Andrew P. Dean

The potential of sustainable algal biofuel production using wastewater resources

The potential of microalgae as a source of renewable energy has received considerable interest, but if microalgal biofuel production is to be economically viable and sustainable, further optimization of mass culture conditions are needed. Wastewaters derived from municipal, agricultural and industrial activities potentially provide cost-effective and sustainable means of algal growth for biofuels. In addition, there is also potential for combining wastewater treatment by algae, such as nutrient removal, with biofuel production. Here we will review the current research on this topic and discuss the potential benefits and limitations of using wastewaters as resources for cost-effective microalgal biofuel production.

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.

A Novel Mechanism by which Silica Defends Grasses Against Herbivory

BACKGROUND AND AIMS Previous studies have shown that silica in grass leaves defends them against small herbivores, which avoid high-silica grasses and digest them less efficiently. This study tested the idea that silica can reduce digestibility by preventing the mechanical breakdown of chlorenchyma cells. METHODS Both the percentage of total chlorophyll liberated from high- and low-silica grass leaves by mechanical grinding and the chlorophyll content of locust faeces were measured. KEY RESULTS High-silica grasses released less chlorophyll after grinding and retained more after passing through the gut of locusts, showing that silica levels correlated with increased mechanical protection. CONCLUSIONS These results suggest that silica may defend grasses at least in part by reducing mechanical breakdown of the leaf, and that mechanical protection of resources in chlorenchyma cells is a novel and potentially important mechanism by which silica protects grasses.

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.

Bioaccumulation of silver nanoparticles into Daphnia magna from a freshwater algal diet and the impact of phosphate availability

Abstract This study assessed the bioavailability, toxicity, and transfer of silver nanoparticles (AgNPs) in comparison with AgNO3 in two model food chain organisms: the alga Chlamydomonas reinhardtii and the grazing crustacean Daphnia magna. The effects of phosphate, a potential Ag+-binding ligand and a determinant of phytoplankton productivity, were evaluated. Nano Ag derived from coated AgNPs and AgNO3 was accumulated at similar concentrations into microalgae during high phosphate treatment, but AgNO3 accumulation was increased by low phosphate availability. After feeding on Ag-containing algae, D. magna equally accumulated AgNO3 and nano-derived Ag. There were significant reductions in feeding when D. magna were fed Ag-contaminated algae, with the AgNO3, low phosphate-exposed cells being ingested the least. Nutritional quality characteristics including fatty acid and trace nutrient content were similar in all algal samples, indicating that feeding reduction is specifically due to the presence of Ag, with AgNO3 being more toxic than nano Ag.

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.

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.

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.

Acclimation of Microalgae to Wastewater Environments Involves Increased Oxidative Stress Tolerance Activity

A wastewater environment can be particularly toxic to eukaryotic microalgae. Microalgae can adapt to these conditions but the specific mechanisms that allow strains to tolerate wastewater environments are unclear. Furthermore, it is unknown whether the ability to acclimate microalgae to tolerate wastewater is an innate or species-specific characteristic. Six different species of microalgae (Chlamydomonas debaryana, Chlorella luteoviridis, Chlorella vulgaris, Desmodesmus intermedius, Hindakia tetrachotoma and Parachlorella kessleri) that had never previously been exposed to wastewater conditions were acclimated over an 8-week period in secondary-treated municipal wastewater. With the exception of C. debaryana, acclimation to wastewater resulted in significantly higher growth rate and biomass productivity. With the exception of C. vulgaris, total chlorophyll content was significantly increased in all acclimated strains, while all acclimated strains showed significantly increased photosynthetic activity. The ability of strains to acclimate was species-specific, with two species, C. luteoviridis and P. kessleri, able to acclimate more efficiently to the stress than C. debaryana and D. intermedius. Metabolic fingerprinting of the acclimated and non-acclimated microalgae using Fourier transform infrared spectroscopy was able to differentiate strains on the basis of metabolic responses to the stress. In particular, strains exhibiting greater stress response and altered accumulation of lipids and carbohydrates could be distinguished. The acclimation to wastewater tolerance was correlated with higher accumulation of carotenoid pigments and increased ascorbate peroxidase activity.

Resolution of codominant phytoplankton species in a eutrophic lake using synchrotron-based Fourier transform infrared spectroscopy

A.P. Dean, M.C. Martin and D.C. Sigee. 2007. Resolution of codominant phytoplankton species in a eutrophic lake using synchrotron-based Fourier transform infrared spectroscopy. Phycologia 46: 151–159. DOI: 10.2216/06-27.1 Synchrotron-based Fourier-transform infrared (FTIR) microspectroscopy was used to distinguish micropopulations of the codominant algae Microcystis aeruginosa (Cyanophyceae) and Ceratium hirundinella (Dinophyceae) in mixed phytoplankton samples taken from the water column of a stratified eutrophic lake (Rostherne Mere, UK). FTIR spectra of the two algae showed a closely similar sequence of 10 bands over the wave-number range 4000–900 cm−1. These were assigned to a range of vibrationally active chemical groups using published band assignments and on the basis of correlation and factor analysis. In both algae, intracellular concentrations of macromolecular components (determined as band intensity) varied considerably within the same population, indicating substantial intraspecific heterogeneity. Interspecific differences were separately analysed in relation to discrete bands and by multivariate analysis of the entire spectral region 1750–900 cm−1. In terms of discrete bands, comparison of individual intensities (normalised to amide I) demonstrated significant (99% probability level) differences in relation to six bands between the two algal species. Key interspecific differences were also noted in relation to the positions of bands 2, 10 (carbohydrate) and 7 (protein) and in the 3-D plots derived by principal component analysis (PCA) of the sequence of band intensities. PCA of entire spectral regions showed clear resolution of species in the PCA plot, with indication of separation on the basis of protein (region 1700–1500 cm−1) and carbohydrate (region 1150–900 cm−1) composition in the loading plot. Hierarchical cluster analysis (Ward algorithm) of entire spectral regions also showed clear discrimination of the two species within the resulting dendrogram.