David C. Sigee

Using FTIR spectroscopy for rapid determination of lipid accumulation in response to nitrogen limitation in freshwater microalgae

In this study Fourier transform infrared micro-spectroscopy (FTIR) was used to determine lipid and carbohydrate content over time in the freshwater microalgae Chlamydomonas reinhardtii and Scenedesmus subspicatus grown in batch culture in limiting concentrations of nitrogen (N). Both algae exhibited restricted cell division and increased cell size following N-limitation. FTIR spectra of cells in N-limited media showed increasing lipid:amide I and carbohydrate:amide I ratios over time. The use of lipid- and starch-staining dyes confirmed that the observed ratio changes were due to increased lipid and carbohydrate synthesis. These results demonstrate rapid metabolic responses of C. reinhardtii and S. subspicatus to changing nutrient availability, and indicate the efficiency of FTIR as a reliable method for high-throughput determination of lipid induction.

Freshwater algae : identification and use as bioindicators

Preface. Copyright Acknowledgements. 1 Introduction to Freshwater Algae. 1.1 General introduction. 1.1.1 Algae - An overview. 1.1.2 Algae as primary producers. 1.1.3 Freshwater environments. 1.1.4 Planktonic and benthic algae. 1.1.5 Size and shape. 1.2 Taxonomic variation - the major groups of algae. 1.2.1 Microscopical appearance. 1.2.2 Biochemistry and cell structure. 1.2.3 Molecular characteristics and identification. 1.3 Blue-green algae. 1.3.1 Cytology. 1.3.2 Morphological and taxonomic diversity. 1.3.3 Ecology. 1.3.4 Blue-green algae as bio-indicators. 1.4 Green algae. 1.4.1 Cytology. 1.4.2 Morphological diversity. 1.4.3 Ecology. 1.4.4 Green algae as bioindicators. 1.5 Euglenoids. 1.5.1 Cytology. 1.5.2 Morphological diversity. 1.5.3 Ecology. 1.5.4 Euglenoids as bioindicators. 1.6 Yellow-green algae. 1.6.1 Cytology. 1.6.2 Morphological diversity. 1.6.3 Ecology. 1.6.4 Yellow-green algae as bioindicators. 1.7 Dinoflagellates. 1.7.1 Cytology. 1.7.2 Morphological diversity. 1.7.3 Ecology. 1.8 Cryptomonads. 1.8.1 Cytology. 1.8.2 Comparison with euglenoid algae. 1.8.3 Biodiversity. 1.8.4 Ecology. 1.8.5 Cryptomonads as bioindicators. 1.9 Chrysophytes. 1.9.1 Cytology. 1.9.2 Morphological diversity. 1.9.3 Ecology. 1.9.4 Chrysophytes as bioindicators. 1.10 Diatoms. 1.10.1 Cytology. 1.10.2 Morphological diversity. 1.10.3 Ecology. 1.10.4 Diatoms as bioindicators. 1.11 Red algae. 1.12 Brown algae. 2 Sampling, Biomass Estimation and Counts of FreshwaterAlgae. A Planktonic Algae. 2.1 Protocol for collection. 2.1.1 Standing water phytoplankton. 2.1.1 River phytoplankton. 2.2 Mode of collection. 2.2.1 Phytoplankton trawl net. 2.2.2 Volume samplers. 2.2.3 Integrated sampling. 2.2.4. Sediment traps. 2.3 Phytoplankton biomass. 2.3.1 Turbidity. 2.3.2 Dry weight and ash-free dry weight. 2.3.3 Pigment concentrations. 2.4 Flow cytometry: automated analysis of phytoplanktonpopulations. 2.5 Microscope counts of species populations. 2.5.1 Sample preservation and processing. 2.5.2 Species counts. 2.5.3 Conversion of species counts to biovolumes. 2.5.4 Chemical cleaning of diatoms. 2.6 Diversity within single-species populations. 2.6.1 Molecular analysis. 2.6.2 Analytical microscopical techniques. B Non-Planktonic Algae. 2.7 Deep water benthic algae. 2.7.1 Benthic-pelagic coupling. 2.7.2 Benthic algae and sediment stability. 2.7.3 Invertebrate grazing of benthic algae. 2.8 Shallow water communities. 2.8.1 Substrate. 2.8.2 Algal communities. 2.9 Algal biofilms. 2.9.1 Mucilaginous biofilms. 2.9.2 Biomass. 2.9.3 Taxonomic composition. 2.9.4 Matrix structure. 2.10 Periphyton - algal mats. 2.10.1 Inorganic substratum. 2.10.2 Plant surfaces. 3 Algae as bioindicators. 3.1 Bioindicators and water quality. 3.1.1 Biomarkers and bioindicators. 3.1.2 Characteristics of bioindicators. 3.1.3 Biological monitoring versus chemical measurements. 3.1.4 Monitoring water quality: objectives. 3.2 Lakes. 3.2.1 Contemporary planktonic and attached algae asbioindicators. 3.2.2 Fossil algae as bioindicators: lake sediment analysis. 3.2.3 Water quality parameters: inorganic and organic nutrients,acidity and heavy metals. 3.3 Wetlands. 3.4 Rivers. 3.4.1 The periphyton community. 3.4.2 River diatoms. 3.4.3 Evaluation of the diatom community. 3.4.4 Human impacts and diatom indices. 3.4.5 Calculation of diatom indices. 3.4.6 Practical applications of diatom indices. 3.5 Estuaries. 3.5.1 Ecosystem complexity. 3.5.2 Algae as estuarine bioindicators. 4 A Key to the More Frequently Occurring FreshwaterAlgae. 4.1 Introduction to the key. 4.1.1 Using the key. 4.1.2 Morphological groupings. 4.2 Key to the main genera and species. 4.3 List of algae included and their occurrence in the key. 4.4 Algal identification: bibliography. Glossary. References. Index.

Impact of phosphorus quota and growth phase on carbon allocation in Chlamydomonas reinhardtii: An FTIR microspectroscopy study

Batch cultures of Chlamydomonas reinhardtii were used to study carbon allocation in relation to growth phase and phosphorus availability. Cultures were grown at initial phosphorus (PO4-P) concentrations of 500 µg l−1 (high-P) and 50 µg l−1 (low-P). Cellular carbon allocation was monitored using Fourier transform infrared (FTIR) microspectroscopy with the ratio of the band intensities at 1736 cm−1 (lipid) and the 1180–950 cm−1 region (carbohydrate) to 1652 cm−1 (amide I) used as an index of changing carbon balance. Cellular phosphorus concentrations (P quota) were measured by energy dispersive X-ray microanalysis (EDXRMA). Both treatments entered stationary phase on day 18. Increased cell counts in the high-P treatment (max. 3.0 × 106 cells ml−1 at stationary phase) led to a rapid decrease in external P availability to <20 µg l−1 during early log phase, with a subsequent decrease in P quota from 0.5% to <0.1% DW. The fall of P quota to <0.1% led to an increase in the lipid/protein ratio (0.13 to 0.23) and carbohydrate/protein ratio (0.37 to 1.57), with ratios increasing further (lipid:protein 1.85; carbohydrate:protein 2.77) in late stationary phase. In the low-P treatment external P concentrations (<20 µg l−1 from day 1) restricted population growth (max. 0.75 × 106 cells ml−1 at stationary phase). P quotas fell to <0.1% in early log phase, with the carbohydrate/protein ratio increasing from 0.15 to 3.7 and remaining high into stationary phase while the lipid/protein ratio increased from 0.2 to 1.2. In both treatments increasing synthesis of lipid and carbohydrate storage products resulted in an increased cell volume. Transfer of P-deficient cells (late stationary) to fresh media led to a rapid stimulation of growth, a rapid reduction in lipid/protein and carbohydrate/protein ratios, and decreased cell volumes.

Ciliate populations and metals in an activated-sludge plant

Abstract Twenty-nine species of ciliate protozoa were recorded in samples of activated sludge collected monthly from tanks at Davyhulme Sewage Works, Manchester, U.K., over a two-year period (1991–1992). The most frequent species present included Aspidisca cicada, Chilodonella uncinata, Vorticella convallaria and Vorticella microstoma . The total ciliate population showed clear seasonal variation, with highest counts between February and August. Only two species showed significant correlation with ambient temperature. Species diversity had little variation, with 8–13 ciliate species being identified in most samples. Significant positive interspecies correlations were noted for ten species, but were not consistent for both years. Mean concentrations of the metals Fe, Ni, Zn, Cu and Cr in the liquid phase of activated sludge varied considerably over the two annual sampling periods, with no seasonal pattern. Major ciliates present were able to tolerate high concentrations of contaminant metals, including Fe (> 2000 ppb), Zn (> 500 ppb), Cu (> 60 ppb) and Cr (100 ppb). Specific positive and negative ciliate species—metal correlations were observed in each annual sampling period, but were not consistent for both years. The lack of consistent correlations over the two-year sampling period between populations of individual protozoan species and single biological (other species) or physicochemical (temperature, metal concentrations) factors is in accordance with a complex (multifactorial) determination of protozoan populations in activated sludge.

Structural DNA and genetically active DNA in dinoflagellate chromosomes

High resolution electron microscope autoradiographs of [3H]adenine incorporation in the dinoflagellate Prorocentrum micans suggest that RNA transcription occurs on extrachromosomal DNA filaments, but not on DNA in the main body of the chromosome. This genetically inactive DNA has an important role, however, in stabilising chromosome structure by its association with protein matrix. Evidence for the importance of this molecular association (which is probably cation-mediated) is provided by alkaline buffer extraction of the protein matrix in chromosomes of Amphidinium carterae , leading to complete destabilisation of the DNA framework. The clear distinction between structural DNA and genetically active DNA in these chromosomes provides a marked contrast to normal eucaryote chromosomes. This distinction is related to the occurrence of high DNA values in these organisms, and the evolutionary status of the dinoflagellate chromosome.

The influence of phosphorus availability on carbon allocation and P quota in Scenedesmus subspicatus: A synchrotron-based FTIR analysis

D.C. Sigee, F. Bahrami, B. Estrada, R.E. Webster and A.P. Dean. 2007. The influence of phosphorus availability on carbon allocation and P quota in Scenedesmus subspicatus: A synchrotron-based FTIR analysis. Phycologia 46: 583–592. DOI: 10.2216/07-14.1 Synchrotron-based Fourier transform infrared (FTIR) microspectroscopy was used to characterise the molecular composition of the freshwater alga Scenedesmus subspicatus, cultured at three different initial concentrations of phosphorus (PO4-P): 0.05 mg l−1, 0.5 mg l−1 and 5 mg l−1. These led, respectively, to limited algal growth due to phosphorus deficiency (low-P culture), maximum algal growth with no luxury consumption (intermediate-P) and maximum algal growth with luxury phosphorus consumption (high-P culture). In all cultures, FTIR spectra had nine major absorbance bands (wavenumber range 1760–900 cm−1), including bands at 1736 cm−1 (lipid), 1652 cm−1 (amide I) and the region from 1180 to 950 cm−1 (carbohydrate). Internal phosphorus concentrations (Qp), determined by energy dispersive X-ray microanalysis, differed markedly between low-P (typically < 0.1% dry wt), intermediate-P (> 0.1%) and high-P (> 0.3%) cultures. During logarithmic growth phase, a rapid change in carbon allocation was observed in the low-P cultures, with increases in both the lipid:protein (0.1–0.34) and carbohydrate:protein (0.4–1.0) ratio. Mean cell volume increased by 60%, and the mean chlorophyll a content remained consistently low (typically < 0.2 pg cell−1). The change in carbon allocation was triggered primarily by low Qp values rather than low external (culture medium) concentrations. Intermediate-P and high-P cultures showed higher chlorophyll a content (> 0.2 pg cell−1) and changes in carbon allocation only after entry into stationary phase. No increase in cell volume occurred, suggesting that a switch in carbon allocation during stationary phase (intermediate- and high-P cultures) rather than log phase (low-P culture) does not result in an increase in cell size. Entry into stationary growth phase occurred simultaneously in all three cultures and was not caused by internal (Qp) or external phosphorus depletion. Medium replacement in late stationary phase (day 35) cultures led to a rapid stimulation of growth, with a reversed carbon allocation (reduced lipid:protein and carbohydrate:protein ratios) and in low-P cultures a decrease in cell volume.

Indirect regulation rule for consecutive stages of ecological succession

Abstract The lake ecosystem model ‘Rostherne’ allowed a theoretical insight into delayed causal relationships in aquatic ecosystems. Model simulations were used to demonstrate the possibility of influencing a species dominant at a later stage of ecological succession, by alleviating growth limitation of a different species, dominant at an earlier stage. Such delayed relationships are characteristic of various types of systems (including ecological), and can be illustrated by using a simple Stella model presented here. The stated indirect regulation rule for consecutive stages of ecological succession provides an important theoretical basis both for certain ecological manipulations and for the better understanding of various environmental relationships. It should, therefore, prove useful for theoretical analysis of system dynamics, studies of terrestrial and aquatic ecosystems, management of natural resources, Environmental Assessment and Auditing.

Fourier-transform infrared spectroscopy of Pediastrum duplex: characterization of a micro-population isolated from a eutrophic lake

Fourier-transform infrared (FTIR) spectroscopy was carried out on single colonies of Pediastrum duplex present in air-dried preparations of mixed phytoplankton samples isolated from a eutrophic freshwater lake. FTIR absorption spectra had 12 distinct bands over the wavenumber range 3300–900 cm−1 which were tentatively assigned to a range of chemical groups, including –OH (residual water, wavenumber 3299 cm−1), –CH2 (lipid, 2924), –C=O (cellulose, 1739), amide (protein, 1650 and 1542), >P=O (nucleic acid, 1077) and –C–O (starch, 1151 and 1077). Measurement of band areas identified residual water, protein and starch as the major detectable constituents. Areas of single bands and combined bands of –CH2, –C–O and >P=O species normalized to protein (to correct for differences in specimen hydration and thickness) showed wide variation between colonies, indicating environmental heterogeneity. Correlation analysis demonstrated close statistical associations between different molecular species. Particularly high levels of correlation between bands 3/4 (CH2), 6/7 (amide) and 8/9 (–CH3) was consistent with their joint origin from the same molecular species. The isolation of bands 11 and 12 in the correlation pattern was confirmed by factor analysis, suggesting that variation in the level of starch is statistically unrelated to other macromolecules being monitored. The use of FTIR spectroscopy to characterize an algal micro-population within mixed phytoplankton has potential for future studies on biodiversity and environmental interactions at the species level.

Expansion of the model 'Rostherne' for fish and zooplankton: Role of top-down effects in modifying the prevailing pattern of ecosystem functioning

Abstract This paper presents a new version of the well-established aquatic ecosystem model ‘Rostherne’, incorporating lake fish and zooplankton. The model currently comprises differential and algebraic equations describing processes and forcing functions most important for a freshwater ecosystem. These include seasonal changes of solar radiation and water temperature, processes of algal and cyanobacterial population dynamics and nutrient uptake, water and chemical budgeting, stratification of the water column and sedimentation of suspended particles, and dynamics of detritus and its chemical constituents. Although it was shown previously for some years that a reasonable simulation of changes in most state variables could be achieved without accounting for fish and zooplankton, in other years, consideration of these compartments improved the fit between observations and model simulations dramatically. This was particularly true for the year 1998, when the high zooplankton numbers in spring appeared to prevent any significant development of the diatom population. The latter, however, bloomed during the first part of the summer (following a decrease in zooplankton counts) causing an unusually delayed major drop in ambient Si levels. As biogoechemical cycles of Si, P and N are interconnected through the dynamics of the primary producers, such a delay has serious implications for the functioning of an aquatic ecosystem model. Since changes in zooplankton dynamics are heavily dependent on the variability in fish recruitment, simultaneous consideration of the two components is helpful both for modelling overall dynamics of the lake ecosystem and for implementation of the biogeochemical regulation proposed previously.

Patterns of cell death in freshwater colonial cyanobacteria during the late summer bloom

D.C. Sigee, A. Selwyn, P. Gallois and A.P. Dean. 2007. Patterns of cell death in freshwater colonial cyanobacteria during the late summer bloom. Phycologia 46: 284–292. DOI: 10.2216/06-69.1 The occurrence of senescence (Evans blue staining) and programmed cell death (Hoechst staining/TUNEL reaction) was studied in the colonial cyanobacteria Anabaena flos-aquae and Microcystis flos-aquae during the late summer bloom in a eutrophic lake. Algae were analysed over a seven-day period (three sampling days) within mixed phytoplankton samples obtained from the upper epilimnion (trawl-net sample) and lower epilimnion (sediment-trap sample). Death of Anabaena in the trawl-net population was attributed to widespread infection by a chytrid fungus, resulting in breakdown and positive Evans blue staining of most filamentous colonies. Associated akinetes were a mixture of senescent cells (30% of total, Evans blue stained) and viable cells (unstained) plus a few dead cells (no contents). Sedimentation resulted in loss of filamentous colonies, leaving groups of akinetes with about 30% nonviable cells (senescent and dead). Over the sampling period, 20–50% of Microcystis colonies in the trawl-net samples showed some cells with clear Evans blue staining, indicating widespread senescence in the algal population. The occurrence of programmed cell death in some colonies of Microcystis was indicated by a positive TUNEL reaction and by condensed regions of nucleoid DNA in Hoechst-stained material. The results suggest that senescence was a general response to adverse environmental conditions (no fungal infection), with at least 30% (minimum count) of cells in affected colonies switching to programmed cell death. The induction of cell death within individual cells occurred randomly throughout the colony (nearest-neighbour comparison to random distribution) and was unrelated to the cell cycle – since both stained and unstained cells included the full range of cell size, from dividing cells to large nondividing cells. Very few colonies of Microcystis were observed in the sediment-trap samples, suggesting that senescence and cell death in the planktonic population of this alga were preceding the major phase of sedimentation.