Ultrasonication to Improve Algae Cell Disruption and Extraction
Algae, macro- and microalgae, contain many valuable compounds, which are used as nutritional foods, food additives or as fuel or fuel feedstock. In order to release the target substances from the algae cell a potent and efficient cell disruption technique is required. Ultrasonic extractors are highly efficient and reliable when it comes to the extraction of bioactive compounds from botanicals, algae and fungi. Available at lab, bench-top and industrial scale, Hielscher ultrasonic extractors are established in the production of cell-derived extracts in food, pharma and bio-fuel production.
Algae as a Valuable Resource for Nutrition and Fuel
Algae cells are a versatile source of bioactive and energy-rich compounds, such as proteins, carbohydrates, lipids and other bio-active substances as well as alkanes. This makes algae a source of food and nutritional compounds as well as for fuels.
Microalgae are a valued source of lipids, which are used for nutrition and as feedstock fo biofuels (e.g., biodiesel). Strains of the marine phytoplankton Dicrateria, such as Dicrateria rotunda, are known as petrol-producing algae, that can synthesize a series of saturated hydrocarbons (n‐alkanes) from C10H22 to C38H78, which are categorized as petrol (C10–C15), diesel oils (C16–C20), and fuel oils (C21–C38).
Due their nutritional value, algae are used as “functional foods” or “nutraceuticals”. Important micro-nutrients extracted from algae include the carotenoids astaxanthin, fucoxanthin and zeaxantin, fucoidan, laminari and other glucans amongst numerous other bioactive substances are used as nutritional supplements and pharmceuticals. Carrageenan, alginate and other hydrocolloids are used as food additives. Algae lipids are used as vegan omega-3 source and also used as fuel or as feedstock for the production of biodiesel.
Algae Cell Disruption and Extraction by Power Ultrasound
Ultrasonic extractors or simply ultrasonicators are used to extract valuable compounds from small samples in the lab as well as for the production on large commercial scale.
Algae cell are protected by complex cell wall matrices, which are composed of lipids, cellulose, proteins, glycoproteins, and polysaccharides. The base of most algae cell walls is built of a microfibrillar network within a gel-like protein matrix; however, some microalgae are equipped with an inorganic rigid wall composed of opaline silica frustules or calcium carbonate. In order to obtain bioactive compounds from algal biomass, an efficient cell disruption technique is necessary. Besides the technological extraction factors (i.e., extraction method and equipment), the efficiency of algae cell disruption and extraction is also strongly influenced by various algae-dependent factors such as composition of cell wall, location of the desired biomolecule in microalgae cells, and growth stage of microalgae during harvesting.
How Does Ultrasonic Algae Cell Disruption and Extraction Work?
When high-intensity ultrasound waves are coupled via an ultrasonic probe (also known as ultrasonic horn or sonotrode) into a liquid or slurry, the sound waves travel through the liquid and create thereby alternating high-pressure / low-pressure cycles. During these high-pressure / low-pressure cycles, minute vacuum bubbles or cavities occur. Cavitation bubbles occur when the local pressure falls during the low pressure cycles far enough below the saturated vapour pressure, a value given by the tensile strength of the liquid at a certain temperature. The which grow over several cycles. When these vacuum bubbles reach a size, where they cannot absorb more energy, the bubble implodes violently during a high-pressure cycle. The implosion of cavitation bubbles is a violent, energy-dense process that generates intense shock waves, turbulences, and micro-jets in the fluid. Additionally, localized very high pressures and very high temperatures are created. These extreme conditions are easily capable to disrupt cell walls and membranes and to release intracellular compounds in an effective, efficacious and rapid manner. Intracellular compounds such as proteins, polysaccharides, lipids, vitamins, minerals, and antioxidants can thereby effectively extracted using power ultrasonics.
Ultrasonic Cavitation for Cell Disruption and Extraction
When exposed to intense ultrasonic energy, the wall or membrane of any kind of cell (including botanical, mammalian, algal, fungal, bacterial etc.) is disrupted and the cell is torn into smaller fragments by the mechanical forces of energy-dense ultrasonic cavitation. When the cell wall is broken, the cellular metabolites such as protein, lipid, nucleic acid and chlorophyll are released from the cell wall matrix as well as from the cell interior and get transferred into the surrounding culture medium or solvent.
The above described mechanism of ultrasonic / acoustic cavitation disrupts whole algal cells or gas and liquid vacuoles within cells severely. The ultrasonic cavitation, vibration, turbulences and micro-streaming promote the mass transfer between the cell interior and the surrounding solvent so that the biomolecules (i.e. metabolites) are efficient and rapidly released. Since sonication is a purely mechanical treatment that does not require harsh, toxic and/or expensive chemicals.
High-intensity, low-frequency ultrasound creates extreme energy-dense conditions, featuring high pressures, temperatures and high shear forces. These physical forces promote the disruption of cell structures in order to release intracellular compounds into the medium. Therefore, low-frequency ultrasound is largely used for the extraction of bioactive substances and fuels from algae. When compared to conventional extraction methods such as solvent extraction, bead-milling or high-pressure homogenization, ultrasonic extraction excels by releasing most of the bioactive compounds (such as lipids, proteins, polysaccharides and micro-nutrients) from the sonoporated and disrupted cell. Applying the right process conditions, ultrasonic extraction gives superior extraction yields within a very short process duration. For instance, high-performance ultrasonic extractors shows excellent extraction performance from algae, when used with a suitable solvent. In an acidic or alkaline medium, the algal cell wall get porous and wrinkled, leading to increased yields at low temperature (below 60°C) in a short sonication time (less than 3 hours). The short extraction duration at mild temperatures prevents fucoidan degradation, so that a highly bioactive polysaccharide is obtained.
Ultrasonication is also a method to transform high-molecular weight fucoidan into low-molecular weight fucoidan, which is significantly more bioactive due to its debranched structure. With its high bioactivity and bioaccessibility, low-molecular weight fucoidan is an interesting compound for pharmaceuticals and drug delivery systems.
Case Studies: Ultrasonic Extraction of Algae Compounds
Ultrasonic extraction efficiency and the optimization of ultrasonic extraction parameters have been widely studied. Below, you can find exemplary results for the extraction results via ultrasonication from various algae species.
Protein Extraction from Spirulina using Mano-Thermo-Sonication
The research group of Prof. Chemat (University of Avignon) investigated the effects of manothermosonication (MTS) on the extraction of proteins (such as phycocyanin) from dry Arthrospira platensis cyanobacteria (also known as spirulina). Mano-Thermo-Sonication (MTS) is the application of ultrasonics combined with elevated pressures and temperatures in order to intensify the ultrasonic extraction process.
“According to experimental results, MTS promoted mass transfer (high effective diffusivity, De) and enabled to get 229% more proteins (28.42 ± 1.15 g/100 g DW) than conventional process without ultrasound (8.63 ± 1.15 g/100 g DW). With 28.42 g of proteins per 100 g of dry spirulina biomass in the extract, a protein recovery rate of 50% was achieved in 6 effective minutes with a continuous MTS process. Microscopic observations showed that acoustic cavitation impacted spirulina filaments by different mechanisms such as fragmentation, sonoporation, detexturation. These various phenomena make extraction, release and solubilization of spirulina bioactive compounds easier.” [Vernès et al., 2019]
Ultrasonic Fucoidan and Glucan Extraction from Laminaria digitata
The TEAGASC research group of Dr. Tiwari investigated the extraction of polysaccharides, i.e. fucoidan, laminarin and total glucans, from the macroalgae Laminaria digitata using the ultrasonicator UIP500hdT. The ultrasonically-assisted extraction (UAE) parameters studied showed significant influence on the levels of fucose, FRAP and DPPH. Levels of 1060.75 mg/100 g ds, 968.57 mg/100 g ds, 8.70 μM trolox/mg fde and 11.02% were obtained for fucose, total glucans, FRAP and DPPH respectively at optimized conditions of temperature (76◦C), time (10 min) and ultrasonic amplitude (100%) using 0.1 M HCl as solvent. The UAE conditions described were then applied successfully to other economically relevant brown macroalgae (L. hyperborea and A. nodosum) to obtain polysaccharide rich extracts. This study demonstrates the applicability of UAE to enhance the extraction of bioactive polysaccharides from various macroalgal species.
Ultrasonic Phytochemical Extraction from F. vesiculosus and P. canaliculata
The research team of García-Vaquero compared various novel extraction techniques including high-performance ultrasonic extraction, ultrasound-microwave extraction, microwave extraction, hydrothermal-assisted extraction and high-pressure-assisted extraction in order to evaluate the extraction efficiency from the brown microalgae species Fucus vesiculosus and Pelvetia canaliculata. For ultrasonication, they used the Hielscher UIP500hdT ultrasonic extractor. The anylsis of the extraction yields revealed that ultrasonic extraction achieved the highest yields of most phytochemicals from both F. vesiculosus. This means, the highest yields of compounds extracted from F. vesiculosus using the ultrasonic extractor UIP500hdT were: total phenolic content (445.0 ± 4.6 mg gallic acid equivalents/g), total phlorotannin content (362.9 ± 3.7 mg phloroglucinol equivalents/g), total flavonoid content (286.3 ± 7.8 mg quercetin equivalents/g) and total tannin content (189.1 ± 4.4 mg catechin equivalents/g).
In their research study, the team concluded that the use of ultrasonically-assisted extraction “combined with 50% ethanolic solution as an extraction solvent could be a promising strategy targeting the extraction of TPC, TPhC, TFC and TTC, while reducing the co-extraction of undesirable carbohydrates from both F. vesiculosus and P. canaliculata, with promising applications when using these compounds as pharmaceuticals, nutraceuticals and cosmeceuticals.” [García-Vaquero et al., 2021]
- High extraction efficiency
- Superior extraction yields
- Rapid process
- Low temperatures
- Suitable to extract thermolabile compounds
- Compatible with any solvent
- Low-energy consumption
- Green extraction technique
- Easy and safe operation
- Low investment and operational costs
- 24/7 operation under heavy-duty
High-Performance Ultrasonic Extractors for Algae Disruption
Hielscher’s state-of-the-art ultrasonic equipment allows for full control over the process parameters such as amplitude, temperature, pressure and energy input.
For ultrasonic extraction, parameters such as raw material particle size, solvent type, solid-to-solvent ratio, and extraction time can be varied and optimized for best results.
Since ultrasonic extraction is a non-thermal extraction method, the thermal degradation of the bioactive ingredients present in the raw material such as algae is avoided.
Overall, advantages such as high yield, short extraction time, low extraction temperature, and the small amounts of solvent makes sonication the superior extraction method.
Ultrasonic Extraction: Established in Lab and Industry
Ultrasonic extraction is widely applied for the extraction of any kind of bioactive compound from botanicals, algae, bacteria and mammalian cells. Ultrasonic extraction has been established as a simple, cost-effective and highly efficient that excels other traditional extraction techniques by higher extraction yields and shorter processing duration.
With lab, bench-top and fully-industrial ultrasonic systems readily available, ultrasonic extraction is nowadays an well-established and trusted technology. Hielscher ultrasonic extractors are installed worldwide in industrial processing facilities that produce food- and pharma-grade bioactive compounds.
Process Standardization with Hielscher Ultrasonics
Algae-derived extracts, which are used in food, pharmaceuticals or cosmetics, must be produced in accordance to Good Manufacturing Practices (GMP) and under standardised processing specifications. Hielscher Ultrasonics’ digital extraction systems come with intelligent software, which makes it easy to set and control the sonication process precisely. Automatic data recording writes all ultrasonic process parameters such as ultrasound energy (total and net energy), amplitude, temperature, pressure (when temp and pressure sensors are mounted) with date and time stamp on the built-in SD-card. This allows you to revise each ultrasonically processed lot. At the same time, reproducibility and continuously high product quality are ensured.
The table below gives you an indication of the approximate processing capacity of our ultrasonicators:
|Batch Volume||Flow Rate||Recommended Devices|
|1 to 500mL||10 to 200mL/min||UP100H|
|10 to 2000mL||20 to 400mL/min||UP200Ht, UP400St|
|0.1 to 20L||0.2 to 4L/min||UIP2000hdT|
|10 to 100L||2 to 10L/min||UIP4000hdT|
|n.a.||10 to 100L/min||UIP16000|
|n.a.||larger||cluster of UIP16000|
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Literature / References
- García-Vaquero, Marco; Rajauria, Gaurav; Brijesh Kumar, Tiwari; Sweeney, Torres; O’Doherty, John (2018): Extraction and Yield Optimisation of Fucose, Glucans and Associated Antioxidant Activities from Laminaria digitata by Applying Response Surface Methodology to High Intensity Ultrasound-Assisted Extraction. Marine Drugs 16(8), 2018.
- Harada, N., Hirose, Y., Chihong, S. et al. (2021): A novel characteristic of a phytoplankton as a potential source of straight‐chain alkanes. Scientific Reports Vol. 11, 2021.
- Halim, Ronald; Hill, David; Hanssen, Eric; Webley, Paul; Blackburn, Susan; Grossman, Arthur; Posten, Clemens; Martin, Gregory (2019): Towards sustainable microalgal biomass processing: Anaerobic induction of autolytic cell-wall self-ingestion in lipid-rich Nannochloropsis slurries. Green Chemistry 21, 2019.
- Garcia-Vaquero, Marco; Rajeev Ravindran; Orla Walsh; John O’Doherty; Amit K. Jaiswal; Brijesh K. Tiwari; Gaurav Rajauria (2021): Evaluation of Ultrasound, Microwave, Ultrasound–Microwave, Hydrothermal and High Pressure Assisted Extraction Technologies for the Recovery of Phytochemicals and Antioxidants from Brown Macroalgae. Marine Drugs 19 (6), 2021.
- Vernès, Léa; Vian, Maryline; Maâtaoui, Mohamed; Tao, Yang; Bornard, Isabelle; Chemat, Farid (2019): Application of ultrasound for green extraction of proteins from spirulina. Mechanism, optimization, modeling, and industrial prospects. Ultrasonics Sonochemistry 54, 2019.
Facts Worth Knowing
Algae: Macroalgae, Microalgae, Phytoplankton, Cyanobacteria, Seaweed
The term algae is an informal one, used for a large and diverse group of photosynthetic eukaryotic organisms. Algae are mostly considered protists, but sometimes they are also classified as a type of plant (botanical) or choromists. Depending on their cell structure, they can be differentiated into macroalgae and microalgae, also known as phytoplankton. Macroalgae are multi-cell organisms, often known as seaweed. The class of macroalgae contains various species of macroscopic, multicellular, marine algae. The term phytoplankton is mainly used for microscopic marine single-celled algae (microalgae), but it can also include cyanobacteria. Phytoplankton is a wide class of various organisms including photosynthesising bacteria as well as microalgae and armour-plated coccolithophores.
As algae can be single-celled or multi-celled with filamentous (string-like) or plant-like structures, they are often difficult to classify.
The most cultivated macroalgae (seaweed) species are Eucheuma spp., Kappaphycus alvarezii, Gracilaria spp., Saccharina japonica, Undaria pinnatifida, Pyropia spp., and Sargassum fusiforme. Eucheuma and K. alvarezii are cultivated for carrageenan, a hydrocolloidal gelling agent; Gracilaria is farmed for agar production; while the other species are foraged for food and nutrition.
Another seaweed type is kelp. Kelps are large brown algae seaweeds that make up the order Laminariales. Kelp is rich in alginate, a carbohydrate, that is used to thicken products such as ice cream, jelly, salad dressing, and toothpaste, as well as an ingredient in some dog food and in manufactured goods. Alginate powder is also used frequently in general dentistry and orthodontics. Kelp polysaccharides such as fucoidan are used in skin care as gelling ingredients.
Fucoidan is a sulfated water-soluble heteropolysaccharides, present in multiple species of brown algae. Commercially produced fucoidan is mainly extracted from the seaweed species Fucus vesiculosus, Cladosiphon okamuranus, Laminaria japonica and Undaria pinnatifida.
Prominent Algae Genera and Species
- Chlorella is a genus of about thirteen species of single-celled green algae (microalga) belonging to the division Chlorophyta. Chlorella cells have a spherical shape, are about 2 to 10 μm in diameter, and have no flagella. Their chloroplasts contain the green photosynthetic pigments chlorophyll-a and -b. One of the most used Chlorella species is Chlorella vulgaris, which is popularly used as in dietary supplement or as protein-rich food additive.
- Spirulina (Arthrospira platensis cyanobacteria) is a filamentous and multicellular blue-green alga.
- Nannochloropsis oculata is a species of a genus Nannochloropsis. It is a unicellular small green algae, found in both marine and freshwater. Nannochloropsis algae is characterized by spherical or slightly ovoid cells with a diameter of 2–5 μm.
- Dicrateria is a genus of haptophytes, comprising the three species Dicrateria gilva, Dicrateria inornata, Dicrateria rotunda, and Dicrateria vlkianum. Dicrateria rotunda (D. rotunda) can synthesize hydrocarbons equivalent to petroleum (saturated hydrocarbons with a carbon number ranging from 10 to 38).