Filtration via Conventional Glass Fiber Filters in 15N2 Tracer Assays Fails to Capture All Nitrogen-Fixing Prokaryotes

Abstract

Biological dinitrogen fixation (BNF) represents a major input of reduced nitrogen (N) to the oceans. Accurate direct measurements of BNF rates are crucial for reliably determining the biogeochemical significance of diazotrophy at local and global scales. Traditionally, borosilicate glass fiber filters (GF/F, Whatman) with a nominal pore size of 0.7 µm are used to collect suspended particles by filtration after incubations with added 15 N 2 tracer. We carried out BNF experiments in the Baltic Sea, Danish coastal waters, and the Pacific Ocean comparing the retentive characteristics of precombusted GF/F filters with newer Advantec glass fiber filters which have a smaller nominal pore size of 0.3 µm. Where BNF was detected, rates were nearly always higher, and sometimes even exclusively detectable, when using Advantec filters. In the majority of samples across tested habitats, significantly more cells were lost to GF/F filtrate (average = 51%, range = 10-70% of cells) than to Advantec filtrate (average = 40%, range = 10-54%). Using Illumina sequencing of nitrogenase (nifH) gene amplicons, we show that diazotroph communities can markedly differ between bulk water and filtrates from GF/F and Advantec filtrations, suggesting that different diazotrophs can pass through the filter types. In order to reduce the potential underestimations of BNF due to filtration loss of diazotrophs, we recommend using Advantec filters or alternatively silver membranes with 0.2 µm pore size, especially in waters expected to be inhabited by relatively small, unicellular diazotrophs.

A critical review of the 15N2 tracer method to measure diazotrophic production in pelagic ecosystems

“…Some researchers have reported that GF/F filters perform as well in retaining phytoplankton as 0.2 μ m pore‐size membrane filters (Li et al 1983; Yentsch ; Goldman and Dennett ; Li ; Taguchi and Laws ; Chavez et al ; Ferrari and Tassan ), whereas others have found 0.2 μ m membrane filters to be superior (Phinney and Yentsch ; Dickson and Wheeler , ). An alternate variety of glass fiber filter has recently become available, the Advantec GF‐75, which claims a nominal pore size of 0.3 μ m. Bombar et al () demonstrated that nonpigmented particles (bacterioplankton) were more abundant in the filtrate of GF/Fs vs. GF‐75, suggesting greater retention of particle mass by the smaller pore‐size GF‐75. N 2 fixation rates were more likely to be detected in their study regime with GF‐75 than GF/F filters, and detected rates were systematically higher with GF‐75 filters.…”

“…Nevertheless, while more retentive than GF/F filters, ~ 40% of bacterioplankton cells were still lost to the filtrate with GF‐75 filters. Moreover, the diazotrophic communities tended to differ markedly between bulk water and filtrates, suggesting differential retention of N 2 ‐fixing communities (Bombar et al ). Similarly, Chaves et al (IOCCG Protocol Series ) reported a small but significant positive mean bias for particulate organic carbon (POC) measured on GF‐75 vs. GF/F in small‐volume sampling off the Peruvian coast where POC was between 10 and 30 μ mol L −1 .…”

Machine Learning Estimates of Global Marine Nitrogen Fixation

“…Sparse and uneven distribution of observations, mismatch of observations with predictors, and methodological biases (i.e., acetylene reduction assays, 15 N 2 gas addition, and dissolved 15 N 2 addition) introduce noise in our estimates. A more detailed discussion of caveats, uncertainties, and future improvements can be found in the supporting information (Bombar et al, 2018;Böttjer et al, 2016;Cassar et al, 2018;Fernandez et al, 2011;Hamersley et al, 2011;Jickells et al, 2017;Landolfi et al, 2015;Luo et al, 2014;Mohr et al, 2010;Monteiro et al, 2011;Mulholland, 2007;Paulsen et al, 2017;Ward et al, 2013;Weber & Deutsch, 2014;Wilson et al, 2012). We tested the sensitivity of our estimates to the limited number of observations used to train the RF and SVR models.…”

Global Marine N2 Fixation Estimates: From Observations to Models

“…The 15 N 2 gas stock contamination by 15 NH 4 + or 15 NO x , may result in N 2 fixation overestimate ( Dabundo et al, 2014 ). Fixation rates may be underestimated if small cells (<2 μm) are lost during filtration, particularly affecting rates where small diazotrophs may dominate the N 2 -fixing community ( Bombar et al, 2018 ). Robust quantification of low rates is currently limited by the low sensitivity and high potential errors of present methods ( Gradoville et al, 2017 ; Moisander et al, 2017 ).…”