Scale-down of oxygen and glucose fluctuations in a tubular photobioreactor operated under oxygen-balanced mixotrophy.

Oxygen-balanced mixotrophy (OBM) is a novel type of microalgal cultivation that improves autotrophic productivity while reducing aeration costs and achieving high biomass yields on substrate. The scale-up of this process is not straightforward, as nonideal mixing in large photobioreactors might have unwanted effects in cell physiology. We simulated at lab scale dissolved oxygen and glucose fluctuations in a tubular photobioreactor operated under OBM where glucose is injected at the beginning of the tubular section. We ran repeated batch experiments with the strain Galdieria sulphuraria ACUF 064 under glucose pulse feeding of different lengths, representing different retention times: 112, 71, and 21 min. During the long and medium tube retention time simulations, dissolved oxygen was depleted 15-25 min after every glucose pulse. These periods of oxygen limitation resulted in the accumulation of coproporphyrin III in the supernatant, an indication of disruption in the chlorophyll synthesis pathway. Accordingly, the absorption cross-section of the cultures decreased steeply, going from values of 150-180 m2 kg-1 at the end of the first batch down to 50-70 m2 kg-1 in the last batches of both conditions. In the short tube retention time simulation, dissolved oxygen always stayed above 10% air saturation and no pigment reduction nor coproporphyrin III accumulation were observed. Concerning glucose utilization efficiency, glucose pulse feeding caused a reduction of biomass yield on substrate in the range of 4%-22% compared to the maximum levels previously obtained with continuous glucose feeding (0.9 C-g C-g-1 ). The missing carbon was excreted to the supernatant as extracellular polymeric substances constituted by carbohydrates and proteins. Overall, the results point out the importance of studying large-scale conditions in a controlled environment and the need for a highly controlled glucose feeding strategy in the scale-up of mixotrophic cultivation.

Towards industrial production of microalgae without temperature control: The effect of diel temperature fluctuations on microalgal physiology.

Regions that offer high levels of sunlight are ideal to produce microalgae. However, as a result of high light intensities, the temperature in photobioreactors can reach temperatures up to 50 °C. Control of temperature is essential to avoid losses on biomass productivity but should be limited to a minimum to avoid high energy requirements for cooling. Our objective is to develop a production process in which cooling is not required. We studied the behaviour of thermotolerant microalgae Picochlorum sp. (BPE23) under four diel temperature regimes, with peak temperatures from 30 °C up to a maximum of 47.5 °C. The highest growth rate of 0.17 h-1 was obtained when applying a daytime peak temperature of 40 °C. Operating photobioreactors in tropical regions, with a maximal peak temperature of 40 °C, up from 30 °C, reduces microalgae production costs by 26.2 %, based on simulations with a pre-existing techno-economic model. Cell pigmentation was downregulated under increasingly stressful temperatures. The fatty acid composition of cell membranes was altered under increasing temperatures to contain shorter fatty acids with a higher level of saturation. Our findings show that the level of temperature control impacts the biomass yield and composition of the microalgae.

Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants.

Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was ∼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 µg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19. IMPORTANCE Vaccination is essential to reduce disease severity and limit the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protein-based vaccines are useful to vaccinate the world population and to boost immunity against emerging variants. Their safety profiles, production costs, and vaccine storage temperatures are advantageous compared to mRNA and adenovirus vector vaccines. Here, we use the versatile and scalable baculovirus expression vector system to generate a two-component nanoparticle vaccine to induce potent neutralizing antibody responses against SARS-CoV-2 variants. These nanoparticle vaccines can be quickly adapted as boosters by simply updating the antigen component.

Analysis of fatty acid content and composition in microalgae.

A method to determine the content and composition of total fatty acids present in microalgae is described. Fatty acids are a major constituent of microalgal biomass. These fatty acids can be present in different acyl-lipid classes. Especially the fatty acids present in triacylglycerol (TAG) are of commercial interest, because they can be used for production of transportation fuels, bulk chemicals, nutraceuticals (ω-3 fatty acids), and food commodities. To develop commercial applications, reliable analytical methods for quantification of fatty acid content and composition are needed. Microalgae are single cells surrounded by a rigid cell wall. A fatty acid analysis method should provide sufficient cell disruption to liberate all acyl lipids and the extraction procedure used should be able to extract all acyl lipid classes. With the method presented here all fatty acids present in microalgae can be accurately and reproducibly identified and quantified using small amounts of sample (5 mg) independent of their chain length, degree of unsaturation, or the lipid class they are part of. This method does not provide information about the relative abundance of different lipid classes, but can be extended to separate lipid classes from each other. The method is based on a sequence of mechanical cell disruption, solvent based lipid extraction, transesterification of fatty acids to fatty acid methyl esters (FAMEs), and quantification and identification of FAMEs using gas chromatography (GC-FID). A TAG internal standard (tripentadecanoin) is added prior to the analytical procedure to correct for losses during extraction and incomplete transesterification.