The adaptive mechanisms of the marine diatom Thalassiosira weissflogii to long-term high CO2 and warming.

While it is known that increased dissolved CO2 concentrations and rising sea surface temperature (ocean warming) can act interactively on marine phytoplankton, the ultimate molecular mechanisms underlying this interaction on a long-term evolutionary scale are relatively unexplored. Here, we performed transcriptomics and quantitative metabolomics analyses, along with a physiological trait analysis, on the marine diatom Thalassiosira weissflogii adapted for approximately 3.5 years to warming and/or high CO2 conditions. We show that long-term warming has more pronounced impacts than elevated CO2 on gene expression, resulting in a greater number of differentially expressed genes (DEGs). The largest number of DEGs was observed in populations adapted to warming + high CO2, indicating a potential synergistic interaction between these factors. We further identified the metabolic pathways in which the DEGs function and the metabolites with significantly changed abundances. We found that ribosome biosynthesis-related pathways were upregulated to meet the increased material and energy demands after warming or warming in combination with high CO2. This resulted in the upregulation of energy metabolism pathways such as glycolysis, photorespiration, the tricarboxylic acid cycle, and the oxidative pentose phosphate pathway, as well as the associated metabolites. These metabolic changes help compensate for reduced photochemical efficiency and photosynthesis. Our study emphasizes that the upregulation of ribosome biosynthesis plays an essential role in facilitating the adaptation of phytoplankton to global ocean changes and elucidates the interactive effects of warming and high CO2 on the adaptation of marine phytoplankton in the context of global change.

Engineered production of isoprene from the model green microalga Chlamydomonas reinhardtii.

Isoprene is a clear, colorless, volatile 5-carbon hydrocarbon that is one monomer of all cellular isoprenoids and a platform chemical with multiple applications in industry. Many plants have evolved isoprene synthases (IspSs) with the capacity to liberate isoprene from dimethylallyl diphosphate (DMADP) as part of cellular thermotolerance mechanisms. Isoprene is hydrophobic and volatile, rapidly leaves plant tissues and is one of the main carbon emission sources from vegetation globally. The universality of isoprenoid metabolism allows volatile isoprene production from microbes expressing heterologous IspSs. Here, we compared heterologous overexpression from the nuclear genome and localization into the plastid of four plant terpene synthases (TPs) in the green microalga Chlamydomonas reinhardtii. Using sealed vial mixotrophic cultivation, direct quantification of isoprene production was achieved from the headspace of living cultures, with the highest isoprene production observed in algae expressing the Ipomoea batatas IspS. Perturbations of the downstream carotenoid pathway through keto carotenoid biosynthesis enhanced isoprene titers, which could be further enhanced by increasing flux towards DMADP through heterologous co-expression of a yeast isopentenyl-DP delta isomerase. Multiplexed controlled-environment testing revealed that cultivation temperature, rather than illumination intensity, was the main factor affecting isoprene yield from the engineered alga. This is the first report of heterologous isoprene production from a eukaryotic alga and sets a foundation for further exploration of carbon conversion to this commodity chemical.

Biomass generation and heterologous isoprenoid milking from engineered microalgae grown in anaerobic membrane bioreactor effluent.

Wastewater (WW) treatment in anaerobic membrane bioreactors (AnMBR) is considered more sustainable than in aerobic reactors. However, outputs from AnMBR are a mixed methane and carbon dioxide gas stream as well as ammonium- (N) and phosphate- (P) containing waters. Using AnMBR outputs as inputs for photoautotrophic algal cultivation can strip the CO2 while removing N and P from effluent which feed algal biomass generation. Recent advances in algal engineering have generated strains that produce high-value side products concomitant with biomass, although only shown in heavily domesticated, lab-adapted strains. Here, it was investigated whether engineered Chlamydomonas reinhardtii could be grown directly in AnMBR effluent with CO2 concentrations found in AnMBR off-gas. The strain was found to proliferate over bacteria in the non-sterile effluent, consume N and P to levels that meet general discharge or reuse limits, and tolerate cultivation in modelled (extreme) outdoor environmental conditions prevalent along the central Red Sea coast. In addition to ∼2.4 g CDW L-1 biomass production in 96 h, a high-value heterologous sesquiterpene co-product could be obtained from 'milking' up to 837 µg L-1 culture in 96 h. This is the first demonstration of a combined bio-process that employs a heavily engineered algal strain to enhance the product generation potentials from AnMBR effluent treatment. This study shows it is possible to convert waste into value through use of engineered algae while also improving wastewater treatment economics through co-product generation.

Multi-trait analysis reveals large interspecific differences for phytoplankton in response to thermal change.

Understanding the responses of multiple traits in phytoplankton, and identifying interspecific variabilities to thermal changes is crucial for predicting the impacts of ocean warming on phytoplankton distributions and community structures in future scenarios. Here, we applied a trait-based approach by examining the patterns in multi-traits variations (eight traits) and interspecific variabilities in five phytoplankton species (two diatoms, three dinoflagellates) in response to a wide range of ecologically relevant temperatures (14-30 °C). Our results show large inter-traits and interspecific variabilities of thermal reaction norms in all of the tested traits. We also found that the interspecific variability exceeded the variations induced by thermal changes. Constrained variations and trade-offs between traits both revealed substantial interspecific differences and shifted as the temperature changed. Our study helps to understand the species-specific response patterns of multiple traits to ocean warming and to investigate the implications of these responses in the context of global change.

Biocompatible fluorocarbon liquid underlays for in situ extraction of isoprenoids from microbial cultures.

Microbial production of heterologous metabolites is now a mature technology in many host organisms, opening new avenues for green production processes of specialty chemicals. At lab scale, petroleum-based hydrophobic bio-compatible solvents like dodecane can be used as a second phase on top of microbial cultures to act as a physical sink for heterologous hydrocarbon products like isoprenoids. However, this approach has significant drawbacks at scale due to the difficulty of handling solvents and their potential contamination with unwanted byproducts of their manufacture. We discovered that synthetic perfluorocarbon liquids (FCs), commonly used for heat transfer, can also act as physical sinks for microbially produced isoprenoid compounds. FCs are stable, inert, and are amenable to direct liquid-liquid extraction with alcohols for rapid product isolation. These liquids are more dense than water and form a lower phase to microbial cultures rather than an upper phase as with other solvents. Their ability to form an under-layer or 'underlay' also enables the cultivation of microbes directly at the FC-culture medium interface via gravity settling, which could open their application for filamentous or mat-forming organisms. We present comparisons of the isoprenoid extraction potential of three commercial FCs: FC-3283, FC-40, and FC-770 with engineered green microalga cultures producing patchoulol, taxadiene, casbene, or 13R(+) manoyl oxide. We demonstrate that FCs are promising alternatives to traditional solvents and open new avenues in bio-process design for microbial heterologous metabolite milking.

Combinatorial Engineering Enables Photoautotrophic Growth in High Cell Density Phosphite-Buffered Media to Support Engineered Chlamydomonas reinhardtii Bio-Production Concepts.

Chlamydomonas reinhardtii has emerged as a powerful green cell factory for metabolic engineering of sustainable products created from the photosynthetic lifestyle of this microalga. Advances in nuclear genome modification and transgene expression are allowing robust engineering strategies to be demonstrated in this host. However, commonly used lab strains are not equipped with features to enable their broader implementation in non-sterile conditions and high-cell density concepts. Here, we used combinatorial chloroplast and nuclear genome engineering to augment the metabolism of the C. reinhardtii strain UVM4 with publicly available genetic tools to enable the use of inorganic phosphite and nitrate as sole sources of phosphorous and nitrogen, respectively. We present recipes to create phosphite-buffered media solutions that enable high cell density algal cultivation. We then combined previously reported engineering strategies to produce the heterologous sesquiterpenoid patchoulol to high titers from our engineered green cell factories and show these products are possible to produce in non-sterile conditions. Our work presents a straightforward means to generate C. reinhardtii strains for broader application in bio-processes for the sustainable generation of products from green microalgae.

Additive impacts of ocean acidification and ambient ultraviolet radiation threaten calcifying marine primary producers.

Ocean acidification (OA) represents a threat to marine organisms and ecosystems. However, OA rarely exists in isolation but occurs concomitantly with other stressors such as ultraviolet radiation (UVR), whose effects have been neglected in oceanographical observations. Here, we perform a quantitative meta-analysis based on 373 published experimental assessments from 26 studies to examine the combined effects of OA and UVR on marine primary producers. The results reveal predominantly additive stressor interactions (69-84% depending on the UV waveband), with synergistic and antagonistic interactions being rare but significantly different between micro- and macro-algae. In microalgae, variations in interaction type frequencies are related to cell volume, with antagonistic interactions accounting for a higher proportion in larger sized species. Despite additive interactions being most frequent, the small proportion of antagonistic interactions appears to have a stronger power, leading to neutral effects of OA in combination with UVR. High levels of UVR at near in situ conditions in combination with OA showed additive inhibition of calcification, but not when UVR was low. The results also reveal that the magnitude of responses is strongly dependent on experimental duration, with the negative effects of OA on calcification and pigmentation being buffered and amplified by increasing durations, respectively. Tropical primary producers were more vulnerable to OA or UVR alone compared to conspecifics from other climatic regions. Our analysis highlights that further multi-stressor long-term adaptation experiments with marine organisms of different cell volumes (especially microalgae) from different climatic regions are needed to fully disclose future impacts of OA and UVR.

Adaptation of a marine diatom to ocean acidification increases its sensitivity to toxic metal exposure.

Most previous studies investigating the interplay of ocean acidification (OA) and heavy metal on marine phytoplankton were only conducted in short-term, which may provide conservative estimates of the adaptive capacity of them. Here, we examined the physiological responses of long-term (~900 generations) OA-adapted and non-adapted populations of the diatom Phaeodactylum tricornutum to different concentrations of the two heavy metals Cd and Cu. Our results showed that long-term OA selected populations exhibited significantly lower growth and reduced photosynthetic activity than ambient CO2 selected populations at relatively high heavy metal levels. Those findings suggest that the adaptations to high CO2 results in an increased sensitivity of the marine diatom to toxic metal exposure. This study provides evidence for the costs and the cascading consequences associated with the adaptation of phytoplankton to elevated CO2 conditions, and improves our understanding of the complex interactions of future OA and heavy metal pollution in marine waters.

Lipid Remodeling Reveals the Adaptations of a Marine Diatom to Ocean Acidification.

Ocean acidification is recognized as a major anthropogenic perturbation of the modern ocean. While extensive studies have been carried out to explore the short-term physiological responses of phytoplankton to ocean acidification, little is known about their lipidomic responses after a long-term ocean acidification adaptation. Here we perform the lipidomic analysis of a marine diatom Phaeodactylum tricornutum following long-term (∼400 days) selection to ocean acidification conditions. We identified a total of 476 lipid metabolites in long-term high CO2 (i.e., ocean acidification condition) and low CO2 (i.e., ambient condition) selected P. tricornutum cells. Our results further show that long-term high CO2 selection triggered substantial changes in lipid metabolites by down- and up-regulating 33 and 42 lipid metabolites. While monogalactosyldiacylglycerol (MGDG) was significantly down-regulated in the long-term high CO2 selected conditions, the majority (∼80%) of phosphatidylglycerol (PG) was up-regulated. The tightly coupled regulations (positively or negatively correlated) of significantly regulated lipid metabolites suggest that the lipid remodeling is an organismal adaptation strategy of marine diatoms to ongoing ocean acidification. Since the composition and content of lipids are crucial for marine food quality, and these changes can be transferred to high trophic levels, our results highlight the importance of determining the long-term adaptation of lipids in marine producers in predicting the ecological consequences of climate change.

Latitudinal Gradient of UV Attenuation Along the Highly Transparent Red Sea Basin.

The tropical and subtropical oceans experience intense incident ultraviolet radiation (280-400 nm) while their water columns are thought to be highly transparent. This combination represents a high potential for harmful effects on organisms, yet only few reports on the UV penetration properties of oligotrophic tropical waters exist. Here, we present the pattern of UV attenuation over a wide latitudinal range of the oligotrophic Red Sea. We recorded spectroradiometer profiles of PAR and UV, together with chlorophyll-a (Chl-a) and light absorption by chromophoric dissolved organic matter (CDOM) to determine the contribution of phytoplankton and CDOM toward UV attenuation. Transparency to UV exhibited a distinct latitudinal gradient, with the lowest and highest diffuse attenuation coefficients at 313 nm (Kd (313)) of 0.130 m-1 and 0.357 m-1 observed at the northern coast off Duba, and in the south close to the Farasan islands, respectively. Phytoplankton and CDOM both modulated UV attenuation, but CDOM was found to be the key driver despite the lack of riverine inputs. We confirm that ultraviolet radiation can reach deeper into the Red Sea than previously described, which means its potential to act as a stressor and selective driver for Red Sea organisms may have been underestimated to date.

Phototoxic effects of PAH and UVA exposure on molecular responses and developmental success in coral larvae.

Exposure to polycyclic aromatic carbons (PAHs) poses a growing risk to coral reefs due to increasing shipping and petroleum extraction in tropical waters. Damaging effects of specific PAHs can be further enhanced by the presence of ultraviolet radiation, known as phototoxicity. We tested phototoxic effects of the PAHs anthracene and phenanthrene on larvae of the scleractinian coral Acropora tenuis in the presence and absence of UVA (320-400 nm). Activity of superoxide dismutase (SOD) enzyme was reduced by anthracene while phenanthrene and UVA exposure did not have any effect. Gene expression of MnSod remained constant across all treatments. The genes Catalase, Hsp70 and Hsp90 showed increased expression levels in larvae exposed to anthracene, but not phenanthrene. Gene expression of p53 was upregulated in the presence of UVA, but downregulated when exposed to PAHs. The influence on stress-related biochemical pathways and gene expresson in A. tenuis larvae was considerably greater for anthracene than phenanthrene, and UVA-induced phototoxicity was only evident for anthracene. The combined effects of UVA and PAH exposure on larval survival and metamorphosis paralleled the sub-lethal stress responses, clearly highlighting the interaction of UVA on anthracene toxicity and ultimately the coral's development.