Understanding marine dissolved organic matter production: Compositional insights from axenic cultures of Thalassiosira pseudonana

Marine dissolved organic matter (DOM) is a key source of carbon and nutrients to microbial life in the oceans, but rapid biological utilization of labile DOM confounds its compositional characterization. In order to characterize potentially bioavailable DOM produced by phytoplankton, DOM from axenic cultures of Thalassiosira pseudonana cultivated in phosphorus (P) replete and low P conditions was extracted using highrecovery electrodialysis (ED) techniques, which resulted in an average dissolved organic carbon (DOC) recovery of 76%67% from all cultures. Low P concentrations resulted in greater cell-normalized production of DOC relative to P replete culture controls at the same growth phase. Despite the different nutrient conditions, DOC composition and DOM molar ratios of carbon to nitrogen (C : N) were similar in all cultures. In contrast, low P concentrations influenced DOM molar carbon to phosphorus (C : P) ratios and dissolved organic phosphorus (DOP) composition. Under P replete and low P conditions, DOM C : P ratios were 130 (6 22) and 2446 (6 519), respectively. P Nuclear Magnetic Resonance (NMR) spectroscopy identified P esters (> 90% of DOP) as the dominant P species in DOM produced under P replete conditions, with small or negligible contributions from phosphonates or glycerol P and polyphosphates. However, based on direct fluorometric analysis, DOP from low P cultures was greater than 8 times enriched in dissolved polyphosphate compared to DOP from replete cultures, which is consistent with the growing evidence that polyphosphate is a dynamic component of total P in low P ocean regions. Dissolved organic matter (DOM) is a source of bioavailable carbon (C) and nutrients, including nitrogen (N) and phosphorus (P). In the ocean, DOM supports the growth of marine bacteria (Azam et al. 1983) and phytoplankton (Bronk et al. 2007; Duhamel et al. 2010), especially in oligotrophic ocean regions (Thomas 1971; Fogg 1983; Ducklow et al. 1995; Karl et al. 1998; Agusti and Duarte 2013). Furthermore, DOM composition influences marine microbial community structure (Neogi et al. 2011; Gomez-Consarnau et al. 2012; Nelson and Carlson 2012; Dinasquet et al. 2013) and ultimately carbon remineralization and export (Letscher and Moore 2015). Despite its critical role in ocean biogeochemistry, the composition of DOM remains poorly understood (Benner 2002). DOM is produced via degradation of non-living organic matter and is also actively produced by living organisms (Carlson and Hansell 2014) including phytoplankton, which are considered a source of labile DOM (Cole et al. 1982; Norrman et al. 1995). Labile DOM is preferentially utilized, predominantly by marine heterotrophs (Jensen 1983; Kirchman et al. 1991; Reinthaler and Herndl 2005; Carlson and Hansell 2014), over timescales of minutes to weeks (Amon et al. 2001; Carlson 2002). However, ambient pools of marine DOM, which are typically characterized in compositional studies (Benner 2002), have radiocarbon ages in the range of thousands of years (Williams and Druffel 1987; Bauer et al. 1992; Loh et al. 2004; Karl and Bjorkman 2014; *Correspondence: [email protected] 1 LIMNOLOGY and OCEANOGRAPHY Limnol. Oceanogr. 00, 2016, 00–00 VC 2016 Association for the Sciences of Limnology and Oceanography doi: 10.1002/lno.10367 Druffel and Griffin 2015) and, thus, may not reflect freshly produced, labile DOM. Ambient pools of marine DOM contain dissolved organic forms of C (DOC), P (DOP), and N (DON), including biomolecules and their derivatives (Repeta 2014). Of these forms, this study focuses on DOP in order to better understand intriguing compositional observations in the ocean. Diverse regions and depths of the ocean contain relatively consistent proportions of polyphosphates (molecules with at least three P atoms joined by P-O-P bonds), 8–13% of DOP; phosphonates (direct C-P bonds), 5–10%; and P esters (P-O-C bonds), 80–85% (Young and Ingall 2010). This uniform composition of DOP, which is distinct from that of marine organisms, has been attributed to microbial decomposition processes (Clark et al. 1998, 1999; Kolowith et al. 2001; Karl and Bjorkman 2014; Young and Ingall 2010). However, despite the critical role of these P forms as potential P sources (Martin et al. 2014; Van Mooy et al. 2015) and as possible drivers of climate (Diaz et al. 2008; Karl et al. 2008), the composition of DOP initially produced by microorganisms remains largely unexplored. In order to characterize the DOM fraction initially supplied to marine systems, previous studies have examined DOM produced in controlled cultures of marine phytoplankton (Helebust 1965; Myklestad 1977; Obernosterer and Herndl 1995; Biddanda and Benner 1997; Biersmith and Benner 1998; Aluwihare and Repeta 1999; Granum et al. 2002). DOM compositional characterization in these studies focused on either specific compounds, such as polysaccharides, or bulk analysis of the high molecular weight fraction of DOM. Compositional differences between phytoplankton-derived DOM and ambient ocean DOM identified in these studies were attributed to selective utilization of certain compounds by bacteria (Biddanda and Benner 1997; Biersmith and Benner 1998; Aluwihare and Repeta 1999). In fact, the composition of phytoplankton-derived DOM can be altered by heterotrophic processing on timescales of days to weeks due to preferential consumption of not only specific compound classes, but also of specific molecules within compound classes (Amon et al. 2001). Limited evidence based on analysis of specific compounds or bacterial utilization of DOM produced in culture studies suggests that nutrient availability influences the composition of phytoplankton-derived DOM (Myklestad and Haug 1972; Myklestad 1977; Obernosterer and Herndl 1995; Puddu et al. 2003). For example, phytoplankton growth under low P conditions can result in the production of DOM that supports a distinct microbial community compared to DOM produced under P replete conditions (Puddu et al. 2003), which may reflect bioavailability differences and, thus, compositional differences between DOM pools produced under different P regimes. Although N is often the limiting nutrient over vast marine areas, spatial and temporal increases in N abundance may shift systems to P limitation (Karl et al. 1995, 1997; Moore et al. 2013). Phytoplankton alter cellular P allocation in response to P scarcity, specifically through the substitution of sulfolipids for phospholipids (Van Mooy et al. 2009; Martin et al. 2011) and modulation of cellular phosphorus pools like polyphosphate compounds (Dyhrman et al. 2012; Martin et al. 2014; Diaz et al. 2016). Whether these Pdependent particulate dynamics translate to similar compositional shifts within the DOP pool remain unknown. For all DOM studies, there are difficulties associated with obtaining a representative sample for chemical analysis (Hedges 1992; Benner 2002). Most chemical characterization techniques require naturally low concentrations of marine DOM ( 1 ppm) to be isolated from the high salt content of seawater ( 35,000 ppm) (Repeta 2014). Isolation difficulties traditionally limit DOM studies to the analysis of a small number of molecules that can be directly measured in a high salt matrix or to the characterization of the partial DOM fractions that can be recovered. For example, many studies have isolated DOM via ultrafiltration, which recovers the high molecular weight fraction of DOM (> 1 kDa) (see references in Benner 2002). Thus, low molecular weight, potentially bioavailable molecules typically escape characterization (Kujawinski 2011). In fact, phytoplankton-derived DOM is dominated by low molecular weight molecules (Jensen 1983; Lancelot 1984), which have been suggested to have a composition distinct from molecules in the high molecular weight fraction (Benner et al. 1992; Biddanda and Benner 1997). Recently developed electrodialysis (ED) extraction techniques have improved DOM recoveries by approximately three times compared to ultrafiltration (Koprivnjak 2009; Green et al. 2014). Higher recovery yields a final sample that is more representative of bulk DOM by diminishing the bias towards the high molecular weight DOM fraction recovered using ultrafiltration (Vetter et al. 2007; Koprivnjak et al. 2009). Thus, ED techniques offer the potential for new insights into the marine DOM composition. For example, the application of ED has recently revealed the presence of dissolved polyphosphates in diverse marine waters (Diaz et al. 2008; Young and Ingall 2010), which typically go undetected in ultrafiltration studies (e.g., Clark et al. 1998, 1999; Kolowith et al. 2001; Sannigrahi et al. 2006). In previous culture studies, the low DOM extraction efficiencies of ultrafiltration required processing large volumes to obtain analytically relevant quantities for studies of bulk DOM composition (Biddanda and Benner 1997). Large volumes made it difficult to evaluate the composition of phytoplankton-derived DOM in the absence of bacteria and fungi (axenic conditions), which can consume the most bioavailable fractions on time scales of minutes to hours (Carlson 2002). The larger recoveries of ED permit the use of smaller culture volumes to obtain analytically useable DOM quantities for bulk compositional analyses. Additionally, Saad et al. T. pseudonana-derived DOM