Non-cyanobacterial diazotrophs mediate dinitrogen fixation in biological soil crusts during early crust formation



Biological soil crusts (BSC) are key components of ecosystem productivity in arid lands and they cover a substantial fraction of the terrestrial surface. In particular, BSC N\(_{2}\)-fixation contributes significantly to the nitrogen (N) budget of arid land ecosystems. In mature crusts, N\(_{2}\)-fixation is largely attributed to heterocystous cyanobacteria, however, early successional crusts possess few N\(_{2}\)-fixing cyanobacteria and this suggests that microorganisms other than cyanobacteria mediate N\(_{2}\)-fixation during the critical early stages of BSC development. DNA stable isotope probing (DNA-SIP) with \(^{15}\)N\(_{2}\) revealed that Clostridiaceae and Proteobacteria are the most common microorganisms that assimilate \(^{15}\)N\(_{2}\) in early successional crusts. The Clostridiaceae identified are divergent from previously characterized isolates, though N\(_{2}\)-fixation has previously been observed in this family. The Proteobacteria identified share \(>\)98.5 %SSU rRNA gene sequence identity with isolates from genera known to possess diazotrophs (e.g. Pseudomonas, Klebsiella, Shigella, and Ideonella). The low abundance of these heterotrophic diazotrophs in BSC may explain why they have not been characterized previously. Diazotrophs play a critical role in BSC formation and characterization of these organisms represents a crucial step towards understanding how anthropogenic change will affect the formation and ecological function of BSC in arid ecosystems.
keywords: microbial ecology / stable isotope probing / nitrogen fixation / biological soil crusts


Biological soil crusts (BSC) are specialized microbial communities that form at the soil surface in arid environments and they fill a variety of important ecological functions. BSCs occupy plant interspaces and cover a wide, global geographic range (Garcia-Pichel et al., 2003). For example, in some regions on the Colorado Plateau BSCs cover 80% of the ground (Karnieli et al., 2003). The global biomass of BSC cyanobacteria alone is estimated at 54 x 10\(^{12}\) g C (Garcia-Pichel et al., 2003). BSD nitrogen fixation (N\(_{2}\)-fixation) is responsible for significant input of nitrogen (N) to arid environments (Evans et al., 1999; Belnap, 2003). Interestingly, much of this fixed N is exported from the crusts in dissolved form through percolation or runoff and little is lost to volatilization (Johnson et al., 2007). The presence of BSC is positively correlated with vascular plant survival due in part to N inputs from BSC (for review of BSC-vascular plant interactions see Belnap et al. 2003). These microbial ecosystems are not immune to climate change and changes in precipitation and temperature could alter BSC microbial community structure/membership and possibly BSC diazotroph diversity and N\(_{2}\)-fixation (Garcia-Pichel et al., 2013).

BSC are highly susceptible to natural and anthropogenic disturbance (Garcia-Pichel et al., 2013). Succession in BSC communities is characterized by transition from early successional “light” crusts to mature “dark” crusts (Yeager et al., 2004; Belnap, 2002). Motile non-heterocystous cyanobacteria(e.g.Microcoleus vaginatus or M. steenstrupii), which cannot fix N\(_{2}\) are pioneer colonizers of early successional crusts and are abundant in all types of BSCs (Garcia-Pichel et al., 2013). Successional development of mature crust is accompanied by a change in color produced by secondary colonization with non-motil N\(_{2}\)-fixing heterocystous cyanobacteria which produce sunscreen compounds that reduce soil albedo . These heterocystous cyanobacteria (e.g. Scytonema, Spirirestis, and Nostoc) increase in abundance during crust development and are more abundant in mature crusts (Yeager et al., 2007; Yeager et al., 2012). Heterocystous cyanobacteria are numerically dominant in surveys of BSC nifH gene diversity \citep{Yeager, Yeager_2004, Yeager_2012}. For example, 89 percent of 693 nifH sequences derived from Colorado Plateau and New Mexico BSC were attributed to heterocystous cyanobacteria (Yeager et al., 2007). Other BSC nifH sequences are attributed to Alpha-, Beta-, and Gammaproteobacteria, as well as a nifH clade (nifH cluster III) that includes diverse anaerobes such as clostridia, sulfate reducing bacteria, and anoxygenic phototrophs (Steppe et al., 1996; Yeager et al., 2007).

Two lines of evidence suggest that nitrogen fixers other than phototrophs are important in early-successional crusts. First, the contributions of early successional BSC to N\(_{2}\)-fixation in arid ecosystems may have been systematically underestimated. The high abundance of heterocystous cyanobacteria at the surface of mature crusts, where acetylene reduction assay rates are often maximal, is generally taken as evidence that BSC N\(_{2}\)-fixation occurs primarily in mature crusts and is dominated by heterocystous cyanobacteria. However, rates of BSC N\(_{2}\)-fixation are typically determined by areal measurements made at the crust surface with the acetylene reduction assay and vary significantly across samples and studies (Evans et al., 2001). The reasons for inter-site and inter-study variability are complex and likely include the spatial heterogeneity of BSC (Evans et al., 2001). The acetylene reduction assay is also subject to methodological artifacts that can complicate comparisons between samples that differ in their physical and biological characteristics (see Belnap 2001 for review). In particular, N\(_{2}\)-fixation in early successional BSC is maximal below the crust surface (Johnson et al., 2005) and hence diffusional limitation (of both acetylene and ethylene) across the crust surface can cause severe underestimates if they do not allow for sufficiently long incubation times (Johnson et al., 2005). If BSC N\(_{2}\)-fixation is instead estimated by integrating rates across a depth profile (which eliminates constraints from diffusional limitation), then total rates of N\(_{2}\)-fixation do not differ significantly between early successional and mature BSC (Johnson et al., 2005). This result suggests that diazotrophs other than heterocystous cyanobacteria may be important contributors to N\(_{2}\)-fixation in early successional BSC communities as early successional BSC possess few heterocystous cyanobacteria and these are present near the crust surface. Second, the bare soils that are colonized during the process of early crust formation are unconsolidated and oligotrophic in many respects, with much lower N content than adjacent crusts (Beraldi-Campesi et al., 2009), and the cyanobacteria that are typical colonization pioneers (Microcoleus spp., Garcia-Pichel et al. 2009), are unable to fix nitrogen as they lack that genetic capacity (Starkenburg et al., 2011; Rajeev et al., 2013).

To determine the agency of nitrogen fixation in early developmental crusts, we conducted \(^{15}\)N\(_{2}\) DNA stable isotope probing (DNA-SIP) experiments with early successional Colorado Plateau BSC conspicuously devoid of significant surface populations of heterocystous cyanobacteria. DNA-SIP with \(^{15}\)N\(_{2}\) has not been previously attempted with BSC. DNA-SIP provides an accounting of active diazotrophs on the basis of \(^{15}\)N\(_{2}\) assimilation into DNA whereas nifH clone libraries merely account for microbes with the genomic potential for N\(_{2}\)-fixation. Further, we investigate the distribution of these active diazotrophs in surveys of microbial diversity conducted on BSC over a range of spatial scales and soil types (Garcia-Pichel et al., 2013; Steven et al., 2013).


DNA buoyant density changes in response to \(^{15}\)