Growth in Lindavia intermedia in Response to Phosphorus and Nitrogen Concentrations and Phylogenetics of the Ferredoxin gene (petF)

The invasive diatom species, Lindavia intermedia produces nuisance extracellular polysaccharides (EPS), termed lake snow, which foul freshwater oligotrophic lakes in New Zealand – many of which are part of National Parks and considered Taonga (treasure) by Māori. Lindavia intermedia was discovered in 2004, resulting in blooms of EPS which have hindered recreational lake activities, as well as obstructed water filtration systems. Lindavia intermedia is poorly understood due to lack of research on the species, despite its recent introduction and yet to be determined effects on native freshwater flora and fauna. For our experiments, Lindavia intermedia was grown in three different media with different concentrations of nitrogen and phosphorus: f/2 ([N] = 882 μM, [P] = 36.2 μM), modified f/2 ([N] = 21.42 μM, [P] = 0.42 μM) and synthetic freshwater media (SFM, [N] = 770 μM, [P] = 13.2 μM). In order to test for differences in growth at different N and P concentrations, triplicate cultures of L. intermedia were grown in each of culture media for a period of 14 weeks. SFM exhibited significantly higher final cell concentration (p-value < 0.05) than f/2 and modified f/2 cultures, possibly due to higher concentrations of trace metals and silica. Growth exhibited between modified f/2 and f/2 during weeks 1-6 and 7-10 were non-significant (p-value = 0.56, 0.94), however, modified f/2 exhibited significantly higher cell density during week 11-14 compared to f/2 (p-value = 0.028). SFM was chosen as the culture media for the following ratio experiment. In the ratio experiments, L. intermedia was grown at 2.5-times increased and decreased nitrogen and phosphorus concentration in SFM, resulting in Set A ratios and Set B ratios of N:P of 136.5:1, 70.5:1, 44.1:1 and 202.1:1, 80.7:1 and 32.3:1, respectively. Growth at week 1-4 and 4-8 at Set A and B ratios were both non-significant (p-value > 0.05), although growth between some ratios during week 4-8 were close to significant, indicating that growth was generally higher at ratios closest to the N:P of SFM at 58.3:1.

Growth between the Set A and B ratios 70.5:1 and 80.7:1 showed no significant difference, which suggests that L. intermedia exhibits identical growth at both ratios, despite differing concentrations of nitrogen and phosphorus (930.8 μM:13.2 μM, and 770 μM:9.54 μM, respectively. Although this result contradicts our hypothesis that L. intermedia is growth limited by phosphorus, it likely indicates that the N and P concentrations satisfied cellular N and P quotas of L. intermedia, and therefore did not significantly influence growth. The iron-sulphur protein ferredoxin is utilised for electron transfer in PSI and essential for the production of NADPH in photosynthetic organisms such as diatoms. Our ferredoxin phylogeny analysis of L. intermedia, consisting of 79 diatom species, were used to produce a maximum likelihood tree with 500 bootstrap replicates. In our secondary structure analysis, we found that the grouping of L. intermedia and Stephanodiscus niagarae had a support value of 88%, both of which belong to the order Stephanodiscales. As an iron-sulphur protein is utilised for electron transfer in PSI, the presence of ferredoxin and absence of its non-iron alternative, flavodoxin, in L. intermedia, implying a potential correlation between its growth and iron concentrations.