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Climate change is increasing water temperature and intensifying the incidence of cyanobacterial blooms worldwide. However, the combined effects of increased temperature and microcystin concentrations as co-stressors on survival and ecological processes in freshwater species are unclear. Here, using purified MC-LR and crude extract of toxigenic Microcystis aeruginosa, we tested the individual and combined effects of three water temperatures (15, 20, 25 °C) and a range of environmentally relevant concentrations of dissolved microcystin and crude extract (0.01–10 µg·L−1) on survival, growth inhibition, grazing and predation rates in three freshwater species: phytoplankton (Scenedesmus quadricauda), zooplankton (Daphnia pulex), and an invertebrate predator (Ischnura elegans). Purified MC-LR exerted a higher growth inhibitory effect on S. quadricauda compared to crude extract with the same concentration of MC-LR, while neither treatment affected its chlorophyll-a content or survival of D. pulex. Crude extract reduced grazing and survival of D. pulex and I. elegans, respectively. The combined effect of higher temperature and crude extract reduced I. elegans survival by 50%. Increased temperature reduced prey handing time in I. elegans by 49%, suggesting a higher predation rate. However, warming together with higher concentrations of crude extract jointly increased zooplankton grazing and reduced damselfly predation. Taken together, these results suggest crude extract, and not necessarily microcystin, can affect survival and productivity in freshwater species, although these effects may vary unevenly across trophic levels. Our findings highlight the importance of complex ecological mechanisms by which warming can exacerbate toxic effects of cyanobacterial bloom extracts on survival and functions among species in eutrophic freshwaters.

Woody plant material represents a vast renewable resource that has the potential to produce biofuels and other bio-based products with favorable net CO 2 emissions. 1 , 2 Its potential has been demonstrated in a recent study that generated novel structural materials from flexible moldable wood. 3 Apple rubbery wood (ARW) disease is the result of a viral infection that causes woody stems to exhibit increased flexibility. 4 Although ARW disease is associated with the presence of an RNA virus 5 known as apple rubbery wood virus (ARWV), how the unique symptoms develop is unknown. We demonstrate that the symptoms of ARWV infections arise from reduced lignification within the secondary cell wall of xylem fibers and result in increased wood digestibility. In contrast, the mid-lamellae region and xylem ray cells are largely unaffected by the infection. Gene expression and proteomic data from symptomatic xylem clearly show the downregulation of phenylalanine ammonia lyase (PAL), the enzyme catalyzing the first committed step in the phenylpropanoid pathway leading to lignin biosynthesis. A large increase in soluble phenolics in symptomatic xylem, including the lignin precursor phenylalanine, is also consistent with PAL downregulation. ARWV infection results in the accumulation of many host-derived virus-activated small interfering RNAs (vasiRNAs). PAL-derived vasiRNAs are among the most abundant vasiRNAs in symptomatic xylem and are likely the cause of reduced PAL activity. Apparently, the mechanism used by the virus to alter lignin exhibits similarities to the RNAi strategy used to alter lignin in genetically modified trees to generate comparable improvements in wood properties. 6-8.

Climate change is increasing water temperature and intensifying the incidence of cyanobacterial blooms worldwide. However, the combined effects of increased temperature and microcystin concentrations as co-stressors on survival and ecological processes in freshwater species are unclear. Here, using purified MC-LR and crude extract of toxigenic Microcystis aeruginosa, we tested the individual and combined effects of three water temperatures (15, 20, 25 °C) and a range of environmentally relevant concentrations of dissolved microcystin and crude extract (0.01-10 µg·L

Background:\ud State-of-the art therapy for recurrent ovarian cancer suitable for platinum-based re-treatment includes bevacizumab-containing combinations (eg, bevacizumab combined with carboplatin–paclitaxel or carboplatin–gemcitabine) or the most active non-bevacizumab regimen: carboplatin–pegylated liposomal doxorubicin. The aim of this head-to-head trial was to compare a standard bevacizumab-containing regimen versus carboplatin–pegylated liposomal doxorubicin combined with bevacizumab.\ud Methods:\ud This multicentre, open-label, randomised, phase 3 trial, was done in 159 academic centres in Germany, France, Australia, Austria, and the UK. Eligible patients (aged ≥18 years) had histologically confirmed epithelial ovarian, primary peritoneal, or fallopian tube carcinoma with first disease recurrence more than 6 months after first-line platinum-based chemotherapy, and an Eastern Cooperative Oncology Group performance status of 0–2. Patients were stratified by platinum-free interval, residual tumour, previous antiangiogenic therapy, and study group language, and were centrally randomly assigned 1:1 using randomly permuted blocks of size two, four, or six to receive six intravenous cycles of bevacizumab (15 mg/kg, day 1) plus carboplatin (area under the concentration curve [AUC] 4, day 1) plus gemcitabine (1000 mg/m 2, days 1 and 8) every 3 weeks or six cycles of bevacizumab (10 mg/kg, days 1 and 15) plus carboplatin (AUC 5, day 1) plus pegylated liposomal doxorubicin (30 mg/m 2, day 1) every 4 weeks, both followed by maintenance bevacizumab (15 mg/kg every 3 weeks in both groups) until disease progression or unacceptable toxicity. There was no masking in this open-label trial. The primary endpoint was investigator-assessed progression-free survival according to Response Evaluation Criteria in Solid Tumors version 1.1. Efficacy data were analysed in the intention-to-treat population. Safety was analysed in all patients who received at least one dose of study drug. This completed study is registered with ClinicalTrials.gov, NCT01837251.\ud Findings:\ud Between Aug 1, 2013, and July 31, 2015, 682 eligible patients were enrolled, of whom 345 were randomly assigned to receive carboplatin–pegylated liposomal doxorubicin–bevacizumab (experimental group) and 337 were randomly assigned to receive carboplatin–gemcitabine–bevacizumab (standard group). Median follow-up for progression-free survival at data cutoff (July 10, 2018) was 12·4 months (IQR 8·3–21·7) in the experimental group and 11·3 months (8·0–18·4) in the standard group. Median progression-free survival was 13·3 months (95% CI 11·7–14·2) in the experimental group versus 11·6 months (11·0–12·7) in the standard group (hazard ratio 0·81, 95% CI 0·68–0·96; p=0·012). The most common grade 3 or 4 adverse events were hypertension (88 [27%] of 332 patients in the experimental group vs 67 [20%] of 329 patients in the standard group) and neutropenia (40 [12%] vs 73 [22%]). Serious adverse events occurred in 33 (10%) of 332 patients in the experimental group and 28 (9%) of 329 in the standard group. Treatment-related deaths occurred in one patient in the experimental group (

© 2020 Access to safe drinking water free from microbial pollution is an issue of global concern. The use of photocatalytic thin films in water treatment has focused on titanium dioxide, which requires UV-activation, proving a potential barrier to upscaling and implementation in the real world. Visible-light-activated photocatalytic thin films, such as bismuth oxide, have recently been shown to have antimicrobial properties. However, more understanding of the photocatalytic effect on the microbial population in water is required. Glass beads coated with bismuth oxide were incubated with either Microcystis aeruginosa, Anabaena sp. or free-floating genomic DNA. The presence of bismuth oxide-coated glass beads was able to rapidly stop a population of cyanobacteria from increasing. The coated beads were also able to degrade genomic DNA. Leachate from the beads showed no increase in toxicity against human liver cells. This data demonstrates the efficacy of bismuth oxide-coated glass beads for controlling potentially dangerous cyanobacterial populations, whilst potentially reducing the amount of free-floating genomic DNA (an essential issue in the face of antimicrobial resistance) – all of which should be essential considerations in emerging water treatment technologies.

Metal transport processes are relatively poorly understood in algae in comparison to higher plants and other eukaryotes. A screen of genomes from 33 taxonomically diverse algal species was conducted to identify members of the Cation Diffusion Facilitator (CDF) family of metal ion transporter. All algal genomes contained at least one CDF gene with four species having >10 CDF genes (median of 5 genes per genome), further confirming that this is a ubiquitous gene family. Phylogenetic analysis suggested a CDF gene organisation of five groups, which includes Zn-CDF, Fe/Zn-CDF and Mn-CDF groups, consistent with previous phylogenetic analyses, and two functionally undefined groups. One of these undefined groups was algal specific although excluded chlorophyte and rhodophyte sequences. The majority of sequences (22 out of 26 sequences) from this group had a putative ion binding site motif within transmembrane domain 2 and 5 that was distinct from other CDF proteins, such that alanine or serine replaced the conserved histidine residue. The phylogenetic grouping was supported by sequence cluster analysis. Yeast heterologous expression of CDF proteins from Chlamydomonas reinhardtii indicated Zn2+ and Co2+ transport function by CrMTP1, and Mn2+ transport function by CrMTP2, CrMTP3 and CrMTP4, which validated the phylogenetic prediction. However, the Mn-CDF protein CrMTP3 was also able to provide zinc and cobalt tolerance to the Zn- and Co-sensitive zrc1cot1 yeast strain. There is wide diversity of CDF transporters within the algae lineage, and some of these genes may be attractive targets for future applications of metal content engineering in plants or microorganisms.

Acid mine drainage (AMD) is a serious environmental problem worldwide that requires efficient and sustainable remediation technologies including the use of biological mechanisms. A key challenge for AMD bioremediation is to provide optimal conditions for microbial-mediated immobilisation of trace metals. Although organic carbon and oxygen can enhance treatment efficiency, the effect on microbial communities is unclear. In this study, surface sediments from a natural wetland with proven efficiency for AMD bioremediation were artificially exposed to oxygen (by aeration) and/or organic carbon (in the form of mixed organic acids) and incubated under laboratory conditions. In addition to measuring changes in water chemistry, a metagenomics approach was used to determine changes in sediment bacterial, archaeal and fungal community structure, and functional gene abundance. The addition of organic carbon produced major changes in the abundance of microorganisms related to iron and sulfur metabolism (including Geobacter and Pelobacter) and increased levels of particulate metals via sulfate reduction. Aeration resulted in an increase in Sideroxydans abundance but no significant changes in metal chemistry were observed. The study concludes that the utilisation of organic carbon by microorganisms is more important for achieving efficient AMD treatment than the availability of oxygen, yet the combination of oxygen with organic carbon addition did not inhibit the improvements to water quality

Metal pollution in freshwater bodies is a long-standing challenge with large expense required to clean-up pollutants such as Cd. There is widespread interest in the potentially low-cost and sustainable use of biological material to perform bioremediation, such as the use of microalgae. Efficient metal bioremediation capacity requires both the ability to tolerate metal stress and metal accumulation. Here, the role of a Chlamydomonas reinhardtii metal tolerance protein (MTP) was examined for enhanced Cd tolerance and uptake. The CrMTP4 gene is a member of the Mn-CDF clade of the cation diffusion facilitator family of metal transporters but is able to provide tolerance and sequestration for Mn and Cd, but not other metals, when expressed in yeast. Over-expression of CrMTP4 in C. reinhardtii yielded a significant increase in tolerance to Cd toxicity and increased Cd accumulation although tolerance to Mn was not increased. In comparison, the metal tolerance of three chlorophyte microalgae strains (Chlorella luteoviridis, Parachlorella hussii, and Parachlorella kessleri) that had previously been adapted to wastewater growth was examined. In comparison to wild type C. reinhardtii, all three natural strains showed significantly increased tolerance to Cd, Cu, Al and Zn, and furthermore their Cd tolerance and uptake was greater than that of the CrMTP4 over-expression strains. Despite CrMTP4 gene over-expression being a successful strategy to enhance the Cd bioremediation potential of a metal-sensitive microalga, a single gene manipulation cannot compete with naturally adapted strain mechanisms that are likely to be multigenic and due in part to oxidative stress tolerance.

Natural flood management interventions, such as Large Wood Debris (LWD) or engineered log jams, are being increasingly deployed throughout the UK and elsewhere. In addition to alleviating flood risk, it is anticipated that they may influence the ecology of freshwater river systems, including macroinvertebrate populations. This study explores macroinvertebrate assemblages, water quality parameters, and sediment size distribution in a headwater stream following the addition of LWD as part of a natural flood management scheme. The study area consists of 6 sites within the intervention zone where LWD log jams were implemented, with comparative control sites upstream and downstream (3 sites each). Macroinvertebrate communities, sediment size distribution, and water chemistry were sampled 3 and 10 months following the addition of LWD. Our findings revealed increased macroinvertebrate abundance and taxa richness in the LWD intervention zone versus control, with an increased BMWP score reflecting the increased taxa richness. Average Score Per Taxon, and water chemistry showed no change, revealing invertebrate changes to be independent of water quality. NMDS and hierarchical clustering analysis on invertebrate data showed a clear separation of communities where LWD was present from those with no LWD, while SIMPER analysis showed that LWD addition led to the rapid establishment of taxa (Hydraenidae, Rhyacophilidae, Scirtidae, and Elmidae) that were otherwise absent. Ten months after LWD addition, improved biodiversity was also found in areas below the intervention zone, suggesting the positive impacts of LWD extend downstream. LWD also altered sediments, with sites immediately upstream of LWD log jams having a greater percentage of fine sediment than those immediately downstream. These results suggest that biological complexity and niche availability increased within the in-channel zone as a result of introduced LWD, thus revealing wider aquatic habitat improvement potential of LWD for natural flood management. The use of LWD as an intervention for flood management is recommended for its additional benefits for ecosystem health.

One of the most widespread approaches for measurement of greenhouse gas emissions from soils involves the use of static chambers. This method is relatively inexpensive, is easily replicated, and is ideally suited to plot-based experimental systems. Among its limitations is the loss of detection sensitivity with increasing chamber height, which creates challenges for deployment in systems including tall vegetation. It is not always possible to avoid inclusion of plants within chambers or to extend chamber height to fully accommodate plant growth. Thus, in many systems, such as perennial forages and biomass crops, plants growing within static chambers must either be trimmed or folded during lid closure. Currently, data on how different types of biomass manipulation affect measured results is limited. Here, we compare the effects of cutting vs. folding of biomass on nitrous oxide measurements in switchgrass ( L.) and alfalfa ( L.) systems. We report only limited evidence of treatment effects during discrete sampling events and little basis for concern that effects may intensify over time as biomass manipulation is repeatedly imposed. However, nonsignificant treatment effects that were consistently present amounted to significant overall trends in three out of the four systems studied. Such minor disparities in flux could amount to considerable quantities over time, suggesting that caution should be exercised when comparing cumulative emission values from studies using different biomass manipulation strategies.

© 2019 Elsevier Ltd Natural recovery and remediation of acid mine drainage (AMD) reduces the generation of acidity and transport of trace elements in the runoff. A natural wetland that receives and remediates AMD from an abandoned copper mine at Parys Mountain (Anglesey, UK) was investigated for better understanding of the remediation mechanisms. Water column concentrations of dissolved Fe and S species, trace metal (loid)s and acidity decreased markedly as the mine drainage stream passed through the wetland. The metal (loid)s were removed from the water column by deposition into the sediment. Fe typically accumulated to higher concentrations in the surface layers of sediment while S and trace metal (loid)s were deposited at higher concentration within deeper (20–50 cm) sediments. High resolution X-ray fluorescence scans of sediment cores taken at three sites along the wetland indicates co–immobilization of Zn, Cu and S with sediment depth as each element showed a similar core profile. To examine the role of bacteria in sediment elemental deposition, marker genes for Fe and S metabolism were quantified. Increased expression of marker genes for S and Fe oxidation was detected at the same location within the middle of the wetland where significant decrease in SO42− and Fe2+ was observed and where generation of particulate Fe occurs. This suggests that the distribution and speciation of Fe and S that mediates the immobilization and deposition of trace elements within the natural wetland sediments is mediated in part by bacterial activity.

© 2018 Elsevier B.V. The diversity and biological characteristics of eukaryotic communities within acid mine drainage (AMD) sites is less well studied than for prokaryotic communities. Furthermore, for many eukaryotic extremophiles the potential mechanisms of adaptation are unclear. This study describes an evaluation of eight highly acidic (pH 1.6–3.1) and one moderately acidic (pH 5.6) metal-rich acid mine drainage ponds at a disused copper mine. The severity of AMD pollution on eukaryote biodiversity was examined, and while the most species-rich site was less acidic, biodiversity did not only correlate with pH but also with the concentration of dissolved and particulate metals. Acid-tolerant microalgae were present in all ponds, including the species Chlamydomonas acidophila, abundance of which was high in one very metal-rich and highly acidic (pH 1.6) pond, which had a particularly high PO4-P concentration. The C. acidophila strain named PM01 had a broad-range pH tolerance and tolerance to high concentrations of Cd, Cu and Zn, with bioaccumulation of these metals within the cell. Comparison of metal tolerance between the isolated strain and other C. acidophila strains previously isolated from different acidic environments found that the new strain exhibited much higher Cu tolerance, suggesting adaptation by C. acidophila PM01 to excess Cu. An analysis of the metabolic profile of the strains in response to increasing concentrations of Cu suggests that this tolerance by PM01 is in part due to metabolic adaptation and changes in protein content and secondary structure.

The metabolism of glycerol-3-phosphate (G3P) is important for environmental stress responses by eukaryotic microalgae. G3P is an essential precursor for glycerolipid synthesis and the accumulation of triacylglycerol (TAG) in response to nutrient starvation. G3P dehydrogenase (GPDH) mediates G3P synthesis, but the roles of specific GPDH isoforms are currently poorly understood. Of the five GPDH enzymes in the model alga

© 2019 Elsevier Inc. Acid mine drainage (AMD) is a significant contributor of metal pollution leading to ecosystem damage. Bioindicator organisms such as intertidal brown macroalgae have an important role in quantifying the risks of metal bioaccumulation in coastal locations exposed to AMD contamination. Measurement of As, Cd, Cu, Fe, Pb, and Zn accumulation was performed in Fucus serratus, Fucus vesiculosus and Ascophyllum nodosum sampled from two marine locations near to an abandoned Cu mine in Anglesey, Wales, UK. Transect samples were taken from a coastal location (Amlwch) that has seen a substantial increase in AMD contamination over 15 years, in comparison to a nearby estuarine location (Dulas Estuary leading to Dulas Bay) with a historic legacy of pollution. These were compared with samples from the same sites taken 30 years earlier. Some of the Dulas macroalgae samples had Cd, Cu and Zn concentrations that were above background but in general indicated a non-polluted estuary in comparison to substantial pollution over previous decades. In contrast, Fucus samples collected from directly below an AMD outflow at Amlwch showed extremely elevated metal bioaccumulation (>250 mg Fe g−1, >6 mg Cu g−1, >2 mg Zn g−1, >190 μg As g−1) and evidence of macroalgae toxicity, indicating severe pollution at this site. However, the pollution dispersed within 200 m of the outflow source. This study has demonstrated the efficiency of three brown macroalgae species as indicators for metal bioavailability at high spatial resolution and over time.

This study describes the first long-term (14-year) evaluation of the efficacy of an established (>100 years) natural wetland to remediate highly acidic mine drainage (AMD). Although natural wetlands are highly valued for their biodiversity, this study demonstrates that they also provide important ecosystem service functions through their ability to consistently and reliably improve water quality by mitigating AMD. The Afon Goch river flows from Parys Mountain copper mine via a natural wetland, and was the major source of Zn and Cu contamination to the Irish Sea. Prior to 2003 the wetland received severe acidic metal contamination and retained a large proportion of the contamination (55, 64, and 37% in dissolved Fe, Zn, and Cu) leading to a greatly reduced metal flow to the Irish Sea. Reduced wetland loadings midway through the sampling period led to a reduction of metals by 83-94% and a pH increase from 2.7 to 5.5, resulting in long-term improvements in the downstream benthic invertebrate community. High root metal accumulation by the dominant wetland plant species and the association of acidophilic bacteria in the wetland rhizosphere indicate that multiple interacting processes provide an efficient and self-sustaining system to remediate AMD.

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.

Many eukaryotic microalgae modify their metabolism in response to nutrient stresses such as phosphorus (P) starvation, which substantially induces storage metabolite biosynthesis, but the genetic mechanisms regulating this response are poorly understood. Here, we show that P starvation-induced lipid and starch accumulation is inhibited in a Chlamydomonas reinhardtii mutant lacking the transcription factor Pi Starvation Response1 (PSR1). Transcriptomic analysis identified specific metabolism transcripts that are induced by P starvation but misregulated in the psr1 mutant. These include transcripts for starch and triacylglycerol synthesis but also transcripts for photosynthesis-, redox-, and stress signaling-related proteins. To further examine the role of PSR1 in regulating lipid and starch metabolism, PSR1 complementation lines in the psr1 strain and PSR1 overexpression lines in a cell wall-deficient strain were generated. PSR1 expression in the psr1 lines was shown to be functional due to rescue of the psr1 phenotype. PSR1 overexpression lines exhibited increased starch content and number of starch granules per cell, which correlated with a higher expression of specific starch metabolism genes but reduced neutral lipid content. Furthermore, this phenotype was consistent in the presence and absence of acetate. Together, these results identify a key transcriptional regulator in global metabolism and demonstrate transcriptional engineering in microalgae to modulate starch biosynthesis.

Fourier transform infra-red (FTIR) spectroscopy was used to assess carbon allocation in selected phytoplankton (Pediastrum duplex, Ceratium hirundinella, Anabaena flos-aquae) in a eutrophic lake. In contrast to bulk-analysis techniques, FTIR spectroscopy can provide information on carbon allocation at the species level within natural mixed populations. Carbon allocation was determined using lipid/protein (L/P) and carbohydrate/protein (C/P) ratios and multivariate analysis of complete spectra and showed considerable intraspecific heterogeneity within samples dates, and clear seasonal changes in the spectra of Pediastrum and Anabaena, with both alga showing increased L/P and C/P ratios on selected sampling dates. Although increased ratios can indicate nutrient deficiency, the alga were not nutrient deficient during these periods, as indicated by continued population growth and high (>0.1%) internal phosphorus concentrations. FTIR spectra from Ceratium did not show any clear seasonal change. In all three algae, the transition from population growth to decline was not accompanied by any significant change in ratios, nor were low epilimnetic nutrients correlated with increased ratios, marking a significant contrast to laboratory batch cultures (previous studies) where low nutrients and the transition to stationary phase led to marked spectral changes.

The characteristics of metal accumulation in freshwater microalgae are important to elucidate for a full understanding of metal cycling and toxicity in a freshwater system. This study has utilized an elemental profiling approach to investigate the impacts of Cd exposure and phosphorus (P) availability on metal accumulation after 7 days in batch culture-grown Chlamydomonas reinhardtii. Multivariate statistical analysis of the elemental data demonstrated distinct responses between both stresses. Sublethal concentrations of Cd (up to 15 μM) caused increased accumulation of Co. Cu, Fe, and Zn content also increased in response to enhanced Cd concentrations but only when P availability was low. While Cd exposure effected the accumulation of a few specific metals, P limitation increased the accumulation of all essential trace metals and macronutrients analyzed including Co, Fe, K, Na, and Zn but not Mn. The accumulation of Cd also markedly increased in response to P limitation. The impact of P availability on essential metal accumulation was the same when either inorganic P or an organic P source (glycerophosphate) was used. These results highlight the potential risks of metal toxicity for freshwater microalgae and aquatic food chains when P availability is limiting and which can be exacerbated by Cd pollution.

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.

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

SUMMARY The response of Anabaena flos-aquae to varying levels of P availability was determined using the combined techniques of Fourier transform infrared (FTIR) microspectroscopy and energy-dispersive X-ray microanalysis (EDXRMA). Batch cultures of Anabaena were grown at initial P concentrations of 50 μg (low-P culture), 500 μg (intermediate-P) and 5000 μg (high-P) (PO4-P L−1) and were monitored for total biovolume, chlorophyll a, media nutrients (PO4-P and NO3-N), cellular P quota (EDXRMA) and carbon allocation (FTIR spectroscopy). The high-P culture showed a sixfold increase in total biovolume over the sampling period. Phosphorus in the media remained at more than ∼4000 μg L−1 and intracellular P concentration (P quota) as determined by EDXRMA showed no decline, remaining at more than 0.20% dry weight (DW). The intermediate-P culture showed a similar increase in total biovolume, but P concentrations in the media fell to ∼20 μg L−1, and this was reflected in a decline in the cellular P quota from 0.24% to 0.06% DW. Although the high- and intermediate-P treatments differed markedly in both internal and external P, analysis of the FTIR spectra from the two treatments, using both hierarchical cluster analysis (HCA) and principal component analysis (PCA), indicated no difference in carbon allocation. Cells from the low-P treatment showed strong evidence for P deficiency. P concentrations in the media were undetectable (

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 samp...

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...

Synchrotron-based Fourier-transform infrared (FTIR) microspectroscopy was used to characterize micropopulations of the cyanobacteria Anabaena flos-aquae and Aphanizomenon flos-aquae in mixed phytoplankton samples taken from the epilimnion and hypolimnion of a stratified eutrophic lake (Rostherne Mere, UK). All FTIR spectra (normalized to amide I) showed a closely similar sequence of 10 bands over the wavenumber range 4,000–900 cm−1 and were assigned to a range of vibrationally active chemical groups. Interspecific differences between Anabaena and Aphanizomenon were revealed using correlation and multivariate analysis (principal component analysis, PCA) of the set of individual band intensities. Differences between species (separately for epilimnion and hypolimnion) were also analysed by comparing band intensity means, and by performing PCA over a broad spectral region (1,750–900 cm−1). Comparison of intensity means showed that spectra obtained from epilimnetic algae had significant differences in bands 5–...

Fourier Transform Infrared (FT-IR) spectroscopy is a robust method for macromolecular analysis and differentiation of microorganisms. However, most studies are performed in controlled conditions and it is unclear whether this tool is appropriate for the identification of eukaryotic microalgae species from variable environments. In order to address this, nine closely-related species of marine and freshwater microalgae were grown under controlled (non-stressed) and variable (non-stressed and stressed) conditions, including nutrient-stressed and wastewater-stressed conditions. Following optimization of data processing methods, FT-IR spectra from all species and conditions were compared. The substantial metabolic changes that were caused by nutrient starvation restricted the ability of FT-IR spectroscopy to differentiate the microalgal species grown under variable conditions efficiently. Comparison of unsupervised and supervised multivariate data analysis methods found that principal component-discriminant function analysis was able best to differentiate between some species under controlled conditions but still gave poor differentiation under variable environmental conditions., Highlights • FT-IR spectroscopy can be used to generate metabolic fingerprints in algae. • Metabolic fingerprinting has been proposed to differentiate species and strains. • Algae exhibit high metabolic plasticity in response to environmental change. • This restricts the use of FT-IR spectroscopy as a discriminatory tool. • Discrimination is improved by multivariate statistical analysis techniques.

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 l...

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, Parachlorella kessleri) that had never previously been exposed to wastewater conditions were acclimated over an eight 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.

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.

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.

The karyotype, larval and pupal morphology of Macropelopia nebulosa group (Tanypodinae) is described. The species is generally identified on the basis of pupal morphology as it is not possible to distinguish the larvae of different Macropelopia species. However, the species has species-specific markers in the salivary gland chromosomes which allow identification at the larval stage. M. nebulosa (Meigen) has a chromosome set 2n = 8, with chromosomes AB CD EF G. Chromosomes AB CD EF are metacentric with large heterochromatin centromeres while chromosome G is acrocentric. For the first time a chromosome map of M. nebulosa has been produced which revealed the divergence of this species from its sibling species M. paranebulosa Fittkau on the basis of fixed homozygous inversions. Specific banding patterns of arms B and E are distinguished from that of M. paranebulosa by two steps of homozygous inversions; arm D—by three steps of homozygous inversions, while arms C and G—by one step of inversion. The significance of the morphology of the salivary gland chromosomes for species identification of subfamily Tanypodinae is emphasized. The first SEM images of pupa are also given.

(1988). Characterizing impure epimorphisms and impure monomorphisms. Communications in Algebra: Vol. 16, No. 4, pp. 849-858.

Microalgae produce metabolites that could be useful for applications in food, biofuel or fine chemical production. The identification and development of suitable strains require analytical methods that are accurate and allow rapid screening of strains or cultivation conditions. We demonstrate the use of Fourier transform infrared (FT-IR) spectroscopy to screen mutant strains of Chlamydomonas reinhardtii. These mutants have knockdowns for one or more nutrient starvation response genes, namely PSR1, SNRK2.1 and SNRK2.2. Limitation of nutrients including nitrogen and phosphorus can induce metabolic changes in microalgae, including the accumulation of glycerolipids and starch. By performing multivariate statistical analysis of FT-IR spectra, metabolic variation between different nutrient limitation and non-stressed conditions could be differentiated. A number of mutant strains with similar genetic backgrounds could be distinguished from wild type when grown under specific nutrient limited and replete conditions, demonstrating the sensitivity of FT-IR spectroscopy to detect specific genetic traits. Changes in lipid and carbohydrate between strains and specific nutrient stress treatments were validated by other analytical methods, including liquid chromatography–mass spectrometry for lipidomics. These results demonstrate that the PSR1 gene is an important determinant of lipid and starch accumulation in response to phosphorus starvation but not nitrogen starvation. However, the SNRK2.1 and SNRK2.2 genes are not as important for determining the metabolic response to either nutrient stress. We conclude that FT-IR spectroscopy and chemometric approaches provide a robust method for microalgae screening. Electronic supplementary material The online version of this article (doi:10.1007/s11306-015-0878-4) contains supplementary material, which is available to authorized users.