How to reach optimal estimates of confidence intervals in microscopic counting of phytoplankton?

Abstract Present practices in the microscopic counting of phytoplankton to estimate the reliability of results rely on the assumption of a random distribution of taxa in sample preparations. In contrast to that and in agreement with the literature, we show that aggregated distribution is common and can lead to over-optimistic confidence intervals, if estimated according to the shortcut procedure of Lund et al. based on the number of counted cells. We found a good linear correlation between the distribution independent confidence intervals for medians and those for parametric statistics so that 95% confidence intervals can be approximated by using a correction factor of 1.4. Instead, the rec…

Development of picoplankton during natural and enhanced mixing under late-winter ice

We studied the development of autotrophic picophytoplankton and heterotrophic bacterioplankton during the tran- sition from winter ice cover to open water under natural and manipulated mixing conditions in eutrophic Lake Vesijarvi. During the melting of the snow and ice cover, a convection layer developed which eventually met the che- mocline at the interface between the oxic and anoxic water masses. However, in the years with mechanically enhanced mixing, the whole water column remained well oxygenated and the deepening of penetrative convection was facilitated. Stochastic variations in weather, primarily the thickness of the snow cover, likely determined the timing of picophytoplankton gr…

Metabolic plasticity of mixotrophic algae is key for their persistence in browning environments

Light availability is the main regulator of primary production, shaping photosynthetic communities and their production of ecologically important biomolecules. In freshwater ecosystems, increasing dissolved organic carbon concentrations, commonly known as browning, leads to lower light availability and the proliferation of mixotrophic phytoplankton. Here, a mixotrophic algal species (Cryptomonas sp.) was grown under five increasing dissolved organic carbon concentrations to uncover the plastic responses behind the success of mixotrophs in browning environments and their effect in the availability of nutritionally important biomolecules. In addition to the browning treatments, phototrophic, …

A European Multi Lake Survey dataset of environmental variables, phytoplankton pigments and cyanotoxins

Under ongoing climate change and increasing anthropogenic activity, which continuously challenge ecosystem resilience, an in-depth understanding of ecological processes is urgently needed. Lakes, as providers of numerous ecosystem services, face multiple stressors that threaten their functioning. Harmful cyanobacterial blooms are a persistent problem resulting from nutrient pollution and climate-change induced stressors, like poor transparency, increased water temperature and enhanced stratification. Consistency in data collection and analysis methods is necessary to achieve fully comparable datasets and for statistical validity, avoiding issues linked to disparate data sources. The Europea…

Variation in ω-3 and ω-6 Polyunsaturated Fatty Acids Produced by Different Phytoplankton Taxa at Early and Late Growth Phase

Phytoplankton synthesizes essential ω-3 and ω-6 polyunsaturated fatty acids (PUFA) for consumers in the aquatic food webs. Only certain phytoplankton taxa can synthesize eicosapentaenoic (EPA; 20:5ω3) and docosahexaenoic acid (DHA; 22:6ω3), whereas all phytoplankton taxa can synthesize shorter-chain ω-3 and ω-6 PUFA. Here, we experimentally studied how the proportion, concentration (per DW and cell-specific), and production (µg FA L-1 day-1) of ω-3 and ω-6 PUFA varied among six different phytoplankton main groups (16 freshwater strains) and between exponential and stationary growth phase. EPA and DHA concentrations, as dry weight, were similar among cryptophytes and diatoms. However, Crypto…

Sample Preservation, DNA or RNA Extraction and Data Analysis for High-Throughput Phytoplankton Community Sequencing

Phytoplankton is the basis for aquatic food webs and mirrors the water quality. Conventionally, phytoplankton analysis has been done using time consuming and partly subjective microscopic observations, but next generation sequencing (NGS) technologies provide promising potential for rapid automated examination of environmental samples. Because many phytoplankton species have tough cell walls, methods for cell lysis and DNA or RNA isolation need to be efficient to allow unbiased nucleic acid retrieval. Here, we analyzed how two phytoplankton preservation methods, three commercial DNA extraction kits and their improvements, three RNA extraction methods, and two data analysis procedures affect…

Stratification strength and light climate explain variation in chlorophyll a at the continental scale in a European multilake survey in a heatwave summer

The authors acknowledge COST Action ES 1105 "CYANOCOST Cyanobacterial blooms and toxins in water resources: Occurrence impacts and management" and COST Action Global Change Biology ES 1201 NETLAKE -Networking Lake Observatories in Europe" for contributing to this study through networking and knowledge sharing with European experts in the field. We acknowledge the members of the Global Lake Ecological Observatory Network (GLEON) for their collaborative spirit and enthusiasm that inspired the grassroots effort of the EMLS. E.M. was supported by a grant from the Swiss State Secretariat for Education, Research and Innovation to Bas Ibelings and by supplementary funding from University of Geneva…

Effects of mechanical mixing on lake water quality, with special emphasis on under-ice phytoplankton

Successful aerobic bioremediation of groundwater contaminated with higher chlorinated phenols by indigenous degrader bacteria

The xenobiotic priority pollutant pentachlorophenol has been used as a timber preservative in a polychlorophenol bulk synthesis product containing also tetrachlorophenol and trichlorophenol. Highly soluble chlorophenol salts have leaked into groundwater, causing severe contamination of large aquifers. Natural attenuation of higher-chlorinated phenols (HCPs: pentachlorophenol + tetrachlorophenol) at historically polluted sites has been inefficient, but a 4-year full scale in situ biostimulation of a chlorophenol-contaminated aquifer by circulation and re-infiltration of aerated groundwater was remarkably successful: pentachlorophenol decreased from 400 μg L−1 to <1 μg L−1 and tetrachlorophen…

Interannual variability of circulation under spring ice in a boreal lake

A small range (, 1uC) of under-ice water temperature is shown to result in remarkably different circulation regimes under spring ice in a deep, oligotrophic boreal lake. With the water column at , 4uC, melting of snow led to deepening vertical convection before ice break and a final depth of convection inversely correlated with earlier deep-water temperature. We attribute that to the nonlinear dependence of water density on temperature, albeit further affected by stochastic weather factors. In four of nine study years, convection led to complete under-ice overturn of the lake, indicating that this may not be uncommon in similar lakes with steep topography. River inflow and more intensive wa…

Pumping of epilimnetic water into hypolimnion improves oxygen but not necessarily nutrient conditions in a lake recovering from eutrophication

To mitigate deep water oxygen depletion and its consequences, epilimnetic water was pumped into deep water of a eutrophic, 26 km 2 subbasin of Lake Vesijarvi, Finland. In winter, the mechanical mixing largely eliminated vertical differences in temperature, oxygen, and nutrients. Although ice cover prevented oxygen flux from the atmosphere, the high proportion of shallows in the basin with high oxygen concentration facilitated the avoidance of hypoxia by mixing. Despite the disappearance of anoxia in deep water, the volume-weighted mean concentrations of nutrients were not affected. In summer, the introduction of epilimnetic water into the hypolimnion improved oxygen conditions, but anoxia o…

Regular build-up of the spring phytoplankton maximum before ice-break in a boreal lake

The development of phytoplankton biomass and composition in a eutrophic boreal lake was studied during the evolution of under-ice convection in spring. The results from 8 yr showed that, within a few weeks before ice-break, phytoplankton biomass regularly increased by up to two or three orders of magnitude, reaching or exceeding the biomass in summer. Accordingly, this may be the most significant single annual phytoplankton episode in the lake. The development of phytoplankton was closely coupled with that of convection created by solar radiation at water temperatures < 4°C. In addition to vertical convection which keeps phytoplankton suspended, there was also horizontal convection which tr…

Rapid Quantification of Microalgae Growth with Hyperspectral Camera and Vegetation Indices

Spectral cameras are traditionally used in remote sensing of microalgae, but increasingly also in laboratory-scale applications, to study and monitor algae biomass in cultures. Practical and cost-efficient protocols for collecting and analyzing hyperspectral data are currently needed. The purpose of this study was to test a commercial, easy-to-use hyperspectral camera to monitor the growth of different algae strains in liquid samples. Indices calculated from wavebands from transmission imaging were compared against algae abundance and wet biomass obtained from an electronic cell counter, chlorophyll a concentration, and chlorophyll fluorescence. A ratio of selected wavebands containing near…

Consistency of Targeted Metatranscriptomics and Morphological Characterization of Phytoplankton Communities

The composition of phytoplankton community is the basis for environmental monitoring and assessment of the ecological status of aquatic ecosystems. Community composition studies of phytoplankton have been based on time-consuming and expertise-demanding light microscopy analyses. Molecular methods have the potential to replace microscopy, but the high copy number variation of ribosomal genes and the lack of universal primers for simultaneous amplification of prokaryotic and eukaryotic genes complicate data interpretation. In this study, we used our previously developed directional primer-independent high-throughput sequencing (HTS) approach to analyze 16S and 18S rRNA community structures. C…

Temperature, phosphorus and species composition will all influence phytoplankton production and content of polyunsaturated fatty acids

Temperature increases driven by climate change are expected to decrease the availability of polyunsaturated fatty acids in lakes worldwide. Nevertheless, a comprehensive understanding of the joint effects of lake trophic status, nutrient dynamics and warming on the availability of these biomolecules is lacking. Here, we conducted a laboratory experiment to study how warming (18–23°C) interacts with phosphorus (0.65–2.58 μM) to affect phytoplankton growth and their production of polyunsaturated fatty acids. We included 10 species belonging to the groups diatoms, golden algae, cyanobacteria, green algae, cryptophytes and dinoflagellates. Our results show that both temperature and phosphorus w…

Calibration of in situ chlorophyll fluorometers for organic matter

AbstractOrganic matter (OM) other than living phytoplankton is known to affect fluorometric in situ assessments of chlorophyll in lakes. For this reason, calibrating fluorometric measurements for OM error is important. In this study, chlorophyll (Chl) fluorescence was measured in situ in multiple Finnish lakes using two sondes equipped with Chl fluorometers (ex.470/em.650–700 nm). OM absorbance (A420) was measured from water samples, and one of the two sondes was also equipped with in situ fluorometer for OM (ex.350/em.430 nm). The sonde with Chl and OM fluorometers was also deployed continuously on an automated water quality monitoring station on Lake Konnevesi. For data from multiple lake…

Assessment of microalgae species, biomass, and distribution from spectral images using a convolution neural network

AbstractEffective monitoring of microalgae growth is crucial for environmental observation, while the applications of this monitoring could also be expanded to commercial and research-focused microalgae cultivation. Currently, the distinctive optical properties of different microalgae groups are targeted for monitoring. Since different microalgae can grow together, their spectral signals are mixed with ambient properties, making estimations of species biomasses a challenging task. In this study, we cultured five different microalgae and monitored their growth with a mobile spectral imager in three separate experiments. We trained and validated a one-dimensional convolution neural network by…

Comparison of epifluorescence microscopy and flow cytometry in counting freshwater picophytoplankton

The smaller the phytoplankton, the greater effort is required to distinguish individual cells by optics-based methods. Flow cytometry is widely applied in marine picophytoplankton research, but in freshwater research its role has remained minor. We compared epifluorescence microscopy and flow cytometry in assessing the composition, abundance and cell sizes of autofluorescent picophytoplankton in epilimnia of 46 Finnish lakes. Phycocyaninrich picocyanobacteria were the most dominant. The two methods yielded comparable total picophytoplankton abundances, but the determination of cell sizes, and thus total biomasses, were on average an order of magnitude higher in the microscopy results. Howev…

Temperature effects explain continental scale distribution of cyanobacterial toxins

Insight into how environmental change determines the production and distribution of cyanobacterial toxins is necessary for risk assessment. Management guidelines currently focus on hepatotoxins (microcystins). Increasing attention is given to other classes, such as neurotoxins (e.g., anatoxin-a) and cytotoxins (e.g., cylindrospermopsin) due to their potency. Most studies examine the relationship between individual toxin variants and environmental factors, such as nutrients, temperature and light. In summer 2015, we collected samples across Europe to investigate the effect of nutrient and temperature gradients on the variability of toxin production at a continental scale. Direct and indirect…

Particle balance and return loops for microplastics in a tertiary-level wastewater treatment plant.

Highlights: Dewatering by centrifugation was a step that removed a high number of MPs from the sludge. Sludge retained especially the fibrous microplactics. Reject water transported microplastics inside a wastewater treatment plant. Disc filter-based tertiary treatment ensured removal of 99% of microplastics in wastewater. Microplastics (MPs) from households, stormwater, and various industries are transported to wastewater treatment plants (WWTPs), where a high proportion of them are captured before discharging their residuals to watersheds. Although recent studies have indicated that the removed MPs are mainly retained in wastewater sludge, sludge treatment processes have gained less atten…

Optical Monitoring of Microplastics Filtrated from Wastewater Sludge and Suspended in Ethanol.

The abundance of microplastics (MPs) in the atmosphere, on land, and especially in water bodies is well acknowledged. In this study, we establish an optical method based on three different techniques, namely, specular reflection to probe the medium, transmission spectroscopy measurements for the detection and identification, and a speckle pattern for monitoring the sedimentation of MPs filtrated from wastewater sludge and suspended in ethanol. We used first Raman measurements to estimate the presence and types of different MPs in wastewater sludge samples. We also used microscopy to identify the shapes of the main MPs. This allowed us to create a teaching set of samples to be characterized …

Original data for article: Particle balance and return loops for microplastics in a tertiary-level wastewater treatment plant

Microplastics (MPs) from households, stormwaters and various industries are transported to wastewater treatment plants (WWTPs) where a high proportion of them are captured before discharging their residuals to watersheds. Although recent studies have indicated that the removed MPs are mainly retained in wastewater sludge, sludge treatment processes have gained less attention in MP research compared to water streams at primary, secondary and tertiary treatments. In this study, we sampled twelve different phases in a tertiary-level municipal WWTP in Central Finland. Our results showed that compared to plant influent load, three times higher number of MPs was circulating via reject water from …

Original data for article: Comparison of epifluorescence microscopy and flow cytometry in counting freshwater picophytoplankton

The dataset is divided into four subfolders: 1) "SEM experiment data" contains Scanning Electron Microscopy data, epifluorescence microscopy data and flow cytometry data of cultured Synechococcus, Chroococcus and Snowella 2) "raw data" contains epifluorescence microscopy and flow cytometry data of picophytoplankton from Finnish lakes. This has two sub folders "flow cytometry raw" and "microscopy raw" 3) "flow cytometry calibration data" contains data for cell size calibration with latex beads and volumetric calibration for the flow cytometer 4) "processed flow and microscopy data" contains excel workbooks for the figures shown in the manuscipt

Research data of article: "Practical calibration of in situ chlorophyll fluorometers for humic lakes"