Innovative vs classical methods for drying heterotrophic Chlorella vulgaris: Impact on protein quality and sensory properties.

Drying is a necessary step in the microalgae production chain to reduce microbial load and oxidative degradation of the end product. Depending on the differences in applied temperature and treatment time, the process of drying can have a substantial impact on protein quality and aroma, important characteristics determining the incorporation potential in food products. In this study, we compared the drying of heterotrophic Chorella vulgaris with both innovative (agitated thin film drying (ATFD), pulse combustion drying (PCD) and solar drying (SolD)) and commonly used drying techniques (spray drying (SprD) and freeze drying (FD)). To evaluate the impact on protein quality, we evaluated techno-functional properties, in vitro digestibility (INFOGEST) as well as protein denaturation using differential scanning calorimetry (DSC). A sensory analysis was performed by a trained expert panel, combined with headspace solid-phase microextraction (HS-SPME) - gas chromatography-mass spectrometry (GC-MS) to determine volatile organic compounds (VOCs). ATFD was found to increase techno-functional properties such as gelling, water holding and solubility as well as in vitro protein digestibility. These observations could be related to induced cell disruption and protein denaturation by ATFD. Sensory analysis indicated an increased earthy off-flavor after ATFD. Interestingly, the high-temperature PCD led to an increase in cacao odor while low-temperature FD resulted in lower flavor, odors and VOCs. These results demonstrate that protein quality and sensorial properties of C. vulgaris can be steered through the type of drying, which could help in the selection of application-specific drying methods. Overall, this work could promote the incorporation of microalgal single cell proteins in different innovative food products.

Microalgae.

Microalgae, in the strictest definition, are eukaryotic, unicellular microorganisms that are photosynthetic and typically have an aquatic lifestyle. Despite the fact that cyanobacteria (or 'blue-green algae') are prokaryotic, and are therefore not true algae, we have included them in this overview because they have a similar physiology and ecology to eukaryotic microalgae, and share many biotechnological applications. In this Primer, we discuss the diversity of microalgae, their evolutionary origin and ecological importance, the role they have played in human affairs so far, and how they can help to accelerate the transition to a more sustainable society.

Combining patterned membrane filtration and flocculation for economical microalgae harvesting.

Membranes have a lot of potential for harvesting microalgae, but mainly membrane fouling and high harvesting costs linked to low fluxes are hampering their breakthrough. Patterned membranes can reduce fouling by enchancing local turbulences close to the membrane surface on one hand, and by increasing the active area per m2 of installed membrane on the other. Flocculation can further increase membrane permeance by increasing microalgal partical size and reducing the fraction of free organic matter in the feed. In current study, the effect of polyethylene glycol (PEG) in the casting solution of patterned polysulfone membranes was investigated to better tune the performance of the patterned membranes, together with the effects of cross-flow velocity and chitosan dosage on membrane fouling. The energy consumption and total harvesting cost, extrapolated to a full-scale microalgal harvesting, were then estimated. The patterned membrane prepared with a 28w% PEG concentration showed the highest clean water permeance (900±22 L/m2 h bar) and membrane permeance in a microalgal suspension (590±17 L/m2 h bar). Patterned membranes showed a lower filtration resistance (15% permeance decline at the end of filtration) than flat membranes (72%) at a cross-flow velocity of 0.0025 m/s. Increasing cross-flow velocity could increase membrane permeance in most cases. The highest stable membrane permeance (110±17 L/m2 h bar) and the lowest filtration resistance were achieved when combining patterned membrane filtration with flocculation at optimized chitosan dosage. A very low energy consumption (0.28 kWh/kg) and harvesting cost (0.16 €/kg) were achieved under these conditions.

Glycine betaine grafted nanocellulose as an effective and bio-based cationic nanocellulose flocculant for wastewater treatment and microalgal harvesting.

Flocculation is a widely used technology in industry including for wastewater treatment and microalgae harvesting. To increase the sustainability of wastewater treatment, and to avoid contamination of the harvested microalgal biomass, there is a need for bio-based flocculants to replace synthetic polymer flocculants or metal salt coagulants. We developed the first cellulose nanocrystalline flocculant with a grafted cationic point charge, i.e. glycine betaine (i.e. N,N,N-trimethylglycine) grafted cellulose nanocrystals (CNCs) effective for the flocculation of kaolin (a model system for wastewater treatment), the freshwater microalgae Chlorella vulgaris, and the marine microalgae Nannochloropsis oculata. We successfully grafted glycine betaine onto CNCs using a one-pot reaction using a tosyl chloride activated esterification reaction with a degree of substitution ranging from 0.078 ± 0.003 to 0.152 ± 0.002. The degree of substitution is controlled by the reaction conditions. Flocculation of kaolin (0.5 g L-1) required a dose of 2 mg L-1, a comparable dose to commercial polyacrylamide-based flocculants. Flocculation was also successful for freshwater as well as marine microalgae (biomass concentration about 300 mg L-1 dry matter), although the flocculation efficiency of the latter remained below 80%. The dose to induce flocculation (DS = 0.152 ± 0.002) was 20 mg L-1 for the freshwater Chlorella vulgaris and 46 mg L-1 for the marine Nannochloropsis oculata, comparable to other bio-based flocculants such as chitosan or TanFloc.

Harvesting microalgal biomass using negatively charged polysulfone patterned membranes: Influence of pattern shapes and mechanism of fouling mitigation.

Membranes have a lot of potential for harvesting microalgae, but membrane fouling is hampering their breakthrough. In this study, the effects of charge and corrugated surface on membrane filtration performance were investigated. The clean water permeance (CWP), the microalgae harvesting efficiency and the membrane flux for a microalgal broth were determined using patterned polysulfone (PSf) membranes with different shapes of the surface patterns and containing different charge densities by blending sulfonated polysulfone (sPSf). The flow behavior near the patterned membrane surface, as well as the interaction energy between membrane and microalgae were investigated using computational fluid dynamics (CFD) simulation and the improved extended "Derjaguin, Landau, Verwey, Overbeek" (XDLVO) theory, respectively. Membrane charge and pattern shape significantly improve the membrane performance. The critical pressures of all sPSf blend patterned membranes were higher than 2.5 bar. A 4.5w% sPSf blend patterned membranes with wave patterns showed the highest CWP (2300 L/m2 h bar) and membrane flux in the microalgal broth (1000 L/m2 h bar) with 100% harvesting efficiency. XDLVO analysis showed that sPSf blend patterned membranes prepared obtained the lowest interaction energy and highest energy barrier for microalgal attachment. CFD simulation showed a higher velocity and wall shear on the pattern apexes.

Isolation and identification of herbivorous ciliates from contaminated microalgal cultures.

Ciliates are a common but understudied group of grazers that can invade microalgal cultures. To estimate the potential impact of ciliates on microalgal culture productivity, the identification of species that can invade these cultures is essential. Furthermore, isolation of these herbivorous ciliates allows to use them in experiments that investigate the impact of ciliate grazing on the productivity of microalgal cultures. The main aims of this study were to isolate and identify ciliates that invade cultures of the freshwater microalgae Chlorella and Chlamydomonas, and to establish a live collection of these ciliates for usage in future experiments. To this end, we optimized a method for isolating ciliates from contaminated microalgal cultures and we developed a new PCR primer set for amplifying the partial 18S rDNA of ciliates belonging to the classes Spirotrichea, Oligohymenophorea and Colpodea. As a result, we isolated 11 ciliates from microalgal enrichment cultures inoculated with non-sterile dust and various freshwater sources. Of these 11 species, 7 were found to be feeding on Chlamydomonas. Ciliate species that fed on Chlorella could not be isolated in this study. Ciliate species feeding on Chlamydomonas were identified based on a combination of morphological observations and molecular analyses of partial 18S rDNA sequences.

The bacterioplankton community composition and a host genotype dependent occurrence of taxa shape the Daphnia magna gut bacterial community.

The assembly of host-associated bacterial communities is influenced by a multitude of biotic and abiotic factors. It is essential to gain insight in the impact and relative strength of these factors if we want to be able to predict the effects of environmental change on the assembly of host-associated bacterial communities, or deliberately modify them. The environmental pool of bacteria, from which the host is colonized, and the genetic background of the host are both considered to be important in determining the composition of host-associated bacterial communities. We experimentally assessed the relative importance of these two factors and their interaction on the composition of Daphnia magna gut bacterial communities. Bacterioplankton originating from natural ponds or a laboratory culture were used to inoculate germ-free Daphnia of different genotypes. We found that the composition of the environmental bacterial community has a major influence on the Daphnia gut bacterial community, both reflected by the presence or absence of specific taxa as well as by a correlation between abundances in the environment and on the host. Our data also indicate a consistent effect of host genotype on the occurrence of specific bacterial taxa in the gut of Daphnia over different environments.

Harvesting of marine microalgae using cationic cellulose nanocrystals.

Flocculation of marine microalgae is challenging because of the high ionic strength of the culture medium. We investigated cationic cellulose nanocrystals (CNCs) as biobased flocculants for the marine microalgae Nannochloropsis oculata, and compared its performance to chitosan. Cationic CNCs induced flocculation at a low dose of 11 mg L-1, while chitosan required a dose of 35 mg L-1. Our cationic CNCs possess a permanent positive charge, allowing flocculation over a wide pH-range (4 to 10). The CNC maximum flocculation efficiency was 90 %, while chitosan achieved > 95 %, attributed to small flocs remaining in suspension for CNCs. However, centrifugation for 1 min at 180 g or gravity filtration using a 30 µm nylon filter after CNC flocculation resulted in the removal of these small, stable flocs (∼150 µm diam) and > 95% harvesting efficiency. Cationic CNCs can thus serve as a sustainable alternative natural flocculant for harvesting both freshwater and marine microalgae.

Optimization of patterned polysulfone membranes for microalgae harvesting.

Membranes with a wave pattern on the membrane surface are now proposed for the first time to alleviate microalgal fouling and increase the membrane flux. The membrane morphology was observed via scanning electron microscope, and the clean water permeance, microalgae harvesting efficiency and membrane flux in a real broth were determined to investigate the effects of polysulfone (PSF) and polyethylene glycol (PEG) concentrations in the membrane casting solution. Furthermore, the influence of the height of the patterned waves and the inter-pattern distance on the fouling prevention were investigated. Higher PSF and PEG concentrations resulted in better pronounced patterns. Patterned membrane showed higher fluxes and critical pressures than the corresponding flat membranes. Larger patterns gave higher membrane fluxes and less fouling. Computational fluid dynamics simulation showed a higher velocity and shear on the pattern apexes.

A highly efficient and energy-saving magnetically induced membrane vibration system for harvesting microalgae.

The optimal operational parameters of a second generation magnetically induced membrane vibration (MMV) system were determined using the response surface methodology (RSM) combined with single-factor experiments. The membrane surfaces were characterized by scanning electron microscopy (SEM) and algae cell states by inverted microscopy. The effect of an intermittent vibration strategy on filtration performance and energy consumption was studied. The results showed that the responses could be fitted by RSM models. High membrane flux, low energy consumption, efficient fouling control and no damage to the microalgae could thus be realized. The filtration strategy tests suggested that an intermittent cycle time of 4 min with 50% vibration rate could be the best vibration strategy for harvesting the microalgae under investigation.

Nanoscale Evidence Unravels Microalgae Flocculation Mechanism Induced by Chitosan.

Microalgae are a promising resource for biofuel production, although their industrial use is limited by the lack of effective harvesting techniques. Flocculation consists in the aggregation and adhesion of cells into flocs that can be more easily removed from water than individual cells. Although it is an efficient harvesting technique, contamination is a major issue as chemical flocculants are often used. An alternative is to use natural biopolymers flocculants such as chitosan. Chitosan is a biobased nontoxic polymer that has been effectively used to harvest Chlorella vulgaris cells at a pH lower than its pKa (6.5). While the reported flocculation mechanism is said to rely on electrostatic interactions between chitosan and the negative cell surface, no molecular evidence has yet confirmed this mechanism. In this study, we performed force spectroscopy atomic force microscopy (AFM) experiments to probe the interactions between C. vulgaris cells and chitosan at the molecular scale to decipher its flocculation mechanism. Our results showed that at pH 6, chitosan interacts with C. vulgaris cell wall through biological interactions rather than electrostatic interactions. These observations were confirmed by comparing the data with cationically modified cellulose nanocrystals, for which the flocculation mechanism, relying on an electrostatic patch mechanism, has already been described for C. vulgaris. Further AFM experiments also showed that a different mechanism was at play at higher pH, based on chitosan precipitation. Thus, this AFM-based approach highlights the complexity of chitosan-induced flocculation mechanisms for C. vulgaris.

Cobalt enrichment enhances the tolerance of Botryococcus braunii to high concentration of CO2.

This work mainly studied B. braunii adapted to different CO2 concentrations with cobalt enrichment, and developed a process for CO2 capture, hydrocarbon production and cobalt removal. The results showed that B. braunii favored rapid growth at 5.0% (v/v) CO2, and the highest biomass was 1.89 g.L-1 with 4.5 mg.L-1 of cobalt. Hydrocarbon productivity in high concentration CO2 (5.0% and 10.0%) with cobalt enrichment was higher than that in Chu 13 medium. The change in cobalt removal efficiency mainly corresponded to the growth of B. braunii. The LCE of B. braunii in cobalt-rich with high CO2 concentration (5.0% and 10.0%) was 15.7%, and 14.9%, respectively, which was higher than that in normal medium. CO2 fixation rates were also higher in cobalt enrichment coupled with high CO2 concentration. This study not only provides ideas for the removal of toxic metal cobalt, but also has great potential for CO2 biofixation.

Quantum yield alterations due to the static magnetic fields action on Arthrospira platensis SAG 21.99: Evaluation of photosystem activity.

Static magnetic fields (SMF) influence the metabolism of microorganisms, however, there is no knowledge explaining how SMF act in cells. This study aimed at evaluating the SMF (30 mT) effect on photosynthetic performance, growth and biomass composition of the cyanobacterium Arthrospira platensis SAG 21.99. A. platensis was cultivated under 30 mT applied for 1 h d-1 and 24 h for 10 d in glass bottles. SMF in both conditions increased cellular growth, achieving a 30% higher biomass concentration. SMF applied for 1 h d-1 increased the pigments and carbohydrate content. The quantum yield was used as an indicator of the photosystem II (PSII) activity and was shown to have been positively affected. SMF for 1 h d-1 had a significant effect on the OJIP curves. This is the first study that evaluated the photosynthetic activity in cyanobacteria cultures under SMF action.

A historical perspective of nutrient change impact on an infectious disease in Daphnia.

Changes in food quality can play a substantial role in the vulnerability of hosts to infectious diseases. In this study, we focused on the genetic differentiation of the water flea Daphnia magna towards food of different quality (by manipulating C:N:P ratios) and its impact on the interaction with a virulent infectious disease, "White Fat Cell Disease (WFCD)". Via a resurrection ecology approach, we isolated two Daphnia subpopulations from different depths in a sediment core, which were exposed to parasites and a nutrient ratio gradient in a common garden experiment. Our results showed a genetic basis for sensitivity towards food deprivation. Both fecundity and host survival was differently affected when fed with low-quality food. This strongly impacted the way both subpopulations interacted with this parasite. A historical reconstruction of nutrient changes in a sediment core reflected an increase in organic material and phosphorus concentration (more eutrophic conditions) over time in the studied pond. These results enable us to relate patterns of genetic differentiation in sensitivity towards food deprivation to an increasing level of eutrophication of the subpopulations, which ultimately impacts parasite virulence effects. This finding was confirmed via a dynamic energy budgets (DEB), in which energy was partitioned for the host and the parasite. The model was tailored to our study by integrating (1) increased growth and a fecundity shift in the host upon parasitism and (2) differences of food assimilation in the subpopulations showing that a reduced nutrient assimilation resulted in increased parasite virulence. The combination of our experiment with the DEB model shows that it is important to consider genetic diversity when studying the impact of nutritional stress on species interactions, especially in the context of changing environments and emerging infectious diseases.

Advanced nutrient removal from surface water by a consortium of attached microalgae and bacteria: A review.

Innovative and cost-effective technologies for advanced nutrient removal from surface water are urgently needed for improving water quality. Conventional biotechnologies, such as ecological floating beds, or constructed wetlands, are not effective in removing nutrients present at low-concentration. However, microalgae-bacteria consortium is promising for advanced nutrient removal from wastewater. Suspended algal-bacterial systems can easily wash out unless the hydraulic retention time is long, attached microalgae-bacteria consortium is more realistic. This critical review summarizes the fundamentals and status of attached microalgae-bacteria consortium for advanced nutrient removal from surface water. Key advantages are the various nutrient removal pathways, reduction of nutrients to very low concentration, and diversified photobioreactor configurations. Challenges include poor identification of functional species, poor control of the community composition, and long start-up times. Future research should focus on the selection and engineering of robust microbial species, mathematical modelling of the composition and functionality of the consortium, and novel photobioreactor configurations.

Deodorization of Arthrospira platensis biomass for further scale-up food applications.

BACKGROUND: Given the importance of A. platensis as a potential food protein source, we describe an affordable deodorization process that does not significantly affect the nutritional value of algae biomass. RESULTS: Ethanol, acetone or hexane were used to deodorize algae biomass and then to identify the profile of volatile compounds associated with its distinctive odor. Sensorial characteristics were improved in the biomass cake after the proposed solvent extraction. Panelists identified the ethanolic extract with the most pronounced algae-related odor. Gas chromatography-mass spectrometry analysis showed that a mixture of 20 different compounds derived from fatty acids and amino acids contributed to the characteristic smell of A. platensis biomass. The results of the present study show that the ethanol solvent-free A. platensis biomass contained > 600 g kg-1 protein, < 10 g kg-1 crude fat and > 65% in vitro protein digestibility, similar to the original biomass. The Fourier transform infrared spectroscopy secondary protein structure was comparable among samples, indicating that the only change after ethanol extraction was a reduction of the algae smell. CONCLUSION: The various extraction procedures investigated in the present study were effective in deodorizing the algae biomass. The most effective protocol was the removal of odoriferous compounds with ethanol. This particular procedure yielded an algae biomass with an improved sensorial traits. The results of the present study should help with the identification of odoriferous compounds derived from fatty acids, pigments and proteins associated with A. platensis. © 2017 Society of Chemical Industry.

Impact of different nitrogen sources on the growth of Arthrospira sp. PCC 8005 under batch and continuous cultivation - A biochemical, transcriptomic and proteomic profile.

The aim of the present study was to evaluate the effects of varying concentrations of different nitrogen sources (individually or in combination) on the biochemical, transcriptomic and proteomic profiles of Arthrospira sp. PCC 8005 under batch and continuous modes. In batch mode, while ammonium showed a repressive effect on nitrate-assimilation pathway of the cyanobacteria; better growth and nutrient uptake rate were observed in presence of urea than nitrate. The inhibitory effect of ammonium was further confirmed by the continuous photobioreactor study wherein the nutrient feed was transiently replaced from nitrate to ammonium (28mM turbiostat regime). The changes in lipid, exopolysaccharide, transcriptomic and proteomic profiles of cyanobacteria on transition from nitrate to ammonium indicated at an onset of nutrient stress.

Two-stage cultivation of Nannochloropsis oculata for lipid production using reversible alkaline flocculation.

Two-stage cultivation for microalgae biomass is a promising strategy to boost lipid accumulation and productivity. Most of the currently described processes use energy-intensive centrifugation for cell separation after the first cultivation stage. This laboratory study evaluated alkaline flocculation as low-cost alternative separation method to harvest Nannochloropsis oculata prior to cultivation in the second nutrient-depleted cultivation stage. Biomass concentration over time and the maximum quantum yield of photosystem II expressed as Fv:Fm ratio showed identical patterns for both harvesting methods in both stages. The composition of total lipids, carbohydrates, and protein was similar for biomass harvested via alkaline flocculation or centrifugation. Likewise, both harvest methods yielded the same increase in total lipid content, to 40% within the first 2days of the nutrient-depleted stage, with an enrichment in C16 fatty acid methyl esters. Centrifugation can therefore be replaced with alkaline flocculation to harvest Nannochloropsis oculata after the first cultivation stage.

Flocculation properties of several microalgae and a cyanobacterium species during ferric chloride, chitosan and alkaline flocculation.

Flocculation holds great potential as a low-cost harvesting method for microalgae biomass production. Three flocculation methods (ferric chloride, chitosan, and alkaline flocculation) were compared in this study for the harvesting of 9 different freshwater and marine microalgae and one cyanobacterium species. Ferric chloride resulted in a separation efficiency greater than 90% with a concentration factor (CF) higher than 10 for all species. Chitosan flocculation worked generally very well for freshwater microalgae, but not for marine species. Alkaline flocculation was most efficient for harvesting of Nannochloropsis, Chlamydomonas and Chlorella sp. The concentration factor was highly variable between microalgae species. Generally, minimum flocculant dosages were highly variable across species, which shows that flocculation may be a good harvesting method for some species but not for others. This study shows that microalgae and cyanobacteria species should not be selected solely based on their productivity but also on their potential for low-cost separation.

How to use Nile Red, a selective fluorescent stain for microalgal neutral lipids.

The use of Nile Red for rapid monitoring of the neutral lipid content in microalgae has gained interest over the last decade, since neutral lipids are feedstock for renewable transportation fuel. In this review, we discuss the main considerations needed to make an NR protocol reliable for staining neutral lipids in microalgae. Cell wall permeability must be enhanced by using stain carriers: DMSO (5% v/v to 25% v/v), glycerol (0.1 to 0.125mg/mL), or EDTA (3.0 to 3.8mg/mL). Temperatures between 30 and 40°C facilitate the diffusion of NR through the cell wall without incurring excess quenching. Good NR-lipid interaction requires using a low NR/cell ratio; the NR concentration must be between 0.25µg/mL and 2.0µg/mL, and the cell concentration >5×10(4)cells/mL. In order to have the maximum and stable NR fluorescence, it is necessary to scan the excitation/emission wavelengths for up to a 40-min of incubation time. We outline a five-step method to customize the Nile Red protocol to a specific strain: 1) Evaluate the strain's suitability by checking for the presence of neutral lipid, 2) Select of the best excitation/emission wavelength, 3) Optimization of incubation time, stain carrier, dye concentration, and temperature, 4) Prepare single-strain algal cultures with different lipid contents to calibrate NR fluorescence with neutral-lipid content, and 5) Correlate NR fluorescence intensity to neutral lipid content for the same strain. Once the protocol is customized, the NR method allows for rapid and reliable monitoring of neutral lipid content of a microalgae strain.