Novel biorefinery supply chains for wastewater valorization and production of high market value bio products using microalgae

Microalgae downstream


After microalgae cultivation is complete, the produced biomass needs to be collected, and the desired compounds and compounds must be extracted and separated. Additionally, a pretreatment method is typically applied to disrupt the microalgae cells to enhance the recovery of the intracellular components. These steps of harvesting, pretreatment, and extraction collectively form what is known as downstream processing.

1. Harvesting

A typical downstream process begins by ‘harvesting’ the biomass. The microalgae are cultivated in very dilute conditions, usually at 0.5 g L-1 solids and the large water volumes make impractical further processing.  There are different techniques to remove the excess water although no one has emerged as clearly superior. Usually a combination of technologies is applied, first a ‘harvesting’ step for the formation of a slurry of 1-5 % wt followed by a ‘dewatering’ step to reach the final highly concentrated paste. These techniques can be mechanical, chemical, biological or electrical. Three main techniques with prospects for upscaling are centrifugation, membrane separation and flocculation.

Centrifugation is a very common technique already applied in other industrial applications. It offers noteworthy advantages such as high efficiencies, fast outcomes and no addition of chemicals. This comes at the cost of high initial capital investments as well as energy consumption. The latter is dependent on the technical characteristics of the centrifuge. The efficiency of the centrifugation is species-related since it is related to the cell size and their density difference to the culture medium.

In membrane separation, microalgae culture passes through filters operating under gravity, pressure or vacuum force to hold back the biomass in a thick paste form. The system can operate either in continuous or batch mode. This approach has the advantages of delivering undamaged biomass and lack of chemical addition. A number of materials (such as polyether sulfone, polyvinylidene fluoride, polytetrafluoroethylene and others) have been tested along with various conditions (microfiltration:0.1–10 μm, microfiltration:10 μm, ultrafiltration: 0.02–0.2 μm etc). The challenges usually associated with these methods include relatively low capacities and rapid fouling of microalgae secretions of organic matter.

During flocculation, the microalgae aggregate through surface charge neutralization, electrostatic patching or bridging after addition of flocculants. The formed agglomerates then separate through simple gravity-induced settling. It has been proposed as a potentially superior technique for harvesting microalgae as it can be used for large scale with a wide range of microalgae species.  Flocculation can be either chemical, auto-flocculation or bioflocculation, depending on the nature of the flocculant that is added. Drawbacks with this method is the relatively high energy cost, the potential flocculant toxicity, or non-feasibility of scaling up.

2. Pretreatment techniques

The characteristic resistance of microalgae cells to intracellular extraction is a significant challenge in the microalgae downstream processing. This is commonly attributed to the rigid cell walls that surround the cell which form a barrier preventing interaction between intracellular molecules and external solvents. To counter this, a pretreatment method is usually necessary in order to rupture the cells and enhance the accessibility to the targeted molecules. This treatment can be physical (mechanical, thermal, electrical, etc), chemical, biological or a combination of the above. An optimal cell disruption technique should operate on wet biomass, be energy efficient and suitable for large scale industrial applications. It is additionally crucial that the applied method will not contaminate or destroy any of the desired compounds.

Physical pretreatment methods include diverse techniques that focus either on the entire destruction of the cell such as bead mill or high-pressure homogenizer (HPH) or milder ones that leave the overall cell structure intact such as microwave treatment.

Bead milling is a technique used routinely for disruption of microalgae and other microorganisms. During milling, biomass is grinded against solid metallic surfaces (beads) under agitation at high frequencies, resulting in the breaking of the cell wall and the total destruction of the cell. It is highly effective for total disruption of cells although it can face scaling challenges in industrial applications and is considered very energy intensive process.

High pressure homogenization (HPH) is another technique aiming to the destruction of the entire cell structure. During operation, the cells are broken up due to high shear stress when forced to flow through a small orifice under high pressures. The end result is an emulsion of proteins, lipids and other cell fragments. The typical range of working pressures is 100-150 MPa with multiple passes required through the homogenizer although, extreme pressures (such as 300 MPa) might be applied to avoid this. A temperature increase of about 2 °C for every 10 MPa of applied pressure is reported which means that a cooling system should be implemented for heat sensitive products such as proteins or pigments. HPH is a well-established technology with a long history in the dairy industry. In order to counter its high energy demand, treatment should take place in high biomass concentrations and ideally with cells not equipped with strong cell walls. However, the resulting emulsion of the different microalgae cell components could pose a significant barrier for a cascade extraction in a biorefinery.

During ultrasound pre-treatment or ultrasonication, acoustic waves of 20 kHz and higher are applied to the microalgae suspension. Disruption is achieved through different pathways, one of which is through cavitation. The applied ultrasound leads to the formulation of microbubbles which expand and contract continuously until they implode disrupting thus the cells. Other direct effects occurring at low power and not involving cavitation, include radiation force and acoustic steaming. Challenges with the implementation of this technique include the temperature rise of the suspension during application and the production of free radicals and side chemical reactions with an uncertain effect on the final product. The exact disruption mechanism is also relatively unexplained with more research work required.

Microwave treatment has also been examined as a pretreatment method. Microwaves are defined as short waves of electromagnetic energy ranging from 300 MHz to 300 GHz and they function by supplying heat to the microalgae biomass which leads to cell rupture. Interestingly, microwave technology has been applied not only as a pre-treatment method but additionally during the actual lipid extraction in order to enhance it. In literature, such techniques are distinguished as ‘microwave assisted extraction’. The major challenges of upscaling microwave treatment would be the limited penetration depth of the microwaves into the absorbing medium  and some concerns regarding the energy requirements.

The main technique in the chemical pretreatment methods is acid/base hydrolysis of the cells. This methodology is particular effective for the production of bio-ethanol from microalgae since the complex cell wall carbohydrates are released and broken down to monosugars with the addition of simple acids such as hydrochloric or sulphuric acid. The disadvantage of these techniques is the volumes of chemicals required as well the fact that the involved extreme conditions, such as high temperatures, might damage other valuable compounds.

Biological pre-treatment involves the addition of a single or several enzymes, specially selected for each microalgae. These enzymes proceed then to digest the cell wall while leaving the intracellular components intact. Typical enzymes include cellulase, protease, lysozyme and others. It is an attractive methodology due to the mild operational conditions (temperature, pressure etc.) and low energy requirements. It is also commonly paired with other techniques such as microwaves in order to reduce costs. The main bottleneck however, is the prohibitive expense of these enzymes at industrial scale and from a more technical point of view, the determination of the cell wall composition of the desired microalgae in order to design the appropriate enzyme mix.

3. Extraction of intracellular components

A number of valuable compounds can be produced and be extracted from microalgae. The ones that are produced in largest quantities include proteins, lipids and carbohydrates. Due to limited industrial applications of microalgae yet, most of the developed extraction techniques are still limited to pilot level demonstrations however. The selection of the appropriate extraction method depends firstly on the targeted molecule (water cannot normally be used for hydrophobic lipids as a solvent for example) and by the final usage of the product.


Proteins are large biomolecules composed of amino acids that are crucial for the metabolism’s proper function. Amino acids are organic compounds with a structure of an amine (-NH2), carboxyl (COOH) and a side chain R. A short chain of 20-30 amino acids linked by peptide bonds is a peptide. A longer chain is called polypeptide or protein. Microalgae can exhibit high protein content and are capable of producing all essential amino acids and are under consideration for human consumption. Protein extraction is typically performed using aqueous, acidic or alkaline methods and the removed protein molecules are separated afterwards through centrifugation, ultrafiltration, precipitation or chromatography. Due to concerns for the bioavailability of the proteins, a common strategy to improve their digestibility is to hydrolyze them into amino acids. This hydrolysis can be either chemical or enzymatic in nature. Industrial scale protein extraction from microalgae is rarely studied. Common challenges with upscaling include the multiple differences between microalgal species, variation in the cell structure, variation in the intracellular protein content and release of protein degrading (protease) enzymes from the cells.


Carbohydrates also known as polysaccharides or sugars are composed mainly of carbon, hydrogen and oxygen. The smaller carbohydrate molecules, such as glucose, are called monosaccharides. Similar to the peptides and polypeptides, longer chains of monosaccharides linked together are known as polysaccharides, and they are mainly encountered as starch. Carbohydrates play an essential role in microalgae processing since they are one of the significant components of their formidable cell walls. Commercially extracted polysaccharides from algae include mainly alginates, agar and carrageenan. They are also studied for the production of biofuels, primarily bioethanol and biomethane (biogas) or increasingly in the bioplastics sector. For analytical purposes, the carbohydrates are typically undergoing acidic hydrolysis according to the National Renewable Energy Laboratory (NREL) procedure. Afterwards, they can be quantified using colorimetric methods like the phenol-sulfuric acid or anthrone.
For the production of bioethanol, the microalgae biomass is pretreated and subsequently hydrolyzed with the addition of enzymes or other microorganisms with the yeast Saccharomyce being a very common one. Hydrolysis can be either enzymatic or acidic at temperatures ranging between 120- 140 °C for 15-30 min with glucose being the monosaccharide most frequently produced. These simple sugars can then be fermented into bioethanol. The carbohydrates can also be used as feedstock for biogas. As the name implies, it is a gaseous biofuel composed mainly by methanol and is produced by anaerobic digestion. Microalgae derived biogas has the advantage of low sulfur content however, the C/N ratio of the feedstock, an important parameter for success of the anaerobic digestion, is relatively low.


Lipids are a large family of organic compounds encountered naturally in liquid and solid phases. Their main constituent is typically glycerides and are characterized by their very low solubility in water. Glycerides or acylglycerols are esters formed from glycerol and fatty acids. Fatty acids are large molecules with a hydrocarbon chain and a carboxylate (ROO-) at their head. Lipids produced by microalgae are neutral (free fatty acids and triglycerides) and polar, which in turn are subdivided into phospholipids (fatty acids with a phosphate group) and glycolipids (fatty acids with an oligosaccharide group). Microalgae lipids are an excellent feedstock for biodiesel products. Certain microalgae species can also produce significant polyunsaturated fatty acids (PUFAs) with a recognized nutritional value. Microalgae lipid extraction has been extensively studied due to its high potential as a renewable biodiesel source.
Industry often uses classical organic solvent extraction techniques like soaking, counter-current, or pressurized liquid extraction. Lipid extraction with organic solvents is the most conventional and examined method to date. For total lipid extraction from microalgae, a ‘co-solvent mixture’ of polar and neutral solvents is usually required. The co-solvent could form a single phase (monophasic extraction) or two phases (biphasic extraction).
Generally, an ‘ideal’ solvent has to fulfill multiple criteria. Apart from compatible polarity, a solvent has to be easy to recycle, nontoxic or harmful to the environment or the end product, the price of bulk solvent and its miscibility with water in case of wet extraction. A number of organic solvents have been examined. Chloroform:methanol is limited to bench-scale applications but is considered a gold standard for lipid estimation. Other common choices are hexane, heptane, ethyl acetate, acetone, ethanol and isopropanol. Hexane and ethanol or acetone are popular choices, due to the fact that their use is already accepted in food processing and hexane’s highly neutral nature and low boiling point. Hexane is in fact already applied industrially for extraction of vegetable oil from oil seed. Consideration should also be given on the emerging trend of Green Chemistry. These so called ‘bio-solvents’ are derived from sustainable sources such as agricultural wastes and not from the petrochemical industry like hexane. 2-Methyltetrahydrofuran (2-MeTHF), Cyclopentyl methyl ether (CPME) or various terpenes such as d-limonene have already been tested successfully and in theory could replace the more conventional solvents.
Other forms of environmentally lipid extraction is the utilization of supercritical carbon dioxide (sCO2). Carbon dioxide is selected due to its inert, nontoxic nature and its almost ambient critical temperature. At relatively modest conditions (7.4 MPa and 31.1 °C), carbon dioxide is above its critical point, with little distinction between physical phases, resulting in a fluid-like density and gas viscosity and diffusivity. At this state, mostly neutral lipids are highly soluble in scCO2 and to some extent polar as well. Once extraction is complete, then a release of pressure, returns CO2 to its gaseous state, leaving the extracted oil behind to be collected. This method is highly promising due to the high selectivity it presents for lipid extraction compared to other components and easy solvent separability. However, it faces high equipment costs and energy usage requirements. Moreover, the affinity to mainly neutral lipids, means that it is either inappropriate for polar lipid extraction or will need to be supplemented with an organic solvent such as ethanol.


Carotenoids are a diverse group of various natural pigments. They are responsible for the yellow, orange and other colors encountered in land plants or animals. They are greatly valued both as color additive to food as well as nutrition supplements due to their reported antioxidant function in the prevention of various diseases. Microalgae are noted for their high output of carotenoids, whose ‘natural’ productivity is considered much higher than synthetic ones. carotenoids are a class of lipophilic terpenoid pigments with a 40-carbon chain of alternating double and single bonds (polyene). Carotenoids are divided into two groups: the carotenes (β-carotene and lycopene) which are pure hydrocarbons and xanthophylls (lutein, astaxanthin etc) that contain oxygen in their structure. Being highly hydrophobic, carotenoids cannot be extracted with water. Their extraction methods therefore share a lot of similarities with the lipid extraction ones. They should be handled with special care and at inert conditions however, due to their highly oxidative nature to prevent their degradation.


In order for the microalgae to break away from limited niche markets and reach more widespread commercialization, the idea of a single product needs to be phased out and be replaced with the biorefinery scheme. This concept, much like the conventional crude oil refinery, aims towards the complete utilization of the biomass through selective and cascade extraction of different components. It is important to note that in this scenario, operating conditions should be mild and not harmful to any of the products. A potential route would be to separate the protein and the lipid fraction first by taking advantage of the natural extruding of the protein in water after a mild pretreatment method such as microwaves. Lipid and pigment extraction can take place then with the addition of solvents. The residual biomass can then be further treated either with anaerobic digestion for production of methane or with high thermal liquefaction (HTL) for biocrude oil.


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