for different prokaryotic and eukaryotic organisms on agar, such as Escherichia coli BL21, Pseudomonas putida KT2440, Streptomyces coelicolor A3(2), Saccharomyces cerevisiae WT, Pichia pastoris WT, and Trichoderma reesei RUT-C30. Furthermore, it is showcased that several potential applications, including the determination of colony forming units, antibiotic diffusion tests, quality control for spore production or for pre-cultures and media optimization, can be quantitatively evaluated by interpretation of the respiration activity.

Electro-fermentation with Clostridium autoethanogenum: Effect of pH and neutral red addition

Clostridium autoethanogenum is a gram-positive anaerobic bacterium that grows heterotrophically using fructose as a substrate, producing organic acids (formic, lactic, and acetic acid) and alcohols (ethanol and 2,3-butanediol). Literature reports show that electro-fermentation (EF) helps achieve higher production yields with Clostridium strains. EF involves manipulating microbial metabolism by applying an external potential using solid-state electrodes with/without redox mediators. However, the decrease in pH value can limit cellular viability and substrate conversion during the fermentation process. This work studies the effect of pH and neutral red as an electron transport on the kinetic of the growth and metabolites distribution during EF of C. autoethanogenum. Fermentation and EF experiments using C. autoethanogenum were performed in two-chamber H-type reactors. Biomass generation and product distribution were recorded in fermentations at pH 5.8 with and without pH control. As a result, when pH was controlled at 5.8, higher substrate consumption and biomass concentration were reached than when an external potential was applied (−600 mV vs. Ag/AgCl), allowing 76.6% more biomass with a 100% fructose conversion. Experiments revealed that redox mediator addition and the application of potential in pH-controlled fermentations mainly affected the production of biomass, lactate, acetate and formate, improving the production of lactate and acetate during the EF. In contrast, the results show no improvement in ethanol and 2,3-butanediol during the EF of fructose.

Validation of mathematical models as a tool for prediction of α-amylase production by Coprinus comatus in a low-cost culture medium

Studying new microbial sources and evaluating the growth kinetics behavior to predict large-scale production become essential. Besides, research with low-cost culture media has gained interest due to the need to decrease enzyme production costs. The literature concerning the use of macromycetes as a source of amylases, and applying mathematical models to predict their behavior is scarce. In this study, four growth kinetics in different compositions of culture media were carried out: two of them using different concentrations of Low-Grade Flour (LGF), one wheat mill by-product and two synthetic ones. In the kinetics mathematical models were used to evaluate the bioprocesses and their scaling feasibility. Mathematical models, such as modified Logistic, modified Gompertz, Baranyi, and Roberts, and modified Luedeking-Piret, were applied to predict mycelial growth, enzyme production, and substrate consumption. The results showed the model’s capability to predict mycelial growth (biomass) and substrate consumption (starch concentration). On the other hand, the mathematical models efficiently describe only the maximum enzymatic activity (α-amylase), not the process's other parameters, because of the complexity and diversity of biomolecules synthesized by Coprinus comatus. The wheat mill by-product was efficient as an ingredient in the culture media composition. Furthermore, it was concluded that B2 formulation (culture medium with 48.5 g/L of LGF) is the batch with the highest potential for the α-amylase production from C. comatus and the expansion of this process on an industrial scale. This study is important and valid for the scientific community since modeling studies for macromycetes are scarce in the literature.

Biohydrogen production from starchy wastewater in upflow anaerobic sludge blanket (UASB) reactor: Possibilities toward circular bioeconomy

The present study investigates the performance of upflow anaerobic sludge blanket (UASB) reactor for stable H2 production from starchy wastewater supplemented with groundnut de-oiled cake using mixed consortia. Insufficient reports are available on H2 production for UASB reactor. Operation of the UASB reactor was divided into two stages i.e., start-up period where desirable sludge height was developed and production period where different hydraulic retention time (HRT) was examined for H2 production. The maximum H2 production rate of 12.3 L L−1 d−1was observed at 3 h HRT. Details physicochemical characteristics of the granular sludge were studied using SEM-EDX image, FTIR spectra, particle size analyser, SVI value, EPS and metal content. Kinetic analysis of the process revealed that Grau, and modified Stover-Kicannon were the most suitable for H2 producing UASB reactor. This is the first report to implement these advanced bio-kinetic models of substrate consumption on hydrogen production in UASB reactor. Furthermore, a potential toward circular bioeconomy model has been discussed for the accomplishment of maximum resource recovery with a minimum waste generation.

Mathematical modeling of bioethanol production from sweet sorghum juice under high gravity fermentation: Applicability of Monod-based, logistic, modified Gompertz and Weibull models

BackgroundMathematical modeling of a fermentation process is crucial in understanding and predicting dynamics of the process, which can be used in process improvement, design and control. The present study aimed to develop Monod-based kinetic models to describe cell growth, substrate consumption and ethanol production by Saccharomyces cerevisiae NP 01 under high gravity (HG) fermentation of sweet sorghum juice (SSJ). ResultsThe fermentation using an initial total sugar (TS) concentration of 240 g/L resulted in 113.3 g/L of ethanol production, with 90.9% TS consumption and a fermentation efficiency of 94.4%. Growth of the yeast in terms of specific growth rate was found to be inhibited at a threshold TS concentration of 65 g/L, and the maximum specific growth rate, Monod constant and inhibition constant were 0.45 1/h, 19.5 g/L and 0.002 L/(g·h), respectively. Monod-based models incorporating substrate and product inhibition terms showed high applicability to describe the changes of cell, TS and ethanol concentrations, based on the values of bias factor, accuracy factor, coefficient of determination and root mean square error. ConclusionsThe Monod-based models fitted the data equally well as compared with the logistic, modified Gompertz, and Weibull models, despite estimating the value of different kinetic parameters. These results demonstrated that all the models tested were applicable in modeling HG ethanol fermentation.How to cite: Salakkam A, Phukoetphim N, Laopaiboon P, et al. Mathematical modeling of bioethanol production from sweet sorghum juice under high gravity fermentation: Applicability of Monod-based, logistic, modified Gompertz and Weibull models. Electron J Biotechnol 2023;64. https://doi.org/10.1016/j.ejbt.2023.03.004.

Insight into polyhydroxyalkanoate (PHA) production from xylose and extracellular PHA degradation by a thermophilic Schlegelella thermodepolymerans

Accumulation of non-degradable plastic waste in the environment might be prevented by the use of biodegradable polyhydroxyalkanoate (PHA). In this study, the thermophile Schlegelella thermodepolymerans produced up to 80 wt% PHA based on dry cell mass. The largest PHA granules were found in the cells within 48 h using 20 g/L xylose, a C/N ratio of 100, an initial pH of 7, at 50 °C. The substrate consumption, pH changes, and cell growth were monitored, revealing the time dependency of PHA production in S. thermodepolymerans. The metabolic pathways from xylose to PHA were identified based on proteomic analysis, revealing involvement of classic phaCAB, de novo fatty acid biosynthesis, and fatty acid β-oxidation. In addition, it was shown that S. thermodepolymerans degraded extracellular PHA with a high efficiency at 50 °C.

Co-Treatment of Winery and Domestic Wastewaters in Municipal Wastewater Treatment Plants: Analysis of Biodegradation Kinetics and Process Performance Impacts

Winery wastewater (WWW) handling strategies often include co-treatment at municipal wastewater treatment plants (WWTPs). Despite this, definitive information regarding oxidation kinetics and process and performance impacts due to co-treatment is lacking. A combined Michaelis–Menten–University of Cape Town kinetic model has been found to best describe the pH-inhibited aerobic biological oxidation of WWW by heterotrophs in activated sludge from four municipal WWTPs. The specific rate of substrate consumption was highest in biomass that had been exposed to WWW (57.3 mg COD/g MLVSS·h) compared to biomass that had not (20.7 mg COD/g MLVSS·h). Bench-scale aerobic co-treatment trials confirm that sorption is a key removal mechanism, with up to 98% chemical oxygen demand and 97% total organic carbon removal after 6 h of reaction time. The WWW solids are quickly incorporated into the biological floc and may improve settleability at loading rates above 75 mg WWW suspended solids/L bioreactor volume at the expense of significantly increasing the observed yield. The aerobic-activated sludge system at municipal WWTPs can effectively co-treat WWW, provided the organic loading rates are limited and the WWTP is designed to accommodate the seasonal loadings of winery wastewater. The identified mitigation measures can be used by co-treating facilities to optimize the co-treatment performance of WWW along with domestic wastewater.

The Impact of Yeast Encapsulation in Wort Fermentation and Beer Flavor Profile.

The food and beverage industry is constantly evolving, and consumers are increasingly searching for premium products that not only offer health benefits but a pleasant taste. A viable strategy to accomplish this is through the altering of sensory profiles through encapsulation of compounds with unique flavors. We used this approach here to examine how brewing in the presence of yeast cells encapsulated in alginate affected the sensory profile of beer wort. Initial tests were conducted for various combinations of sodium alginate and calcium chloride concentrations. Mechanical properties (i.e., breaking force and elasticity) and stability of the encapsulates were then considered to select the most reliable encapsulating formulation to conduct the corresponding alcoholic fermentations. Yeast cells were then encapsulated using 3% (w/v) alginate and 0.1 M calcium chloride as a reticulating agent. Fourteen-day fermentations with this encapsulating formulation involved a Pilsen malt-based wort and four S. cerevisiae strains, three commercially available and one locally isolated. The obtained beer was aged in an amber glass container for two weeks at 4 °C. The color, turbidity, taste, and flavor profile were measured and compared to similar commercially available products. Cell growth was monitored concurrently with fermentation, and the concentrations of ethanol, sugars, and organic acids in the samples were determined via high-performance liquid chromatography (HPLC). It was observed that encapsulation caused significant differences in the sensory profile between strains, as evidenced by marked changes in the astringency, geraniol, and capric acid aroma production. Three repeated batch experiments under the same conditions revealed that cell viability and mechanical properties decreased substantially, which might limit the reusability of encapsulates. In terms of ethanol production and substrate consumption, it was also observed that encapsulation improved the performance of the locally isolated strain.

Treatment of wastewater using Black Soldier Fly larvae: Effect of organic concentration and load

Recent studies have investigated the use of Black Soldier Fly (BSF) larvae as a promising biological treatment process for high organic content wastewater (i.a. Leachate from municipal solid waste landfill, food processing effluents), achieving both high treatment efficiency and production of secondary resources from larval biomass (i.a. Proteins and lipids). The present study was aimed at achieving a better understanding of how organic concentration and load might influence treatment performance. Larvae were fed with three artificial wastewaters characterised by same organic substances quality (degree of biodegradability and oxidation of the organic content measured respectively as BOD/COD and TOC/COD ratios) but different organic concentrations. Each type of wastewater was tested at four different loads. Treatment performance was assessed by monitoring both larval growth (in terms of weight variation, mortality and prepupation), and variation of wastewater quality and quantity to determine organic substrate consumption (measured in terms of Total Organic Carbon, TOC). Larval starvation was observed in all tests when TOC concentrations dropped below approx. 1000 mg C/L, which, for the tested wastewater, could be assumed as the limit value for adopting BSF larvae process. Substrate concentration in the feed (mgC/L) influenced larval growth (in terms of maximum wet weight, prepupation and mortality) only when organic load was above 10 mgC/larva: the higher the load, the higher the positive impact of the substrate concentration. On the contrary, the specific substrate consumption rate (vS, mgC/larva/day) appeared not to be influenced by substrate concentration but only by the organic load, with a Michaelis Menten like relationship. Accordingly, substrate load can be assumed as a design parameter for BSF treatment process, while substrate concentration might only influence potential resource recovery from larval biomass.

Relationship between denitrification and anammox rates and N2 production with substrate consumption and pH in a riparian zone

ABSTRACT Denitrification and anaerobic ammonium oxidation (anammox) are the key processes to quantitatively remove nitrate (NO3 −) and balance the nitrogen (N) budget of the ecosystem. In this paper, a slurry-based 15N tracer approach was used to study the correlation and quantitative relation of substrate consumption and pH with rates of denitrification and anammox in a riparian zone. The results showed that the fastest rates of 0.93 µg N h−1 and 0.32 µg N h−1 for denitrification (Denitrif-N2) and anammox (Denitrif-N2), respectively. N2 produced by denitrification occupied 74.04% and produced by anammox occupied 25.96% of the total N2, proving denitrification is the dominant process to remove NO3 −. The substrate content (NO3 −, NH4 + and TOC) and pH varied during incubation and were significantly correlated with Dentrif-N2 and Anammox-N2. Nitrate and TOC as the substrates of denitrification demonstrated a significant correlation with Anammox-N2, which was associated with the products of denitrification involved in the anammox process. This proved a coupling of denitrification and anammox. A quantitative relationship was observed between Dentrif-N2 and Anammox-N2 in the range of 2.75–2.90 when TOC, NH4 + and NO3 − consumption per unit mass or pH changed per unit. Nitrogen mass balance analysis showed that 1 mg N substrate (NO3 −+NH4 +) consumption in the denitrification and anammox can produce 1.05 mg N2 with a good linear relationship (r 2 = 0.9334). This could be related to other processes that produced extra N2 in denitrification and anammox system.

Effects of citric acid and heterofermentative inoculants on anaerobic co-fermentation of Chinese cabbage waste and wheat bran

The effects of Lactobacillus buchneri, Lactobacillus hilgardii and citric acid on organic acid production, substrate consumption, protein degradation and microbial community were investigated in this study. The results indicated that combined inoculants induced a significant increase in levels of lactic acid (43 g/kg dry matter), acetic acid (14 g/kg dry matter), butyric acid (5 g/kg dry matter), total organic acid (60 g/kg dry matter) and ammonia nitrogen (20 g/kg total nitrogen). Furthermore, citric acid addition into the combined inoculants caused a significant increase in levels of acetic acid (12 g/kg dry matter), water-soluble carbohydrate (12 g/kg dry matter) and a reduction in ammonia nitrogen formation (22 g/kg total nitrogen). Microbiologically, combining inoculants and citric acid enriched Lactobacillus buchneri and Lactobacillus hilgardii and upregulated the functional pathways related to acid production and resistance. Collectively, combining citric acid and heterofermentative inoculants was beneficial to recycle Chinese cabbage waste in producing organic acids.

Monitoring biotechnological processes through quantitative image analysis: Application to 2-phenylethanol production by Yarrowia lipolytica

Quantitative image analysis (QIA) is a simple and automated tool for process monitoring that, when combined with chemometric techniques, enables the association of changes in microbiota morphology to various operational parameters. To that effect, principal component analysis, multilinear regression, and ordinary least squares methods were applied to the obtained dataset of the biotransformation conditions for Y. lipolytica through the monitor of yeast morphology, substrates (glycerol, L-phenylalanine - L-Phe) consumption and metabolites (2-phenylethanol – 2-PE) production was developed. Glycerol and L-Phe were successfully monitored by the proposed approach, though with a lower monitoring ability for 2-PE, and mostly related to yeast and cluster size and proportion, yeasts contents and cluster morphology. The chemometric approach also allowed to identify significant morphological modifications related with the change in the stirring speed in the experiments at 600 rpm, 600/400 rpm (600 rpm for 24 h, and 400 rpm until the end of the experiment) and in pH from 5.5 to 7.5. This work demonstrated, for the first time, that QIA combined with chemometric analysis can be considered a valuable tool to monitor biotechnological processes, namely the 2-PE production by Y. lipolytica, by analyzing yeast and cluster morphology.

Stochasticity causes high β-diversity and functional divergence of bacterial assemblages in closed systems.

Stochasticity is a major cause of compositional β-diversity in communities that develop under similar environmental conditions. Such communities may exhibit functional similarity due to sympatric taxa with equivalent metabolic capacities in the source assemblage. However, the redundancy of individual physiological traits may differ in the original source community, which in turn might lead to more or less pronounced variability of single functions among newly formed communities. We analyzed the degree of stochasticity during the primary assembly of bacterial communities originating from the same source and growing under identical conditions. We tested the links between community composition and functioning in parallel microcosms containing glucose and its dimer cellobiose. Bacteria from prefiltered lake water were diluted in artificial lake water and grown to the stationary phase. The resulting assemblages exhibited high compositional variability of taxa that were rare in the source communities. Simulations showed that the observed richness and incidence-based β-diversity could be reproduced by dispersal limitation, or by low dispersal rates associated with the ecological drift of the colonizers. Further null model analysis supported an important influence of stochasticity, as well as a synergy between dispersal limitation and both, heterogeneous and homogeneous selection. The communities functionally differed and the magnitude of functional variability depended on the substrate: more communities consumed glucose than cellobiose. However, there was no relationship between community structure and growth kinetics or substrate consumption. Thus, both structural and functional variability may be a consequence of stochastic processes during initial colonization in closed microbial communities.

Immobilization of mixed cells by Flaxseeds (Linum usitatissimum) extract as new nonconventional biocarrier for biodegradation of sodium dodecyl sulfate

Flaxseeds as a new nonconventional biocarrier was used for the first time to immobilize mixed bacterial cells for biodegradation of sodium dodecyl sulfate (SDS) in actual wastewaters. The results revealed that SDS elimination efficiency in domestic wastewater (DWW) and household detergents manufacturing wastewater (HWW) were 98.8% and 94.5%, respectively. The immobilized cells were repeatedly used for 60 batches without notable loss of their bioactivity, mechanical and chemical stability. Seven kinetic models were applied to depict the interrelation between cells growth and substrate consumption which were; Blackman, Monod, Haldane, Teissier, Moser, Han-Levenspiel, and Contois Models. Teissier model fit well the experimental data with lowest sum of squares error (SSE) of 2.41 × 10−14 indicating exponential growth of biocatalyst inside the biocarrier. Endogenous rate constant (Kd), and yield coefficient (Y) were found to be 0.0039 h−1, and 0.44 h−1, respectively. Values of first order rate constant (K1), effective diffusivity (De), mass transfer coefficient, effectiveness factor (η), and Thiele modulus (Φ) were found to be 1.55 × 10−5 s−1, 6.44×10−7 cm2/s, 2.14×10−6 cm/s, 0.88, and 0.49, respectively. Values of oxygen uptake rate (OUR), volumetric oxygen mass transfer coefficient (KLa) and oxygen transfer rate (OTR) were 0.0013 mg O2/L. s, 0.0222 s−1 and 0.00111 mg O2/L. s, respectively. Experimental and predicted results of (OUR) were in a good agreement with determination coefficient (R2) of 0.94.

Physiological and Biochemical Characteristics, and Biotechnological Potential of Hydrolitic Haloalkalitolerant Bacteria of Soda Sludge Storage

Peculiarities of manifestation of hydrolytic activity of haloalkalitolerant bacteria Pseudomonas peli 3-T, Microbacterium kitamiense 16-DB, and Bacillus aequororis 5-DB, isolated from the soil of the territory of the soda sludge storage and the soda sludge of the functioning sludge reservoir of Berezniki Soda Plant (Perm Territory, Russia) were studied. It was shown that the maximum activity of extracellular lipases and amylases of the three studied bacteria were manifested at pH 11 and a high concentration of sodium chloride (50–200 g/L). Cultivation of P. peli 3-T on a mineral medium with 0.5% glycerol (source of carbon) and 0.03% urea (source of nitrogen) makes it possible to obtain biomass (6.9 g/L) and activity of extracellular lipase (1.26 U/L) and lipase associated with cells (3.02 U/mg dry cells) with a high economic ratio of substrate consumption (138%). The efficiency of immobilization of P. peli 3-T and B. aequororis 5-DB cells for use in lipid and starch biotransformation was shown. P. peli 3-T cells immobilized by adsorption on kaolin and incorporated into the structure of barium alginate and agarose gels retained from 40.4 to 63.8% of the cell lipase activity in suspension. B. aequororis 5-DB cells immobilized by adsorption on kaolin retained 42.5% of lipase and 90.7% of amylase activity as compared to the initial ones. At the same time, the activities of the P. peli 3-T and B. aequororis 5-DB enzymes were preserved during 6 consecutive reactions. The amylase activity of immobilized M. kitamiense 16-DB cells by adsorption on kaolin and chitosan decreased to 2.7–3.5% of the initial value and was completely inhibited upon immobilization by cell’s entrapment into barium alginate gels or agarose gels.

Potential Novel Role of Membrane-Associated Carbonic Anhydrases in the Kidney.

Carbonic anhydrases (CAs), because they catalyze the interconversion of carbon dioxide (CO2) and water into bicarbonate (HCO3-) and protons (H+), thereby influencing pH, are near the core of virtually all physiological processes in the body. In the kidneys, soluble and membrane-associated CAs and their synergy with acid-base transporters play important roles in urinary acid secretion, the largest component of which is the reabsorption of HCO3- in specific nephron segments. Among these transporters are the Na+-coupled HCO3- transporters (NCBTs) and the Cl--HCO3- exchangers (AEs)-members of the "solute-linked carrier" 4 (SLC4) family. All of these transporters have traditionally been regarded as "HCO3-" transporters. However, recently our group has demonstrated that two of the NCBTs carry CO32- rather than HCO3- and has hypothesized that all NCBTs follow suit. In this review, we examine current knowledge on the role of CAs and "HCO3-" transporters of the SLC4 family in renal acid-base physiology and discuss how our recent findings impact renal acid secretion, including HCO3- reabsorption. Traditionally, investigators have associated CAs with producing or consuming solutes (CO2, HCO3-, and H+) and thus ensuring their efficient transport across cell membranes. In the case of CO32- transport by NCBTs, however, we hypothesize that the role of membrane-associated CAs is not the appreciable production or consumption of substrates but the minimization of pH changes in nanodomains near the membrane.

Steering the product spectrum in high-pressure anaerobic processes: CO2 partial pressure as a novel tool in biorefinery concepts

BackgroundElevated CO2 partial pressure (pCO2) has been proposed as a potential steering parameter for selective carboxylate production in mixed culture fermentation. It is anticipated that intermediate product spectrum and production rates, as well as changes in the microbial community, are (in)directly influenced by elevated pCO2. However, it remains unclear how pCO2 interacts with other operational conditions, namely substrate specificity, substrate-to-biomass (S/X) ratio and the presence of an additional electron donor, and what effect pCO2 has on the exact composition of fermentation products. Here, we investigated possible steering effects of elevated pCO2 combined with (1) mixed substrate (glycerol/glucose) provision; (2) subsequent increments in substrate concentration to increase the S/X ratio; and (3) formate as an additional electron donor.ResultsMetabolite predominance, e.g., propionate vs. butyrate/acetate, and cell density, depended on interaction effects between pCO2–S/X ratio and pCO2–formate. Individual substrate consumption rates were negatively impacted by the interaction effect between pCO2–S/X ratio and were not re-established after lowering the S/X ratio and adding formate. The product spectrum was influenced by the microbial community composition, which in turn, was modified by substrate type and the interaction effect between pCO2–formate. High propionate and butyrate levels strongly correlated with Negativicutes and Clostridia predominance, respectively. After subsequent pressurized fermentation phases, the interaction effect between pCO2–formate enabled a shift from propionate towards succinate production when mixed substrate was provided.ConclusionsOverall, interaction effects between elevated pCO2, substrate specificity, high S/X ratio and availability of reducing equivalents from formate, rather than an isolated pCO2 effect, modified the proportionality of propionate, butyrate and acetate in pressurized mixed substrate fermentations at the expense of reduced consumption rates and increased lag-phases. The interaction effect between elevated pCO2 and formate was beneficial for succinate production and biomass growth with a glycerol/glucose mixture as the substrate. The positive effect may be attributed to the availability of extra reducing equivalents, likely enhanced carbon fixating activity and hindered propionate conversion due to increased concentration of undissociated carboxylic acids.

Co-feeding enhances the yield of methyl ketones.

By combining computational and experimental work, we demonstrate that feeding ethanol in addition to glucose improves the yield of biotechnologically produced methyl ketones.

Calcium and bicarbonate signaling control mitochondrial ATP production.

Two unexpected findings surfaced from a recent careful investigation of ATP production by cardiac mitochondria. First, the Ca2+ sensitivity of ATP production under physiological conditions involved neither direct Ca2+-sensitivity of Krebs cycle enzymes nor Ca2+-sensitivity of the electron transport chain proteins. Instead the Ca2+ sensitivity came from the matrix proteins pyruvate dehydrogenase and glutamate dehydrogenase. Second, only half of the dynamic range of ATP production could be attributed to changes in matrix [Ca2+]m. Here we report on the huge contribution to ATP production of an unlikely source -- the consumption of energy substrates. This contribution occurs as high-energy substrates are metabolized by mitochondria and eventually oxidized to CO2. We show that it is the water-soluble CO2 equilibrium-product, bicarbonate (HCO3-), that regulates the second half of physiological ATP production. This regulation is shown to occur through soluble adenylyl cyclase (sAC) which is directly activated by bicarbonate to locally produce the second messenger cAMP. Additionally, we show that the sAC signalling occurs within the mitochondria intermembrane space where its activation leads to the augmentation of ATP production. This signaling arm of the cardiac mitochondrial ATP production machinery is also shown to work in conjunction with [Ca2+]m-dependent ATP production to meet the energy needs of cellular activity in both health and disease.

Investigating ethanol production using the Zymomonas mobilis crude extract

Cell-free systems have become valuable investigating tools for metabolic engineering research due to their easy access to metabolism without the interference of the membrane. Therefore, we applied Zymomonas mobilis cell-free system to investigate whether ethanol production is controlled by the genes of the metabolic pathway or is limited by cofactors. Initially, different glucose concentrations were added to the extract to determine the crude extract's capability to produce ethanol. Then, we investigated the genes of the metabolic pathway to find the limiting step in the ethanol production pathway. Next, to identify the bottleneck gene, a systemic approach was applied based on the integration of gene expression data on a cell-free metabolic model. ZMO1696 was determined as the bottleneck gene and an activator for its enzyme was added to the extract to experimentally assess its effect on ethanol production. Then the effect of NAD+ addition at the high concentration of glucose (1 M) was evaluated, which indicates no improvement in efficiency. Finally, the imbalance ratio of ADP/ATP was found as the controlling factor by measuring ATP levels in the extract. Furthermore, sodium gluconate as a carbon source was utilized to investigate the expansion of substrate consumption by the extract. 100% of the maximum theoretical yield was obtained at 0.01 M of sodium gluconate while it cannot be consumed by Z. mobilis. This research demonstrated the challenges and advantages of using Z. mobilis crude extract for overproduction.