Influence of temperature on the δ13C values and distribution of methanotroph‐related hopanoids in Sphagnum‐dominated peat bogs

Methane emissions from peat bogs are mitigated by methanotrophs, which live in symbiosis with peat moss (e.g. Sphagnum). Here, we investigate the influence of temperature and resultant changes in methane fluxes on Sphagnum and methanotroph‐related biomarkers, evaluating their potential as proxies in ancient bogs. A pulse‐chase experiment using ¹³C‐labelled methane in the field clearly showed label uptake in diploptene, a biomarker for methanotrophs, demonstrating in situ methanotrophic activity in Sphagnum under natural conditions. Peat cores containing live Sphagnum were incubated at 5, 10, 15, 20 and 25°C for two months, causing differences in net methane fluxes. The natural δ¹³C values of diploptene extracted from Sphagnum showed a strong correlation with temperature and methane production. The δ¹³C values ranged from ?34‰ at 5°C to ?41‰ at 25°C. These results are best explained by enhanced expression of the methanotrophic enzymatic isotope effect at higher methane concentrations. Hence, δ¹³C values of diploptene, or its diagenetic products, potentially provide a useful tool to assess methanotrophic activity in past environments. Increased methane fluxes towards Sphagnum did not affect δ¹³C values of bulk Sphagnum and its specific marker, the C₂₃ n‐alkane. The concentration of methanotroph‐specific bacteriohopanepolyols (BHPs), aminobacteriohopanetetrol (aminotetrol, characteristic for type II and to a lesser extent type I methanotrophs) and aminobacteriohopanepentol (aminopentol, a marker for type I methanotrophs) showed a non‐linear response to increased methane fluxes, with relatively high abundances at 25°C compared to those at 20°C or below. Aminotetrol was more abundant than aminopentol, in contrast to similar abundances of aminotetrol and aminopentol in fresh Sphagnum. This probably indicates that type II methanotrophs became prevalent under the experimental conditions relative to type I methanotrophs. Even though BHP concentrations may not directly reflect bacterial activity, they may provide insight into the presence of different types of methanotrophs.     

Correcting mass isotopomer distributions for naturally occurring isotopes

In one method of metabolic flux analysis, simulated mass spectrometry data is fitted to measured mass distributions of metabolites that are isolated from cultures with defined feeding of (13)C-labeled substrates. Doing so, simulated mass distributions must be corrected for the presence of naturally occurring isotopes. A method that was recently introduced for this purpose consists of consecutive correction steps for each isotope of each element in the considered compound. Here we show that all isotopes of each individual element must, however, be corrected in one single step. Furthermore, it is shown that the source of information with respect to isotopic compositions of the elements needs to be chosen with care.     

Quantitative analysis of the microbial metabolome by isotope dilution mass spectrometry using uniformly 13C-labeled cell extracts as internal standards

A novel method was developed for the quantitative analysis of the microbial metabolome using a mixture of fully uniformly (U) (13)C-labeled metabolites as internal standard (IS) in the metabolite extraction procedure the subsequent liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS) analysis. This mixture of fully U (13)C-labeled metabolites was extracted from biomass of Saccharomyces cerevisiae cultivated in a fed-batch fermentation on fully U (13)C-labeled substrates. The obtained labeled cell extract contained, in principle, the whole yeast metabolome, allowing the quantification of any intracellular metabolite of interest in S. cerevisiae. We have applied the labeled cell extract as IS in the analysis of glycolytic and tricarboxylic acid (TCA) cycle intermediates in S. cerevisiae sampled in both steady-state and transient conditions following a glucose pulse. The use of labeled IS effectively reduced errors due to variations occurring in the analysis and sample processing. As a result, the linearity of calibration lines and the precision of measurements were significantly improved. Coextraction of the labeled cell extract with the samples also eliminates the need to perform elaborate recovery checks for each metabolite to be analyzed. In conclusion, the method presented leads to less workload, more robustness, and a higher precision in metabolome analysis.     

Characterization of an experimental miniature bioreactor for cellular perturbation studies

A mini bioreactor (3.0 mL volume) has been developed and shown to be a versatile tool for rapidly screening and quantifying the response of organisms on environmental perturbations. The mini bioreactor is essentially a plug flow device transformed into a well-mixed reactor by a recycle flow of the broth. The gas and liquid phases are separated by a silicone membrane. Dynamic mass transfer experiments were performed to determine the mass transfer capacities for oxygen and carbon dioxide. The mass transfer coefficients for oxygen and carbon dioxide were found to be 1.55 +/- 0.17 x 10(-5) m/s and 4.52 +/- 0.60 x 10(-6) m/s, respectively. Cultivation experiments with the 3.0 mL bioreactor show that (i) it can maintain biomass in the same physiological state as the 4.0 L lab scale bioreactor, (ii) reproducible perturbation experiments such as changing substrate uptake rate can be readily performed and the physiological response monitored quantitatively in terms of the O2 and CO2 uptake and production rates.     

Generating short-term kinetic responses of primary metabolism of Penicillium chrysogenum through glucose perturbation in the bioscope mini reactor

A first study of the in vivo kinetic properties of primary metabolism of Penicillium chrysogenum is presented. Dynamic metabolite data have been generated by rapidly increasing the extracellular glucose concentration of cells cultivated under well-defined conditions in an aerobic glucose-limited chemostat followed by measurement of the fast dynamic response of the primary metabolite levels (glucose pulse experiment). These experiments were carried out directly in the chemostat as well as in a mini plug flow reactor (BioScope) outside the chemostat. The results of the glucose pulse experiments carried out in the chemostat and the Bioscope were highly similar. During the 90 s time window of the pulse experiment, the glucose consumption rate increased to a value twice as high as in the steady state, a much lower increase than observed for the fermenting yeast Saccharomyces cerevisiae under similar conditions. Although the observed metabolite patterns in P. chrysogenum were comparable to S. cerevisiae large differences in the magnitude of the dynamic behavior were observed between both organisms. During the pulse experiment the level of glycolytic and TCA cycle intermediates, and adenine nucleotides changed between two- and five-fold. Furthermore, a highly similar five-fold increase in the cytocolic NADH/NAD ratio could be calculated from two independent equilibrium assumptions (fructose 1,6 bis-phosphate to the pool of 2 and 3PG and oxaloacetate to fumarate with glutamate transaminase). It was also found that the C4 pool (aspartate, fumarate, and malate) became much more reduced due to this increase in NADH/NAD ratio. Equilibrium conditions were confirmed to exist in the hexose-P pool, the glycolysis between F16bP and 2+3PG and in the C4 pool of the TCA cycle (fumarate, malate, oxaloacetate and aspartate).