FEMS Microbiology Ecology, 94, 2018, fiy191 doi: 10.1093/femsec/fiy191 Advance Access Publication Date: 27 September 2018 Minireview MINIREVIEW Impact of hydrologic boundaries on microbial planktonic and biofilm communities in shallow terrestrial subsurface environments H.J. Smith1,9, A.J. Zelaya1,2,9, K.B. De Leo´n3,9, R. Chakraborty4,9, D.A. Elias5,9, T.C. Hazen6,9, A.P. Arkin7,9, A.B. Cunningham1,8 and M.W. Fields1,2,9,*,† 1Center for Biofilm Engineering, Montana State University, Bozeman, MT, 2Department of Microbiology & Immunology, Montana State University, Bozeman, MT, 3Department of Biochemistry, University of Missouri, Columbia, MO, 4Climate and Ecosystems Science Division, Lawrence Berkeley National Laboratory, Berkeley, CA, 5Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, 6Department of Civil and Environmental Engineering, University of Tennessee, Knoxville, TN, 7Department of Bioengineering, Lawrence Berkeley National Laboratory, Berkeley, CA, 8Department of Civil Engineering, Montana State University, Montana State University, Bozeman, MT and 9ENIGMA (www.enigma.lbl.gov) Environmental Genomics and Systems Biology Division, Biosciences Area, Lawrence Berkeley National Laboratory, 1 Cyclotron Road, MS:977, Berkeley, CA 94720 ∗Corresponding author: 366 Barnard Hall, Center for Biofilm Engineering, Montana State University, Bozeman, MT 59717. Tel: 406-994-7340; E-mail: matthew.fields@biofilm.montana.edu One sentence summary: The current information on the diversity and activity of shallow freshwater subsurface habitats is discussed within the context of the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and geological gradients, and how biofilms may respond and impact shallow terrestrial subsurface aquifers. Editor: Marcus Horn †M.W. Fields, http://orcid.org/0000-0001-9053-1849 ABSTRACT Subsurface environments contain a large proportion of planetary microbial biomass and harbor diverse communities responsible for mediating biogeochemical cycles important to groundwater used by human society for consumption, irrigation, agriculture and industry. Within the saturated zone, capillary fringe and vadose zones, microorganisms can reside in two distinct phases (planktonic or biofilm), and significant differences in community composition, structure and activity between free-living and attached communities are commonly accepted. However, largely due to sampling constraints and the challenges of working with solid substrata, the contribution of each phase to subsurface processes is largely unresolved. Here, we synthesize current information on the diversity and activity of shallow freshwater subsurface habitats, discuss the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and Received: 30 March 2018; Accepted: 26 September 2018 C© FEMS 2018. This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs licence (http://creativecommons.org/licenses/by-nc-nd/4.0/), which permits non-commercial reproduction and distribution of the work, in any medium, provided the original work is not altered or transformed in any way, and that the work is properly cited. For commercial re-use, please contact journals.permissions@oup.com 1 D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 2 FEMS Microbiology Ecology 2018, Vol. 94, No. 12 geological gradients, and discuss how biofilms may be constrained within shallow terrestrial subsurface aquifers. We suggest that merging traditional activity measurements and sequencing/-omics technologies with hydrological parameters important to sediment biofilm assembly and stability will help delineate key system parameters. Ultimately, integration will enhance our understanding of shallow subsurface ecophysiology in terms of bulk-flow through porous media and distinguish the respective activities of sessile microbial communities from more transient planktonic communities to ecosystem service and maintenance. Keywords: groundwater; sediment; aquifer; ecology; activity INTRODUCTION The terrestrial, shallow subsurface is a complex andmicrobially- active habitat located beneath the surface soil layers, com- prised of sediments (inorganic or organic unconsolidated mate- rial that comes from theweathering of rock transported bywind, water or ice), rocks, gas, porewater and groundwater (Atekwana, Werkema and Atekwana 2006). Typically, subsurface environ- ments contain less labile organic matter (OM) compared to sur- face soils, and the degree of hydrological connectivity to the sur- face is routinely used to delineate between shallow and deep biospheres rather than depth alone (Lovley and Chapelle 1995). Although water covers 70% of the Earth’s surface, roughly 1% is readily available for human use, and a vast majority (∼95%) of the Earth’s consumable and available freshwater is groundwa- ter (Danielopol et al. 2008; Griebler et al. 2014; Dennehy, Reilly and Cunningham 2015). Despite the importance for the world’s pop- ulation, the role ofmicrobial communities in themaintenance of groundwater ecosystems is not fully understood. Case in point, the recent increase of artificially recharging natural aquifers via managed aquifer recharge to meet the global demand for water availability is concerning because of the potential to drastically alter groundwater systems (Lee and Lee 2017). This mini-review will focus on aspects of ‘shallow’ freshwater subsurface envi- ronments (mainly porous/granular) which typically have higher rates of recharge and flow as well as have a high degree of con- nectedness with the surface as opposed to ‘deep’ subsurface environments that are much less connected with the surface and receive limited surficial inputs of water and/or nutrients. Primary motivations for studying the subsurface are to expand what is known about Earth’s microbial diversity and the subsurface microorganisms under low nutrient conditions that significantly impact C, S, N, P and mineral cycles. Microbial life is thought to vary from the terrestrial surface to the deep subsurface dependent upon water, nutrient inputs and envi- ronmental stressors. Upwards of 40% of the microbial biomass and 1016–1017 g C on Earth resides within the terrestrial subsur- face (Whitman, Coleman and Weibe 1998; Griebler and Lued- ers 2009; McMahon and Parnell 2014). Over the last 30 years, there has been an increasing interest in surveying the taxonomic and functional biodiversity of subsurface environments, largely due to the concern over biodiversity and subsequent ecosys- tem function loss (Danielopol et al. 2003; Hancock, Boulton and Humphreys 2005; Wall and Nielsen 2012; Lijzen, Otte and van Dreumel 2014). However, on an ecosystem scale, there is limited information regarding the exact relationship between micro- bial diversity, environmental parameters and biogeochemical processes between groundwater and subsurface porous media. Studies focusing on subsurface habitats have revealedmany sig- nificant roles that microorganisms play in shallow subsurface processes (e.g. Chapelle 2000; Atekwana, Werkema and Atek- wana 2006; Hwang et al. 2009; Mitchell et al. 2010; Akob and Ku¨sel 2011; Griebler and Avramov 2015). In the environment, microorganisms can be observed in two distinct phases: free-living (planktonic) and associated with a surface as single cells to multicellular aggregates (i.e. biofilm). Biofilms are often composed of diverse taxonomic lin- eages attached to surfaces and each other, typically surrounded by extracellular polymeric substances (Hall-Stoodley, Costerton and Stoodley 2004; Gross et al. 2007; Stewart and Franklin 2008). Biofilms have not been explicitly studied within the subsur- face; however, because biofilms have been described at liquid– solid, liquid–gas or solid–solid interfaces, it is becoming increas- ingly clear that biofilms more closely resemble in situ conditions for microorganisms from diverse environments (Hall-Stoodley, Costerton and Stoodley 2004). Therefore, it is likely that attached modes of growth are a universal feature presenting an impor- tant physiology to explore within the subsurface in addition to typically conducted planktonic cell studies (Dunne 2002; Kolter 2005). Cells growing on a surface (i.e. biofilms) are known to have physiologies and properties distinct from planktonic cells including increased resistance to external stresses such as antimicrobials, heavy metals, desiccation and substrate depri- vation (e.g. Clark et al. 2012; Kurczy et al. 2015; Stylo et al. 2015). Most microbial environments are physically dynamic habitats where fluxes in water, nutrients, temperature, pH and osmolar- ity can create challenges for survival. Altered flow conditions can limit motility/dispersal and nutrient availability can result in decreased microbial activity and altered population distri- bution (Or et al. 2007). Biofilm matrices can retain water, sorb nutrients and protect against rapid changes in local geochem- istry, attributes that significantly improvemicrobial viability and activity. Additional ecophysiological advantages from residing within biofilms include metabolic cooperation, the exchange of genetic material and the development of regulatory mecha- nisms and social behaviors (Dang and Lovell 2016 and references therein). Traditionally, subsurface habitats were analyzed through bulk activity assays and total and viable cell enumerations (mainly with groundwater samples). Recent studies have relied on sequencing and -omics techniques to identify new diversity and functionality. Unfortunately, little overlap exists between more traditional quantitative activity measurements and newer sequencing capabilities. Such overlap is neces- sary to link phylogenies to quantitative functionality, although systems approaches have been used for bioremediation sites (Chakraborty, Wu and Hazen 2012). The objectives of this review are to synthesize the current understanding of (i) microbial pop- ulation distributions and activities spanning shallow subsurface habitats (with a focus on freshwater systems when possible), (ii) discuss the challenges associated with sampling planktonic and biofilm communities across spatial, temporal and geologi- cal gradients, (iii) identify subsurface geochemical and physical properties that potentially constrain biofilm development and D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 Smith et al. 3 (iv) give recommendations and considerations for future stud- ies. Additionally, Table S1 (Supporting Information) provides a quick summary of sample and environmental details, including lithologic information when available, for the relevant sources cited in this review. CHARACTERISTICS OF SHALLOW SUBSURFACE ENVIRONMENTS Although estimates vary, the shallow subsurface environment can extend from beneath the OM rich soil layers (A and B hori- zons) to tens of meters (Atekwana, Werkema and Atekwana 2006; Pepper and Brusseau 2006). In the shallow subsurface envi- ronment of an aquifer, sediments are assumed to lie below the vertically weathered top soil profiles. In the B horizon (below O, A and E), minerals, clays and organic material are leached from the upper horizons. The C horizon (below B) is characterized by unweathered minerals that were the parent material from which the upper soils were formed while at deeper depths the R horizon is the native bedrock material (Pepper and Brusseau 2006). The shallow subsurface is typically described as being below the surface soil horizons (typically 1–10 cm) and above bedrock (<50 m in depth) (Chu et al. 2016), and can have a high degree of hydrological connectedness with the surface com- pared to the deep subsurface (Toth 1963; Lovley and Chapelle 1995). By contrast, deep subsurface systems have been distin- guished by arbitrary depth measurements ranging from hun- dreds to thousands of meters below the surface (Balkwill 1989; Lovley and Chapelle 1995; Head, Jones and Larter 2003) or by a lack of surface connectivity (Toth 1963; Lovely and Chapelle 1995). The designation of ‘shallow’ versus ‘deep’ can be variable dependent upon respective geology and environment. Addition- ally, albeit not within the scope of this minireview, there are different concepts to categorize aquifers (e.g. aquifer, aquiclude and aquitard) in the context of hydrology. Traditionally, the shallow subsurface can be separated into three distinct zones based on moisture content in relation- ship to water table configuration termed the vadose, capillary fringe and saturated zones (Fig. 1). The vadose zone represents the upper most boundary of the subsurface comprised of the upper horizons (O–B) and contains unweathered and weath- ered materials. Following precipitation events, the vadose zone experiences high saturation levels as vertical infiltration pro- ceeds downward to the water table, yet residual pore water can persist creating varying levels of water and gas saturation (Jones and Bennett 2014). The capillary fringe exists at the inter- face of the saturated and vadose zone and is highly dependent upon fluctuations of the local water table. The capillary fringe is dynamic overtime with varying physicochemical conditions resulting from water table fluctuations (Griebler and Lueders 2009). This fluctuating interface has been shown to be a ‘hotspot’ of subsurface activity especially with respect to biogeochemical cycling (Silliman et al. 2002; Berkowitz, Silliman and Dunn 2004). The saturated zone (i.e. at/below water table) of most aquifers consists of porous parent material (C and R horizons) and voids are filled with water. Generally, the direction of water flow in the saturated zone can be 3-dimensional depending on hydraulic gradients and porous media properties (e.g. clay lenses). With respect to the impact on microbial communities, much attention has been given to the water table position and sedi- ments transported in the saturated and capillary fringe zones. Figure 1. Conceptual illustration of representative shallow subsurface environ- ment that includes the vadose, capillary fringe and saturated zones. Arrows depict themovement of water through infiltration, evapotranspiration, capillary rise and re-charge, and the movement of water within and between these zones creates dynamic conditions for the formation and maintenance of subsurface biofilms. The latter transitional boundary between the vadose and satu- rated zones is capable of drastic changes in geochemical param- eters [e.g. pH and dissolved oxygen] that impact ecosystem func- tion in terms of geochemical cycling, biotic/abiotic filtering and buffering processes (Rainwater et al. 1993; Reddi, Han and Banks 1998; Dobson, Schroth and Zeyer 2007; Pilloni 2011; Chakraborty, Wu and Hazen 2012). However, the vadose zone can also have soils and weathered particles (sediments) impacted by water movement, likely dictated by surface infiltration and evapotran- spiration. In addition, clays and clay lenses are also thought to impact water and gas flow that could significantly impactmicro- bial processes (Faybishenko et al. 2000). In addition, particle structure can impact community com- position and activity, for example, the turnover of matrix- associated NOM (natural organic matter) correlates to the pro- portion of fine-grained particles (Keil and Mayer 2014). Physi- cal properties of sediments (e.g. particle size) is also thought to impact microbial activity and distribution although most stud- ies have been done with surface or near-surface soils/sediments D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 4 FEMS Microbiology Ecology 2018, Vol. 94, No. 12 (e.g. Jackson and Weeks 2008; Hemkemeyer et al. 2015, 2018). Individual aggregates in groundwater and soil as well as soil pores can have discrete microenvironments with distinct activ- ities and conditions (Keil and Mayer 2014) that likely contribute to spatial and temporal areas of high metabolic activities or ‘hot spots and hot moments’ (McClain et al. 2003). Pores and aggre- gates are continuously changing due to biogeochemical and physical processes (Schlu¨ter andVogel 2016), andwetting/drying cycles (i.e. capillary fringe) can greatly impact pore size distri- butions (Bodner, Scholl and Kaul 2013). Sediment–groundwater– cell interactions can occur at the pore scale (≤micrometer) where diffusion and dispersal can be limited. However, little is known about how microbially relevant scales ultimately impact field scale behavior and function, and few studies have deter- mined the proper scale to delineate these relationships. While the subsurface begins below the humus rich soil horizons, NOM (including particulate and dissolved fractions excluding organic contaminants) is a primary source of C/N that supports microbial life in the shallow subsurface. Despite sea- sonal shifts, there is a natural gradient of decreasing nutrient and oxygen concentrations with depth leading to oligotrophic and anoxic conditions within the saturated zone (Danielopol, Pospisil and Rouch 2000; Awoyemi, Achudume and Okoya 2014). Additionally, NOM is thought to decline with depth, and recent comparisons of water-extractable organicmatter from a shallow subsurface core showed total organic carbon was ∼19 mg/g and inorganic carbon was 8 mg/g in shallow sediment (Chakraborty et al., unpublished data). Due to nutrient limiting conditions, microorganisms in subsurface habitats have most likely devel- oped strategies to use NOM and other reduced compounds (e.g. Mn(II), Fe(II), ammonia, sulfide, methane and hydrogen) as part of directly or indirectly coupled processes in the groundwater, pore water and sediment surfaces. SHALLOW SUBSURFACE MICROBIAL BIODIVERSITY: PROGRESS AND CHALLENGES The relationship between biodiversity and ecosystem func- tioning has been well studied above ground (Cardinale et al. 2006; Ives and Carpenter 2007); however, similar studies are in the early stages for subsurface environments. While perceived functional redundancy could have a limited role in subsurface ecosystem functioning, studies also indicate that microbial tax- onomic diversity plays a role in mitigating ecosystem collapse and contributing to faster functional recovery (Wagg et al. 2014; Delgado-Baquerizo et al. 2016; Louca and Doebeli 2016). Subsur- face groundwaters and sediments have been shown to harbor far more taxonomic and functional diversity than previously inferred by cultivation attempts and microscopic observations (Brown et al. 2015; Lynch and Neufeld 2015; Lennon and Locey 2016). In addition, these environments exhibit a wide diversity of previously undescribed bacteria and archaea (Castelle et al. 2013; Brown et al. 2015; Anantharaman et al. 2016; Lazar et al. 2017). While specific taxonomic lineages can be prevalent in several types of underground ecosystems (Griebler and Lueders 2009; Akob and Ku¨sel 2011; Hubalek et al. 2016), thus far no true ‘endemic’ shallow subsurface populations have been identified (Griebler and Lueders 2009). To date, the debate regarding the influence of biodiversity and ecosystem functioning, especially within the subsurface, has yet to be thoroughly explored. Any resolution will most likely be challenging at best due to extreme spatial heterogeneity. It remains unresolved whether our current understanding of subsurface microbial biodiversity is real or merely an artifact of the following topics: (i) technological approaches (i.e. short reads lengths from next generation sequencing), (ii) low relative diver- sity and/or abundances of oligotrophic systems, (iii) the use of bulk sampling techniques compared to the retrieval of samples representing discrete phases (planktonic vs. biofilm) and/or dis- crete zones (i.e. vadose, capillary fringe, and saturated zones) which could further delineate spatial differences, and/or (iv) temporal dynamics that have been poorly resolved. For example, recent work has shown the potential importance of microbial biomass for protozoan food webs in shallow aquifers (Hutchins et al. 2016) and differences in carbon cycling between ground- water and shallow sediments over time and space (<1 m) (Long- necker and Kujawinski 2013). Given these types of observations, the roles of biofilm diversity in the shallow subsurface for resis- tance to predation pressures and ultimately on resource alloca- tion are not known. Therefore, as discussed below, future studies should combine technological approaches at appropriate tem- poral and spatial scales for both groundwater and matrix mate- rial. Technological approaches Studies of microbial biodiversity have historically been per- formed via traditionalmicrobiological techniques (Goldscheider, Hunkeler and Rossi 2006 and references therein; Sinclair and Ghiorse 1989). Profound advancements have been made in the application of next-generation sequencing (Tringe and Hugen- holtz 2008), high-throughput -omics approaches (Lo´pez-Garcı´a and Moreira 2008; Prosser 2015), single-cell methods (Lasken andMcLean 2014) andmethods encompassing untargeted func- tional potential (Lo´pez-Garcı´a andMoreira 2008; Rajendhran and Gunasekaran 2011). Up until July 2015, a total of ∼1.4 × 106 and ∼5.4 × 105 full-length bacterial and archaeal 16S rRNA refer- ence sequences, respectively, have been deposited into Silva- ARB (www.arb-silva.de) and IMG (img.jgi.doe.gov), comprising a total of 65 bacterial and 20 archaeal phyla (Schloss et al. 2016). Interestingly, it was estimated that only 7.8% and 16.5% of all reference sequences originated from soil and aquatic environ- ments, respectively (Schloss et al. 2016). As the above estimates include surface waters (e.g. lakes and rivers), marine environ- ments and surface soils, the percentage of sequences specific to groundwater, and more so for shallow subsurface sediments, is quite low. The drastic under-sampling of the subsurface has led to a scarcity of reference sequences specific to these environ- ments, leading to the high risk of mis-identification of retrieved sequences and an under estimation of subsurface biodiver- sity and biochemical capacity. As the number of non-targeted (DNA/RNA-based) and targeted metagenomes (SIP/activity) are increased for shallow subsurface groundwater and sediments, it is likely that unique lineages with novel capability will be dis- covered across all three domains, and thus an improved repre- sentation of in situ diversity can be achieved. Low relative diversity/abundances of oligotrophic systems Based upon a limited number of studies that survey diversity as a function of depth, it has been observed that species rich- ness declines over depth, with transient increases at transition zones. Currently, it remains unclear if this trend is merely a con- sequence of the combination of limited/recalcitrant resources D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 Smith et al. 5 (C) and energy restriction (anoxic) or other more specific selec- tion mechanisms that may differ from surface environments (Musslewhite et al. 2003; Lin et al. 2012b; Chu et al. 2016). Recent studies suggest that the large fraction of lowly abundant or ‘rare’ organisms observed in subsurface environments may play important ecological roles. For example, they may contribute to biogeochemical reactions (Pester et al. 2010) while also serving as a ‘microbial bank’ that can ‘seed’ environments when con- ditions change (Lynch and Neufeld 2015). Biofilms could play a major role across the shallow subsurface zones in which changing conditions (e.g. pH, conductivity and flow) could drive dispersal and/or invasion (discussed below). As oligotrophy is inherent to most subsurface systems, techniques that couple high-throughput manipulation with small volumes and -omics methodology (e.g. micro-droplet fluidics and flow cytometry) should be included in future work to enable cultivation and activity measurements of slower growing microorganisms (e.g. Wilkins et al. 2014). Spatial variability: diversity of discrete zones While mechanisms that affect population distributions have been formulated based on surface habitats (i.e. biotic interac- tions, dispersal limitation and environmental filtering) (Mar- tiny et al. 2006; O’Malley 2007; Griebler and Lueders 2009; Shoe- maker, Locey and Lennon 2017), it remains unclear whether these mechanisms hold true for the distribution of microor- ganisms within the oligotrophic subsurface (Musslewhite et al. 2003; Chu et al. 2016). It is hypothesized that transition zones (macro- as well asmicro-transition zones, such as between indi- vidual particles and surrounding pore water) are important eco- tones or ‘hotspots’ of microbial diversity and activity (Zhang et al. 1998; McClain et al. 2003; Goldscheider, Hunkeler and Rossi 2006; Bougon et al. 2012; Campbell et al. 2012; Jones and Ben- nett 2014) and deserve more careful attention. There is evi- dence of spatial (vertical and horizontal) taxonomic variation of groundwater (Lin et al. 2011; Lin et al. 2012a; Herrmann et al. 2015) and sediments (Lin et al. 2012b). Results typically show a decline in microbial richness and diversity over vertical depth. The extent thatmicrobial communities vary in relation to depth, even with application of newer sequencing technologies, is still poorly resolved for the variety of geological strata that represent the shallow subsurface. Therefore, increased spatial resolution is needed to better understand the implications of micro-scale heterogeneity on microbial population distributions. It is not known if observed variation is a consequence of geophysical, geochemical or hydrogeochemical constraints, or a combination thereof. Whereas various scales have been sur- veyed (cm, m, km) whenmeasuring spatial β-diversity of micro- bial communities, studies that span over several cm are more common,with deeper samplings that spanmeters being less fre- quent. Pronounced effects of horizontal spatial dissimilarity on β-diversity increasing with depth have been shown for surface and subsurface soils (Chu et al. 2016), suggesting that, at least down to the saturated zone, subsurface sediment biofilms could be more greatly affected by dispersal limitation than commu- nities of surface soils. Moreover, the proper scale at varied spa- tial resolution to capturemicroscale heterogeneity or the proper scale for different geologic strata is currently unknown. Spatial variability: diversity of discrete phases In order to further investigate microbial diversity in the sub- surface, it is essential to differentiate between planktonic and attached populations. Historically, the ease of groundwater sam- pling via well-pumping has resulted in the majority of sub- surface datasets. However, inferences made about subsurface communities based solely on the planktonic fraction may not adequately represent all microbial members of the subsurface ecosystem (Hug et al. 2015). Studies that have attempted to compare planktonic versus biofilm communities have resorted to the use of surrogate sedi- ments (native and/or artificial material incubated in situ down well) that represent the geology of the aquifer (Reardon et al. 2004; Flynn, Sanford and Bethke 2008; Flynn et al. 2012; Con- verse et al. 2015; Graham et al. 2017). These surrogates include laboratory microcosms (Lee and Lee 2017), in field biofilm reac- tors (King et al. 2017; Christensen et al. 2018), or sediment fines from backwashed pumps (Cardenas et al. 2008; Li et al. 2018). Early studies that compared the planktonic versus attached frac- tions have generally observed a subset of the planktonic com- munity in the attached fraction (Hazen et al. 1991). Several stud- ies have corroborated these findings over the years (Reardon et al. 2004; Brad et al. 2008; Anneser et al. 2010; Zhou, Kellermann and Griebler 2012). Studies to differentiate planktonic versus biofilm functions may be able to capture the transitional states of planktonic com- munities (from planktonic to biofilm and vice versa), but there are unique limitations to each approach. For example, samplers (e.g. sampling coupons) typically contain unconsolidated sedi- ments that may not accurately mimic the hydrological effects of consolidated or saturated sediments. Thus, borehole arti- facts must be considered (Lehman 2007a). In addition, coloniza- tion of the native matrix material is dependent upon surround- ing groundwater/porewater. The colonization no doubt occurs in situ, but the studies are over short time periods compared to in situ conditions and may not achieve the diversity of the natural setting. However, the down-well incubations of solid material does enable the capture of somemicrobial populations typically missed by groundwater sampling and could capture interaction dynamics across the aqueous/solid matrix boundary under in situ conditions (Barnhart et al. 2013). We recently used a revised microbial sampler (patent pend- ing) in a coal-bed aquifer packed with native coal material incubated down-well for ∼3 months and compared the bac- terial communities (SSU rRNA gene libraries) between sam- pled groundwater, native coal core and coal material from the same formation incubated in microbial samplers (Schweitzer et al., unpublished data). Preliminary analyses suggest that some family-level operational taxonomic units were common to all three samples while other operational taxonomic units were common to groundwater and the surrogate matrix material (n = 3). Two operational taxonomic units were unique to the sampled groundwater and four unique to the surrogate matrix material. Not surprisingly, the native coal material had themost unique operational taxonomic units (n= 6).Whereas differences between the samples were expected, surrogate matrix material could be used in future studies to capture additional diversity from the subsurface and delineate ecology dynamics in terms of core (i.e. consistent) and transient populations between ground- water and matrix material. The inability to distinguish between different phases (i.e. groundwater vs. sediments) for key biogeochemical processes poses a challenge for answering basic ecological questions. Recent -omics approaches applied to samples from the subsur- face are revealing the range of possible activities within the sub- surface and the potential for broad biochemical functionalities (Griebler and Lueders 2009; Akob and Ku¨sel 2011; Flynn et al. D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 6 FEMS Microbiology Ecology 2018, Vol. 94, No. 12 2013; Hubalek et al. 2016). Through genomic surveys, microor- ganisms have been linked to the transformations of carbon com- pounds, the nitrogen cycle and sequestration of greenhouse gases (Hemme et al. 2015; Trivedi, Delgado-Baquerizo and Trivedi 2016). Typically, functionality has been inferred from the pres- ence of specific functional genes in a sample set (e.g. Yan et al. 2003; Fields et al. 2006; Winderl, Schaefer and Lueders 2007) or, more holistically, across a subsurface ecosystem. Usually the larger scale sampling needed to characterize the potential microbial function in an ecosystem relies on testing the more easily accessible groundwater (Hug et al. 2015; Smith et al. 2015; He et al. 2018). Therefore, despite any novel and potential activ- ity identified in groundwater, the full extent of possible differ- ences in the sediments are unknown. Due to the challenges of getting intact sediment samples, previous studies have used surge blocks (Wu et al. 2013) on groundwater pumps to col- lect sediment fines. This procedure allowed delineation of the groundwater populations from those associated with the sedi- ment fines (Cardenas et al. 2008; Li et al. 2018), and the reported results showed definite differences between the groundwater and sampled sediments. The potential differences in functional- ity between the planktonic and sediment microorganisms have implications (e.g. selection pressures and dispersal) for ecosys- tem stability and resiliency, particularly with dynamic hydrolog- ical cycles that can change over varying time-scales. Effects of temporal fluctuations on biodiversity Taxonomic diversity in relation to spatiotemporal fluctuations in physicochemical and geochemical properties of the shallow subsurface is largely understudied. Physicochemical and geo- chemical relationships over time may drive phylogenetic and functional microbial diversity changes that are often associ- ated with both short-term (e.g. diel cycles; extreme weather) and long-term (e.g. seasonal) alterations. Spatiotemporal fluc- tuations in conjunction with porosity and permeability modi- fications can lead to varying degrees of hydrogeochemical mix- ing. The role of mixing in aquatic systems has been implicated in the creation of patchy distributions of both nutrients and biomass (Ebrahimi and Or 2016; 2018). Depending on the aquifer system, highly mixed waters can be found in shallow areas, where there are higher rates of infiltration from rain or surface waters. Alternatively, at the water table, seasonal fluctuations can result in mixing groundwater with sediments of the vari- ably saturated zone (Fig. 2). In the shallow subsurface, mixing is thought to cause instability due to faster and shorter local flow paths; whereas, more stable and predictable diversity may result from slower regional flow significantly below the water table (Ben Maamar et al. 2015). Most spatiotemporal studies of subsurface environments focus on the changes of geochemistry and corresponding groundwater communities collected via sampling wells over time across different depths in the water table (Lin et al. 2012a; Brad et al. 2013; Sirisena et al. 2013; Ben Maamar et al. 2015; Schwab et al. 2016). Studies have consistently shown that mixed groundwaters have higher diversity and variability than ground- water that undergoes less mixing (Bougon et al. 2012; Hug et al. 2015; Danczak et al. 2016; Hubalek et al. 2016). Additionally, it has been shown that the degree of hydrogeochemical mixing can greatly impact microbial assemblage compositions due to the influx of nutrients and migration of transient populations (Haack et al. 2004; Fields et al. 2006; Hwang et al. 2009; Velasco- Ayuso et al. 2009; Lin et al. 2012a; Hubalek et al. 2016). The impact of mixing on in situ sediment biofilms is largely unknown, mainly because temporal sampling in the same loca- tion is nearly impossible for native matrix material. Surrogate sediments incubated down-well have enabled some degree of temporal sampling of attached microorganisms (Zhou, Keller- mann and Griebler 2012), as well as successional events when monitored longer times. Comparisons of cataloged cored sed- iments separated by months or years have also been per- formed (Hug et al. 2015). It is likely that feedbacks between res- ident microorganisms and hydrogeochemistry exist and impact subsurface ecosystem structure and function at a larger-scale (Mendoza-Lera and Mutz 2013; Lee and Lee 2017) particularly for sediment biofilms where impacts at the microscale are more likely compared to bulk-phase changes. ACTIVITY IN THE SHALLOW SUBSURFACE There are significantly fewer studies that have simultaneously compared microbial activities in the sediment and groundwater fractions. Quantifying activity fromsubsurface samples is a non- trivial task, as the retrieval of ‘undisturbed’ samples in combina- tionwith ‘representative’ incubation times necessary for activity assays can potentially lead to artifacts which greatly influences downstream analyses and interpretations. While sequencing capabilities have produced substantial insight about the poten- tial functionality of porous subsurface aquifers, traditional - omics studies struggle to make quantitative estimations about activity (Hemme et al. 2015; Hug et al. 2015). Subsurface activity is typically measured utilizing traditional approaches (e.g. extra- cellular enzyme assays, radioisotope tracers, viable plate counts, most probable numbers and direct counts with fluorescent com- pounds indicative of activity), all of which have been shown to have inherent biases (Kepner and Pratt 1994; Stewart et al. 1994). Historically, not only were there greater densities of total cells in the sediments, but a higher proportion of active cells are associ- ated with sediment compared to planktonic cells (Hazen et al. 1991; Alfreider, Kro¨ssbacher and Psenner 1997). However, the contribution of free-living and biofilm cells to subsurface pro- cesses on a per cell basis is unclear. Recently, it has been pro- posed that microbial competition selects against rapid growth in biofilm populations (Coyte et al. 2016). These findings offer a unique and contradictory perspective as to the role of free- living organisms compared to biofilms that may alter our cur- rent understanding of colonization, maintenance and dispersal of microbial populations in porous environments. Activity in groundwater Most researchers have now concluded that attached bacteria dominate oligotrophic subsurface environments in terms of biomass and activity and thatmost planktonic cells are ‘inactive’ subsets of benthic organisms (Goldscheider, Hunkeler and Rossi 2006 and references therein). Initially, indications that ground- water samples had a low proportion of active cells came from microscopic evaluation of pristine aquifers which observed cells between 0.4 and 0.9 μm in size, suggesting that these bacte- ria were in a starved state with reduced activity (Balkwill and Ghiorse 1985). However, a recent study identified novel ultra- microbacteria that are inherently small (<0.1 μm) in groundwa- ter but activity was not reported (Luef et al. 2015). Groundwater habitats have been shown to be able to vary drastically over time and space. For example, in a two-year study, all tested extracellular enzyme assays were found to vary signif- icantly both spatially and temporally (Velasco-Ayuso et al. 2011). D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 Smith et al. 7 Figure 2. Conceptual model of subsurface flow and mixing zones and potential effects on biofilm life-cycle dynamics. Subsurface porous media habitats can be conceptually divided into three zones (I, II and III) with respect to ground water flow and mixing. (I) The vadose zone (including the capillary fringe) is variably saturated depending on infiltration episodes and degree of vertical water table fluctuation, (II) Zone II is the ‘shallow’ groundwater zone wherein ground water flow, together with seasonal changes in water table elevation, can cause multi-directional flow (i.e. vertical and horizontal fluctuations) that can result in greater mixing, (III) The ‘deeper’ groundwater zone (zone III) lies below the depth affected by seasonal water table fluctuations. The degree of mixing in zone III is related mainly to the ground water flow field. In zone II the higher level of seasonal mixing could result in a ‘hot spot’ of greater relative biofilm diversity and activity (represented by multi-color sections; biofilm not depicted at scale) (Bougon et al. 2012). Zone I could have lower biofilm diversity/activity due to limited and transientmixing, although it is possible that diversity and activity in zone I would bemore similar to zone II than region III. In zone III, which is deeper and has a more consistent ground water flow regime, biofilms would be less diverse/active. The roles of adhesion/detachment/dispersion could vary with the extent of mixing in the different zones and suggests that different mechanisms of microbial community assembly and diversification impact in situ biofilms. Recent advances are moving away from relying solely on bulk activity measurements. Quantitative studies that are capable of linking individual microorganisms to biogeochemical processes have been applied to groundwater from carbonate-rock aquifers. Although these results are not from a porous aquifer, the com- bination of metabolic labeling (i.e. D2O) with Raman microspec- troscopy, metaproteomics and carbon amendments quantita- tively showed that naturally occurring heterotrophic organisms preferentially assimilated lignin derivatives over biomass degra- dation products (Taubert et al. 2017) and are therefore involved in subsurface carbon cycling processes. Activity in sediment The overwhelming density of sediment associated organisms presents a compelling case that sediment core samples are likely the most representative samples for biomass analysis in the shallow subsurface. Studies based on cored samples have looked at the microbial activity of attached communities as a function of depth and particle size. Not only are cell num- bers higher in shallower depths compared to deeper depths, the same holds true for activity (Beloin, Sinclair and Ghiorse 1988; Martino et al. 1998). This trend has been shown, regardless of the methodology used [ATP assays, MPN, viable plate counts and the tetrazolium reduction method (INT)]; however, activity (specifically within the saturated zone) was shown to vary sea- sonally dependent on the method utilized (Beloin, Sinclair and Ghiorse 1988). When comparing similar depth profiles (<50 m), other researchers have observed only slight variations in total cell abundances over depth and the largest differences were observed in the active fraction (determined with viable plate counts) which decreased with depth (Balkwill and Ghiorse 1985; Balkwill 1989). Studies utilizing radioisotope tracers have found higher metabolic activities in shallower depths as well as spikes in activity within the saturated zone (Phelps et al. 1988). Interest- ingly, anaerobic bacteria have also been found to decrease in via- bility with depth and have been reported to be a 100-fold lower than aerobic organisms (Balkwill and Ghiorse 1985). Conversely, in low conductivity ecosystems, studies have found that anaero- bicmicroorganisms have greater viability at deeper depths (Mar- tino et al. 1998). The discrepancies between studies are likely attributed to differences in hydraulic conductivitywhich directly impacts microbial and nutrient sources and local geochemistry, the exclusion of temporal analysis and differences in method- ologies. Activity measurements comparing biofilm and planktonic populationswithin sedimentmesocosms observed a higher pro- portion of activity in sediments (0.25 m columns with shal- low sediments) compared to the planktonic communities (Long- necker and Kujawinski 2013), a finding that corroborates results from field studies (Thomas, Lee and Ward 1987; Hazen et al. D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 8 FEMS Microbiology Ecology 2018, Vol. 94, No. 12 1991; Holm et al. 1992; Alfreider, Kro¨ssbacher and Psenner 1997; Anneser et al. 2010). Some studies have observed total cellu- lar abundances to be highest with coarse particles (Albrecht- sen 1994), while others have demonstrated a greater number of microorganisms associated with fine silt particles (<20 μm) (Harvey, Smith and George 1984). However, the study that found higher total densities of organisms associated with coarse parti- cles showed with multiple methodologies that the greatest pro- portion of active organisms (91.9–100% of viable bacteria) were associated with smaller size particles (1.2–100 μm) (Albrechtsen 1994). An additional attribute that is largely unknown for sedi- ment biofilms is cell density per given surface area and/or co- occurrence of cells or populations in more oligotrophic environ- ments. Paired studies comparing activity in groundwater to sediment In order to accurately determine the contribution of free-living and attached, paired groundwater and core samples are neces- sary albeit these studies are significantly fewer in number.Multi- ple methodologies have shown that total measured activity (e.g. general metabolic activity, degradation of a specific compound) is greater per gram of sediment than for comparable adjacent groundwater (per mL or L) for both contaminated and pristine aquifers (Thomas, Lee and Ward 1987; Hazen et al. 1991; Holm et al. 1992; Alfreider, Kro¨ssbacher and Psenner 1997; Anneser et al. 2010). A variety of radioisotope tracers are routinely used to infer activity from environmental samples, and a study on a sandy aquifer that used 3H-thymidine in combination with 14C- leucine was not able to detect significant activity in any tested groundwater samples while activity was readily quantified for colonized material from down-well sampling devices (Alfrei- der, Kro¨ssbacher and Psenner 1997). The greatest activity was observed for smaller grain size particles (125–250 μm) compared to larger particle sizes (250–500 μm) (Alfreider, Kro¨ssbacher and Psenner 1997). The percentage of respiring bacteria as deter- mined with INT has been shown to range between 1.0–24.9% of total cells for pumped groundwater and 6.0–41.1% of total cells for sandy sedimentswith on average three-fold fewer active cells in groundwater than associated with sediment.While this study demonstrated higher totals and higher numbers of metaboli- cally active cells as well as greater rates of general metabolic activity in sediment samples, the results potentially stem from an artifact due to the use of sterile sediments suspended down- well rather than the use of actual sediment cores. An early study used direct counts (cellular DNA stained via acridine orange) and viable plate counts to compare subsurface sediment to adjacent groundwater. Total and viable bacterial abundances for sediments collected from three individual bore- holes were higher than for any adjacent groundwater samples from discrete depths (Hazen et al. 1991). The study also observed higher densities of total and active cells associated with aquifer sediments than groundwater and concluded that attached bac- terial communities are not reflected in groundwater samples. In addition, recovered sediment isolates were capable of utilizing a broader range of carbon sources than planktonic cells from groundwater which the authors concluded relevant for in situ bioremediation efforts. More recently, froma tar oil contaminated aquifer, sediments contained greater than 97.7% of all bacterial cells and displayed six-fold greater enzyme activities than groundwater, although groundwater dominated more specific processes such as sul- fate and iron reduction (Anneser et al. 2010). These observa- tions corroborate previous studies: the highest density of organ- isms is associated with sediments but groundwater commu- nities have the potential to dominate certain redox reactions when sampled at the appropriate resolution. The idea that some microorganisms reside mainly in the planktonic phase of sub- surface porous environments is supported by the predominance of methanogenic microorganisms observed in the planktonic phase compared to sediments (Lehman 2007b). Using a combi- nation of laboratory and field-based studies Holm et al. (1992) examined the role of planktonic and biofilm associated cells on the biodegradation of hydrocarbons and concluded thatwhereas there were substantially lower rates of degradation for ground- water samples, the planktonic phase significantly contributed to the biodegradation of organic contaminants. While reaction rates within the groundwater are typically lower on a per vol- ume basis, the studies demonstrate the importance of sampling and studying both groundwater and subsurface biofilms. A possible explanation for differences in activities between attached and free-living populations is likely due to differ- ences in cell abundances. However, it remains unresolved whether free-living cells in porous subsurface habitats are in fact metabolically slower or faster. Recently, models have pre- dicted that attached cells can be selected to grow at slower rates as to avoid mass transport limitations (Coyte et al. 2016). While this concept has not been directly shown under natural condi- tions, a study that combined cellular abundances and volumet- ric rates of degradation found that some cell specific activities for planktonic bacteria may be higher than for sediment associ- ated organisms (Lehman 2007a). SUBSURFACE BIOFILMS Subsurface communities are traditionally discussed in terms of the planktonic or attached phases with little reference to attached communities as ‘biofilms’; therefore, the following sec- tion aims to merge the available information from biofilm stud- ies (often done in laboratory settings with single model organ- isms) with properties and constraints relevant to subsurface processes.Within these attached communities,microorganisms with varied metabolic functionalities can coexist and have cell- to-cell contact (Stewart and Franklin 2008), periodically detach- ing to become part of the planktonic phase (McDougald et al. 2012). It is likely these cells colonize new environments and are a primary mechanism for translocation from one surface to another (Watnick and Kolter 2000). Thus, the solid sediment matrix potentially could act as a seed bank of pelagic bacte- ria that can then be translocated. Despite the ecological signif- icance of biofilms, the relationships between source diversity (and activity) within the groundwater and local diversity (and activity) in the sediments are difficult to ascertain based upon logistics of sampling intact sediment material. Major logistics include expensive sampling that only provides a single time and space point that cannot be replicated and the subsequent impact on water flow through the disturbed matrix (discussed above). Life cycle stages of subsurface biofilms Biofilms appear to be an inherent phenotype for most microor- ganisms studied to date. The basic cycle of at least bacterial biofilms is attachment or adhesion,maturation and detachment (Hall-Stoodley, Costerton and Stoodley 2004). Typically, surface D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 Smith et al. 9 properties control cellular attachment while mass transport of substrates (influx) and/or products (efflux) limits overall biofilm maturation/growth. Detachment can be caused by a variety of conditions that result in desorption, detachment and/or disper- sion dependent upon varying geochemical and geophysical con- ditions. Adhesion Typically,microbial attachment is reversible (Dowd, Herman and Maier 2000), which could be beneficial for subsurface microor- ganisms subjected to environmental perturbations. The exact conditions that promote some microorganisms in the shallow subsurface to initiate attachment is unknown, but most likely includes physical (e.g. cell charge and flow) and chemical (e.g. pH and conductivity) parameters as well as biological (e.g. aggre- gation). While this has not been explicitly explored in shallow, subsurface biofilms, it is likely that the attachment of biofilms within the subsurface enhances survival in nutrient limited con- ditions by creating a microenvironment distinct from surround- ing conditions (Coombs et al. 2010). Previous work mostly with Pseudomonas but also others (e.g. Shewanella), has compared ver- tical and horizontal attachment in reference to flow forces and the ability of cells to attach or detach (Conrad et al. 2011; Ben- nett et al. 2016). Initial attachment and any subsequent cell divi- sion has a direct impact on the architecture of the biofilm. For biofilms on porous media similar to shallow subsurface sedi- ments and for those with slow flow regimes that impact mass transport variables (e.g. advection, dispersion andmass flux), the studies are quite limited. It is likely that biofilms in the oligotrophic subsurface are non-continuous or patchy and that surface substrate and com- munity composition impact adhesion. Biofilms from injections of radiolabeled cells into intact sediment cores dominated by quartz showed cell attachment around particularmineral grains suggesting amineral preference for the adsorbed bacteria (Dong et al. 1999). Bacterial adhesion has been shown in laboratory studies to increase in areas where quartz sands have been arti- ficially coated with metal oxyhydroxides (Scholl et al. 1990; Mills et al. 1994). Other studies have demonstrated different popula- tions are enriched on different materials (Reardon et al. 2004; Bollmann et al. 2010; Converse et al. 2015). Furtherwork is needed to better understand the physical forces under low fluidization that can promote or deter microbial biofilms under oligotrophic conditions and different mixing regimes (Fig. 2). Growth/Dispersal The formation and growth of biofilms in subsurface habitats is likely dependent on the microbial assemblages present, nutri- ent availability, substrate composition and hydraulic residence time (Coombs et al. 2010). Biofilm thickness is highly variable and ranges from the thickness of single cells to thicker micro- colonies that are adhered together by extracellular polymeric substances. Thin and/or patchy biofilms are usually not limited by diffusion (Rittman 1993) and most likely represent biofilms in undisturbed, oligotrophic (i.e. pristine) subsurface environ- ments. However, sediment biofilms can be thicker under differ- ent conditions that change over time and space, for instance, during biostimulation when nutrients are added. Total cell numbers of bacteria that have been documented in groundwater ecosystems have ranged between 102 and 106 cells/mL of groundwater (Griebler and Lueders 2009); however, these values may be dependent on hydrological fluctuations overtime (Velasco-Ayuso et al. 2009). While micro-eukaryotes have also been found within subsurface groundwater environ- ments, reported observations have shown that the majority of cellular biomass in the subsurface is bacterial and archaeal (Griebler and Lueders 2009; Valster et al. 2009; Zinger, Gobeta and Pommiers 2012). In comparison for subsurface matrix material, cell counts range from 104 to 109 cells/g sediment (Turco and Sadowsky 1995; Balkwill and Boone 1997; Griebler and Lueders 2009). These ranges typically vary with depth (Lin et al. 2012a), pH (Fierer and Jackson 2006), soil and sediment texture and porosity (Schwo- erbel 1961; Balkwill and Ghiorse 1985; Strayer 1994; Hahn 2006), redox conditions, dissolved oxygen, mineral content and mois- ture content (Sirisena et al. 2014). More recently, the distribution of viruses within subsurface groundwaters and the impact on microbial abundance has been studied (Pan et al. 2017). Thor- ough reviews on cell count abundances within different areas of the subsurface have been synthesized (Goldscheider, Hun- keler and Rossi 2006; Akob and Ku¨sel 2011) and cell count sur- veys have consistently shown differences in the abundances between attached and free-living phases (Griebler and Lueders 2009) dependent upon variations in physicochemical parame- ters (Sinclair and Ghiorse 1989). For example, microbial popula- tion density estimates correlated positively with sand content and pore-water pH and declined with clay content and pore- water heavy metals (Sinclair and Ghiorse 1989). In laboratory experiments, biofilm growth within granu- lar/porous reactor systems has been shown to reduce pore spaces that leads to the blockage of pores and flow (Taylor and Jaffe´ 1990a,b; Cunningham et al. 1991), alteration of water reten- tion (Or et al. 2007) and significantly reduces hydraulic con- ductivity (Rodriguez-Escales et al. 2016). Biofilms can further reduce permeability by the entrapment of fine grained or col- loidal materials that block flow (Hama 1997; Hama et al. 2001). It has also been shown that fine textured materials have higher occurrences of clogging compared to coarse textured materi- als (Vandevivere et al. 1995). Undoubtedly, biofilms have signifi- cant impacts on the porosity and permeability in natural porous aquifer systems; however, it is likely that biofilm heterogeneity and distribution in situ will be different than observed in reac- tor and consolidated aquifer studies. Environmental biofilms in situ have been shown to be patchy rather than uniform in distribution and thickness, and conceptual models have been applied to microbial growth and transport in subsurface habi- tats (Vandevivere et al. 1995; Clement, Hooker and Skeen 1996; Ebigbo et al. 2010). Historically, different models have been used to estimate unsaturated (Farthing and Ogden 2017) and satu- rated (Molnar et al. 2015) water flow in porous media relevant to the shallow subsurface; however, the lack of data and under- standing of microbial processes in the shallow subsurface chal- lenges the incorporation of microbial ecology and physiology into these models. Hopefully in the future, drivers of microbial biofilm assembly and maintenance (e.g. selection, dispersal and drift) can be investigated andmodeled with respect to hydrolog- ical parameters (e.g. porosity, permeability and mixing). Laboratory approaches to the study of subsurface biofilms While not a focus of this mini-review, there are numerous examples of using laboratory experiments to study subsurface microbial transport under unsaturated and saturated condi- tions (Tufenkji, Redman and Elimelech 2003; Jordan et al. 2004; D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 10 FEMS Microbiology Ecology 2018, Vol. 94, No. 12 Gargiulo et al. 2007; Harvey, Harms and Landkamer 2007; Brad- ford, Schijven and Harter 2015). Laboratory experiments are routinely used to mimic and investigate environmental sub- surface processes (i.e. grain size distribution, biofilm thick- ness/diffusion, biodegradation, pore clogging, flow, mass trans- port and hydraulic conductivity), and different reactor and incu- bation conditions (e.g. column, flat plate and serum bottles) and surrogate sediments (e.g. silica beads/sand or collected sedi- ment core material) are used in various combinations depend- ing on the process being investigated. When sediment cores are taken for laboratory studies, the pore structure may be altered by packing and repacking that results in porous media flow and transport properties significantly different from in situ condi- tions. Thus, an iterative approach combining field and labora- tory studies is beneficial for ensuring laboratory findings that hold relevance to the natural system while maintaining con- trolled laboratory conditions necessary for developing and test- ing predictive models. For example, with column reactors filled with different-sized silica beads (coarse and fine), coarse sedi- ments had higher biofilm biomass and activity although overall functionality was impaired (activity and diversity) (Perujo et al. 2017). Fine beads constrained biofilm activity and biomass while bead-size transitions promoted increased OM degradation and biomass at the interface (Perujo et al. 2017). The results corrobo- rate the notion that particle size impacts interstitial fluxes and mixing, and thereby biofilm growth and activity for sandy sedi- ment. Future work is needed to further elucidate these relation- ships under various conditions of flow, substrate flux and biofilm accumulation/activity. The hydrological impacts in the capillary fringe and water table boundary could affect biofilm dynamics in different ways that result in varying levels of biofilm diversity and activity (Fig. 2). Hydrogeochemical mixing As stated above, mixing can impact taxonomic diversity in groundwater communities and recent studies suggest it also affects sediment associated communities in similar ways (Ebrahimi and Or 2016, 2018). However, due to chemical (min- eralogy) and physical (e.g. size, arrangement) heterogeneity of the sediment matrix, niche partitioning and species filtering are likely additional factors that impact the composition of attached communities. In addition, mixing in the shallow subsurface due to faster and shorter local flow paths could impact local hydrodynamic dispersion and thus biofilms. Therefore, sedi- ment biofilms likely have distinct zone-specific responses (e.g. vadose to capillary fringe to saturated zones) (Fig. 1). Also, the subsurface sediment zones likely experience different degrees and rates of flow that impact the formation and stability (chem- ical, physical, and biological) of sediment-associated biofilms, and the biofilms are impacted by fluctuations of the water table and associated re-distribution in the capillary fringe (Moser et al. 2003; Stegen et al. 2016). Based on the available information from diversity-based studies, bulk activities and biofilm studies, hydrodynamics likely affects biofilm structure, function and dispersal in shallow sub- surface aquifers both by vertical and horizontal mixing. In high shear environments (e.g. water distribution lines), high shear stress is observed to decrease biofilm diversity and thickness (Rochex et al. 2008); however, the relationship between mix- ing and biofilm diversity is not known for low-shear condi- tions analogous to shallow subsurface environments that can have unsteady groundwater flow (Sposito 2006). If required resource ratios are not available, microbial activity cannot be sustained and continued non-growth could promote dispersal and/or death. Some work has attempted to explain the occur- rence of microbial populations in terms of a resource ratio the- ory, where a given level of resource is needed to sustain a pop- ulation (i.e. a consumption rate that is greater than a death rate at a given resource concentration) (Smith 1993; De Mazan- court and Schwartz 2010). However, this relationship does not account for varying substrate affinities, interacting populations or different behavior across phase boundaries under mixing conditions. Recently, it has been hypothesized that biofilm cells with restricted growth can outcompete populations with faster growth in the bulk-phase based upon a laboratory model (Coyte et al. 2016). This is an interesting hypothesis to test relevant con- ditions for the shallow subsurface that includes different popu- lations, interactions and/or activities in a porous medium with dynamic mixing in the shallow and deep saturated zone (Fig. 2). Microbial interactions within biofilms Many shallow subsurface biofilms are likely comprised of multi- ple species as in numerous other environments; however, few studies have delineated the spatial arrangement of microbial cells on particles from the shallow subsurface nor have deci- phered potential metabolic interactions in situ. Certainly, labo- ratory studies of multispecies biofilms observe that populations are not always randomly distributed but organized based on needs (Møller et al. 1998; Watnick and Kolter 2000). Conducting such studies with native material under in situ conditions are challenging. Recent work with upper layer soil/sediment par- ticles have shown particle-specific communities (Jackson and Weeks 2008; Hemkemeyer et al. 2015, 2018), but similar work for shallow subsurface sediments (>1 m depth) is sparse. From cell counts, one cannot determine whether attached populations reside as individual cells separated by micrometers of space, as clonal microcolonies or as multispecies biofilms over preferred locations (e.g. nutrient/mineral availability). This is also an issue with community analyses via amplicon or shotgun sequencing. Samples large enough to yield sufficient DNA quantities often encompass too much physical space to confidently infer repre- sentative microscale interactions, although progress has been made with upper layer soil particles. Future work is needed to elucidate whether sediments from the shallow subsurface are amenable to the same methods and if microscopy methods can be usedwith intact sediment samples in order to retain inherent physical structure. FUTURE DIRECTIONS It has become increasingly apparent that free-living and biofilm associated cells have distinct physiologies and function but the potential impacts on shallow subsurface systems is not well understood (Hall-Stoodley, Costerton and Stoodley 2004; Anneser et al. 2010). Many questions remain regarding the biofilm ‘life-cycle’ including attachment transitions, the dis- tribution and rate of specialized and general activity, coop- erative/competitive interactions, and mechanisms of dispersal in the shallow subsurface mixing zones (Fig. 2). Due to sam- pling challenges and the complexity of the heterogeneous sub- surface matrix that ranges across the vadose, capillary fringe and saturated zones, few field sites have been comprehensively described and studied despite the important ecosystem services associated with shallow subsurface systems. The shallow sub- surface has historically been considered a stable environment, D ow nloaded from https://academ ic.oup.com /fem sec/article-abstract/94/12/fiy191/5107865 by Libraries-M ontana State U niversity, Bozem an user on 09 January 2019 Smith et al. 11 but it is now clear that temporal and seasonal dynamics influ- ence hydrological mixing, particularly between and within the saturated and capillary fringe zones. Aquifer recharge and fluc- tuating water table can occur via seasonal patterns, and not sur- prisingly, the transition zones between the variably saturated and saturated zones has been shown to be an important eco- tone for microbial diversity and activity. Due to the complex- ity of the system and logistical challenges in sampling, there is much yet to be learned about the distribution of shallow sub- surface biofilms, the physiological activities/responses to envi- ronmental disturbances related to geochemical cycling and the roles these systems play in groundwater maintenance and sta- bility. Technological advances for sample retrieval and fine- scale analyses (spatial, temporal, cellular) of subsurface samples are needed, including samplers that can retrieve intact porous media and associated biofilms. Similarly, engineered reactor systems need to be modified to accurately simulate subsurface environmental conditions and address inconsistencies in repro- ducibility that currently exist. In order to understand and predict the role of microorgan- isms accurately within an environmental context, it is essen- tial to distinguish between active and inactive organisms. The majority of studies on activity in subsurface porous aquifers are from the 1980–1990’s and very few recent studies incorporate activity measurements with sequencing technologies. While the more recently adopted metagenomics-based sequencing approaches have opened new windows as to the functional diversity present within porous aquifers, activity is seldom linked to phylogeny. Of the open reading frames recovered with metagenomic sequencing, typically the functions from a small fraction can be linked to known genes and only a few of these genes have been studied in depth (Ferrer et al. 2016). Currently, untargeted metagenomic sequencing predominantly retrieves genomic sequences from dominant organisms and does not allow active organisms to be differentiated from inac- tive. While this is more suitable for low diversity habitats (Tyson et al. 2004; Woyke et al. 2006), subsurface environments can be highly diverse (Hug et al. 2015). Therefore, in order to accu- rately capture rare, underexplored and possibly environmen- tally significant metabolic processes, it will be imperative to apply functional/targeted metagenomic approaches. While the use of sequencing technologies has allowedmicrobial ecologists to glean the taxonomic compositions of microbial communities as never before, it is important to note that such methodologies contain inherent problems (Wintzingerode, Go¨bel and Stacke- brandt 1997; Bent and Forney 2008; Fraser et al. 2009). Recently, Carini et al. (2016) showed that extracellular, or ‘relic’, DNA was abundant in a variety of soil and sediment types and that this DNA can skew diversity measurements. Due to the rapid evolution of technologies capable of work- ing with small DNA and transcript quantities, the application of targeted strategies (i.e. sequencing data within a functional context) has increased for environmental studies (Lueders et al. 2016). Approaches already exist that target active fractions of microbial communities (e.g. bioorthagonal non-canonical amino acid tagging (BONCAT) (Hatzenpichler and Orphan 2015), DNA andRNA stable isotope probing (SIP) (Lueders et al. 2016) and pro- pidiummonoazide (PMA)-Seq (Carini et al. 2016)) which can then be combined with metagenomic or rRNA sequencing strategies. In the near future, these methods will be combined with micro- scopic/spectroscopic techniques that allow physical structure to be maintained and the proper scale for sediment-associated biofilms to be determined. The ability to infer subsurface-specific functional capabil- ities from genetic information, as well as the generation of testable hypotheses that can be confirmed at ecologically rel- evant microscales, is limited by the current lack of subsurface- specific reference sequences. In addition, laboratory studies are needed at the microscale with field-relevant isolates to confirm hypotheses generated from sequencing data. Of the currently available subsurface isolates, many have slow growth rates and/or most likely use forms of C, N and P associated with sedi- ments not typically used in cultivation/microcosms. It is becom- ing increasingly crucial to frame in situ experimentation to eco- logical questions and conditions pertinent to the respective environment (e.g. subsurface transport through porous media with intermittent inputs of temporally- and spatially-relevant OM). As noted by Prosser (2012), many microbial ecology ques- tions require studies that focus on smaller spatial scale, phe- notypic diversity, temporality and activity/rates. Through these combined approaches, a more complete understanding of shal- low subsurface ecosystems will be gained that includes biofilm dynamics at zone interfaces. 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