Bench-scale calorimetry – application in biotechnology

Two calorimetric techniques are described allowing us to monitor and control microbial processes at relatively high cell densities: dynamic and continuous calorimetry. These techniques have been applied to study microbial systems, e.g. the dependence of thermodynamic efficiency and heat yield on specific growth rate, the heat yield of mesophilic and thermophilic microorganisms, the oxy-caloric coefficient during aerobic microbial growth, as well as the energetics of the yeast-cell cycle. Such studies are of importance for optimizing biotechnical processes.     

Calorimetric investigation of aerobic fermentations

A modified bench scale calorimeter has been employed to determine the heat generated by various microbial strains growing on a range of different substrates, covering degrees of reduction from 3 to 6.13. The results are analyzed, and interpreted in the light of coupled enthalpy and elemental balances. The heat released by the microbial cultures has been found to correlate linearly with other process variables, such as biomass generation and oxygen uptake. The ratio between the heat generated and the biomass formed, the so-called "heat yield" (Y(Q/x)), has been shown both on theoretical and experimental grounds to increase with increasing degree of reduction of the substrate and to decrease with increasing biomass yield. The two effects could be combined into a simple model which permits the amount of heat released per unit of biomass formed to be predicted from the degree of reduction of the substrate as the only independent variable. The ratio between the heat generated and the oxygen taken up was constant at 440 kJ (mol O(2))(-1) throughout all experiments as expected from theoretical considerations for strongly aerobic processes.     

Experimental bioenergetics ofSaccharomyces cerevisiae in respiration and fermentation

Aerobic growth of Saccharomyces cerevisiae on glucose was investigated, focusing on the heat evolution as it relates to biomass and ethanol synthesis. “Aerobic fermentation” and “aerobic respiration” were established respectively in the experimental system by performing batch and fed-batch experiments. “Balanced growth” batch cultivations were carried out with initial sugar concentrations ranging from 10 to 70 g/L, resulting in different degrees of catabolite repression. The fermentative heat generation was continuously monitored in addition to the key culture parameters such as ethanol production rate, CO₂ evolution rate, O₂ uptake rate, specific growth rate, and sugar consumption rate. The respective variations of the above quantities reflecting the variations in the catabolic activity of the culture were studied. This was done in order to evaluate the microbial regulatory system, the energetics of microbial growth including the rate of heat evolution and the distribution of organic substrate between respiration and fermentation. This study was supported by closing C, energy, and electron balances on the system. The comparison of the fractions of substrate energy evolved as heat (δ_h) with the fraction of available electrons transferred to oxygen (?_O2) indicated equal values of the two (0.46) in the aerobic respiration (fed-batch cultivation). However, the glucose effect in batch cultivations resulted in smaller ?_O2 than δ_h, while both values decreased in their absolute values. The evaluation of the heat energetic yield coefficients, together with the fraction of the available electrons transferred to O, contributed to the estimation of the extent of heat production through oxidative phosphorylation.     

A thermodynamic assessment of energy requirements for biomass synthesis by chemolithoautotrophic micro-organisms in oxic and anoxic environments

The flow of metabolic energy is arguably the most fundamental property governing ecosystem structure. In many microbial communities, particularly those that inhabit environments with little input of exogenous organic matter such as submarine hydrothermal systems and deep subsurface environments, chemolithoautotrophic organisms generate most of the organic matter available to support heterotrophic growth. In these environments, inorganic chemical reactions constitute the main source of energy input to the system, and the conversion of chemical energy to biomass by chemolithoautotrophs exerts a prominent control on the size, composition, and trophic structure of the biological community. A rigorous accounting of energy flow would aid in understanding the potential biological productivity of chemolithoautotrophic communities and help clarify the limits to habitability in geothermal and subsurface environments. In a step towards achieving a more complete accounting of energy flow in such communities, we present here computations to quantify the amount of thermodynamic energy required to synthesize the molecular components of biomass and to compare the relative energy requirements under oxic and anoxic conditions. The results suggest that only about 10% or less of the overall energy consumed during growth by chemolithoautotrophs is transformed directly into biomass. In addition, the results indicate aerobic organisms require approximately 17 kJ (g cells)⁻¹ more energy than anaerobes to synthesize the same biomass. This advantage may help explain why anaerobic organisms appear to yield greater biomass per unit energy input than aerobic organisms in laboratory growth studies, and why anaerobic micro-organisms can exist where the energy yield from catabolism is extremely low.     

Estimating the maintenance energy and biomass concentration of Saccharomyces cerevisiae by continuous calorimetry

Continuous calorimetry has been applied to monitoring the heat evolution of Saccharomyces cerevisiae grown on d-glucose. The heat evolution, together with the energy and carbon balances, was used to evaluate the energetic efficiency of biomass, by-product biosynthesis, fermentative heat evolution as well as the maintenance energy of S. cerevisiae in ‘aerobic fermentation’ and ‘aerobic respiration’. In aerobic fermentation, under catabolite repression, the fraction of substrate energy converted to heat evolution, maintenance requirement, and biomass decreased with the increase of d-glucose concentration. The fraction of substrate energy converted to ethanol is the highest value and it could contribute up to 70% of the total substrate energy. In aerobic respiration, 43% of the total substrate energy was evolved as heat. While 50% of the total substrate energy was converted into biomass, only 7% of the total substrate energy was used for maintenance functions. The maintenance energy coefficient of S. cerevisiae was determined to be 0.427 MJ kg^?1 cell h^?1 (0.102 kcal g^?1 cell h^?1). For the first time, heat evolution together with yield-maintenance energy was used to predict biomass concentration during the fed-batch cultivation of S. cerevisiae.     

Simultaneous evaluation of on-line microcalorimetry and fluorometry during batch culture of Pseudomonas putida-ATCC 11172 and Saccharomyces cerevisiae ATCC 18824

Application of on-line sensors (flow calorimeter, fluorescence probe, dissolved oxygen and CO₂ probes) was assessed to monitor microbial biomass and physiological state of cells during a biological process. Two systems were studied; diauxic growth of Pseudomonas putida ATCC 11172 on glycerol and phenol, and the aerobic growth of Saccharomyces cerevisiae ATCC 18824 on glucose. The results showed that the cells produced a heat output which could be quantitatively related to the various phases of the growth cycle. The initial stage of enzymatic induction and substrate mobilization during the diauxic growth of P. putida was easily detected, and a clear oscillation phenomenon was observed during enzymatic rearrangement in shifting from phenol to glycerol metabolism. Glucose oxidation in ethanol and then in acetate was also clearly delineated from the growth of S cerevisiae. NADH (fluorescence probe) measurements gave a strong correlation with various biomass indicators such as optical density, dry weight, ATP content and cellular protein. The fluorescence signal appeared to be very sensitive to the quenching effect of the culture medium and of the cells themselves. The fluorescence emitted from the NADH molecules in a culture medium can be reduced from 30–70% depending on the chemical composition and the optical density.     

Application of monoclonal antibodies in quantifying fungal growth dynamics during aerobic spoilage of silage

Proliferation of filamentous fungi following ingress of oxygen to silage is an important cause of dry matter losses, resulting in significant waste. In addition, the production of mycotoxins by some filamentous fungi poses a risk to animal health through mycotoxicosis. Quantitative assessment of fungal growth in silage, through measurement of ergosterol content, colony-forming units or temperature increase is limiting in representing fungal growth dynamics during aerobic spoilage due to being deficient in either representing fungal biomass or being able to identify specific genera. Here, we conducted a controlled environment aerobic exposure experiment to test the efficacy of a monoclonal antibody-based enzyme-linked immunosorbent assay (ELISA) to detect the proliferation of fungal biomass in six silage samples. We compared this to temperature which has been traditionally deployed in such experiments and on-farm to detect aerobic deterioration. In addition, we quantified ergosterol, a second marker of fungal biomass. After 8 days post-aerobic exposure, the ergosterol and ELISA methods indicated an increase in fungal biomass in one of the samples with a temperature increase observed after 16 days. A comparison of the methods with Pearson's correlation coefficient showed a positive association between temperature and ergosterol and both markers of fungal biomass. This work indicates that the technology has potential to be used as an indicator of microbial degradation in preserved forage. Consequently, if it developed as an on-farm technique, this could inform forage management decisions made by farmers, with the goal of decreasing dry matter losses, improving resource and nutrient efficiency and reducing risks to animal health.     

Growth kinetics of aerobic granules developed in sequencing batch reactors

AIMS: This paper attempts to develop a kinetic model to describe the growth of aerobic granules developed under different operation conditions. METHODS AND RESULTS: A series of experiments were conducted by using four-column sequencing batch reactors to study the formation of aerobic granules under different conditions, e.g. organic loading rates, hydrodynamic shear forces and substrate N/COD ratios. A simple kinetic model based on the Linear Phenomenological Equation was successfully derived to describe the growth of aerobic granules. It was found that the growth of aerobic granules in terms of equilibrium size and size-dependent growth rate were inversely related to shear force imposed to microbial community, while a high organic loading favoured the growth of aerobic granules, leading to a large size granule. The effect of substrate N/COD ratio on the growth kinetics of aerobic granules was realized through change in microbial populations, and enriched nitrifying population in aerobic granules developed at high substrate N/COD ratio resulted in a low overall growth rate of aerobic granules. CONCLUSIONS: The proposed model can provide good prediction for the growth of aerobic granules indicated by the correlation coefficient >0.95. SIGNIFICANCE AND IMPACT OF THE STUDY: The kinetic model proposed could offer a useful tool for studying the growth kinetics of cell-to-cell immobilization process. The study confirmed that the growth of aerobic granules and biofilms are subject to a similar kinetic pattern. This work would also be helpful for better understanding the mechanism of aerobic granulation.     

Effect of excess ethanol on the growth of yeasts of the genus Candida during continuous cultivation

The effect of flow rates and a specific ethanol load on the growth of Candida utilis and Candida krusei was studied in the process of one-step and three-step cultivation. The productive capacity of fermenters and the economic coefficient of yeast biomass production were shown to depend on the ability of microbial populations to assimilate a certain quantity of a carbon substrate per unit time. When a specific ethanol load exceeds the optimal one, the respiratory activity of a population and the economic coefficient of growth fall down whereas the accumulation of metabolites in the cultural broth increases. The steady state of biomass can be maintained in the process of continuous cultivation by inhibiting the yeast growth with an excess of ethanol.     

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.     

Der Einfluß der Flockenbildung auf die Versorgung der Hefezellen mit Sauerstoff bei der Fermentation flüssiger Kohlenwasserstoffe

Floc formation, especially the influence of floe diameter variations on the total velocity of the process, was investigated in aerobic growth processes of yeast on the hydrocarbons of crude oil. The experimental results show that the diameter of the flocs is a function of the rheological properties of the fluids and the flow conditions. The floc diameter varies between 0,1 mm and a few millimeters. About 90% of the total yeast cells are situated in the interior of the flocs. Since oxygen must be transferred to all yeast cells their oxygen supply was studied. Thus, the yeast cells in the floc interior were not sufficiently supplied with oxygen, if the floc diameter reached a critical value. In such cases a decrease of the biomass formation rate was observed, although the dissolved oxygen concentration of the aquaeous fermentation medium was greater than zero. Therefore, aerobic microbial growth processes in multicomponent systems must be carried out without floc formation or under such conditions as cause very small floc diameters.     

Detachment of multi species biofilm in circulating fluidized bed bioreactor

In this study, the detachment rates of various microbial species from the aerobic and anoxic biofilms in a circulating fluidized bed bioreactor (CFBB) with two entirely separate aerobic and anoxic beds were investigated. Overall detachment rate coefficients for biomass, determined on the basis of volatile suspended solids (VSS), glucose and protein as well as for specific microbial groups, i.e., for nitrifiers, denitrifiers, and phosphorous accumulating organisms (PAOs), were established. Biomass detachment rates were found to increase with biomass attachment on carrier media in both beds. The detachment rate coefficients based on VSS were significantly affected by shear stress, whereas for protein, glucose and specific microbial groups, no significant effect of shear stress was observed. High detachment rates were observed for the more porous biofilm structure. The presence of nitrifiers in the anoxic biofilm and denitrifiers in the aerobic biofilm was established by the specific activity measurements. Detachment rates of PAOs in aerobic and anoxic biofilms were evaluated.     

Biomass and porosity profiles in microbial granules used for aerobic wastewater treatment

AIMS: To obtain biomass and porosity profiles for aerobically grown granules of different diameters and to determine a suitable range of granule diameters for application in wastewater treatment. METHODS AND RESULTS: Microbial granules were cultivated in an aerobic granulated sludge reactor with model wastewaters containing acetate, or ethanol plus acetate, or glucose as the main carbon source. Granules were formed by retaining microbial aggregates using a settling time of 2 min. Sampled granules had diameters ranging from 0.45 to 3 mm. Microbial biomass in the granules was detected with the nucleic acid stain SYTO 9 and confocal laser scanning microscopy. The thickness of the microbial biomass layer was proportional to the granule diameter, and had a maximum value of 0.8 mm. The thickness of the microbial biomass layer correlated with the penetration depth of 0.1 microm fluorescent beads into the granule. CONCLUSIONS: The microbial biomass and porosity studies suggest that aerobically grown microbial granules should have diameters less than a critical diameter of 0.5 mm, if deployed for wastewater treatment applications. This critical diameter is based on the assumption that whole granules should have a porous biomass-filled matrix. SIGNIFICANCE AND IMPACT OF THE STUDY: This work could contribute to the development of aerobic granulation technology for effective biological wastewater treatment.     

Microscopic observation of aerobic granulation in sequential aerobic sludge blanket reactor

AIMS: This paper attempts to provide visual evidence of how aerobic granulation evolves in sequential aerobic sludge blanket reactors. METHODS AND RESULTS: A series of experiments were conducted in two column-type sequential aerobic sludge reactors fed with glucose and acetate as sole carbon source, respectively. The evolution of aerobic granulation was monitored using image analysis and optical and scanning electron microscopy. The results indicated that the formation of aerobic granules was a gradual process from seed sludge to compact aggregates, further to granular sludge and finally to mature granules with the sequential operation proceeding. Glucose- and acetate-fed granules have comparable characteristics in terms of settling velocity, size, shape, biomass density and microbial activity. However, the microbial diversity of the granules was associated with the carbon source supplied. In this work, an important aerobic starvation phase was identified during sequential operation cycles. It was found that periodical aerobic starvation was an effective trigger for microbial aggregation in the reactor and further strengthened cell-cell interaction to form dense aggregates, which was an essential step of granulation. The periodical starvation-induced aggregates would finally be shaped to granules by hydrodynamic shear and flow. CONCLUSION: Aerobic granules can be formed within 3 weeks in the systems. The periodical starvation and hydrodynamic conditions would play a crucial role in the granulation process. SIGNIFICANCE AND IMPACT OF THE STUDY: Aerobic granules have excellent physical characteristics as compared with conventional activated sludge flocs. This research could be helpful for the development of an aerobic granule-based novel type of reactor for handling high strength organic wastewater.     

Investigation of the potential of biocalorimetry as a process analytical technology (PAT) tool for monitoring and control of Crabtree-negative yeast cultures

Biological reaction calorimetry, also known as biocalorimetry, has led to extensive applications in monitoring and control of different bioprocesses. A simple real-time estimator for biomass and growth rate was formulated, based on in-line measured metabolic heat flow values. The performance of the estimator was tested in a unique bench-scale calorimeter (BioRC1), improved to a sensitivity range of 8 mW l^− 1 in order to facilitate the monitoring of even weakly exothermic biochemical reactions. A proportional–integral feedback control strategy based on these estimators was designed and implemented to control the growth rate of Candida utilis, Kluyveromyces marxianus and Pichia pastoris by regulating an exponential substrate feed. Maintaining a particular specific growth rate throughout a culture is essential for reproducible product quality in industrial bioprocesses and therefore a key sequence for the step from quality by analysis to quality by design. The potential of biocalorimetry as a reliable biomass monitoring tool and as a key part of a robust control strategy for aerobic fed-batch cultures of Crabtree-negative yeast cells in defined growth medium was investigated. Presenting controller errors of less than 4% in the best cases, the approach paves the way for the development of a generally applicable process analytical technology platform for monitoring and control of microbial fed-batch cultures.     

Microbial activity in pig slurry-amended soils under aerobic incubation

A 120-day aerobic incubation experiment was conducted to study the effects of pig slurry application on soil microbial activity. Pig slurry was added to soil at rates of 0 (control treatment), 150 and 300 m³ ha⁻¹. Soil samples were taken after 0, 7, 14, 30, 45, 60, and 120 days of incubation and analyzed for total organic C and microbial biomass C contents, and basal respiration. Most of the organic C applied to soil with pig slurry was readily decomposed within 30 days. During the first phase (0 to 14–30 days), the addition of pig slurry to the soil, especially at the larger rate, increased microbial biomass C content, microbial biomass C/total organic C ratio, basal respiration, and metabolic quotient. The microbial growth and the increase of their activity that these results reflected were not persistent, since the initially measured values in pig slurry-amended soils decreased and reached those of the control soil in a relatively short time.     

Microcalorimetric study of Escherichia coli aerobic growth: theoretical aspects of growth on succinic acid.

Two methods of investigation were used to evaluate the heat quantity associated with anabolic processes (qan) during the aerobic growth of Escherichia coli in a minimal medium containing succinic acid as the sole energy and carbon source. The study of the contribution of biosynthetic reactions from succinic acid and ammonia were investigated by both methods. The two qan values obtained were in excellent agreement and were found to be significant. Thus it was demonstrated that the contribution of anabolism strongly influenced the quantity of heat associated with microbial aerobic growth. The qan calculated as above explained the experimental enthalpy change which was recently reported.     

Integrating mixed microbial population dynamics into modeling energy transport during the initial stages of the aerobic composting of a switchgrass mixture

A mathematical model which integrates empirically derived microbial growth kinetics with heat and mass transfer phenomena and substrate degradation kinetics has been developed to capture the dynamics of the aerobic composting of a switchgrass and dog food mixture over a period of 64 h. The model incorporated three microbial populations of yeasts, bacteria and fungi that metabolized composting material consisting of sugars and starches, cellulose and hemicelluloses to produce heat and utilize oxygen in a static, cylindrical reactor employing forced aeration. Model predictions captured well the dynamics obtained experimentally between physical and microbial variables and the model has the potential to become a predictive tool for substrate degradation during aerobic composting processes.     

Microbial degradation of hydrocarbons in soil during aerobic/anaerobic changes and under purely aerobic conditions

Microbial hydrocarbon degradation in soil was studied during periodical aerobic/anaerobic switching and under purely aerobic conditions by using a pilot-scale plant with diesel-fuel-contaminated sand. The system worked according to the percolation principle with controlled circulation of process water and aeration. Periodical switching between 4 h of aerobic and 2 h of anaerobic conditions was achieved by repeated saturation of the soil with water. Whatever the cultivation mode, less than 50% of the diesel was degraded after 650 h because the hydrocarbons were adsorbed. Contrary to expectations, aerobic/anaerobic changes neither accelerated the rate of degradation nor reduced the residual hydrocarbon content of the soil. Obviously the pollutant degradation rate was determined mainly by transport phenomena and less by the efficiency of microbial metabolism. The total mass of oxygen consumed and carbon dioxide produced was greater under aerobic/anaerobic changing than under aerobic conditions, although the mass of hydrocarbons degraded was nearly the same. As shown by an overall balance of microbial growth and by a carbon balance, the growth yield coefficient was smaller during aerobic/anaerobic changes than under aerobic conditions. Received: 25 November 1997 /  Received revision: 15 January 1998 / Accepted: 18 January 1998