Use of microcalorimetric monitoring in establishing continuous energy balances and in continuous determinations of substrate and product concentrations of batch-grown Saccharomyces cerevisiae

Energy balance calculations were performed for different physiological states during batch growth of Saccharomyces cerevisiae with glucose as carbon and energy source. For the different physiological states, energy recoveries close to one were obtained, which permitted a continuous control that the constantly changing growth process was quantified accurately. During the respiro-fermantative phase of growth, during which glucose served as the carbon and energy source, a low-heat-yield value (DeltaQ(x)) of -8.6 kJ/g dry biomass formed was obtained. This low-heat-yield value was due to the mainly fermentative metabolism during the middle of this phase of growth. After a transition phase, the ethanol produced during the respiro-fermentative growth was respired. During this respiratory phase, the heat yield values increased markedly, resulting in a lowest value of -42.7 kJ/g. The low-heat-yield values of the respiro-fermentative growth is not a reflection of the most efficient metabolism of S. cerevisiae. On the contrary, during the middle of this phase, 74% of the energy input was dissipated as ethanol, 6% was dissipated as heat, and the energy conserved as biomass was just 13%, while during the early respiratory phase, 69% of the energy input was dissipated as heat, and 22% of the energy input was conserved as biomass. By mathematical modeling and direct monitoring on-line of the rate of heat production, continuous calculations of (1) glucose consumption, and (3) biomass production were performed, and were shown to correlate closely with measured values for the continuously changing growth process.     

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.     

Application of mass and energy balance regularities in fermentation. Reprinted from Biotechnology and Bioengineering, Vol. XX, No. 10, Pages 1595-1621 (1978)

Material and energy balances for fermentation processes are developed 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. Mass-energy balance equations are developed which relate the biomass energetic yield coefficient to sets of variables which may be determined experimentally. Organic substrate consumption, biomass production, oxygen consumption, carbon dioxide production, heat evolution, and nitrogen consumption are considered as measured variables. Application of the balances using direct and indirect methods of yield coefficient estimation is illustrated using experimental results from the literature. Product formation is included in the balance equations and the effect of product formation on biomass yield estimates is examined. Application of mass-energy balances in the optimal operation of continuous single-cell protein production facilities is examined, and the variation of optimal operating conditions with changes in yield are illustrated for methanol as organic substrate.     

Application of mass and energy balance regularities in fermentation

Material and energy balances for fermentation processes are developed 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. Mass–energy balance equations are developed which relate the biomass energetic yield coefficient to sets of variables which may be determined experimentally. Organic substrate consumption, biomass production, oxygen consumption, carbon dioxide production, heat evolution, and nitrogen consumption are considered as measured variables. Application of the balances using direct and indirect methods of yield coefficient estimation is illustrated using experimental results from the literature. Product formation is included in the balance equations and the effect of product formation on biomass yield estimates is examined. Application of mass–energy balances in the optimal operation of continuous single-cell protein production facilities is examined, and the variation of optimal operating conditions with changes in yield are illustrated for methanol as organic substrate.     

Impact of temperature on growth and metabolic efficiency of Penicillium roqueforti– correlations between produced heat, ergosterol content and biomass

The influence of temperature on the growth of the mould Penicillium roqueforti growing on malt extract agar was studied by correlating the produced heat (measured by isothermal calorimetry), ergosterol content (quantified by GC-MS/MS) and biomass of the mould at 10, 15, 20, 25 and 30°C. The results were analysed with a simple metabolic model from which the metabolic efficiency was calculated. The results show that the impact of temperature on growth rate and metabolic efficiency are different: although the mould fungus had the highest growth rate (in terms of thermal power, which was continuously measured) at 25°C, the substrate carbon conversion efficiency (biomass production divided by substrate consumption, both counted as moles carbon) was the highest at 20°C. The temperature of the most rapid growth did not therefore equal the temperature of the most efficient growth.     

Oxygen enriched air supply in Escherichia coli processes: production of biomass and recombinant human growth hormone

In order to investigate the impact of high oxygen and carbon dioxide concentrations, Escherichia coli was grown in batch cultivations where the air supply was enriched with either oxygen or carbon dioxide. The effect of elevated concentrations of oxygen and carbon dioxide on stochiometric and kinetic constants was studied this way. The maximum growth rate was significantly reduced, the production of acetic acid and the biomass yield coefficient on glucose increased in cultures with carbon dioxide enriched air, compared to reference cultivations and cultivations with oxygen enriched air. The application of oxygen enriched air was studied in high cell density cultivations of Escherichia coli. Two production processes were chosen to investigate the impact of oxygen enrichment. Biomass concentration, specific growth rate, yield coefficient, respiration, mixed acid fermentation products and the product yield and quality for the recombinant product were investigated. First, a process for the production of biomass was investigated. Exponential growth could proceed for a longer time and higher growth rates could be maintained with oxygen enriched air supply. However, a higher specific oxygen consumption rate per glucose was measured after the start of the oxygen enrichment, indicating higher maintenance and consequently the growth rate and yield coefficient decreased drastically in the end of the process. Second, a process for the production of recombinant human growth hormone (rhGH) was investigated. Although the glucose feed rate and all medium components were doubled, the amount of produced biomass could only be increased by 77% when oxygen enriched air (40% oxygen) supply was applied. This was due to a decreased yield coefficient of biomass per glucose. The total amount of produced product was decreased by almost 50% compared to the control, although less proteolytically degraded variants were produced.     

Effect of operating conditions on solid substrate fermentation

In this work the effects of environmental parameters on the performance of solid substrate fermentation (SSF) for protein production are studied. These parameters are (i) air flow rate, (ii) inlet air relative humidity, (iii) inlet air temperature, and (iv) the heat transfer coefficient between the outer wall of the fermentor and the air in the incubator. The air flow is supplied to effect cooling of the fermented mass by evaporation of water. A dynamic model is developed, which permits estimation of biomass content, total dry matter, moisture content, and temperature of the fermented matter. The model includes the effects of temperature and moisture content on both the maximum specific growth rate and the maximum attainable biomass content. The results of the simulation are compared with actual experimental data and show good agreement with them. The most important conclusions are that (i) the evaporative cooling of the biomass is very effective for temperature control and (ii) the air flow rate and the heat transfer coefficient have strong effects but they affect the biomass morphology and are not controllable easily. Also, a simple technique for the determination of the optimum temperature and moisture content profile for cell protein production is applied. The simulated biomass production increases considerably employing the optimum temperature and moisture content profiles. The ultimate goal is to implement the determined effects of the environmental parameters on the SSF biomass production and the temperature and moisture variation profiles to effectively control the SSF and optimize the biomass production. (c) 1993 John Wiley & Sons, Inc.     

Heat flow measurements in aerobic microbial growth processes with a nonisothermal calorimeter operating directly in the fermenter

A nonisothermal flow calorimeter operating directly in the fermenter was used for heat flow measurements of aerobic microbial growth processes with high biomass productivities. The measuring arrangement makes it possible to describe transitional stages of aerobic yeast cell growth by the ratio of heat production to oxygen consumption (oxy-caloric coefficient). The oxy-caloric coefficient was not constant under the described conditions. The results refer to the existence of an additional energy-delivering mechanism in microbial systems with aerobic carbon source utilization. The mechanism can involve polyphosphate bond division coupled to biomass synthesis.     

Response of plankton to nutrients,planktivory and terrestrial organic matter: a model analysis of whole‐lake experiments

Terrestrial organic matter can be assimilated by aquatic consumers but implications for biomass and production are unresolved. An ecosystem model was fit to estimate effects of phosphorus (P) load, planktivory, and supply rate of terrestrial particulate organic carbon (TPOC) on phytoplankton and zooplankton in five whole‐lake experiments. Phytoplankton biomass increased with P load and planktivory and decreased with TPOC supply rate. Zooplankton biomass increased with P load and responded weakly to planktivory and TPOC supply rate. Zooplankton allochthony (proportion of carbon from terrestrial sources) decreased with P load and planktivory and increased with TPOC supply rate. Lakes with low allochthony (< 0.3) had wide ranges of phytoplankton and zooplankton biomass and production, depending on P load and planktivory. Lakes with high allochthony (> 0.3) had low biomass and production of both phytoplankton and zooplankton. In summary, terrestrial OC inhibits primary production and is a relatively low‐quality food source for zooplankton.     

Bacteria and heterotrophic microflagellate production in the Santa Rosa Sound, Florida

Bacterial and microflagellate biomass and production and grazing onbacteria were compared weekly at a fixed station in Santa Rosa Sound,Florida, starting in February and ending in October. For bothpopulations the weekly variation in biomass and production was aslarge as the seasonal variation. Cycles for biomass and production ofthese organisms were generally out of phase, rendering it difficultto estimate the net grazing of bacteria by microflagellates atindividual time points. For evaluation of factors that control thefate of carbon cycled by bacterial, experiments were conducted toexamine bacterial growth rates in the absence of predators. Thisexamination resulted in low bacterial growth rates when biomass washigh, and rapid growth rates typically occurred near minimumpopulations. Further analysis suggested that microflagellatepredation was greater than bacterial production during minimumbacterial growth rates. With integration of production and grazingrates over the study period, factors controlling bacterial growthwere examined. Using this approach, 71% of the bacterial productionwas grazed by < 8.0µm predators. The microflagellate biomassproduction was 41% of the grazing rate on bacteria. The total amountof bacterial production assimilated into microflagellate biomass was29%. However, based on the variations in biomass and activity of themicrobial assemblages, it appears that substrate and predation exertalternating control on bacterial abundance and production.     

Optimal Locations for Methanol and CHP Production in Eastern Finland

Finland considers energy production from woody biomass as an efficient energy planning strategy to increase the domestic renewable energy production in order to substitute fossil fuel consumption and reduce greenhouse gas emissions. Consequently, a number of developmental activities are implemented in the country, and one of them is the installation of second generation liquid biofuel demonstration plants. In this study, two gasification-based biomass conversion technologies, methanol and combined heat and power (CHP) production, are assessed for commercialization. Spatial information on forest resources, sawmill residues, existing biomass-based industries, energy demand regions, possible plant locations, and a transport network of Eastern Finland is fed into a geographically explicit Mixed Integer Programming model to minimize the costs of the entire supply chain which includes the biomass supply, biomass and biofuel transportation, biomass conversion, energy distribution, and emissions. The model generates a solution by determining the optimal number, locations, and technology mix of bioenergy production plants. Scenarios were created with a focus on biomass and energy demand, plant characteristics, and cost variations. The model results state that the biomass supply and high energy demand are found to have a profound influence on the potential bioenergy production plant locations. The results show that methanol can be produced in Eastern Finland under current market conditions at an average cost of 0.22??/l with heat sales (0.34??/l without heat sales). The introduction of energy policy tools, like cost for carbon, showed a significant influence on the choice of technology and CO₂ emission reductions. The results revealed that the methanol technology was preferred over the CHP technology at higher carbon dioxide cost (>145??/t_CO2). The results indicate that two methanol plants (360?MW_biomass) are needed to be built to meet the transport fuel demand of Eastern Finland.     

Inorganic carbon promotes photosynthesis,growth, and maximum biomass of phytoplankton in eutrophic water bodies

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Modeling aerobic carbon oxidation and storage by integrating respirometric, titrimetric, and off-gas CO2 measurements

A method for detailed investigation of aerobic carbon degradation processes by microorganisms is presented. The method relies on an integrated use of the respirometric, titrimetric, and off-gas CO(2) measurements. The oxygen uptake rate (OUR), hydrogen ion production rate (HPR), and the carbon dioxide transfer rate (CTR) resulting from the biological as well as physicochemical processes, coupled with a metabolic model characterizing both the growth and carbon storage processes, enables the comprehensive study of the carbon degradation processes. The method allows the formation of carbon storage products and the biomass growth rates to be estimated without requiring any off-line biomass or liquid-phase measurements, although the practical identifiability of the system could be improved with additional measurements. Furthermore, the combined yield for biomass growth and carbon storage is identifiable, along with the affinity constant with respect to the carbon substrate. However, the individual yields for growth and carbon storage are not identifiable without further knowledge about the metabolic pathways employed by the microorganisms in the carbon conversion. This is true even when more process variables are measured. The method is applied to the aerobic carbon substrate degradation by a full-scale sludge using acetate as an example carbon source. The sludge was able to quickly take up the substrate and store it as poly-beta-hydroxybutyrate (PHB). The PHB formation rate was a few times faster than the biomass growth rate, which was confirmed by off-line liquid- and solid-phase analysis. The estimated combined yield for biomass growth and carbon storage compared closely to that determined from the theoretical yields reported in literature based on thermodynamics. This suggests that the theoretical yields may be used as default parameters for modeling purposes.     

Specific production rate of VHH antibody fragments by Saccharomyces cerevisiae is correlated with growth rate, independent of nutrient limitation

Saccharomyces cerevisiae carrying a multicopy integrated expression vector containing the gene encoding a Llama antibody fragment, has been cultivated in continuous cultures both under carbon and nitrogen limiting conditions with galactose as the sole carbon source. VHH-R2 expression was under control of the inducible GAL7 promoter. Induction however, was independent of the galactose consumption rate and maximal at all growth rates. VHH-R2 was secreted with 70% efficiency at all growth rates and under both limitations. The specific production rate increased linear with increasing growth rate in a growth-associated manner. However, when grown under nitrogen limitation at growth rates above 0.09 h(-1), the extracellular VHH-R2 was less active or part of the VHH-R2 was in an inactive form. From our results we conclude that to obtain a maximal amount of VHH per kilogram biomass per hour, VHH production should be done in carbon limited continuous cultures at high specific growth rates.     

Bacterial and phosphorus dynamics in profundal Lake Erken sediments following the deposition of diatoms: a laboratory study

The benthic microbial response to the deposition of naturalseston and the microbial impact on nutrient dynamics wasstudied in an experimental system using whole sediment coresequipped with flow-through systems for the overlying water. For20 days, changes in sediment bacterial activity, totalmetabolic activity (heat production), bacterial biomass,phosphorus fractions and basic chemistry were followed, as wellas the exchange of nutrients between sediment and water.Microbial activity and biomass increased immediately inresponse to the deposition of seston, peaked after seven daysand then decreased linearly over the remaining time of theexperiment. Co-settled bacteria were suggested to play animportant role in the microbial response. Changes in bacterialbiomass production, bacterial biomass and the NaOH-nrPextractable phosphorus fraction were concurrent in response toseston additions. The sediment acted as a trap for SRP from theoverlying water when bacterial activity was high and as asource when the bacterial activity decreased. Altogether, theresults suggest an important role of bacteria in theregeneration of seston P. Mineralization rates estimated fromsediment heat production showed that ca. 11% of the addedseston carbon was oxidized in the sediments during theexperiment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Carbon dioxide utilisation of Dunaliella tertiolecta for carbon bio-mitigation in a semicontinuous photobioreactor

Bio-fixation of carbon dioxide (CO₂) by microalgae has been recognised as an attractive approach to offset anthropogenic emissions. Biological carbon mitigation is the process whereby autotrophic organisms, such as microalgae, convert CO₂ into organic carbon and O₂ through photosynthesis; this process through respiration produces biomass. In this study Dunaliella tertiolecta was cultivated in a semicontinuous culture to investigate the carbon mitigation rate of the system. The algae were produced in 1.2-L Roux bottles with a working volume of 1 L while semicontinuous production commenced on day 4 of cultivation when the carbon mitigation rate was found to be at a maximum for D. tertiolecta. The reduction in CO₂ between input and output gases was monitored to predict carbon fixation rates while biomass production and microalgal carbon content are used to calculate the actual carbon mitigation potential of D. tertiolecta. A renewal rate of 45 % of flask volume was utilised to maintain the culture in exponential growth with an average daily productivity of 0.07 g L^?1 day^?1. The results showed that 0.74 g L^?1 of biomass could be achieved after 7 days of semicontinuous production while a total carbon mitigation of 0.37 g L^?1 was achieved. This represented an increase of 0.18 g L^?1 in carbon mitigation rate compared to batch production of D. tertiolecta over the same cultivation period.     

Needle biomass turnover rates of Scots pine (Pinus sylvestris L.) derived from the needle-shed dynamics

To understand carbon cycle and flows of forests, accurate information on tree-component-specific litter production of trees is needed. In the ecosystem models, the litterfall of living trees is usually predicted by the biomass component by average amounts corresponding to site conditions or by multiplying the biomass of the growing stock by the component-specific biomass turnover rate. In this study, the rates of needle biomass turnover of Scots pine (Pinus sylvestris L.) were derived from the needle-shed dynamics. When the rates for needle litter production were modelled, the weighting and yellowing effects were taken into account. The annual biomass turnover rates of needles for southern and northern Finland are 0.21 and 0.10, respectively. Species-specific estimation of litter production is essential for understanding the carbon cycle and flows of forests. Biomass turnover rates can provide useful litter production estimates for large areas with average biomass values as a source of data.This revised version was published online in March 2005 with corrections to the figures. Owing to technical problems, the wrong figures were published.     

Carbon dioxide uptake efficiency by outdoor microalgal cultures in tubular airlift photobioreactors

The influence of solar irradiance and carbon dioxide molar fraction of injected CO(2)-air mixtures on the behavior of outdoor continuous cultures of the microalga Phaeodactylum tricornutum in tubular airlift photobioreactors was analyzed. Instantaneous solar irradiance, pH, dissolved oxygen, temperature, biomass concentration, and the mass flow rates of both the inlet and outlet oxygen and carbon with both the liquid and gas phases were measured. In addition, elemental analysis of the biomass and the cell-free culture medium was performed. The oxygen production rate and carbon dioxide consumption rate increased hyperbolically with the incident solar irradiance on the reactor surface. Carbon losses showed a negative correlation with the daily variation of the carbon dioxide consumption rate. The maximum CO(2) uptake efficiency was 63% of the CO(2) supplied when the CO(2) concentration in the gas supplied was 60% v/v. Carbon losses were >100% during the night, due to CO(2) production by respiration, and hyperbolically decreased to values of 10% to 20% in the midday hours. An increase in the carbon fixed in the biomass with the solar cycle was observed. A slight daily decrease of carbon content of the cell-free culture medium indicated the existence of carbon accumulation in the culture. A decrease in CO(2) molar fraction in the injected gas had a double benefit: first, the biomass productivity of the system was enhanced from 2.05 to 2.47 g L(-1) day(-1) by reduction of CO(2) inhibition and/or pH gradients; and second, the carbon losses during the daylight period were reduced by 60%. The fluid dynamics in the reactor also influenced the carbon losses: the higher the liquid flow rate the higher the carbon losses. By using a previous mass transfer model the experimental results were simulated and the usefulness of this method in the evaluation and scale-up of tubular photobioreactors was established.     

Calorimetric control of the specific growth rate during fed-batch cultures of Saccharomyces cerevisiae

The specific growth rate of a Saccharomyces cerevisiae strain with glucose as limiting C-source was estimated from the measured heat flow produced by the cells. For the cultivation a standard 30 l laboratory bioreactor was used, which was extended in such a way that heat balancing is possible. The feed rate was adjusted by a feedforward/feedback controller such that the specific growth rate was kept on the desired set-point value. On the basis of experimental investigations it was demonstrated that the specific growth rate can be controlled at a given set point value below the critical value to prevent the production of growth-inhibitory ethanol due to the Crabtree effect. With this control strategy high biomass concentrations of more than 110 g l(-1) can be obtained.