Diffusion-based process for carbon dioxide uptake and isoprene emission in gaseous/aqueous two-phase photobioreactors by photosynthetic microorganisms

Photosynthesis for the generation of fuels and chemicals from cyanobacteria and microalgae offers the promise of a single host organism acting both as photocatalyst and processor, performing sunlight absorption and utilization, as well as CO(2) assimilation and conversion into product. However, there is a need to develop methods for generating, sequestering, and trapping such bio-products in an efficient and cost-effective manner that is suitable for industrial scale-up and exploitation. A sealed gaseous/aqueous two-phase photobioreactor was designed and applied for the photosynthetic generation of volatile isoprene (C(5)H(8)) hydrocarbons, which operates on the principle of spontaneous diffusion of CO(2) from the gaseous headspace into the microalgal or cyanobacterial-containing aqueous phase, followed by photosynthetic CO(2) assimilation and isoprene production by the transgenic microorganisms. Volatile isoprene hydrocarbons were emitted from the aqueous phase and were sequestered into the gaseous headspace. Periodic replacement (flushing) of the isoprene (C(5)H(8)) and oxygen (O(2)) content of the gaseous headspace with CO(2) allowed for the simultaneous harvesting of the photoproducts and replenishment of the CO(2) supply in the gaseous headspace. Reduction in practice of the gaseous/aqueous two-phase photobioreactor is offered in this work with a fed-batch and a semi-continuous culturing system using Synechocystis sp. PCC 6803 heterologously expressing the Pueraria montana (kudzu) isoprene synthase (IspS) gene. Constitutive isoprene production was observed over 192 h of experimentation, coupled with cyanobacterial biomass accumulation. The diffusion-based process in gaseous/aqueous two-phase photobioreactors has the potential to be applied to other high-value photosynthetically derived volatile molecules, emanating from a variety of photosynthetic microorganisms.     

Ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography with cryogenic oven trapping

We have established an ultra-sensitive method for determination of ethanol in whole blood by headspace capillary gas chromatography (GC) with cryogenic oven trapping. After heating a blood sample containing ethanol and isobutyl alcohol (internal standard, IS) in a 7.0-ml vial at 55°C for 15 min, 5 ml of the headspace vapor was drawn into a glass syringe and injected into a GC port. All vapor was introduced into an Rtx-BAC2 wide-bore capillary column in the splitless mode at −60°C oven temperature to trap entire analytes, and then the oven temperature was programmed up to 240°C for GC measurements with flame ionization detection. The present method gave sharp peaks of ethanol and IS, and low background noise for whole blood samples. The mean partition into the gaseous phase for ethanol and IS was 3.06±0.733 and 8.33±2.19%, respectively. The calibration curves showed linearity in the range 0.02–5.0 μg/ml whole blood. The detection limit was estimated to be 0.01 μg/ml. The coefficients of intra-day and inter-day variation for spiked ethanol were 8.72 and 9.47%, respectively. Because of the extremely high sensitivity, we could measure low levels of endogenous ethanol in whole blood of subjects without drinking. The concentration of endogenous ethanol measured for 10 subjects under uncontrolled conditions varied from 0 to 0.377 μg/ml (mean, 0.180 μg/ml). Data on the diurnal changes of endogenous ethanol in whole blood of five subjects under strict food control are also presented; they are in accordance with the idea that endogenous blood ethanol is of enteric bacterial origin.     

Effects of alcohols on the respiration and fermentation of aerated suspensions of baker's yeast.

The immediate effects of externally added alcohols on CO2 production and O2 consumption of suspensions of washed, aerated baker's yeast were studied by stopped-flow membrane inlet mass spectrometry. Glucose-supported fermentation was progressively inhibited by increasing ethanol concentration (0-20%, v/v). The inhibition by ethanol was quite different from that observed for acetaldehyde; thus it is unlikely that toxicity of the latter can account for the observed effects. For five different alkanols (methanol, ethanol, 1-propanol, 2-propanol and 1-butanol) increasing inhibition of anaerobic fermentation was correlated with increased partition coefficients into a hydrophobic milieu. This suggests that the action of ethanol is primarily located at a hydrophobic site, possibly at a membrane. Results for respiratory activities were not as definite as for those for anaerobic metabolism because some alkanols act as respiratory substrates as well as giving inhibitory effects.     

Diffusion of short chain alcohols from amorphous maltose-water mixtures above and below their glass transition temperature

The apparent diffusion coefficient for short chain alcohols in undercooled maltose-water mixtures close to the calorimetric glass transition temperature, Tg, was measured by following desorption using headspace gas chromatography. The plasticising effect of the alcohols on Tg was characterised using differential scanning calorimetry. The initial appearance of alcohol in the headspace showed a linear dependence on the square root of time, allowing it to be modelled as a Fickian diffusive process. The diffusion coefficient decreased with increasing molecular size of alcohol and proximity to Tg. Close to the glass transition the variation of diffusion coefficient with temperature and composition does not follow that of viscosity and, for ethanol, divergence was observed at Tg/T> 0.88.     

Respiration of 13C-labeled substrates added to soil in the field and subsequent 16S rRNA gene analysis of 13C-labeled soil DNA

Our goal was to develop a field soil biodegradation assay using (13)C-labeled compounds and identify the active microorganisms by analyzing 16S rRNA genes in soil-derived (13)C-labeled DNA. Our biodegradation approach sought to minimize microbiological artifacts caused by physical and/or nutritional disturbance of soil associated with sampling and laboratory incubation. The new field-based assay involved the release of (13)C-labeled compounds (glucose, phenol, caffeine, and naphthalene) to soil plots, installation of open-bottom glass chambers that covered the soil, and analysis of samples of headspace gases for (13)CO(2) respiration by gas chromatography/mass spectrometry (GC/MS). We verified that the GC/MS procedure was capable of assessing respiration of the four substrates added (50 ppm) to 5 g of soil in sealed laboratory incubations. Next, we determined background levels of (13)CO(2) emitted from naturally occurring soil organic matter to chambers inserted into our field soil test plots. We found that the conservative tracer, SF(6), that was injected into the headspace rapidly diffused out of the soil chamber and thus would be of little value for computing the efficiency of retaining respired (13)CO(2). Field respiration assays using all four compounds were completed. Background respiration from soil organic matter interfered with the documentation of in situ respiration of the slowly metabolized (caffeine) and sparingly soluble (naphthalene) compounds. Nonetheless, transient peaks of (13)CO(2) released in excess of background were found in glucose- and phenol-treated soil within 8 h. Cesium-chloride separation of (13)C-labeled soil DNA was followed by PCR amplification and sequencing of 16S rRNA genes from microbial populations involved with (13)C-substrate metabolism. A total of 29 full sequences revealed that active populations included relatives of Arthrobacter, Pseudomonas, Acinetobacter, Massilia, Flavobacterium, and Pedobacter spp. for glucose; Pseudomonas, Pantoea, Acinetobacter, Enterobacter, Stenotrophomonas, and Alcaligenes spp. for phenol; Pseudomonas, Acinetobacter, and Variovorax spp. for naphthalene; and Acinetobacter, Enterobacter, Stenotrophomonas, and Pantoea spp. for caffeine.     

Biochemical basis for carbon monoxide tolerance and butanol production by Butyribacterium methylotrophicum

The biochemical mechanisms for growth tolerance to a 100% CO headspace in cultures, and butanol plus ethanol production from CO by Butyribacterium methylotrophicum were assessed in the wild-type and CO-adapted strains. The CO-adapted strain grew on glucose or CO under a 100% CO headspace, whereas, the growth of the wild-type strain was severely inhibited by 100% CO. The CO-adapted strain, unlike the wild-type, also produced butyrate, from either pyruvate or CO. The CO-adapted strain was a metabolic mutant having higher levels of ferredoxin–NAD oxidoreductase activity, which was not inhibited by NADH. Consequently, only the CO-adapted strain can grow on CO because CO oxidation generates reduced ferredoxin which, via the mutated ferredoxin–NAD reductase activity, forms reduced NADH required for catabolism. When the CO-adapted strain was grown at pH 6.0 it produced butanol (0.33 g/l) and ethanol (0.5 g/l) from CO and the cells contained the following NAD-linked enzyme activities (μmol min⁻¹ mg protein⁻¹): butyraldehyde dehydrogenase (227), butanol dehydrogenase (686), acetaldehyde dehydrogenase (82) and ethanol dehydrogenase (129). Received: 15 September 1998 / Received revision: 12 February 1999 / Accepted: 19 February 1999     

Gas phase ethyl acetate production in a batch bioreactor

Gas phase ethyl acetate production was studied using a porcine pancreatic lipase powder. It was observed that gaseous ethyl acetate was produced from gaseous ethanol and acetic acid. Accordingly, the effects of amount of lipase powder, gaseous ethanol and acetic acid concentrations, and reaction temperature on the performance of a batch bioreactor were investigated. Apparent Michaelis-Menten constant of ethanol was 0.163 [μM] and there was no inhibition by ethanol over the range investigated. As acetic acid concentration increased, ethyl acetate production increased to a maximum, then decreased, thus suggesting the inhibition effects by acetic acid. Over the reaction temperature of 25–55?°C, activation energy was calculated as 3.93 kcal/gmol and initial reaction rate was obtained as follows: r?=?75.7 exp(?1975.7/T) [μM/mg of lipase/hr]     

Online measurement of dissolved carbon monoxide concentrations reveals critical operating conditions in gas fermentation experiments

Syngas fermentation is one possible contributor to the reduction of greenhouse gas emissions. The conversion of industrial waste gas streams containing CO or H₂, which are usually combusted, directly reduces the emission of CO₂ into the atmosphere. Additionally, other carbon‐containing waste streams can be gasified, making them accessible for microbial conversion into platform chemicals. However, there is still a lack of detailed process understanding, as online monitoring of dissolved gas concentrations is currently not possible. Several studies have demonstrated growth inhibition of Clostridium ljungdahlii at high CO concentrations in the headspace. However, growth is not inhibited by the CO concentration in the headspace, but by the dissolved carbon monoxide tension (DCOT). The DCOT depends on the CO concentration in the headspace, CO transfer rate, and biomass concentration. Hence, the measurement of the DCOT is a superior method to investigate the toxic effects of CO on microbial fermentation. Since CO is a component of syngas, a detailed understanding is crucial. In this study, a newly developed measurement setup is presented that allows sterile online measurement of the DCOT. In an abiotic experiment, the functionality of the measurement principle was demonstrated for various CO concentrations in the gas supply (0%–40%) and various agitation rates (300–1100 min^?1). In continuous stirred tank reactor fermentation experiments, the measurement showed reliable results. The production of ethanol and 2,3‐butanediol increased with increasing DCOT. Moreover, a critical DCOT was identified, leading to the inhibition of the culture. Thus, the reported online measurement method is beneficial for process understanding. In future processes, it can be used for closed‐loop fermentation control.     

Dynamic properties of monomeric insect erythrocruorin III from Chironomus thummi-thummi: relationships between structural flexibility and functional complexity.

We have investigated the kinetics of geminate carbon monoxide binding to the monomeric component III of Chironomus thummi-thummi erythrocruorin, a protein that undergoes pH-induced conformational changes linked to a pronounced Bohr effect. Measurements were performed from cryogenic temperatures to room temperature in 75% glycerol and either 0.1 M potassium phosphate (pH 7) or 0.1 potassium borate (pH 9) after nanosecond laser photolysis. The distributions of the low temperature activation enthalpy g(H) for geminate ligand binding derived from the kinetic traces are quite narrow and are influenced by temperature both below and above approximately 170 K, the glass transition temperature. The thermal evolution of the CO binding kinetics between approximately 50 K and approximately 170 K indicates the presence of some degree of structural relaxation, even in this temperature range. Above approximately 220 K the width of the g(H) progressively decreases, and at 280 K geminate CO binding becomes exponential in time. Based on a comparison with analogous investigations of the homodimeric hemoglobin from Scapharca inaequivalvis, we propose a link between dynamic properties and functional complexity.     

Glass transition temperatures and fermentative activity of heat-treated commercial active dry yeasts

Differential scanning calorimetry thermograms of various samples of commercial instant active dry yeasts revealed a clear glass transition typical of amorphous carbohydrates and sugars. The resulting glass transition temperatures were found to decrease with increasing moisture content. The observed glass curve was similar to that of pure trehalose, which is known to accumulate in large amounts in baker's yeast. The effect of heat treatment at various temperatures on the fermentative activity (as measured by the metabolic production of CO(2)) of dry yeast was studied. First-order plots were obtained representing the loss of fermentative activity as a function of heating time at the various temperatures assayed. Significant losses of fermentative activity were observed in vitrified yeast samples. The dependence of rate constants with temperature was found to follow Arrhenius behavior. The relationship between the loss of fermentative activity and glass transition was not verified, and the glass transition was not reflected on the temperature dependence of fermentative activity loss.     

Optical and IR absorption as probe of dynamics of heme proteins

The spectroscopy of horseradish peroxidase with and without the substrate analogue benzohydroxamic acid (BHA) was monitored in different solvents as a function of the temperature in the interval from 10 to 300 K. Thermal broadening of the Q(0,0) optical absorption band arises mainly from interaction of the electronic pi --> pi transition with the heme vibrations. In contrast, the width of the IR absorption band of CO bound to heme is controlled by the coupling of the CO transition moment to the electric field of the protein matrix. The IR bandwidth of the substrate free enzyme in the glycerol/H2O solvent hardly changes in the glassy matrix and strongly increases upon heating above the glass transition. Heating of the same enzyme in the trehalose/H2O glass considerably broadens the band. The binding of the substrate strongly diminishes the temperature broadening of the CO band. This result is consistent with the view that the BHA strongly reduces the amplitude of vibrations of the heme pocket environment. Unusually strong thermal broadening of the CO band above the glass transition is interpreted to be caused by thermal population of a very flexible excited conformational substate. The thermal broadening of the same band in the trehalose glass is caused by an increase of the protein vibrational amplitude in each of the conformational substates, their population being independent of the temperature in the glassy matrix.     

Effect of aeration and carbon dioxide on cell morphology of Candida utilis

The effect of CO2 availability on cell size, shape, and aggregation in continuous cultures of Candida utilis was studied in minimal medium with glucose or ethanol as the sole carbon and energy source. Enrichment with CO2 was achieved (i) by using the substrate with more C atoms, (ii) by using pure oxygen and thus decreasing aeration intensity at the same dissolved-oxygen concentration, or (iii) by adding CO2 to the aeration gas. The cells were always of yeast shape, and no filaments were formed. In cultures with a biomass concentration above 6 g (dry weight) per liter, no cell aggregates were observed. In cultures with a lower biomass, the daughter cells failed to separate from the parent cells and formed aggregates with thickened walls. The average cell number per aggregate was found to be higher, and the average protoplast volume lower, under conditions of probable CO2 limitation. Simultaneously, the ratio of total dry weight to wet weight of protoplasts was considerably higher, indicating an increased share of wall or extracellular material. The possible effect of the observed morphological changes for maintaining a suitable concentration gradient of CO2 around the cell is discussed.     

Effect of aeration and carbon dioxide on cell morphology of Candida utilis.

The effect of CO2 availability on cell size, shape, and aggregation in continuous cultures of Candida utilis was studied in minimal medium with glucose or ethanol as the sole carbon and energy source. Enrichment with CO2 was achieved (i) by using the substrate with more C atoms, (ii) by using pure oxygen and thus decreasing aeration intensity at the same dissolved-oxygen concentration, or (iii) by adding CO2 to the aeration gas. The cells were always of yeast shape, and no filaments were formed. In cultures with a biomass concentration above 6 g (dry weight) per liter, no cell aggregates were observed. In cultures with a lower biomass, the daughter cells failed to separate from the parent cells and formed aggregates with thickened walls. The average cell number per aggregate was found to be higher, and the average protoplast volume lower, under conditions of probable CO2 limitation. Simultaneously, the ratio of total dry weight to wet weight of protoplasts was considerably higher, indicating an increased share of wall or extracellular material. The possible effect of the observed morphological changes for maintaining a suitable concentration gradient of CO2 around the cell is discussed.     

Carbon dioxide concentrations are very high in developing oilseeds.

A new method has been developed to rapidly determine the total inorganic carbon concentration (gaseous [CO2] + aqueous [CO(2)] + [HCO3-] + [CO3(2)-]) in developing seeds. Seeds are rapidly dissected and homogenized in 1 N HCl in gas-tight vials. The headspace gas is then analyzed by infrared gas analysis. Developing rapeseed (Brassica napus L.) and soybean [Glycine max (L.) Merr.] seeds were analyzed and found to have up to 40 and 12 mM total inorganic carbon, respectively. These concentrations are ca. 600-2000-fold higher than in ambient air or values reported for leaves. Carbon dioxide concentrations in rapeseed peaked during the stage of maximum oil synthesis and declined as seeds matured. The consequences for seed metabolism, physiology and carbon economy are discussed.     

Assaying CO2 release for determination of formate dehydrogenase activity in entrapment matrices and aqueous-organic two-phase systems

Formate dehydrogenase is an important enzyme for NADH-regeneration in enzyme-catalysed reductions. Methods to determine the activity of this biocatalyst during reaction in aqueous-organic two-phase systems or after immobilisation were therefore investigated. Determination of gaseous CO2 in the headspace of reaction vessels either by gas chromatography or by pressure sensors was found to be a suitable way for deduction of FDH-activity in either of these reaction systems. In the presence of organic solvents, gas chromatography yielded more precise data than pressure sensors, while pressure measurements offer the opportunity to assay continuously the activity of entrapped FDH throughout the whole course of reaction.     

Effects of ethanol and diabetes on galactose oxidative metabolism and elimination in rats.

Blood galactose clearance after an intravenous galactose load has been widely used for years as an index of liver function. We developed a noninvasive [13C]galactose breath test, which explores galactose oxidative metabolism; this test is well correlated with liver fibrosis in patients with chronic viral hepatitis. The goal of this study was to evaluate the influence of nonhepatic factors such as diabetes and ethanol on whole-body galactose clearance (measured as the serum galactose elimination capacity test) and oxidative metabolism (measured as the [13C]galactose-induced breath 13CO2 production) in rats. Acute ethanol administration induced a significant decrease of galactose clearance and 13CO2 production. There was a significant correlation between the amount of ethanol given and the inhibition of galactose metabolism (R2 = 0.72, p < 0.0001). In streptozotocin-induced diabetic rats, the [13C]galactose-induced breath 13CO2 production was significantly reduced (p < 0.0001) and normalized by insulin treatment. However, diabetes did not decrease whole-body galactose clearance, indicating an isotopic dilution of [13C]glucose produced from [13C]galactose metabolism into the enlarged glucose pool. These results must be taken into account when using the [13C]galactose breath test as a quantitative liver function test.     

Effect of gas phase composition on formation of hydrocarbons by Desulfovibrio desulfuricans

Changes in the synthesis of extracellular metabolic products generated by sulfate-reducing bacteria Desulfovibrio desulfuricans grown on a lactate-containing mineral medium in the presence of H2 and CO2 at various volume ratios in the gaseous phase were studied. An increase in the amount of extracellular products synthesized by the bacteria was observed at an H2/CO2 ratio of 3:1. High concentrations of molecular hydrogen (80-95%) in the presence of 5-20% CO2 facilitated the synthesis of hydrocarbons (alkanes) whose highest concentrations were produced at an H2/CO2 ratio of 9:1. An increase in the initial CO2 concentration in the gaseous phase above 20% increased the amount of oxygenated compounds in the culture.     

Control of Penicillium martensii Development and Penicillic Acid Production by Atmospheric Gases and Temperatures

The effects of various gaseous environments and temperatures on development of Penicillium martensii NRRL 3612 and production of penicillic acid (PA) were determined. Accumulation of PA in mold-inoculated corn was measured following incubation under air; 20% CO(2), 20% O(2), 60% N(2); 40% CO(2), 20% O(2), 40% N(2); and 60% CO(2), 20% O(2), 20% N(2). Although reduced temperature initially inhibited PA production, at the end of the trial the largest quantity of PA (120 mug/g of corn) was found in air-incubated corn at the lowest test temperature (5 C). Atmospheres enriched with 60% CO(2) reduced PA accumulation below a detectable level at 5 and 10 C after a 4-week incubation period. Spore germination tests were carried out in a liquid growth medium incubated for 16 hr under several test conditions. Germ tube outgrowth at 30 C ranged from 36% in air to 2% in 60% CO(2), whereas no germination was observed in CO(2)-enriched gases at 10 C. When spore respiration rates were measured in air and O(2) in a liquid growth medium, complete removal of CO(2) from the reaction atmosphere did not reduce O(2) uptake.     

Growth of Seliberia carboxydohydrogena carboxy bacteria with an altered composition of the gas mixture

The growth and gas exchange of Seliberia carboxydohydrogena Z-1062 were studied in the regime of turbidostat when the conditions of gaseous nutrition were changed: a decrease in hydrogen concentration and an increase in carbon monoxide concentration, growth on two carbon sources (CO+CO2) and on two energy sources (H2+CO). The inhibition of the bacterial growth by CO was expressed in a decrease of the specific growth rate and in the reduced effectiveness of using a gaseous substrate. When the concentration of carbon monoxide was elevated from 0 to 40% and that of hydrogen was reduced from 80 to 40%, the specific growth rate of the cells was decreased from 0.4 to 0.04 h-1; here, the economic coefficient in terms of hydrogen fell from 3.6 to 0.62 g/g. The CO-oxidizing system of the bacterium was shown to be resistant. The rate of CO oxidation by the culture was from 0.6 to 0.8 L/h per 1 g of the synthesized biomass at the following concentration of gases in the medium (%); H2, 80-40; CO2, 5; O2, 15; CO, 10-40. The rate of CO oxidation by the culture rose when hydrogen concentration was decreased and CO concentration was increased.     

Hot spot kinetics of the sonolysis of aqueous acetate solutions

Water and acetate solutions were irradiated under argon by 300 kHz ultrasonic waves. Oxygen was found to be generated besides the products H2 and H2O2, already known. In the presence of acetate the O2 yield decreased rapidly while that of H2O2 decreased more slowly. Succinic acid was found as a product of the attack of OH radicals on acetate. Appreciable amounts of glyoxylic and glycolic acid and smaller amounts of formaldehyde and carbon dioxide were also detected. They resulted from the reaction of sonolytically generated oxygen with CH2CO2- radicals, produced upon attack of OH on acetate. Methane was a minor product of sonolysis. At acetate concentrations above 0.4 mol dm-3 CO2 and CO became the predominant products of sonolysis. This is explained by a second kind of action of ultrasound on dissolved acetate, i.e. by a thermal decomposition. This decomposition is possibly facilitated by radical attack on acetate. The results are discussed in terms of a 'structured hot spot' model, in which three regions for the occurrence of chemical reactions are postulated: a hot gaseous nucleus, an interfacial region with radial gradient in temperature and local radical density; and the bulk solution at ambient temperature.