Measurement of heat evolution and correlation with oxygen consumption during microbial growth

A procedure for measuring the rate of heat production from a fermentation has been developed. The method is based on measuring the rate of temperature rise of the fermentation broth resulting from metabolism, when the temperature controller is turned off. The heat accumulation measured in this manner is then corrected for heat losses and gains. A sensitive thermistor is used to follow the temperature rise with time. This procedure is shown to be as accurate as previous methods but much simpler in execution. Using this technique, the rate of heat production during metabolism was found to correlate with the rate of oxygen consumption. Experiments were performed using bacteria (E. coli and B. subtilis), a yeast (C. intermedia), and a mold (A. niger). The substrates investigated included glucose, molasses, and soy bean meal. The proportionality constant for the correlation is independent of the growth rate, slightly dependent on the substrate, and possibly dependent On the type of organism growth. This correlation has considerable potential for predicting heat evolution from the metabolism of microorganisms on simple or complex substrates and providing quantitative parameters necessary for heat removal calculations.     

The effect of succinate on respiration, transamination, and pyruvate formation in cells of the yeast Dipodascus magnusii

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

The Effect of Succinate on Respiration,Transamination, and Pyruvate Formation in Cells of the Yeast <Emphasis Type="Italic">Dipodascus magnusii</Emphasis>

The effect of succinate on the growth and respiration of the yeast Dipodascus magnusii VKM Y-1072, which is auxotrophic for thiamine and biotin, was studied. The addition of succinate to a culture grown on glucose was found to activate the respiration of cells on various substrates by enhancing the processes related to transamination reactions. In this case, aerobic fermentation (ethanol production) decreased, whereas pyruvate production increased. When succinate was added to the medium as the sole carbon source, it supported yeast growth in the absence of one of the two vitamins, thiamine or biotin, but not both. The yeast metabolism was completely respiratory, without any signs of aerobic fermentation. A drastic rise in pyruvate production in the yeast grown on glucose in the presence of succinate and the absence of biotin are also indicative of metabolic changes.     

Involvement of nitrogen metabolism in the triggering of ethanol fermentation in aerobic chemostat cultures of Saccharomyces cerevisiae.

We have investigated whether central nitrogen metabolism may influence the triggering of ethanol fermentation in Saccharomyces cerevisiae strain CEN.PK122 grown in the presence of different N-sources (ammonia, glutamate, or glutamine) under conditions in which the carbon to nitrogen (C : N) ratio was varied. An exhaustive quantitative evaluation of yeast physiology and metabolic behavior through metabolic flux analysis (MFA) was undertaken. It is shown that ethanol fermentation is triggered at dilution rates, D (growth rate), significantly lower (D=0.070 and 0.074 h(-1) for glutamate and glutamine, respectively, and D=0.109 h(-1) for ammonia) under N- than C-limitation (approximately 0.18 h(-1) for all N-sources). A characteristic specific rate of glucose influx, q(Glc), for each N-source at Dc, i.e., just before the onset of respirofermentative metabolism, was determined (approximately 2.0, 1.5, and 2.5, for ammonia, glutamate, and glutamine, respectively). This q(Glc) was independent of the nutritional limitation though dependent on the nature of the N-source. The onset of fermentation occurs when this "threshold q(Glc)" is overcome. The saturation of respiratory activity appears not to be associated with the onset of fermentation since q(O(2)) continued to increase after Dc. It was remarkable that under respirofermentative conditions in C-limited chemostat cultures, the glucose consumed was almost completely fermented with biomass being synthesized from glutamate through gluconeogenesis. The results obtained show that the enzyme activities involved in central nitrogen metabolism do not appear to participate in the control of the overflow in carbon catabolism, which is driven toward ethanol production. The role of nitrogen metabolism in the onset of ethanol fermentation would rather be realized through its involvement in setting the anabolic fluxes directed to nitrogenous macromolecules. It seems that nitrogen-related anabolic fluxes would determine when the threshold glucose consumption rate is achieved after which ethanol fermentation is triggered.     

生物热对传统固态发酵菌群演替及其代谢影响的研究进展

解析传统固态发酵中产生的生物热对微生物菌群代谢的影响,是认识发酵机制、调控发酵过程、保证发酵效率的关键之一。固态发酵过程中,微生物菌群代谢活动所产生的生物热及传热效率低等问题引起微环境温度升高,进而影响微生物的生长与代谢。然而,关于传统固态发酵微生物受生物热的影响及其适应机制仍不明晰。因此,本文以传统固态发酵体系为研究对象,阐述持续生物热介导的高温对固态发酵过程中微生物群落演替和代谢功能的影响,并提出复杂群落中具有多层次调控微生物代谢以适应高温环境的方式,主要从微生物群体与个体层面介绍可能存在的耐热机制。了解生物热对传统固态发酵微生物的影响及潜在的耐热机制,有助于靶向调控发酵过程、强化高温发酵等,以满足未来的工业化需求。     

Temperature control in solid substrate fermentation through discontinuous rotation

A laboratory-scale system for controlled dynamic solid substrate fermentation was developed and tested. The fermentation takes place in a stainless steel discontinuously rotating drum reactor, under controlled conditions of temperature, gas composition, relative humidity and direction and rate of rotation. The system was tested on a model fermentation of soya beans with Rhizopus oligosporus. In contrast to the traditional tempe fermentation, a granular product is obtained and build-up of heat and mass gradients is restricted. Despite the discontinuous rotation, the fungal growth continues, as evidenced by the production of heat. The rate of cooling depends on the temperature of the gas flushed through the reactor, the gas flow rate and the lenght of the rotation period. As a consequence of the homogeneous temperature control, the fungal heat development continued up to 70 h of fermentation. This is in clear contrast with the traditional tempe fermentation, which is already limited after 36 h by its own heat accumulation. Correspondence to: M. J. R. Nout     

Central alpha-MSH,energy balance,thermal balance,and antipyresis

Intracerebroventricular administration of alpha-MSH in young adult rats enhanced metabolic rate and caused a dose-dependent suppression of food intake, exhibiting a coordinated catabolic pattern. However, the thermoregulatory effects did not seem to be coordinated: the rising heat production was accompanied by a practically simultaneous tendency for rise in heat loss (skin vasodilatation), and the final core temperature either increased or decreased depending on which rise prevailed. The effect on heat loss possibly explains the antipyretic properties of the peptide.     

Mathematical modeling of Kluyveromyces marxianus growth in solid-state fermentation using a packed-bed bioreactor

This work investigated the growth of Kluyveromyces marxianus NRRL Y-7571 in solid-state fermentation in a medium composed of sugarcane bagasse, molasses, corn steep liquor and soybean meal within a packed-bed bioreactor. Seven experimental runs were carried out to evaluate the effects of flow rate and inlet air temperature on the following microbial rates: cell mass production, total reducing sugar and oxygen consumption, carbon dioxide and ethanol production, metabolic heat and water generation. A mathematical model based on an artificial neural network was developed to predict the above-mentioned microbial rates as a function of the fermentation time, initial total reducing sugar concentration, inlet and outlet air temperatures. The results showed that the microbial rates were temperature dependent for the range 27–50°C. The proposed model efficiently predicted the microbial rates, indicating that the neural network approach could be used to simulate the microbial growth in SSF.     

Beyond purified dietary fibre supplements: Compositional variation between cell wall fibre from different plants influences human faecal microbiota activity and growth in vitro

Dietary fibre is a major energy source for the human gut microbiota, but it is unclear to what extent the fibre source and complexity affect microbial growth and metabolite production. Cell wall material and pectin were extracted from five different dicotyledon plant sources, apples, beet leaves, beetroots, carrots and kale, and compositional analysis revealed differences in the monosaccharide composition. Human faecal batch incubations were conducted with 14 different substrates, including the plant extracts, wheat bran and commercially available carbohydrates. Microbial activity was determined for up to 72 h by measuring gas and fermentation acid production, total bacteria (by qPCR) and microbial community composition by 16S rRNA amplicon sequencing. The more complex substrates gave rise to more microbiota variation compared with the pectins. The comparison of different plant organs showed that the leaves (beet leaf and kale) and roots (carrot and beetroot) did not give rise to similar bacterial communities. Rather, the compositional features of the plants, such as high arabinan levels in beet and high galactan levels in carrot, appear to be major predictors of bacterial enrichment on the substrates. Thus, in-depth knowledge on dietary fibre composition should aid the design of diets focused on optimizing the microbiota.     

Influence of ambient air temperature on the cooling/heating load of a single cell protein jacketed fermenter operating on cheese whey under continuous conditions

The heat generated by mixing and lactose metabolism, during the continuous production of single cell protein from cheese whey lactose using a jacketed fermenter with running cooling water, was calculated using a heat balance equation. The technique quantified the heat produced in and lost from the fermentation unit. Most of the heat generated by mixing in the cell-free system (97.47%) was lost with exhaust gas, while a very small amount (2.53%) was lost through the fermenter lid, wall, and bottom. The heat generated by mixing was significant (26.31% of the total heat generated in the fermentation system with an active yeast population present) and, therefore, cannot be ignored in heat balance calculations. About 19.71% of the total heat generated in the reactor was lost through the coolant at an ambient temperature of 22 +/- 0.5 degrees C, showing the need for a cooling system. A yeast population size of 986 million cells/mL and a lactose removal efficiency of 95.6% were observed. About 72.5% and 27.5% of the lactose consumed were used for growth and respiration, respectively. A yield of 0.66 g of cells/g of lactose was achieved. The heat released by unit biomass was 7.05 kJ/g of cells. The results showed the significant impact of ambient air temperature on the cooling load. The heat to be removed from the medium by the cooling system varied from 3.46 to 281.56 kJ/h when the temperature increased from 16 to 30 degrees C. A heating system is needed to maintain the medium temperature at 34 degrees C when the ambient air temperature is below 16 degrees C.     

High temperature stimulates acetic acid accumulation and enhances the growth inhibition and ethanol production by Saccharomyces cerevisiae under fermenting conditions

Cellular responses of Saccharomyces cerevisiae to high temperatures of up to 42 °C during ethanol fermentation at a high glucose concentration (i.e., 100 g/L) were investigated. Increased temperature correlated with stimulated glucose uptake to produce not only the thermal protectant glycerol but also ethanol and acetic acid. Carbon flux into the tricarboxylic acid (TCA) cycle correlated positively with cultivation temperature. These results indicate that the increased demand for energy (in the form of ATP), most likely caused by multiple stressors, including heat, acetic acid, and ethanol, was matched by both the fermentation and respiration pathways. Notably, acetic acid production was substantially stimulated compared to that of other metabolites during growth at increased temperature. The acetic acid produced in addition to ethanol seemed to subsequently result in adverse effects, leading to increased production of reactive oxygen species. This, in turn, appeared to cause the specific growth rate, and glucose uptake rate reduced leading to a decrease of the specific ethanol production rate far before glucose depletion. These results suggest that adverse effects from heat, acetic acid, ethanol, and oxidative stressors are synergistic, resulting in a decrease of the specific growth rate and ethanol production rate and, hence, are major determinants of cell stability and ethanol fermentation performance of S. cerevisiae at high temperatures. The results are discussed in the context of possible applications.     

Air pressure pulsation solid state fermentation of feruloyl esterase by Aspergillus niger

Air pressure pulsation solid state fermentation (APP-SSF) was applied to produce feruloyl esterase (FAE) by Aspergillus niger. With the optimization of some variables by orthogonal design, the optimal condition obtained was 0.2 MPa (gauge pressure) of high pressure intensity, 30 min of low pressure duration and 20s of high pressure duration. Based on the optimized condition, the APP-SSF achieved the reasonable enzyme yield of 881 mU/g at 48 h, which was 58% more than that by static solid state fermentation (static SSF) at 72 h. By comparison of two fermentation methods in temperature, O(2) and CO(2) concentration, and respiration intensity, it was concluded that APP-SSF enhanced heat and mass transfer of fermentation system and strengthened the metabolism of microorganisms. The APP-SSF had a greatly positive effect on FAE production by A. niger, by enhancing mass and heat transfer and activating growth and metabolism.     

Production of polyhydroxyalkanoates by mixed culture: recent trends and biotechnological importance

Polyhydroxyalkanoates (PHAs) are the polymers of hydroxyalkanoates that accumulate as carbon/energy or reducing-power storage material in various microorganisms. PHAs have been attracting considerable attention as biodegradable substitutes for conventional polymers. To reduce their production cost, a great deal of effort has been devoted to developing better bacterial strains and more efficient fermentation/recovery processes. The use of mixed cultures and cheap substrates can reduce the production cost of PHA. Accumulation of PHA by mixed cultures occurs under transient conditions mainly caused by intermittent feeding and variation in the electron donor/acceptor presence. The maximum capacity for PHA storage and the PHA production rate are dependent on the substrate and the operating conditions used. This work reviews the development of PHA research. Aspects discussed include metabolism and various mechanisms for PHA production by mixed cultures; kinetics of PHA accumulation and conversion; effects of carbon source and temperature on PHA production using mixed cultures; PHA production process design; and characteristics of PHA produced by mixed cultures.     

Single-cell analysis of <Emphasis Type="Italic">S. cerevisiae</Emphasis> growth recovery after a sublethal heat-stress applied during an alcoholic fermentation

Interest in bioethanol production has experienced a resurgence in the last few years. Poor temperature control in industrial fermentation tanks exposes the yeast cells used for this production to intermittent heat stress which impairs fermentation efficiency. Therefore, there is a need for yeast strains with improved tolerance, able to recover from such temperature variations. Accordingly, this paper reports the development of methods for the characterization of Saccharomyces cerevisiae growth recovery after a sublethal heat stress. Single-cell measurements were carried out in order to detect cell-to-cell variability. Alcoholic batch fermentations were performed on a defined medium in a 2 l instrumented bioreactor. A rapid temperature shift from 33 to 43°C was applied when ethanol concentration reached 50 g l⁻¹. Samples were collected at different times after the temperature shift. Single cell growth capability, lag-time and initial growth rate were determined by monitoring the growth of a statistically significant number of cells after agar medium plating. The rapid temperature shift resulted in an immediate arrest of growth and triggered a progressive loss of cultivability from 100 to 0.0001% within 8 h. Heat-injured cells were able to recover their growth capability on agar medium after a lag phase. Lag-time was longer and more widely distributed as the time of heat exposure increased. Thus, lag-time distribution gives an insight into strain sensitivity to heat-stress, and could be helpful for the selection of yeast strains of technological interest.     

Effects of temperature on cell growth and xanthan production in batch cultures of Xanthomonas campestris

Batch xanthan fermentations by Xanthomonas campestris NRRL B-1459 at various temperatures ranging between 22 degrees C and 35 degrees C were studied. At 24 degrees C or lower, xanthan formation lagged significantly behind cell growth, resembling typical secondary metabolism. However, at 27 degrees C and higher, xanthan biosynthesis followed cell growth from the beginning of the exponential phase and continued into the stationary phase. Cell growth at 35 degrees C was very slow; the specific growth rate was near zero. The specific growth rate had a maximum value of 0.26 h(-1) at temperatures between 27 degrees C and 31 degrees C. Cell yield decreased from 0.53 g/g glucose at 22 degrees C to 0.28 g/g glucose at 33 degrees C, whereas xanthan yield increased from 54% at 22 degrees C to 90% at 33 degrees C. The specific xanthan formation rate also increased with increasing temperature. The pyruvate content of xanthan produced at various temperatures ranged between 1.9% and 4.5%, with the maximum occurring between 27 degrees C and 30 degrees C. These results suggest that the optimal temperatures for cell growth are between 24 degrees C and 27 degrees C, whereas those for xanthan formation are between 30 degrees C and 33 degrees C. For single-stage batch fermentation, the optimal temperature for xanthan fermentation is thus dependent on the design criteria (i. e., fermentation rate, xanthan yield, and gum qualities). However, a two-stage fermentation process with temperature shift-up from 27 degrees C to 32 degrees C is suggested to optimize both cell growth and xanthan formation, respectively, at each stage, and thus to improve overall xanthan fermentation.     

Intracellular accumulation of AMP as a cause for the decline in rate of ethanol production by Saccharomyces cerevisiae during batch fermentation.

A general hypothesis is presented for the decline in the rate of ethanol production (per unit of cell protein) during batch fermentation. Inhibition of ethanol production is proposed to result from the intracellular accumulation of AMP during the transition from growth to the stationary phase. AMP acts as a competitive inhibitor of hexokinase with respect to ATP. When assayed in vitro in the presence of ATP and AMP concentrations equivalent to those within cells at different stages of fermentation, hexokinase activity declined in parallel with the in vivo decline in the rate of ethanol production. The coupling of glycolytic flux and fermentation to cell growth via degradation products of RNA may be of evolutionary advantage for Saccharomyces cerevisiae. Such a coupling would reduce the exposure of nongrowing cells to potentially harmful concentrations of waste products from metabolism and would conserve nutrients for future growth under more favorable conditions.     

Effect of temperature up-shift on fermentation and metabolic characteristics in view of gene expressions in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Escherichia coli induces heat shock genes to the temperature up-shift, and changes the metabolism by complicated mechanism. The heat shock response is of practical importance for the variety of applications such as temperature-induced heterologous protein production, simultaneous saccharification and fermentation (SSF) etc. However, the effect of heat shock on the metabolic regulation is not well investigated. It is strongly desired to understand the metabolic changes and its mechanism upon heat shock in practice for the efficient metabolite production by temperature up-shift. In the present research, therefore, we investigated the effect of temperature up-shift from 37°C to 42°C on the metabolism in view of gene expressions.     

Dialysis fermentation. II. Kinetic analysis of salicylic acid production via cell growth and maintenance

A kinetic analysis was made of the relationship between salicylate production from naphthalene and growth of Pseudomonas fluorescens in semicontinuous dialysis culture. The specific rates both of product formation and growth initially were increased by the diffusional withdrawal of salicylate, but subsequently were reduced to low levels despite continued salicylate removal. Productivity and growth were correlated by the Luedeking-Piret equation in an initial nondialysis period and in the early stages of dialysis fermentation, when specific growth rates exceeded. 005 hr^?1. Below this level of growth at later stages of dialysis fermentation, the specific production rate was correlated only with total cell mass by a proportionality constant of .035 hr^?1, which was attributed to maintenance metabolism. Maintenance accounted for about 84% of the total salicylate produced, while growth-associated metabolism accounted for the remainder.     

Application of dynamic calorimetry for monitoring fermentation processes

The rate of heat evolution (kcal/liter-hr) in mycelial fermentations for novobiocin and cellulase production with media containing noncellular solids was measured by an in situ dynamic calorimetric procedure. Thermal data so obtained have proved significant both in monitoring cell concentration during the trophophase (growth phase) and in serving as a physiological variable in the fermentation process. The validity of this technique has been demonstrated by closing the overall material and energy balances. The maintenance energy in a batch fermentation can also be calculated by integrating heat evolution data. This integration method is applicable to a fermentation lacking a precise cell growth curve. The maintenance coefficient, obtained for the novobiocin fermentation by Streptomyces niveus, is equal to 0.028 g glucose equivalent/g cell-hr. The production of novobiocin in the idio-phase (production phase) also correlates well with the amount of energy catabolixed for maintenance and this results in an observed conversion yield of glucose to novobiocin of 11.8 mg of novobiocin produced per gram of glucose catabolized. A new physiological variable, kilocalories of heat evolved per millimole of oxygen consumed, has been proposed to monitor the state of cells during the fermentation. This method may provide a simple way to monitor on-line shifts in the efficiency of cell respiration and changes in growth yields during a microbial process.     

Specific heat flow rate: an on-line monitor and potential control variable of specific metabolic rate in animal cell culture that combines microcalorimetry with dielectric spectroscopy

One of the requirements for enhanced productivity by the animal culture systems used in biotechnology is the direct assessment of the metabolic rate by on-line biosensors. Based on the fact that cell growth is associated with an enthalpy change, it is shown that the specific heat flow rate is stoichiometrically related to the net specific rates of substrates, products, and indeed to specific growth rate, and therefore a direct reflection of metabolic rate. Heat flow rate measured by conduction calorimetry has a technical advantage over estimates for many material flows which require assays at a minimum of two discrete times to give the rate. In order to make heat flow rate specific to the amount of the living cellular system, it would be advantageous to divide it by viable biomass. This requirement has been fulfilled by combining a continuous flow microcalorimeter ex situ with a dielectric spectroscope in situ, the latter measuring the viable cell mass volume fraction. The quality of the resulting biosensor for specific heat flow rate was illustrated using batch cultures of Chinese hamster ovary cells (CHO 320) producing recombinant human interferon-gamma (IFN-gamma) during growth in a stirred tank bioreactor under fully aerobic conditions. The measuring scatter of the probe was decreased significantly by applying the moving average technique to the two participant signals. It was demonstrated that the total metabolic rate of the cells, as indicated by the specific heat flow rate sensor, decreased with increasing time in batch culture, coincident with the decline in the two major substrates, glucose and glutamine, and the accumulation of the by-products, ammonia and lactate. Furthermore, the specific heat flow rate was an earlier indicator of substrate depletion than the flow rate alone. The calorimetric-respirometric ratio showed the intensive participation of anaerobic processes during growth and the related IFN-gamma production. Specific heat flow rate was monotonically related to specific cell growth rate and associated with specific IFN-gamma production. Specific heat flow rate is potentially a valid control variable for the growth of genetically engineered cell lines producing target proteins.