HOMOLACTIC FERMENTATION PDF

Lactic acid fermentation is a metabolic process by which glucose and other six-carbon sugars also, disaccharides of six-carbon sugars, e. It is an anaerobic fermentation reaction that occurs in some bacteria and animal cells , such as muscle cells. If oxygen is present in the cell, many organisms will bypass fermentation and undergo cellular respiration ; however, facultative anaerobic organisms will both ferment and undergo respiration in the presence of oxygen. In homolactic fermentation , one molecule of glucose is ultimately converted to two molecules of lactic acid. Heterolactic fermentation , in contrast, yields carbon dioxide and ethanol in addition to lactic acid, in a process called the phosphoketolase pathway.

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Derek A. Abbott, Joost van den Brink, Inge M. Minneboo, Jack T. Pronk, Antonius J. Conversion of glucose to lactic acid is stoichiometrically equivalent to ethanol formation with respect to ATP formation from substrate-level phosphorylation, redox equivalents and product yield. However, anaerobic growth cannot be sustained in homolactate fermenting Saccharomyces cerevisiae. In an effort to understand the mechanisms behind the decreased lactic acid production rate in anaerobic homolactate cultures of S.

Intracellular measurements of adenosine phosphates confirmed ATP depletion and decreased energy charge immediately upon anaerobicity. Unexpectedly, readily available sources of carbon and energy, trehalose and glycogen, were not activated in homolactate strains as they were in reference strains that produce ethanol.

Finally, the anticipated increase in maximal velocity V max of glycolytic enzymes was not observed in homolactate fermentation suggesting the absence of protein synthesis that may be attributed to decreased energy availability. Essentially, anaerobic homolactate fermentation results in energy depletion, which, in turn, hinders protein synthesis, central carbon metabolism and subsequent energy generation.

Lactic acid, used for food preservation, production of cosmetics and pharmaceuticals and traditionally produced using various species of lactobacilli Benninga, , can also be used for the production of the bio-based biodegradable polymer polylactic acid Benninga, ; Datta, The pH sensitivity and limited ability to synthesize B-vitamins and amino acids of these lactobacilli, increases the cost of lactic acid production due to the requirement of complex nutrients and the formation of large amounts of gypsum as a byproduct Benninga, ; Chopin, The deletion of one or more of the functional genes encoding pyruvate decarboxylase in combination with the expression of a heterologous lactate dehydrogenase results in S.

Considering the intracellular stoichiometry, production of lactic acid is equivalent to ethanol formation with respect to energy and redox metabolism. As with ethanol formation, the conversion of 1 mol of glucose to 2 mol of lactic acid via glycolysis results in the formation of 2 moles of ATP.

As such, anaerobic lactic acid production by S. Indeed, the exposure of homofermentative lactate-producing S. However, under anaerobic conditions the strain was incapable of sustaining growth, and the lactate production rate rapidly decreased van Maris, b.

Subsequently, oxygen-limited chemostat cultivation showed that lactic acid production did not result in the net formation of ATP in lactic acid-producing S. Moreover, the positive influence of oxygenation on the lactate production was observed with a 2. The high intracellular pH ensures that the majority of lactic acid is present as the lactate anion, which is incapable of diffusing across the plasma membrane.

Therefore, analogous to the export of other weak organic acid anions Piper, ; Fernandes, , it is likely that ATP-dependent export of the lactate anion is required.

At the very least, ATP is required to export the dissociated proton in order to maintain intracellular pH. Although not in agreement with the above mentioned observations under oxygen limitation, in the worst case scenario ATP-dependent mechanisms may be involved in both proton and anion export.

These intriguing observations make one wonder about the intracellular processes that occur during anaerobic homolactic fermentation. Perturbations in oxygen availability and especially the glucose concentrations are known to affect the intracellular concentrations of the adenosine phosphates in wild-type strains Kresnowati, The drastic differences between homolactic and alcoholic fermentation might therefore either originate from, or cause, differences in the energy charge of the cells.

Not only these conserved moieties, but also the activities of the glycolytic enzymes themselves are known to respond to perturbations in the glucose concentration van den Brink, a. Additionally, in wild-type S. Consequently, glycogen accumulated during aerobic carbon-limited chemostat cultivation may initially provide a source of free energy to drive lactic acid production and cellular maintenance under anaerobic conditions.

In this study, aerobic carbon-limited chemostats were used as a reproducible starting point for comparison of the physiological response of homolactic RWB and alcoholic-fermenting CEN.

PK D S. Finally, the glycolytic pathway was measured for changes in maximal velocity V max to evaluate the effects of anaerobic homolactate fermentation on central carbon metabolism.

The S. Synthetic medium was prepared as described previously Verduyn, Glucose 6. The dilution rate was set at 0. A stirrer speed of r.

Steady-state samples were withdrawn after c. Culture dry weights were determined in duplicate via filtration onto dry, preweighed nitrocellulose membranes. Samples were dried in a microwave oven for 20 min at W. Anaerobic glucose-pulse experiments were started by sparging a steady-state carbon-limited aerobic chemostat culture with 0.

Two minutes after nitrogen sparging started and just before adding the glucose, the medium and effluent pumps were switched off.

The mM added as 60 g of glucose monohydrate and 60 mL water glucose pulse was injected aseptically through a rubber septum, and samples were removed periodically for analysis.

Viability of RWB was determined by plating appropriate dilutions prepared in sterile 0. Triplicate plates containing between 20 and colonies were counted for each of the duplicate fermentations. One milliliter of cell suspension was added to safelock tubes with 0. The tubes were placed in a FastPrepA machine Thermo Scientific and shaken in four bursts of 20 s, at speed 6 6. Samples were placed on ice between bursts. The supernatant was stored on ice and used for determination of enzyme activities.

To assure reproducibility, all assays were performed in duplicates for two different concentrations of cell extracts. Each in vitro enzyme assay for the glycolytic pathway was performed as described previously Jansen, Protein concentrations in cell-free extracts were determined by the Lowry method Lowry, with dried bovine serum albumin fatty acid free, Sigma as a standard.

Concentrations of intracellular ADP and AMP were determined enzymatically according to Mashego based on myokinase, pyruvate kinase and lactate dehydrogenase.

The energy charge EC Atkinson, was calculated as shown in Eqn. Aerobic steady-state carbon-limited chemostat cultures of the reference strain, CEN. PK D, and the isogenic lactate-producing strain, RWB , were used to obtain well defined, reproducible and comparable starting conditions for the study of the dynamic response to anaerobic glucose excess.

The culture dry weights in these steady-state cultures were identical with values of 3. PK D. In contrast to the almost identical behavior of both strains in aerobic chemostat cultures, large differences became apparent shortly after introduction of anaerobicity and glucose excess mM. The growth of the reference strain as measured by OD nm began c.

In sharp contrast, the biomass concentration of RWB had not increased after 8 h. In agreement with previous research van Maris, b , growth of the reference strain was accompanied by formation of ethanol, carbon dioxide and glycerol with complete glucose consumption occurring after c.

Although lactic acid production excluding export is theoretically equal to ethanol formation with respect to ATP generation and redox metabolism, the glucose consumption rate in the lactic acid-producing strain was lower and even more importantly, the rate of lactate production continually decreased over time Fig.

Viability was not measured for the reference strain. PK D bottom row after exposure to mM glucose and anaerobicity. The average data of two independent fermentations is presented.

To study this dramatic difference in response to anaerobic glucose excess and to determine the possible impact of lactate export on the cellular energetics, intracellular levels of ATP, ADP and AMP were measured at the aerobic steady state and throughout the dynamic anaerobic phase Fig. During aerobic, carbon-limited growth, the levels of intracellular ATP were identical for each strain at 7. In these steady states, the energy charge of both strains had a value of c.

Five minutes after the shift to glucose-excess conditions both strains showed a drastic decrease in the ATP concentration to 3. However, the ATP concentration in the reference strain quickly recovered to a new pseudo-steady state, while ATP levels in the RWB strain decreased for the first 30 min of the pulse experiment, subsequently leveled of at the low concentration of 1.

Error bars of the adenosine concentrations represent SDs between duplicate analyses of two independent culture samples. When ATP concentrations decrease, the concentrations of the other two forms of the adenosine phosphate conserved moieties are expected to increase.

Indeed, although the intracellular concentrations of ADP were almost constant for both strains, the response of the AMP concentrations to the glucose pulse was strikingly different in both strains. This decrease in AXP has been described previously Kresnowati, As a result of these concentration changes, the energy charge for the reference strain decreased the first 5 min after the shift before it recovered to the initial steady-state value of 0.

In contrast, the energy charge of the RWB strain mirrored the drop in ATP concentration and decreased to the extremely low value of 0. As expected, the levels of the storage carbohydrates, trehalose and glycogen, were approximately equal in aerobic chemostats for both the reference strain and the lactic acid-producing strain. However, the dynamic responses to the anaerobic glucose excess conditions were dramatically different.

As previously observed van den Brink, b , both the glycogen and trehalose concentrations in the reference strain decreased quickly from 1. Surprisingly, these storage carbohydrates were not mobilized in RWB throughout the experiment Fig. Therefore, the initially higher rate of lactic acid production was not explained by additional ATP provided by consumption of glycogen. Error bars represent SDs between duplicate analyses of two independent culture samples.

The activity of several glycolytic enzymes was measured for changes in maximal velocity V max to determine the influence of anaerobic lactate production on central carbon metabolism. Predictably, pyruvate decarboxylase activity was absent in RWB and lactate dehydrogenase was absent in the reference strain.

In the reference strain, substantial increases in activity were observed for the majority of the measured glycolytic enzymes, including the activities of phosphoglucose isomerase, fructose-bisphosphate aldolase, triose-phosphate isomerase, glyceraldehydephosphate dehydrogenase, phosphoglycerate kinase, phosphoglycerate mutase and pyruvate kinase Fig.

The increased activity of these glycolytic enzymes has been well documented and is directly correlated to changes in transcript levels van den Brink, a, b. In contrast to both the observations and documented response of wild-type S.

The stable activity of these enzymes in anaerobic lactate-producing cultures suggests a problem with de novo protein synthesis or alternatively a drastic change in protein turnover. In vitro glycolytic enzyme activity in CEN. For PFK, notorious for its sensitivity to many allosteric effectors, such as fructose-2,6-biphosphate and ATP Bartrons, , relating in vitro activities to in vivo fluxes is precarious.

The activity of both HXK and ADH was already lower during the steady states, even though the physiological parameters under steady-state conditions were similar between the strains. This might indicate different ratios of the HXK and ADH iso-enzymes in each strain even under nonfermentative conditions van den Brink, a.

The observed rapid decrease of the intracellular ATP concentration within the first few minutes after the anaerobic glucose pulse to homolactic S. As transcription and especially translation are energetically expensive processes Warner, , the depletion of the ATP concentration may also hinder protein synthesis.

This conclusion is supported by the absence of increased glycolytic enzyme activities in RWB , and resulted in reduced metabolic rates, which further amplified the energetic constraints in these lactate-producing cultures. These observations are consistent with previous publications, which hypothesized that lactic-acid production in this engineered S.

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Lactic acid fermentation

Many cells are unable to carry out respiration because of one or more of the following circumstances:. Whereas lack of an appropriate inorganic final electron acceptor is environmentally dependent, the other two conditions are genetically determined. Thus, many prokaryotes, including members of the clinically important genus Streptococcus , are permanently incapable of respiration, even in the presence of oxygen. Conversely, many prokaryotes are facultative, meaning that, should the environmental conditions change to provide an appropriate inorganic final electron acceptor for respiration, organisms containing all the genes required to do so will switch to cellular respiration for glucose metabolism because respiration allows for much greater ATP production per glucose molecule. Some living systems use an organic molecule commonly pyruvate as a final electron acceptor through a process called fermentation. Fermentation does not involve an electron transport system and does not directly produce any additional ATP beyond that produced during glycolysis by substrate-level phosphorylation. Organisms carrying out fermentation, called fermenters, produce a maximum of two ATP molecules per glucose during glycolysis.

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SparkNotes is here for you with everything you need to ace or teach! Find out more. Glycolysis, as we have just described it, is an anaerobic process. None of its nine steps involve the use of oxygen. However, immediately upon finishing glycolysis, the cell must continue respiration in either an aerobic or anaerobic direction; this choice is made based on the circumstances of the particular cell.

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