Staff Publications

Staff Publications

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    'Staff publications' is the digital repository of Wageningen University & Research

    'Staff publications' contains references to publications authored by Wageningen University staff from 1976 onward.

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Potential of a liquid foam-bed photobioreactor for microalgae cultivation
Janoska, Agnes ; Andriopoulos, Vasilis ; Wijffels, Rene H. ; Janssen, Marcel - \ 2018
Algal Research 36 (2018). - ISSN 2211-9264 - p. 193 - 208.
Energy requirement - Foam-bed - Growth modeling - Microalgae - Photobioreactor

The liquid foam-bed photobioreactor is a novel photobioreactor for microalgae cultivation. A mathematical model was developed to evaluate its potential, and to optimize the design and operation of a large-scale unit. This model describes light limited microalgal growth in a rising foam column in a foam-bed photobioreactor, which is continuously operated at constant biomass density. The microalgae-containing liquid is recirculated from the bottom of the reactor and dispersed equally on the top of the foam column, in order to ensure homogenous microalgae distribution and a wet and stable foam. The model combines calculations of liquid fraction gradient, light penetration, microalgal growth, and gas transfer in the foam-bed. The liquid fraction and light model was experimentally validated. The areal productivity of a 5 cm deep foam-bed photobioreactor operated at 30 g L−1 microalgae and 1500 μmol photons m−2 s−1 was estimated to be 67.7 g m−2 d−1. This productivity is slightly lower compared to what is achievable in flat panels, which is related to light scattering in the foam-bed. Nevertheless, the advantages of the foam-bed photobioreactor, such as high gas transfer rate and high biomass densities, were confirmed with the simulations. In addition, it was calculated that a CO2 uptake efficiency of 97% can be obtained ensuring minimal CO2 loss. These benefits result in reduced gas supply requirement and reduced energy required for downstream processing. The total energy required for the production and separation of 1 g biomass in liquid foam-beds is only 8.5% of what is required in flat panels with suspended biomass. These results highlight the potential of foam-bed photobioreactors for large scale application for microalgae production.

Metabolic modelling and energy parameter estimation of Tetradesmus obliquus
León-Saiki, G.M. ; Ferrer Ledo, Narcís ; Lao-Martil, David ; Veen, Douwe van der; Wijffels, René H. ; Martens, Dirk E. - \ 2018
Algal Research 35 (2018). - ISSN 2211-9264 - p. 378 - 387.
Compartmentalized metabolism - Flux Balance Analysis (FBA) - Maintenance requirement - Microalgae - Scenedesmus obliquus

We developed a metabolic network describing the primary metabolism of Tetradesmus obliquus aimed to get a better understanding of metabolism to improve industrial production. The network includes 351 reactions with 183 metabolites distributed over 4 compartments: cytosol, chloroplast, mitochondria, and extracellular space. The energy requirements for biomass assembly and maintenance (Kx and mATP, respectively) were experimentally determined from batch cultures and included in the model. The determined values were 121.02 mmolATP·gDW −1 for Kx and 0.66 mmolATP·gDW −1·h−1 for the mATP. The maintenance value found for T. obliquus is, to our knowledge, one of the lowest reported in literature for microalgae. This low value is also in agreement with the photon maintenance requirement found experimentally for T. obliquus (1.18 mmolph·gDW −1·h−1). Finally, the theoretical maximum yields based on the model for biomass, triacylglycerides (TAG), and starch yield on light were calculated to be 1.15 g·molph −1, 1.05 gTAG·molph −1, and 2.69 gstarch·molph −1.

Simultaneous production of antioxidants and starch from the microalga Chlorella sorokiniana
Petruk, Ganna ; Gifuni, Imma ; Illiano, Anna ; Roxo, Mariana ; Pinto, Gabriella ; Amoresano, Angela ; Marzocchella, Antonio ; Piccoli, Renata ; Wink, Michael ; Olivieri, Giuseppe ; Monti, Daria M. - \ 2018
Algal Research 34 (2018). - ISSN 2211-9264 - p. 164 - 174.
Antioxidants - C. elegans - Chlorella sorokiniana - Eukaryotic cells - Microalgae

In recent years, microalgae have gained considerable importance as potential source of biofuels and bioplastics. However, these markets are still developing, as the high costs of cultivation ask for exploiting microalgae into new areas and with a biorefinery approach towards a multicomponent cascade extraction process. Here, a sequential processing strategy was used to extract starch with high yield from Chlorella sorokiniana under biocompatible conditions. The extract residue was then tested as a potential source of antioxidants. We found a strong protective activity of the extract residue towards oxidative stress in vitro on human colon cancer cells and in vivo on Caenorhabditis elegans nematodes, by inhibiting ROS production and activating DAF-16/FOXO transcription factor pathway. A pool of molecules from three different classes (fatty acids, photosynthetic pigments and carotenoids) was identified as responsible for the antioxidant activity. To our knowledge, this is the first report on the obtainment, from a “waste” fraction, of high value products endowed with antioxidant activity tested in cell-based models and in vivo.

Transcriptome analysis reveals the genetic foundation for the dynamics of starch and lipid production in Ettlia oleoabundans
Sturme, Mark H.J. ; Gong, Yanhai ; Heinrich, Josué Miguel ; Klok, Anne J. ; Eggink, Gerrit ; Wang, Dongmei ; Xu, Jian ; Wijffels, Rene H. - \ 2018
Algal Research 33 (2018). - ISSN 2211-9264 - p. 142 - 155.
Carbon-partitioning - Lipids - Microalgae - Nitrogen starvation - Starch - Transcriptome

The oleaginous microalga Ettlia oleoabundans accumulates both starch and lipids to high levels under stress conditions such as nitrogen starvation (N−). To steer biosynthesis towards starch or lipids only, it is important to understand the regulatory mechanisms involved. Here physiological and transcriptional changes under nitrogen starvation were analysed in controlled flat-panel photobioreactors at both short and long time-scales. Starch accumulation was transient and occurred rapidly within 24 h upon starvation, while lipid accumulation was gradual and reached a maximum after 4 days. The major fraction of accumulated lipids was composed of de novo synthesized neutral lipids - triacylglycerides (TAG) - and was characterized by a decreased composition of the polyunsaturated fatty acids (PUFAs) C18:3 and C16:3 and an increased composition of the mono-unsaturated (MUFAs) and saturated (SFAs) fatty acids C18:1/C16:1 and C18:0/C16:0, respectively. RNA-sequencing revealed that starch biosynthesis and degradation genes show different expression dynamics from lipid biosynthesis ones. An immediate rapid increase in starch synthetic transcripts was followed by an increase in starch degrading transcripts and a decrease in the starch synthetic ones. In contrast, increased gene expression for fatty acid and TAG synthesis was initiated later and occurred more gradually. Expression of several fatty acid desaturase (FAD) genes was decreased upon starvation, which corresponds to the observed changes to higher levels of MUFAs and SFAs. Moreover, several homologs of transcription regulators that were implicated in controlling starch and lipid metabolism in other microalgae showed differential gene expression and might be key regulators of starch and lipid metabolism in E. oleoabundans as well. Our data provide insights into the genetic foundation of starch and lipid metabolism in E. oleoabundans under nitrogen starvation and should facilitate metabolic engineering towards tailored strains with desired storage compound composition.

Improved liquid foam-bed photobioreactor design for microalgae cultivation
Janoska, Agnes ; Barten, Robin ; Nooy, Sam de; Rijssel, Piotr van; Wijffels, René H. ; Janssen, Marcel - \ 2018
Algal Research 33 (2018). - ISSN 2211-9264 - p. 55 - 70.
Cultivation - Foam - Foam-bed photobioreactor - Microalgae - Surfactant
The liquid foam-bed photobioreactor is a recently developed photobioreactor concept that allows for cost reduction in microalgae cultivation. Long term operation was not yet achieved, due to degradation of the surfactant. In this study, Pluronic F68 was applied for foam stabilization. In order to compensate for the low algae partitioning to Pluronic F68 stabilized foam, liquid recirculation was introduced into the reactor design. The microalgal suspension was continuously pumped from the bottom of the reactor to the top of the foam column where it was allowed to drain down again through the foam. This new design allowed increased mixing and, thereby, a homogenous algae distribution within the reactor. The volumetric mass transfer coefficient for CO2 in the foam-bed was 0.14 s−1, revealing that the gas transfer rate is an order of magnitude higher compared to bubble column reactors. This characteristic, together with a very high gas residence time, allows for a dramatic reduction in gas flow rate and a high carbon dioxide utilization efficiency. Long-term cultivation (>500 h) of Chlorella sp. was achieved in a stable foam-bed. The areal productivity of the foam-bed photobioreactor was 57 g m−2 d−1, which is slightly lower than maximally achieved in flat panels under similar conditions. This is possibly related to substantial light scattering taking place in the foam leading to a steeper light gradient and increased reflection. The reactor dilution rate and the liquid recirculation rate were not optimized during our experiments and there is room for further improvement. During continuous reactor operation, biomass densities of >20 g L−1 could be maintained. This biomass density is a factor of 10 higher compared to traditional, liquid phase photobioreactors, thereby, contributing to reduced energy requirements for microalgae harvesting.
The impact of day length on cell division and efficiency of light use in a starchless mutant of Tetradesmus obliquus
León-Saiki, G.M. ; Cabrero Martí, Tània ; Veen, Douwe van der; Wijffels, René H. ; Martens, Dirk E. - \ 2018
Algal Research 31 (2018). - ISSN 2211-9264 - p. 387 - 394.
Cell division - Day/night cycle - Diurnal biochemical changes - Microalgae - Photosynthetic efficiency - Scenedesmus obliquus
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.
Techno-economic evaluation of microalgae harvesting and dewatering systems
Fasaei, F. ; Bitter, J.H. ; Slegers, P.M. ; Boxtel, A.J.B. van - \ 2018
Algal Research 31 (2018). - ISSN 2211-9264 - p. 347 - 362.
Cost - Dewatering - Energy - Harvesting - Microalgae - System analysis
Microalgal biomass is processed into products by two main process steps: 1) harvesting and dewatering; and 2) extraction, fractionation and conversion. The performance of unit operations for harvesting and dewatering is often expressed in qualitative terms, like “high energy consumption” and “low in operational cost”. Moreover, equipment is analysed as stand-alone unit operations, which do not interact in a chain of operations. This work concerns a quantitative techno-economic analysis of different large-scale harvesting and dewatering systems with focus on processing cost, energy consumption and resource recovery. Harvesting and dewatering are considered both as a single operation and as combinations of sequential operations. The economic evaluation shows that operational costs and energy consumption are in the range 0.5–2 €·kg− 1 algae and 0.2–5 kWh·kg− 1 of algae, respectively, for dilute solutions from open cultivation systems. Harvesting and dewatering of the dilute systems with flocculation results in the lowest energy requirement. However, due to required chemicals and loss of flocculants, these systems end at the same cost level as mechanical harvesting systems. For closed cultivation systems the operational costs decrease to 0.1–0.6 €·kg− 1 algae and the energy consumption to 0.1–0.7 kWh·kg− 1 algae. For all harvesting and dewatering systems, labour has a significant contribution to the total costs. The total costs can be reduced by a high level of automation, despite the higher associated investment costs. The analysis shows that a single step operation can be satisfactory if the operation reaches high biomass concentrations. Two-step operations, like pressure filtration followed by spiral plate technology or centrifugation, are attractive from an economic point of view, just as the operation chain of flocculation followed by membrane filtration and a finishing step with spiral plate technology or centrifugation.
Surfactant selection for a liquid foam-bed photobioreactor
Janoska, Agnes ; Vázquez, María ; Janssen, Marcel ; Wijffels, René H. ; Cuaresma, María ; Vílchez, Carlos - \ 2018
Biotechnology Progress 34 (2018)3. - ISSN 8756-7938 - p. 711 - 720.
Foam-bed reactor - Liquid-foam - Microalgae - Surfactant
A novel liquid foam-bed photobioreactor has been shown to hold potential as an innovative technology for microalgae production. In this study, a foam stabilizing agent has been selected which fits the requirements of use in a liquid foam-bed photobioreactor. Four criteria were used for an optimal surfactant: the surfactant should have good foaming properties, should not be rapidly biodegradable, should drag up microalgae in the foam formed, and it should not be toxic for microalgae. Ten different surfactants (nonionic, cationic, and anionic) and two microalgae genera (Chlorella and Scenedesmus) were compared on the above-mentioned criteria. The comparison showed the following facts. Firstly, poloxameric surfactants (Pluronic F68 and Pluronic P84) have acceptable foaming properties described by intermediate foam stability and liquid holdup and small bubble size. Secondly, the natural surfactants (BSA and Saponin) and Tween 20 were easily biodegraded by bacteria within 3 days. Thirdly, for all surfactants tested the microalgae concentration is reduced in the foam phase compared to the liquid phase with exception of the cationic surfactant CTAB. Lastly, only BSA, Saponin, Tween 20, and the two Pluronics were not toxic at concentrations of 10 CMC or higher. The findings of this study indicate that the Pluronics (F68 and P84) are the best surfactants regarding the above-mentioned criteria. Since Pluronic F68 performed slightly better, this surfactant is recommended for application in a liquid foam-bed photobioreactor.
Can We Approach Theoretical Lipid Yields in Microalgae?
Remmers, Ilse M. ; Wijffels, René H. ; Barbosa, Maria J. ; Lamers, Packo P. - \ 2018
Trends in Biotechnology 36 (2018)3. - ISSN 0167-7799 - p. 265 - 276.
Cultivation strategies - Metabolism - Microalgae - Outdoor production - Strain improvement - Triacylglycerol (TAG)
Can we approach theoretical lipid yields in microalgae? Yes: we can substantially reduce the gap between current and theoretical maximum yield. A realistic maximum is approximately 0.5. g triacylglycerol (TAG) per mol photons, about five times higher than what is currently achieved in outdoor cultivation. Achieving this realistic maximum will require several breakthroughs. First, outdoor operation typically has low yields, mainly caused by fluctuating insolation. Future adaptive control models will help increase these yields. Additionally, the lipid production capacity of currently used strains needs to increase. Powerful strain-specific molecular toolboxes are being developed, shifting the bottleneck towards understanding metabolism and identifying target genes. Finally, strains and processes should be improved concurrently to fully exploit the potential lipid production from microalgae. To fulfill the necessity for improved yields in microalgal lipid production, current research is moving towards advanced cultivation strategies, including adaptation to fluctuating outdoor weather conditions.Recent developments of targeted gene-editing technology (e.g., CRISPR/Cas9) and improved high-throughput screening techniques in microalgae contribute to fast and automatized strain improvement approaches.Industrial needs and higher oil productivities of microalgae compared with plants will foster the development of tailored microalgal oils.
Identification of an industrial microalgal strain for starch production in biorefinery context : The effect of nitrogen and carbon concentration on starch accumulation
Gifuni, Imma ; Olivieri, Giuseppe ; Pollio, Antonino ; Marzocchella, Antonio - \ 2018
New Biotechnology 41 (2018). - ISSN 1871-6784 - p. 46 - 54.
Biorefinery - CO supply - Microalgae - Nitrate demand - Starch
The recent trends in microalgal cultures are focused on the biorefinery of the biomass components. Some of them are not completely valorised, for example starch. Since there is a wide market for starch products in food and non-food industries, the exploitation of microalgal starch fractions could improve the economic sustainability of microalgae production. In this perspective, the optimization of nitrogen and carbon source uptake for starch accumulation is a critical point for reducing the nitrogen requirement footprint and to increase CO2 capture. In this study, four robust microalgal strains, already known as starch-accumulating strain, were investigated: Chlorella sorokiniana, Scenedesmus vacuolatus, Dunaliella tertiolecta, and Tetraselmis chuii. C. sorokiniana was selected as the best starch producer in the biorefinery context, and the role nitrogen and CO2 concentration had on the starch production was investigated. For light irradiance of 300 μmol m−2 s−1 the optimal nitrogen concentration for growth and starch accumulation resulted 32 mg L−1. The CO2 concentration clearly does not influence the starch accumulation, but concentrations distant from 2% negatively influence microalgal growth, affecting the final starch productivity. The biomass composition during the batch growth of C. sorokiniana was also analysed in order to explicitly characterise the dynamic of starch accumulation during the different growth phases. Protein content decreased during N-depletion, carbohydrates were mainly produced during the early N-depletion, followed by the accumulation of lipids in the late depletion.
Rubisco separation using biocompatible aqueous two-phase systems
Suarez Ruiz, C.A. ; Berg, C. van den; Wijffels, R.H. ; Eppink, M.H.M. - \ 2018
Separation and Purification Technology 196 (2018). - ISSN 1383-5866 - p. 254 - 261.
Aqueous two phase systems - Ionic liquids - Microalgae - Proteins - Rubisco - Separation
Mild and efficient separation processes have to be developed to convert microalgal biomass into high valuable products. Aqueous two-phase system (ATPS) was adopted as a new approach in microalgae to separate hydrophilic from hydrophobic components. In this work, three biocompatible ATPSs polyethylene glycol (PEG) 400-Potassium citrate, Iolilyte 221PG-potassium citrate and PEG 400-Cholinium dihydrogen phosphate ATPS were selected based on their interaction with Ribulose-1,5-biphosphate carboxylase/oxygenase (Rubisco), a protein predominantly present in microalgae and used as ingredient in human and animal food. Binodal curves were constructed for each system and the parameters influencing phase formation were investigated. Iolilyte 221PG-potassium citrate has a stronger ability to form ATPS compared with the PEG-based systems. This stronger ability was attributed to hydrophobic and electrostatic interactions between the phase-forming components. After characterization, we investigated the performance of the ATPSs in the partitioning of Rubisco. In this study, the effect of the tie-line length (TLL), pH and type of phase-forming components on Rubisco extraction efficiency (%) was analyzed. In a single step, the appropriate parameters lead to extraction efficiencies between 80 and 100%. Additionally, stability studies were performed to see if ATPS retain the native protein structure. Iolilyte 221PG-Citrate was found to be the most efficient ATPS in Rubisco separation. However, stability studies indicated that PEG-based ATPSs have a better performance in retaining the Rubisco integrity.
Bioeconomy with algae - Life cycle sustainability assessment including biophysical climate impacts (ALBEDO) of an algae-based biorefinery
Hingsamer, Maria ; Bird, Neil ; Kaltenegger, Ingrid ; Jungmeier, Gerfried ; Kleinegris, Dorinde ; Lamers, Packo ; Boussiba, Sammy ; Rodolfi, Liliana ; Norsker, Niels Henrik ; Jacobs, Fons ; Fenton, Marcus ; Ranjbar, Reza ; Hujanen, Mervi ; Sanz, Macarena - \ 2017
European Biomass Conference and Exhibition Proceedings 2017 (2017)25thEUBCE. - ISSN 2282-5819 - p. 1453 - 1455.
Albedo - Biorefinery - Greenhouse gases (GHG) - Life cycle sustainability assessment - Microalgae
The viability of using microalgae for energy production depends on the overall sustainability (environmental, economic, social). The project FUEL4ME applies a life cycle sustainability assessment (LCSA) providing scientific indicators for economic (e.g. operational costs, investment cost, trade effects, effects on employment), environmental (global warming potential, cumulated primary energy demand, land use) and social aspects (e.g. regional cooperations, product responsibility, labour practices) of an algae-based biorefinery (production of biofuels and PUFAs). Climate change impacts usually focus only on the bio/chemical contribution (i.e. greenhouse gases) but within FUEL4ME biophysical climate impacts due to albedo change were included in the assessment of the global warming potential. A comparison using the LCSA results of a modelled full scale commercial FUEL4ME process was made to analyse the gap between the current TRL and guiding values for TRL 9. Due to actual demonstrated state of technology (current TRL) further technology development is needed.
Comparison of Protein Extracts from Various Unicellular Green Sources
Teuling, Emma ; Wierenga, Peter A. ; Schrama, Johan W. ; Gruppen, Harry - \ 2017
Journal of Agricultural and Food Chemistry 65 (2017)36. - ISSN 0021-8561 - p. 7989 - 8002.
amino acid composition - carbohydrate composition - cyanobacteria - Microalgae - physicochemical properties - single-cell protein
Photosynthetic unicellular organisms are considered as promising alternative protein sources. The aim of this study is to understand the extent to which these green sources differ with respect to their gross composition and how these differences affect the final protein isolate. Using mild isolation techniques, proteins were extracted and isolated from four different unicellular sources (Arthrospira (spirulina) maxima, Nannochloropsis gaditana, Tetraselmis impellucida, and Scenedesmus dimorphus). Despite differences in protein contents of the sources (27-62% w/w) and in protein extractability (17-74% w/w), final protein isolates were obtained that had similar protein contents (62-77% w/w) and protein yields (3-9% w/w). Protein solubility as a function of pH was different between the sources and in ionic strength dependency, especially at pH < 4.0. Overall, the characterization and extraction protocol used allows a relatively fast and well-described isolation of purified proteins from novel protein sources.
Outdoor performance of Chlorococcum littorale at different locations
Cabanelas, Iago Teles Dominguez ; Slegers, Petronella M. ; Böpple, Hanna ; Kleinegris, Dorinde M.M. ; Wijffels, René H. ; Wijffels, René H. ; Barbosa, Maria J. - \ 2017
Algal Research 27 (2017). - ISSN 2211-9264 - p. 55 - 64.
Chlorococcum Littorale - Microalgae - Modelling carbon partitioning - Outdoor productivities - Strain improvement - Tag - Year round productivities
Our goal in the present study was to evaluate the potential for lipid production of two cell populations of the marine microalgae Chlorococcum littorale under different climate conditions. We selected, in a previous study and via fluorescence activated cell sorting (FACS), a new cell population of Chlorococcum littorale, namely S5. S5 showed a stable doubled triacylglycerol (TAG) productivity in comparison with the original population. A previously developed model was expanded to include day:night cycles and validated to predict biomass and outdoor TAG productivities at different locations. Four different locations were chosen to simulate the response of C. littorale to different day lengths and light intensities (the Netherlands, Norway, Brazil and Spain). Indoor experiments (simulated summer) were carried out with Original and S5, showing that S5 had a doubled TAG productivity under N-starvation. Finally, simulations of biomass and TAG productivities of Original and S5 at different locations were performed. At locations with lower light intensities, Norway and the Netherland s, biomass productivities were higher than at locations with higher light intensities, Brazil/Spain. Such results might be associated with light-saturation effects. TAG productivities, however, showed no effect of local light intensity. Locations at higher latitudes, Norway/Netherlands, cannot sustain phototrophic year-round production, hence, the yearly average TAG productivities were doubled in Brazil/Spain (from 1.4–1.6 to 3.0–3.2 g m − 2 d − 1 ). Likewise, C. littorale S5 was simulated with doubled TAG productivities when compared with Original, at all locations (2.5–2.7 (low light) to 4.7–5.2 g m − 2 d − 1 (high light)). The present results confirm the industrial potential of Chlorococcum littorale, both Original and S5, as a source of TAG. Furthermore, our results can be used for comparison and to estimate future production scenarios.
Towards microalgal triglycerides in the commodity markets
Benvenuti, Giulia ; Ruiz, Jesús ; Lamers, Packo P. ; Bosma, Rouke ; Wijffels, René H. ; Barbosa, Maria J. - \ 2017
Biotechnology for Biofuels 10 (2017)1. - ISSN 1754-6834
Microalgae - Production costs - Techno-economic analysis - Triglyceride production
Background: Microalgal triglycerides (TAGs) hold great promise as sustainable feedstock for commodity industries. However, to determine research priorities and support business decisions, solid techno-economic studies are essential. Here, we present a techno-economic analysis of two-step TAG production (growth reactors are operated in continuous mode such that multiple batch-operated stress reactors are inoculated and harvested sequentially) for a 100-ha plant in southern Spain using vertically stacked tubular photobioreactors. The base case is established with outdoor pilot-scale data and based on current process technology. Results: For the base case, production costs of 6.7 € per kg of biomass containing 24% TAG (w/w) were found. Several scenarios with reduced production costs were then presented based on the latest biological and technological advances. For instance, much effort should focus on increasing the photosynthetic efficiency during the stress and growth phases, as this is the most influential parameter on production costs (30 and 14% cost reduction from base case). Next, biological and technological solutions should be implemented for a reduction in cooling requirements (10 and 4.5% cost reduction from base case when active cooling is avoided and cooling setpoint is increased, respectively). When implementing all the suggested improvements, production costs can be decreased to 3.3 € per kg of biomass containing 60% TAG (w/w) within the next 8 years. Conclusions: With our techno-economic analysis, we indicated a roadmap for a substantial cost reduction. However, microalgal TAGs are not yet cost efficient when compared to their present market value. Cost-competiveness strictly relies on the valorization of the whole biomass components and on cheaper PBR designs (e.g. plastic film flat panels). In particular, further research should focus on the development and commercialization of PBRs where active cooling is avoided and stable operating temperatures are maintained by the water basin in which the reactor is placed.
Mild and Selective Protein Release of Cell Wall Deficient Microalgae with Pulsed Electric Field
Lam, Gerard 't; Kolk, Jelmer A. van der; Chordia, Akshita ; Vermuë, Marian H. ; Olivieri, Giuseppe ; Eppink, Michel H.M. ; Wijffels, René H. - \ 2017
ACS sustainable chemistry & engineering 5 (2017)7. - ISSN 2168-0485 - p. 6046 - 6053.
Microalgae - Mild cell disruption - Multiproduct biorefinery - Selective release

Pulsed electric field (PEF) is considered to be a very promising technology for mild cell disruption. The application of PEF for microalgae that have a rigid cell wall, however, is hampered by the presence of that rigid outer cell wall. A cell wall free mutant of C. reinhardtii was used to mimic pretreated microalgae with removed cell wall, to investigate the possibility of using PEF for protein release from microalgae. A complete release of hydrophilic proteins from the cell wall free mutants was observed whereas PEF treatment on the cell wall containing species resulted in substantially lower protein yields. Additional experiments showed that even at low energy input (0.05 kWh/kgbiomass), still about 70% of the proteins could be released with respect to bead beating as reference. These released proteins were water-soluble while the hydrophobic chlorophyll remained mainly entrapped in cell particles. SEM-analysis of these cell particles showed that PEF only opened the cells, instead of completely fragmenting them into smaller particles. These results indicate that PEF is an energy-efficient cell disruption method for selective release of water-soluble proteins, after the microalgal outer cell wall is removed. Enzymatic pretreatment to degrade the cell walls before PEF treatment was shown to be an efficient method to remove the cell wall.

The influence of day/night cycles on biomass yield and composition of Neochloris oleoabundans
Winter, Lenneke de; Dominguez Teles, Iago ; Martens, Dirk E. ; Wijffels, René H. ; Barbosa, Maria J. - \ 2017
Biotechnology for Biofuels 10 (2017). - ISSN 1754-6834 - 10 p.
Biomass composition - Cell cycle - Circadian clock - Day/night cycle - Microalgae

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 role of starch as transient energy buffer in synchronized microalgal growth in Acutodesmus obliquus
Leon Saiki, Mitsue ; Remmers, Ilse M. ; Martens, Dirk E. ; Lamers, Packo P. ; Wijffels, René H. ; Veen, Douwe van der - \ 2017
Algal Research 25 (2017). - ISSN 2211-9264 - p. 160 - 167.
Day/night cycles - Diurnal biochemical changes - Microalgae - Photosynthetic efficiency - Scenedesmus obliquus - Starchless mutant

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.

Continuous versus batch production of lipids in the microalgae Acutodesmus obliquus
Remmers, I.M. ; Hidalgo-Ulloa, A. ; Brandt, B.P. ; Evers, W.A.C. ; Wijffels, R.H. ; Lamers, P.P. - \ 2017
Bioresource Technology 244 (2017). - ISSN 0960-8524 - p. 1384 - 1392.
Diurnal LD cycles - Microalgae - Scenedesmus obliquus - Starch - Triacylglycerol (TAG)
This work provides a novel quantitative comparison of batch versus continuous microalgal lipid production in the wild type and starchless mutant strain of Acutodesmus obliquus. Both strains showed higher TAG yields on light under batch operation compared to continuous nitrogen limitation. The starchless mutant showed 0.20gTAGmolph -1 for batch and 0.12gTAGmolph -1 for continuous operation, while the wildtype only showed 0.16gTAGmolph -1 for batch and 0.08gTAGmolph -1 for continuous operation. Also, higher TAG contents were found under batch starvation (26% of dry weight for the wildtype and 43% of dry weight for starchless mutant) compared to continuous cultivations (16% of dry weight for the wildtype and 33% of dry weight for starchless mutant). Starch acts as the favoured storage metabolite during nitrogen limitation in A. obliquus, whereas TAG is only accumulated after starch reaches a cellular maximum of 40% of dry weight.
Polar snow algae as a valuable source of lipids?
Hulatt, Chris J. ; Berecz, Orsolya ; Egeland, Einar Skarstad ; Wijffels, René H. ; Kiron, Viswanath - \ 2017
Bioresource Technology 235 (2017). - ISSN 0960-8524 - p. 338 - 347.
Arctic - Microalgae - Photobioreactor - Snow algae - Triacylglycerol (TAG)

Microalgae offer excellent opportunities for producing food and fuel commodities, but in colder climates the low growth rates of many varieties may hamper production. In this work, extremophilic Arctic microalgae were tested to establish whether satisfactory growth and lipid production could be obtained at low water temperature. Five species of snow/soil algae originating from Svalbard (78–79°N) were cultivated at 6 °C, reaching high cell densities (maximum dry weight 3.4 g·L−1) in batch cultivations, and high productivity (maximum 0.63 g·L−1·d−1). After 20 days of cultivation total lipids ranged from 28% to 39% of the dry weight, and diverse patterns of neutral lipid (triacylglycerol; TAG) accumulation were observed. The five species largely accumulated unsaturated fatty acyl chains in neutral lipids, especially polyunsaturated C16 series fatty acids, C18:1n-9 and C18:3n-3. The results indicate that polar microalgae could provide an opportunity to increase the yields of microalgal biomass and oil products at low temperatures.

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