At the scale of microorganisms, marine environments are heterogeneous — environmental conditions and bacterial communities vary on the scale of microns to millimeters (Long and Azam 2001; Simon et al. 2002; Azam and Malfatti 2007; Stocker 2012). A main contributor to the ocean’s microscale heterogeneity are particles that vary in size, shape, density, and chemical composition (Alldredge and Silver 1988). These particles are habitats for microorganisms whose metabolic and behavioral niches differ from those of free living organisms (Simon et al. 2002; Leu et al. 2022). Microbial communities can differ between individual particles (Bižić-Ionescu et al. 2018; Farnelid et al. 2018). Particles of different sizes harbor distinct microbial communities (Duret et al. 2015; Mestre et al. 2017). Highly size resolved measurements from global sampling efforts have shown that particle size variability promotes microbial diversity globally (Mestre et al. 2018). Additionally, chemical species such as oxygen and nitrate have diffusion gradients in large particles, allowing anoxic zones under low oxygen or sulfidic zones under low nitrate (Ploug et al. 1997; Stief et al. 2016; Bianchi et al. 2018; Fuchsman et al. 2019b; Saunders et al. 2019; Raven et al. 2021). Previous analyses of bacterial communities along the particle size spectrum have been semi-quantitative, providing relative abundance estimates (fraction of the total community), rather than estimates of quantitative abundance (cells per liter of water or milligram of particle). Therefore, we implemented a novel size fractionation approach to allow collection of DNA, microscopic samples and concentrations of the particles themselves in each size fraction, and utilized standards to quantitatively describe microbial distributions along the particle size spectrum.

The Chesapeake Bay is the largest estuary in the United States and provides a well-studied model system characterized by high production and active biogeochemical processes (Turk et al. 2021). The Bay is characterized by abundant particles, which transport nutrients and carbon through the system (Sanford et al. 2001; Malpezzi et al. 2013; Palinkas et al. 2019). Particles transport organic carbon to the middle of the Bay, where it fuels microbial respiration, depleting the mid-Bay of oxygen (Wang and Hood 2020) and creating a seasonally oxygen-starved environment (Testa et al. 2018). Bacteria in the anoxic Bay are known to produce greenhouse gasses including methane (Gelesh et al. 2016) and nitrous oxide (Ji et al. 2018; Laperriere et al. 2019), as well as hydrogen sulfide (Luther et al. 1988) which is toxic to marine life (Kang 1997; Boyd 2014). Sulfur oxidizing bacteria, responsible for the removal of hydrogen sulfide and other reduced sulfur species have been identified in the hypoxic Bay (Crump et al. 2007; Findlay et al. 2015), potentially using nitrogen species as terminal electron acceptors (Arora-Williams et al. 2022). Methane appears to be produced in the sediments, but is oxidized in the water column (Reeburgh 1969; Hagen and Vogt 1999; Gelesh et al. 2016). However, it is unknown whether and on what sizes of particles methane and sulfur cycling bacteria associate. Bay microbial communities vary across space and season (Kan et al. 2006, 2007; Wang et al. 2020; Arora-Williams et al. 2022). Furthermore, in general, particle and free-living microbial communities differ (DeLong et al. 1993; Bidle and Fletcher 1995). However, no investigation of the spatial variability of particle associated bacterial communities using modern techniques has been done in the Bay. In this manuscript we describe particle size resolved measurements of microbial communities and how they vary across space in the Chesapeake Bay.