RETRACTED ARTICLE: A Review on Microalgae Cultivation and Harvesting, and Their Biomass Extraction Processing Using Ionic Liquids

Open pond cultivation has been one of the oldest and simplest ways to cultivate microalgae in large scale. Open pond is widely used in the industry due to its relatively cheaper construction, maintenance and operation cost. Other advantages of using open pond system include simplistic operation and maintenance, low energy demand, and ease to scale up [7]. There are few types of open pond, which includes natural water such as lakes and ponds, and artificial water bodies such as circular and raceway ponds. At some cases, a container such as a tank can also be used to culture microalgae [8]. Open pond system has the advantage of being the most cost-efficient cultivation system. However, despite the large cultivation area, cultivation of microalgae from natural water has relatively lower cell concentration and thus a highly efficient harvesting method is required [9]. In addition, issues such as rainwater runoff which affects the growth condition of microalgae such as salinity and pH, erosion of banks that resulting in leakage and increased water turbidity might significantly affect the productivity of microalgae in open ponds [10]. Another issue that presents in open pond cultivation system is the possibility of contamination by protozoa and bacteria which causes the products to be toxic and unusable. The current available way to circumcise this problem is by cultivating microalgae which are capable of surviving at extreme alkaline or saline condition since only a few contaminants are able to thrive under this condition [11,12]. Besides, due to the open nature of the system, it is harder to control certain growth parameters such as temperature and light intensity which might affect the growth rate of microalgae [11].

Harvesting of microalgae from naturally occurring sources has been one of the oldest microalgae cultivation techniques known to man. The oldest record of microalgae harvesting was done by Aztec peoples where they harvest Spirulina from Lake Texcoco located at Mexico [13]. This cultivation method highly depends on naturally occurring water bodies to provide right condition and nutrients for the growth of microalgae. Currently, one of the largest commercial cultivation of microalgae in natural water is located at Hutt Lagoon, Australia which is capable of producing 6 tons of β-carotene every year form Dunaliella using its 700-hectare ponds [10].

Circular ponds are the first artificial pond to be used in large-scale cultivation of microalgae. This cultivation system got its name from its circular-shaped culture tank, and typically have the depth of 30–70 cm and width of 45 m along with a rotating agitator located at the center of the pond [14]. The rotating agitator is being used to ensure efficient mixing and prevent sedimentation of algae biomass [15]. However, the design of this cultivation system is limited by its size since bigger pond might introduce stronger water resistance, and therefore causes strain on the mechanical parts of agitator [16]. Moreover, this design has disadvantages for high energy usage in the agitation process and high construction cost [13]. Currently, this cultivation system has been used in Japan and Taiwan to culture Chlorella for consumption [14,17].

Raceway pond is one of the most frequently used open pond types for the cultivation of microalgae. It consists of a series of closed loop channel around 30-cm deep and paddlewheel which enable recirculation of microalgae biomass to ensure equal distribution of nutrients and prevent sedimentation of microalgae biomass. Raceway pond has been perceived as one of the best open pond cultivation design available due to its energy efficiency, as a single paddlewheel is sufficient enough to properly agitate a 5-hectare raceway pond [18]. One of the successful raceway pond cultivation is by Sapphire Energy’s Columbus Algal Biomass Farm located at Columbus, United States, which has successfully produced 520 metric tonnes of dried microalgae biomass during 2 years of its operation without any technical issue [19].

Photobioreactor is a bioreactor system used to culture phototrophs such as microalgae in an enclosed system which does not allow direct exchange of material between the culture and environment. Photobioreactor is able to overcome several constraints faced commonly by open pond culture design. First, the size of bioreactor is more compact compared to open pond, therefore providing more efficient land usage. Second, the system provides a closed and highly controlled growth condition for the culture, thus able to produce a contamination free, single strain microalgae culture [20]. In addition, the highly controlled culture condition can also translate into higher nutrient and metabolic efficiency which results in higher biomass production per unit of substrate. However, the bottleneck of practical usage of photobioreactor is its limited scalability due to various design flaws, rendering it uneconomical to be used in large-scale production [21]. Moreover, highly controlled growth condition of photobioreactor always comes with high capital and operating costs.

Tubular photobioreactor is comprised of transparent long tubes made out of glass or transparent plastics which are arranged in horizontal, vertical, helix, or slanted orientation to maximize capture of sunlight [13,22]. The microalgae culture is circulated within the loop by the means of mechanical pump or air lift system [23]. However, the biggest drawback of tubular photobioreactor design is in its poor mass transfer across the system since the long tube used in the bioreactor design might result in the differences in concentration of substrate and product along the tubes. Commercial usage of large-scale tubular photobioreactor is being used to produce Haematococcus and Chlorella at Germany and Israel, respectively [24].

Vertical column bioreactor is constructed by a transparent vertical cylindrical tubing and a sparger which pumps in air bubbles to enable homogenization of the culture and allow transfer of carbon dioxide and oxygen between air and microalgae culture [25]. This culture system offers the best gas-liquid mass transfer efficiency compared to other system owing to the capability of the sparger used in this system to generate smaller bubbles which provide larger total surface area for more efficient transfer of substance [26]. In addition, the simplicity of the design allows it to have lower energy demand and simpler operating procedure. However, the cylindrical-shaped container does not provide adequate amount of light required by the microalgae to perform photosynthesis efficiently. Moreover, high construction cost and difficulties in cleaning the reactor make commercial usage of vertical column photobioreactor difficult [27]. Commercial usage of this photobioreactor design has yet to be found, however, numerous large-scale experimental reactor has been made including a 40-liter vertical column outdoor photobioreactor for the cultivation of Chlorella zofingiensis at Guangdong, China [28].

Flat-plate photobioreactor on the other hand is characterized by its rectangular-shaped compartment made out of transparent material with the depth between 1 and 5 cm. The culture inside the reactor is mixed via the means of recirculating airlift system [29]. This design supports the largest total surface area for illumination and low oxygen build up, thus achieving the highest photosynthetic efficiency out of all photobioreactor design [30]. However, the aeration design of this photobioreactor causes stress damage to the microalgae cells [31]. Numerous innovative design has been implemented to further improve the efficiency of the flat-plate photobioreactor, such as twin layer flat-plate and plastic sheets photobioreactors [32]. Commercial usage of flat-plate photobioreactor has been used to produce astaxanthin by Algamo company located at Krkonoše, Czech Republic.

Filtration process utilizes a semipermeable membrane which can retain microalgae on the membrane while allowing the liquid media to pass through, leaving the algae biomass behind to be collected [50]. This method can harvest high concentration of cell from the medium, and the varying pore size of the filter membrane enables the system to suit the need of different microalgae and are able to handle the more delicate species which are prone to damage due to shearing. However, this method is very prone to fouling and clogging and therefore requires frequent change of fresh filter or membrane that might contribute significantly to its processing cost [51]. In view of this constraint, filter membrane using cheap and easily accessible material has been developed. Bejor and his colleagues [52] have successfully developed a filter membrane made out of stretch cotton which can achieve harvesting efficiency of 66–93%.

Centrifugation operation separates microalgae cells from the culture media based on each component’s density and particle size using centrifugal force [53]. This technique has high cell harvesting efficiency, but the process is time consuming and energy intensive [54,55]. Moreover, high gravitational force used in centrifugation might cause cellular damage making it unfavorable for certain applications since the sensitive nutrients might be lost [56,57]. Several types of centrifugal systems have been used in the industry; these include disk stack centrifuges, perforated basket centrifuges, imperforated basket centrifuges, decanters, and hydro-cyclones [35].

Flocculation is a process where free floating unicellular microalgae cells aggregate together to form a larger particle known as floc by the addition of flocculating agent to remove the surface charge of cells [58]. Flocculating agents can be grouped into two major types, namely chemical flocculants and bio-flocculants. Low cost and highly available chemical flocculants such as iron and aluminum salts have been used widely in industry [59]. The study carried out by Chatsungnoen and Chistt [60] has demonstrated that the metal salts such as aluminum sulfate and iron chloride are able to remove 95% of the microalgae biomass at a standard condition. However, the chemicals are not eco-friendly due to their high toxicity, and they must be removed by additional treatment processes which add to the production cost [61].

Bio-flocculants on the other hand are much safer and eco-friendly when compared to their chemical counter parts. They are also cheaper to be used, and typically there is no pretreatment required before further downstream processing of microalgae and recycling of culture media [62,63]. Most of the bio-flocculants used are biopolymers such as acrylic acid and chitosan which exist naturally or produce artificially [64]. It is reported that chitosan at a lower dosage is capable of reaching 90% cell recovery when compared to chemical flocculants such as aluminum sulfate which requires higher concentration to achieve same results [65].

Flotation utilizes small bubbles which attach on microalgae cells to promote the floating of cells on the surface of the culture media for easy harvesting [66]. The advantages associated with the flotation system include relatively high harvesting efficiency, easy operating procedure, and high processing throughput at low cost [67].

There are 3 main types of flotation systems which generate air bubbles required by using different mechanisms. Dissolved air flotation system generates air bubbles by the means of saturating the culture with compressed air and then discharging the culture at atmospheric condition. This method has been widely used in wastewater treatment but is hindered by high cost due to power consumption and usage of chemicals [68]. Dispersed air flotation on the other hand uses a sparger to generate air bubble which in turn have lower energy demand when compared to dissolved air flotation [69]. The third method is electro-flotation, which applies electrolysis operation to generate microbubbles from its electrode to trap free floating microalgae [70]. Apart from harvesting, this method also allows simultaneous cell disruption operation when alternating current is being used [71]. However, the respective system is extremely energy consuming and frequent replacement of electrode is required due to fouling which might then bring up the production cost [35].

ILs are described as molten organic salts which has melting point below 100℃ [76,77]. The properties of ILs vary greatly depending on their molecular size, functional groups attached, and also the type of cation and anion pairs. Nevertheless, several common characteristics have made IL as an ideal candidate for green applications. First, the nonvolatile nature makes ILs ideal when compared to other organic solvents such as ethanol and methanol which are extremely combustible. Second, some ILs have low viscosity which is a plus for any IL that is intended to be used for large-scale industrial application since low fluid viscosity facilitates pumping and agitation operations and reduce the power consumption [78]. Additionally, the low vapor pressure of ILs makes them relatively safe to use especially when they have low emission rate and consequently reduce exposure toward operators. The negligible vapor pressure of ILs also translate into less air pollution since there is lesser ILs will be liberated into the atmosphere. Furthermore, ILs can be specifically tailored-made to suit specific conditions and to solubilize specific compounds by altering their functional groups or selection of appropriate cation and anion combinations. Moreover, ILs have high thermal and chemical stability, high conductivity, and remarkable solubilizing capacity for various organic and inorganic compounds [79,80].

Amongst the commercially available ILs, ILs from imidazolium cation family are widely investigated for their potential to extract bioproducts from microalgae. Imidazolium cation has 5 membered, heterocyclic structure with a pair of nitrogen at the 1st and 3rd position in the ring [81]. Its peculiar structure allows it to be used for various applications. Apart from imidazolium-based ILs, cholinium-based ILs that are highly recognized for their marginally toxicity have been applied for the microalgae extraction studies [82]. However, there is still lack of study in this field using other ILs such as pyridinium- and phosphonium-based ILs.

Several extraction methods using ILs have been studied. Although direct dissolution of microalgae biomass to extract their intracellular compounds is within the realm of possibility, most of the time additional external force is used to aid the extraction process [83]. Microwave, ultrasound, electrical pulse field, and bead milling can be coupled with the use of ILs to generate additional force to intensify the extraction process. These methods are mainly developed to work around the tough cell wall of microalgae that is being a hindrance to the full utilization of microalgae-derived compounds.

Microalgae-derived bioproducts can be categorized into four major groups, namely lipids, carbohydrates, proteins, and bioactive compounds. Table 3 summarizes the recent studies utilizing ILs to extract these bioproducts from microalgae.

Despite the various studies that emphasize on the benefits of ILs for the extraction of microalgae derived bio-compounds, ILs are still to this date not being used commercially. This may be attributed to the toxicity and non-biodegradability of some traditional ILs such as imidazolium-based ILs, which may pose as an environmental risk when they are not properly treated prior to discharge to the environment. In fact, several toxicology studies have reported that conventional ILs are able to disrupt cell membrane and increase reactive oxygen species production in various organisms such as bacteria, fungus, plants, and animal cell lines in-vitro and ultimately lead to death. Moreover, due to the complexity of ILs’ molecular structure, they tend to be much harder to be synthesized, and therefore contribute to high production cost and are generally 5–20 times more expensive than conventional solvents. On-going efforts need to be continued to lower down the production cost of ILs, and also optimize their usage.