Application of bench-scale biocalorimetry to photoautotrophic cultures

Bench-scale biocalorimetry (≥1 L) allows for the determination of the metabolic heat flow during bioprocesses under complete control of all process conditions for extended periods of time. It can be combined with a number of on-line and off-line measurement techniques. This combination can significantly improve insight into the metabolism of microorganisms and the optimization of bioprocesses. In this study it is demonstrated that bench-scale biocalorimetry can also be applied to phototrophic microorganisms. The green microalga Chlorella vulgaris CCAP 211/11B was cultivated in a Mettler-Toledo RC1 calorimeter adapted for high-sensitivity biological calorimetry (BioRC1). Heat production was monitored in 1.5 L batch cultures. In the linear phase of growth, inhibitors of photosynthetic electron transport (DCMU, 3-(3,4-dichlorophenyl)-1,1-dimethylurea, and DBMIB, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone), were used to stop photosynthesis and to monitor the resulting increase in the energy dissipating heat flux. This resulted in a calculated storage of light energy as chemical energy, i.e. biomass, of 141 ± 12.2 mW L⁻¹ (±S.D.). In addition, it was demonstrated that calorimetric determination of the total amount of light energy absorbed within the reactor was accurate by comparing two different calorimetric techniques. Using both the value of the total light input and the quantity stored as chemical energy, the photosynthetic efficiency could be calculated as 10.5% in this example.     

Parallel factor analysis combined with PLS regression applied to the on-line monitoring of Pichia pastoris cultures

Various key variables (biomass, substrate and product) of bioprocesses should be monitored in order to retrieve useful information on the system, with the biomass (the cell density) the principal target. Although several analytical methods have been adapted and used to monitor the evolution of cell density evolution in cultures, a general method for performing this determination has not yet been established, as each technique has its own advantages and drawbacks. In the present work, noninduced glycerol batch cultures (for which biomass and substrate are the key variables) were monitored using multiwavelength fluorescence spectroscopy. The data gathered were modelled via PARAFAC-PLS chemometric methodologies, resulting in important qualitative and quantitative information about the behaviours of different biogenic fluorophors in batch cultures of the yeast Pichia pastoris. This information was used to predict the target process variables in such cultures; this permitted the applicability of this combined technique to bioprocess monitoring to be assessed.     

Production of Poly (3-Hydroxybutyric Acid) by <Emphasis Type="Italic">Ralstonia eutropha</Emphasis> 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 13.56 kJ/g, respectively. The oxycalorific value obtained for the PHB production was 443.80 kJ/mol of O₂. The concentration of PHB obtained in BioRC1e was 4.33 g/L with a production rate of 0.09 g/L/h. The chemical structure of the extracted PHB by R. eutropha was confirmed using fourier transform infrared spectroscopy (FT-IR) and ¹H and ¹³C nuclear magnetic resonance (NMR) analysis.     

Monitoring alcoholic fermentation by joint use of soft and hard modelling methods

The strengths of hard and soft modelling were exploited by using both types of methods in combination to monitor alcoholic fermentations under Saccharomyces cerevisiae yeasts. Experimental work was performed in two steps. In the first, fermentation processes were conducted under identical conditions except for the initial glucose concentration in order to tests various previously reported empirical hard modelling methods. The product inhibition model of Hinshelwood was found to provide the best results in terms of goodness of fit and consistency in parameter values between runs under these conditions. In the second step, fermentation processes conducted at variable temperature and pH were monitored in-line by using an immersion NIR probe. The results were processed by using multivariate curve resolution-alternating least-squares (MCR-ALS) methodology in combination with the hard modelling information obtained in the first step as spectral equality constraints. Notwithstanding the complexity of the fermentation matrix introduced by variability in the species involved in yeast metabolism, the extracted profiles exhibited highly significant correlation with the reference values provided by a validated reference method for the determination of glucose, ethanol and biomass. The results testify to the efficiency of the joint use of soft and empirical hard modelling for studying evolving biological systems and opens up new avenues for application to other bioprocesses.     

Oil Production Towards Biofuel from Autotrophic Microalgae Semicontinuous Cultivations Monitorized by Flow Cytometry

Two microalgae species (Scenedesmus obliquus and Neochloris oleoabundans) were cultivated in closed sleeve photobioreactors in order to select the best oil producer for further large-scale open raceway pond cultivations, aiming at biofuel production. Scenedesmus obliquus reached a higher maximum biomass concentration (1.41 g l⁻¹) with a lower lipid content (12.8% w/w), as compared to N. oleoabundans [maximum biomass concentration of 0.92 g l⁻¹ with 16.5% (w/w) lipid content]. Both microalgae showed adequate fatty acid composition and iodine values as substitutes for diesel fuel. Based on these results, N. oleoabundans was selected for further open raceway pond cultivations. Under these conditions, N. oleoabundans reached a maximum biomass concentration of 2.8 g l⁻¹ with 11% (w/w) of lipid content. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional gravimetric lipid analysis was found for both microalgae, making this method a suitable and quick technique for the screening of microalgae strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of microalgal oil production is now in progress.     

Using Multi-parameter Flow Cytometry to Monitor the Yeast <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> CCMI 145 Batch Growth and Oil Production Towards Biodiesel

Multi-parameter flow cytometry was used to monitor cell intrinsic light scatter, viability, and lipid content of Rhodotorula glutinis CCMI 145 cells grown in shake flasks. Changes in the side light scatter and forward light scatter were detected during the yeast batch growth, which were attributed to the different yeast growth phases. A progressive increase in the proportion of cells stained with PI (cells with permeabilized cytoplasmic membrane) was observed during the yeast growth, attaining 79% at the end of the fermentation. A high correlation between the Nile Red fluorescence intensity measured by flow cytometry and total lipid content assayed by the traditional gravimetric lipid analysis was found for this yeast, making this method a suitable and quick technique for the screening of yeast strains for lipid production and optimization of biofuel production bioprocesses. Medium growth optimization for enhancement of the yeast oil production is now in progress.     

Comparative Performance of Decoupled Input–Output Linearizing Controller and Linear Interpolation PID Controller: Enhancing Biomass and Ethanol Production in Saccharomyces cerevisiae

A decoupled input–output linearizing controller (DIOLC) was designed as an alternative advanced control strategy for controlling bioprocesses. Simulation studies of its implementation were carried out to control ethanol and biomass production in Saccharomyces cerevisiae and its performance was compared to that of a proportional–integral–derivative (PID) controller with parameters tuned according to a linear schedule. The overall performance of the DIOLC was better in the test experiments requiring the controllers to respond accurately to simultaneous changes in the trajectories of the substrate and dissolved oxygen concentration. It also exhibited better performance in perturbation experiments of the most significant parameters q _S,max, q _O2,max, and k _s , determined through a statistical design of experiments involving 730 simulations. DIOLC exhibited a superior ability of constraining the process when implemented in extreme metabolic regimes of high oxygen demand for maximizing biomass concentration and low oxygen demand for maximizing ethanol concentration.     

Quantitative monitoring of yeast fermentation using Raman spectroscopy

Compared to traditional IR methods, Raman spectroscopy has the advantage of only minimal interference from water when measuring aqueous samples, which makes this method potentially useful for in situ monitoring of important industrial bioprocesses. This study demonstrates real-time monitoring of a Saccharomyces cerevisiae fermentation process using a Raman spectroscopy instrument equipped with a robust sapphire ball probe. A method was developed to correct the Raman signal for the attenuation caused by light scattering cell particulate, hence enabling quantification of reaction components and possibly measurement of yeast cell concentrations. Extinction of Raman intensities to more than 50 % during fermentation was normalized with approximated extinction expressions using Raman signal of water around 1,627 cm^?1 as internal standard to correct for the effect of scattering. Complicated standard multi-variant chemometric techniques, such as PLS, were avoided in the quantification model, as an attempt to keep the monitoring method as simple as possible and still get satisfactory estimations. Instead, estimations were made with a two-step approach, where initial scattering correction of attenuated signals was followed by linear regression. In situ quantification measurements of the fermentation resulted in root mean square errors of prediction (RMSEP) of 2.357, 1.611, and 0.633 g/L for glucose, ethanol, and yeast concentrations, respectively.     

Simultaneous determination of glycerol and clavulanic acid in an antibiotic bioprocess using attenuated total reflectance mid infrared spectroscopy

Attenuated total reflectance mid infrared (ATR-MIR) spectroscopy is a potential technique for the near real-time monitoring of filamentous bioprocesses. Here we investigate the utility of ATR-MIR to monitor and predict concentrations of glycerol and product (clavulanic acid) in a complex antibiotic bioprocess. Streptomyces clavuligerus exhibits filamentous growth, thus, as biomass accumulates the process fluid becomes much more viscous, and develops pronounced non-Newtonian behaviour. A multivariate statistical technique, partial least square (PLS) has been used to develop models for the key analytes over the time course of the bioprocess. These models were then validated externally using unseen samples, not used in the original modelling exercise. Despite the heterogeneous nature of the bioprocess and the resulting complexity of the spectra, the models developed had high correlation coefficient values and low prediction error values of 0.302 and 0.009 for glycerol and clavulanic acid, respectively. The findings extend the use of ATR-MIR in these difficult fluids which are typical of filamentous industrial bioprocesses, and demonstrate the practical utility of the technique in the measurement of a range of analyte types, including those present at relatively modest levels compared to the concentrations of biomass and major substrates.     

Thermokinetic responses of the metabolic activity of <Emphasis Type="Italic">Staphylococcus lentus</Emphasis> cultivated in a glucose limited mineral salt medium

Biocalorimetric experiments were performed to investigate the metabolic thermal responses of the halotolerant species Staphylococcus lentus in glucose limited mineral salt medium. Growth factors were optimized in both shaker flask and calorimetric experiments. A limiting value of 5 g/L glucose was found to be the optimum for S. lentus growth. The heat flux profiles, OUR, biomass growth, and substrate depletion profiles were compared to deduce the metabolic activity of S. lentus. Shifts in heat flux due to the shifts in substrate uptake and three distinct phases of growth are noticeable in heat profile curves. Respirogram (OUR) and heat profiles are seen to follow the biomass growth curve. Oxycalorific coefficient is validated with the theoretical studies and those noticed in published literature.     

Yeast Biomass Production in Brewery’s Spent Grains Hemicellulosic Hydrolyzate

Yeast single-cell protein and yeast extract, in particular, are two products which have many feed, food, pharmaceutical, and biotechnological applications. However, many of these applications are limited by their market price. Specifically, the yeast extract requirements for culture media are one of the major technical hurdles to be overcome for the development of low-cost fermentation routes for several top value chemicals in a biorefinery framework. A potential biotechnical solution is the production of yeast biomass from the hemicellulosic fraction stream. The growth of three pentose-assimilating yeast cell factories, Debaryomyces hansenii, Kluyveromyces marxianus, and Pichia stipitis was compared using non-detoxified brewery’s spent grains hemicellulosic hydrolyzate supplemented with mineral nutrients. The yeasts exhibited different specific growth rates, biomass productivities, and yields being D. hansenii as the yeast species that presented the best performance, assimilating all sugars and noteworthy consuming most of the hydrolyzate inhibitors. Under optimized conditions, D. hansenii displayed a maximum specific growth rate, biomass yield, and productivity of 0.34 h⁻¹, 0.61 g g⁻¹, and 0.56 g l⁻¹ h⁻¹, respectively. The nutritional profile of D. hansenii was thoroughly evaluated, and it compares favorably to others reported in literature. It contains considerable amounts of some essential amino acids and a high ratio of unsaturated over saturated fatty acids.     

Advanced Calorimetric Techniques in Polymer Engineering

Summary: In polymer synthesis, reaction calorimetry (RC) is an appropriate technique for on-line process monitoring, since polymerization reactions are highly exothermic. Measurements are noninvasive, rapid, and straightforward. Nowadays RC is the technique recognized as the most powerful way to study such process in near-to-the- industrial conditions. Our approach was focused on temperature oscillation calorimetry (TOC). Two different reaction calorimeters were used, i.e. a isoperibolic calorimeter and a Calvet type high sensitivity differential calorimeter, respectively. A special attention was paid to the interpretation of the measured signals in order to obtain reliable calorimetric data. The evolution of heat transfer coefficient UA was followed by performing appropriate Joule effect calibrations, before and after the reaction. A convolution differential method of the measured heat flow by the generated one was used for determining the time constants and deconvoluting the measured heat flow.     

Lipid Production by Culturing Oleaginous Yeast and Algae with Food Waste and Municipal Wastewater in an Integrated Process

Food waste and municipal wastewater are promising feedstocks for microbial lipid biofuel production, and corresponding production process is to be developed. In this study, different oleaginous yeast strains were tested to grow in hydrolyzed food waste, and growths of Cryptococcus curvatus, Yarrowia lipolytica, and Rhodotorula glutinis in this condition were at same level as in glucose culture as control. These strains were further tested to grow in municipal primary wastewater. C. curvatus and R. glutinis had higher production than Y. lipolytica in media made from primary wastewater, both with and without glucose supplemented. Finally, a process was tested to grow C. curvatus and R. glutinis in media made from food waste and municipal wastewater, and the effluents from these processes were further treated with yeast culture and phototrophic algae culture; 1.1 g/L C. curvatus and 1.5 g/L R. glutinis biomass were further produced in second-step yeast cultures, as well as 1.53 and 0.58 g/L Chlorella sorokiniana biomass in phototrophic cultures. The residual nitrogen concentrations in final effluents were 33 mg/L and 34 mg/L, respectively, and the residual phosphorus concentrations were 1.5 and 0.6 mg/L, respectively. The lipid contents in the produced biomass were from 18.7% to 28.6%.     

Detection of volatile metabolites of <Emphasis Type="Italic">Escherichia coli</Emphasis> by multi capillary column coupled ion mobility spectrometry

Detection and immediate quantification of microbial metabolic activities is of high interest in fields as diverse as biotechnology and infection biology. Interestingly, the most direct signals of microbial metabolism, the evolution of volatile metabolites, is largely ignored in the literature, and rather, metabolite concentrations in the microbial surrounding or even disruptive methods for intracellular metabolite measurements (i.e., metabolome analysis) are favored. Here, the development of a multi capillary column coupled ion mobility spectrometer (MCC-IMS) was described for the detection of volatile organic compounds from microbes and the MCC-IMS was used for characterization of metabolic activity of growing Escherichia coli. The MCC-IMS chromatogram of the microbial culture off-gas of the acetone-producing E. coli strain BL21 pLB4 revealed four analytes that positively correlated with growth, which were identified as ethanol, propanone (acetone), heptan-2-one, and nonan-2-one. The occurrence of these analytes was cross-validated by solid-phase micro-extraction coupled with gas chromatography mass spectrometry analysis. With this information in hand, the dynamic relationship between the E. coli biomass concentration and the metabolite concentrations in the headspace was measured. The results suggest that the metabolic pathways of heptan-2-one and nonan-2-one synthesis are regulated independent of each other. It is shown that the MCC-IMS in-line off-gas analysis is a simple method for real-time detection of microbial metabolic activity and discussed its potential for application in metabolic engineering, bioprocess control, and health care.     

Microcalorimetric studies of the effects of artesunate liposomes on the metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> during growth

A thermal dynamic model of nanoformulations entrapped in artesunate liposomes was established and biological thermodynamics was applied for investigation of the drug formulations. Effects of artesunate liposomes on the growth metabolism of Escherichia coli were studied by microcalorimetry. The results showed that (1) Comparison of artesunate and artesunate liposomes, the thermogenesis curves of E. coli were significant different in the metabolic process: lag phase (AB), log phase (BC), stationary phase (CD), and decline phase (DE); (2) Linear fit of the data of total metabolic heat of E. coli effected by different concentration artesunate (1–300 μg), the equation can be obtained as follows: Y = 364720.61−1075.25x, R = 0.9985; Linear fit of the data of total metabolic heat of E. coli effected by different concentration artesunate liposomes (30–120 μg), the linear equation can be obtained as follows: Y = 54251.5765−35.71122x, R = 0.98345; (3) The half inhibitory concentration I _C50 was 50.05 μg/mL, the relative sensitivity was obviously different; (4) Artesunate liposomes having better sustained release properties as compare to artesunate.     

Flow-Batch Method with a Sequential Injection System for Spectrophotometric Determination of Selenium(IV) in Selenium-Enriched Yeast Using o-Phenylenediamine

A precise, accurate, and reliable flow-batch spectrophotometric method for the determination of selenium (IV) was developed using o-phenylenediamine as a reagent with a sequential injection monosegmented flow system incorporating a simple heating unit. The reaction zones of selenium(IV) and o-phenylenediamine were mixed and heated in a chamber at 62°C for 5 minutes. The piaselanol complexes were then detected at a maximum absorption wavelength of 335 nm. In-line single standard calibration and standard addition procedures were developed employing the monosegmented flow technique. Under the optimized conditions, a linear calibration graph in a range of 0.1–4.0 mg L^?1 selenium (IV) was obtained with limits of detection and quantitation of 0.01 and 0.1 mg L^?1, respectively. Relative standard deviations were 2% [for both 0.1 and 0.5 mg L^?1 selenium (IV) (n = 11)]. A sample throughput of 2 h^?1 using four standard addition levels was achieved. The developed system was successfully applied to raw selenium-enriched yeast samples. The analyses performed by the developed method agreed well with those obtained from a standard inductively coupled plasma mass spectrometry method.     

Microcalorimetric study on the growth and metabolism of <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis>

Microcalorimetry is an experimental technique which allows us to precisely measure the energy released as a consequence of any transformation process. All organisms produce heat as a consequence of metabolism. The rate of heat production is an adequate measurement of metabolic activity of organisms and their constituent parts, cells and sub-cellular levels. Microorganisms produce small amounts of heat, in the order of 1–3 pW per cell. Despite the low quantity of heat produced by bacteria, their exponential replication in culture medium allows their detection using microcalorimetry. This study is a microcalorimetric study of the growth and metabolism of the bacterium Pseudomonas aeruginosa, using the heat liberated as a consequence of bacterial metabolism. With this aim, we used a Calvet microcalorimeter, inside which two Teflon screw-capped stainless steel cells were located (sample and reference). Experiments were carried out at final concentrations of 10⁶, 10⁵, 10³ and 10 CFU/mL, and a constant temperature of 309.65 K was maintained within the microcalorimeter. Recording the difference in calorific potential over time we obtained P. aeruginosa’s growth curves. The shape of these curves is characteristic and has a single phase. Thus, the heat flow curves were mathematically studied to calculate the growth constant and generation time of this bacterium.     

Biotechnological Utilization of Biodiesel-Derived Glycerol for the Production of Ribonucleotides and Microbial Biomass

Ten yeast strains were evaluated concerning their capabilities to assimilate biodiesel-derived glycerol in batch cultivation. The influence of glycerol concentration, temperature, pH and yeast extract concentration on biomass production was studied for the yeast selected. Further, the effect of agitation on glycerol utilization by the yeast Hansenula anomala was also studied. The yeast H. anomala CCT 2648 showed the highest biomass yield (0.30?g?g^?1) and productivity (0.19?g?L^?1?h^?1). Citric acid, succinic acid, acetic acid and ethanol were found as the main metabolites produced. The increase of yeast extract concentration from 1 to 3?g?L^?1 resulted in high biomass production. The highest biomass concentration (21?g?L^?1), yield (0.45?g?g^?1) and productivity (0.31?g?L^?1?h^?1), as well as ribonucleotide production (13.13?mg?g^?1), were observed at 700?rpm and 0.5?vvm. These results demonstrated that glycerol from biodiesel production process showed to be a feasible substrate for producing biomass and ribonucleotides by yeast species.     

Thermal characteristics of the combustion process of biomass and sewage sludge

The combustion of two kinds of biomass and sewage sludge was studied. The biomass fuels were wood biomass (pellets) and agriculture biomass (oat). The sewage sludge came from waste water treatment plant. The biomass and sludge percentage in blends with coal were 10 %. The studied materials were characterised in terms of their proximate and ultimate analysis and calorific value. The composition of the ash of the studied fuels was also carried out. The behaviour of studied fuels was investigated by thermogravimetric analysis (TG, DTG and DTA). The samples were heated from an ambient temperature up to 1,000 °C at a constant three rates: 10, 40 and 100 °C min^?1 in 40 mL min^?1 air flow. TG, DTG and DTA analysis showed differences between coal, biomass fuels and sewage sludge. 10 % addition of studied fuels to the mixture with coal changed its combustion profile in the case of sewage sludge addition. The combustion characteristics of fuel mixtures showed, respectively, qualitative summarise behaviour based on single fuels. Evolved gaseous products from the decomposition of studied samples were identified. This study showed that thermogravimetric analysis connected with mass spectrometry is useful techniques to investigate the combustion and co-combustion of biomass fuels, and sewage sludge, together with coal. Non-isothermal kinetic analysis was used to evaluate the Arrhenius activation energy and the pre-exponential factor. The kinetic parameters were calculated using Kissinger–Akahira–Sunose model.     

Microcalorimetric study of the growth of <Emphasis Type="Italic">Enterococcus faecalis</Emphasis> in an enriched culture medium

Enterococcus faecalis is a Gram-positive bacteria, considered one of the most common causes of nosocomial infections. Bacterial cultures produce an exchange of energy as a result of the bacteria metabolisms. The rate of heat production is an adequate measure of the metabolic activity of the organisms and their constituent parts. Microorganisms produce small amounts of heat: 1–3 pW per cell. Although the heat produced by bacteria is very small, their exponential reproduction in a culture medium permits heat detection through microcalorimetry. In this study, we analyzed the microcalorimetric behavior of Enterococcus faecalis. A thermal Calvet microcalorimeter was used. The inside of the calorimeter contains two stainless steel cells (experimental and reference). Experiments were carried out at final concentrations of 10⁶,10⁵,10³, and 10 CFU/mL and a constant temperature of 309.65 K was maintained within the microcalorimeter. Recording the difference in calorific potential over time we obtained E. faecalis’s growth curves. Thermograms were analyzed mathematically allowing us to calculate the constant growth, generation time and the amount of heat exchanged over the culture time.