Energetics of Saccharomyces cerevisiae CBS 426: Comparison of anaerobic and aerobic glucose limitation

Saccharomyces cerevisiae CBS 426 was grown aerobically and anaerobically in a glucose-limited chemostat. The flows of biomass, glucose, ethanol, carbon dioxide, oxygen, glycerol, and the elemental composition of the biomass were measured. Models for anaerobic and aerobic growth are constructed. Values for Y_ATP and P/O are obtained from continuous culture data for aerobic growth; this Y_ATP value is compared with that obtained from the anaerobic growth results. The ratio between the heat produced and the oxygen consumed increases if more glucose in fermented to ethanol and carbon dioxide. An equation for ?_H/?_O as a function of the respiratory quotient is given.     

Data consistency,yield and maintenance coefficient for the growth of Candida lypoytica and Pseudomonas aeruginosa on n-hexadecane

Summary When more than the minimum number of variables are measured, and measurement error is taken into account, the results of parameter estimation depend on which of the measured variables are selected for this purpose. The reparameterization of Pirt's models for growth produces multiresponse models with common parameters. By using the covariate adjustment technique, a unit variate linear model with covariates is obtained. This allows a combined point and interval estimates of biomass energetic yield and maintenance coefficient to be obtained using standard multiple regression programmes. When this method was applied using form I and form II of the Pirt's models, good combined estimates were obtained and compared. Using data from the literature for Candida lipolytica produced reliable results. However, for Pseudomonas aeruginosa, which has been known to produce intermediate products, a modified Pirt's model is required for a good estimate of the biomass energetic yield.Nomenclature a Mole of ammonia per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - b Moles of oxygen per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - c Moles of water per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon; no of covariates included in model - d Moles of carbon dioxide per quantity of organic substrate containing 1 g atom carbon, g mole/g atom carbon - e _i Error terms in Eqs. (6–8) - l Atomic ratio of oxygen to carbon in organic substrate, dimensionless - m Atomic ratio of hydrogen to carbon in organic substrate, dimensionless - m _e Rate of organic substrate consumption for maintenance, g equiv. of available electrons in biomass (h) or kcal/Kcal of biomass(h) - n Atomic ratio of oxygen to carbon in biomass, dimensionless - p Atomic ratio of hydrogen to carbon in biomass, dimensionless - Q _{CO 2} Rate of evolution of carbon dioxide, g moles/g dry wt (h) - Q _{O 2} Rate of oxygen consumption, g moles/g dry wt (h) - Q _s Rate of organic substrate consumption g/g dry wt (h) - q Atomic ratio of nitrogen to carbon in biomass, dimensionless - r Atom ratio of hydrogen to carbon in products, dimensionless; the number of parameters of interest - s Atomic ratio of oxygen to carbon in products, dimensionless - t Atomic ratio of nitrogen to carbon in products, dimensionless - r Mean of k responses in Eq. (10) - x _ki Kth response in the ith observation - y _c Biomass carbon yield (fraction of organic substrate carbon in biomass), dimensionless - z _i Covariate matrix - z Fraction of organic substrate carbon in products, dimensionless - a _i Parameters associated with covariates - _s Reductance degree of biomass, equivalents of available electrons per gram atom carbon - Reductance degree of organic substrate, equivalents of available electrons per gram atom carbon - Fraction of energy in organic substrate which is evolved as heat, dimensionless - Fraction of available electrons transferred to biomass; biomass energetic yield - True growth yield - Specific growth rate, h^-1 - _p Fraction of available electrons incorporated into products; product energetic yield - Correlation coefficient - Mass fraction carbon - ² Mean square error of model (10)     

Modeling of microbial substrate conversion,growth and product formation in a recycling fermentor

Paracoccus denitrificans and Bacillus licheniformis were grown in a carbon- and energy source-limited recycling fermentor with 100% biomass feedback. Experimental data for biomass accumulation and product formation as well as rates of carbon dioxide evolution and oxygen consumption were used in a parameter optimization procedure. This procedure was applied on a model which describes biomass growth as a linear function of the substrate consumption rate and the rate of product formation as a linear function of the biomass growth rate. The fitting procedure yielded two growth domains for P. denitrificans. In the first domain the values for the maximal growth yield and the maintenance coefficient were identical to those found in a series of chemostat experiments. The second domain could be described best with linear biomass increase, which is equal to a constant growth yield. Experimental data of a protease producing B. licheniformis also yielded two growth domains via the fitting procedure. Again, in the first domain, maximal growth yield and maintenance requirements were not significantly different from those derived from a series of chemostat experiments. Domain 2 behaviour was different from that observed with P. denitrificans. Product formation halts and more glucose becomes available for biomass formation, and consequently the specific growth rate increases in the shift from domain 1 to 2. It is concluded that for many industrial production processes, it is important to select organisms on the basis of a low maintenance coefficient and a high basic production of the desired product. It seems less important that the maximal production becomes optimized, which is the basis of most selection procedures.     

The effects of carbon sources and micronutrients in fermented whey on the biodegradation of n-hexadecane in diesel fuel contaminated soil

Fermented whey has previously been shown to stimulate biodegradation of n-hexadecane in diesel contaminated soils. The proposed explanation for the stimulatory effect is that fermented whey provides easily accessible carbon and micronutrients, which give rise to an increased degrading biomass.The objective of this work has been to investigate the role of the different carbon sources and vitamins in fermented whey on the microbial degradation of n-hexadecane in soil.The effects of lactose, lactate, vitamins and free amino acids were tested in combinations according to a full factorial design experiment, at concentrations corresponding to those present in fermented whey. The target substance was ¹⁴C-labeled n-hexadecane in nutrient amended soil microcosms contaminated with 5000 mg diesel fuel kg⁻¹ dw. Biodegradation was monitored by determination of evolved ¹⁴CO₂.Significant effects on the biodegradation of n-hexadecane were observed for lactate and amino acids additions in a sandy soil. Lactate showed both an inhibitory effect in the early phase of the experiment and a stimulatory effect in the later phase. The effect of amino acids was slightly stimulatory, mainly evident as a shortening of the lag time.The degree of n-hexadecane degradation at the end of the experiment was correlated with the total concentration of organic compounds added to the soil.

Scientific relevance

There are a handful papers describing the potential of using organic amendments (often industrial by-products) with a content of both easily accessible carbon and micronutrients, to enhance the bioremediation of polluted soils. Enhanced biodegradation is often reported and the proposed explanations are that the combination of easily accessible carbon and micronutrients increases the degrading biomass.In this paper, we examine the effect of fermented whey on the degradation of n-hexadecane and correlate the observed effects on the biodegradation with the main components lactate, amino acids, lactose and B-vitamins. This has to our knowledge never been done before.     

Indirect estimation of cell mass and substrate concentration using a computer-coupled mass spectrometer

On-line estimation of cell mass and substrate concentration based on exhaust gas analysis was developed. The O₂, CO₂, H₂O, and N₂ contents at the inlet and outlet of fermentor, analyzed by a computer-coupled quadrupole mass spectrometer, were used to calculate the oxygen uptake rate and carbon dioxide evolution rate, and these rates were further used to evaluate cell mass and substrate concentration in a recombinant Escherichia coli fermentation. Cell mass, glucose concentration, specific growth rate, and specific consumption rate of glucose were well estimated by this method; the oxygen uptake rate gave more accurate estimates for these state variables than did the carbon dioxide evolution rate.     

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures

The ferrous iron oxidation kinetics of Thiobacillus ferrooxidans in batch cultures was examined, using on-line off-gas analyses to measure the oxygen and carbon dioxide consumption rates continuously. A cell suspension from continuous cultures at steady state was used as the inoculum. It was observed that a dynamic phase occurred in the initial phase of the experiment. In this phase the bacterial ferrous iron oxidation and growth were uncoupled. After about 16 h the bacteria were adapted and achieved a pseudo-steady state, in which the specific growth rate and oxygen consumption rate were coupled and their relationship was described by the Pirt equation. In pseudo-steady state, the growth and oxidation kinetics were accurately described by the rate equation for competitive product inhibition. Bacterial substrate consumption is regarded as the primary process, which is described by the equation for competitive product inhibition. Subsequently the kinetic equation for the specific growth rate, μ, is derived by applying the Pirt equation for bacterial substrate consumption and growth. The maximum specific growth rate, μ _max, measured in the batch culture agrees with the dilution rate at which washout occurs in continuous cultures. The maximum oxygen consumption rate, q _O2,max, of the cell suspension in the batch culture was determined by respiration measurements in a biological oxygen monitor at excess ferrous iron, and showed changes of up to 20% during the course of the experiment. The kinetic constants determined in the batch culture slightly differ from those in continuous cultures, such that, at equal ferric to ferrous iron concentration ratios, biomass-specific rates are up to 1.3 times higher in continuous cultures. Received: 8 February 1999 / Accepted: 17 February 1999     

Utilization of mass–energy balance regularities in the analysis of continuous-culture data

Material and energy balances for continuous-culture processes are described based on the facts that the heat of reaction per electron transferred to oxygen for a wide variety of organic molecules, the number of available electrons per carbon atom in biomass, and the weight fraction carbon in biomass are relatively constant. Energy requirements for growth and maintenance are investigated and related to the biomass energetic yield. The consistency of experimental data is examined using material and energy balances and the regularities identified above. When extracellular products are absent, the consistency of yield models containing separate terms for growth and maintenance may be investigated using organic substrate consumption, biomass production, oxygen consumption (or heat evolution), and carbon dioxide evolution rate data for a series of dilution rates. The consistency of continuous-culture data in the published literature is examined.     

Production of Fluorescent Compounds from Guanine by Microorganisms

One species of hydrocarbon utilizing bacteria was isolated from soil. This strain was named as Achromobacter petrophilum No. 4017. This bacterial species utilizes normal hydrocarbons with carbon chains of nC₁₀ to nC₁₈, but does not utilize glucose or other carbohydrates. Achromobacter petrophilum forms small amounts of green-yellow, green-blue and violet fluorescent compounds in the medium containing n-hexadecane (nC₁₆) as a carbon source. The mutant strain, No. 4510, which requires hypoxanthine and thiamine for growth, was obtained from Achromobacter petrophilum No. 4017 by ultraviolet irradiation and formed considerable amounts of green-yellow fluorescent compound by the addition of guanine to the n-hexadecane medium. This fluorescent compound was crystallized from culture broth.     

A quantitative description of the growth of Saccharomyces cerevisiae CBS 426 on a mixed substrate of glucose and ethanol

Saccharomyces cerevisiae CBS 426 was grown aerobically in continuous culture with a mixture of glucose and ethanol as the carbon source. The flows of biomass, glucose, ethanol, oxygen, and carbon dioxide were measured. A model for growth with two substrates was derived. Application of this model to the above-mentioned system yielded values for Y_ATP and P/O. The joint confidence regions for these parameters were calculated. The relevance to industrial production of bakers' yeast is discussed.     

Bubble free gaseous transfer in bioreactors using perfluorocarbons

In this work the concept of bubble-free oxygenation that was able to ensure oxygen supply and carbon dioxide extraction for a chemostat culture of Escherichia coli was experimentally demonstrated. It was operated at the dilution rate of 0.275 h^–1 under atmospheric pressure and at 35.5 °C. Foralkyl, a commercial perfluorocarbon, added in the influent medium under emulsified form and at 50% volumic fraction, was able to provide 0.17 g O₂/l/h and extract 0.23 g CO₂/l/h for a culture at 0.74 g/l of biomass. This oxygen supply was close to the maximum oxygenation Foralkyl was theoretically able to provide at this pressure when imposing a minimum oxygen concentration of 1 mg/l in the water phase. The quantification of transfer was not done from a direct measurement of oxygen transfer rates because conventional oxygen concentration measurement by membrane polarographic probe in an emulsion was not judged reliable. This evaluation was done by referring to conventional aerated culture whose measurable parameters (biomass and product concentrations) were found unaffected when shifting to the novel oxygenation device.List of Symbols C _LV ^* , C _LV g/l dissolved oxygen concentration in the vector phase at equilibrium and in the reactor - C _LW ^* , C _LW g/l dissolved oxygen concentration in the water phase at equilibrium and in the reactor - C _LWinput , C _LWoutput g/l dissolved oxygen concentrations in aqueous medium in the reactor input and output flow - D h^–1 dilution rate - ^DMM_{o 2}, MM_{co 2} g/mol oxygen and carbon dioxide molar masses - %O_2input, %O_2output oxygen percentages in the reactor input and output flow - %CO_2input, %CO_2output carbon dioxide percentages in the reactor input and output flow - %N _2input %N _2output nitrogen percentages in the reactor input and output flow - Q _G.i ⁿ Q _G.o ⁿ l/h gas flow rates at the reactor input and output at normal conditions (273 K, 1 atm) - Q _L l/h liquid flow rate - Q _LW , Q _LV l/h water and vector flow rates - r_{O 2} g/l/h oxygen consumption rate - r _x g/l/h biomass production rate - r _{CO 2} g/l/h carbon dioxide production rate - V _L l fermentor aqueous volume - V _LW , V _LV l water phase and vector phase volume - V _{O 2}, V _{CO 2}, l/mol oxygen and carbon dioxide molar volume under gaseous form at normal conditions (273 K, 1 atm) - Y _{O 2 x} gO₂/g cell oxygen consumption yield for biomass growth - Y _sx g glucose/g cell glucose consumption yield for biomass growth - vector volumic fraction - h^–1 growth rate This work was totally financed by the European Space Agency.     

Über den Einfluß niedriger und hoher O2-Partialdrucke auf den Sauerstoff- und Kohlendioxidumsatz von Amaranthus und Phaseolus während der Lichtphase

Summary Carbon dioxide and oxygen gas exchange of illuminated Amaranthus and Phaseolus leaves was measured from 0–600 ppm of CO₂ in an open system.At low oxygen concentration (2% O₂) the ratio of CO₂ uptake to O₂ evolution came close to 1.At high oxygen partial pressure (42% O₂) the O₂ compensation point of an Amaranthus leaf was increased and oxygen evolution was depressed. Accordingly the CO₂/O₂ quotients were variable; the lowest value of 1,9 differed significantly from 1,0.The oxygen and carbon dioxide compensation points of a Phaseolus leaf were increased at high oxygen concentration (42% O₂) and oxygen evolution as well as carbon dioxide uptake were reduced. Therefore the ratios CO₂ over O₂ varied and differed greatly from 1,0.It was concluded that the nature of photosynthates is regulated by the gas composition around the leaves.     

Effects of oxygen on Pseudomonas nautica growth on n-alkane with or without nitrate

The denitrifying marine bacterium, Pseudomonas nautica 617, can grow on lactate aerobically or anaerobically in presence of nitrate with generation times of 1.5 and 3 h respectively. The growth on heptadecane occurs only in presence of oxygen whatever its concentration with a genrration time of 8.5 h. The influence of oxygen, carbon sources (lactate or heptadecane) and nitrate was examined on O₂, NO₃ ^-, NO₂ ^- consumption, on nitrate and nitrite reductases activities, on cell yields, and on the ratio of CO₂ produced per unit of biomass. Pseudomonas nautica metabolizes hydrocarbons under denitrifying conditions in the presence of oxygen. Nitrate and nitrite are used during growth on lactate and heptadecane up to oxygen concentrations corresponding to 50 and 30% of air-saturation, respectively. When growth on n-alkane was not oxygen-limited (above 50% of air-saturation) the catabolism decreases in favour of carbon incorporation into the cell. Nitrate and nitrite reductases were strongly inhibited after 20% of airsaturation in the presence of lactate as growth substrate. With n-alkane, only the nitrate reductase activity was greatly reduced.     

The effects of carbon sources and micronutrients in fermented whey on the biodegradation of n-hexadecane in diesel fuel contaminated soil

Fermented whey has previously been shown to stimulate biodegradation of n-hexadecane in diesel contaminated soils. The proposed explanation for the stimulatory effect is that fermented whey provides easily accessible carbon and micronutrients, which give rise to an increased degrading biomass.The objective of this work has been to investigate the role of the different carbon sources and vitamins in fermented whey on the microbial degradation of n-hexadecane in soil.The effects of lactose, lactate, vitamins and free amino acids were tested in combinations according to a full factorial design experiment, at concentrations corresponding to those present in fermented whey. The target substance was ¹⁴C-labeled n-hexadecane in nutrient amended soil microcosms contaminated with 5000 mg diesel fuel kg⁻¹ dw. Biodegradation was monitored by determination of evolved ¹⁴CO₂.Significant effects on the biodegradation of n-hexadecane were observed for lactate and amino acids additions in a sandy soil. Lactate showed both an inhibitory effect in the early phase of the experiment and a stimulatory effect in the later phase. The effect of amino acids was slightly stimulatory, mainly evident as a shortening of the lag time.The degree of n-hexadecane degradation at the end of the experiment was correlated with the total concentration of organic compounds added to the soil.Scientific relevanceThere are a handful papers describing the potential of using organic amendments (often industrial by-products) with a content of both easily accessible carbon and micronutrients, to enhance the bioremediation of polluted soils. Enhanced biodegradation is often reported and the proposed explanations are that the combination of easily accessible carbon and micronutrients increases the degrading biomass.In this paper, we examine the effect of fermented whey on the degradation of n-hexadecane and correlate the observed effects on the biodegradation with the main components lactate, amino acids, lactose and B-vitamins. This has to our knowledge never been done before.     

Protein-like Activator for n-Alkane Oxidation by Pseudomonas aeruginosa S7B1

During the study of the n-alkane oxidation by Pseudomonas aeruginosa S₇B₁, a nondialysable activator for n-alkane oxidation was discovered in the culture broth of the strain. The activator was purified, as judged by cellulose acetate membrane electrophoresis, by ammonium sulfate precipitation and chromatography on DEAE-Sephadex, CM-Sephadex and Sephadex G-75 columns.

The purified activator, which was positive in protein color reactions, remarkable stimulated only the oxidation of n-hexadecane, though it was not observed in case of palmitic acid or glucose oxidation.

Co-operative action between the activator and rhamnolipid, which had been isolated as a growth stimulant of P. aeruginosa S₇B₁ on n-hexadecane by the authors, was observed not only in the oxidation of n-hexadecane but also in the growth on n-hexadecane.     

Carbon dioxide inhibition of yeast growth in biomass production

Saccharomyces cerevisiae was grown under aerobic and substrate-limiting conditions for efficient biomass production. Under these conditions, where the sugar substrate was fed incrementally, the growth pattern of the yeast cells was found to be uniform, as indicated by a constant respiratory quotient during the entire growing period. The effect of carbon dioxide was investigated by replacing portions of the nitrogen in the air stream with carbon dioxide, while maintaining the oxygen content at the normal 20% level, so that identical oxygen transfer rate and atmospheric pressure were maintained for all experiments with different partial pressures of carbon dioxide. Inhibition of yeast growth was negligible below 20% CO₂ in the aeration mixture. Slight inhibition was noted at the 40% CO₂ level and significant inhibition was noted above the 50% CO₂, level, corresponding to 1.6 × 10^?2M of dissolved CO₂ in the fermentor broth. High carbon dioxide content in the gas phase also inhibited the fermentation activity of baker's yeast.     

Oxygen effect on batch cultures of Leuconostoc mesenteroides: relationship between oxygen uptake,growth and end-products

Growth and lactose metabolism of a Leuconostoc mesenteroides strain were studied in batch cultures at pH 6.5 and 30° C in 101 modified MRS medium sparged with different gases: nitrogen, air and pure oxygen. In all cases, growth occurred, but in aerobiosis there was oxygen consumption, leading to an improvement of growth yield Y _x/s and specific growth rate compared to anaerobiosis. Whatever the extent of aerobic growth, oxygen uptake and biomass production increased with the oxygen transfer rate so that the oxygen growth yield, Y _x/o2, remained at a constant value of 11 g dry weight of biomass/mol oxygen consumed. Pure oxygen had a positive effect on Leuconostoc growth. Oxygen transfer was limiting under air, but pure oxygen provided bacteria with sufficient dissolved oxygen and leuconostocs were able to consume large amounts of oxygen. Acetate production increased progressively with oxygen consumption so that the total molar concentration of acetate plus ethanol remained constant. Maximal Y _x/s was obtained with a 120 l/h flow rate of pure oxygen: the switch from ethanol to acetate was almost complete. In this case, a 46.8 g/mol Y _x/s and a 0.69 h^–1 maximal growth rate could be reached.     

Contribution of the polychaete,Neanthes japonica (Izuka), to the oxygen uptake and carbon dioxide production of an intertidal mud-flat of the Nanakita River estuary,Japan

The benthic oxygen consumption and carbon dioxide production of undisturbed and sieved sediment cores with various values for the biomass of polychaetes collected from the intertidal mud-flat of Nanakita River estuary of Japan were measured simultaneously. The benthic oxygen consumption and carbon dioxide production increased in proportion to the biomass of a dominant polychaete species Neanthes japonica (Izuka). This increase was not explained by the respiration of the animals alone. The residual increase in benthic O₂ and CO₂ fluxes may be due to mineralization processes in the burrow wall and enhanced diffusion caused by the pumping activity of the worms. From the average biomass of polychaetes at the study site, total benthic O₂ and CO₂ fluxes were estimated to be 5.2 mmol·m⁻²·h⁻¹ and 7.3 mmol·m⁻²·h⁻¹, respectively, at 20 ° C. The worms were responsible for 79% of the total O₂ flux and 73% of the total CO₂ flux but the respiration of the worms accounted for only 53% of the total O₂ flux and 36% of the total CO₂ flux. The residual enhanced fluxes were 26% and 37% for the total O₂ and CO₂ fluxes, respectively.     

Production of biomass from untreated orange peel by Fusarium avenaceum

Summary The growth behaviour of Fusarium avenaceum (Sect. Roseum Wr.) in slurry fermentation systems using untreated orange peel as substrate was studied in a laboratory-fermenter scale to reproduce the results obtained in a shakenflask fermenter. The eventual effect of impeller speed on mechanical disruption of mycelial hyphae was then assessed by determining mycelial growth, total reducing sugars consumption, TOC reduction, carbon dioxide evolution and oxygen absorption rates. In particular, the main biomass yield coefficient, as well as the apparent specific growth rate, appeared to be independent of the impeller speed, at least within the experimental range of 450 and 900 min^–1 (equivalent to peripheral impeller speeds of 3.8–7.5 m sec^–1.     

Respiratory activity of the bacteriumAcinetobacter calcoaceticus TM-31 during assimilation of alkane hydrocarbons

The respiratory activity ofAcinetobacter calcoaceticus TM-31 with resect to alkane hydrocarbons was studied. The dynamics of oxygen consumption by the cells while assimilatingn-hexadecane was assayed by a modified technique using an oxygen electrode. The dependence of cell respiratory activity on the amount ofn-hexadecane within the concentration range of 0.03–0.66% was determined. It was demonstrated that the cells also displayed respiratory activity towards other medium-chainn-alkanes: hexane, octane, decane, tridecane, and heptadecane. Thus, we demonstrated the possibility of determining alkanes by measuring the respiratory activities of microorganisms.     

Synthesis of dl-Phyllostine

“Protein-like activator (PA)” for n-alkane oxidation was formed by Pseudomonas aeruginosa S₇B₁ from long-chain n-alkanes, 1-hexadecene and cetyl alcohol but not from glucose, glycerol and palmitic acid. The molecular weight and the total amino acid residues of PA were estimated at about 14,300 and 147, respectively. PA was relatively stable to low pH and high temperature, and completely inactivated upon heating at 98°C for 45 min. The cultural fluid obtained from n-hexadecane medium stimulated the growth of the strain on n-hexadecane. The degree of the growth stimulation by the fluid depended on the amount of PA and rhamnolipid (RL) in the fluid. The heat-treated PA lost the growth-stimulaing effect and the emulsifying power on the n-hexadecane medium in the presence of RL.