The effects of environmental conditions on growth and protease inhibitor production of the cyanobacterium Microcystis aeruginosa

Cyanobacteria are photoautotrophic organisms that proliferate in lakes, ponds and estuaries under favorable conditions. The nutrient pollution of waterbodies, together with increasing temperatures and CO2 levels, has led to an increased frequency of cyanobacterial mass developments (‘blooms’) in the last decades, with severe economic and ecological consequences. Therefore, cyanobacteria became an important topic within limnological research. Special emphasis lies on two central question: Which conditions control the development of cyanobacterial blooms and what is the role of the variety of secondary metabolites produced by cyanobacteria in the success of cyanobacteria? Some of those metabolites have proven to be toxic for other aquatic organisms, but have also detrimental effects on human health. Much research was focused on microcystins, one of the most toxic groups of cyanobacterial metabolites. Other secondary metabolites, such as protease inhibitors, have received less attention. Protease inhibitors, like nostpeption 920 and cyanopeptolin 954, specifically inhibit serine proteases, for example in the gut of the water flea Daphnia, which results in a limitation by amino acids and leads subsequently to other negative consequences (e.g. reduced growth). This in turn has detrimental effects on the whole ecosystem, because Daphnia are the main consumers of phytoplankton (including cyanobacteria) and serve additionally as food for a number of predators and are therefore essential for the transfer of nutrients and energy from the level of primary production to higher trophic levels. The investigation of protease inhibitors is therefore necessary to understand the development and consequences of cyanobacterial blooms. In particular, knowledge about the influence of environmental factors on cyanobacteria, their growth and the production of protease inhibitors is required. This dissertation investigated the effect of three major environmental factors on the cyanobacterium Microcystis aeruginosa, one of the most common bloom-forming cyanobacteria. The fundamental question was: How do different concentrations of nitrogen and phosphorus as well as different light intensities affect the growth and more interestingly the production and content of protease inhibitors in M. aeruginosa? To answer this question, I performed growth experiments (batch cultures) with M. aeruginosa NIVA Cya 43 in which the cyanobacterium was grown at different initial nitrogen and phosphorus concentrations as well as under different light intensities. The cultures were sampled regularly to measure the growth, stoichiometry and the content of two protease inhibitors (nostopeptin 920 and cyanopeptolin 954) of M. aeruginosa. The samples were analyzed using a combination of classical (e.g. microscopic cell counting) and modern analytic methods (e.g. high-pressure liquid chromatography and high resolution mass spectrometry). Additionally, I used mathematical models, including a new developed dynamic process-based model, to describe and analyze the growth and the dynamics of the protease inhibitor content. In this dissertation, I showed that M. aeruginosa can grow under various experimental conditions and that in particular high nutrient concentrations and low light intensities support cyanobacterial growth. This thesis, furthermore, shows that the protease inhibitors are constitutively produced, but that the inhibitor content changes with the growth phase and the tested experimental conditions. Accordingly, I found coherences between the inhibitor content and the growth rate, respectively the stoichiometry, of the cyanobacterium. Additionally, I showed that protease inhibitors are especially produced under nutrient-rich conditions during the early growth phases, while they are degraded in the late growth phase, when the external nutrients are depleted and the nutrient limitation increases. These dynamics indicate that both investigated inhibitors are involved in intracellular processes. A potential involvement of protease inhibitors in the storage of nitrogen is, according to my results, plausible and should be investigated in the future. The results of this dissertation support the already existing knowledge that M. aeruginosa benefits from the anthropogenic nutrient input and adds new insights about the influence of environmental factors on cyanobacteria, in particular on protease inhibitors. This dissertation also provides insights into the functional role of protease inhibitors in cyanobacteria, but also discusses the ecological consequences of changes in the protease inhibitor content. Especially nutrient-rich conditions will most likely result in an increase of the cyanobacterial biomass and a simultaneously increase in the inhibitor content, which will reduce the potential control of cyanobacteria by Daphnia and could promote the development of cyanobacterial blooms. Hence, we should increase the efforts to reduce the nutrient input (nitrogen, phosphorus) into lakes and should additionally put more focus on the variety of metabolites produced by cyanobacteria to mitigate the ecological consequences of cyanobacterial blooms in the future

Phosphate Limitation Increases Content of Protease Inhibitors in the Cyanobacterium Microcystis aeruginosa

Increased anthropogenic nutrient input has led to eutrophication of lakes and ponds, resulting worldwide in more frequent and severe cyanobacterial blooms. In particular, enhanced availability of phosphorus (P) can promote cyanobacterial mass developments and may affect the content of secondary metabolites in cyanobacteria, such as protease inhibitors (PIs). PIs are common among cyanobacteria and have been shown to negatively affect herbivorous zooplankton. Here, we test the hypothesis that P-limitation reduces the growth of Microcystis, but increases the content of PIs. In batch culture experiments with eight different initial phosphate concentrations (5-75 mu M) we determined growth, stoichiometry, and PI content of Microcystis aeruginosa NIVA Cya 43. This strain produces the protease inhibitor BN920 that is converted by chlorination to CP954, which constitutes the major PI in this strain. C:N:P-ratios of the biomass indicated variation of P-limitation with treatment and time. When normalized to biomass, the PI content varied up to nearly nineteen-fold with treatment and time and was highest in the low-P treatments, especially during the mid-exponential growth phase. However, these effects were alleviated under nitrogen co-limitation. The content of CP954 showed an inverse u-shaped response to growth rate and C:N-ratio of the cyanobacterial biomass, whereas it increased with cyanobacterial C:P. The results indicate that P-limitation supports a higher content of defensive PIs and may indirectly foster cyanobacterial blooms by increasing the negative interference of cyanobacteria with their consumers

Nitrate determines growth and protease inhibitor content of the cyanobacterium Microcystis aeruginosa

The eutrophication of lakes and ponds through anthropogenic nutrient input has resulted in higher frequencies of cyanobacterial blooms worldwide. The increased availability of nitrate (NO3-) plays an important role for the development of such blooms and also affects the content of secondary metabolites in cyanobacteria. Cyanobacterial protease inhibitors (PIs) are widespread, nitrogen (N)-rich metabolites that can be considered potential defense molecules, as they have detrimental effects on herbivorous zooplankton, the major consumer of cyanobacteria. In batch culture experiments, we investigated the effect of varying NO3- concentrations on the growth, stoichiometry, and PI content of the cyanobacterium Microcystis aeruginosa NIVA Cya 43, which synthesizes the two PIs nostopeptin 920 (BN920) and cyanopeptolin 954 (CP954). The dynamics of particulate organic nitrogen (PON) and the concentration of the two PIs indicate that BN920 and CP954 serve as temporary N storage compounds and are suggested to be degraded under N limitation. When related to the cyanobacterial biomass, the inhibitor content varied by more than 80% with NO3- concentration and time. The PI content increased with growth rate and N content of the cyanobacterium, which indicates that increased N availability supports higher cyanobacterial biomass with a higher content of defensive PIs. Therefore, increased NO3- concentrations foster cyanobacterial blooms directly by providing more nutrients and indirectly by increasing the negative interference of cyanobacteria with their consumers due to an increased content of PIs