Enabling large-scale production of algal oil in continuous output mode.

Large-scale algal oil production requires continuous outputs and a trade-off between growth and oil content. Two unrelated marine algae (Nannochloropsis oceanica [CCAP 849/10] and Chlorella vulgaris [CCAP 211/21A]) that showed high oil production under batch culture were studied under controlled semicontinuous cultivation conditions. Three essential attributes maximized oil productivity: (i) downregulation of cell size to maximize light absorption under N limitation; (ii) low nutrient-depletion thresholds to trigger oil induction; (iii) a means of carbohydrate suppression in favor of oil. N. oceanica responded better to input N/P variations and is more suited to continuous oil production. A low N/P ratio was effective in both suppressing carbohydrate and reducing cell size concomitant with oil production. In C. vulgaris, nutrient starvation thresholds for oil were higher and carbohydrate was preferentially induced, which impeded stress-level optimization for oil. These differences, which impact continuous oil production at scale, are driven by species adaptation to specific marine habitats.

Effects of cryopreservation on viability and functional stability of an industrially relevant alga.

As algal biotechnology develops, there is an increasing requirement to conserve cultures without the cost, time and genetic stability implications of conventional serial transfers, including issues regarding potential loss by failure to regrow, contamination on transfer, mix up and/or errors in the documentation on transfer. Furthermore, it is crucial to ensure both viability and functionality are retained by stored stock-cultures. Low temperature storage, ranging from the use of domestic freezers to storage under liquid nitrogen, is widely being used, but the implication to stability and function rarely investigated. We report for the first time, retention of functionality in the maintenance of master stock-cultures of an industrially relevant, lipid-producing alga, under a variety of cryopreservation regimes. Storage in domestic (-15 °C), or conventional -80 °C freezers was suboptimal, with a rapid reduction in viability observed for samples at -15 °C and a >50% loss of viability, within one month, for samples stored at -80 °C. No reduction in viability occurred at -196 °C. Post-thaw culture functional performance was also influenced by the cryopreservation approach employed. Only samples held at -196 °C responded to nitrogen limitation in terms of growth characteristics and biochemical profiles (lipid production and chlorophyll a) comparable to the untreated control, cultured prior to cryopreservation. These results have important implications in microbial biotechnology, especially for those responsible for the conservation of genetic resources.

Snow algae communities in Antarctica: metabolic and taxonomic composition.

Snow algae are found in snowfields across cold regions of the planet, forming highly visible red and green patches below and on the snow surface. In Antarctica, they contribute significantly to terrestrial net primary productivity due to the paucity of land plants, but our knowledge of these communities is limited. Here we provide the first description of the metabolic and species diversity of green and red snow algae communities from four locations in Ryder Bay (Adelaide Island, 68°S), Antarctic Peninsula. During the 2015 austral summer season, we collected samples to measure the metabolic composition of snow algae communities and determined the species composition of these communities using metabarcoding. Green communities were protein-rich, had a high chlorophyll content and contained many metabolites associated with nitrogen and amino acid metabolism. Red communities had a higher carotenoid content and contained more metabolites associated with carbohydrate and fatty acid metabolism. Chloromonas, Chlamydomonas and Chlorella were found in green blooms but only Chloromonas was detected in red blooms. Both communities also contained bacteria, protists and fungi. These data show the complexity and variation within snow algae communities in Antarctica and provide initial insights into the contribution they make to ecosystem functioning.

Energetic equivalence underpins the size structure of tree and phytoplankton communities.

The size structure of autotroph communities - the relative abundance of small vs. large individuals - shapes the functioning of ecosystems. Whether common mechanisms underpin the size structure of unicellular and multicellular autotrophs is, however, unknown. Using a global data compilation, we show that individual body masses in tree and phytoplankton communities follow power-law distributions and that the average exponents of these individual size distributions (ISD) differ. Phytoplankton communities are characterized by an average ISD exponent consistent with three-quarter-power scaling of metabolism with body mass and equivalence in energy use among mass classes. Tree communities deviate from this pattern in a manner consistent with equivalence in energy use among diameter size classes. Our findings suggest that whilst universal metabolic constraints ultimately underlie the emergent size structure of autotroph communities, divergent aspects of body size (volumetric vs. linear dimensions) shape the ecological outcome of metabolic scaling in forest vs. pelagic ecosystems.

Effect of ammonium and high light intensity on the accumulation of lipids in Nannochloropsis oceanica (CCAP 849/10) and Phaeodactylum tricornutum (CCAP 1055/1).

BACKGROUND: Microalgae accumulate lipids when exposed to stressful conditions such as nutrient limitation that can be used to generate biofuels. Nitrogen limitation or deprivation is a strategy widely employed to elicit this response. However, this strategy is associated with a reduction in the microalgal growth, leading to overall poor lipid productivities. Here, we investigated the combined effect of a reduced source of nitrogen (ammonium) and super-saturating light intensities on the growth and induction of lipid accumulation in two model but diverse microalgal species, Phaeodactylum tricornutum and Nannochloropsis oceanica. We hypothesized that the lower energy cost of assimilating ammonium would allow the organisms to use more reductant power for lipid biosynthesis without compromising growth and that this would be further stimulated by the effect of high light (1000 µmol m-2 s-1) stress. We studied the changes in growth and physiology of both species when grown in culture media that either contained nitrate or ammonium as the nitrogen source, and an additional medium that contained ammonium with tungsten in place of molybdenum and compared this with growth in media without nitrogen. We focused our investigation on the early stages of exposure to the treatments to correspond to events relevant to induction of lipid accumulation in these two species. RESULTS: At super-saturating light intensities, lipid productivity in P. tricornutum increased twofold when grown in ammonium compared to nitrogen free medium that increased further when tungsten was present in the medium in place of molybdenum. Conversely, N. oceanica growth and physiology was not compromised by the high light intensities used, and the use of ammonium had a negative effect on the lipid productivity, which was even more marked when tungsten was present. CONCLUSIONS: Whilst the use of ammonium and super-saturating light intensities in P. tricornutum was revealed to be a good strategy for increasing lipid biosynthesis, no changes in the lipid productivity of N. oceanica were observed, under these conditions. Both results provide relevant direction for the better design of processes to produce biofuels in microalgae by manipulating growth conditions without the need to subject them to genetic engineering manipulation.

Physiological responses of coccolithophores to abrupt exposure of naturally low pH deep seawater.

Upwelling is the process by which deep, cold, relatively high-CO2, nutrient-rich seawater rises to the sunlit surface of the ocean. This seasonal process has fueled geoengineering initiatives to fertilize the surface ocean with deep seawater to enhance productivity and thus promote the drawdown of CO2. Coccolithophores, which inhabit many upwelling regions naturally 'fertilized' by deep seawater, have been investigated in the laboratory in the context of ocean acidification to determine the extent to which nutrients and CO2 impact their physiology, but few data exist in the field except from mesocosms. Here, we used the Porcupine Abyssal Plain (north Atlantic Ocean) Observatory to retrieve seawater from depths with elevated CO2 and nutrients, mimicking geoengineering approaches. We tested the effects of abrupt natural deep seawater fertilization on the physiology and biogeochemistry of two strains of Emiliania huxleyi of known physiology. None of the strains tested underwent cell divisions when incubated in waters obtained from <1,000 m (pH = 7.99-8.08; CO2 = 373-485 p.p.m; 1.5-12 µM nitrate). However, growth was promoted in both strains when cells were incubated in seawater from ~1,000 m (pH = 7.9; CO2 ~560 p.p.m.; 14-17 µM nitrate) and ~4,800 m (pH = 7.9; CO2 ~600 p.p.m.; 21 µM nitrate). Emiliania huxleyi strain CCMP 88E showed no differences in growth rate or in cellular content or production rates of particulate organic (POC) and inorganic (PIC) carbon and cellular particulate organic nitrogen (PON) between treatments using water from 1,000 m and 4,800 m. However, despite the N:P ratio of seawater being comparable in water from ~1,000 and ~4,800 m, the PON production rates were three times lower in one incubation using water from ~1,000 m compared to values observed in water from ~4,800 m. Thus, the POC:PON ratios were threefold higher in cells that were incubated in ~1,000 m seawater. The heavily calcified strain NZEH exhibited lower growth rates and PIC production rates when incubated in water from ~4,800 m compared to ~1,000 m, while cellular PIC, POC and PON were higher in water from 4,800 m. Calcite Sr/Ca ratios increased with depth despite constant seawater Sr/Ca, indicating that upwelling changes coccolith geochemistry. Our study provides the first experimental and field trial of a geoengineering approach to test how deep seawater impacts coccolithophore physiological and biogeochemical properties. Given that coccolithophore growth was only stimulated using waters obtained from >1,000 m, artificial upwelling using shallower waters may not be a suitable approach for promoting carbon sequestration for some locations and assemblages, and should therefore be investigated on a site-by-site basis.

Proteome response of Phaeodactylum tricornutum, during lipid accumulation induced by nitrogen depletion.

Nitrogen stress is a common strategy employed to stimulate lipid accumulation in microalgae, a biofuel feedstock of topical interest. Although widely investigated, the underlying mechanism of this strategy is still poorly understood. We examined the proteome response of lipid accumulation in the model diatom, Phaeodactylum tricornutum (CCAP 1055/1), at an earlier stage of exposure to selective nitrogen exclusion than previously investigated, and at a time point when changes would reflect lipid accumulation more than carbohydrate accumulation. In total 1043 proteins were confidently identified (≥ 2 unique peptides) with 645 significant (p < 0.05) changes observed, in the LC-MS/MS based iTRAQ investigation. Analysis of significant changes in KEGG pathways and individual proteins showed that under nitrogen starvation P. tricornutum reorganizes its proteome in favour of nitrogen scavenging and reduced lipid degradation whilst rearranging the central energy metabolism that deprioritizes photosynthetic pathways. By doing this, this species appears to increase nitrogen availability inside the cell and limit its use to the pathways where it is needed most. Compared to previously published proteomic analysis of nitrogen starvation in Chlamydomonas reinhardtii, central energy metabolism and photosynthesis appear to be affected more in the diatom, whilst the green algae appears to invest its energy in reorganizing respiration and the cellular organization pathways.

Resource supply overrides temperature as a controlling factor of marine phytoplankton growth.

The universal temperature dependence of metabolic rates has been used to predict how ocean biology will respond to ocean warming. Determining the temperature sensitivity of phytoplankton metabolism and growth is of special importance because this group of organisms is responsible for nearly half of global primary production, sustains most marine food webs, and contributes to regulate the exchange of CO2 between the ocean and the atmosphere. Phytoplankton growth rates increase with temperature under optimal growth conditions in the laboratory, but it is unclear whether the same degree of temperature dependence exists in nature, where resources are often limiting. Here we use concurrent measurements of phytoplankton biomass and carbon fixation rates in polar, temperate and tropical regions to determine the role of temperature and resource supply in controlling the large-scale variability of in situ metabolic rates. We identify a biogeographic pattern in phytoplankton metabolic rates, which increase from the oligotrophic subtropical gyres to temperate regions and then coastal waters. Variability in phytoplankton growth is driven by changes in resource supply and appears to be independent of seawater temperature. The lack of temperature sensitivity of realized phytoplankton growth is consistent with the limited applicability of Arrhenius enzymatic kinetics when substrate concentrations are low. Our results suggest that, due to widespread resource limitation in the ocean, the direct effect of sea surface warming upon phytoplankton growth and productivity may be smaller than anticipated.

Unimodal size scaling of phytoplankton growth and the size dependence of nutrient uptake and use.

Phytoplankton size structure is key for the ecology and biogeochemistry of pelagic ecosystems, but the relationship between cell size and maximum growth rate (µ(max) ) is not yet well understood. We used cultures of 22 species of marine phytoplankton from five phyla, ranging from 0.1 to 10(6) µm(3) in cell volume (V(cell) ), to determine experimentally the size dependence of growth, metabolic rate, elemental stoichiometry and nutrient uptake. We show that both µ(max) and carbon-specific photosynthesis peak at intermediate cell sizes. Maximum nitrogen uptake rate (V(maxN) ) scales isometrically with V(cell) , whereas nitrogen minimum quota scales as V(cell) (0.84) . Large cells thus possess high ability to take up nitrogen, relative to their requirements, and large storage capacity, but their growth is limited by the conversion of nutrients into biomass. Small species show similar volume-specific V(maxN) compared to their larger counterparts, but have higher nitrogen requirements. We suggest that the unimodal size scaling of phytoplankton growth arises from taxon-independent, size-related constraints in nutrient uptake, requirement and assimilation.

Isometric size-scaling of metabolic rate and the size abundance distribution of phytoplankton.

The relationship between phytoplankton cell size and abundance has long been known to follow regular, predictable patterns in near steady-state ecosystems, but its origin has remained elusive. To explore the linkage between the size-scaling of metabolic rate and the size abundance distribution of natural phytoplankton communities, we determined simultaneously phytoplankton carbon fixation rates and cell abundance across a cell volume range of over six orders of magnitude in tropical and subtropical waters of the Atlantic Ocean. We found an approximately isometric relationship between carbon fixation rate and cell size (mean slope value: 1.16; range: 1.03-1.32), negating the idea that Kleiber's law is applicable to unicellular autotrophic protists. On the basis of the scaling of individual resource use with cell size, we predicted a reciprocal relationship between the size-scalings of phytoplankton metabolic rate and abundance. This prediction was confirmed by the observed slopes of the relationship between phytoplankton abundance and cell size, which have a mean value of -1.15 (range: -1.29 to -0.97), indicating that the size abundance distribution largely results from the size-scaling of metabolic rate. Our results imply that the total energy processed by carbon fixation is constant along the phytoplankton size spectrum in near steady-state marine ecosystems.