Insights into cell wall disintegration of Chlorella vulgaris

With their ability of CO2 fixation using sunlight as an energy source, algae and especially microalgae are moving into the focus for the production of proteins and other valuable compounds. However, the valorization of algal biomass depends on the effective disruption of the recalcitrant microalgal cell wall. Especially cell walls of Chlorella species proved to be very robust. The wall structures that are responsible for this robustness have been studied less so far. Here, we evaluate different common methods to break up the algal cell wall effectively and measure the success by protein and carbohydrate release. Subsequently, we investigate algal cell wall features playing a role in the wall’s recalcitrance towards disruption. Using different mechanical and chemical technologies, alkali catalyzed hydrolysis of the Chlorella vulgaris cells proved to be especially effective in solubilizing up to 56 wt% protein and 14 wt% carbohydrates of the total biomass. The stepwise degradation of C. vulgaris cell walls using a series of chemicals with increasingly strong conditions revealed that each fraction released different ratios of proteins and carbohydrates. A detailed analysis of the monosaccharide composition of the cell wall extracted in each step identified possible factors for the robustness of the cell wall. In particular, the presence of chitin or chitin-like polymers was indicated by glucosamine found in strong alkali extracts. The presence of highly ordered starch or cellulose was indicated by glucose detected in strong acidic extracts. Our results might help to tailor more specific efforts to disrupt Chlorella cell walls and help to valorize microalgae biomass.

Comparative transcriptomics reveals the molecular toolkit used by an algivorous protist for cell wall perforation

Microbial eukaryotes display a stunning diversity of feeding strategies, ranging from generalist predators to highly specialised parasites. The unicellular protoplast feeders represent a fascinating mechanistic intermediate, as they penetrate other eukaryotic cells (algae, fungi) like some parasites, but then devour their cell contents by phagocytosis. Besides prey recognition and attachment, this complex behaviour involves the local, pre-phagocytotic dissolution of the prey cell wall, which results in well-defined perforations of species-specific size and structure. Yet, the molecular processes that enable protoplast feeders to overcome cell walls of diverse biochemical composition remain unknown. We used the flagellate Orciraptor agilis (Viridiraptoridae, Rhizaria) as a model protoplast feeder, and applied differential gene expression analysis to examine its penetration of green algal cell walls. Besides distinct expression changes that reflect major cellular processes (e.g. locomotion, cell division), we found lytic carbohydrate-active enzymes that are highly expressed and upregulated during the attack on the alga. A putative endocellulase (family GH5_5) with a secretion signal is most prominent, and a potential key factor for cell wall dissolution. Other candidate enzymes (e.g. lytic polysaccharide monooxygenases) belong to families that are largely uncharacterised, emphasising the potential of non-fungal micro-eukaryotes for enzyme exploration. Unexpectedly, we discovered various chitin-related factors that point to an unknown chitin metabolism in Orciraptor, potentially also involved in the feeding process. Our findings provide first molecular insights into an important microbial feeding behaviour, and new directions for cell biology research on non-model eukaryotes.

Relationship between β-Carotene Accumulation and Geranylgeranyl Pyrophosphate Synthase in Different Species of Dunaliella

To study the relationship between β-carotene synthesis and geranylgeranyl pyrophosphate synthase (GGPS) activity, 15 species of Dunaliella were used to determine the changes in photosynthetic pigment contents, chlorophyll fluorescence parameters, β-carotene content, and GGPS activity. By observing the morphology and size of 15 species of Dunaliella, D8 has the largest individual algal cell and D9 has the smallest individual. Growth was relatively slow during days one through seven. After about eight days, the cells entered the logarithmic growth period and grew rapidly to a high density. After about 45 days, they entered a mature period, and growth slowed down. The contents of chlorophyll, carotenoids, and β-carotene increased during growth. D1 has the highest accumulation of β-carotene, and GGPS enzyme activity has a positive linear relationship with the β-carotene synthesis content. Phylogenetic analysis showed that the GGPS proteins of the 15 species were highly homologous, and the GGPS protein was not part of the membrane.

SNICAR-ADv3: a community tool for modeling spectral snow albedo

The Snow, Ice, and Aerosol Radiative (SNICAR) model has been used in various capacities over the last 15 years to model the spectral albedo of snow with light-absorbing constituents (LACs). Recent studies have extended the model to include an adding-doubling two-stream solver and representations of non-spherical ice particles; carbon dioxide snow; snow algae; and new types of mineral dust, volcanic ash, and brown carbon. New options also exist for ice refractive indices and solar-zenith-angle-dependent surface spectral irradiances used to derive broadband albedo. The model spectral range was also extended deeper into the ultraviolet for studies of extraterrestrial and high-altitude cryospheric surfaces. Until now, however, these improvements and capabilities have not been merged into a unified code base. Here, we document the formulation and evaluation of the publicly available SNICAR-ADv3 source code, web-based model, and accompanying library of constituent optical properties. The use of non-spherical ice grains, which scatter less strongly into the forward direction, reduces the simulated albedo perturbations from LACs by ∼9 %–31 %, depending on which of the three available non-spherical shapes are applied. The model compares very well against measurements of snow albedo from seven studies, though key properties affecting snow albedo are not fully constrained with measurements, including ice effective grain size of the top sub-millimeter of the snowpack, mixing state of LACs with respect to ice grains, and site-specific LAC optical properties. The new default ice refractive indices produce extremely high pure snow albedo (>0.99) in the blue and ultraviolet part of the spectrum, with such values only measured in Antarctica so far. More work is needed particularly in the representation of snow algae, including experimental verification of how different pigment expressions and algal cell concentrations affect snow albedo. Representations and measurements of the influence of liquid water on spectral snow albedo are also needed.

Does the freshwater mussel Anodonta anatina remove the fish pathogen Flavobacterium columnare from water?

Global decline of freshwater mussels (Unionoida) is threatening biodiversity and the essential ecosystem services that mussels provide. As filter-feeding organisms, freshwater mussels remove phytoplankton and suspended particles from the water. By filtering bacteria, freshwater mussels also decrease pathogen loads in the water. The objective of this study was to evaluate whether the common freshwater bivalve Anodonta anatina (duck mussel) could remove the bacterial fish pathogen Flavobacterium columnare from the water. Mussels reduced bacteria in both of the two experiments performed, so that the bacterial concentration at the end of the 96-h monitoring in mussel treatments was only 0.3–0.5 times that of the controls. Surprisingly, mussels did not reduce algal cell concentration statistically significantly. Mussel behavior (shell openness, foot position, and movement) was not affected by the presence of bacteria or algae, except for biodeposition formation, which was greatest in algal-fed treatments, followed by bacterial-fed treatments and controls, respectively. The intestines of bacteria-incubated A. anatina harbored F. columnare, suggesting that mussels ingested the bacteria. Present results suggest that freshwater mussels may also have a potential to mitigate aquaculture pathogen problems, as well as play a role in water quality management.

The Control of Carbon Translocation in a Sea Anemone-Dinoflagellate Symbiosis from New Zealand

<p>Anthopleura aureoradiata, a common sea anemone of New Zealand's intertidal mudflats and rocky shores, hosts symbiotic dinoflagellates of the genus Symbiodinium. This study investigated the control of photosynthetic carbon translocation in this symbiosis, and in particular the presence and operation of socalled 'host release factor' (HRF). Evidence for HRF exists in a number other algalinvertebrate symbioses, where tissue extracts of the host stimulate carbon release by isolated algal symbionts. However, its identity remains elusive and it has never been studied before in A. aureoradiata. Translocation of photosynthetically-fixed carbon in the intact symbiosis and in the presence of host tissue extract was measured using a 14C label. Zooxanthellae in the intact symbiosis released around 40% of their photosynthetically-fixed carbon to the anemone. Isolated zooxanthellae, however, translocated only 8%, even less than the amount of photosynthate liberated by zooxanthellae in FSW alone (11%). Photosynthetic rates per algal cell were similar in the intact symbiosis and both host homogenate and FSW incubations, meaning that the total amount of photosynthetically-fixed carbon released (in pg C/cell/h) by the zooxanthellae in these different situations reflected the %translocation values. Given the failure of homologous zooxanthellae (i.e. those from A. aureoradiata) to respond to homogenized host tissue, it was tested whether zooxanthellae from other host species (i.e. cultured heterologous algae) responded. Heterologous zooxanthellae representing 5 clades (A-E) of Symbiodinium were incubated in host tissue homogenate and photosynthate release again measured with 14C. The %translocation varied from 12-51% in A. aureoradiata homogenate and 17-67% in FSW, again suggesting a lack of an active HRF in the homogenized tissues of this sea anemone. Photosynthetic rates amongst the different heterologous algae also varied widely with, for instance, freshly isolated zooxanthellae from A. aureoradiata having 6-fold higher photosynthetic rates than cultured algae from the same clade (clade A). The zooxanthellae of A. aureoradiata are known to be N-sufficient in the field, and studies with other species have demonstrated that N-deficient zooxanthellae release more photosynthate in response to HRF than do N-sufficient ones. Therefore, induction of an HRF effect was attempted by starving sea anemones, and hence their zooxanthellae, prior to incubation of freshly isolated zooxanthellae in homogenized tissue. However, even after 8 weeks of starvation, the zooxanthellae showed no signs of N-deficiency (as indicated by the extent to which ammonium enhanced the rate of dark 14C fixation), meaning that the relationship with HRF activity could not be examined. The ability of these temperate zooxanthellae to maintain their Nsufficiency, even after relatively long periods of food deprivation, may indicate a lower reliance on host feeding for nitrogen than is seen in tropical zooxanthellae, or a greater capacity to use internal stores of nitrogen. The lack of photosynthate release by both homologous and heterologous zooxanthellae in host homogenate, as opposed to substantial carbon released in the intact symbiosis, suggests that control of carbon translocation in A. aureoradiata is not related to the activity of an HRF; alternatively, if an HRF is present, its activity is hindered when the symbiosis is disrupted. Further study is needed to determine what is responsible for the control of photosynthate translocation in the A. aureoradiata-Symbiodinium symbiosis.</p>

The Control of Carbon Translocation in a Sea Anemone-Dinoflagellate Symbiosis from New Zealand

<p>Anthopleura aureoradiata, a common sea anemone of New Zealand's intertidal mudflats and rocky shores, hosts symbiotic dinoflagellates of the genus Symbiodinium. This study investigated the control of photosynthetic carbon translocation in this symbiosis, and in particular the presence and operation of socalled 'host release factor' (HRF). Evidence for HRF exists in a number other algalinvertebrate symbioses, where tissue extracts of the host stimulate carbon release by isolated algal symbionts. However, its identity remains elusive and it has never been studied before in A. aureoradiata. Translocation of photosynthetically-fixed carbon in the intact symbiosis and in the presence of host tissue extract was measured using a 14C label. Zooxanthellae in the intact symbiosis released around 40% of their photosynthetically-fixed carbon to the anemone. Isolated zooxanthellae, however, translocated only 8%, even less than the amount of photosynthate liberated by zooxanthellae in FSW alone (11%). Photosynthetic rates per algal cell were similar in the intact symbiosis and both host homogenate and FSW incubations, meaning that the total amount of photosynthetically-fixed carbon released (in pg C/cell/h) by the zooxanthellae in these different situations reflected the %translocation values. Given the failure of homologous zooxanthellae (i.e. those from A. aureoradiata) to respond to homogenized host tissue, it was tested whether zooxanthellae from other host species (i.e. cultured heterologous algae) responded. Heterologous zooxanthellae representing 5 clades (A-E) of Symbiodinium were incubated in host tissue homogenate and photosynthate release again measured with 14C. The %translocation varied from 12-51% in A. aureoradiata homogenate and 17-67% in FSW, again suggesting a lack of an active HRF in the homogenized tissues of this sea anemone. Photosynthetic rates amongst the different heterologous algae also varied widely with, for instance, freshly isolated zooxanthellae from A. aureoradiata having 6-fold higher photosynthetic rates than cultured algae from the same clade (clade A). The zooxanthellae of A. aureoradiata are known to be N-sufficient in the field, and studies with other species have demonstrated that N-deficient zooxanthellae release more photosynthate in response to HRF than do N-sufficient ones. Therefore, induction of an HRF effect was attempted by starving sea anemones, and hence their zooxanthellae, prior to incubation of freshly isolated zooxanthellae in homogenized tissue. However, even after 8 weeks of starvation, the zooxanthellae showed no signs of N-deficiency (as indicated by the extent to which ammonium enhanced the rate of dark 14C fixation), meaning that the relationship with HRF activity could not be examined. The ability of these temperate zooxanthellae to maintain their Nsufficiency, even after relatively long periods of food deprivation, may indicate a lower reliance on host feeding for nitrogen than is seen in tropical zooxanthellae, or a greater capacity to use internal stores of nitrogen. The lack of photosynthate release by both homologous and heterologous zooxanthellae in host homogenate, as opposed to substantial carbon released in the intact symbiosis, suggests that control of carbon translocation in A. aureoradiata is not related to the activity of an HRF; alternatively, if an HRF is present, its activity is hindered when the symbiosis is disrupted. Further study is needed to determine what is responsible for the control of photosynthate translocation in the A. aureoradiata-Symbiodinium symbiosis.</p>

The World of Algae Reveals a Broad Variety of Cryptochrome Properties and Functions

Algae are photosynthetic eukaryotic (micro-)organisms, lacking roots, leaves, and other organs that are typical for land plants. They live in freshwater, marine, or terrestrial habitats. Together with the cyanobacteria they contribute to about half of global carbon fixation. As primary producers, they are at the basis of many food webs and they are involved in biogeochemical processes. Algae are evolutionarily distinct and are derived either by primary (e.g., green and red algae) or secondary endosymbiosis (e.g., diatoms, dinoflagellates, and brown algae). Light is a key abiotic factor needed to maintain the fitness of algae as it delivers energy for photosynthesis, regulates algal cell- and life cycles, and entrains their biological clocks. However, excess light can also be harmful, especially in the ultraviolet range. Among the variety of receptors perceiving light information, the cryptochromes originally evolved as UV-A and blue-light receptors and have been found in all studied algal genomes so far. Yet, the classification, biophysical properties, wavelength range of absorbance, and biological functions of cryptochromes are remarkably diverse among algal species, especially when compared to cryptochromes from land plants or animals.

Microcystin bioaccumulation in Limnoperna fortunei following Microcystis aeruginosa exposure, analysis of in vivo enzymatic phosphatase, acetylcholinesterase and carboxylesterase effects and in vitro experiments

Toxic cyanobacteria blooms have been reported in freshwater sources worldwide and may lead to aquatic biota toxin accumulation and trophic chain transfer, resulting in ecological and public health concerns. To assess cyanobacteria effects on microcystin uptake and accumulation and on phosphatase, acethylcholinesterase (AChE) and carboxylesterase (CarbE) enzymatic activities, an in vivo experiment was carried out employing the golden mussel Limnoperna fortunei. These mussels were exposed to a Microcystis aeruginosa NPLJ-4 strain (NPLJ-4) for 48 hours at different cell densities. Subsequently, algal cell counts were carried out and enzymatic activities were assayed. All three enzymes (Phosphatase, AChE and CarbE) were inhibited at the end of the exposure experiment. Mussels exposed to higher in vivo M. aeruginosa densities exhibited microcystin uptake and accumulation. In vitro assays were also carried out, exposing soluble L. fortunei enzyme fractions to M. aeruginosa extracts containing microcystin, and phosphatase inhibition was observed, whereas acetylcholinesterase and carboxylesterase were not inhibited. The results indicate that metabolites other than mycrocystin probably caused the observed in vivo esterase inhibitions, requiring further investigations.