The influence of potassium and sodium ions on the negative pasteur effect in Brettanomyces claussenii custers

Summary The present article deals with the negative Pasteur effect in Brettanomyces claussenii Custers, i.e. with the inhibition of the alcoholic fermentation under anaerobic conditions and its stimulation in presence of atmospheric oxygen.As distinct from the negative Pasteur effect in resting cells of Saccharomyces species the effect in Br. claussenii is not specific for cell suspensions prepared with succinic acid-succinate buffer but may at Ph 4.5–4.6 in addition be demonstrated in resting cells suspended in distilled water or phosphate buffer as well as in buffer solutions of a great number of organic acids and their alkali salts, e. g. in acetic acid-acetate, propionic acid-propionate, oxalic acid-oxalate, malonic acid-malonate, fumaric acid-fumarate, malic acid-malate, d-tartaric acid-tartrate, and citric acid-citrate mixtures.The aerobic fermentation of glucose by resting cells of Br. claussenii is quite sensitive to potassium and sodium ions. In all systems examined, except the succinic acid-succinate buffer and the buffer solutions inhibiting the fermentation completely or practically completely, the rate of the aerobic fermentation is considerably increased on increasing the concentration of the potassium ions and decreasing that of the sodium ions. Under anaerobic conditions the alcoholic fermentation is insusceptible to the ions mentioned.Because of the fact that the influence of the potassium ions or of the K⁺/Na⁺ ratio upon the rate of fermentation is comparatively large under aerobic conditions but small or even negligible under anaerobic conditions, the magnitude of the negative Pasteur effect will under the proper conditions be determined by the potassium ion concentration or by the ratio between the concentrations of the potassium and sodium ions. The negative Pasteur effect obtained in a buffer of an acid and its potassium salt may be considerably larger than that observed in a buffer of the same acid and its sodium salt. In solutions containing a mixture of the potassium and sodium salts in addition to an acid the magnitude of the negative Pasteur effect will increase with increasing potassium ion concentration and decreasing sodium ion concentration at constant total molar concentration of the alkali ions.To Professor Dr. E. G. Pringsheim with best wishes on his 80th birthday.     

On a disturbance of the normal Pasteur reaction in baker's yeast

Resting cells of baker's yeast, suspended in phosphate buffer pH 5.0 with glucose give initially a normal Pasteur reaction, which means that fermentation is repressed under aerobic conditions by the respiratory process.However, after 1 to 2 hours fermentation a disturbance of the Pasteur reaction sets in, the aerobic fermentation rising to the anaerobic level or sometimes above this level without a corresponding decrease in respiration. It is demonstrated that this disturbance is closely related to an aerobic growth pattern in which the yeast in its final growth stage before harvesting obtains its energy exclusively from the respiratory process.The interrelation of fermentation and respiration is discussed. In this discussion the aerobic fermentation is defined as the metabolism of the excess of intermediates formed along the Embden-Meyerhof pathway and unable to enter the Krebs cycle due to the limited capacity of the electron transfer system.The author is greatly indebted to Prof. Dr. T. O. Wikén for his interest in this study and for offering him the opportunity to conduct the investigations in his laboratory.     

Adaptation to Stochastic Temporal Variations in Intratumoral Blood Flow: The Warburg Effect as a Bet Hedging Strategy

While most cancers promote ingrowth of host blood vessels, the resulting vascular network usually fails to develop a mature organization, resulting in abnormal vascular dynamics with stochastic variations that include slowing, cessation, and even reversal of flow. Thus, substantial spatial and temporal variations in oxygen concentration are commonly observed in most cancers. Cancer cells, like all living systems, are subject to Darwinian dynamics such that their survival and proliferation are dependent on developing optimal phenotypic adaptations to local environmental conditions. Here, we consider the environmental stresses placed on tumors subject to profound, frequent, but stochastic variations in oxygen concentration as a result of temporal variations in blood flow. While vascular fluctuations will undoubtedly affect local concentrations of a wide range of molecules including growth factors (e.g., estrogen), substrate (oxygen, glucose, etc.), and metabolites (\(\hbox {H}^{+})\), we focus on the selection forces that result solely from stochastic fluctuations in oxygen concentration. The glucose metabolism of cancer cells has been investigated for decades following observations that malignant cells ferment glucose regardless of oxygen concentration, a condition termed the Warburg effect. In contrast, normal cells cease fermentation under aerobic conditions and this physiological response is termed the Pasteur effect. Fermentation is markedly inefficient compared to cellular respiration in terms of adenosine triphosphate (ATP) production, generating just 2 ATP/glucose, whereas respiration generates 38 ATP/glucose. This inefficiency requires cancer cells to increase glycolytic flux, which subsequently increases acid production and can significantly acidify local tissue. Hence, it initially appears that cancer cells adopt a disadvantageous metabolic phenotype. Indeed, this metabolic “hallmark” of cancer is termed “energy dysregulation.” However, if cancers arise through an evolutionary optimization process, any common observed property must confer an adaptive advantage. In the present work, we investigate the hypothesis that aerobic glycolysis represents an adaptation to stochastic variations in oxygen concentration stemming from disordered intratumoral blood flow. Using mathematical models, we demonstrate that the Warburg effect evolves as a conservative metabolic bet hedging strategy in response to stochastic fluctuations of oxygen. Specifically, the Warburg effect sacrifices fitness in physoxia by diverting resources from the more efficient process of respiration, but preemptively adapts cells to hypoxia because fermentation produces ATP anaerobically. An environment with sufficiently stochastic fluctuations of oxygen will select for the bet hedging (Warburg) phenotype since it is modestly successful irrespective of oxygen concentration.     

Evaluation and Modeling of Bioethanol Yield Efficiency from Sweet Sorghum Juice

One of the challenges with using sweet sorghum as an energy crop is that although fermentation of the juice to ethanol does not require enzymes, the juice can easily spoil. One strategy to avoid spoilage is to harvest the juice in the field, place it into a tanker for transport, and add the yeast immediately to initiate the fermentation process to begin during transport. Hence, it is also important to understand how the fermentation process is influenced by pH, temperature, and dissolved oxygen, since these parameters would not be “controlled” during transport. A full factorial design was applied to examine and optimize yield efficiency of ethanol production for the fermentation of sweet sorghum juice. Bioethanol yield efficiency was modeled using a linear equation. Under optimal pH (5.5), temperature (28 °C), and dissolved oxygen (0%) conditions, a maximum theoretical yield efficiency of 0.75 was achieved for bioethanol produced from M81E variety of sweet sorghum.     

Physiological, biochemical, and mathematical studies of micro-aerobic continuous ethanol fermentation by Saccharomyces cerevisiae. I: hysteresis, oscillations, and maximum specific ethanol productivities in chemostat culture

The growth and metabolism of Saccharomyces cerevisiae was studied in steady-state chemostat cultures under conditions of scarce oxygen and excess glucose. The specific ethanol productivity and specific glucose uptake rate were stimulated by 50% within a narrow range of air/nitrogen mixtures to the fermentor. Fermentation was inhibited at slightly higher and lower air/nitrogen ratios, confirming similar results by previous investigators. This stimulation could not be caused by obvious mechanisms, such as the Pasteur or Crabtree effects. Since this maximum in the fermentation rate occurred in a steady-state chemostat and at a constant dilution rate, the ATP yield of the culture necessarily attained a minimum. Thus, changes in the energetic efficiency of growth or the degree of wasting of ATP were surmised. The steady-state biomass concentration at various oxygenation rates exhibited hysteresis phenomena. Ignition and extinction of the biomass concentration occurred as critical oxygen feed rates were passed. The hysteresis was prevented by adding yeast extract to or reducing the antifoam concentration in the medium. These medium alterations had the simultaneous effect of stimulating the fermentation rate, suggesting that ATP has a critical role in dictating the biomass concentration in micro-aerobic culture. Silicone polymer antifoam was found to stimulate glycerol production at the expense of ethanol production, having consequences for the energy generation and the biomass concentration of the culture.     

Mitochondrial bioenergetics in aging

Mitochondria are strongly involved in the production of reactive oxygen species, considered as the pathogenic agent of many diseases and of aging. The mitochondrial theory of aging considers somatic mutations of mitochondrial DNA induced by oxygen radicals as the primary cause of energy decline; experimentally, complex I appears to be mostly affected and to become strongly rate limiting for electron transfer. Mitochondrial bioenergetics is also deranged in human platelets upon aging, as shown by the decreased Pasteur effect (enhancement of lactate production by respiratory chain inhibition). Cells counteract oxidative stress by antioxidants; among lipophilic antioxidants, coenzyme Q is the only one of endogenous biosynthesis. Exogenous coenzyme Q, however, protects cells from oxidative stress by conversion into its reduced antioxidant form by cellular reductases.     

Pasteur, oxygen and the anaerobes revisited

As a biochemist, Louis Pasteur focused on fermentation, demonstrating that it was a vital process. In 1860, he discovered anaerobic life and the strict anaerobes, particularly those responsible for butyric fermentation. Then, in spite of his lack of medical background, Pasteur turned to investigating the role of bacteria in human and animal diseases. In 1877, Pasteur and Joubert described for the first time a pathogenic anaerobe, the 'septic vibrio' (now Clostridium septicum). Not only was the bacterium cultivated, but the disease symptoms described and the disease experimentally reproduced. Pasteur also described what are now known as mixed anaerobic infections. A historical review of Pasteur's work is made in the light of our present knowledge of this field.     

Solid-state fermentation-supported enhanced production of α-galactosidase by a deoxyglucose-resistant mutant of <Emphasis Type="Italic">Aspergillus niger</Emphasis> and thermostabilization of the production process

The aim of the present investigation was to comparatively evaluate the behaviour of A. niger and its derepressed mutant in production of α-galactosidase in submerged (SmF) and solid state fermentation (SSF) using basal Vogel’s medium or corn steep liquor as nitrogen source and observe the response of latter source under both cultural techniques under different temperature regimes, and determine if SSF can be exploited in a wide range of temperature expected to vary in this fermentation system. All studies were performed in both systems under pre-optimized cultural conditions. Higher melting temperature and negative values of entropy of activation in SSF indicated that the genetic system of both organisms was thermodynamically resistant in the presence of corn steep liquor but sensitive to inactivation in the presence of Vogel’s nitrogen sources in submerged fermentation. This was reflected as the organisms demanded higher magnitudes of energy for product formation in the presence of ammonium salts. Studies on influence of corn steep liquor revealed that it had stabilizing effect too in both fermentation systems but the mutant strain was more stable in both fermentation systems. Because of these properties, the mutant organism may be exploited for bulk production of α-galactosidase in SSF under condition where temperature may fluctuate during fermentation.     

Interaction of flooding with carbon metabolism of forest trees

Waterlogging and flooding cause oxygen deprivation in the root system of trees. Since oxygen is essentially for mitochondrial respiration, this process cannot be maintained under anoxic conditions and must be replaced by other pathways. For the roots it is therefore a matter of survival to switch from respiration to alcoholic fermentation. Due to the low efficiency of this process to yield energy equivalents (ATP), energy and carbon metabolism of trees are usually strongly affected by oxygen deprivation, even if a rapid switch from respiration to fermentation is achieved. The roots can compensate for the low energy yield of fermentation either (1) by decreasing the demand for energy by a reduction of energy-dependent processes such as root growth and/or nutrient uptake, or (2) by consuming more carbohydrates per unit time in order to generate sufficient energy equivalents. In the leaves of trees, flooding and waterlogging cause a decline in the rates of photosynthesis and transpiration, as well as in stomatal conductance. It is assumed that, due to reduced phloem transport, soluble sugars and starch accumulate in the leaves of flooded trees, thereby negatively affecting the sugar supply of the roots. Thus, root growth and survival is negatively affected by both changes in root internal carbon metabolism and impaired carbon allocation to the roots by phloem transport. In addition, accumulation of toxic products of fermentation in the roots, such as acetaldehyde, can further impair root metabolism. A main feature of tolerance against flooding and waterlogging of trees seems to be the steady supply of carbohydrates to the roots in order to maintain alcoholic fermentation; in addition, roots of tolerant trees seem to avoid accumulation of fermentation-derived ethanol and acetaldehyde. From studies with flooding tolerant and non-tolerant tree species, it is hypothesized that (1) the transport of ethanol produced in the roots under hypoxic conditions into the leaves via the transpiration stream, (2) its conversion into acetyl-CoA in the leaves, and (3) its use in the plant's general metabolism, are mechanisms of flooding tolerance of trees.     

Designing photosystem II: molecular engineering of photo-catalytic proteins

Biological photosynthesis utilizes membrane-bound pigment/protein complexes to convert light into chemical energy through a series of electron-transfer events. In the unique photosystem II (PSII) complex these electron-transfer events result in the oxidation of water to molecular oxygen. PSII is an extremely complex enzyme and in order to exploit its unique ability to convert sunlight into chemical energy it will be necessary to make a minimal model. Here we will briefly describe how PSII functions and identify those aspects that are essential in order to catalyze the oxidation of water into O(2), and review previous attempts to design simple photo-catalytic proteins and summarize our current research exploiting the E. coli bacterioferritin protein as a scaffold into which multiple cofactors can be bound, to oxidize a manganese metal center upon illumination. Through the reverse engineering of PSII and light driven water splitting reactions it may be possible to provide a blueprint for catalysts that can produce clean green fuel for human energy needs.     

Studies on Oxygen Transfer in Submerged Fermentations

In conventional shaken culture system, control of oxygen supply is performed by changing liquid volume in flasks and it necessarily introduces variation in the effectiveness of agitation and in the partial pressure of carbon dioxide. In jar or tank culture system, also, the changes in mechanical agitation and in the flow rate of air for control of aeration induce similar problems. It is impossible, therefore, to isolate the effects of oxygen on microbial metabolism from these accompanying ones. Hence, there is a basic requirement of making clear distinction among them, and in this paper the effects of agitation and carbon dioxide on product formation are presented in glutamic acid fermentation using the apparatus of controlling the level of dissolved oxygen throughout the fermentation.

To obtain fundamental knowledge required for attaining adequate aeration, the rate of oxygen demand in glutamic acid fermentation was discussed in connection with its fermentation rates. On the basis of specific rates, rates of change per unit mass of cells, glutamic acid fermentation was found to fall in the process pattern of Gaden’s type II, in which a constant rate of oxygen demand was sustained for a considerable time. On the basis of volumetric rates, rates of change per unit volume of broths, oxygen demand was recognized to be correlated with growth, sugar utilization and product formation, and it was pointed out particularly that the oxygen demand was closedly related with sugar utilization. In the particular cases where rapid utilization of sugar occurred, therefore, oxygen deficiency was liable to be evoked being unable to fill the growing oxygen demand. This finding might be useful for scale-up studies or process design.     

The Pasteur effect and catabolite repression in an oxidative yeast,Kluyveromyces lactis

The presence of the Pasteur effect in Kluyveromyces lactis grown in glucose was shown by azide-stimulated glucose fermentation. Extracts from these cells contained ATP-sensitive phosphofructokinase activity. Cells grown on succinate oxidized glucose slowly at first without azide-stimulated rates of fermentation. Phosphofructokinase in these cells was ATP-insensitive. The activity of NAD+-isocitrate dehydrogenase in cell extracts did not require AMP activation. These results suggested the presence of a Pasteur effect in glucose-grown but not in succinate-grown K. lactis, mediated by (a) ATP inhibition of phosphofructokinase (b) possibly via feedback control of glucose transport, but not by AMP activation of isocitrate dehydrogenase. Azide inhibition of the Pasteur effect during growth of the cells did not lead to catabolite repression of respiratory activity. The results therefore suggest that the Pasteur effect does not inhibit the development of a Crabtree effect in oxidative yeasts.     

The heuristic function of 'error' in the scientific methodology of Louis Pasteur: the case of the silkworm diseases

With the aid of the Cahiers de laboratoire, the Correspondence and, of course, the Oeuvre de Pasteur, this work reconstructs the extraordinary scientific undertakings of the great French scientist in his study of silkworm diseases. The focus of this study consists in the attempt to explain the initial perplexing behaviour of Pasteur, even in the presence of correct interpretations regarding the causes of these diseases (cfr. the results obtained by Béchamp); for a good three years he insisted on maintaining that the aetiology of silkworm diseases could not be attributed to pathogenetic germs from outside. And this was in spite of the fact that previously (through fermentation and spontaneous generation) he had been able to demonstrate the importance of microorganisms in biological processes. Finally it is intended to highlight the extraordinary methodological depth of that initial 'error', which was capable of paving the way for the future conquests of Pasteur in the field of aetiology and the prevention of infectious diseases.     

Remarks on the metabolism of kluyveromyces lactis van der walt

Kluyveromyces lactis CBS 683, showed no Crabtree effect but the Pasteur effect was very strong. It could be seen that the metabolism of glucose was adaptative. The anaerobic fermentation of lactose was always greater than the anaerobic fermentation on glucose and galactose. The transport across wall and cell membrane of glucose and galactose limits the fermentation rate of these substrates.     

The mechanism of energy conservation and transduction by mitochondrial cytochrome c oxidase.

Oxidation of ferrocytochrome c by molecular oxygen catalysed by cytochrome c oxidase (cytochrome aa3) is coupled to translocation of H+ ions across the mitochondrial membrane. The proton pump is an intrinsic property of the cytochrome c oxidase complex as revealed by studies with phospholipid vesicles inlayed with the purified enzyme. As the conformation of cytochrome aa3 is specifically sensitive to the electrochemical proton gradient across the mitochondrial membrane, it is likely that redox energy is primarily conserved as a conformational "strain" in the cytochrome aa3 complex, followed by relaxation linked to proton translocation. Similar principles of energy conservation and transduction may apply on other respiratory chain complexes and on mitochondrial ATP synthase.     

A robust soft sensor based on artificial neural network for monitoring microbial lipid fermentation processes using Yarrowia lipolytica

Microbial oils produced by Yarrowia lipolytica offer an environmentally friendly and sustainable alternative to petroleum as well as traditional lipids from animals and plants. The accurate measurement of fermentation parameters, including the substrate concentration, dry cell weight, and lipid accumulation, is the foundation of process control, which is indispensable for industrial lipid production. However, it remains a great challenge to measure the complex parameters online during the lipid fermentation process, which is nonlinear, multivariate, and characterized by strong coupling. As a type of AI technology, the artificial neural network model is a powerful tool for handling extremely complex problems, and it can be employed to develop a soft sensor to monitor the microbial lipid fermentation process of Y. lipolytica. In this study, we first analyzed and emphasized the volume of sodium hydroxide and dissolved oxygen concentration as central parameters of the fermentation process. Then, a soft sensor based on a four-input artificial neural network model was developed, in which the input variables were fermentation time, dissolved oxygen concentration, initial glucose concentration, and additional volume of sodium hydroxide. This provides the possibility of online monitoring of dry cell weight, glucose concentration, and lipid production with high accuracy, which can be extended to similar fermentation processes characterized by the addition of bases or acids, as well as changes of the dissolved oxygen concentration.