Diazotrophy modulates cyanobacteria stoichiometry through functional traits that determine bloom magnitude and toxin production

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.

Methods
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.

有害蓝藻水华（Harmful Cyanobacterial Blooms）正对水环境质量构成日益严峻的威胁。蓝藻的两种生态生理功能性状——固氮营养型（diazotrophy，即氮（N）固定作用）与富氮蓝藻毒素合成——之间的相互作用，此前从未以化学计量学明确方式开展过研究。本研究探讨了资源氮（N）:磷（P）梯度如何影响固氮蓝藻束丝藻属（Aphanizomenon）的生物量、氮磷化学计量、光捕获色素以及柱孢藻毒素（cylindrospermopsin）的合成情况。低氮磷比培养基中的束丝藻培养物，其生物量与高氮磷比培养基中的培养物相当。以碳为基准的生物量积累结果表明，低氮磷比条件下的束丝藻并未出现固氮作用的生长权衡现象，这与其他部分为维持化学计量氮稳态而牺牲生长速率的固氮生物不同。然而，与非固氮种群相比，固氮束丝藻种群的颗粒态柱孢藻毒素产量更低，且未检出溶解态柱孢藻毒素。在固氮型柱孢藻毒素产生菌中，随氮磷比梯度变化的蓝藻毒素细胞配额模式，与非固氮蓝藻的蓝藻毒素细胞配额响应模式相似。本研究提出，固氮蓝藻可划分为两大类宽泛的功能群：氮储存策略型与生长策略型，二者对固氮作用的利用方式存在差异，或可决定自然环境中水华规模与毒素产生的模式。

方法
实验室生物测定实验。本实验采用水华束丝藻（Aphanizomenon flos-aquae，菌株PCC 7905），将其置于11组不同氮磷比（摩尔比分别为1、2、4、8、12、16、20、30、50、75、100）的培养基中培养37天。培养过程置于温度为26℃的培养箱中进行，光照强度为140 μmol·m⁻²·s⁻¹，光暗周期为14h:10h。每两日对培养物进行振荡与旋转操作，以避免细胞沉降及培养箱内位置差异带来的影响。每3~4天测定一次活体叶绿素a（in vivo chlorophyll a）含量，以追踪培养物的生长情况。分别在培养第10、17、21、25、29、33天时进行取样，用于测定颗粒态碳与氮含量。培养37天后，取样测定颗粒态碳、氮、磷、柱孢藻毒素、叶绿素a以及藻胆蛋白色素，取样时采用24 mm玻璃纤维滤膜（GF/F）进行过滤收集。收集所得的滤液留存，用于测定溶解态氮（硝酸盐、亚硝酸盐、铵盐）、磷以及总溶解态氮与磷含量。每份培养物取一份子样本用鲁戈氏碘液（Lugol’s iodine）固定，随后在400倍放大倍率的复合光学显微镜下进行计数，以获取细胞密度数据。本研究采用质量平衡法计算总固氮速率。

活体叶绿素a测定：采用Tuner Designs荧光光度计搭配活体叶绿素a检测模块完成。
颗粒态碳、氮、磷测定：采用0.7μm GF/F Whatman滤膜进行取样。颗粒态碳与氮的滤膜样品经60℃烘干24小时后，使用元素分析仪进行同步测定，方法参照Wagner等人（2019）的报道。颗粒态磷的测定采用过硫酸盐热消解（3% w/v）结合钼蓝比色法，方法参照APHA（2002）标准。
叶绿素a测定：参照美国环境保护署（EPA）方法445.0执行。滤膜样品经90%丙酮水溶液在4℃下过夜萃取后，使用Tuner Designs Trilogy荧光光度计搭配酸性叶绿素a检测模块完成测定。
藻胆蛋白色素测定：参照Wang等人（2021）的方法执行。简要步骤为：将滤膜置于5 mL 0.1M磷酸盐缓冲液中，经过两轮冻融循环以促进细胞裂解；随后将滤膜超声破碎7分钟，并在4℃下过夜存放。采用紫外-可见分光光度计在625、615及562 nm波长下读取藻胆蛋白色素的吸光度值，浓度计算方法参照Wang等人（2021）的报道。
总柱孢藻毒素与溶解态柱孢藻毒素测定：颗粒态与溶解态柱孢藻毒素的提取与分析采用同位素稀释法结合液相色谱-串联质谱（LC-MS/MS），方法参照Haddad等人（2019）的报道，并由Osburn等人（2022）优化实施。总柱孢藻毒素的含量通过颗粒态与溶解态柱孢藻毒素的含量之和计算得到。