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.

    Publications authored by the staff of the Research Institutes are available from 1995 onwards.

    Full text documents are added when available. The database is updated daily and currently holds about 240,000 items, of which 72,000 in open access.

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    The complete mitogenome and plastome of the haptophyte Pavlova lutheri NIVA-4/92
    Hulatt, Chris J. ; Wijffels, René H. ; Viswanath, Kiron ; Posewitz, Matthew C. - \ 2020
    Mitochondrial DNA Part B: Resources 5 (2020)3. - p. 2748 - 2749.
    aquaculture - DHA - Haptophyte - lipid metabolism - metabolic model

    The complete mitochondrial and plastid genomes of the microalga Pavlova lutheri strain NIVA-4/92 are reported. The circular-mapping mitogenome is 36,202 bp in length, contains 22 protein-coding genes, 24 tRNAs, and has a GC content of 37.5%. Like other haptophytes the mitogenome contains a single large, complex repeat region of approximately 5.4 kbp. The plastome is 95,281 bp in length and has a GC content of 35.6%. It contains 111 protein-coding genes and 27 tRNAs.

    Process optimization of fucoxanthin production with Tisochrysis lutea
    Gao, Fengzheng ; Teles (Cabanelas, ITD), Iago ; Wijffels, René H. ; Barbosa, Maria J. - \ 2020
    Bioresource Technology 315 (2020). - ISSN 0960-8524
    Absorbed light - Dilution rate - Fucoxanthin - Temperature - Tisochrysis lutea

    To optimize fucoxanthin production in Tisochrysis lutea, the effect of different process parameters on fucoxanthin productivity (Pfx) were evaluated using batch and continuous experiments. In batch, the highest Pfx was found at 30 °C and 300 μmol m−2 s−1, allowing to design continuous experiments to optimize the dilution rate. The highest ever reported Pfx (9.43–9.81 mg L−1 d−1) was achieved at dilution rates of 0.53 and 0.80 d−1. Irradiance was varied (50–500 μmol m−2 s−1) to result in a range of absorbed light between 2.23 and 25.80 mol m−2 d−1 at a fixed dilution rate (0.53 d−1). These experiments validated the hypothesis that light absorbed can be used to predict fucoxanthin content, resulting in 2.23 mol m−2 d−1 triggering the highest fucoxanthin content (16.39 mg/g). The highest Pfx was found with 18.38 mol m−2 d−1. These results can be used to achieve high Pfx or fucoxanthin content during cultivation of Tisochrysis lutea.

    Production of carbohydrates, lipids and polyunsaturated fatty acids (PUFA) by the polar marine microalga Chlamydomonas malina RCC2488
    Morales-Sánchez, Daniela ; Schulze, Peter S.C. ; Kiron, Viswanath ; Wijffels, René H. - \ 2020
    Algal Research 50 (2020). - ISSN 2211-9264
    Chlamydomonas - Light intensity - Nitrogen deprivation - Polar microalgae - PUFA - Salinity

    Polar microalgae that are highly productive in cold climates can produce large amounts of biomass and polyunsaturated fatty acids (PUFA). The polar Chlamydomonas malina RCC2488, grows at low temperatures and produces high amounts of lipids, which are mainly composed of PUFA. However, not much is known about its phylogenetic relationship with other strains within the order Chlamydomonadales and the optimum growth conditions for maximum biomass productivity have not yet been identified. In this study, a phylogenetic analysis was performed to determine the closest relatives of C. malina within the Chlamydomonadales order. To select the best growth conditions for maximum biomass productivities in cultivations performed at 8 °C, different salinities (0–80) and light intensities (70–500 μmol photons m−2 s−1) were tested, using bubble column and flat-panel photobioreactors. The effect of nitrogen limitation was tested to determine if C. malina can accumulate energy reserve metabolites (carbohydrates and lipids). Phylogenetic analysis confirmed that C. malina, which belongs to the Chlamydomonales order, is closely related to the psychrophilics Chlamydomonas sp. UWO 241 and Chlamydomonas sp. SAG 75.94, as well as to the mesophilic C. parkeae MBIC 10599. The highest biomass (527 mg L−1 day−1), lipid (161.3 mg L−1 day−1) and polyunsaturated fatty acids (PUFA; 85.4 mg L−1 day−1) productivities were obtained at a salinity of 17.5, light intensity of 250 μmol photons m−2 s−1 and nitrogen replete conditions. Strikingly, the marine C. malina can grow even in fresh water, but the biomass productivity was reduced. While the intracellular lipid content remained unchanged under nitrogen deprivation, the carbohydrate content increased (up to 49.5% w/w), and the protein content decreased. The algal lipids were mainly comprised of neutral lipids, which were primarily composed of PUFA. Chlamydomonas malina RCC2488 is a polar marine microalga suitable for high biomass, carbohydrate, lipid and PUFA productivities at low temperatures.

    Bioprospecting and characterization of temperature tolerant microalgae from Bonaire
    Barten, Robin J.P. ; Wijffels, Rene H. ; Barbosa, Maria J. - \ 2020
    Algal Research 50 (2020). - ISSN 2211-9264
    Biomass composition - Bioprospecting - Fatty acids - Growth rate - Microalgae - Thermo-tolerant

    Control of temperature is a major challenge for industrial microalgae production in photobioreactors outdoors. Strains with tolerance for high temperatures can reduce the cost of production as active temperature control is not required. In this study, marine photoautotrophic microorganisms were isolated to reduce the need for control of high temperature. Twenty-two samples were taken from different saline waters on the Caribbean island Bonaire. During strain enrichment, a temperature of 40 °C was used as selective pressure and strains with the highest growth rate were selected. We isolated and identified 59 strains, after which 5 were selected for characterization on growth rate and biomass composition. Picochlorum sp. and Leptolyngbya sp. showed optimal growth at 40 °C and 35 °C with a growth rate of 0.12 h−1 during daytime, respectively. The strains contain 62.1% and 68.2% of protein and have varying fatty acid compositions suitable for application as edible oil and biofuel.

    How to improve the process of forming biobased R&D collaborations
    Israël-Hoevelaken, Brenda T.P.M. ; Wubben, Emiel F.M. ; Bos, Harriëtte L. ; Wijffels, René H. ; Omta, Onno S.W.F. - \ 2020
    Biofuels Bioproducts and Biorefining (2020). - ISSN 1932-104X - 19 p.
    biobased - innovation - models - multi-partner - R&D collaboration

    The transition towards a biobased economy requires innovations. In addition to the usual challenges of innovation trajectories, the characteristics of biobased innovations cause extra difficulties. To lower the failure rate of innovation trajectories in general, companies tend to form R&D collaborations. Choices made during the formation of such R&D collaborations play a key role in the project's success. Here, one may benefit from the social sciences. This paper presents a perspective on what the social sciences may bring to analyze and improve the formation process of biobased R&D collaborations. The paper also provides an overview of relevant innovation and transition models, and lists the dominant variables in such formation processes (biobased characteristics and general determinants), and the guidelines that seem useful. Although each model has its advantages, none of the innovation and transition models studied addresses both the phases of a formation process of a biobased R&D collaboration and the variables involved in each phase. Concerning the formation process of biobased R&D collaborations, the literature addresses social, organizational, technological, economic, and environmental variables. The key determinants of multi-partner R&D collaborations are partner properties, motives to join a consortium, appropriability of a firm, and project properties. The descriptions of their influence on an R&D collaboration presented here can be used as guidelines, as recommendations, in processes for the formation of relatively less complex R&D collaborations. The influence of biobased characteristics – such as type of innovation (drop-ins versus novel materials), biorefinery, biomass supply and technological challenges – on R&D collaboration have not been studied systematically as yet.

    Neochloris oleoabundans biorefinery : Integration of cell disruption and purification steps using aqueous biphasic systems-based in surface-active ionic liquids
    Suarez Ruiz, C.A. ; Martins, M. ; Coutinho, J.A.P. ; Wijffels, R.H. ; Eppink, M.H.M. ; Berg, C. van den; Ventura, S.P.M. - \ 2020
    Chemical Engineering Journal 399 (2020). - ISSN 1385-8947
    Aqueous biphasic systems - Microalgae disruption - Multi-product approach - Purification - Tensioactive compounds

    In this work, an approach to integrate the downstream processing of bioactive compounds present in the microalgae cells by combining the use of tensioactive compounds and aqueous biphasic systems (ABS) is proposed. For this purpose, several aqueous solutions using solvents with and without tensioactive nature were investigated on their capacity to disrupt the microalgae cells as well as to extract the different classes of biomolecules, namely pigments (chlorophylls a and b, and lutein), proteins and carbohydrates. Cationic tensioactive compounds were selected due to their high ability to simultaneously extract the different classes of compounds present in the Neochloris oleoabundans biomass. To fractionate pigments, proteins and carbohydrates extracted from the microalgae, ABS formed by polyethylene glycol (PEG 8000) and sodium polyacrylate (NaPA 8000) were used, with the solvent selected to disrupt the cells acting as electrolyte. This allowed to tune the biomolecule's partition reaching a selective fractionation. This approach provided the simultaneous extraction of different biomolecules (pigments, protein and carbohydrates) from the cells and, the subsequent origin of two fractions, one rich in proteins (extraction efficiencies of 100%) and carbohydrates (extraction efficiency of 80%) and the second concentrated in pigments (e.g. lutein, extraction efficiency of 98%). The further isolation of the biomolecules from the ABS forming solvents is proposed aiming at the development of an integrated downstream process, including the cell disruption/compounds extraction, the fractionation, and the isolation of the biomolecules.

    Production of Rhodomonas sp. at pilot scale under sunlight conditions
    Oostlander, P.C. ; Latsos, C. ; Houcke, J. van; Wijffels, R.H. ; Barbosa, M.J. - \ 2020
    Algal Research 48 (2020). - ISSN 2211-9264
    Biomass productivity - Biomass yield on light - Microalgae production - Photobioreactors - Pilot-scale - Sunlight

    Rhodomonas sp., is an important microalga for aquaculture feed applications and gained increased research interest over the past few years. While efforts to optimise cultivation of the strain have been studied in detail under laboratory conditions, Rhodomonas sp. has never been grown in photobioreactors at large scale under outdoor light conditions. To study the industrial potential of this strain, we cultivated Rhodomonas sp. in three identical tubular photobioreactors with 200 l working volume each, located in a greenhouse using sunlight conditions only. Growth experiments were performed from February with winter light conditions (<10 mol m−2 d−1) up to high light conditions of summer (>50 mol m−2 d−1) in July, representing all sunlight conditions in the Netherlands. All nutrients were supplied in surplus and temperature and pH were maintained at optimum values for growth of Rhodomonas sp., based on lab data. The total light per reactor was calculated using a ray-tracing analysis to allow calculations based on the light reaching each individual reactor. Rhodomonas sp. grew under all tested light conditions. Biomass yield on light decreased with increasing light conditions from 0.43 ± 0.21 g mol−1 to 0.18 ± 0.04 g mol−1 at 0–10 molph m−2 d−1 to 30–40 molph m−2 d−1. Biomass productivities increased with increasing light from 0.09 ± 0.04 g l−1 d−1 to 0.19 ± 0.06 g l−1 d−1, for 0–10 and 30–40 molph m−2 d−1. We obtained a 2–5 fold increase in biomass productivity compared to previous reports on Rhodomonas sp. cultivation using artificial light at large scale. Our results show that Rhodomonas sp. can be grown at pilot scale using sunlight conditions and further improvements can be reached in the future.

    Evaluation of diurnal responses of Tetradesmus obliquus under nitrogen limitation
    León-Saiki, G.M. ; Carreres, Benoit M. ; Remmers, Ilse M. ; Wijffels, René H. ; Martins dos Santos, Vitor A.P. ; Veen, Douwe van der; Schaap, Peter J. ; Suarez-Diez, Maria ; Martens, Dirk E. - \ 2020
    Algal Research 49 (2020). - ISSN 2211-9264
    Day/night cycles - Diurnal transcription changes - Microalgae - Nitrogen limitation - Scenedesmus obliquus - Starchless mutant

    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.

    From Eat to trEat : Engineering the mitochondrial Eat1 enzyme for enhanced ethyl acetate production in Escherichia coli
    Kruis, Aleksander J. ; Bohnenkamp, Anna C. ; Nap, Bram ; Nielsen, Jochem ; Mars, Astrid E. ; Wijffels, Rene H. ; Oost, John Van Der; Kengen, Servé W.M. ; Weusthuis, Ruud A. - \ 2020
    Biotechnology for Biofuels 13 (2020)1. - ISSN 1754-6834
    Alcohol acetyl transferase (AAT) - Eat1 - Escherichia coli - Ethyl acetate - Mitochondria

    Background: Genetic engineering of microorganisms has become a common practice to establish microbial cell factories for a wide range of compounds. Ethyl acetate is an industrial solvent that is used in several applications, mainly as a biodegradable organic solvent with low toxicity. While ethyl acetate is produced by several natural yeast species, the main mechanism of production has remained elusive until the discovery of Eat1 in Wickerhamomyces anomalus. Unlike other yeast alcohol acetyl transferases (AATs), Eat1 is located in the yeast mitochondria, suggesting that the coding sequence contains a mitochondrial pre-sequence. For expression in prokaryotic hosts such as E. coli, expression of heterologous proteins with eukaryotic signal sequences may not be optimal. Results: Unprocessed and synthetically truncated eat1 variants of Kluyveromyces marxianus and Wickerhamomyces anomalus have been compared in vitro regarding enzyme activity and stability. While the specific activity remained unaffected, half-life improved for several truncated variants. The same variants showed better performance regarding ethyl acetate production when expressed in E. coli. Conclusion: By analysing and predicting the N-terminal pre-sequences of different Eat1 proteins and systematically trimming them, the stability of the enzymes in vitro could be improved, leading to an overall improvement of in vivo ethyl acetate production in E. coli. Truncated variants of eat1 could therefore benefit future engineering approaches towards efficient ethyl acetate production.

    Doubling of Microalgae Productivity by Oxygen Balanced Mixotrophy
    Abiusi, Fabian ; Wijffels, Rene H. ; Janssen, Marcel - \ 2020
    ACS sustainable chemistry & engineering 8 (2020)15. - ISSN 2168-0485 - p. 6065 - 6074.
    Biomass yield on substrate - Carbon balance - Microalgae productivity - Mixotrophic cultivation - Oxygen balance

    Microalgae productivity was doubled by designing an innovative mixotrophic cultivation strategy that does not require gas-liquid transfer of oxygen or carbon dioxide. Chlorella sorokiniana SAG 211/8K was cultivated under continuous operation in a 2 L stirred-tank photobioreactor redesigned so that respiratory oxygen consumption was controlled by tuning the acetic acid supply. In this mixotrophic setup, the reactor was first operated with aeration and no net oxygen production was measured at a fixed acetic acid supply rate. Then, the aeration was stopped and the acetic acid supply rate was automatically regulated to maintain a constant dissolved oxygen level using process control software. Respiratory oxygen consumption was balanced by phototrophic oxygen production, and the reactor was operated without any gas-liquid exchange. The carbon dioxide required for photosynthesis was completely provided by the aerobic conversion of acetic acid. Under this condition, the biomass/substrate yield was 0.94 C-molx·C-molS -1. Under chemostat conditions, both reactor productivity and algal biomass concentration were doubled in comparison to a photoautotrophic reference culture. Mixotrophic cultivation did not affect the photosystem II maximum quantum yield (Fv/Fm) and the average-dry-weight-specific optical cross section of the microalgal cells. Only light absorption by chlorophylls over carotenoids decreased by 9% in the mixotrophic culture in comparison to the photoautotrophic reference. Our results demonstrate that photoautotrophic and chemoorganotrophic metabolism operate concurrently and that the overall yield is the sum of the two metabolic modes. At the expense of supplying an organic carbon source, photobioreactor productivity can be doubled while avoiding energy intensive aeration.

    Multilevel optimisation of anaerobic ethyl acetate production in engineered Escherichia coli
    Bohnenkamp, Anna C. ; Kruis, Aleksander J. ; Mars, Astrid E. ; Wijffels, Rene H. ; Oost, John Van Der; Kengen, Servé W.M. ; Weusthuis, Ruud A. - \ 2020
    Biotechnology for Biofuels 13 (2020)1. - ISSN 1754-6834
    Alcohol acetyl transferase (AAT) - Anaerobic - Bioreactor - Eat1 - Escherichia coli - Ethyl acetate - Fermentation

    Background: Ethyl acetate is a widely used industrial solvent that is currently produced by chemical conversions from fossil resources. Several yeast species are able to convert sugars to ethyl acetate under aerobic conditions. However, performing ethyl acetate synthesis anaerobically may result in enhanced production efficiency, making the process economically more viable. Results: We engineered an E. coli strain that is able to convert glucose to ethyl acetate as the main fermentation product under anaerobic conditions. The key enzyme of the pathway is an alcohol acetyltransferase (AAT) that catalyses the formation of ethyl acetate from acetyl-CoA and ethanol. To select a suitable AAT, the ethyl acetate-forming capacities of Atf1 from Saccharomyces cerevisiae, Eat1 from Kluyveromyces marxianus and Eat1 from Wickerhamomyces anomalus were compared. Heterologous expression of the AAT-encoding genes under control of the inducible LacI/T7 and XylS/Pm promoters allowed optimisation of their expression levels. Conclusion: Engineering efforts on protein and fermentation level resulted in an E. coli strain that anaerobically produced 42.8 mM (3.8 g/L) ethyl acetate from glucose with an unprecedented efficiency, i.e. 0.48 C-mol/C-mol or 72% of the maximum pathway yield.

    Combined bead milling and enzymatic hydrolysis for efficient fractionation of lipids, proteins, and carbohydrates of Chlorella vulgaris microalgae
    Alavijeh, Razieh Shafiei ; Karimi, Keikhosro ; Wijffels, Rene H. ; Berg, Corjan van den; Eppink, Michel - \ 2020
    Bioresource Technology 309 (2020). - ISSN 0960-8524
    Bead milling - Combined treatment - Enzymatic hydrolysis - Fractionation - Microalgae

    A combined bead milling and enzymatic hydrolysis process was developed for fractionation of the major valuable biomass components, i.e., proteins, carbohydrates, and lipids from the microalgae Chlorella vulgaris. The cells were treated by bead milling followed by hydrolysis with different hydrolytic enzymes, including lipase, phospholipase, protease, and cellulase. Without enzymatic hydrolysis, the recovery yield of lipids, carbohydrates, and proteins for bead milled biomass was 75%, 31%, and 40%, respectively, while by applying enzymatic treatments these results were improved significantly. The maximum recovery yield for all components was obtained after enzymatic hydrolysis of bead milled biomass by lipase at 37 °C and pH 7.4 for 24 h, yielding 88% lipids in the solid phase while 74% carbohydrate and 68% protein were separated in the liquid phase. The recovery yield of components after enzymatic hydrolysis of biomass without bead milling was 44% lower than that of the milled biomass.

    Optimization of Rhodomonas sp. under continuous cultivation for industrial applications in aquaculture
    Oostlander, P.C. ; Houcke, J. van; Wijffels, R.H. ; Barbosa, M.J. - \ 2020
    Algal Research 47 (2020). - ISSN 2211-9264
    Biomass production rate - Biomass yield on light - Fatty acid composition - Growth parameter optimization - Rhodomonas sp. - Turbidostat production

    The microalgae species Rhodomonas sp. is commonly used in aquaculture for its high nutritional value due to the eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) content. Understanding the effect of cultivation parameters on biomass production rate and composition is presently limited, however essential in further commercialization of this strain. Under nutrient replete conditions, light intensity and temperature are the main factors determining biomass growth and composition. Therefore, the combined effect of light and temperature on the biomass production rate and biomass composition of Rhodomonas sp. was studied using a statistical Design of Experiment approach. Rhodomonas sp. was cultivated under continuous (turbidostat) conditions in lab-scale reactor systems (1.8 l) under different temperature (15–20–25–30 °C) and light conditions (60–195–330–465–600 μmol m−2 s−1). Stable biomass production was observed under all conditions except experiments performed at 30 °C, which led to cell death. Under optimized growth conditions, high growth rates (>1.0d−1) and high biomass production rates, up to 1.5 g l−1 d−1, were obtained in this study. The biomass production rate reported here is >10-fold higher than values reported in literature on Rhodomonas sp. The optimal temperature for maximal growth was found at T = 22–24 °C under all light conditions. The maximum biomass yield on light (Yx,ph – 0.87 g mol−1) was found at light levels between 110 and 220 μmol m−2 s−1. The fatty acid profile was only significantly influenced by temperature, with higher EPA and DHA contents at lower temperatures (15 °C). A total fatty acid (TFA) content of 8–10% of the total dry-weight was found for all tested conditions. The EPA content fluctuated between 9 and 16% of TFA and DHA content between 6 and 9% of TFA, only affected by temperature. A maximum EPA + DHA production rate of 114 mg l −1 d−1 was obtained at 20 °C and high light (600 μmol m−2 s−1) conditions.

    Modeling of industrial-scale anaerobic solid-state fermentation for Chinese liquor production
    Jin, Guangyuan ; Uhl, Philipp ; Zhu, Yang ; Wijffels, René H. ; Xu, Yan ; Rinzema, Arjen - \ 2020
    Chemical Engineering Journal 394 (2020). - ISSN 1385-8947
    Chinese liquor - Heat transfer - Mathematical modeling - Product inhibition - Solid-state fermentation - Temperature modeling

    Traditional solid-state fermentation processes can give fluctuating product quality and quantity due to difficulties in control and scale up. This paper describes an engineering study of an industrial-scale anaerobic solid-state fermentation process for Chinese liquor (Baijiu) production, aimed at better understanding of the traditional process, as an initial step for future optimization. This mixed-culture fermentation is done in 0.44-m3 vessels embedded in the soil. At this scale, the fermentation is limited by product inhibition. We developed mathematical models based on the Han-Levenspiel equation for product inhibition, with parameters derived from measured data. The models accurately predicted the concentrations of starch and dry matter. A model with radial conduction into a small soil volume around the fermenter and consecutive vertical conduction into the underlying soil accurately predicted the pit temperature in the heating and cooling phases. This model is very sensitive to the values used for the enthalpies of combustion, meaning that direct measurement of the heat production rate would be preferable. In the industry practice, the fermenter volume can be from around 0.20 to 15.00 m3. The model predicts that overheating will occur not only in larger fermenters, but also in the 0.44-m3 fermenters when the soil temperature is high in summer. Our model predictions are consistent with observed behavior in the industry. Our findings can be used to improve this traditional process, as well as similar systems.

    Microalgae production cost in aquaculture hatcheries
    Oostlander, P.C. ; Houcke, J. van; Wijffels, R.H. ; Barbosa, M.J. - \ 2020
    Aquaculture 525 (2020). - ISSN 0044-8486
    Aquaculture hatcheries - Cost price - Cost reduction - Microalgae production - Techno-economic analysis

    Microalgae are a crucial part in many aquaculture feed applications processes, mainly in hatcheries. Many aquaculture hatcheries maintain a small scale microalgae production facility in-house for the production of live feed. Microalgae are usually grown in non-automated bubble-column systems at unknown production costs. Other reactor systems or scenarios utilizing artificial light or sunlight and at different scales could result in a more cost efficient production processes. To determine the cost-price and cost-distribution of microalgae production facilities in Dutch aquaculture industry and identify the most efficient cost reducing strategies a techno-economic analysis for small scale microalgae production facilities (25-1500 m2) was developed. Commercially available reactors commonly used in aquaculture were compared; tubular photobioreactors (TPBR) and bubble-columns (BC) in two placement possibilities; using artificial light in an indoor facility (AL) and utilizing sunlight in a greenhouse (GH) under Dutch climate conditions. Data from commercial microalgae facilities in the Netherlands are used to model reference scenarios describing the cost price of microalgae production with state of the art technology in aquaculture for a biomass production capacity of 125 kg year−1. The reference cost price for algae biomass (on the basis of dry matter) is calculated at €290,- kg−1 and € 329 kg−1 for tubular reactors under artificial light and a greenhouse, respectively and €587,- kg−1 and €573 kg−1 for bubble-columns under artificial light and a greenhouse, respectively. The addition of more artificial light will significantly reduce production costs (by 33%) in all small-scale systems modelled. Biomass yield on light (Yx,ph) showed the largest effect on cost price when not considering a different scale of the production process. Process parameters like temperature control should be aimed at optimizing Yx,ph rather than other forms of cost reduction. The scale of a microalgae production facility has a very large impact on the cost price. With state of the art technologies a cost price reduction of 92% could be achieved by changing the scale from 25m2 to 1500m2, resulting in a cost price of €43,- kg−1, producing 3992 kg year−1 for tubular reactors in a greenhouse. The presented techno-economic model gives valuable insights in the cost price distribution of microalgae production in aquaculture. This allows to focus research efforts towards the most promising cost reduction methods and to optimize existing production facilities in aquaculture companies to achieve economically sustainable microalgae production for live feed in hatcheries.

    Production of phycocyanin by Leptolyngbya sp. in desert environments
    Schipper, Kira ; Fortunati, Filippo ; Oostlander, Pieter C. ; Muraikhi, Mariam Al; Jabri, Hareb Mohammed S.J. Al; Wijffels, René H. ; Barbosa, Maria J. - \ 2020
    Algal Research 47 (2020). - ISSN 2211-9264
    Cyanobacteria - Extract purity - Extraction - Light intensity - Phycocyanin - Thermotolerance

    Leptolyngbya sp. QUCCCM 56 was investigated as a possible alternative to A. platensis, for the production of phycocyanin-rich biomass under desert conditions. Under elevated temperatures and light intensities, of up to 40 °C and 1800 μmol·m−2·s−1, the strain's biomass productivity was up to 45% higher as compared to reported productivities for A. platensis, with comparable phycocyanin content. Increasing temperatures were found to improve the biomass productivity and phycocyanin content, which, at 40 °C, were 1.09 ± 0.03 gX·L−1·d−1 and 72.12 ± 3.52 mgPC·gX −1, respectively. The optimum biomass productivity was found at a light intensity of 300 μmol·m−2·s−1, with higher light intensities causing a decrease of 15%. Furthermore, of the various phycocyanin extraction methods tested, bead-beating in phosphate buffer had the highest combined phycocyanin yield (169.9 ± 3.6 mgPC·gX) and purity (7.37 ± 0.16) for Leptolyngbya sp. For A. platensis, this extraction method also resulted in the highest extract purities (3.78 ± 0.04). The extract purities obtained for Leptolyngbya sp. are considerably higher than other reported phycocyanin purities, and further investigation is recommended to study the scale-up of both Leptolyngbya sp. and bead-beating for commercial scale high-grade phycocyanin production under desert conditions.

    Cultivation of Bacteria From Aplysina aerophoba: Effects of Oxygen and Nutrient Gradients
    Gutleben, Johanna ; Loureiro, Catarina ; Ramírez Romero, Laura Adriana ; Shetty, Sudarshan ; Wijffels, René H. ; Smidt, Hauke ; Sipkema, Detmer - \ 2020
    Frontiers in Microbiology 11 (2020). - ISSN 1664-302X
    antibiotic resistance - Aplysina aerophoba - environmental microbes - marine sponge - microbial cultivation - viability PCR

    Sponge-associated bacteria possess biotechnologically interesting properties but as yet have largely evaded cultivation. Thus, “omics”-based information on the ecology and functional potential of sponge symbionts is awaiting its integration into the design of innovative cultivation approaches. To cultivate bacteria derived from the marine sponge Aplysina aerophoba, nine novel media formulations were created based on the predicted genomic potential of the prevalent sponge symbiont lineage Poribacteria. In addition, to maintain potential microbial metabolic interactions in vitro, a Liquid-Solid cultivation approach and a Winogradsky-column approach were applied. The vast majority of microorganisms in the inoculum appeared viable after cryopreservation of sponge specimen as determined by selective propidium monoazide DNA modification of membrane-compromised cells, however, only 2% of the initial prokaryotic diversity could be recovered through cultivation. In total, 256 OTUs encompassing seven prokaryotic phyla were cultivated. The diversity of the cultivated community was influenced by the addition of the antibiotic aeroplysinin-1 as well as by medium dilution, rather than carbon source. Furthermore, the Winogradsky-column approach reproducibly enriched distinct communities at different column depths, amongst which were numerous Clostridia and OTUs that could not be assigned to a known phylum. While some bacterial taxa such as Pseudovibrio and Ruegeria were recovered from nearly all applied cultivation conditions, others such as Bacteroidetes were specific to certain medium types. Predominant sponge-associated prokaryotic taxa remained uncultured, nonetheless, alternative cultivation approaches applied here enriched for previously uncultivated microbes.

    Pilot kweken van algen als duurzame grondstof veelbelovend
    Wijffels, Rene - \ 2020
    Multistep Fractionation of Microalgal Biomolecules Using Selective Aqueous Two-Phase Systems
    Suarez Ruiz, Catalina A. ; Kwaijtaal, Jennifer ; Peinado, Oriol Cabau ; Berg, Corjan Van Den; Wijffels, Rene H. ; Eppink, Michel H.M. - \ 2020
    ACS sustainable chemistry & engineering 8 (2020)6. - ISSN 2168-0485 - p. 2441 - 2452.
    Aqueous two-phase systems - Biomolecules - Cholinium-based ionic liquids - Downstream processes - Microalgae

    We aim to develop liquid-liquid extraction processes for the fractionation of microalgal components (proteins, pigments, lipids, and carbohydrates). The partitioning behavior of microalgal pigments and proteins in aqueous two-phase systems (ATPS) composed of the polymer polypropylene glycol with molecular weight 400 (PPG 400) + various cholinium based-ionic liquids was studied. A process for fractionation of multiple components from disrupted Neochloris oleoabundans was developed and evaluated. Results show that cholinium dihydrogen phosphate (Ch DHp) allows the fractionation of pigments in the PPG 400-rich phase and proteins in the Ch DHp-rich phase with high selectivity. It was demonstrated that a multiproduct approach can fractionate free glucose, and proteins in the ionic liquid-rich phase, pigments in the polymer-rich phase, while starch and lipids are recovered at the interface.

    Time-dependent transcriptome profile of genes involved in triacylglycerol (TAG) and polyunsaturated fatty acid synthesis in Nannochloropsis gaditana during nitrogen starvation
    Janssen, Jorijn H. ; Spoelder, Jacco ; Koehorst, Jasper J. ; Schaap, Peter J. ; Wijffels, René H. ; Barbosa, Maria J. - \ 2020
    Journal of Applied Phycology 32 (2020). - ISSN 0921-8971 - p. 1153 - 1164.
    Microalgae - Nannochloropsis gaditana - Nitrogen starvation - Polyunsaturated fatty acids - Transcriptome - Triacylglycerol

    In this research, the gene expression of genes involved in lipid metabolism of the eustigmatophyte alga Nannochloropsis gaditana was measured by transcriptomic data. This microalga can be used as a source of triacylglycerol (TAG) and the omega-3 fatty acid eicosapentaenoic acid (EPA). Insight in TAG and EPA production and regulation are needed to improve their productivity. Nitrogen starvation induces TAG accumulation in N. gaditana. Previous research showed that during nitrogen starvation, EPA was translocated from the polar lipids to TAG and de novo synthesized in N. gaditana. Therefore, the expression levels of genes involved in fatty acid translocation and de novo TAG synthesis were measured. Furthermore, the genes involved in de novo EPA synthesis such as elongases and desaturases were studied. The expression levels were measured during the first hours of nitrogen starvation and the subsequent period of 14 days. One phospholipid:diacylglycerol acyltransferase (PDAT) gene involved in translocation of fatty acids from membrane lipids to TAG was upregulated. In addition, several lipases were upregulated, suggesting that these enzymes might be responsible for the translocation of EPA to TAG. Most desaturases and elongases involved in de novo EPA synthesis were downregulated during nitrogen starvation, except for Δ9 desaturase which was upregulated. This upregulation correlates with the increase in oleic acid. Due to the presence of many hypothetical genes, improvement in annotation is needed to increase our understanding of these pathways and their regulation.

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