INTRODUCTION
Globally, soils store approximately 1,500 Pg of soil organic carbon
(SOC) in the upper meter of the soil profile, with 50-67% of SOC
occurring below 20 cm (Jobbágy & Jackson 2000). The persistence of this
C pool is in part controlled by extracellular enzymes (EEs) primarily
released by soil microorganisms that decompose soil organic matter
(Burns et al. 2013). However, even though the majority of SOC
occurs in the subsoil, most studies of soil microorganisms and the EEs
they secrete focus on the upper soil layers. While the age (and thus
persistence) of SOC increases with depth (Trumbore et al. 1996;
Paul et al. 1997; Rumpel et al. 2002), recent studies have
shown that subsoil (>20 cm depth) C is still vulnerable to
decomposition. Indeed, subsurface microbial communities have resource
demands that rival those of surface soils when normalized to a microbial
biomass (MB) basis (Jones et al. 2018). Understanding subsurface
processes is critical in an age of global change because vulnerability
of SOC to EE attack could be enhanced by increased temperatures or
wetting/drying cycles (Schimel et al. 2011; Hicks Pries et
al. 2017). This means that if subsoils are disturbed (either physically
or through altered environmental conditions), portions of the soil
organic matter pool at depth could become accessible to EEs, resulting
in the mineralization of significant quantities of C and nutrients.
Therefore, increased understanding of EE patterns at depth could help
elucidate the mechanisms of subsoil organic matter decomposition and aid
in predicting how pools of SOC and nutrients will be affected by ongoing
global change factors.
Because EEs both respond to and influence soil properties, the study of
EEs has led to greater insights into soil C persistence (Billings &
Ballantyne 2013; Birge et al. 2015; Dove et al. 2019),
nitrogen (N) and phosphorus (P) mineralization (Weintraub & Schimel
2003; Waring et al. 2014; Chen et al. 2018), ecosystem
development (Olander & Vitousek 2000; Selmants & Hart 2010; Turneret al. 2014), and microbial metabolism (Sinsabaugh & Shah 2011,
2012; Sinsabaugh et al. 2013). Given that the methods for
measuring EE activity in soils are relatively high-throughput,
inexpensive, and reproducible across laboratories (Dick et al.2018), it is one of the most common soil biogeochemical measurements
(‘Soil extracellular enzyme activity’ resulted in 2,013 records using
Clarivate Analytics Web of Science as of Jan. 28, 2020). However,
despite the widespread measurement of soil EEs, most studies have
focused on EE activities in surface horizons, with few studies exploring
EE activity patterns in soil horizons below 20 cm (but see Tayloret al. 2002; Kramer et al. 2013; Stone et al. 2014;
Taş et al. 2014; Schnecker et al. 2015; Loeppmann et
al. 2016; Jing et al. 2017).
Numerous soil physical and biogeochemical properties change with depth.
As organic matter (both SOC and organically bound nutrients) moves into
the subsoil, it becomes increasingly more microbially processed and
sorbed onto charged mineral surfaces (Rumpel & Kögel-Knabner 2010),
which concomitantly increase with depth. Soil pH may also increase with
depth in instances where the parent material is enriched in base cations
(Brubaker et al. 1993). These gradients in soil properties result
in subsoil microbial communities that are vastly different than their
surface soil counterparts (Eilers et al. 2012; Brewer et
al. 2019). Soil pH (Sinsabaugh et al. 2008; Kivlin & Treseder
2014), substrate availability and demand (Olander & Vitousek 2000; Doveet al. 2019), and microbial community composition (Schneckeret al. 2015) influence EE activities in surface soils. Because
these factors change along soil profiles, EE activities should also
change with soil depth. Two main generalizations have emerged from the
few studies that have investigated EE activities in subsoils: 1) EE
activities decline with depth in association with decreases in soil
organic matter concentrations and decreases in microbial biomass (Tayloret al. 2002; Stone et al. 2014; Loeppmann et al.2016), and 2) EE activities at depth are less responsive to surface
conditions, manipulations, and management practices (Kramer et
al. 2013; Jing et al. 2017; Yao et al. 2019). However,
our ability to quantify the total EE pool and elucidate the controls on
EEs in subsoils has been hindered by unstandardized ancillary
measurements, assay parameters, and depths of sampling across studies
(Nannipieri et al. 2018).
Systematic, continental- and global-scale assessments and meta-analyses
of EEs in surface soils have begun to clarify controls and correlates of
EE activity (Sinsabaugh et al. 2008, 2009; Xiao et al.2018). For instance, EE stoichiometry (the ratio of C-, N-, and
P-acquiring enzymes), which can represent the relative C, N, and P
demand (Sinsabaugh & Shah 2012), scales at 1:1:1 (C:N:P) globally
across soil, freshwater, and saltwater ecosystems, suggesting that the
plasticity of microbial resource demand is somewhat constrained
(Sinsabaugh et al. 2008, 2009). These large-scale assessments
also confirm that pH, substrate availability, and microbial demand
influence EE activity in surface soils (Sinsabaugh et al. 2008,
2009; Xiao et al. 2018). However, it is currently unknown if
these controls in surface soils extend into the subsoil. We posit that
EE activities at depth may follow different patterns than in the surface
horizons given that EEs at depth are less responsive to environmental
perturbations (Jing et al. 2017), subsoils have greater
heterogeneity of organic substrates than at the surface (Salomé et
al. 2010), and the microbial communities at depth are dominated by
oligotrophic microorganisms (Brewer et al. 2019).
To quantify EE activities and elucidate their controls throughout the
soil profile, we sampled the upper meter of mineral soil at 10 cm
increments in 19 soil pits across the 10 United States National Science
Foundation-supported Critical Zone Observatories (CZOs) in the United
States of America (USA). We hypothesized that EE activities per gram (g)
soil would decline with depth due to decreased SOC and MB
concentrations; however, a significant proportion of EE activity in the
top meter of soil would occur below 20 cm depth. We also hypothesized
that the fundamental controls on EE activities would differ between
surface and subsoil horizons due to shifting biological, chemical, and
physical conditions throughout the soil profile. Specifically, as
organically bound microbial resources decrease with depth, mineral
sorption of both substrates and EEs will become a more dominant control
of potential EE activity. Our overall goal was to quantify potential EE
activity in the subsoil over a diverse set of soils, ecosystems, and
climates to elucidate how EE activity mediates subsoil C and limiting
nutrient availabilities in order to improve predictive understanding.
METHODS