DOI: 10.5061/dryad.zcrjdfnh4
title: Diazotrophy modulates cyanobacteria stoichiometry through functional traits that determine bloom magnitude and toxin production

description: Harmful cyanobacterial blooms are an increasing threat to water quality.
 The interactions between two eco-physiological functional traits of
 cyanobacteria, diazotrophy (nitrogen (N)-fixation) and N-rich cyanotoxin
 synthesis, have never been examined in a stoichiometric explicit manner.
 We explored how a gradient of resource N:phosphorus (P) affects the
 biomass, N, P stoichiometry, light-harvesting pigments, and
 cylindrospermopsin production in a N-fixing
 cyanobacterium, Aphanizomenon. Low N:P Aphanizomenon cultures
 produced the same biomass as populations grown in high N:P cultures. The
 biomass accumulation determined by carbon, indicated low N:P Aphanizomenon
 cultures did not have a N-fixation growth tradeoff, in contrast to some
 other diazotrophs that maintain stoichiometric N homeostasis at the
 expense of growth. However, N-fixing Aphanizomenon populations produced
 less particulate cylindrospermopsin and had undetectable dissolved
 cylindrospermopsin compared to non-N-fixing populations. The pattern of
 low to high cyanotoxin cell quotas across an N:P gradient in the
 diazotrophic cylindrospermopsin producer is similar to the cyanotoxin cell
 quota response in non-diazotrophic cyanobacteria. We suggest that
 diazotrophic cyanobacteria may be characterized into two broad functional
 groups, the N-storage-strategists and the growth-strategists, which use
 N-fixation differently and may determine patterns of bloom magnitude and
 toxin production in nature.
description: Laboratory bioassay experiment. Aphanizomenon flos-aquae (PCC
 7905) was grown under 11 Nitrogen:Phosphorus media conditions (1, 2, 4, 8,
 12, 16, 20, 30, 50, 75 and 100 by mol) for 37 days. Cultures were grown in
 incubators held at 26°C with a light intensity of 140 µmol m-2 s-1 on a
 14h:10h light:dark cycle. Every other day cultures were shaken and rotated
 to prevent settling and incubator placement effects. We measured in-vivo
 chlorophyll a every 3-4 days to track growth. Subsampling for particulate
 carbon and nitrogen was done after 10, 17, 21, 25, 29, 33 days of growth.
 After 37 days of growth, we sampled for particulate carbon, nitrogen,
 phosphorus, cylindrospermopsin, chlorophyll a, and phycobilin pigments on
 to 24 mm glass fiber filters (GF/F). The filtrate was saved for dissolved
 nitrogen (nitrate, nitrite, ammonium), phosphorus, and total dissolved
 nitrogen and phosphorus. A subsample of each culture was preserved in
 Lugol’s iodine and counted on a compound microscope at 400x magnification
 to obtain cell densities. We used a mass balance approach to determine
 gross-nitrogen-fixation rates. In vivo chlorophyll-a –
 performed using a Tuner Designs fluorometer with the in vivo chlorophyll a
 module Particulate carbon, nitrogen, and phosphorus -
 sampled using 0.7µm GF/F Whatman filters. Particulate carbon and nitrogen
 filters were dried at 60 °C for 24 hours, and then analyzed simultaneously
 on an elemental analyzer as described by Wagner et al (2019). Particulate
 phosphorus was analyzed by hot persulfate digestion (3% w/v) and analyzed
 using the molybdate blue method (APHA 2002).  
 Chlorophyll a was analyzed according to EPA method 445.0. Filters
 were extracted in 90% acetone:water over night at 4°C and analyzed on a
 Tuner Designs Trilogy fluorometer using the acid chlorophyll a
 module.   Phycobilin pigments were
 analyzed as previously described by Wang et al. (2021). Briefly, filters
 were placed in 5 mL of 0.1M phosphate buffer with two rounds of
 freeze-thaw cycle to promote cell lysis. After filters were sonicated for
 7 min and stored at 4°C overnight. Phycobilin pigments were read on a
 UV/Visible spectrophotometer at 625, 615, and 562 nm. Concentrations were
 calculated as in Wang et al. 2021. Total and dissolved
 cylindrospermopsin – Particulate and dissolved cylindrospermopsin were
 extracted and analyzed using an isotope dilution method coupled with
 LC-MS/MS as described in Haddad et al. (2019) and implemented in Osburn et
 al. (2022). Total cylindrospermopsin was calculated by the sum of
 particulate and dissolved.