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.

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.