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.     

A portable EAG system for the measurement of pheromone concentrations in the field

A robust and portable apparatus for the measurement of pheromoneconcentrations under field conditions has been developed. Ituses the insect antenna (Lobesia botrana Hb.) itself as a sensitiveand specific pheromone detector. Shock-proof contact with theelectrodes is maintained by fixing the antenna in a specially-shapedplexiglass holder mounted within a glass tube. This allows measurementsto be made while moving the apparatus. Continuous airflow throughthe tube is generated by a suction pump and the incoming aircan be purified by passage through a charcoal filter. This allowsto readjust the offset and to calibrate the instrument by theapplication of pheromone pulses of known concentrations. Removalof the filter allows the direct access of ambient air over theantenna which responds by generating an electro-antennogram(EAG) as a measure of the pheromone concentration. Using the calibration curve, relative pheromone concentrationsin ambient air in a vineyard can be determined. Sample measurementsfrom areas treated with artificial pheromone for pest controlare presented.     

In situ recalibration of probes of dissolved gas concentrations in bioreactors without effect on metabolism and growth

This article describes a general method for the in situ recalibration of a probe for dissolved gas concentrations without changing any parameter but the gas composition. Recalibration of an oxygen probe is used as an example.The recalibration procedure can be used for any probe, provided the culture's rate of metabolism and growth is independent of the substrate concentration in the range being measured by the probe during recalibration. The method was tested using a galvanic oxygen electrode in an oxygen-consuming culture of Klebsiella oxytoca and an oxygen-evolving culture of Anabaena variabilis. The recalibration caused no detectable effect on the metabolism and growth of the cultures, provided the dissolved oxygen tension was above 3muM.     

Gas phase hydrogen/deuterium exchange reactions of peptide ions in a quadrupole ion trap mass spectrometer

Hydrogen/deuterium exchange reactions of protonated and sodium cationized peptide molecules have been studied in the gas phase with a MALDI/quadrupole ion trap mass spectrometer. Unit-mass selected precursor ions were allowed to react with deuterated ammonia introduced into the trap cell by a pulsed valve. The reactant gas pressure, reaction time, and degree of the internal excitation of reactant ions were varied to explore the kinetics of the gas phase isotope exchange. Protonated peptide molecules exhibited a high degree of reactivity, some showing complete exchange of all labile hydrogen atoms. On the contrary, peptide molecules cationized with sodium exhibited only very limited reactivity, indicating a vast difference between the gas phase structures of the two ions. © 1997 Wiley-Liss Inc.     

Application and evaluation of limited mass monitoring with a gas chromatograph mass spectrometer computer system

The technique of scanning a preselected set of ions employing a combined gas chromatography mass spectrometer computer system has been investigated to ascertain the advantages and disadvantages of such a procedure. This technique allows one to determine gas chromatographic retention data with with a high degree of precision and accuracy, in rapid temperature programming operation, due to shortening of the mas spectral scanning interval. Signal-to-noise ratio in ion abundance recordings can be enhanced by increasing the dwell time for as many as 100 ions without lenghtening the scanning interval. The utility of such an approach was demonstrated by analysis of complex mixtures isolated form human urine and cerebrospinal fluid.     

Diurnal cycling of dissolved gas concentrations in a willow vegetation filter treating landfill leachate

Membrane inlet mass spectrometry (MIMS) was used to monitor continuously and simultaneously the concentrations of dissolved gases (O₂, CO₂, CH₄), in the willow root zone at the laboratory scale, and within the treatment bed of a willow vegetation filter treating leachate at a landfill site in mid Wales. These results demonstrate that willows are able to release oxygen into the root zone which accumulates during daylight. Diurnal cycles of oxygen, carbon dioxide, and methane were observed, whereby CO₂ and CH₄ varied reciprocally in relation to O₂. The intensity of these cycles appeared to be related to light intensity and temperature. Oxygen was shown to fluctuate between completely anaerobic and fully aerated (300 μM), between day and night in sunny conditions.     

Spatial variation of dissolved gas concentrations in a willow vegetation filter treating landfill leachate

Membrane inlet mass spectrometry (MIMS) was used to monitor continuously and simultaneously the concentrations of dissolved gases (O₂, CO₂, CH₄) within the treatment bed of a willow vegetation filter treating leachate at a landfill site in mid Wales. The distribution of dissolved gasses within the bed was shown to be highly heterogeneous at the small spatial scale with considerable variation between vertical profiles measured simultaneously at different locations. In general, aerobic conditions were observed above the water table with reduced levels of oxygen and increasing levels of carbon dioxide and methane below it. Distinct pockets of oxygen (up to 200 μM) were observed in anaerobic zones and pockets of reduced oxygen and elevated carbon dioxide were observed in the aerobic zone. Pockets of methane were observed in some profiles coexisting with up to 200 μM oxygen at 5 cm depth. These observations confirm the hypothesis that micro-sites exists within the soil/root matrix where aerobic organic matter decomposition and anaerobic processes such as methanogenesis can occur in relatively close proximity to each other. We hypothesise that the distribution of dissolved gases is determined by rapid diffusion of air maintaining aerobic conditions above the water table, removal of oxygen by microbial processes creating anaerobic conditions below the water table and the distribution of willow roots in the soil which create local aerobic zones by oxygen release.     

In situ determination of nitrogen fixation in Antarctica using a high sensitivity portable gas chromatograph

A portable gas chromatograph was employed in the Vestfold Hills, Antarctica, during the austral summer of 1979-80 for determining nitrogenase activity of the blue-green alga Nostoc commune Vaucher by the acetylene reduction assay. Acetylene reduction was measured in samples taken along a transect where the vegetation changed with respect to differing topography and water availability. Submerged colonies of Nostoc recorded the highest fixation rates (6.89 nmol C₂H₄. cm^-2 h^-1). Damp mosscyanophyte associations growing on shallow slopes showed moderate rates of acetylene reduction (1.99 nmol C₂H₄. cm^-2 h^-1) whilst the drier vegetation of the steeper terrain was the least active (0.19 nmol C₂H₄. cm^-2 h^-1. The employment of a high sensitivity portable gas chromatograph provided an accurate and reliable method of measuring acetylene reduction.     

In vitro maturation of ovine oocytes in a portable incubator

Immature ovine oocytes were collected from ovaries obtained from an abattoir and assigned to one of three treatment groups for in vitro maturation. For Treatment 1 (T1), oocytes were matured in a conventional incubator, in tissue culture wells in an atmosphere of 5% CO(2) and air. Maturation medium consisted of bicarbonate buffered Tissue Culture Medium 199 (TCM199) supplemented with fetal calf serum (FCS), follicle stimulating hormone (FSH), luteinizing hormone (LH), and penicillin/streptomycin (pen/strep). For Treatment 2 (T2), oocytes were matured in a portable incubator, in plastic tubes containing the same medium as T1. The medium was equilibrated with 5% CO(2) and overlayed with oil. For Treatment 3 (T3) oocytes were matured in the portable incubator without CO(2) equilibration, in tubes containing HEPES buffered TCM 199 supplemented as in T1. After 24 hours at 39 degrees C, the percentage of oocytes undergoing normal nuclear maturation was 72.55, 68.14 and 66.96% for T1, T2 and T3, respectively (P >0.05). In a second experiment oocytes were matured in the 3 treatments described, then fertilized in vitro using frozen-thawed ram sperm. Fertilization rates were 44.09, 58.62 and 55.69% for T1, T2 and T3, respectively. T1 and T2 were significantly different (P < 0.05). For Experiment 3, oocytes matured and fertilized as described were cultured in drops of Modified Brinster's Mouse Ova Culture (MBMOC) containing bovine oviductal cells. These were incubated at 39 degrees C in an atmosphere of 5% CO(2) and air for 7 days. T1, T2 and T3 resulted in 20.26, 16.94 and 24.43% development to morulae, and 4.01, 3.06 and 1.85% development to blastocysts, respectively (P >0.05). The results of these experiments indicate that maturation, fertilization, and developmental rates of ovine oocytes matured in the portable incubator are similar to those of oocytes matured in a conventional incubator. This technique shows promise for transportation of oocytes to laboratories where abattoirs are not in close proximity, and holds promise for transportation of oocytes from non-domestic animals collected in the field or remote locations, to facilities capable of utilizing and preserving the gametes.     

Comparison of bovine rumen liquor and bovine faeces as inoculum for an in vitro gas production technique for evaluating forages

Two experiments were undertaken using the in vitro gas production technique of Theodorou et al. [Anim. Feed Sci. Technol. 48 (1994) 185] to compare rumen liquor (RL) and faeces (FA) as inocula for fermenting gramminaceous forages over 96 h periods. Experiment 1 used 12 forages of differing in vivo digestibility (ammonia treated wheat straw, field-cured hay (Lolium perenne) and 10 artificially dried grasses (L. perenne) harvested at different maturities). Experiment 2 used seven maize-silage based forages (whole plant, stover, leaf, lower stem, middle stem, upper stem and husk). In both experiments, rumen liquor and faeces were obtained from two cows in early lactation, each fed daily with 9.4 kg DM of grass silage and 9.0 kg DM of concentrate. Rumen contents were sampled through the fistula, before morning feeding; faeces were sampled from the rectum, immediately afterwards. Rumen liquor (250 ml) was prepared by straining contents through two layers of muslin, adding the solids after blending with 250 ml of buffer and re-straining. Faeces were prepared by mixing (300 ml) with 150 ml of buffer and straining through two layers of muslin and adding a homogenate of the solids and 150 ml of buffer after straining. Data were fitted to the model of France et al. [J. Theor. Biol. 163 (1993) 99]. All model parameters showed FA to have a poorer fermentation capacity than RL. In both experiments, potential gas production volumes (A) were lower (on average 52.9 ml (18.5%)) and lag times longer (on average 2.9 h) for FA compared to RL. Fractional rate of fermentation at half asymptote (T/2) was generally greater for RL than FA (overall means, 0.042, 0.028) and the time required to T/2 being less (overall means, 21.9, 35.4 h). However, potential gas production (A) was highly correlated between RL and FA: Experiment 1 (r²=0.94, 11 forages, excluding ammonia treated straw) and Experiment 2 (r²=0.83, six forages, excluding middle stem). In Experiment 1, organic matter digestibility in vivo (OMD_IV) was also highly correlated with both OMD_FA (r²=0.77, 11 forages) and OMD_RL (r²=0.89, 11 forages); OMD_RL and OMD_FA were also highly correlated (r²=0.81). Similar correlations occurred in Experiment 2. It is concluded that faeces have potential as an alternative inoculum to rumen liquor for in vitro gas production techniques, but methods of overcoming the longer lag phase with faeces require further research.     

Mass spectrometry as en ecological tool for in situ measurement of dissolved gases in sediment systems

Abstract The use of membrane-inlet quadrupole mass spectrometry, as a method for quantitative monitoring of dissolved gases in natural or semi-natural environments, is described. Its advantages over other methods lie in the fact that it provides an accurate, sensitive means for non-invasive, continuous analysis of several dissolved gases simultaneously. The potential of mass spectrometry as an ecological tool is illustrated by representative results from measurements made on undisturbed and experimentally amended estuarine and fresh-water sediments.
Dissolved gas profiles from the surface to a depth of 10 cm in the estuarine sediment showed that the dissolved oxygen decreased gradually until at 10 cm it was undetectable (< 0.25 μM); dinitrogen reached a maximum at 6 cm, where oxygen was 20 μM. In a fresh-water sediment, methane reached 1.5 mM at 10 cm depth. NO_x was also detected; quantitation of carbon dioxide necessitates a correction for the contribution of NO_x. Manipulation of conditions (gas phase, nitrogen and carbon sources) permitted ecological modelling.     

Membrane inlet ion trap mass spectrometry for the direct measurement of dissolved gases in ecological samples

The use of an ion trap mass spectrometer with three different membrane inlet probes is described. Two methods of removing water from the sample are compared. One is the use of a PTFE-silicone rubber double membrane, PTFE is relatively impermeable to water and so reduces the amount entering with the gas sample (Probe A). The second is the use of a silicone rubber membrane covered long probe, which condenses water out of the sample (Probe B). Response times (100%) for dissolved N2O, O2, Ar and CO2 without He in the chamber vary from between 158 and 684 s with Probe A. For the same probe with He, the response times were between 283 and 551 s. In the gas phase response times were between 99 and 153 s with He and 117 and 122 s without He. Probe B had 100% response of between 122 and 152 s for dissolved gases. Further extension of the probe by 2 m slowed response times as did increasing the ionisation time. Response times for Probe B increased to between 99 and 340 s when ionisation time increased from 1000 to 24,930 microseconds. Plots of output against concentration showed the steepest line of response for the short single membrane covered probe with 1000 microseconds ionisation time. Increasing the ionisation time, extending the probe and the use of a double membrane all reduced the gradient of output against concentration for every gas tested. In an intact sediment core, concentrations of O2, N2O and CO2 rose at the start and the concentration of N2 fell. As the disturbed sediment settled, this was reversed. The initial increase in O2 concentration stimulated respiration and inhibited the final pathway in dentrification producing higher concentrations of N2O and reducing the concentration of N2.     

Membrane inlet mass spectrometry — measurement of dissolved gases in fermentation liquids

The measurement of gas concentrations in fermenters is vital for monitoring and controlling a great variety of biotechnological processes. At present, most systems analyse the content of inflowing and outflowing gas streams. The information obtained in this way has proved to be very useful, but gas phase analyses suffer from a considerable disadvantage; the gas composition of the outflow does not precisely reflect the gas composition of the microbes' environment at the time the measurements are made. To overcome this problem, new systems have been developed which measure the concentration of gases (particularly O₂, CO₂ and H₂) dissolved in fermentation liquids.     

Hydration of oleic acid by a Selenomonas strain from the ovine rumen

J.A. HUDSON, C.A.M. MACKENZIE AND K.N. JOBLIN. 1996. A Selenomonas sp., isolated from the ovine rumen, was characterized with regard to its ability to hydrate oleic acid to 10-hydroxystearic acid. Hydration occurred only in stationary phase in a medium containing 0.1%, 0.5% (w/v) galactose or 0.5% (w/v) glucose, but not in a medium containing 1% galactose. Growth under a hydrogen headspace did not result in the production of stearic acid, the biohydrogenated product of oleic acid. Linoleic and linolenic acids (0.1% v/v) were not hydrated. It is concluded that the growing bacterium is unlikely to contribute to oleic acid hydration in the rumen.