Microbial remineralization processes during post-spring-bloom with excess phosphate available in the northern Baltic Sea.

The phosphorus (P) concentration is increasing in parts of the Baltic Sea following the spring bloom. The fate of this excess P-pool is an open question and here we investigate the role of microbial degradation processes in the excess P assimilation phase. During a 17-day-long mesocosm experiment in the south-west Finnish archipelago, we examined nitrogen, phosphorus and carbon acquiring extracellular enzyme activities in three size fractions (<0.2 µm, 0.2-3 µm, and >3 µm), bacterial abundance, production, community composition and its predicted metabolic functions. The mesocosms received carbon (C) and nitrogen (N) amendments individually and in combination (NC) to distinguish between heterotrophic and autotrophic processes. Alkaline phosphatase activity occurred mainly in the dissolved form and likely contributed to the excess phosphate conditions together with grazing. At the beginning of the experiment, peptidolytic and glycolytic enzymes were mostly produced by free-living bacteria. However, by the end of the experiment, the NC-treatment induced a shift in peptidolytic and glycolytic activities and degradation of phosphomonoesters towards the particle-associated fraction, likely as a consequence of higher substrate availability. This would potentially promote retention of nutrients in the surface as opposed to sedimentation, but direct sedimentation measurements are needed to verify this hypothesis.

Diatom-mediated food web functioning under ocean artificial upwelling.

Enhancing ocean productivity by artificial upwelling is evaluated as a nature-based solution for food security and climate change mitigation. Fish production is intended through diatom-based plankton food webs as these are assumed to be short and efficient. However, our findings from mesocosm experiments on artificial upwelling in the oligotrophic ocean disagree with this classical food web model. Here, diatoms did not reduce trophic length and instead impaired the transfer of primary production to crustacean grazers and small pelagic fish. The diatom-driven decrease in trophic efficiency was likely mediated by changes in nutritional value for the copepod grazers. Whilst diatoms benefitted the availability of essential fatty acids, they also caused unfavorable elemental compositions via high carbon-to-nitrogen ratios (i.e. low protein content) to which the grazers were unable to adapt. This nutritional imbalance for grazers was most pronounced in systems optimized for CO2 uptake through carbon-to-nitrogen ratios well beyond Redfield. A simultaneous enhancement of fisheries production and carbon sequestration via artificial upwelling may thus be difficult to achieve given their opposing stoichiometric constraints. Our study suggest that food quality can be more critical than quantity to maximize food web productivity during shorter-term fertilization of the oligotrophic ocean.

Respiration rate scales inversely with sinking speed of settling marine aggregates.

Sinking marine aggregates have been studied for a long time to understand their role in carbon sequestration. Traditionally, sinking speed and respiration rates have been treated as independent variables, but two recent papers suggest that there is a connection albeit in contrasting directions. Here we collected recently formed (<2 days old) aggregates from sediment traps mounted underneath mesocosms during two different experiments. The mesocosms were moored off Gran Canaria, Spain (~ 27.9 N; 15.4 E) in a coastal, sub-tropical and oligotrophic ecosystem. We determined the respiration rates of organisms (mainly heterotrophic prokaryotes) attached to aggregates sinking at different velocities. The average respiration rate of fast sinking aggregates (>100 m d-1) was 0.12 d-1 ± 0.08 d-1 (SD). Slower sinking aggregates (<50 m d-1) had on average higher (p <0.001) and more variable respiration rates (average 0.31 d-1 ± 0.16 d-1, SD). There was evidence that slower sinking aggregates had higher porosity than fast sinking aggregates, and we hypothesize that higher porosity increase the settlement area for bacteria and the respiration rate. These findings provide insights into the efficiency of the biological carbon pump and help resolve the apparent discrepancy in the recent studies of the correlation between respiration and sinking speed.

Brownification affects phytoplankton community composition but not primary productivity in eutrophic coastal waters: A mesocosm experiment in the Baltic Sea.

Climate change is projected to cause brownification of some coastal seas due to increased runoff of terrestrially derived organic matter. We carried out a mesocosm experiment (15 d) to test the effect of this on the planktonic ecosystem expecting reduced primary production and shifts in the phytoplankton community composition. The experiment was set up in 2.2 m3 mesocosm bags using four treatments, each with three replicates: control (Contr) without any manipulation, organic carbon additive HuminFeed (Hum; 2 mg L-1), inorganic nutrients (Nutr; 5.7 µM NH4 and 0.65 µM PO4), and combined Nutr and Hum (Nutr + Hum) additions. Measured variables included organic and inorganic nutrient pools, chlorophyll a (Chla), primary and bacterial production and particle counts by flow cytometry. The bags with added inorganic nutrients developed a phytoplankton bloom that depleted inorganic N at day 6, followed by a rapid decline in Chla. Brownification did not reduce primary production at the tested concentration. Bacterial production was lowest in the Contr, but similar in the three treatments receiving additions likely due to increased carbon available for heterotrophic bacteria. Picoeukaryotes clearly benefited by brownification after inorganic N depletion, which could be due to more effective nutrient recycling, nutrient affinity, light absorption, or alternatively lower grazing pressure. In conclusion, brownification shifted the phytoplankton community composition towards smaller species with potential effects on carbon fluxes, such as sinking rates and export to the sea floor.

Dataset from a mesocosm experiment on brownification in the Baltic Sea.

Climate change is projected to cause brownification of some coastal seas due to increased runoff of terrestrially derived organic matter. We carried out a mesocosm experiment over 15 days to test the effect of this on the planktonic ecosystem. The experiment was set up in 2.2 m3 plastic bags moored outside the Tvärminne Zoological Station at the SW coast of Finland. We used four treatments, each with three replicates: control (Contr) without any manipulation; addition of a commercially available organic carbon additive called HuminFeed (Hum; 2 mg L-1); addition of inorganic nutrients (Nutr; 5.7 µM NH4 and 0.65µM PO4); and a final treatment of combined Nutr and Hum (Nutr+Hum) additions. Water samples were taken daily, and measured variables included water transparency, organic and inorganic nutrient pools, chlorophyll a (Chla), primary and bacterial production and particle counts by flow cytometry.

Basic Methods for Isolating and Culturing Microalgae.

In this chapter, I go through the very basic methods for isolating and setting up a microalgae culture. Isolation can be done from local algal strains with a micropipette and microscope. Determining algal growth is important and can be done by counting cells under a microscope or by determining the change in dry weight (DW).

Large Scale Cultivation of Microalgae: Open and Closed Systems.

There are two main approaches for cultivating microalgae on a large scale: open or closed cultivation. The main difference between open and closed systems is related to how they operate (e.g., cooling and gas exchange), vulnerability for outside influence (e.g., rainwater and introduction of unwanted species), and costs for building and operating the system. In this chapter we introduce the main cultivation technologies and discuss their main advantages and disadvantages when cultivating microalgae.

Measurement of Fluorescence for Monitoring Algal Growth and Health.

Measuring fluorescence is a noninvasive, inexpensive, and quick way of determining biomass concentration and health of the algae. Fluorescence is generally positively correlated with chlorophyll a and can as such be used as a proxy for biomass. In addition, the proportion variable fluorescence of maximal fluorescence is a measure of photochemical efficiency, which is affected by stress in a very early stage and can as such be used as a proxy for algal health.

Total Nitrogen Determination by a Spectrophotometric Method.

Being able to measure total nitrogen (TN) is important for following the nitrogen budget. In this chapter, we present the spectrophotometric method we use for determining TN. The method relies on oxidation and reduction steps, involving persulfate digestion of nitrogen compounds into nitrate followed by spectrophotometric determination.

Determining Inorganic and Organic Nitrogen.

Nitrogen (N) is one of the key nutrients for algal growth and is an integral part of many cellular components, for example in proteins. Being able to determine the inorganic and organic pools of N is consequently critical for algal cultivation. In this chapter we present the methods we use for determining dissolved inorganic nitrogen (DIN), dissolved organic nitrogen (DON), and particulate organic nitrogen (PON). The methods presented here for DIN rely on colorimetric methods and those of DON and PON on filtration and high temperature catalytic oxidation.

Determining Dissolved and Biogenic Silica.

Most algae do not use silicon in any form with one notable exception, diatoms. Silicon is a major constituent of diatoms. Diatoms are characterized by high growth rates and are often one of the key groups in forming algal blooms in natural waters, and as such it is an interesting group for cultivation. In this chapter we present methods for determining dissolved silica (DSi) and biogenic silica (BSi), oxide forms of silicon, based on colorimetric methods. BSi is determined after filtration and alkaline digestion.

Determining Inorganic and Organic Phosphorus.

Phosphorus (P) is a macronutrient for all microalgal species, and the main form of uptake is orthophosphate (PO4). In this chapter we present a colorimetric method for determining the PO4 concentration and dissolved organic phosphorus (DOP) based on total phosphorus (TP) measurements. We also describe a method for determining particulate organic phosphorus (POP) based on the same principles.

Determining Inorganic and Organic Carbon.

Carbon is the element which makes up the major fraction of lipids and carbohydrates, which could be used for making biofuel. It is therefore important to provide enough carbon and also follow the flow into particulate organic carbon and potential loss to dissolved organic forms of carbon. Here we present methods for determining dissolved inorganic carbon, dissolved organic carbon, and particulate organic carbon.

Correction to: Determining Inorganic and Organic Phosphorus.

There are three corrections for this chapter. On page 90, 12th line from top should read as '(see step 6) with ultrapure water (e.g., 0.1 ml' instead of '(see step 6) with ultrapure water (e.g., 1 ml'.

Correction to: Determining Inorganic and Organic Nitrogen.

There are three corrections for this chapter. On page 74, 10th line from the bottom in Sect. 3.1, point 3 appears incorrect as '85 ml ultrapure water and 8.5 ml' instead of '85 ml ultrapure water and 8.4 ml'.

Light and Primary Production Shape Bacterial Activity and Community Composition of Aerobic Anoxygenic Phototrophic Bacteria in a Microcosm Experiment.

Phytoplankton is a key component of aquatic microbial communities, and metabolic coupling between phytoplankton and bacteria determines the fate of dissolved organic carbon (DOC). Yet, the impact of primary production on bacterial activity and community composition remains largely unknown, as, for example, in the case of aerobic anoxygenic phototrophic (AAP) bacteria that utilize both phytoplankton-derived DOC and light as energy sources. Here, we studied how reduction of primary production in a natural freshwater community affects the bacterial community composition and its activity, focusing primarily on AAP bacteria. The bacterial respiration rate was the lowest when photosynthesis was reduced by direct inhibition of photosystem II and the highest in ambient light condition with no photosynthesis inhibition, suggesting that it was limited by carbon availability. However, bacterial assimilation rates of leucine and glucose were unaffected, indicating that increased bacterial growth efficiency (e.g., due to photoheterotrophy) can help to maintain overall bacterial production when low primary production limits DOC availability. Bacterial community composition was tightly linked to light intensity, mainly due to the increased relative abundance of light-dependent AAP bacteria. This notion shows that changes in bacterial community composition are not necessarily reflected by changes in bacterial production or growth and vice versa. Moreover, we demonstrated for the first time that light can directly affect bacterial community composition, a topic which has been neglected in studies of phytoplankton-bacteria interactions.IMPORTANCE Metabolic coupling between phytoplankton and bacteria determines the fate of dissolved organic carbon in aquatic environments, and yet how changes in the rate of primary production affect the bacterial activity and community composition remains understudied. Here, we experimentally limited the rate of primary production either by lowering light intensity or by adding a photosynthesis inhibitor. The induced decrease had a greater influence on bacterial respiration than on bacterial production and growth rate, especially at an optimal light intensity. This suggests that changes in primary production drive bacterial activity, but the effect on carbon flow may be mitigated by increased bacterial growth efficiencies, especially of light-dependent AAP bacteria. Bacterial activities were independent of changes in bacterial community composition, which were driven by light availability and AAP bacteria. This direct effect of light on composition of bacterial communities has not been documented previously.

Organic matter export to the seafloor in the Baltic Sea: Drivers of change and future projections.

The impact of environmental change and anthropogenic stressors on coastal marine systems will strongly depend on changes in the magnitude and composition of organic matter exported from the water column to the seafloor. Knowledge of vertical export in the Baltic Sea is synthesised to illustrate how organic matter deposition will respond to climate warming, climate-related changes in freshwater runoff, and ocean acidification. Pelagic heterotrophic processes are suggested to become more important in a future warmer climate, with negative feedbacks to organic matter deposition to the seafloor. This is an important step towards improved oxygen conditions in the near-bottom layer that will reduce the release of inorganic nutrients from the sediment and hence counteract further eutrophication. The evaluation of these processes in ecosystem models, validated by field observations, will significantly advance the understanding of the system's response to environmental change and will improve the use of such models in management of coastal areas.

Contrasting seasonality in optical-biogeochemical properties of the Baltic Sea.

Optical-biogeochemical relationships of particulate and dissolved organic matter are presented in support of remote sensing of the Baltic Sea pelagic. This system exhibits strong seasonality in phytoplankton community composition and wide gradients of chromophoric dissolved organic matter (CDOM), properties which are poorly handled by existing remote sensing algorithms. Absorption and scattering properties of particulate matter reflected the seasonality in biological (phytoplankton succession) and physical (thermal stratification) processes. Inherent optical properties showed much wider variability when normalized to the chlorophyll-a concentration compared to normalization to either total suspended matter dry weight or particulate organic carbon. The particle population had the largest optical variability in summer and was dominated by organic matter in both seasons. The geographic variability of CDOM and relationships with dissolved organic carbon (DOC) are also presented. CDOM dominated light absorption at blue wavelengths, contributing 81% (median) of the absorption by all water constituents at 400 nm and 63% at 442 nm. Consequentially, 90% of water-leaving radiance at 412 nm originated from a layer (z90) no deeper than approximately 1.0 m. With water increasingly attenuating light at longer wavelengths, a green peak in light penetration and reflectance is always present in these waters, with z90 up to 3.0-3.5 m depth, whereas z90 only exceeds 5 m at biomass < 5 mg Chla m-3. High absorption combined with a weakly scattering particle population (despite median phytoplankton biomass of 14.1 and 4.3 mg Chla m-3 in spring and summer samples, respectively), characterize this sea as a dark water body for which dedicated or exceptionally robust remote sensing techniques are required. Seasonal and regional optical-biogeochemical models, data distributions, and an extensive set of simulated remote-sensing reflectance spectra for testing of remote sensing algorithms are provided as supplementary data.

Interaction Effects of Light, Temperature and Nutrient Limitations (N, P and Si) on Growth, Stoichiometry and Photosynthetic Parameters of the Cold-Water Diatom Chaetoceros wighamii.

Light (20-450 µmol photons m(-2) s(-1)), temperature (3-11 °C) and inorganic nutrient composition (nutrient replete and N, P and Si limitation) were manipulated to study their combined influence on growth, stoichiometry (C:N:P:Chl a) and primary production of the cold water diatom Chaetoceros wighamii. During exponential growth, the maximum growth rate (~0.8 d(-1)) was observed at high temperature and light; at 3 °C the growth rate was ~30% lower under similar light conditions. The interaction effect of light and temperature were clearly visible from growth and cellular stoichiometry. The average C:N:P molar ratio was 80:13:1 during exponential growth, but the range, due to different light acclimation, was widest at the lowest temperature, reaching very low C:P (~50) and N:P ratios (~8) at low light and temperature. The C:Chl a ratio had also a wider range at the lowest temperature during exponential growth, ranging 16-48 (weight ratio) at 3 °C compared with 17-33 at 11 °C. During exponential growth, there was no clear trend in the Chl a normalized, initial slope (α*) of the photosynthesis-irradiance (PE) curve, but the maximum photosynthetic production (P(m)) was highest for cultures acclimated to the highest light and temperature. During the stationary growth phase, the stoichiometric relationship depended on the limiting nutrient, but with generally increasing C:N:P ratio. The average photosynthetic quotient (PQ) during exponential growth was 1.26 but decreased to <1 under nutrient and light limitation, probably due to photorespiration. The results clearly demonstrate that there are interaction effects between light, temperature and nutrient limitation, and the data suggests greater variability of key parameters at low temperature. Understanding these dynamics will be important for improving models of aquatic primary production and biogeochemical cycles in a warming climate.

Nile Red staining of phytoplankton neutral lipids: species-specific fluorescence kinetics in various solvents.

Nile Red (NR) staining potentially offers a simple method for monitoring lipid accumulation in microalgal cultivation. However, variable staining efficiencies and methods have been reported. The effect of dimethyl sulfoxide (DMSO), ethylene glycol (EG) and glycerol on NR penetration with four different phytoplankton species representing different taxonomical groups was studied. Treatment with the solvents enhanced the NR fluorescence of the diatom Phaeodactylum tricornutum during kinetic fluorescence measurements, but high concentrations of solvents were needed. None of the solvents improved NR staining of the green alga Chlorella pyrenoidosa and Scenedesmus obliquus, which are known to be difficult to stain due to their thick and rigid cell walls. The naked Isochrysis sp. cells stained best without solvents. The results confirm that NR staining protocol needs to be optimized for each species.