Feedback from the Science Café at the 7th European Bioanalysis Forum Young Scientist Symposium.

The 7th Young Scientist Symposium, a meeting again organized as a hybrid online event by young scientists for young scientists under the umbrella of the European Bioanalysis Forum and in collaboration with the Universities of Bologna and Ghent, included a variety of interesting presentations on cutting-edge bioanalytical science and processes. On the morning of day 2, the meeting hosted their traditional Science Café around the theme: 'How has COVID-19 changed our future?' in which the Young Scientist Symposium organizing committee engaged with the delegates on how the COVID-19 pandemic has impacted the careers of young scientists working in a bioanalytical (industry or academic) laboratory, that is, things they lost, for good or for bad - things they gained, wanted or unwanted, things they learned about themselves and their industry. This manuscript provides feedback from those discussions.

Feedback from the Sixth European Bioanalysis Forum Young Scientist Symposium.

As part of the European Bioanalysis Forum mission to provide development opportunities for scientists, a Young Scientist Symposium has been organized every year since 2014. The meetings, organized by and for young scientists, aim at immersing talent from industry and academia in the scientific and process challenges important for their (future) professional environment. In an ideal world, the setting of an interactive symposium in stimulating auditorium sets the foundation for long lasting peer scientific relationship. This year, a pandemic has descended across all continents, changing the dynamics of the meeting. This manuscript summarizes the discussions at the Sixth EBF Young Scientist Symposium, originally planned as a face-to face event in March 2020 in Bologna, Italy but finally executed as a hybrid meeting in Cyberspace and on location in a few regions across Europe between 24-25 September 2020.

Feedback from the Science Café from the Sixth European Bioanalysis Forum Young Scientist Symposium.

The 6th Young Scientist Symposium, a meeting organized by young scientists for young scientists under the umbrella of the European Bioanalysis Forum vzw and in collaboration with the Universities of Bologna and Ghent, included a variety of interesting presentations on cutting edge bioanalytical science and processes. Integrated in the meeting, an interactive round table session, the Science Café, discussed the challenges related to sustainability for bioanalytical lab activities. This manuscript reflects conclusions from these discussions. They can provide our community a compass for future business practices to embrace more sustainable laboratory activities considerate of smarter use of a wide array of resources and laboratory tools, resulting in increased wellbeing for our next generations and our planet.

Ocean acidification has little effect on the biochemical composition of the coccolithophore Emiliania huxleyi.

Owing to the hierarchical organization of biology, from genomes over transcriptomes and proteomes down to metabolomes, there is continuous debate about the extent to which data and interpretations derived from one level, e.g. the transcriptome, are in agreement with other levels, e.g. the metabolome. Here, we tested the effect of ocean acidification (OA; 400 vs. 1000 µatm CO2) and its modulation by light intensity (50 vs. 300 µmol photons m-2 s-1) on the biomass composition (represented by 75 key metabolites) of diploid and haploid life-cycle stages of the coccolithophore Emiliania huxleyi (RCC1216 and RCC1217) and compared these data with interpretations from previous physiological and gene expression screenings. The metabolite patterns showed minor responses to OA in both life-cycle stages. Whereas previous gene expression analyses suggested that the observed increased biomass buildup derived from lipid and carbohydrate storage, this dataset suggests that OA slightly increases overall biomass of cells, but does not significantly alter their metabolite composition. Generally, light was shown to be a more dominant driver of metabolite composition than OA, increasing the relative abundances of amino acids, mannitol and storage lipids, and shifting pigment contents to accommodate increased irradiance levels. The diploid stage was shown to contain vastly more osmolytes and mannitol than the haploid stage, which in turn had a higher relative content of amino acids, especially aromatic ones. Besides the differences between the investigated cell types and the general effects on biomass buildup, our analyses indicate that OA imposes only negligible effects on E. huxleyi´s biomass composition.

Synthetic metabolic pathways for photobiological conversion of CO2 into hydrocarbon fuel.

Liquid fuels sourced from fossil sources are the dominant energy form for mobile transport today. The consumption of fossil fuels is still increasing, resulting in a continued search for more sustainable methods to renew our supply of liquid fuel. Photosynthetic microorganisms naturally accumulate hydrocarbons that could serve as a replacement for fossil fuel, however productivities remain low. We report successful introduction of five synthetic metabolic pathways in two green cell factories, prokaryotic cyanobacteria and eukaryotic algae. Heterologous thioesterase expression enabled high-yield conversion of native fatty acyl-acyl carrier protein (ACP) into free fatty acids (FFA) in Synechocystis sp. PCC 6803 but not in Chlamydomonas reinhardtii where the polar lipid fraction instead was enhanced. Despite no increase in measurable FFA in Chlamydomonas, genetic recoding and over-production of the native fatty acid photodecarboxylase (FAP) resulted in increased accumulation of 7-heptadecene. Implementation of a carboxylic acid reductase (CAR) and aldehyde deformylating oxygenase (ADO) dependent synthetic pathway in Synechocystis resulted in the accumulation of fatty alcohols and a decrease in the native saturated alkanes. In contrast, the replacement of CAR and ADO with Pseudomonas mendocina UndB (so named as it is responsible for 1-undecene biosynthesis in Pseudomonas) or Chlorella variabilis FAP resulted in high-yield conversion of thioesterase-liberated FFAs into corresponding alkenes and alkanes, respectively. At best, the engineering resulted in an increase in hydrocarbon accumulation of 8- (from 1 to 8.5 mg/g cell dry weight) and 19-fold (from 4 to 77 mg/g cell dry weight) for Chlamydomonas and Synechocystis, respectively. In conclusion, reconstitution of the eukaryotic algae pathway in the prokaryotic cyanobacteria host generated the most effective system, highlighting opportunities for mix-and-match synthetic metabolism. These studies describe functioning synthetic metabolic pathways for hydrocarbon fuel synthesis in photosynthetic microorganisms for the first time, moving us closer to the commercial implementation of photobiocatalytic systems that directly convert CO2 into infrastructure-compatible fuels.

Metabolic survey of Botryococcus braunii: Impact of the physiological state on product formation.

The microalga Botryococcus braunii is widely regarded as a potential renewable and sustainable source for industrial applications because of its capability to produce large amounts of metabolically expensive (exo-) polysaccharides and lipids, notably hydrocarbons. A comprehensive and systematic metabolic characterization of the Botryococcus braunii race A strain CCAP 807/2 was conducted within the present study, including the detailed analysis of growth-associated and physiological parameters. In addition, the intracellular metabolome was profiled for the first time and showed growth- and product-specific fluctuations in response to the different availability of medium resources during the cultivation course. Among the identified metabolites, a constant expression of raffinose was observed for the first time under standard conditions, which has until now only been described for higher plants. Overall, the multilayered analysis during the cultivation of strain CCAP 807/2 allowed the differentiation of four distinct physiological growth phases and revealed differences in the production profiles and content of liquid hydrocarbons and carbohydrates with up to 84% of organic dry weight (oDW). In the process, an enhanced production of carbohydrates with up to 63% of oDW (1.36±0.03 g L-1) could be observed during the late linear growth phase, whereas the highest accumulation of extracellular hydrocarbons with up to 24% of oDW (0.66±0.12 g L-1) occurred mainly during the stationary growth phase. Altogether, the knowledge obtained is potentially useful for the general understanding of the overall physiology of Botryococcus braunii and provide important insights into the growth behavior and product formation of this microalga, and is thus relevant for large scale biofuel production and industrial applications.

Label-free in vivo analysis of intracellular lipid droplets in the oleaginous microalga Monoraphidium neglectum by coherent Raman scattering microscopy.

Oleaginous photosynthetic microalgae hold great promise as non-food feedstocks for the sustainable production of bio-commodities. The algal lipid quality can be analysed by Raman micro-spectroscopy, and the lipid content can be imaged in vivo in a label-free and non-destructive manner by coherent anti-Stokes Raman scattering (CARS) microscopy. In this study, both techniques were applied to the oleaginous microalga Monoraphidium neglectum, a biotechnologically promising microalga resistant to commonly applied lipid staining techniques. The lipid-specific CARS signal was successfully separated from the interfering two-photon excited fluorescence of chlorophyll and for the first time, lipid droplet formation during nitrogen starvation could directly be analysed. We found that the neutral lipid content deduced from CARS image analysis strongly correlated with the neutral lipid content measured gravimetrically and furthermore, that the relative degree of unsaturation of fatty acids stored in lipid droplets remained similar. Interestingly, the lipid profile during cellular adaption to nitrogen starvation showed a two-phase characteristic with initially fatty acid recycling and subsequent de novo lipid synthesis. This works demonstrates the potential of quantitative CARS microscopy as a label-free lipid analysis technique for any microalgal species, which is highly relevant for future biotechnological applications and to elucidate the process of microalgal lipid accumulation.

Efficient phototrophic production of a high-value sesquiterpenoid from the eukaryotic microalga Chlamydomonas reinhardtii.

The heterologous expression of terpene synthases in microbial hosts has opened numerous possibilities for bioproduction of desirable metabolites. Photosynthetic microbial hosts present a sustainable alternative to traditional fermentative systems, using freely available (sun)light and carbon dioxide as inputs for bio-production. Here, we report the expression of a patchoulol synthase from Pogostemon cablin Benth in the model green microalga Chlamydomonas reinhardtii. The sesquiterpenoid patchoulol was produced from the alga and was used as a marker of sesquiterpenoid production capacity. A novel strategy for gene loading was employed and patchoulol was produced up to 922±242µgg-1 CDW in six days. We additionally investigated the effect of carbon source on sesquiterpenoid productivity from C. reinhardtii in scale-up batch cultivations. It was determined that up to 1.03mgL-1 sesquiterpenoid products could be produced in completely photoautotrophic conditions and that the alga exhibited altered sesquiterpenoid production metabolism related to carbon source.