Measurement of heat evolution and correlation with oxygen consumption during microbial growth. Reprinted from Biotechnology and Bioengineering, Vol. XI, Issue 3, Pages 269-281 (1968)

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.     

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     

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.     

Evaluation of methane-utilising bacteria products as feed ingredients for monogastric animals

Bacterial proteins represent a potential future nutrient source for monogastric animal production because they can be grown rapidly on substrates with minimum dependence on soil, water, and climate conditions. This review summarises the current knowledge on methane-utilising bacteria as feed ingredients for animals. We present results from earlier work and recent findings concerning bacterial protein, including the production process, chemical composition, effects on nutrient digestibility, metabolism, and growth performance in several monogastric species, including pigs, broiler chickens, mink (Mustela vison), fox (Alopex lagopus), Atlantic salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss), and Atlantic halibut (Hippoglossus hippoglossus). It is concluded that bacterial meal (BM) derived from natural gas fermentation, utilising a bacteria culture containing mainly the methanotroph Methylococcus capsulatus (Bath), is a promising source of protein based on criteria such as amino acid composition, digestibility, and animal performance and health. Future research challenges include modified downstream processing to produce value-added products, and improved understanding of factors contributing to nutrient availability and animal performance.     

Regulatory aspects of bakers' yeast metabolism in aerobic fed-batch cultures

A highly instrumented computer-coupled bioreactor is used to investigate metabolic changes of Saccharomyces cerevisiae in aerobic fed-batch systems which are generally applied in bankers' yeast manufacture. The four types of metabolism (oxidation of glucose, aerobic fermentation, oxidation of glucose and ethanol, and oxidation of ethanol) appearing in such systems are characterized by four significant fermentation parameters: Respiratory quotient (RQ), glucose uptake rate (Q_g), ethanol turnover rate (Q_EtOH), and growth yield on glucose (Y_g). Below the critical glucose concentration glucose and ethanol are utilized simultaneously. The shift from aerobic fermentation to nondiauxic growth on glucose and ethanol is not only dependent on glucose concentration. but also on the precultivation on cells. The uptake of ethanol is controlled by the glucose supply except in the case when ethanol is limiting; the oxygen uptake rate (Q_o2), however, is unaffected by the ratio of Q_g and Q_EtOH. Critical glucose concentration is not a constant value for a particular strain, but varies corresponding to the nutritional state of the cells.     

Effects of gas periodic stimulation on key enzyme activity in gas double-dynamic solid state fermentation (GDD-SSF)

The heat and mass transfer have been proved to be the important factors in air pressure pulsation for cellulase production. However, as process of enzyme secretion, the cellulase formation has not been studied in the view of microorganism metabolism and metabolic key enzyme activity under air pressure pulsation condition. Two fermentation methods in ATPase activity, cellulase productivity, weight lose rate and membrane permeability were systematically compared. Results indicated that gas double-dynamic solid state fermentation had no obviously effect on cell membrane permeability. However, the relation between ATPase activity and weight loss rate was linearly dependent with r = 0.9784. Meanwhile, the results also implied that gas periodic stimulation had apparently strengthened microbial metabolism through increasing ATPase activity during gas double-dynamic solid state fermentation, resulting in motivating the production of cellulase by Trichoderma reesei YG3. Therefore, the increase of ATPase activity would be another crucial factor to strengthen fermentation process for cellulase production under gas double-dynamic solid state fermentation.     

Insulin‐like receptor substrate gene chico regulates octopamine metabolism in Drosophila melanogaster

Octopamine, one of the main insect biogenic amines, plays an important role in the control of fitness in Drosophila melanogaster Meigen. The present study examines the effects of a null mutation of the gene of the insulin‐like receptor substrate (chico), in the heterozygous state, on octopamine metabolism, heat stress resistance and fecundity of D. melanogaster. A rise in the activity of one of the key enzymes of octopamine synthesis, tyrosine decarboxylase, as well as that of an enzyme of its degradation, octopamine‐dependent N‐acetyl transferase, is observed in chico^1/+ females. It is also found that the resistance to heat stress is decreased and fecundity is reduced dramatically in chico^1/+ flies. Such changes in these parameters in D. melanogaster females result from a rise in octopamine titre, which suggests that chico affects the octopamine level by regulating the activity of tyrosine decarboxylase.     

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.     

Bioprocess optimization for production of thermoalkali-stable protease from Bacillus subtilis K-1 under solid-state fermentation

Cost-effective production of proteases, which are robust enough to function under harsh process conditions, is always sought after due to their wide industrial application spectra. Solid-state production of enzymes using agro-industrial wastes as substrates is an environment-friendly approach, and it has several advantages such as high productivity, cost-effectiveness, being less labor-intensive, and less effluent production, among others. In the current study, different agro-wastes were employed for thermoalkali-stable protease production from Bacillus subtilis K-1 under solid-state fermentation. Agricultural residues such as cotton seed cake supported maximum protease production (728?U?ml^?1), which was followed by gram husk (714?U?ml^?1), mustard cake (680?U?ml^?1), and soybean meal (653?U?ml^?1). Plackett–Burman design of experiment showed that peptone, moisture content, temperature, phosphates, and inoculum size were the significant variables that influenced the protease production. Furthermore, statistical optimization of three variables, namely peptone, moisture content, and incubation temperature, by response surface methodology resulted in 40% enhanced protease production as compared to that under unoptimized conditions (from initial 728 to 1020?U?ml^?1). Thus, solid-state fermentation coupled with design of experiment tools represents a cost-effective strategy for production of industrial enzymes.     

Fermentation production of keratinase from Bacillus licheniformisPWD-1 and a recombinant B. subtilis FDB-29

Bacillus licheniformis PWD-1, the parent strain, and B. subtilis FDB-29, a recombinant strain. In both strains, keratinase was induced by proteinaceous media, and repressed by carbohydrates. A seed culture of B. licheniformis PWD-1 at early age, 6–10 h, is crucial to keratinase production during fermentation, but B. subtilis FDB-29 is insensitive to the seed culture age. During the batch fermentation by both strains, the pH changed from 7.0 to 8.5 while the keratinase activity and productivity stayed at high levels. Control of pH, therefore, is not necessary. The temperature for maximum keratinase production is 37°C for both strains, though B. licheniformis is thermophilic and grows best at 50°C. Optimal levels of dissolved oxygen are 10% and 20% for B. licheniformis and B. subtilis respectively. A scale-up procedure using constant temperature at 37°C was adopted for B. subtilis. On the other hand, a temperature-shift procedure by which an 8-h fermentation at 50°C for growth followed by a shift to 37°C for enzyme production was used for B. licheniformis to shorten the fermentation time and increase enzyme productivity. Production of keratinase by B. licheniformis increased by ten-fold following this new procedure. After respective optimization of fermentation conditions, keratinase production by B. licheniformis PWD-1 is approximately 40% higher than that by B. subtilis FDB-29. Received 16 July 1998/ Accepted in revised form 07 March 1999     

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.     

Modelling and optimization of fermentation factors for enhancement of alkaline protease production by isolated Bacillus circulans using feed-forward neural network and genetic algorithm

Aim: Modelling and optimization of fermentation factors and evaluation for enhanced alkaline protease production by Bacillus circulans. Methods and Results: A hybrid system of feed‐forward neural network (FFNN) and genetic algorithm (GA) was used to optimize the fermentation conditions to enhance the alkaline protease production by B. circulans. Different microbial metabolism regulating fermentation factors (incubation temperature, medium pH, inoculum level, medium volume, carbon and nitrogen sources) were used to construct a ‘6‐13‐1’ topology of the FFNN for identifying the nonlinear relationship between fermentation factors and enzyme yield. FFNN predicted values were further optimized for alkaline protease production using GA. The overall mean absolute predictive error and the mean square errors were observed to be 0·0048, 27·9, 0·001128 and 22·45 U ml^?1 for training and testing, respectively. The goodness of the neural network prediction (coefficient of R²) was found to be 0·9993. Conclusions: Four different optimum fermentation conditions revealed maximum enzyme production out of 500 simulated data. Concentration‐dependent carbon and nitrogen sources, showed major impact on bacterial metabolism mediated alkaline protease production. Improved enzyme yield could be achieved by this microbial strain in wide nutrient concentration range and each selected factor concentration depends on rest of the factors concentration. The usage of FFNN–GA hybrid methodology has resulted in a significant improvement (>2·5‐fold) in the alkaline protease yield. Significance and Impact of the Study: The present study helps to optimize enzyme production and its regulation pattern by combinatorial influence of different fermentation factors. Further, the information obtained in this study signifies its importance during scale‐up studies.     

Studies on the growth metabolism of Bacillus thuringiensis and its vegetative insecticidal protein engineered strains by microcalorimetry

The metabolic power-times curves of Bacillus thuringiensis and its vegetative insecticidal protein-engineered strains were determined at 30°C using a thermal activity monitor, air Isothermal Microcalorimeter, and ampoule method. From the power-times curves, the maximum power (P _max) in the log phase, growth rate constant (k), generation times (t _G), time of the maximum power (t _max), heat effects (Q _log) for log phase, and the total heat effect in 45 h (Q _total) of. B. thuringiensis strains can be obtained. The results indicate that their power-times curves are different. The relationship between their metabolic power-times curves and character of bacteria metabolism, and thermokinetics and gene expression were analyzed and discussed. The character of the bacteria power-times curves reflected the physiologic character of gene expression. The microcalorimetric method proved to be a reliable and sensitive tool for the assessment of growth metabolism, heat output in bacteria and its engineered strains. The determination of the thermokinetic character is beneficial to the control of fermentation. The text was submitted by the authors in English.     

Anaerobic metabolism in the leech (Hirudo medicinalis L.): direct and indirect calorimetry during severe hypoxia

Anaerobic metabolism in the limnic annelid Hirudo medicinalis L. was investigated by direct and indirect calorimetry. During long-term severe hypoxia, the rate of heat dissipation was reduced up to 13% of the aerobic rate. At the same time, the rate of ATP turnover was reduced to about 30% of the aerobic rate, indicating that metabolic depression is an important mechanism to ensure survival of the leech during environmental anaerobiosis. Heat dissipation during hypoxia was monitored under two experimental conditions, favouring either concomitant hypocapnia (continuous N₂ bubbling) or hypercapnia (self-induced hypoxia). The reduction in heat dissipation during hypocapnic hypoxia was less pronounced than during hypercapnic hypoxia, indicating that the different experimental conditions may influence anaerobic metabolism and the extent of metabolic depression. Biochemical analysis of known anaerobic substrates and endproducts provided the basis for indirect calorimetry during self-induced hypoxia. From changes in metabolites, the expected heat dissipation was calculated for initial (0–8 h) and long-term severe hypoxia (8–72 h). During the initial period, the calculated heat dissipation fully accounted for direct calorimetric determination. During long-term hypoxia, only 71% of the measured heat production could be explained from biochemical analysis of metabolites. Therefore, an additional unknown endproduct cannot be excluded, especially when anaerobic ammonia production and analysis of the carbohydrate balance are considered.Abbreviations APW artificial pond water - HPLC high-performance liquid chromatography - fw fresh weight - HP heat production - HD heat dissipation - MR metabolic rate     

甲烷菌对厌氧真菌不同碳源代谢的影响

【目的】探讨碳源和甲烷菌对厌氧真菌碳代谢的影响。【方法】利用体外批次厌氧发酵法,比较厌氧真菌纯培养(Orpinomyces sp.和Neocallimastix sp.)及其与甲烷菌共培养(F1:Orpinomyces sp.+Methanobrevibacter sp.和N3:Neocallimastix sp.+Methanobrevibacter sp.)发酵不同类型碳水化合物代谢产物的差异。【结果】对厌氧真菌和甲烷菌共培养F1和N3的研究显示,F1发酵木薯粉[(26.44±0.22)mmol/L]的乳酸产量是发酵玉米芯[(1.31±0.04)mmol/L]的20.18倍,是N3发酵木薯粉[(1.59±0.03)mmol/L]的16.63倍,玉米芯[(0.79±0.08)mmol/L]的33.47倍。当F1和N3中的厌氧真菌纯培养时,各组乳酸产量均1.90 mmol/L。对F1进一步研究,结果显示发酵体系中木薯粉添加量在0.8%–2.0%之间时,乳酸产量随木薯粉添加量增加而增加。当含量在1.0%–2.4%之间时,随木薯粉添加量增加,甲烷和乙酸产量逐渐降低。比较F1发酵大米粉、木薯粉、玉米粉、小麦粉和土豆粉的发酵结果,发现乳酸产量与底物中支链淀粉的含量成正相关(R2=0.9554)。当F1发酵葡萄糖和麦芽糖时,乳酸产量5.00 mmol/L。当以麦芽糊精为底物时,乳酸产量高达(28.00±0.95)mmol/L。【结论】本文首次报道碳源和甲烷菌能够增强厌氧真菌的乳酸代谢途径并且这种增强存在种属特异性。     

Optimization of metabolic pathways for bioconversion of lignocellulose to ethanol through genetic engineering

The optimization of metabolic pathways is of fundamental importance for strategies aimed at improving the economics and yield of the lignocellulose-to-ethanol processes. Although Escherichia coli is capable of metabolizing a wide variety of substrates including hexoses and pentoses, its hexose metabolism is inferior to that of Zymomonas mobilis, an obligate, ethanologenic bacterium. We therefore inserted and expressed Z. mobilis genes encoding essential enzymes involved in the fermentation pathway, alcohol dehydrogenase II (adh II) and pyruvate decarboxylase (pdc), into E. coli, resulting in increased cell growth and ethanol production. Ethanol concentrations of > 30 g/L were obtained on 10% glucose. Additionally, since pyruvate is mainly assimilated through pyruvate formate lyase (pfl) and forms formic acid and acetyl coenzyme A, metabolic redirection was attempted through gene knockout by Red-mediated recombination to decrease the byproducts of pyruvate metabolism. Under microaerobic conditions, pflA- and pflB-mutants produced more ethanol (163% and 207%, respectively) relative to the parent strain, using glucose as a carbon source.     

Daily rhythm of oxygen consumption and thermoregulatory responses in some European winter- or summer-acclimatized finches at different ambient temperatures

The oxygen consumption of European finches, the siskin (Carduelis spinus), the brambling (Fringilla montifringilla), the bullfinch (Pyrhulla pyrhulla), the greenfinch (Carduelis chloris) and the hawfinch (Coccothraustes coccothraustes), was recorded continuously while ambient temperature was decreased stepwise from +30 down to-75°C. The oxygen consumption, body temperature (telemetrically), and shivering (integrated pectoral electromyography) of greenfinches were measured simultaneously at ambient temperatures between +30 and-75°C. Maximum heat production, cold limit, lower critical temperature, basal metabolic rate and thermal conductance (of the greenfinch) were determined. The diurnal variation of oxygen consumption of siskins and greenfinches was recorded at thermoneutrality and below the thermoneutral zone in winter- and summer-acclimatized birds. The diurnal variation of body temperature and thermal conductance of greenfinches were also determined. The diurnal variation of heat production was not seasonal or temperature dependent in the siskin and in the greenfinch. Nocturnal reduction of oxygen consumption saved 15–33% energy in the siskin and greenfinch. Body temperature of the greenfinch was lowered by 2.5–3.4°C. The nocturnal reduction of thermal conductance in the greenfinch was 39–48%. The basal metabolic rate was lowest in the largest bird (hawfinch) and highest in the smallest bird (siskin). The values were in the expected range. The heat production capacity of finches in winter was 4.7 times basal metabolic rate in the siskin, 4.2 times in the brambling, 3.5 times in the greenfinch and 2.9 times in the bullfinch and hawfinch. The heat production capacity of the siskin and greenfinch was not significantly lower in summer. The cold limit temperatures (°C) in winter were-61.2 in the siskin,-41.3 in the greenfinch,-37.0 in the bullfinch,-35.7 in the brambling and-28.9 in the hawfinch. The cold limit was 14.3°C higher in summer than in winter in the siskin and 8.7°C in the greenfinch. Thermal insulation of the greenfinch was significantly better in winter than in summer. The shivering of the greenfinch increased linearly when ambient temperature was decreased down to-40°C. Maintenance of shivering was coincident with season. In severe cold integrated pectoral electromyography did not correlate with oxygen consumption as expected. The possible existence of non-shivering thermogenesis in birds is discussed. It is concluded that the acclimatization of European finches is primarily metabolic and only secondly affected by insulation.Abbreviations AAT avian adipose tissue - bm body mass - BMR basal metabolic rate - C _t thermal conductance - EMG electromyogram - HP heat production - HP _max maximum heat production - MR metabolic rate - NST non-shivering thermogenesis - RMR resting metabolic rate - RQ respiratory quotient - T _a ambient temperature - T _b body temperature - T _c colonic temperature - T _1c lower critical temperature - TNZ thermoneutral zone - T _st shivering threshold temperature - V oxygen consumption     

Optimization of culture conditions for the production of an extracellular ribonuclease by <Emphasis Type="Italic">Aspergillus niger</Emphasis> in a benchtop bioreactor

SummarySelf-directing optimization was successfully employed to determine the optimal combination of engineering parameters, viz., pH, aeration rate and agitation rate, for extracellular ribonuclease production by Aspergillus niger SA-13-20 in a batch bioreactor. Maximal RNase production of 5.38 IU ml^–1 was obtained at controlled pH of 2.33, aeration rate of 1.67 v/v/m and agitation rate of 850 rev/min. The effect of oxygen on the fermentation was also investigated. With increase in volumetric oxygen transfer coefficients (K_La), cell growth and RNase production first increased and then decreased. RNase production was further increased to 7.10 IU ml^–1 and the fermentation time was shortened from 96 to 72 h by controlling dissolved oxygen concentration at 10% saturation by aerating oxygen after about 28 h of fermentation under the above optimal condition. The kinetic model showed that RNase production by A. niger SA-13-20 was growth-associated.     

Tourmaline ceramic balls stimulate growth and metabolism of three fermentation microorganisms

Effects of tourmaline ceramic balls on growth and metabolism of Saccharomyces cerevisiae, Lactobacillus acidophilus and Aspergillus oryzae were studied. Treatments with 3, 6, 9 or 12 g of tourmaline ceramic balls in a 50 ml culture showed significant stimulation of the growth of the three microorganisms. In optimal treatments with 12 g of tourmaline balls, the growth of S. cerevisiae, L. acidophilus, and A. oryzae was increased by 34, 32 and 10%, respectively. After 72 h fermentation of S. cerevisiae, total carbohydrate content in the culture medium was decreased by 65% and ethanol production was increased by 150%. Total carbohydrate content was decreased by 80% and the pH value was decreased by 0.3, as a result of organic acid production in the medium of L. acidophilus after 72 h fermentation. In the case of A. oryzae, enzyme activities of protease and amylase were increased by 90 and 31%, respectively, after 96 h fermentation. Results indicated that tourmaline stimulates initiation of growth in the early lag stage and increases production of metabolites at a later stage of fermentation. The strong stimulatory effect of tourmaline on growth, utilization of substrates and production of metabolites in the three microorganisms suggests a potential application in the fermentation industry.