Conditions
7.4 Results and Discussion
7.4.3 Isotope labeling in continuous culture
We applied hydrogen isotope labeling in continuous culture experiments to estimate microbial activity as a proof of concept, and to assess various parameters that could affect the interpretation of data from environmental applications. Continuous culture, or chemically static (chemostat) experiments provide the ideal setting for testing any tracer of microbial activity. The chemostat was developed independently around 1950 by Jacques Monod at the Pasteur Institute (MONOD,1950), who coined the term continuous culture (and called the device a bactogène), and by Aaron Novick and Leo Szilard at the University of Chicago (Novick and SZILARD, 1950), who gave it its modern name.
The theory and application of chemostats was refined in subsequent years, and led to fascinating new insights into fundamental aspect of microbial physiology (e.g. Novick (1955); Herbert et al. (1956)). The molecular revolution saw its use diminish for several decades, but the importance of continuous culture to physiological research remains as important today as it was 60 years ago, which is increasingly re-recognized for a wide range of research applications (Hoskisson, 2005). Briefly, a chemostat provides a chemically constant environment for microbial culture by continuously feeding fresh medium at a constant rate into a reactor vessel that is kept at a constant volume by removing the overflow. Assuming that cells are either not actively degraded, or if they are, their (partly
labeled) cellular constituents are recycled by the remainder of the population (i.e. cells are not completely lost to the dissolved organic matter pool), the only form of actual removal of biomass is the dilution rate imposed by the continuous culture setup. This sets a fixed dilution rate of the microbial culture (equivalent conceptually to removal rate ddiscussed in Section 7.2), which the population needs to counteract by doubling at the same rate (µ=d) once at steady-state7. Here, we take advantage of a culturing environment with an independently controlled growth rate to test incorporation of an isotopic spike of D2O administered at steady-state as a function of growth. Towards this end, we expand on the equations and concepts derived in Sections 7.2 and 7.4.2.
In the case of an isotope labeling experiment in an environmental setting, the overall metabolic mode (heterotrophy vs. autotrophy, chemotrophy vs. phototrophy) can likely be constrained, and laboratory values for the water fraction factorxw·↵l/w (Eq. 7.11, Table 7.1 and Figure 7.4) provide reasonable estimates for the corresponding environmental values. The exact nature and hydrogen isotope composition of the environmental growth substrate(s)Fsis rarely known, providing limited information on how to apply the substrate offset or substrate fraction factor (if known). However, with an estimate of the water fraction factor and knowledge of the natural isotopic composition of the source water Fwnat, the medium/environment specific substrate offset can be estimated simply from observing the natural isotopic composition of the lipids 2Flipid(t0) before application of an isotopic spike:
(1 xw)·↵l/s·2Fs =2Flipid(to) xw·↵l/w·2Fwnat (7.12)
7A tremendous amount of literature exists on the theory and application of chemostats; please see e.g. Smith(1995) for details.
which, substituted back into Eq. 7.11to provide an expression for the isotopic composition of newly synthesized lipids, yields:
2Flipidnew =xw· ↵l/w · 2Fw 2Fwnat +2Flipid(t0) (7.13) Using Eq. 7.13 to describe the isotopic composition of newly-formed lipids, with a dilution model for the isotopically spiked water 2Fw(t) = 2Fwspiked · e kt (with initial isotopic composition of the spiked medium water 2Fwspiked and dilution rate constantk) and substituting back into Equation7.8 finally yields:
2Flipid(t) = Bnew·2Fnew
B + Bold·2Fold
B
=
xw·↵l/w·2Fwspiked ·fS· µ+!
µ+!+k · 1 e (µ+!+k)·t + 2Flipid(t0) xw·↵l/w·2Fwnat ·fS· 1 e (µ+!)·t ⇤ +⇥2
Flipid(t0)· 1 fS· 1 e (µ+!)·t ⇤
=xw·↵l/w ·fS·
2Fwspiked· µ+!
µ+!+k · 1 e (µ+!+k)·t 2Fwnat · 1 e (µ+!)·t +2Flipid(t0)
(7.14) which in the case of a constant isotopic label (no dilution or other variation of the water isotopic composition over the course of the isotope labeling experiment), would simplify to:
2Flipid(t) = xw ·↵l/w·fS· 2Fwspiked
2Fwnat · 1 e (µ+!)·t +2Flipid(t0) (7.15) where xw · ↵l/w is the water fraction factor, fS the fraction of de novo biosynthesis (vs. recycling of exogenous lipids), 2Fwspiked the isotopic composition of the spiked
medium/environmental source water,2Fwnatthe natural isotopic composition of the source water, µ the growth rate of the population, ! the turnover rate and 2Flipid the isotopic composition of the microbial lipids at time t (all parameters as described previously).
120 160 200 240
doubling: ~2.29 hours
0.00 0.05 0.10 0.15
Time [fraction of one doubling]
2Ffatty acid[ppm]
Figure 7.6 – Isotope labeling of E. coli. This figure illustrates the isotopic enrichment of major membrane components (>5%) of E. coli after an isotopic D2O spike into a culture grown continuously at a fixed dilution rate equivalent to doubling every⇠2.3 hours. Time is shown as the fraction of a doubling for ease of comparison to other experiments. Colors indicate the different fatty acids, symbol sizes represent the relative pool sizes (% of all fatty acids in the membrane). The thick dashed line indicates the average isotopic composition of the whole membrane. The dotted thin line indicates the predicted enrichment from theoretical considerations, and is based on the weighted average water fraction factor determined for this medium (Table 7.1). The gray shaded band indicates the maximal range of predicated labeling considering the maximal range of measured water fraction factors (of any fatty acid of this organism) in combination with the 95% confidence interval on the measurement of the initial water isotopic composition of the D2O spiked reactor.
Figure 7.6 shows the incorporation of 2H into the major fatty acid components of E.
coli over the course of ⇠1/5 of a doubling after an isotopic spike with D2O (2Fwspiked= 1208ppm) during growth in continuous culture at a constant growth rate of ⇠7.3 day-1(a doubling time of ⇠2.3 hours) in Minimal medium (see Table D.14 for the data). These data show immediate incorporation of the water tracer into all membrane components, consistent with rapid osmotic equilibration predicted to occur in microorganisms due to
their high surface-to-volume ratio (Verkman,2013). Additionally, the different fatty acids show substantial divergence in their labeling patterns, with C16:1 getting over-labeled with respect to the major membrane component C16:0, and C18:1 and cyclo-C17:0 getting under-labeled (significantly so in the case of cyclo-C17:0). This divergence pattern provides potential insight into dynamics within this microbial population, and is discussed in some detail in Section 7.4.5. Here, we focus on the overall isotopic labeling of the membrane as a whole (the weighted isotopic average, or mass balance, of all membrane components), as shown in Figure 7.6 by the thick dashed line. Since the growth rate µ is controlled by the chemostat setup, we can model the expected isotopic enrichment using Equation 8.2 with the following measured parameters: 2Fwspiked is the isotopic composition of the medium water after the spike (diluted with dilution rate k set by the chemostat), 2Fwnat is the natural isotopic composition of the medium water,xw ·↵l/w is the weighted average water fraction factor for E. coli in this medium (see Section 7.4.2 for details on individual fatty acids),2Flipid(t0)the weighted average isotopic composition of the membrane measured at the onset of the labeling experiment, and fS = 1 (the medium is well-defined, and contains no exogenous fatty acid sources). The dotted line in Figure 7.6 represents the resulting theoretical level of enrichment, which closely matches the actual labeling pattern of the major fatty acid (C16:0), but slightly overestimates the observed labeling of the membrane as a whole. The single largest source of error in the prediction stems from uncertainty in the water fraction factor (see Figure 7.4).
This is illustrated in Figure 7.6 by the gray band, which represents the maximal and minimal labeling prediction based on the entire range of water fraction factors of all membrane components observed forE. coli combined with uncertainty in the water isotope measurement (the latter is a minor component). The observed isotope labeling of the membrane matches the theoretical prediction well within this uncertainty, indicating that this method could be used in reverse for its original purpose (predicting growth rates from isotopic enrichment).
It is important to note that the slight under-labeling of the membrane as a whole with respect to the model prediction could also hint at a role of metabolically produced water in diluting the water label within the cells (Kreuzer-Martin et al.,2006). This effect could explain the observed enrichment if the heavy water label is effectively diluted by 5-10% within the cells due to water produced from metabolic activity. However, given the uncertainty in the water fraction factor, the current data does not reveal whether this effect occurs in this experiment or what its exact magnitude might be. If present, it is reasonable to assume that its importance would increase at faster growth rates and decrease at slower growth rates due to its direct dependence on metabolic water production. This merits careful evaluation when applying this technique to even faster-growing cultures.