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Real-time, detailed online information on cell cultures is essential for understanding modern biopharmaceutical production processes. The determination of key parameters, such as cell density and viability, is usually based on the offline sampling of bioreactors. Gathering offline samples is invasive, has a low time resolution, and risks altering or contaminating the production process. In contrast, measuring process parameters online provides more safety for the process, has a high time resolution, and thus can aid in timely process control actions. We used online double differential digital holographic microscopy (D3HM) and machine learning to perform non-invasive online cell concentration and viability monitoring of insect cell cultures in bioreactors. The performance of D3HM and the machine learning model was tested for a selected variety of baculovirus constructs, products, and multiplicities of infection (MOI). The results show that with online holographic microscopy insect cell proliferation and baculovirus infection can be monitored effectively in real time with high resolution for a broad range of process parameters and baculovirus constructs. The high-resolution data generated by D3HM showed the exact moment of peak cell densities and temporary events caused by feeding. Furthermore, D3HM allowed us to obtain information on the state of the cell culture at the individual cell level. Combining this detailed, real-time information about cell cultures with methodical machine learning models can increase process understanding, aid in decision-making, and allow for timely process control actions during bioreactor production of recombinant proteins.

The potential of sponge-derived chemicals for pharmaceutical applications remains largely unexploited due to limited available biomass. Although many have attempted to culture marine sponge cells in vitro to create a scalable production platform for such biopharmaceuticals, these efforts have been mostly unsuccessful. We recently showed that Geodia barretti sponge cells could divide rapidly in M1 medium. In this study we established the first continuous marine sponge cell line, originating from G. barretti. G. barretti cells cultured in OpM1 medium, a modification of M1, grew more rapidly and to a higher density than in M1. Cells in OpM1 reached 1.74 population doublings after 30 min, more than twofold higher than the already rapid growth rate of 0.74 population doublings in 30 min in M1. The maximum number of population doublings increased from 5 doublings in M1 to at least 98 doublings in OpM1. Subcultured cells could be cryopreserved and used to inoculate new cultures. With these results, we have overcome a major obstacle that has blocked the path to producing biopharmaceuticals with sponge cells at industrial scale for decades.

Chikungunya virus (CHIKV) is a rapidly emerging mosquito-borne virus that causes a severe febrile illness with long-lasting arthralgia in humans. As there is no vaccine to protect humans and limit CHIKV epidemics, the virus continues to be a global public health concern. The CHIKV envelope glycoproteins E1 and E2 are important immunogens; therefore, the aim of this study is to produce trimeric CHIKV spikes in insect cells using the baculovirus expression system. The CHIKV E1 and E2 ectodomains were covalently coupled by a flexible linker that replaces the 6K transmembrane protein. The C-terminal E1 transmembrane was replaced by a Strep-tag II for the purification of secreted spikes from the culture fluid. After production in Sf9 suspension cells (product yields of 5.8–7.6 mg/L), the CHIKV spikes were purified by Strep-Tactin affinity chromatography, which successfully cleared the co-produced baculoviruses. Bis(sulfosuccinimidyl)suberate cross-linking demonstrated that the spikes are secreted as trimers. PNGase F treatment showed that the spikes are glycosylated. LC–MS/MS-based glycoproteomic analysis confirmed the glycosylation and revealed that the majority are of the mannose- or hybrid-type N-glycans and

Process intensification is increasingly used in the mammalian biomanufacturing industry. The key driver of this trend is the need for more efficient and flexible production strategies to cope with the increased demand for biotherapeutics predicted in the next years. Therefore, such intensified production strategies should be designed, established, and characterized. We established a CHO cell process consisting of an intensified fed-batch (iFB), which is inoculated by an N-1 perfusion process that reaches high cell concentrations (100 × 106 c ml−1). We investigated the impact of butyric acid (BA) supplementation in this iFB process. Most prominently, higher cellular productivities of more than 33% were achieved, thus 3.5 g L−1 of immunoglobulin G (IgG) was produced in 6.5 days. Impacts on critical product quality attributes were small. To understand the biological mechanisms of BA in the iFB process, we performed a detailed transcriptomic analysis. Affected gene sets reflected concurrent inhibition of cell proliferation and impact on histone modification. These translate into subsequently enhanced mechanisms of protein biosynthesis: enriched regulation of transcription, messenger RNA processing and transport, ribosomal translation, and cellular trafficking of IgG intermediates. Furthermore, we identified mutual tackling points for optimization by gene engineering. The presented strategy can contribute to meet future requirements in the continuously demanding field of biotherapeutics production.

Outer membrane vesicles (OMVs) are nanoparticles produced by Gram-negative bacteria that can be used as vaccines. The application of OMVs as vaccine component can be expanded by expressing heterologous antigens on OMVs, creating an OMV-based antigen presenting platform. This study aims to develop a production process for such OMV-based vaccines and studies a production method based on meningococcal OMVs that express heterologous antigens on their surface. As a proof of concept, the Borrelia burgdorferi antigens OspA and OspC were expressed on Neisseria meningitidis OMVs to create a concept anti-Lyme disease vaccine. Production of OMVs released in the culture supernatant was induced by high dissolved oxygen concentrations and purification was based on scalable unit operations. A crude recovery of 90 mg OMV protein could be obtained per liter culture. Expressing heterologous antigens on the OMVs did result in minor reduction of bacterial growth, while OMV production remained constant. The antigen expression did not alter the OMV characteristics. This study shows that production of well characterized OMVs containing heterologous antigens is possible with high yields by combining high oxygen concentrations with an optimized purification process. It is concluded that heterologous OMVs show potential as a vaccine platform. Unit Licence Agreement

Besides being considered pathogens, viruses are important drivers of evolution and they can shape large ecological and biogeochemical processes, by influencing host fitness, population dynamics, and community structures. Moreover, they are simple systems that can be used and manipulated to be beneficial and useful for biotechnological applications. In this context, microalgae biotechnology is a growing field of research, which investigated the usage of photosynthetic microorganisms for the sustainable production of food, fuel, chemical, and pharmaceutical sectors. Viruses infecting microalgae have become important subject of ecological studies related to marine and aquatic environments only four decades ago when virus-like-particles associated with bloom-forming algae were discovered. These first findings have opened new questions on evolution and identity. To date, 63 viruses that infect eukaryotic microalgae have been isolated and cultured. In this short review we briefly summarize what is known about viruses infecting eukaryotic microalgae, and how acknowledging their importance can shape future research focussed not only on marine ecology and evolutionary biology but also on biotechnological applications related to microalgae cell factories.

Current CHO cell production processes require an optimized space-time-yield. Process intensification can support achieving this by enhancing the productivity and improving facility utilization. The use of perfusion at the last stage of the seed train (N-1) for high cell density inoculation of the fed-batch N-stage production culture is a relatively new approach with few industry applicable examples. Within this work, the impact of the cell-specific perfusion rate (CSPR) of the N-1 perfusion and the relevance of its control for the quality of generated inoculation cells was evaluated using an automated perfusion rate (PR) control based on online biomass measurements. Precise correlations (R² = 0.99) between permittivity and viable cell counts were found up to the high densities of 100⋅106 c·mL−1. Cells from N-1 perfusion were cultivated at a high and low CSPR with 50 and 20 pL·(c·d)−1, respectively. Lowered cell growth and an increased apoptotic reaction was found as a consequence of the latter due to nutrient limitations and reduced uptake rates. Subsequently, batch cultivations (N-stage) from the different N-1 sources were inoculated to evaluate the physiological state of the inoculum. Successive responses resulting from the respective N-1 condition were uncovered. While cell growth and productivity of approaches inoculated from high CSPR and a conventional seed were comparable, low CSPR inoculation suffered significantly in terms of reduced initial cell growth and impaired viability. This study underlines the importance to determine the CSPR for the design and implementation of an N-1 perfusion process in order to achieve the desired performance at the crucial production stage.

Currently, the mammalian biomanufacturing industry explores process intensification (PI) to meet upcoming demands of biotherapeutics while keeping production flexible but, more importantly, as economic as possible. However, intensified processes often require more development time compared with conventional fed-batches (FBs) preventing their implementation. Hence, rapid and efficient, yet straightforward strategies for PI are needed. In this study we demonstrate such a strategy for the intensification of an N-stage FB by implementing N-1 perfusion cell culture and high inoculum cell densities resulting in a robust intensified FB (iFB). Furthermore, we show successful combination of such an iFB with the addition of productivity enhancers, which has not been reported so far. The conventional CHO cell FB process was step-wise improved and intensified rapidly in multi-parallel small-scale bioreactors using N-1 perfusion. The iFBs were performed in 15 and 250 ml bioreactors and allowed to evaluate the impact on key process indicators (KPI): the space–time yield (STY) was successfully doubled from 0.28 to 0.55 g/L d, while product quality was maintained. This gain was generated by initially increasing the inoculation density, thus shrinking process time, and second supplementation with butyric acid (BA), which reduced cell growth and enhanced cell-specific productivity from ~25 to 37 pg/(cell d). Potential impacts of PI on cell metabolism were evaluated using flux balance analysis. Initial metabolic differences between the standard and intensified process were observed but disappeared quickly. This shows that PI can be achieved rapidly for new as well as existing processes without introducing sustained changes in cellular and metabolic behavior.

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 similar to 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 mu g 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.

Baculovirus expression vectors are successfully used for the commercial production of complex (glyco)proteins in eukaryotic cells. The genome engineering of single-copy baculovirus infectious clones (bacmids) in E. coli has been valuable in the study of baculovirus biology, but bacmids are not yet widely applied as expression vectors. An important limitation of first-generation bacmids for large-scale protein production is the rapid loss of gene of interest (GOI) expression. The instability is caused by the mini-F replicon in the bacmid backbone, which is non-essential for baculovirus replication in insect cells, and carries the adjacent GOI in between attTn7 transposition sites. In this paper, we test the hypothesis that relocation of the attTn7 transgene insertion site away from the mini-F replicon prevents deletion of the GOI, thereby resulting in higher and prolonged recombinant protein expression levels. We applied lambda red genome engineering combined with SacB counterselection to generate a series of bacmids with relocated attTn7 sites and tested their performance by comparing the relative expression levels of different GOIs. We conclude that GOI expression from the odv-e56 (pif-5) locus results in higher overall expression levels and is more stable over serial passages compared to the original bacmid. Finally, we evaluated this improved next-generation bacmid during a bioreactor scale-up of Sf9 insect cells in suspension to produce enveloped chikungunya virus-like particles as a model vaccine.

Sponges and their associated microorganisms are the most prolific source of marine natural products, and many attempts have been made at creating a marine sponge cell line to produce these products efficiently. However, limited knowledge on the nutrients sponge cells require to grow and poor genetic accessibility have hampered progress toward this goal. Recently, a new sponge-specific nutrient medium M1 has been shown to stimulate sponge cells in vitro to divide rapidly. In this study, we demonstrate for the first time that sponge cells growing in M1 can be genetically modified using a CRISPR/Cas12a gene editing system. A short sequence of scrambled DNA was inserted using a single-stranded oligodeoxynucleotide donor template to disrupt the 2′,5′-oligoadenylate synthetase gene of cells from the boreal deep-sea sponge Geodia barretti. A blue fluorescent marker gene appeared to be inserted in an intron of the same gene and expressed by a small number of G. barretti cells. Our results represent an important step toward developing an optimized continuous sponge cell line to produce bioactive compounds.

Genetic transformation of microalgae remains a challenge due to poor intracellular delivery of exogenous molecules. This limitation is caused by the structure and composition of the cell wall and cell membrane of each species. Moreover, successful delivery of proteins or nucleic acids cannot be assessed by determining transformability since their functionality is not always known in the studied microorganism. We propose a quick and effective screening tool for the prediction and optimization of electroporation settings by monitoring cell permeability and viability using Sytox Green and propidium iodide respectively. We determined voltage settings for the microalgae Chlamydomonas reinhardtii, Chlorella vulgaris, Neochloris oleoabundans and Acutodesmus obliquus. To evaluate the predicted settings, we delivered labelled DNA and proteins into the cells. We demonstrated that high transformation efficiencies can be accomplished when predicted values were applied with functional plasmids. Additionally, we increased transformation efficiencies by testing cell concentrations, light intensities and fragment sizes. This method can be used to determine suitable transformation conditions for non-transformed microalgae species and to increase the insight on established transformation protocols.

Normally, the growth profile of a CHO cell fed-batch process can be divided into two main phases based on changes in cell concentration, being an exponential growth phase and a stationary (non-growth) phase. In this study, an additional phase is observed during which the cell division comes to a halt but the cell growth continues in the form of an increase in cell size. The cell size increase (SI) phase occurs between the exponential proliferation phase (also called the number increase or NI phase) and the stationary phase. During the SI phase, the average volume and dry weight per cell increase threefold linearly with time. The average mAb specific productivity per cell increases linearly with the cell volume and therefore is on average two times higher in the SI phase than in the NI phase. The specific essential amino acids consumption rates per cell remain fairly constant between the NI and the SI phase, which agrees with the similar biomass production rate per cell between these two phases. Accumulation of fatty acids and formation of lipid droplets in the cells are observed during the SI phase, indicating that the fatty acids synthesis rate exceeds the demand for the synthesis of membrane lipids. A metabolic comparison between NI and SI phase shows that the cells with a larger size produce more mAb per unit of O2 and nutrient consumed, which can be used for further process optimization. Electronic supplementary material The online version of this article (10.1007/s00253-017-8531-y) contains supplementary material, which is available to authorized users.

In this study the influence of day length on circadian rhythms of N. oleoabundans was investigated. N. oleoabundans was grown in a photobioreactor continuously operated (turbidostat), under various day/night (D/N) cycles; 20D4N, 16D8N and 12D12N. The following variables showed to be regulated by the circadian clock: maximum growth rate, start of starch synthesis and DNA replication to 4 and 8 copies. Timing of these processes was not influenced by day length. The length of the photoperiod caused changes in biomass composition, especially due to variations in starch content. In longer days, more starch was accumulated. Starch was used for cell division probably also when cell division occurred in the light. Therefore, also the timing of cell division should be considered in the production of microalgae biomass. This timing mechanism can be explored to produce biomass with a desired concentration of protein, lipids, carbohydrates or pigments.

In a Chinese Hamster Ovary (CHO) cell fed-batch process, arrest of cell proliferation and an almost threefold increase in cell size occurred, which is associated with an increase in cell-specific productivity. In this study, transcriptome analysis is performed to identify the molecular mechanisms associated with this. Cell cycle analysis reveals that the cells are arrested mainly in the G0 /G1 phase. The cell cycle arrest is associated with significant up-regulation of cyclin-dependent kinases inhibitors (CDKNs) and down-regulation of cyclin-dependent kinases (CDKs) and cyclins. During the cell size increase phase, the gene expression of the upstream pathways of mechanistic target of rapamycin (mTOR), which is related to the extracellular growth factor, cytokine, and amino acid conditions, shows a strongly synchronized pattern to promote the mTOR activity. The downstream genes of mTOR also show a synchronized pattern to stimulate protein translation and lipid synthesis. The results demonstrate that cell cycle inhibition and stimulated mTOR activity at the transcriptome level are related to CHO cell size increase. The cell size increase is related to the extracellular nutrient conditions through a number of cascade pathways, indicating that by rational design of media and feeds, CHO cell size can be manipulated during culture processes, which may further improve cell growth and specific productivity.

Outer membrane vesicles (OMVs) are nanoparticles secreted by Gram-negative bacteria that can be used for diverse biotechnological applications. Interesting applications have been developed, where OMVs are the basis of drug delivery, enzyme carriers, adjuvants, and vaccines. Historically, OMV research has mainly focused on vaccines. Therefore, current OMV production processes have been based on batch processes. The production of OMVs in batch mode is characterized by relatively low yields and high costs. Transition of OMV production processes from batch to continuous processes could increase the volumetric productivity, reduce the production and capital costs, and result in a higher quality product. Here, we study the continuous production of Neisseria meningitidis OMVs to improve volumetric productivity. Continuous cultivation of N. meningitidis resulted in a steady state with similar high OMV concentrations as are reached in current batch processes. The steady state was reproducible and could be maintained for at least 600 h. The volumetric productivity of a continuous culture reached 4.0 × 1014 OMVs per liter culture per day, based on a dilution rate of 1/day. The tested characteristics of the OMVs did not change during the experiments showing feasibility of a continuous production process for the production of OMVs for any application. Electronic supplementary material The online version of this article (10.1007/s00253-019-10163-z) contains supplementary material, which is available to authorized users.

Sponges are rich sources of novel natural products. Production in cell cultures may be an option for supply of these compounds but there are currently no sponge cell lines. Because there is a lack of understanding about the precise conditions and nutritional requirements that are necessary to sustain sponge cells in vitro, there has yet to be a defined, sponge-specific nutrient medium. This study utilized a genetic algorithm approach to optimize the amino acid composition of a commercially available basal cell culture medium in order to increase the metabolic activity of cells of the marine sponge Dysidea etheria. Four generations of the algorithm were carried out in vitro in wet lab conditions and an optimal medium combination was selected for further evaluation. When compared to the basal medium control, there was a twofold increase in metabolic activity. The genetic algorithm approach can be used to optimize other components of culture media to efficiently optimize chosen parameters without the need for detailed knowledge on all possible interactions. Electronic supplementary material The online version of this article (10.1007/s11626-018-00317-0) contains supplementary material, which is available to authorized users.

Outer membrane vesicles (OMVs) produced by bacteria are interesting vaccine candidates. OMVs are nanoparticles that contain many immunogenic components, are self-adjuvating, and non-replicative. Despite recent insights in the biogenesis of OMVs, there is no consensus on a conserved mechanism of OMV release and the OMV yield from bacterial cultures remains low. For Neisseria meningitidis, a Gram-negative human pathogen causing meningitis and sepsis, a feasible OMV production method based on triggering OMV release by cysteine depletion has been described. In this study, we investigated the mechanism behind this external trigger for OMV release to improve the production process. Since enhanced OMV release upon cysteine depletion was associated with oxidative stress and redox responses, we investigate the influence of more oxidized sulfur sources on OMV release. We show that N. meningitidis grows similarly on sulfate, the most oxidized sulfur source, and OMV release is triggered by sulfur depletion in general. Sulfate depletion induced increased release of OMVs over cysteine depletion. Proteomics showed that sulfur depletion resulted in oxidative stress responses and upregulated phospholipid and LPS biosynthesis. Furthermore, OMVs produced by sulfur depletion were enriched in phospholipids. Mechanistically, we hypothesize that sulfur depletion results in overproduction of phospholipids causing increased bulging of the outer membrane and subsequent OMV release.

Sponges (Phylum Porifera) are among the oldest Metazoa and considered critical to understanding animal evolution and development. They are also the most prolific source of marine-derived chemicals with pharmaceutical relevance. Cell lines are important tools for research in many disciplines, and have been established for many organisms, including freshwater and terrestrial invertebrates. Despite many efforts over multiple decades, there are still no cell lines for marine invertebrates. In this study, we report a breakthrough: we demonstrate that an amino acid-optimized nutrient medium stimulates rapid cell division in 9 sponge species. The fastest dividing cells doubled in less than 1 hour. Cultures of 3 species were subcultured from 3 to 5 times, with an average of 5.99 population doublings after subculturing, and a lifespan from 21 to 35 days. Our results form the basis for developing marine invertebrate cell models to better understand early animal evolution, determine the role of secondary metabolites, and predict the impact of climate change to coral reef community ecology. Furthermore, sponge cell lines can be used to scale-up production of sponge-derived chemicals for clinical trials and develop new drugs to combat cancer and other diseases.

Tetradesmus obliquus is an oleaginous microalga with high potential for triacylglycerol production. We characterized the biochemical composition and the transcriptional landscape of T. obliquus wild-type and the starchless mutant (slm1), adapted to 16:8 h light dark (LD) cycles under nitrogen limitation. In comparison to the nitrogen replete conditions, the diurnal RNA samples from both strains also displayed a cyclic pattern, but with much less variation which could be related to a reduced transcription activity in at least the usually highly active processes. During nitrogen limitation, the wild-type continued to use starch as the preferred storage compound to store energy and carbon. Starch was accumulated to an average content of 0.25 g·gDW−1, which is higher than the maximum observed under nitrogen replete conditions. Small oscillations were observed, indicating that starch was being used as a diurnal energy storage compound, but to a lesser extent than under nitrogen replete conditions. For the slm1 mutant, TAG content was higher than for the wild-type (average steady state value was 0.26 g·gDW−1 for slm1 compared to 0.06 g·gDW−1 for the wild-type). Despite the higher TAG content in the slm1, the conversion efficiency of photons into biomass components for the slm1 was only half of the one obtained for the wild-type. This is related to the observed decrease in biomass productivity (from 1.29 gDW·L−1·day−1 for the wild-type to 0.52 gDW·L−1·day−1 for the slm1). While the transcriptome of slm1 displayed clear signs of energy generation by degrading TAG and amino-acids during the dark period, no significant variation of these metabolites could be measured. When looking through the diurnal cycle, the photosynthetic efficiency was lower for the slm1 mutant compared to the wild-type especially during the second half of the light period, where starch accumulation occurred in the wild-type. Paid Open Access

Tetradesmus obliquus is a promising oleaginous microalga. We functionally annotated its genome and characterized the transcriptional landscape of T. obliquus adapted to 16:8h light dark (LD) cycles in turbidostat culture conditions at very high temporal resolution (1h intervals). Revealing a cycle of cellular events, six distinct expression profiles were obtained, each with transcriptional phenotypes correlating with measurements of biochemical composition.The impact of starch deficiency was studied using the starchless mutant slm1. Significant changes in the transcriptional landscape were observed. Starch deficiency resulted in incapacity to supply energy during dark period, resulting in early or late time shift for energy demanding processes. Our study provides new perspectives on the role of starch and the adaptation to LD cycles of oleaginous microalgae.

Neochloris oleoabundans is an oleaginous microalgal species that can be cultivated in fresh water as well as salt water. Using salt water gives the opportunity to reduce production costs and the fresh water footprint for large scale cultivation. Production of triacylglycerols (TAG) usually includes a biomass growth phase in nitrogen-replete conditions followed by a TAG accumulation phase under nitrogen-deplete conditions. This is the first report that provides insight in the saline resistance mechanism of a fresh water oleaginous microalgae. To better understand the osmoregulatory mechanism of N. oleoabundans during growth and TAG accumulating conditions, the transcriptome was sequenced under four different conditions: fresh water nitrogen-replete and -deplete conditions, and salt water (525 mM dissolved salts, 448mM extra NaCl) nitrogen-replete and -deplete conditions. In this study, several pathways are identified to be responsible for salt water adaptation of N. oleoabundans under both nitrogen-replete and -deplete conditions. Proline and the ascorbate-glutathione cycle seem to be of importance for successful osmoregulation in N. oleoabundans. Genes involved in Proline biosynthesis were found to be upregulated in salt water. This was supported by Nuclear magnetic resonance (NMR) spectroscopy, which indicated an increase in proline content in the salt water nitrogen-replete condition. Additionally, the lipid accumulation pathway was studied to gain insight in the gene regulation in the first 24 hours after nitrogen was depleted. Oil accumulation is increased under nitrogen-deplete conditions in a comparable way in both fresh and salt water. The mechanism behind the biosynthesis of compatible osmolytes can be used to improve N. oleoabundans and other industrially relevant microalgal strains to create a more robust and sustainable production platform for microalgae derived products in the future.

Nitrogen deprivation increases the triacylglycerol (TAG) content in microalgae but also severely decreases the growth rate. Most approaches that attempted to increase TAG productivity by overexpression or knockdown of specific genes related to the regulation of the lipid synthesis have reported only little success. More insight into the molecular mechanisms related to lipid accumulation and impaired growth rate is needed to find targets for improving TAG productivity. By using the emerging “omics” approach, we comprehensively profiled the physiology, transcriptome, proteome and metabolome of the diatom Phaeodactylum tricornutum during steady state growth at both nitrogen limited and replete levels during light:dark cycles. Under nitrogen limited conditions, 22% (2699) of the total identified transcripts, 17% (543) of the proteins and 44% (345) of the metabolites were significantly differentially regulated compared to nitrogen replete growth conditions. Although nitrogen limitation was responsible for the majority of significant differential transcript, protein and metabolite accumulation, we also observed differential expression over a diurnal cycle. Nitrogen limitation mainly induced an upregulation of nitrogen fixation, central carbon metabolism and TCA cycle, while photosynthetic and ribosomal protein synthesis are mainly downregulated. Regulation of the lipid metabolism and the expression of predicted proteins involved in lipid processes suggest that lipid rearrangements may substantially contribute to TAG distribution. However, TAG synthesis is also limited by the reduced carbon flux through central metabolism. Future strain improvements should therefore focus on understanding and improving the carbon flux through central carbon metabolism, selectivity and activity of DGAT isoforms and lipase enzymes.

Large scale microalgal production will be primarily done under natural sunlight conditions, where microalgae will be exposed to diurnal cycles of light and dark (LD) and to differences in the length of both periods (photoperiod). Tetradesmus obliquus (formerly known as Scenedesmus obliquus), a strain with potential for biofuel production, and the starchless mutant slm1 were grown under 3 different LD periods: 16:8 h, 14:10 h and 12:12 h. Cell division started a fix number of hours after the light went on (sunrise), independently of the length of the photoperiod. For the wild-type, cell division started approximately 14 h after the beginning of the day and occurred mainly at night. For the starchless mutant slm1, timing of cell division was also independent of the photoperiod length (starting 10–12 h after sunrise). However, as opposed to the wild-type, cell division always started during the day. For both strains, growth rate increased with increased length of the light period. The slm1 mutant is capable of surviving long dark periods (up to 12 h) despite the lack of starch. In general, the slm1 mutant has a lower photosynthetic efficiency than the wild-type, with the 12:12 h LD resulting into even less efficiency than the other two LD cycles.

Transcriptome and metabolism analysis were performed to evaluate the scale-down of a CHO cell fed-batch process from a 10 L bioreactor to an ambr 15® (ambr) system. Two different agitation scale-down principles were applied, resulting in two different agitation rates in the ambr system: 1300 RPM based on the agitator tip speed, and 800 rpm based on the volumetric power input (P/V). Culture performance including cell growth, product titer, glycosylation, and specific consumption/production rates of metabolites was the same for both agitation rates in the ambr and was comparable to that of the 10 L system. The initial variation in gene expression between the inocula for the ambr and 10 L system was no longer present after three days of culture, indicating comparable culture conditions in both systems. Based on principal component analysis, changes in gene expression over time were similar between both scales with less than 6% variation. 2455 genes were uniquely regulated in the ambr system compared to 1604 genes in the 10 L system. Functional analysis of these genes did not reveal their relations with scale or cellular function. This study further strengthens that the ambr system gives representative culture performance for the 10 L bench-scale bioreactor.

This work investigates how the photoacclimated state at the onset of nitrogen starvation and the light intensity during nitrogen starvation influence carbon partitioning and photosynthetic efficiency in nitrogen-starved Scenedesmus obliquus . Nitrogen-depleted batch cultivations were performed at an incident light intensity of 200 and 1000 μmol m − 2 s − 1 . These nitrogen-depleted batch cultivations were started with biomass that was photoacclimated to an incident light intensity of either 200 or 1000 μmol m − 2 s − 1 under nitrogen replete conditions. During initial nitrogen starvation, fatty acids and starch were produced in a 1:4 ratio. This ratio progressively increased towards only fatty acid synthesis. Hereafter, the initially accumulated starch was degraded and likely used as a substrate for fatty acid synthesis. The ratio between starch and fatty acid synthesis correlated strongly to the biomass nitrogen content. Slightly more carbon was partitioned to starch synthesis at high light intensities. Carbon partitioning was not affected by the photoacclimated state at the onset of nitrogen starvation. The photosynthetic efficiency decreased 2-fold at high versus low light intensities but was not affected by photoacclimation. Likely, the observed carbon partitioning is caused by competition between fatty acid and starch synthesis for a common carbon pre-cursor.

Preventing or delaying cell death is a challenge in mammalian cell cultures for the development and optimization of production processes for biopharmaceuticals. Cell cultures need to be maintained highly viable for extended times in order to reach maximum production yields. Moreover, programmed cell death through apoptosis is often believed to occur without being detected by classical viability measurements. In this study, we characterized cell death in PER.C6® batch and perfusion cultures using three flow cytometry techniques measuring different steps of the apoptosis cascade: DNA fragmentation, caspases activation and phosphatidylserine externalization. We showed that apoptosis is the main pathway of PER.C6® cell death in batch cultures after depletion of main carbon sources. In high cell density perfusion cultures fed at a constant specific perfusion rate, both high viability and very limited apoptosis were observed. When extending this perfusion process far beyond standard operations, cultures were exposed to suboptimal process conditions, which resulted in an increase of apoptotic cell death. Moreover, we showed that the reference viability measurement using trypan blue exclusion properly assesses the level of cell death in PER.C6® cultures. This study is a first step in understanding the mechanisms of PER.C6® cell death, which will be helpful to support applications of the cell line.

The effect of nitrogen replenishment on the kinetics of secondary carotenoids, triacylglycerol (TAG) and primary cell components was studied in nitrogen-starved Chromochloris zofingiensis (Chlorophyta), an oleaginous and carotenogenic microalga. Nitrogen resupplied after a period of starvation was initially consumed at a more than four times higher rate than in an equivalent nitrogen-replete culture. Simultaneously, chlorophylls, primary carotenoids, polar (membrane) lipids and proteins were rapidly produced. After 2 days, the contents of these primary metabolites, as well as the nitrogen consumption rate and the overall biomass production rate, had returned to values equivalent to those of cells grown under nitrogen-replete conditions, indicating that culture recovery required 2 days. Nitrogen resupply was immediately followed by rapid degradation of TAG and starch, suggesting that these metabolites served as carbon and energy source for the recovery process. Also, the secondary carotenoids canthaxanthin and ketolutein were rapidly degraded upon nitrogen resupply, whereas degradation of astaxanthin, the main secondary carotenoid, started only when the cells were fully recovered 2 days after nitrogen resupply. This is the first time that such culture recovery has been described in detail and, moreover, that astaxanthin was found to be not immediately degraded after nitrogen resupply. The observed rapid recovery of C. zofingiensis and the delay in astaxanthin degradation suggest that a repeated batch cultivation may result in a higher secondary carotenoid productivity than a series of classical single batch cultivations.

Outer membrane vesicles (OMVs) are spherical membrane nanoparticles released by Gram-negative bacteria. OMVs can be quantified in complex matrices by nanoparticle tracking analysis (NTA). NTA can be performed in static mode or with continuous sample flow that results in analysis of more particles in a smaller time-frame. Flow measurements must be performed manually despite the availability of a sample changer on the NanoSight system. Here we present a method for automated measurements in flow mode. OMV quantification in flow mode results in lower variance in particle quantification (coefficient of variation (CV) of 6%, CV static measurements of 14%). Sizing of OMVs was expected to be less favorable in flow mode due to the increased movement of the particles. However, we observed a CV of 3% in flow mode and a CV of 8% in static measurements. Flow rates of up to 5 µL/min displayed correct size and particle measurements, however, particle concentration was slightly lower than in static measurements. The automated method was used to assess OMV release of batch cultures of Neisseria meningitidis. The bacteria released more OMVs in stationary growth phase, while the size of the vesicles remained constant throughout the culture. Taken together, this study shows that automated measurements in flow mode can be established with advanced scripting to reduce the workload for the user.

Photosynthetic organisms have evolved to use light efficiently by scheduling their cellular processes, such as growth and cell division, at specific times of the day. During the day, fixed carbon is used for growth and is partially stored as carbohydrates (e.g., starch). It is commonly assumed that this accumulated starch is essential for fuelling up cell division at night. To test this hypothesis, this study investigates growth, cell division and presence of a transitory energy storage (TES) in both the wild-type and starchless mutant strain of Acutodesmus obliquus under light/dark (LD) cycles and nitrogen replete conditions. A. obliquus (formerly known as Scenedesmus obliquus) wild-type utilized light 20% more efficiently under LD regimes compared with continuous light. When exposed to LD regimes, the wild-type scheduled cell division in a 4-hour period starting 2 h before ‘sunset’. Starch acted as the major transitory energy storage (TES) compound: it was accumulated during the last part of the light period and was consumed throughout the entire dark period. The slm1 mutant, with a blocked starch synthesis pathway, showed diurnal rhythms in growth and cell division. However, no other carbohydrates nor triacylglycerols took over the role of TES compound in slm1. Therefore, in contrast to what is generally acknowledged, this study shows that neither starch nor any other major alternative TES is required for synchronized growth and cell division in A. obliquus. The starchless mutant did show a reduced growth and cell division rate compared to the wild-type. Starch, thus, plays a major role in efficient harnessing of light energy over LD cycles, likely because the ability to accumulate starch enhances biomass production capacity and accelerates cell division rate in A. obliquus.

Background Day/night cycles regulate the circadian clock of organisms to program daily activities. Many species of microalgae have a synchronized cell division when grown under a day/night cycle, and synchronization might influence biomass yield and composition. Therefore, the aim of this study was to study the influence of day/night cycle on biomass yield and composition of the green microalgae Neochloris oleoabundans. Hence, we compared continuous turbidostat cultures grown under continuous light with cultures grown under simulated day/night cycles. Results Under day/night cycles, cultures were synchronized as cell division was scheduled in the night, whereas under continuous light cell division occurred randomly synchronized cultures were able to use the light 10–15% more efficiently than non-synchronized cultures. Our results indicate that the efficiency of light use varies over the cell cycle and that synchronized cell division provides a fitness benefit to microalgae. Biomass composition under day/night cycles was similar to continuous light, with the exception of starch content. The starch content was higher in cultures under continuous light, most likely because the cells never had to respire starch to cover for maintenance during dark periods. Day/night cycles were provided in a ‘block’ (continuous light intensity during the light period) and in a ‘sine’ (using a sine function to simulate light intensities from sunrise to sunset). There were no differences in biomass yield or composition between these two ways of providing light (in a ‘block’ or in a ‘sine’). Conclusions The biomass yield and composition of N. oleoabundans were influenced by day/night cycles. These results are important to better understand the relations between research done under continuous light conditions and with day/night cycle conditions. Our findings also imply that more research should be done under day/night cycles.

Lipid production in microalgae is highly dependent on the applied light intensity. However, for the EPA producing model-diatom Phaeodactylum tricornutum, clear consensus on the impact of incident light intensity on lipid productivity is still lacking. This study quantifies the impact of different incident light intensities on the biomass, TAG and EPA yield on light in nitrogen starved batch cultures of P. tricornutum. The maximum biomass concentration and maximum TAG and EPA contents were found to be independent of the applied light intensity. The lipid yield on light was reduced at elevated light intensities (>100 μmol m-2 s-1). The highest TAG yield on light (112 mg TAG molph -1) was found at the lowest light intensity tested (60 μmol m-2 s-1), which is still relatively low to values reported in literature for other algae. Furthermore, mass balance analysis showed that the EPA fraction in TAG may originate from photosynthetic membrane lipids.

In this study, stearoyl-ACP desaturase (SAD), the enzyme that converts stearic acid into oleic acid, is silenced by artificial microRNA in the green microalga Chlamydomonas reinhardtii. Two different constructs, which target different positions on the mRNA of stearoyl-ACP desaturase, were tested. The mRNA levels for SAD were reduced after the silencing construct was induced. In one of the strains, the reduction in SAD mRNA resulted in a doubling of the stearic acid content in triacylglycerol molecules, which shows that stearic acid production in microalgae is possible.

The microalgae Tetradesmus obliquus is able to maintain a high photosynthetic efficiency under nitrogen limitation and is considered a promising green microalgae for sustainable production of diverse compounds, including biofuels. Here, we report the first draft whole-genome shotgun sequencing of T. obliquus . The final assembly comprises 108,715,903 bp with over 1,368 scaffolds.

Outer membrane vesicles (OMVs) are naturally non-replicating, highly immunogenic spherical nanoparticles derived from Gram-negative bacteria. OMVs from pathogenic bacteria have been successfully used as vaccines against bacterial meningitis and sepsis among others and the composition of the vesicles can easily be engineered. OMVs can be used as a vaccine platform by engineering heterologous antigens to the vesicles. The major advantages of adding heterologous proteins to the OMV are that the antigens retain their native conformation, the ability of targeting specific immune responses, and a single production process suffices for many vaccines. Several promising vaccine platform concepts have been engineered based on decorating OMVs with heterologous antigens. This review discusses these vaccine concepts and reviews design considerations as the antigen location, the adjuvant function, physiochemical properties, and the immune response.

The effect of biomass-specific photon absorption rate on triacylglycerol (TAG) and secondary carotenoid yield was studied. Chlorella zofingiensis (Chlorophyta) was cultivated batch-wise with sufficient initial nitrogen to produce 2.5, 3.4 and 4.1 g L− 1 prior to nitrogen depletion, which resulted in biomass-specific photon absorption rates of 4.7, 3.5 and 2.9 μmolph g dry weight− 1 s− 1, respectively. During nitrogen starvation, the TAG as well as secondary carotenoid yield on light did not differ between the cultures, which indicates that in the studied range the biomass-specific photon absorption rate did not affect the amounts of energy used for TAG and secondary carotenoid production. The maximal TAG yield on light (320 mg molph− 1) was higher than values reported before for microalgae. Besides, for the first time the secondary carotenoid yield on light was reported (maximally 2.75 mg molph− 1).

Background Day/night cycles regulate the circadian clock of organisms to program daily activities. Many species of microalgae have a synchronized cell division when grown under a day/night cycle, and synchronization might influence biomass yield and composition. Therefore, the aim of this study was to study the influence of day/night cycle on biomass yield and composition of the green microalgae Neochloris oleoabundans. Hence, we compared continuous turbidostat cultures grown under continuous light with cultures grown under simulated day/night cycles. Results Under day/night cycles, cultures were synchronized as cell division was scheduled in the night, whereas under continuous light cell division occurred randomly synchronized cultures were able to use the light 10–15% more efficiently than non-synchronized cultures. Our results indicate that the efficiency of light use varies over the cell cycle and that synchronized cell division provides a fitness benefit to microalgae. Biomass composition under day/night cycles was similar to continuous light, with the exception of starch content. The starch content was higher in cultures under continuous light, most likely because the cells never had to respire starch to cover for maintenance during dark periods. Day/night cycles were provided in a ‘block’ (continuous light intensity during the light period) and in a ‘sine’ (using a sine function to simulate light intensities from sunrise to sunset). There were no differences in biomass yield or composition between these two ways of providing light (in a ‘block’ or in a ‘sine’). Conclusions The biomass yield and composition of N. oleoabundans were influenced by day/night cycles. These results are important to better understand the relations between research done under continuous light conditions and with day/night cycle conditions. Our findings also imply that more research should be done under day/night cycles.

The process analytical technology (PAT) initiative is now 10 years old. This has resulted in the development of many tools and software packages dedicated to PAT application on pharmaceutical processes. However, most applications are restricted to small molecule drugs, mainly for the relatively simple process steps like drying or tableting where only a limited number of parameters need to be controlled. A big challenge for PAT still lies in applications for biopharmaceuticals and then especially in the cultivation process step, where the quality of a biopharmaceutical product is largely determined. This review gives an overview of the currently available tools for monitoring and controlling the biopharmaceutical cultivation step and of the main challenges for the most common cell platforms (i.e. Escherichia coli, yeast, and mammalian cells) used in biopharmaceutical manufacturing. The real challenge is to understand how intracellular mechanisms (from synthesis to excretion) influence the quality of biopharmaceuticals and how these mechanisms can be monitored and controlled to yield the desired end product quality. Modern “omics” tools and advanced process analyzers have opened up the way for PAT applications for the biopharmaceutical cultivation process step.

Microalgae-derived lipids in the form of triacylglycerols (TAGs) are considered an alternative resource for the production of biofuels and food commodities. Large scale production of microalgal TAGs is currently uneconomical. The cost price could be reduced by improving the areal and volumetric TAG productivity. The economic value could be increased by enhancing the TAG quality. To improve these characteristics, the impact of light intensity, and the combined impact of pH and temperature on TAG accumulation were studied for Scenedesmus obliquus UTEX 393 under nitrogen starved conditions. The maximum TAG content was independent of light intensity, but varied between 18% and 40% of dry weight for different combinations of pH and temperature. The highest yield of fatty acids on light (0.263 g/mol photon) was achieved at the lowest light intensity, pH 7 and 27.5 °C.