Pressure measurement to evaluate ethanol or lactic acid production during glucose fermentation by yeast or heterofermentative bacteria in pure and mixed culture

A rapid and simple technique to follow CO2 release during fermentation of glucose by heterofermentative bacteria or yeasts was used in order to evaluate ethanol and lactate production in pure and mixed cultures of yeast and bacteria. In pure cultures, good correlations were found between gas pressure variations (deltaP) and ethanol or lactate production by yeasts or heterofermentative bacteria, and ratios between deltaP and ethanol or lactate produced could be established. In mixed cultures, ratios between maximal deltaP and total amount of glucose consumed were determined. It was thus possible to evaluate the amount of glucose that was consumed by each strain and then deduce the bacterial lactate production. Good results were obtained for mixed cultures of yeast and homofermentative bacteria. This technique may be useful to evaluate the activity of strains in mixed cultures of yeast and lactic acid bacteria.     

An on-line technique for monitoring propionic acid fermentation

An on-line technique, based on measuring the increase in pressure due to CO₂ release in a closed air-tight reactor, was used to evaluate the fermentation of lactate by propionibacteria. The method was applied to batch cultures of Propionibacterium shermanii grown in yeast extract/sodium lactate medium containing lactate as a carbon source under micro-aerophilic conditions. Gas pressure evolution was compared both with substrate consumption and metabolites production and with acidification and growth. Linear relationships were found between gas pressure variation, lactate consumption and propionate and acetate production. The technique also enabled the evaluation of total CO₂ produced, by taking account of pressure, oxygen and pH measurements. These results tend to show that this simple and rapid method could be useful to monitor propionic acid bacteria growth.     

Influence of Several Process Parameters on Sourdough Fermentation

The influence of several process parameters (starter, sourdough refreshment, dough yield, ingredients) on sourdough fermentation was investigated. This was realised by online measurement of both pH and gas pressure in sourdough in a closed air‐tight reactor. Firstly, the effect of sourdough refreshment on both gas pressure and acidification was evaluated and shown to be accompanied by an increase in both acidification and gas pressure evolution rates. Secondly, two commercial starters (BRS and BIO) differing in their microflora were compared as regards to their gassing power and acidifying properties. BRS exhibited faster acidification but less gassing power. Moreover, the variation of sourdough yield for BRS starter induced a different evolution of the acidification profile. Sourdough fermentation varied also depending on the presence of NaCl. 2% of NaCl induced a lower gassing power in sourdough by inhibiting the yeasts.     

Ascopore formation in yeasts during active growth on hydrocarbons

Summary Induction of ascospore formation in hydrocarbon utilizing ascosporogenous yeasts was observed during the growth of the yeasts on gas oil, diesel oil, white kerosene and n-alkanes. Studies of relationships between cell morphology and cell growth showed that ascospores were formed during the active growth phase on gas oil but not on glucose. Contact of yeast cells with hydrocarbons may be the possible reason for sporogenesis on hydrocarbons.     

Carboxylase Levels and Carbon Dioxide Fixation in Baker''s Yeast

Levels of pyruvate carboxylase (PC), phosphopyruvate carboxylase (PEPC), and malate dehydrogenase (decarboxylating) were compared in wild-type bakers' yeast (I), a cytoplasmic-respiratory mutant (II), a biotin-deficient wild-type yeast (III), and a biotin-deficient respiratory mutant (IV). PC activities were greatly reduced in III and IV, whereas PEPC was reduced in II and IV. Malate dehydrogenase (decarboxylating) could not be detected in any of the yeasts. With yeast I growing on glucose as the sole carbon source, PEPC decreased to negligible levels during the logarithmic phase of growth (glucose repression effect), whereas PC increased. Both enzymes reverted to their original levels during the stationary phase, when glucose in the medium was exhausted. In agreement with the leading role of PC for CO(2) assimilation, the rates of (14)CO(2) fixation in yeasts I and II were approximately equal and were much higher than that in yeast IV. With I and II, most of the (14)C was distributed similarly in oxalacetate derivatives; with yeast IV, most of (14)C appeared in a compound apparently unrelated to CO(2) fixation via C(4)-dicarboxylic acids.     

High-pressure inactivation of dried microorganisms

Dried microorganisms are particularly resistant to high hydrostatic pressure effects. In this study, the survival of Saccharomyces cerevisiae was studied under pressure applied in different ways. Original processes and devices were purposely developed in our laboratory for long-term pressurization. Dried and wet yeast powders were submitted to high-pressure treatments (100-150 MPa for 24-144 h at 25 degrees C) through liquid media or inert gas. These powders were also pressurized after being vacuum-packed. In the case of wet yeasts, the pressurization procedure had little influence on the inactivation rate. In this case, inactivations were mainly due to hydrostatic pressure effects. Conversely, in the case of dried yeasts, inactivation was highly dependent on the treatment scheme. No mortality was observed when dried cells were pressurized in a non-aqueous liquid medium, but when nitrogen gas was used as the pressure-transmitting fluid, the inactivation rate was found to be between 1.5 and 2 log for the same pressure level and holding time. Several hypotheses were formulated to explain this phenomenon: the thermal effects induced by the pressure variations, the drying resulting from the gas pressure release and the sorption and desorption of the gas in cells. The highest inactivation rates were obtained with vacuum-packed dried yeasts. In this case, cell death occurred during the pressurization step and was induced by shear forces. Our results show that the mechanisms at the origin of cell death under pressure are strongly dependent on the nature of the pressure-transmitting medium and the hydration of microorganisms.     

Energy metabolism in cultured human fibroblasts during aging in vitro

To explore the relationship between energy metabolism and the limited replicative life span of cultured human fibroblasts, we studied several bioenergetic parameters in normal fibroblasts at early passage (young cells) and at late passage (old cells) and early passage cells from a subject with the Hutchinson-Gilford (progeria) syndrome. Old cells consumed more glucose and produced more lactate during growth, but O2 consumption, both basal and following maximum uncoupling of oxidative phosphorylation by SF-6847, was the same as in young cells. Progeria cells produced the most lactate but did not consume more glucose, while their basal and uncoupled O2 consumption was similar to that of young and old cells during both log and confluent states. Consumption of glutamine, a source of both oxidative energy and lactate, was approximately the same in all three cell types as was 14CO2 production from 2- 14C-pyruvate and 5- 14C-glutamate. ATP and ADP concentrations were similar in all cell types with a rise in the ATP/ADP ratio during growth from log to confluent state. Thus, old and progeria cells, in contrast to young cells, produce more lactate during growth consistent with a rise in energy demand and/or inefficiency of oxidative phosphorylation. Although limitations in total energy output do not appear to be causal to the loss of replicative capacity in normal cells after serial passage, they could play a role in the curtailed replicative capacity of progeria cells.     

乙酸菌糖磷酸化作用的研究

对7种乙酸菌(Acetitomaculum ruminis, Acetobacterium woodii, Eubacterium limosum和分离株A2、A4、A10、H3HH)的葡萄糖和2脱氧葡萄糖磷酸化作用进行了研究。尽管所有机体都存在磷酸化反应,但它们在依赖PEP和ATP的比例上有实质性区别。分离菌株A10具有最高的依赖PEP的葡萄糖磷酸化活力(1162nmol·L-1·mg-1·min-1),A10、H3HH和E.limosum都具有葡萄糖磷酸转移酶系统(phosphotransferase systemPTS)。相反,Ａ.ruminis、A.woodii、A2和A4则不具有PTS活力。这七株菌的葡萄糖依赖ATP的磷酸化活力都高于依赖PEP的磷酸化活力,但其程度有所不同。A10和H3HH的葡萄糖PTS可通过胞外葡萄糖诱导,并且其比活在对数期随培养时间延长而增加。此外,还检测到A10和H3HH对麦芽糖和果糖的依赖ATP和PEP的磷酸化活力。     

Glucose transport in crabtree-positive and crabtree-negative yeasts

The kinetic parameters of glucose transport in four Crabtree-positive and four Crabtree-negative yeasts were determined. The organisms were grown in aerobic glucose-limited chemostats at a dilution rate of 0.1 h-1. The results show a clear correlation between the presence of high-affinity glucose transport systems and the absence of aerobic fermentation upon addition of excess glucose to steady-state cultures. The presence of these H+-symport systems could be established by determination of intracellular accumulation of 6-deoxy-[3H]glucose and alkalinization of buffered cell suspensions upon addition of glucose. In contrast, the yeasts that did show aerobic alcoholic fermentation during these glucose pulse experiments had low-affinity facilitated-diffusion carriers only. In the yeasts examined the capacity of the glucose transport carriers was higher than the actual glucose consumption rates during the glucose pulse experiments. The relationship between the rate of sugar consumption and the rate of alcoholic fermentation was studied in detail with Saccharomyces cerevisiae. When S. cerevisiae was pulsed with low amounts of glucose or mannose, in order to obtain submaximal sugar consumption rates, fermentation was already occurring at sugar consumption rates just above those which were maintained in the glucose-limited steady-state culture. The results are interpreted in relation with the Crabtree effect. In Crabtree-positive yeasts, an increase in the external glucose concentration may lead to unrestricted glucose uptake by facilitated diffusion and hence, to aerobic fermentation. In contrast, Crabtree-negative yeasts may restrict the entry of glucose by their regulated H+-symport systems and thus prevent the occurrence of overflow metabolism.     

Influence of Metronidazole, CO, CO(2), and Methanogens on the Fermentative Metabolism of the Anaerobic Fungus Neocallimastix sp. Strain L2

The effects of metronidazole, CO, methanogens, and CO(2) on the fermentation of glucose by the anaerobic fungus Neocallimastix sp. strain L2 were investigated. Both metronidazole and CO caused a shift in the fermentation products from predominantly H(2), acetate, and formate to lactate as the major product and caused a lower glucose consumption rate and cell protein yield. An increased lactate dehydrogenase activity and a decreased hydrogenase activity were observed in cells grown under both culture conditions. In metronidazole-grown cells, the amount of hydrogenase protein was decreased compared with the amount in cells grown in the absence of metronidazole. When Neocallimastix sp. strain L2 was cocultured with the methanogenic bacterium Methanobrevibacter smithii, the fermentation pattern changed in the opposite direction: H(2) and acetate production increased at the expense of the electron sink products lactate, succinate, and ethanol. A concomitant decrease in the enzyme activities leading to these electron sink products was observed, as well as an increase in the glucose consumption rate and cell protein yield, compared with those of pure cultures of the fungus. Low levels of CO(2) in the gas phase resulted in increased H(2) and lactate formation and decreased production of formate, acetate, succinate, and ethanol, a decreased glucose consumption rate and cell protein yield, and a decrease in most of the hydrogenosomal enzyme activities. None of the tested culture conditions resulted in changed quantities of hydrogenosomal proteins. The results indicate that manipulation of the pattern of fermentation in Neocallimastix sp. strain L2 results in changes in enzyme activities but not in the proliferation or disappearance of hydrogenosomes.     

Gas exchange across avian eggshells oscillates in phase with heartbeat

Rahn et al. (J. Appl. Physiol. 69: 1546-1548, 1990) showed that the gas pressure in a plethysmograph containing an intact egg oscillates in phase with electrocardiogram (ECG) and that this pressure variation could be used as a noninvasive way to determine the heart rate of an avian embryo. One possible mechanism to account for the pressure oscillation is the mechanical movement of the embryonic heart, which leads to volume shifts of gas within the plethysmograph. Another possibility is that the oscillation of gas pressure with heartbeat is pulsatile gas exchange resulting from pulsatile blood flow. If gas exchange were transiently stopped, a pressure signal dependent on gas exchange should disappear, while a pressure signal dependent on cardiovascular motion should persist. Using a number of late-age hen eggs (at days 15-20 of incubation), we tested these hypotheses by suddenly changing the gas composition surrounding an egg and measuring the effect of the pressure oscillation. We found that 1) after 5% CO2-95% N2 was flushed into the plethysmograph (presumably halting gas exchange), pressure oscillations went almost to zero and the ECG signal remained; after air was flushed back to the plethysmograph, the pressure signal returned to control level; 2) after 20% CO2-20% O2-60% N2 was flushed into the plethysmograph (presumably increasing net gas exchange), the pressure signal increased 2.5-fold compared with that in air; and 3) after 1% CO2-99% N2 was flushed into the plethysmograph (presumably reversing gas exchange), the oscillation pressure decreased to one-fourth of that in air and the phase of pressure relative to ECG reversed compared with the phase in air.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enrichment of CO2-fixing bacteria in cylinder-type electrochemical bioreactor with built-in anode compartment

Bacterial assimilation of CO2 into stable biomolecules using electrochemical reducing power may be an effective method to reduce atmospheric CO2 without fossil fuel combustion. For the enrichment of the CO2-fixing bacteria using electrochemical reducing power as an energy source, a cylinder-type electrochemical bioreactor with a built-in anode compartment was developed. A graphite felt cathode modified with neutral red (NR-graphite cathode) was used as a solid electron mediator to induce bacterial cells to fix CO2 using electrochemical reducing power. Bacterial CO2 consumption was calculated based on the variation in the ratio of CO2 to N2 in the gas reservoir. CO2 consumed by the bacteria grown in the electrochemical bioreactor (2,000 ml) reached a maximum of approximately 1,500 ml per week. Time-coursed variations in the bacterial community grown with the electrochemical reducing power and CO2 in the mineral-based medium were analyzed via temperature gradient gel electrophoresis (TGGE) of the 16S rDNA variable region. Some of the bacterial community constituents noted at the initial time disappeared completely, but some of them observed as DNA signs at the initial time were clearly enriched in the electrochemical bioreactor during 24 weeks of incubation. Finally, Alcaligenes sp. and Achromobacter sp., which are capable of autotrophically fixing CO2, were enriched to major constituents of the bacterial community in the electrochemical bioreactor.     

Influence of CO2 and low concentrations of O2 on fermentative metabolism of the rumen ciliate Dasytricha ruminantium

The effects of ruminal concentrations of CO2 and O2 on glucose-stimulated and endogenous fermentation of the rumen isotrichid ciliate Dasytricha ruminantium were investigated. Principal metabolic products were lactic, butyric and acetic acids, H2 and CO2. Traces of propionic acid were also detected; formic acid present in the incubation supernatants was found to be a fermentation product of the bacteria closely associated with this rumen ciliate. 13C NMR spectroscopy revealed alanine as a minor product of glucose fermentation by D. ruminantium. Glucose uptake and metabolite formation rates were influenced by the headspace gas composition during the protozoal incubations. The uptake of exogenously supplied D-glucose was most rapid in the presence of O2 concentrations typical of those detected in situ (i.e. 1-3 microM). A typical ruminal gas composition (high CO2, low O2) led to increased butyrate and acetate formation compared to results obtained using O2-free N2. At a partial pressure of 66 kPa CO2 in N2, increased cytosolic flux to butyrate was observed. At low O2 concentrations (1-3 microM dissolved in the protozoal suspension) in the absence of CO2, increased acetate and CO2 formation were observed and D. ruminantium utilized lactate in the absence of extracellular glucose. The presence of both O2 and CO2 in the incubation headspaces resulted in partial inhibition of H2 production by D. ruminantium. Results suggest that at the O2 and CO2 concentrations that prevail in situ, the contribution made by D. ruminantium to the formation of ruminal volatile fatty acids is greater than previously reported, as earlier measurements were made under anaerobic conditions.     

Cultivation of microplantlets derived from the marine red alga Agardhiella subulata in a stirred tank photobioreactor

Macrophytic marine red algae are a diverse source of bioactive natural compounds. "Microplantlet" suspension cultures established from red algae are potential platforms for biosynthesis of these compounds, provided suitable bioreactor configurations for mass culture can be identified. The stirred tank bioreactor offers high rates of gas-liquid mass transfer, which may facilitate the delivery of the CO(2) in the aeration gas to the phototrophic microplantlet suspension culture. Therefore, the effects of impeller speed and CO(2) delivery on the long-term production of microplantlet biomass of the model red alga Agardhiella subulata was studied within a stirred tank photobioreactor equipped with a paddle blade impeller (D(i)/D(T) = 0.5). Nutrient medium replacement was required for sustained biomass production, and the biomass yield coefficient based on nitrate consumption was 1.08 +/- 0.09 g dry biomass per mmol N consumed. Biomass production went through two exponential phases of growth, followed by a CO(2) delivery limited growth phase. The CO(2)-limited growth phase was observed only if the specific growth rate in the second exponential phase of growth was at least 0.03 day(-)(1), the CO(2) delivery rate was less than 0.258 mmol CO(2) L(-)(1) culture h(-)(1), and the plantlet density was at least 10 g fresh mass L(-)(1). Increasing the aeration gas CO(2) partial pressure from 0.00035 to 0.0072 atm decreased the cultivation pH from 8.8 to 7.8, prolonged the second exponential phase of growth by increasing the CO(2) delivery rate, and also increased the photosynthetic oxygen evolution rate. Impeller speeds ranging from 60 to 250 rpm, which generated average shear rates of 2-10 s(-)(1), did not have a significant effect on biomass production rate. However, microplantlets cultivated in a stirred tank bioreactor ultimately assumed compact spherical shape, presumably to minimize exposure to hydrodynamic stress.     

Calorimetric and stoichiometric analysis of growth of Kluyveromices fragilis in continuous culture: nitrogen limitation imposed upon carbon-limited growth

The calorimetric response of the yeast Kluyveromices fragilis was investigated for growth in continuous culture where nitrogen limitation was imposed on a carbon-limited culture. Calorimetric measurements were combined with off gas analysis, measurements of biomass, substrate and product concentrations, elemental biomass composition, and heat production to study the physiological response of K. fragilis. Regions where both carbon and nitrogen limited growth, were found over a broad range of dilution rates and feed carbon-to-nitrogen ratios. The principle mechanism by which K. fragilis accommodated regions of dual carbon and nitrogen limitation was by partial decoupling of the anabolic and catabolic pathways. When the culture was only nitrogen-limited, increased decoupling of the two pathways was observed. The principal effect of the decoupling was an increased catabolic consumption of glucose, generating an increased heat yield. The preferred way to process the excess glucose was through respiration but the cells were also capable of fermenting a small percentage of the excess glucose in specific cases where the dissolved oxygen partial pressure approached zero. In addition, these results were qualitatively compared to similar studies on Saccharomices cerevisiae. The two yeasts were similar in their ability to accommodate dual limitation by uncoupling anabolic biomass formation from substrate consumption. The two yeasts were dissimilar in how the catabolic substrate was processed. For S. cerevisiae the presence of a bottleneck in the respiration pathway dictated that the majority of the catabolic glucose consumption was by fermentation. For K. fragilis, the lack of a bottleneck in the respiration pathway dictated that the majority of catabolic glucose substrate consumption was by respiration.     

Interaction between CO2-mass transfer, light availability, and hydrodynamic stress in the growth of phaeodactylum tricornutum in a concentric tube airlift photobioreactor

The microalga Phaeodactylum tricornutum was grown in a concentric tube airlift photobioreactor. A maximum specific growth rate of 0. 023 h-1 was obtained using a superficial gas velocity around 0.055 m/s. Lower or higher gas flow rates limited the culture performance. To establish if the observed limitation was due to CO2 or to the photosynthetically active irradiance, characteristic times for mixing, mass transfer and CO2 consumption, and the photon flux absorbed by the culture were analyzed. The CO2-gradients in the culture were shown to be responsible for the limitation during the exponential growth phase, and both CO2 and light irradiance were limiting in the linear growth phase. The decrease in specific growth rate relative to the maximum was found to be related to the specific gas-liquid interfacial area, the length scale of the microeddies and the shear rate. Copyright 1998 John Wiley & Sons, Inc.     

Effect of L-amino acids on Mucor rouxii dimorphism.

Mucor rouxii organisms growing aerobically and exponentially on a well-defined minimal medium are able to differentiate as yeasts or as mycelia, depending on the amino acid as the nitrogen source. When certain amino acids were used as the nitrogen source, spores differentiated only as hyphae, whereas other amino acids gave rise to other morphological forms having different ratios of yeasts to hyphae. In both hyphal and yeast cultures, an aerobic metabolism was predominant, as shown by determining several metabolic parameters such as oxygen tension, glucose consumption, ethanol production, and CO2 release. A complete conversion of yeasts to hyphae was obtained by the appropriate change in the amino acid used as nitrogen source. By preparing spheroplasts from mycelial cultures and transferring them to media with amino acids that induce yeast formation, a 50% yield in the reverse transformation was achieved. A correlation between the change in pH of the medium and cell morphology was observed in different growth conditions. Decrease in the pH of the medium preceded the appearance of hyphae. Also, when the initial pH of the medium was increased, aspartate-containing cultures developed mainly as mycelia, instead of yeasts, with a corresponding decrease in the final pH.     

Response to high-pressure, low-temperature treatment in vegetables: determination of survival rates of microbial populations using flow cytometry and detection of peroxidase activity using confocal microscopy

Application of high hydrostatic pressure (200, 300, 350 and 400 MPa) at 5 degrees C for 30 min to different micro-organisms, including Gram-positive and Gram-negative bacteria, moulds and yeasts, proved to be more effective in inactivating these organisms than treatments at 20 degrees C for 10 min and at 10 degrees C for 20 min. Moulds, yeasts, Gram-negative bacteria and Listeria monocytogenes were most sensitive, and their populations were completely inactivated at pressures between 300 and 350 MPa. The same conditions of pressure, temperature, and time were applied to different vegetables (lettuce, tomato, asparagus, spinach, cauliflower and onion), achieving reductions of from 2-4 log units in both viable mesophiles and moulds and yeasts at pressures of between 300 and 400 MPa. Sensory characteristics were unaltered, especially in asparagus, onion, tomato and cauliflower, though slight browning was observed in cauliflower at 350 MPa. Flow cytometry was applied to certain of the microbial populations used in the above experiment before and after the pressurization treatment. The results were indicative of differing percentage survival rates depending on micro-organism type, with higher survival rates for Gram-positive bacteria, except L. monocytogenes, than in the other test micro-organisms. Growth of survivors was undetectable using the plate count method, suggesting that micro-organisms suffering from pressure stress were metabolically inactive though alive. The pressurization treatments did not inactivate the peroxidase responsible for browning in vegetables. Confocal microscopic examination of epidermal tissue from onion showed that the enzyme had been displaced to the cell interior. Use of low temperatures and moderately long pressurization times yielded improved inactivation of micro-organisms and better sensorial characteristics of the vegetables, and should lower industrial costs.     

Identification of mass-transfer parameters and process simulation of SCP production process in airlift tower reactors with an external loop

A distributed parameter model for simulation of SCP-production processes in tower reactors with an outer loop was developed by considering substrate, cell, and CO(2) balances in the liquid phase, and O(2) and CO(3) balances in the ges phase and taking into account variations of dissolved oxygen concentration, pressure, and k(L)a along the column, as well as double substrate Monod kinetics. This model was used to describe the cultivation of Hansenula polymorpha in a tower-loop reactor (height 275 cm, diameter 15 cm). Parameter identification and process simulation were carried out by a hybrid computer. The variation of identified mass transfer parameters with fermentation time and operation mode is considered employing ethanol and glucose substrate, respectively. Relationships among k(L)a, substrate concentration, and superficial gas velocity were developed to facilitate the layout and simulation of pilot-plant reactors.     

H2/CO2 metabolism in acetogenic bacteria isolated from the human colon

The present work reports on autotrophic metabolism in four H2/CO2-utilizing acetogenic bacteria isolated from the human colon (two Clostridium species, one Streptococcus species, and Ruminococcus hydrogenotrophicus). H2/CO2-utilization by these human acetogenic strains occurred during both exponential and stationary phases of growth. Acetate was the major metabolite produced by all isolates following the stoichiometric equation of reductive acetogenesis. Furthermore, the ability of these acetogenic bacteria to incorporate 13CO2 into acetate in the presence of H2 in the gas phase demonstrated the utilization of the reductive pathway of acetate formation from a one-carbon compound. Energy conservation during the autotrophic metabolism in colonic acetogens might involve sodium- or proton-chemiosmotic mechanisms. A sodium-dependent ATP generation was only demonstrated in one Clostridium species, whereas sodium could be replaced by potassium in other strains. The minimal thresholds of hydrogen uptake were determined and varied from 1100 to 3680 ppm depending on the acetogenic strain. These values appeared higher than those measured for the colonic methanogen,Methanobrevibacter smithii.