Uptake of Spartina-derived humic nitrogen by estuarine phytoplankton in nonaxenic and axenic culture

Humic substances are a collection of colored organic acids characterized by high molecular weight and low nitrogen (N) content that are thought to be biologically recalcitrant. We examined a suite of nonaxenic estuarine phytoplankton isolates to determine their ability to take up 15N-labeled humic substances formed in the laboratory and supplied as the sole N source. All 17 estuarine and coastal strains took up the added humic N, but the one polar isolate did not. Two of the coastal isolates (Heterosigma akashiwo and Fibrocapsa japonica) could take up the humic N in nonaxenic culture but not in axenic culture, suggesting that bacterial remineralization played a role in making humic N accessible to these species. The ability of nonaxenic phytoplankton isolates to use humics of different ages (1 week to 1 yr old) was tested using three strains capable of taking up humic N at high rates. Younger, fresher humics were taken up by the phytoplankton strains at higher rates than older, more fulvic-like compounds, and at rates higher than inorganic N uptake run in parallel. Time-course results indicate that while uptake of the inorganic N forms was sustained, high rates of humic N uptake declined after the first few hours of incubation. Additional humic substances were labeled with both 15N and 13C, and the relative incorporation of N versus carbon (C) was used to infer potential uptake mechanisms. None of the isolates took up humic C, suggesting that uptake of the humic N followed breakdown of the humic molecule by bacteria or via extracellular enzyme cleavage of humic N. Regardless of the mode of uptake, the observation that humic N can be rapidly used by phytoplankton suggests that the importance of humic N as a source of phytoplankton N nutrition should be reevaluated. On average, approximately 7.3 3 1012 g of dissolved organic nitrogen (DON) is discharged into the coastal ocean annually (Meybeck 1982; Sarmiento and Sundquist 1992), with 40–80% of this nitrogen (N) considered to be humic in nature (Beck et al. 1974; Thurman 1985). However, the ability of estuarine and coastal phytoplankton strains to use humic substances as an N source has not been thoroughly investigated. Owing to their size and chemical composition, humics have traditionally been perceived as biologically recalcitrant, largely because they are high molecular weight (HMW), carbon (C)-rich compounds with N making up only 0.5–6% of the humic molecule (Rashid 1985; Thurman 1985; Hedges and Hare 1987). Furthermore, only 50% of humic N is in the amine form and therefore likely to be labile (Schnitzer 1985). More recently, however, the refractory nature of humic substances has been challenged, and evidence is accumulating that indicates that coastal phytoplankton may have the ability to take up humic N, either directly or after remineralization by bacteria (Carlsson et al. 1995, 1998, 1999). More recently, the uptake of laboratory produced 15N-labeled humic compounds into the .0.7-mm size fraction has been observed in both riverine and coastal ecosystems (Bronk et al. unpubl. data), humic substances were shown to be a potential source of C and/or N to the toxic dinoflagellate Alexandrium catenella (Doblin et al. 2000), and growth of the toxic dinoflagellate Alexandrium tamarense was shown to increase when exposed to humic substances (Gagnon et al. 2005). There is also evidence that humic substances are not as N poor in natural waters as currently believed. It has been shown that during the process of isolating humic substances with macroporous resins N is stripped from the humic structure, resulting in 2 Present address: NOAA, Center for Sponsored Coastal Ocean Research, Silver Spring, Maryland 20910.