Carbon Dioxide Evolution Rate Research Articles

Production of Poly (3-Hydroxybutyric Acid) by Ralstonia eutropha in a Biocalorimeter and its Thermokinetic Studies.

Bioplastic production from microbial sources is an emerging area which provides opportunities even to convert the wastes into bioplastics. Poly (3-hydroxybutyric acid), commonly called as PHB, is a bioplastic, which is stored as intracellular cytoplasmic inclusions in microorganisms. The objectives of this study are to calorimetrically monitor the PHB production and evaluate the thermokinetic data in a bioreaction calorimeter (BioRC1e). Thus, a well-known PHB-producing bacteria Ralstonia eutropha was selected for batch process in a bioreaction calorimeter. The metabolic heat generated was found to be correlated with the biomass, substrate consumption, oxygen uptake rate (OUR), carbon dioxide evolution rate (CER) and PHB production. The OUR pattern explained the oxidative metabolism of the strain R. eutropha. The heat yields due to biomass and glucose consumption during PHB production were found to be 12.56 and 13.56kJ/g, respectively. The oxycalorific value obtained for the PHB production was 443.80kJ/mol of O2. The concentration of PHB obtained in BioRC1e was 4.33g/L with a production rate of 0.09g/L/h. The chemical structure of the extracted PHB by R. eutropha was confirmed using fourier transform infrared spectroscopy (FT-IR) and (1)H and (13)C nuclear magnetic resonance (NMR) analysis.

The effect of Tween 80 on the polymalic acid and pullulan production by Aureobasidium pullulans CCTCC M2012223.

The effect of Tween 80 on the fermentative production of polymalic acid (PMA) and pullulan using Aureobasidium pullulans CCTCC M2012223 was investigated. Tween 80 is beneficial for the biosynthesis of PMA and pullulan, and can regulate the ratio of PMA to pullulan in a dose-dependent manner. After adding 0.05% Tween 80 to the media, the maximal PMA and pullulan production was 46.45 and 28.8g/L at 60h in a 5L fermenter, with an increase of 75.08 and 27.21% when compared to the control. Tween 80 could regulate and enhance oxygen uptake rate and carbon dioxide evolution rate in the early phase of fermentation, and change the cell morphology. The transcription levels of mitochondrial dicarboxylate transporter and transmembrane transporter were also dramatically upregulated. The present work will be helpful in deeply understanding the mechanism of Tween 80 on the effect of PMA and pullulan production.

Evolution of chemical and biological characterization during thermophilic composting of vegetable waste using rotary drum composter

Vegetable waste usually contains high levels of organic matter, moisture and nutrients that make the waste unsuitable for disposal in municipal landfills. Composting of vegetable waste is in practice by many urban local bodies, and therefore, it was composted along with cow dung and sawdust in a 550-L batch scale rotary drum composter. Four different trials of varying waste combinations of vegetable waste, cow dung and sawdust, i.e., trial 1 (5:4:1), trial 2 (6:2:1), trial 3 (7:2:1) and trial 4 (8:1:1) were composted by adding 10 kg of dry leaves as bulking agent with a total mass of 100 kg. With proper combinations of organic waste mix, a maximum temperature of 66.5 °C was observed in trial 1 and 61.4 °C in trial 2, when compared to other two trials with prolonged thermophilic period. Due to such elevated temperature, higher degradation was observed in trials 1 and 2 with inactivation of pathogens to considerable amounts. Furthermore, final compost had total nitrogen of 2.31 and 3.01 %, total phosphorous of 4.30 and 3.27 % and final carbon-to-nitrogen ratio of 15 and 12, in trials 1 and 2, respectively. Carbon dioxide evolution and oxygen uptake rate of compost samples was analyzed for its stability and was observed to reduce completely at the end of 20 days with lower emission rates.

The effect of gas double-dynamic on mass distribution in solid-state fermentation

The mass distribution regularity in substrate of solid-state fermentation (SSF) has rarely been reported due to the heterogeneity of solid medium and the lack of suitable instrument and method, which limited the comprehensive analysis and enhancement of the SSF performance. In this work, the distributions of water, biomass, and fermentation product in different medium depths of SSF were determined using near-infrared spectroscopy (NIRS) and the developed models. Based on the mass distribution regularity, the effects of gas double-dynamic on heat transfer, microbial growth and metabolism, and product distribution gradient were systematically investigated. Results indicated that the maximum temperature of substrate and the maximum carbon dioxide evolution rate (CER) were 39.5°C and 2.48mg/(hg) under static aeration solid-state fermentation (SASSF) and 33.9°C and 5.38mg/(hg) under gas double-dynamic solid-state fermentation (GDSSF), respectively, with the environmental temperature for fermentation of 30±1°C. The fermentation production (cellulase activity) ratios of the upper, middle, and lower levels were 1:0.90:0.78 at seventh day under SASSF and 1:0.95:0.89 at fifth day under GDSSF. Therefore, combined with NIRS analysis, gas double-dynamic could effectively strengthen the solid-state fermentation performance due to the enhancement of heat transfer, the stimulation of microbial metabolism and the increase of the homogeneity of fermentation products.

A FUNCTION DESCRIBING NITROGEN AVAILABILITY FROM ORGANIC SOURCES

A simple first-order mathematical function is presented to describe net nitrogen (N) immobilization and mineralization from organic materials spread on or incorporated into horticultural soils. To facilitate the representation of immobilization, kinetics are determined by a conveniently measured carbon dioxide (CO2-C) evolution rate. Other parameters include a substrate conversion efficiency, the C:N ratio of microbial decomposers, and the initial C and N contents of the materials. The model was parameterized using a previously reported incubation study of uncomposted lawn trimming materials (Sullivan et al., 2004). To avoid overfitting, parameters are lumped between the different treatments whenever reasonable. All parameters were lumped except for two labile/recalcitrant partition factors, one for C and one for N. Results for each of eight lawn trimming materials represented evolving C very well (r2=0.99). Nitrogen immobilization and release fits were reasonable (r2=0.71), predicting immobilization and mineralization appropriately. Parameterization options based on proximate carbon analysis were also introduced.

Reconstruction of the oxygen uptake and carbon dioxide evolution rates of microbial cultures at near-neutral pH during highly dynamic conditions

The reconstruction of the dynamically changing rates of O2 uptake and CO2 production during short term bioreactor perturbation experiments, from dissolved oxygen and offgas O2 and CO2 measurements, requires a model describing the mass transfer and dispersion of O2 and CO2 in the system as well as the sensor dynamics. Additionally, if perturbation experiments are carried out at near-neutral pH levels, also the inter conversion of dissolved carbon dioxide and bicarbonate needs to be taken into account. While developing such a model we found that for an accurate description of the systems dynamic response on a time-scale of seconds, it is required to incorporate not only the mass transfer between gas bubbles and broth, but also between headspace and broth. The delay and dispersion in the measurements of the gas analyser, due to the length and complexity of the offgas system, was accounted for by determining the impulse response of the offgas system. This model could be fitted excellently to an identification data set. Finally the model was successfully applied to reconstruct the dynamic rates of O2 uptake and CO2 production of a culture of Penicillium chrysogenum, which was perturbed by a glucose pulse.

A sensitive monitoring system for mammalian cell cultivation processes: a PAT approach

Biopharmaceuticals such as antibodies are produced in cultivated mammalian cells, which must be monitored to comply with good manufacturing practice. We, therefore, developed a fully automated system comprising a specific exhaust gas analyzer, inline analytics and a corresponding algorithm to precisely determine the oxygen uptake rate, carbon dioxide evolution rate, carbon dioxide transfer rate, transfer quotient and respiratory quotient without interrupting the ongoing cultivation, in order to assess its reproducibility. The system was verified using chemical simulation experiments and was able to measure the respiratory activity of hybridoma cells and DG44 cells (derived from Chinese hamster ovary cells) with satisfactory results at a minimum viable cell density of ~2.0×10(5) cells ml(-1). The system was suitable for both batch and fed-batch cultivations in bubble-aerated and membrane-aerated reactors, with and without the control of pH and dissolved oxygen.

Biochemical Parameters ofSaccharopolyspora erythraeaduring Feeding Ammonium Sulphate in Erythromycin Biosynthesis Phase

The physiology of feeding ammonium sulphate in erythromycin biosynthesis phase of Saccharopolyspora erythraea on the regulation of erythromycin A (Er-A) biosynthesis was investigated in 50 L fermenter. At an optimal feeding ammonium sulphate rate of 0.03 g/L per h, the maximal Er-A production was 8281 U/mL at 174 h of growth, which was increased by 26.3% in comparison with the control (6557 U/mL at 173 h). Changes in cell metabolic response of actinomycete were observed, i.e. there was a drastic increase in the level of carbon dioxide evolution rate and oxygen consumption. Assays of the key enzyme activities and organic acids of S. erythraea and amino acids in culture broth revealed that cell metabolism was enhanced by ammonium assimilation, which might depend on the glutamate transamination pathway. The enhancement of cell metabolism induced an increase of the pool of TCA cycle and the metabolic flux of erythromycin biosynthesis. In general, ammonium assimilation in the erythromycin biosynthesis phase of S. erythraea exerted a significant impact on the carbon metabolism and formation of precursors of the process for dramatic regulation of secondary metabolites biosynthesis.

Modeling and Fault Monitoring of Bioprocess Using Generalized Additive Models (GAMs) and Bootstrap

Fault monitoring of bioprocess is important to ensure safety of a reactor and maintain high quality of products. It is difficult to build an accurate mechanistic model for a bioprocess, so fault monitoring based on rich historical or online database is an effective way. A group of data based on bootstrap method could be resampling stochastically, improving generalization capability of model. In this paper, online fault monitoring of generalized additive models (GAMs) combining with bootstrap is proposed for glutamate fermentation process. GAMs and bootstrap are first used to decide confidence interval based on the online and off-line normal sampled data from glutamate fermentation experiments. Then GAMs are used to online fault monitoring for time, dissolved oxygen, oxygen uptake rate, and carbon dioxide evolution rate. The method can provide accurate fault alarm online and is helpful to provide useful information for removing fault and abnormal phenomena in the fermentation.

Improving the production of human‐like collagen by pulse‐feeding glucose during the fed‐batch culture of recombinant Escherichia coli

To increase the target protein production and reduce acetic acid accumulation during fed-batch cultivation of recombinant Escherichia coli BL21 in a 30-L bioreactor, 12 different models of pulse feeding were performed to evaluate the effect of pulse feeding at different cultivation phases and pulse frequency on cell growth, acetic acid accumulation, and human-like collagen (HLC) synthesis. The results showed that the acetate concentration was kept at a low level (below 0.5 g/L) in all cases when pulse feeding was introduced before induction, whereas the pulse frequency affected cytoactivity significantly through cell growth rate, oxygen uptake rate, carbon dioxide evolution rate, and the synthesis of the target protein. The final biomass and HLC reached 75.46 and 7.26 g/L, respectively, in the model of 8-Sec feedings per 188 Sec. After induction, the pulse frequency had a great effect on HLC synthesis after high-temperature induction; low frequency was adverse to microorganisms. The model of 3-Sec feeding per 27 Sec was best and resulted in the highest biomass and HLC production. Compared to the pseudo-exponential feeding, pulse feeding reduced acetic acid accumulation effectively.

Simultaneous measurement of the TCA cycle and respiration in isolated mitochondria and intact cells with the XF24‐3 Analyzer

Measuring mitochondrial dysfunction is increasingly important in the study of neurodegenerative and cardiovascular disease, metabolic syndrome, diabetes, cancer, and aging. Mitochondrial function is usually measured by oxygen consumption rates using respirometry methods. However, a critical aspect of mitochondrial function may be overlooked when only O2 consumption is employed: the tricarboxylic acid (TCA) or Krebs cycle, a central metabolic pathway. Measuring the TCA cycle function requires monitoring the flux of an additional analyte, such as carbon dioxide (CO2). The reported method describes simultaneous monitoring of carbon dioxide evolution rates (CDER) and oxygen consumption rates (OCR) using isolated mitochondria and intact cells, in real‐time, using the XF24‐3 Analyzer. This technology employs fluorescent sensors specific for CO2 and O2, which operate reversibly, and reveal details of mitochondrial respiration and TCA cycle activities. Results indicate that O2 consumption and CO2 evolution may be monitored simultaneously, and that data agrees with attributes of TCA cycle and mitochondrial function obtained by other methods. Differential rates of O2 and CO2 flux can be identified relative to substrate utilization and interdependency among the TCA cycle, electron transport, and oxidative phosphorylation systems. Support for this research is provided by Seahorse Bioscience.

Robust artificial intelligence tool for automatic start-up of the supplementary medium feeding in recombinant E. coli cultivations

One of the most important events in fed-batch fermentations is the definition of the moment to start the feeding. This paper presents a methodology for a rational selection of the architecture of an artificial intelligence (AI) system, based on a neural network committee (NNC), which identifies the end of the batch phase. The AI system was successfully used during high cell density cultivations of recombinant Escherichia coli. The AI algorithm was validated for different systems, expressing three antigens to be used in human and animal vaccines: fragments of surface proteins of Streptococcus pneumoniae (PspA), clades 1 and 3, and of Erysipelothrix rhusiopathiae (SpaA). Standard feed-forward neural networks (NNs), with a single hidden layer, were the basis for the NNC. The NN architecture with best performance had the following inputs: stirrer speed, inlet air, and oxygen flow rates, carbon dioxide evolution rate, and CO2 molar fraction in the exhaust gas.

Introducing process analytical technology (PAT) in filamentous cultivation process development: comparison of advanced online sensors for biomass measurement

The recent process analytical technology (PAT) initiative has put an increased focus on online sensors to generate process-relevant information in real time. Specifically for fermentation, however, introduction of online sensors is often far from straightforward, and online measurement of biomass is one of the best examples. The purpose of this study was therefore to compare the performance of various online biomass sensors, and secondly to demonstrate their use in early development of a filamentous cultivation process. Eight Streptomyces coelicolor fed-batch cultivations were run as part of process development in which the pH, the feeding strategy, and the medium composition were varied. The cultivations were monitored in situ using multi-wavelength fluorescence (MWF) spectroscopy, scanning dielectric (DE) spectroscopy, and turbidity measurements. In addition, we logged all of the classical cultivation data, such as the carbon dioxide evolution rate (CER) and the concentration of dissolved oxygen. Prediction models for the biomass concentrations were estimated on the basis of the individual sensors and on combinations of the sensors. The results showed that the more advanced sensors based on MWF and scanning DE spectroscopy did not offer any advantages over the simpler sensors based on dual frequency DE spectroscopy, turbidity, and CER measurements for prediction of biomass concentration. By combining CER, DE spectroscopy, and turbidity measurements, the prediction error was reduced to 1.5 g/l, corresponding to 6% of the covered biomass range. Moreover, by using multiple sensors it was possible to check the quality of the individual predictions and switch between the sensors in real time.

An assessment of seed quality on erythromycin production by recombinant Saccharopolyspora erythraea strain

An assessment of seed quality on erythromycin production by recombinant strain Saccharopolyspora erythraea ZL1004 was investigated in 15 l fermenter. Adding 10 g/l corn steep liquor and 30 g/l soybean flour in seed medium were beneficial to improve cell growth, and the maximal biomass reached 36% at 40 h. Enzyme activity in cell showed that the maximal protease and minimum amylase were appeared in this stage. Compared with the control in 50 l fermenter, the cell metabolism with inoculation of the optimized seed cultivation was obviously quicker, and physiological response such as oxygen uptake rate (OUR) and carbon dioxide evolution rate (CER) were also improved. The maximal erythromycin A production was 9160 U/ml at 215 h, which was increased by 21.63% with respect to the control. It was the first report to integrate cell growth characteristics and physiological response method to assess the seed quality for erythromycin production.

Synchronized populations of Escherichia coli using simplified self-cycling fermentation

Self-cycling fermentation (SCF) was developed as a method to continuously produce synchronized microbial populations at high cell densities. The present study demonstrates the application of this process to populations of Escherichia coli, making use of a simpler and non-intrusive approach to the control strategy. The carbon dioxide evolution rate (CER) could be easily monitored during growth in batch experiments and its first derivative was found to be a good indicator of the transition from exponential growth to stationary phase. This, in turn, was shown to be a suitable control parameter for stable SCF operation. Earlier approaches to the volume changes during the harvest and refilling steps of SCF using load cells and intermediate vessels have been cumbersome and subject to errors in these crucial measurements. In the improved set up, these were replaced with electro-optic level sensors, which resulted in <2% error in volume. This helped to generate stable synchrony with a high degree of inter-cycle reproducibility. The new system was more robust, reliable, cheaper, not subject to probe fouling and easier to scale up. The cultures of E. coli demonstrated significant synchrony as early as the third cycle and, in every experiment, stable synchrony was observed by the sixth cycle. The stable pattern had a synchrony index between 0.68 and 0.74 and the cycle time was 2.47 h ± 0.08. Unexpectedly, the cells doubled in the middle of each synchronized cycle and the length of these cycles was noticeably longer than the doubling time calculated from a batch culture. Neither of these led to a significant loss of cell productivity.

1-MCP SHORTENS PEONY VASE LIFE

Ethylene action and synthesis inhibitors were evaluated for their effects on cut peony flower longevity and quality. Field-grown 'Karl Rosenfield' and 'Sarah Bernhardt' cut peony flowers were treated for two hours with either 0.5 mM silver thiosulfate (STS) or 0.60 μl L -1 1-methycyclopropene (1-MCP) ethylene action inhibitors, Floralife ® (FLO) at the label recommended dosage, 0.60 μl L -1 ethylene (Eth), or deionized water (CON) before being stored for nine weeks. In addition, cut peony flowers temporarily held for three weeks in refrigerated storage were treated for two hours with either 100 μl L -1 aminoethoxyvinylglycine (AVG), an ethylene synthesis inhibitor or deionized water and monitored for an additional seven week storage period. Vase life, flower weight, bud diameter, degree of petal openness, petal color, rate of ethylene and carbon dioxide (CO 2 ) evolution, and percent disease incidence were monitored. 'Karl Rosenfield' flowers were unaffected by all treatments over the storage time. 'Sarah Bernhardt' flowers responded to STS and 1-MCP treatments during CO 2 evolution, petal color and percent disease incidence measurements. Flowers treated with AVG had slowed reproductive organ development and increased disease incidence. The Floralife ® treatment promoted the longest flower vase life; 1-MCP-treated flowers had the shortest vase life. Flowers treated with STS were lighter and less vivid in color, whereas FLO-treated flowers had darker and more vivid petal color. AVG had little to no effect on flower quality or vase life, with the exception of delayed flower development and increased disease incidence. The decline of vase life and flower quality was significantly related to the length of storage time. The ethylene antagonists used in this study were ineffective at increasing storage longevity and quality of cut peony flowers.

Two-stage, self-cycling process for the production of bacteriophages

BackgroundA two-stage, self-cycling process for the production of bacteriophages was developed. The first stage, containing only the uninfected host bacterium, was operated under self-cycling fermentation (SCF) conditions. This automated method, using the derivative of the carbon dioxide evolution rate (CER) as the control parameter, led to the synchronization of the host bacterium. The second stage, containing both the host and the phage, was operated using self-cycling infection (SCI) with CER and CER-derived data as the control parameters. When each infection cycle was terminated, phages were harvested and a new infection cycle was initiated by adding host cells from the SCF (first stage). This was augmented with fresh medium and the small amount of phages left from the previous cycle initiated the next infection cycle. Both stages were operated independently, except for this short period of time when the SCF harvest was added to the SCI to initiate the next cycle.ResultsIt was demonstrated that this mode of operation resulted in stable infection cycles if the growth of the host cells in the SCF was synchronized. The final phage titers obtained were reproducible among cycles and were as good as those obtained in batch productions performed under the same conditions (medium, temperature, initial multiplicity of infection, etc.). Moreover, phages obtained in different cycles showed no important difference in infectivity. Finally, it was shown that cell synchronization of the host cells in the first stage (SCF) not only maintained the volumetric productivity (phages per volume) but also led to higher specific productivity (phage per cell per hour) in the second stage (SCI).ConclusionsProduction of bacteriophage T4 in the semi-continuous, automated SCF/SCI system was efficient and reproducible from cycle to cycle. Synchronization of the host in the first stage prior to infection led to improvements in the specific productivity of phages in the second stage while maintaining the volumetric productivity. These results demonstrate the significant potential of this approach for both upstream and downstream process optimization.

Comprehensive characterization of recombinant Pichia pastoris using dynamic process conditions and process analytical technology

HRP_ FB I HRP_FB II HRP_B II Fig 3. Induction by methanol pulses in a batch culture. Cells were grown using D-glucose as C-source. When the substrate was exhausted, induction of protein expression was carried out by successive pulses of methanol: 1 adaptation pulse with 0,5 % methanol (in gray), successive pulses with 1 % methanol (in orange) and 1 pulse with 2 % methanol (not shown). The on-line calculated carbon dioxide evolution rate (CER) and the oxygen uptake rate (OUR) are shown. These calculations were performed by a metabolic balance analyzer implemented on-line. HRP activity was detected both in the absence and in the presence of D-Ala with slightly better results in the latter case, therefore D-Ala was added to the fed-batch cultures.

Model‐based data evaluation of polyhydroxybutyrate producing mixed microbial cultures in aerobic sequencing batch and fed‐batch reactors

The production of polyhydroxyalkanoates (PHAs) with mixed microbial cultures is a promising approach for the sustainable production of bioplastics. Usually a two-step process is employed consisting of (i) the enrichment of a PHA producing mixed culture in a sequencing batch reactor (SBR) and (ii) the subsequent PHA production in a fed-batch reactor. Both reactors are highly dynamic systems, particularly if the SBR is working at low sludge residence times (SRTs) or if growth is (partly) permitted in fed-batch systems. Under these conditions the concentrations of substrate, PHA and biomass change rapidly, complicating the identification of biomass specific conversion rates as required for process characterization. We developed a structured approach for the evaluation of such SBR and fed-batch experiments consisting of five steps: (1) Measurement of a sufficiently large set of parameters including off-gas concentrations, (2) Corrections of measurements for effects of sampling and addition of liquids (pH control, substrate), (3) Calculation of oxygen uptake and carbon dioxide evolution rates, the latter including inorganic carbon dissolved in the liquid phase, (4) Balancing of the measured conversions, (5) Evaluation of the measurements by means of a metabolic model. This approach has been successfully applied to a large number of data sets. Steps 1-4 ensured that data sets of high quality were obtained. Step 5 allowed to find the best estimates for all conversions and biomass specific rates for the measured data sets, while complying with material balances. Conversions of the substrate acetate, the nitrogen source ammonia and of the storage polymer PHA (here polyhydroxybutyrate (PHB)) were described very accurately by the model. Modeled off-gas conversions often deviated somewhat from measured conversions, which might be partly due to an inaccurate model stoichiometry. Nonetheless, the described approach proved to be a very useful tool for the evaluation and comparison of PHB producing cultures.

Control of continuous fed-batch fermentation process using neural network based model predictive controller

Cell growth and metabolite production greatly depend on the feeding of the nutrients in fed-batch fermentations. A strategy for controlling the glucose feed rate in fed-batch baker's yeast fermentation and a novel controller was studied. The difference between the specific carbon dioxide evolution rate and oxygen uptake rate (Qc - Qo) was used as controller variable. The controller evaluated was neural network based model predictive controller and optimizer. The performance of the controller was evaluated by the set point tracking. Results showed good performance of the controller.