Elsevier

Geochimica et Cosmochimica Acta

Volume 261, 15 September 2019, Pages 368-382
Geochimica et Cosmochimica Acta

CO2-dependent carbon isotope fractionation in Archaea, Part I: Modeling the 3HP/4HB pathway

https://doi.org/10.1016/j.gca.2019.06.042Get rights and content

Abstract

The 3-hydroxypropionate/4-hydroxybutyrate (3HP/4HB) pathway of carbon fixation is found in thermophilic Crenarchaeota of the order Sulfolobales and in aerobic, ammonia-oxidizing Thaumarchaeota. Unlike all other known autotrophic carbon metabolisms, this pathway exclusively uses HCO3 rather than CO2 as the substrate for carbon fixation. Biomass produced by the 3HB/4HP pathway is relatively 13C-enriched compared to biomass fixed by other autotrophic pathways, with total biosynthetic isotope effects (εAr) of ca. 3‰ in the Sulfolobales and ca. 20‰ in the Thaumarchaeota. Explanations for the difference between these values usually invoke the dual effects of thermophily and growth at low pH (low [HCO3-]) for the former group vs. mesophily and growth at pH > 7 (high [HCO3-]) for the latter group. Here we examine the model taxa Metallosphaera sedula and Nitrosopumilus maritimus using an isotope flux-balance model to argue that the primary cause of different εAr values more likely is the presence of carbonic anhydrase in M. sedula and its corresponding absence in N. maritimus. The results suggest that the pool of HCO3- inside N. maritimus is out of isotopic equilibrium with CO2 and that the organism imports < 10% HCO3- from the extracellular environment. If correct and generalizable, the aerobic, ammonia-oxidizing marine Thaumarchaeota are dependent on passive CO2 uptake and a slow rate of intracellular conversion to HCO3-. Values of εAr should therefore vary in response to growth rate (μ) and CO2 availability, analogous to eukaryotic algae, but in the opposite direction: εAr becomes smaller as [CO2(aq)] increases and/or μ decreases. Such an idea represents a testable hypothesis, both in the laboratory and in natural systems. Sensitivity to μ and CO2 implies that measurements of εAr may hold promise as a pCO2 paleobarometer.

Introduction

Three new pathways of carbon fixation have been discovered in recent decades: the 3HP cycle of Chloroflexi (Strauss and Fuchs, 1993), the 3HP/4HB cycle of Archaea (Berg et al., 2007, Könneke et al., 2014), and the dicarboxylic/4-HB (Di/4HB) cycle of Archaea (Huber et al., 2008). These three, plus the globally dominant Calvin-Benson-Bassham pathway (CBB; Calvin and Benson, 1948), the reverse tricarboxylic acid (rTCA) cycle (Evans et al., 1966), and the Wood-Ljungdahl, or Acetyl-CoA, pathway (e.g., Wood et al., 1986) represent current knowledge of microbial autotrophy.

The rTCA, 3HP, 3HP/4HP, and Di/4HB pathways share the common property of being catalytic cycles in which one or more of the carbon-fixing enzymes is specific for HCO3- as a substrate. The biomass of organisms containing these pathways is enriched in 13C (has higher values of δ13C) relative to biomass produced via the CBB and Acetyl-CoA pathways (Sirevag et al., 1977, Holo and Sirevag, 1986, van der Meer et al., 2001a, van der Meer et al., 2001b, House et al., 2003, van der Meer et al., 2003, Könneke et al., 2012, Jennings et al., 2014). Such organism-level biosynthetic isotope effects, or ε values, reflect the difference between total dissolved inorganic carbon (DIC) and the resulting biomass, and are reported as ε ≈ Δδ = δ13CDIC – δ13Cbiomass (Hayes, 1993, Hayes, 2001). The δ13C characteristics of this family of autotrophic pathways reflect both the enrichment of 13C in HCO3- when in equilibrium with CO2 (Mook et al., 1974), plus the small (2–3‰) kinetic isotope effect (KIE) associated with enzymes specific for HCO3- (O’Leary et al., 1981, McNevin et al., 2006). Here we specify εAr to mean the ε value for biomass of Archaea using the 3HP/4HB pathway.

The ca. 20‰ value of εAr for marine 3HP/4HB Thaumarchaeota, specifically the model organism N. maritimus (Könneke et al., 2005, Könneke et al., 2012), is markedly different from the ca. 3‰ value for the 3HP/4HB Sulfolobales (van der Meer et al., 2001a, House et al., 2003, Jennings et al., 2014). Genomic (Walker et al., 2010) and enzymatic (Könneke et al., 2014) evidence verify the 3HP/4HB metabolism in N. maritimusi.e., it contains a single carbon-fixing enzyme known to be solely dependent on HCO3-, not CO2 – yet isotopically, it is distinct.

The marked difference in εAr values between thermoacidophilic Sulfolobales and the ammonia-oxidizing marine Thaumarchaeota is well recognized (e.g., Könneke et al., 2012, Könneke et al., 2014) but has not yet been quantitatively explained. Explanation of the 20‰ εAr value in Thaumarchaeota generally invokes both direct enzymatic fractionation during carbon fixation (Könneke et al., 2012) and fractional contributions to biomass from 13C-depleted organic carbon, either by true mixotrophy or in communities that contain heterotrophs in addition to autotrophs (e.g., Herndl et al., 2005, Ingalls et al., 2006, Pearson et al., 2016). However, recent work indicates that N. maritimus and other ammonia-oxidizing marine Thaumarchaeota do not assimilate organic carbon into biomass and lack genes to acquire inorganic carbon from organic substrates like urea (Walker et al., 2010, Santoro et al., 2015, Kim et al., 2016). Therefore the ca. 20‰ value of εAr for N. maritimus in pure culture as determined by Könneke et al. (2012) must solely reflect autotrophy. By analogy, similar values of εAr in the marine environment (Schouten et al., 2013, Pearson et al., 2016, Hurley et al., 2019) suggest dominantly autotrophic processes as well. If so, prior reports of mixo- or heterotrophy may be attributed to artifacts of sampling or analysis.

Here we seek to reconcile these issues by modeling εAr for both N. maritimus and the well-studied member of the thermoacidophilic Sulfolobales, M. sedula. The results suggest that the extent of intracellular inorganic carbon disequilibrium may be the critical factor determining εAr values expressed by the 3HP/4HB cycle. Because the intracellular carbon budget depends on the supply of CO2, εAr should be sensitive to changes in carbon availability and growth rate. Our accompanying Part II paper supports this idea, showing that εAr as recorded in thaumarchaeal membrane lipids isolated from marine suspended particulate organic matter (POM) correlates significantly with the in-situ CO2 concentration (Hurley et al., 2019).

Section snippets

Isotope flux-balance model

We constructed a carbon isotope and flux-balance model for bulk cellular carbon resulting from HCO3- fixation in the 3HP/4HB cycle (Fig. 1). The model follows established principles (e.g., Hayes, 2001, Tang et al., 2017) for interrogating stable isotope distributions in open systems and assumes steady state. Fluxes (φ) and kinetic isotope effects (KIEs, ε) are combined in a system of dependent linear equations of the form:tXi=jφj=0;tδXi=jφjδi-εj=0,where Xi is the fraction of carbon in

Simplified model, M. sedula

The predicted flux balances for M. sedula and N. maritimus are notably different in their magnitude of intracellular conversion between CO2 and HCO3- (Fig. 2a). When normalized to carbon fixation rate, the hydration flux (φ4) for M. sedula is nearly 200-fold faster than for N. maritimus, resulting in dehydration backflow (B) ratios > 0.99 vs. ≤ 0.47, respectively (Table 1).

Rapid exchange of DIC species within M. sedula has isotopic consequences, namely, that Hi and Ci are predicted to approach

Physiological and evolutionary role of the 3HP/4HB pathway

Growth under conditions of extreme resource limitation is thought to be a common feature not only of the Archaea, but of Earth’s microbial biosphere in general (Valentine, 2007, Lever et al., 2015). While typically discussed in the context of energy limitation, here we argue that in the case of marine Thaumarchaeota, carbon limitation also likely plays an important role in their physiology, due to the apparent absence of a functional CA. The ammonia-oxidizing, marine Thaumarchaeota have a low

Conclusions

The flux-balance model developed here provides a simple explanation for why the carbon isotope signatures for 3HP/4HB pathway Sulfolobales and Thaumarchaeota are so different, despite both taxonomic groups of Archaea utilizing the same enzymatic pathway of autotrophic carbon fixation. The key observation is that the total biosynthetic isotope effect (εAr) in Thaumarchaeota is larger than the probable magnitude of the isotope effect for the carbon-fixing enzyme (εfix). This requires that the

Acknowledgements

We thank Martin Könneke, Jed Fuhrman, David Johnston, Emma Bertran, Itay Halevy, Rich Pancost, and Anne Dekas for valuable discussions. We are grateful to Joe Werne for editorial assistance and to three anonymous reviewers for their valuable comments. Funding from NSF-1129343, NSF-1702262, and the Gordon and Betty Moore Foundation (to A.P.); from National Science Foundation Graduate Research Fellowship 1144152 (to E.B.W.); and from the Agouron Institute (to S.J.H.) supported this work.

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