Interference of extracellular soluble algal organic matter on flocculation-sedimentation harvesting of Chlorella sp.

Extracellular soluble algal organic matter (AOM) significantly interferes with microalgae flocculation. This study investigated the effects of various AOM fractions on Chlorella sp. flocculation using ferric chloride (FeCl3), sodium hydroxide (NaOH), and chitosan. All flocculants achieved high separation efficiency (87-99 %), but higher dosages were required in the presence of AOM. High molecular weight (>50 kDa) AOM fraction was identified as the primary inhibitor of flocculation across different pH levels, whereas low/medium molecular weight (<3 and <50 kDa) AOM had minimal impact. Compositional analysis revealed that the inhibitory AOM fraction is a glycoprotein rich in carbohydrates, including neutral, amino, and acidic sugars. The significance of this study is in identifying carboxyl groups (-COOH) from acidic monomers in >50 kDa AOM that inhibit flocculation. Understanding AOM composition and the interaction dynamics between AOM, cells, and flocculants is crucial for enhancing the techno-economics and sustainability of flocculation-based microalgae harvesting.

The Biophysical Properties of Microalgal Cell Surfaces Govern Their Interactions with an Amphiphilic Chitosan Derivative Used for Flocculation and Flotation.

Microalgae show great promise for producing valuable molecules like biofuels, but their large-scale production faces challenges, with harvesting being particularly expensive due to their low concentration in water, necessitating extensive treatment. While methods such as centrifugation and filtration have been proposed, their efficiency and cost-effectiveness are limited. Flotation, involving air-bubbles lifting microalgae to the surface, offers a viable alternative, yet the repulsive interaction between bubbles and cells can hinder its effectiveness. Previous research from our group proposed using an amphiphilic chitosan derivative, polyoctyl chitosan (PO-chitosan), to functionalize bubbles used in dissolved air flotation (DAF). Molecular-scale studies performed using atomic force microscopy (AFM) revealed that PO-chitosan's efficiency correlates with cell surface properties, particularly hydrophobic ones, raising the question of whether this molecule can in fact be used more generally to harvest different microalgae. Evaluating this, we used a different strain of Chlorella vulgaris and first characterized its surface properties using AFM. Results showed that cells were hydrophilic but could still interact with PO-chitosan on bubble surfaces through a different mechanism based on specific interactions. Although force levels were low, flotation resulted in 84% separation, which could be explained by the presence of AOM (algal organic matter) that also interacts with functionalized bubbles, enhancing the overall separation. Finally, flocculation was also shown to be efficient and pH-independent, demonstrating the potential of PO-chitosan for harvesting microalgae with different cell surface properties and thus for further sustainable large-scale applications.

High-Temperature Hydrothermal Extraction of Phenolic Compounds from Brewer's Spent Grain and Malt Dust Biomass Using Natural Deep Eutectic Solvents.

Aligned with the EU Sustainable Development Goals 2030 (EU SDG2030), extensive research is dedicated to enhancing the sustainable use of biomass waste for the extraction of pharmaceutical and nutritional compounds, such as (poly-)phenolic compounds (PC). This study proposes an innovative one-step hydrothermal extraction (HTE) at a high temperature (120 °C), utilizing environmentally friendly acidic natural deep eutectic solvents (NADESs) to replace conventional harmful pre-treatment chemicals and organic solvents. Brewer's spent grain (BSG) and novel malt dust (MD) biomass sources, both obtained from beer production, were characterized and studied for their potential as PC sources. HTE, paired with mild acidic malic acid/choline chloride (MA) NADES, was compared against conventional (heated and stirred maceration) and modern (microwave-assisted extraction; MAE) state-of-the-art extraction methods. The quantification of key PC in BSG and MD using liquid chromatography (HPLC) indicated that the combination of elevated temperatures and acidic NADES could provide significant improvements in PC extraction yields ranging from 251% (MD-MAC-MA: 29.3 µg/g; MD-HTE-MA: 103 µg/g) to 381% (BSG-MAC-MA: 78 µg/g; BSG-HTE-MA: 375 µg/g). The superior extraction capacity of MA NADES over non-acidic NADES (glycerol/choline chloride) and a traditional organic solvent mixture (acetone/H2O) could be attributed to in situ acid-catalysed pre-treatment facilitating the release of bound PC from lignin-hemicellulose structures. Qualitative 13C-NMR and pyro-GC-MS analysis was used to verify lignin-hemicellulose breakdown during extraction and the impact of high-temperature MA NADES extraction on the lignin-hemicellulose structure. This in situ acid NADES-catalysed high-temperature pre-treatment during PC extraction offers a potential green pre-treatment for use in cascade valorisation strategies (e.g., lignin valorisation), enabling more intensive usage of available biomass waste stream resources.

Short-term effects of cadmium on leaf growth and nutrient transport in rice plants.

Consumption of rice grains contaminated with high concentrations of cadmium (Cd) can cause serious long-term health problems. Moreover, even low Cd concentrations present in the soil can result in the abatement of plant performance, leading to lower grain yield. Studies examining the molecular basis of plant defense against Cd-induced oxidative stress could pave the way in creating superior rice varieties that display an optimal antioxidative defense system to cope with Cd toxicity. In this study, we showed that after one day of Cd exposure, hydroponically grown rice plants exhibited adverse shoot biomass and leaf growth effects. Cadmium accumulates especially in the roots and the leaf meristematic region, leading to a disturbance of manganese homeostasis in both the roots and leaves. The leaf growth zone showed an increased amount of lipid peroxidation indicating that Cd exposure disturbed the oxidative balance. We propose that an increased expression of genes related to the glutathione metabolism such as glutathione synthetase 2, glutathione reductase and phytochelatin synthase 2, rather than genes encoding for antioxidant enzymes, is important in combating early Cd toxicity within the leaves of rice plants. Furthermore, the upregulation of two RESPIRATORY BURST OXIDASE HOMOLOG genes together with a Cd concentration-dependent increase of abscisic acid might cause stomatal closure or cell wall modification, potentially leading to the observed leaf growth reduction. Whereas abscisic acid was also elevated at long term exposure, a decrease of the growth hormone auxin might further contribute to growth inhibition and concomitantly, an increase in salicylic acid might stimulate the activity of antioxidative enzymes after a longer period of Cd exposure. In conclusion, a clear interplay between phytohormones and the oxidative challenge affect plant growth and acclimation during exposure to Cd stress.

Surface Chemistry of Oil-Filled Organic Nanoparticle Coated Papers Analyzed Using Micro-Raman Mapping.

Raman spectroscopy and micro-Raman mapping have been used to study the distribution of different chemical components at the surface of coated papers. The paper coatings contain organic nanoparticles with a structure of poly(styrene- co-maleimide) and encapsulated vegetable oils. Raman spectroscopy is able to differentiate between various types of oil, i.e., polyunsaturated, monounsaturated, or saturated, and indicates that the degree of imidization and reactivity of the oil (amount of free oil) complement each other. The surface mapping over large areas (5 × 5 mm2) illustrates good homogeneity of the coating layer and even surface coverage. The imide and oil are homogeneously distributed within the coating itself without a tendency for agglomeration. The covered areas of imide and oil mostly overlap for polyunsaturated oils, while larger amounts of oil occur outside the imide zones for monounsaturated and saturated oils. The latter indicates that the oil is partly "free" within the coating and acts as a continuous binder phase. The surface mapping over smaller areas (1 × 1 mm2) shows the coating and cellulose covered areas are complementary. The surface maps confirm that interaction between the coating and paper substrate happens through hydrogen bonding. Heterogeneities in the coating are due to the presence of remaining ammonolyzed maleic anhydride precursors forming amic acid moieties. The organic phase, oil phase, and cellulose substrate can also be differentiated by principal component analysis of the surface maps.

Selective separation of microalgae cells using inertial microfluidics.

Microalgae represent the most promising new source of biomass for the world's growing demands. However, the biomass productivity and quality is significantly decreased by the presence of bacteria or other invading microalgae species in the cultures. We therefore report a low-cost spiral-microchannel that can effectively separate and purify Tetraselmis suecica (lipid-rich microalgae) cultures from Phaeodactylum tricornutum (invasive diatom). Fluorescent polystyrene-microbeads of 6 µm and 10 µm diameters were first used as surrogate particles to optimize the microchannel design by mimicking the microalgae cell behaviour. Using the optimum flowrate, up to 95% of the P. tricornutum cells were separated from the culture without affecting the cell viability. This study shows, for the first time, the potential of inertial microfluidics to sort microalgae species with minimal size difference. Additionally, this approach can also be applied as a pre-sorting technique for water quality analysis.

A 3D-printed mini-hydrocyclone for high throughput particle separation: application to primary harvesting of microalgae.

The separation of micro-sized particles in a continuous flow is crucial part of many industrial processes, from biopharmaceutical manufacturing to water treatment. Conventional separation techniques such as centrifugation and membrane filtration are largely limited by factors such as clogging, processing time and operation efficiency. Microfluidic based techniques have been gaining great attention in recent years as efficient and powerful approaches for particle-liquid separation. Yet the production of such systems using standard micro-fabrication techniques is proven to be tedious, costly and have cumbersome user interfaces, which all render commercialization difficult. Here, we demonstrate the design, fabrication and evaluation based on CFD simulation as well as experimentation of 3D-printed miniaturized hydrocyclones with smaller cut-size for high-throughput particle/cell sorting. The characteristics of the mini-cyclones were numerically investigated using computational fluid dynamics (CFD) techniques previously revealing that reduction in the size of the cyclone results in smaller cut-size of the particles. To showcase its utility, high-throughput algae harvesting from the medium with low energy input is demonstrated for the marine microalgae Tetraselmis suecica. Final microalgal biomass concentration was increased by 7.13 times in 11 minutes of operation time using our designed hydrocyclone (HC-1). We expect that this elegant approach can surmount the shortcomings of other microfluidic technologies such as clogging, low-throughput, cost and difficulty in operation. By moving away from production of planar microfluidic systems using conventional microfabrication techniques and embracing 3D-printing technology for construction of discrete elements, we envision 3D-printed mini-cyclones can be part of a library of standardized active and passive microfluidic components, suitable for particle-liquid separation.

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.

Inhibition of alkaline flocculation by algal organic matter for Chlorella vulgaris.

Alkaline flocculation is a promising strategy for the concentration of microalgae for bulk biomass production. However, previous studies have shown that biological changes during the cultivation negatively affect flocculation efficiency. The influence of changes in cell properties and in the quality and composition of algal organic matter (AOM) were studied using Chlorella vulgaris as a model species. In batch cultivation, flocculation was increasingly inhibited over time and mainly influenced by changes in medium composition, rather than biological changes at the cell surface. Total carbohydrate content of the organic matter fraction sized bigger than 3 kDa increased over time and this fraction was shown to be mainly responsible for the inhibition of alkaline flocculation. The monosaccharide identification of this fraction mainly showed the presence of neutral and anionic monosaccharides. The addition of 30-50 mg L(-1) alginic acid, as a model for anionic carbohydrate polymers containing uronic acids, resulted in a complete inhibition of flocculation. These results suggest that inhibition of alkaline flocculation was caused by interaction of anionic polysaccharides leading to an increased flocculant demand over time.

CO2 controlled flocculation of microalgae using pH responsive cellulose nanocrystals.

Cellulose nanocrystals were grafted with imidazole functionalities up to DS 0.06 using a one-pot functionalization strategy. The resulting nanocrystals were shown to have a pH responsive surface charge which was found to be positive below pH 6 and negative above pH 7. These imidazolyl cellulose nanocrystals were tested for flocculation of Chlorella vulgaris using CO2 to induce flocculation. Up to 90% flocculation efficiency was achieved with 200 mg L(-1) dose. Furthermore, the modified cellulose nanocrystals showed good compatibility with the microalgae during cultivation, giving potential for the production of reversible flocculation systems.

Alkaline flocculation of Phaeodactylum tricornutum induced by brucite and calcite.

Alkaline flocculation holds great potential as a low-cost harvesting method for marine microalgae biomass production. Alkaline flocculation is induced by an increase in pH and is related to precipitation of calcium and magnesium salts. In this study, we used the diatom Phaeodactylum tricornutum as model organism to study alkaline flocculation of marine microalgae cultured in seawater medium. Flocculation started when pH was increased to 10 and flocculation efficiency reached 90% when pH was 10.5, which was consistent with precipitation modeling for brucite or Mg(OH)2. Compared to freshwater species, more magnesium is needed to achieve flocculation (>7.5mM). Zeta potential measurements suggest that brucite precipitation caused flocculation by charge neutralization. When calcium concentration was 12.5mM, flocculation was also observed at a pH of 10. Zeta potential remained negative up to pH 11.5, suggesting that precipitated calcite caused flocculation by a sweeping coagulation mechanism.

Highly charged cellulose-based nanocrystals as flocculants for harvesting Chlorella vulgaris.

This study presents a novel flocculant for harvesting Chlorella vulgaris as model species for freshwater microalgae based on cellulose nanocrystals (CNCs), thus synthesized from a renewable and biodegradable resource. Cationic pyridinium groups were grafted onto CNCs by two separate one-pot simultaneous esterification and nucleophilic substitution reactions. Both types of modified CNCs were positively charged in the pH range 4-11. Both reactions yielded CNCs with a high degree of substitution (up to 0.38). A maximum flocculation efficiency of 100% was achieved at a dosage of 0.1 g g(-1) biomass. In contrast to conventional polymer flocculants, cationic CNCs were relatively insensitive to inhibition of flocculation by algal organic matter. The present results highlight the potential of these new type of nanocellulose-based flocculants for microalgae harvesting.

Cultivation of Chlorella vulgaris and Arthrospira platensis with recovered phosphorus from wastewater by means of zeolite sorption.

In this study, zeolite was employed for the separation and recovery of P from synthetic wastewater and its use as phosphorus (P) source for the cultivation of the green microalga Chlorella vulgaris and the cyanobacterium Arthrospira (Spirulina) platensis. At P-loaded zeolite concentration of 0.15-1 g/L, in which P was limited, the two species displayed quite different behavior regarding their growth and biomass composition. C. vulgaris preferred to increase the intracellular P and did not synthesize biomass, while A. platensis synthesized biomass keeping the intracellular P as low as possible. In addition under P limitation, C. vulgaris did display some little alteration of the biomass composition, while A. platensis did it significantly, accumulating carbohydrates around 70% from about 15%-20% (control). Both species could desorb P from zeolite biologically. A. platensis could recover over 65% and C. vulgaris 25% of the P bounded onto zeolite. When P-loaded zeolite concentration increased to 5 g/L, P was adequate to support growth for both species. Especially in the case of C. vulgaris, growth was stimulated from the presence of P-loaded zeolite and produced more biomass compared to the control.

Microalgal and cyanobacterial cultivation: the supply of nutrients.

Microalgae and cyanobacteria are a promising new source of biomass that may complement agricultural crops to meet the increasing global demand for food, feed, biofuels and chemical production. Microalgae and cyanobacteria cultivation does not interfere directly with food production, but care should be taken to avoid indirect competition for nutrient (fertilizer) supply. Microalgae and cyanobacteria production requires high concentrations of essential nutrients (C,N,P,S,K,Fe, etc.). In the present paper the application of nutrients and their uptake by microalgae and cyanobacteria is reviewed. The main focus is on the three most significant nutrients, i.e. carbon, nitrogen and phosphorus; however other nutrients are also reviewed. Nutrients are generally taken up in the inorganic form, but several organic forms of them are also assimilable. Some nutrients do not display any inhibition effect on microalgal or cyanobacterial growth, while others, such as NO2 or NH3 have detrimental effects when present in high concentrations. Nutrients in the gaseous form, such as CO2 and NO face a major limitation which is related mainly to their mass transfer from the gaseous to the liquid state. Since the cultivation of microalgae and cyanobacteria consumes considerable quantities of nutrients, strategies to improve the nutrient application efficiency are needed. Additionally, a promising strategy to improve microalgal and cyanobacterial production sustainability is the utilization of waste streams by recycling of waste nutrients. However, major constraints of using waste streams are the reduction of the range of the biomass applications due to production of contaminated biomass and the possible low bio-availability of some nutrients.

Ammonia inhibition on Arthrospira platensis in relation to the initial biomass density and pH.

In this study the combined effect of total ammoniacal nitrogen (TAN) concentration, initial biomass density and initial pH of the cultivation medium on growth of Arthrospira platensis was studied. The results indicate that TAN inhibition in relation to the initial biomass in unregulated pH cultures is neither a clearly biomass-independent nor biomass-dependent phenomenon. However, low biomass densities are more susceptible to ammonia inhibition than higher biomass densities. Higher biomass densities seems to mitigate ammonia inhibition through rapider assimilation of TAN. In all cases studied the growth rates were lower compared to the cultures with nitrate as nitrogen source. It was observed that at low TAN concentration, although no ammonia inhibition occured the growth rates were decreased due to nitrogen limitation. Low TAN concentration triggered the accumulation of carbohydrates affecting thus significantly the biomass composition. Ammonia losses from the cultivation system were also determined. Ammonia losses ranged between 17% and 80%.

Using natural zeolite for ammonia sorption from wastewater and as nitrogen releaser for the cultivation of Arthrospira platensis.

Herein a new approach for the application of wastewater nutrients for the cultivation of cyanobacteria or microalgae is described. Natural zeolite was used as medium for the sorption of ammonia from wastewater and subsequently as nitrogen releaser in cultures of Arthrospira platensis. The main scope of the present approach was to isolate ammonia from the wastewater and to transfer it into the culture medium excluding thus the suspended solids, the dissolved colored compounds or any other possible contaminant of the wastewater. The results demonstrate that the indirect use of ammonia derived from wastewater using zeolite as sorption and releasing medium for the cultivation of A. platensis is promising. This is the first time that a medium was used for indirect application of wastewater nutrient for the production of cyanobacterial or microalgal biomass.

Flocculation as a low-cost method for harvesting microalgae for bulk biomass production.

The global demand for biomass for food, feed, biofuels, and chemical production is expected to increase in the coming decades. Microalgae are a promising new source of biomass that may complement agricultural crops. Production of microalgae has so far been limited to high-value applications. In order to realize large-scale production of microalgae biomass for low-value applications, new low-cost technologies are needed to produce and process microalgae. A major challenge lies in the harvesting of the microalgae, which requires the separation of a low amount of biomass consisting of small individual cells from a large volume of culture medium. Flocculation is seen as a promising low-cost harvesting method. Here, we overview the challenges and possible solutions for flocculating microalgae.

Influence of organic matter generated by Chlorella vulgaris on five different modes of flocculation.

Microalgae excrete relatively large amounts of algal organic matter (AOM) that may interfere with flocculation. The influence of AOM on flocculation of Chlorella vulgaris was studied using five different flocculation methods: aluminum sulfate, chitosan, cationic starch, pH-induced flocculation and electro-coagulation-flocculation (ECF). The presence of AOM was found to inhibit flocculation for all flocculation methods resulting in an increase of dosage demand. For pH-induced flocculation, the dosage required to achieve 85% flocculation increased only 2-fold when AOM was present, while for chitosan, this dosage increased 9-fold. For alum, ECF and cationic starch flocculation, the dosage increased 5-6-fold. Interference by AOM is an important parameter to consider in the assessment of flocculation-based harvesting of microalgae.

Flocculation of Chlorella vulgaris induced by high pH: role of magnesium and calcium and practical implications.

Microalgae hold great potential as a feedstock for biofuels or bulk protein or treatment of wastewater or flue gas. Realising these applications will require the development of a cost-efficient harvesting technology. Here, we explore the potential of flocculation induced by high pH for harvesting Chlorella vulgaris. Our results demonstrate that flocculation can be induced by increasing medium pH to 11. Although both calcium and magnesium precipitated when pH was increased, only magnesium (≥0.15 mM) proved to be essential to induce flocculation. The costs of four different bases (sodium hydroxide, potassium hydroxide, calcium hydroxide, magnesium hydroxide and sodium carbonate) were calculated and evaluated and the use of lime appeared to be the most cost-efficient. Flocculation induced by high pH is therefore a potentially useful method to preconcentrate freshwater microalgal biomass during harvesting.