Production and harvesting of microalgae biomass

Environmental Technology Reviews

ISSN: 2162-2515 (Print) 2162-2523 (Online) Journal homepage: http://www.tandfonline.com/loi/tetr20

Production and harvesting of microalgae biomass from wastewater: a critical review Nor Fadzilah Pahazri, RMSR Mohamed, AA Al-Gheethi & Amir Hashim Mohd Kassim To cite this article: Nor Fadzilah Pahazri, RMSR Mohamed, AA Al-Gheethi & Amir Hashim Mohd Kassim (2016) Production and harvesting of microalgae biomass from wastewater: a critical review, Environmental Technology Reviews, 5:1, 39-56, DOI: 10.1080/21622515.2016.1207713 To link to this article: http://dx.doi.org/10.1080/21622515.2016.1207713

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Date: 23 December 2016, At: 06:20

Environmental Technology Reviews, 2016 Vol. 5, No. 1, 39–56, http://dx.doi.org/10.1080/21622515.2016.1207713

Production and harvesting of microalgae biomass from wastewater: a critical review Nor Fadzilah Pahazri, RMSR Mohamed ∗ , AA Al-Gheethi∗ and Amir Hashim Mohd Kassim Department of Water and Environmental Engineering, Faculty of Civil & Environmental Engineering, Universiti Tun Hussein Onn Malaysia, Batu Pahat, Johor, Malaysia (Received 11 January 2016; accepted 26 June 2016 ) The wide range of microalgae applications has increased in the last decade, due to their importance as the source of biofuel and biomass. The potential of wastewater as a culture media lies in the presence of high contents of nutrients and elements required to improve the growth of microalgae and, thus, the high quantity of biomass. However, these properties might be the limitations in the harvesting of microalgae biomass from wastewater. This review discussed the potential of wastewater as the production media for biomass and focused on the harvesting methods, because it represented a major challenge in the quality and quantity of microalgal cells. It can be concluded that among several technologies used for harvesting microalgae biomass from wastewater, the natural flocculant method was the most efficient due to the absence of toxic by-products and secondary effects on the quality of biomass yield, as well as the high biomass quantity. Keywords: wastewater; flocculation; harvesting methods; quantification; microalgae

1. Introduction Microalgae are one of the microorganisms used for the biotreatment of wastewater, since it can contribute to the reduction of nutrients such as phosphorus and nitrogen in the wastewater and produce a detectable biomass simultaneously during the phycoremediation process, which is called the green technology.[1–3] The production of biomass from phycoremediation of wastewater is acceptable with the term of zero discharge in which wastewater is reused as the production medium for microalgae biomass production. Zero discharge is a term raised in the 1980 for industrial wastewater, which aims to recycle or reuse wastes.[4] The production and application of algae biomass have been investigated since the 1970s. However, these applications have increased significantly since 2008. Figure 1 shows the numbers of publications related to the production of microalgae biomass. In the period between 2008 and 2012, most researchers focused on the potential of biofuel production from microalgae biomass. This might be due to the increase in oil price during that time. However, many publications in 2013 focused on the harvesting methods of microalgae biomass from the culture media. In 2014, the publication focused on the application of advanced methods to extract lipids and protein from microalgae biomass. Furthermore, the biomass produced during the phycoremediation process has several applications such as the source of valuable chemicals, pharmaceuticals, and food additives. However, the quality

and quantity of biomass yield rely on the characteristics of wastewater, microalgae species, and the harvesting method.[5] The overall process of microalgae production, harvesting, quantification, and final utilization is presented in Figure 2. In this paper, the potential of wastewater as a production media for microalgae biomass has been reviewed. In the literature, there are no adequate review studies on the microbiological aspects of the harvesting process of microalgae biomass as well as the final utilization and quality of microalgae biomass. Therefore, this paper is important to contribute more knowledge on the microbial contamination and the competition between microalgae and indigenous microorganisms in wastewater. The efficiency of wastewater as a production media and the efficiency of different harvesting methods for microalgae biomass were also reviewed.

2.

Characteristics of wastewater as production media for microalgae biomass Wastewater is a general term used to represent many types of liquid wastes generated from activities associated with the utilization of a huge amount of water. The present review focused on meat processing wastewater and the potential of these wastes to be used as a production media for microalgae biomass, based on their characteristics in comparison with other wastewaters. Among several types of industrial activities in food production,

*Corresponding authors. Emails: [email protected]; [email protected]

© 2016 Informa UK Limited, trading as Taylor & Francis Group

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Figure 1. to 2014.

N.F. Pahazri et al.

Distribution of 314 publications on the production and harvesting of microalgae biomass during the period from 2000

Figure 2. Microalgae productions, harvesting and quantification processes, and final utilization.

meat manufacturing is the most difficult in the production and preservation processes. It is the nature of these foods which contain high contents of proteins that makes it more susceptible for microbial contamination. Therefore, the production process of meat is associated with the usage of high quantity of water to remove suspended nutrients, and this process leads to the increase in the quantity of discharged wastewater.[6] Meat processing wastewater has a high level of nitrogen (protein) and fats, as well as large amounts of separable materials. Over 90% of the meat processing wastewater is characterized by high biochemical oxygen demand (BOD5 ) and chemical oxygen demand (COD), as well as high concentrations of suspension, biogenic, and dissolved

substances. The main characteristics of meat processing wastewater compared to that of other wastewaters are illustrated in Table 1. It can be noted that the characteristics of wastewater vary from one type to another. Meat processing wastewater has a high content of COD; in contrast, the dairy industry wastewater is heavily contaminated with total suspended solids (TSS). Meat processing wastewater contains high turbidity than dairy industry wastewater. Most of the studies on meat processing wastewater have been focusing on total nitrogen (TN) and total phosphorus (TP), because both significantly contribute to the increase in microbial contaminations due to their role in the enhancement of pathogenicity and enzyme activity among pathogenic

Environmental Technology Reviews Table 1.

Characteristics of meat processing wastewater in comparison with different types of wastewaters. Meat processing wastewater

Parameters pH Turbidity (NTU) TSS Oil and grease BOD5 COD Fats TN TP

41

Caixeta et al. [7]

Bohdziewicz Sena et al. et al.[8] [6]

6.3–6.6 NS

6.0–7.2 NS

850–6300 NS

112–1743 NS

1300–2300 2000–6000 40–600 NS NS

1200–3000 2780–6720 NS 49–287 15–70

6.5–6.7 1000–12,000

Dairy industry wastewater

Textile industries wastewater

Qazi et al. Tikahara and Patil et al. Hussain Savin and Imtiazuddin [9] Sahu [10] [11] et al. [12] Butnaru [13] et al. [14] 7.2–7.5 NS

6.1–7.7 35.9–97.1

6.5 NS

7.0–9.0 NS

11 NS

7.5–11.5 NS

2300–7000 7200–8000 180.2–445.4 820–1050 NS NS

729 NS

830–1580 NS

288 NS

934–1875 NS

1200–1760 1300–1600 2800–3230 2500–3000 NS NS NS NS NS NS

530 790 NS NS NS

500–1010 1600–3200 NS NS NS

184 2688.5 NS NS NS

125–653 115–705 NS NS NS

741–9033 613–4958 0.01–0.06 NS NS

Note: TSS, total suspended solid; BOD5 , biochemical oxygen demand; COD, chemical oxygen demand; TN, total nitrogen; TP, total phosphorus; NS, not stated. All values, except pH and turbidity, are expressed in mg L−1 .

microorganisms.[15] Therefore, the discharge of these wastes into the environment flags the quality of the surface water.[8] The recycle of wastewater as a production media has been reported in the literature.[16,17] In terms of the potential of microalgae to grow in wastewater, it has been reported that microalgae have a high potential to adapt and utilize nitrogen, phosphate, and other nutrients available in wastewater.[18] The utilization of wastewater as the nutrient resource for microalgae biomass has been reported in the literature.[19] However, the nutrients available in the wastewater are not the sole requirement for microalgal growth, since many factors including light, temperature, aeration and mixing, as well as pH might contribute to the quality and quantity of the growth. Moreover, these factors might be considered as secondary factors, which might have no significant effect in the absence of nutrients where microalgal growth will be very weak, even at the optimal conditions of these factors. Some factors including light, temperature, aeration and mixing, and salinity can be adjusted to be within the optimum range of microalgae growth. In contrast, other factors such as pH have to be adjusted using chemical additives. Nevertheless, any chemical additions into wastewater might negatively affect the effectiveness of wastewater as a culture media for microalgae. In general, the pH range for most algal species is between 7 and 9, and the optimum range is between 8.2 and 8.7. Based on the pH of the wastewater, it appears that the textile industries wastewater with a pH range between 7.5 and 11.5 is the most favourable for microalgal growth, more than the dairy industry wastewater (pH 6.5–7.7) and meat processing wastewater (pH 6.7–7.2) (Table 1). Furthermore, it should be noted that the pH of the production media depends on the aeration, microalgae initial inoculum, as well as the nitrogen compounds. The availability of CO2 , H2 CO3 , and HCO− 3 which occur rapidly during photosynthesis process

of algae plays an important role to maintain and control the pH to be within the optimal range for algal cultivation.[20] Nitrogen is an essential element to produce high quantity of biomass yield, and it is usually presented in wastew+ ater as NO− 3 or NH4 , and both compounds contribute significantly to the changes of pH value. The utilization of ammonia by microalgae as a nitrogen source is associated with the decline in pH values due to the release of H+ ions. Conversely, degradation of NO− 3 contributes to the increase in pH of wastewater during the microalgal growth. Moreover, nitrate is the preferred form of nitrogen source for Scenedesmus bijugatus, which does mean that in order to achieve the high maximum production of microalgae biomass, the chemical characteristics of wastewater should be known first and then the microalgae used is selected based on that composition.[21] Phosphorus is an essential nutrient for algal growth due to its role in the metabolic and anabolic pathways. Therefore, the presence or absence of phosphorus in wastewater should be considered when these wastes are used as production media. Moreover, the form of phosphorus in wastewater might limit microalgae growth. The preferred form of phosphorus for most algae is orthophosphate (PO3− 4 ). One of the advantages of phosphorus in wastewater is it easily binds to iron. Therefore, the wastewater with a high concentration of phosphorus and low amount of Fe can be a good medium for microalgae growth.[22] By referring to the characteristics of different wastewaters (Table 1), it can be noted that both TN and TP in the meat processing wastewater are usually determined by the researchers, since they reflect the quantity of nutrients. The concentrations of TN and TP in these wastes range from 49 to 287 mg TN L−1 and 15–70 mg TP L−1 , respectively. As aforementioned, it can be concluded that pH is not critical or it is an independent factor that might lead to significant limitations for microalgae growth in wastewater, as long as the pH value is a secondary factor that varies

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depending on the type of nutrients. Conversely, TN and TP are the critical requirements to use wastewater as a culture medium for microalgae. The high content of TN and TP in meat processing wastewater makes it more preferred to be used as the production media for microalgae biomass. Macronutrients such as calcium, potassium, and magnesium as well as the trace elements including nickel, manganese, and copper are important for microalgal growth in the culture media.[23] Microalgae has the potential to grow and multiply in different wastewaters with heavy metal contents. In industrial and agricultural wastewaters, the concentration of heavy metals might be high, depending on the source of these wastes. Heavy metals are a group of transition elements and contribute in an important role as trace elements in a sophisticated biochemical reaction. However, some heavy metal ions such as Hg, Cd, and Ag have toxic effects on the metabolic pathways of several organism cells where the presence these metals leads to the formation of strong toxic complexes and make them too dangerous for any physiological function. Microalgae show the potential to resist heavy metals; therefore, they are used to remove heavy metals from different wastewaters.[24] Hence, the presence of heavy metals in wastewater might not limit the wastewater usage as the production medium for microalgae biomass. Temperature and light play a major role in algal growth. Temperature affects the biochemical composition and reactions of microalgae biomass. Microalgae growth has an optimum temperature which differs from species to species.[20] A high temperature might lead to the inactivation and denaturation of metabolic enzymes of the organism cells.[25] Light is an essential factor for microalgae growth, because macroalgae cells convert light into energy via the photosynthesis process. The effect of light on the utilization of wastewater as the production media for microalgae biomass lies in the presence of particulate matter, which may prevent microalgae that are not floating from getting the optimum light for energy buildup.[22] Moreover, both temperature and light factors might be overcome by using indigenous microalgae strains that have been acclimated to local environments. 3. Phycoremediation technique Phycoremediation is a technique that depends on the activity of algae in removing pollutants from wastewater. This technique is eco-friendly compared to other chemical and physical treatment processes in terms of toxic by-products and secondary pollution. The conventional technologies are associated with health hazards and environmental pollution, rather than reducing infectious pollutants in the wastewater.[4] Therefore, the investigators received a great opportunity to investigate the alternative technology with a high-efficiency and eco-friendly technique. Phycoremediation of wastewater takes advantage of the algae’s properties

in terms of their ability to survive and degrade organic matter and nutrients from wastewater by using it as the source for their growth.[26] The application of phycoremediation technology includes the removal of contaminants which include phosphorus and nitrogen from wastewater, biosorption and bioaccumulation of heavy metals from industrial wastewater, biodegradation and biotransformation of biodegradable compounds, and use of biosensors for toxic compounds.[3] In this section, the application of phycoremediation technology for treating wastewater is discussed based on the efficiency of this technology in comparison to other traditional technologies and the role of microalgae species in this process. 3.1.

Microalgae used in phycoremediation technology

Phycoremediation depends on the facts that microalgae are photosynthetic organisms and have the ability to utilize carbon and energy from sunlight. The basic assumption for phycoremediation as a safe biotechnology is that microalgae will turn a few ingredients into harmless substances that allow wastewater to be treated and then reused or safely disposed.[27] Algae are a diverse group of species, and the selection of the most potent strains based on natural adaption or by recent advances in genetic engineering and material science might provide efficient strategies for the development of wastewater treatment.[3] However, not all microalgae strains are suitable to be used in phycoremediation technology, since many microalgae are harmful algae such as cyanobacteria, diatoms, and prymnesiophytes, which are also known as harmful algal blooms and have multiple problems in the aquaculture environment. Therefore, the microalgae strains used in the phycoremediation process should be non-pathogenic. Other aspects need to be considered for utilizing microorganisms, including microalgae, in the wastewater treatment which comprise the composition of wastewater, insufficiency of nutrients, and the competition of introducing organisms with indigenous ones.[28] Hence, in a biotechnology application, the selection of organisms is a critical step in order to get a high effective technique due to the differences in the ability of organism strains to grow and survive under extreme conditions and their affinity.[16,29] The selected microorganism should have several properties. Among them are the potential to survive under extreme conditions (e.g. nutrient, redox, pH, and osmotic factors) and the potential to compete with indigenous and predatory organisms in the wastewater.[30,31] Microalgae have the potential to adapt and grow in different environmental conditions as well as survive for a long time. This is due to their ability to form resting cysts, which remain in a dormant state in unfavourable conditions.[32] For their growth, trace elements and nitrogen source should be available. Wastewaters are rich in organic and inorganic pollutants; thus, the application of

Environmental Technology Reviews phycoremediation for wastewater treatment is associated with the reduction of these contaminants and the biomass produced. Besides, the release of free oxygen during the metabolism process of algae is a major significance of promoting which induces the bioremediation of biodegradable compounds by indigenous microorganisms, including bacteria and fungi. Furthermore, algal species are relatively easy to grow, adapt, and manipulate within a laboratory setting and appear to be the ideal organisms for remediation studies.[26] The most common microalgae used in the phycoremediation process are illustrated in Table 2. The table shows that Chlorella vulgaris is the most common and efficient microalgae in the phycoremediation of wastewaters. Recently, the use of Chlorella spp. in bioremediation and as a pollution control agent has increased due to its fast growth rate and high nutrient and inorganic elements removal capabilities.[26,38]

3.2. Advantages of phycoremediation The main advantage of using microalgae-based technology is the ability of microalgae to reproduce and multiply in the wastewater, which improves the treatment process compared to other physical and chemical treatment methods. Table 2.

43

The phycoremediation process might start with an initial inoculum of microalgae, which increases in wastewater due to the presence of nutrients required for their growth. In contrast, chemical treatment should start with high concentrations of chemical substances, because they will react with the soluble substances in the wastewater. Bioremediation process which takes place during the phycoremediation and depends on the metabolic potential of microorganisms to detoxify or transform pollutants is one of the advantages of phycoremediation technology.[38] Theoretically, a perfect treatment process for wastewater comprises a natural treatment without toxic by-products and chemical additives, as well as having the ability to remove a wide range of challenging contaminants simultaneously.[45] These characteristics are available in the phycoremediation technology, which has the potential to remove organic and inorganic pollutants from wastewater simultaneously, whereas this process does not take place during the conventional chemical processes. Moreover, the consumption of atmospheric CO2 by microalgae during the photosynthesis process plays an important role in global warming. Phycoremediation is flexible in handling fluctuations in the quality and quantity of effluent feed, has easy implementation and maintenance, as well as is sustainable and eco-friendly. Finally, in contrast to

Most common microalgae used in phycoremediation of different wastewaters.

Microalgae C. vulgaris Chlorella minutissima, Scendesmus spp.

Types of wastewater Chemical manufacturing wastewater Sewage

Chlamydomonas polypyrenoideum

Dairy industry wastewater

Chlorella sorokiniana

Alcohol distillery wastewater

Halochlorella rubescens

Municipal wastewater

Desmodesmus sp.

Anaerobic digestion wastewater

Nostoc spp., Oscillatonia spp.

Sewage

Chlorella pyrenoidosa

Textile wastewater

Chlorella vulgaris, Pseudokirchneriella subcapitata Scenedesmus obliquus

Synthetic wastewater

C. vulgaris, Scenedesmus obliquus, Consortium C Chlorella kessleri, C. vulgaris, Nannochloropsis oculata C. vulgaris

Urban wastewater Urban wastewater Urban wastewater Saline wastewater

Notes

References

Has exhibited appreciable nutrient Rao et al. [26] removal capacities They were very effective in the reducSharma and Khan [33] tion of BOD5 , COD, NO3 ,− NH4 , PO3− 4 , and TDS The algae can reduce pollution load of Kothari et al. [34] nitrate, nitrite, phosphate, chloride, fluoride, and ammonia Effective in reducing nitrate and Solovchenko et al. [35] phosphate Microalgae can reduce P and N Shi et al. [36] concentrations up to 99% High efficiency in removal of nitrogen Ji et al. [37] and phosphorus Both algae have the potential to remove Azarpira et al. [38] Ca+2 , Mg+2 , K+ , and Na+ The algae has the potential to reduce Pathak et al. [39] phosphate, nitrate, and BOD Both algae have high removal Silva et al. [40] efficiencies for ammonia, nitrate, and phosphate The microalgae were able to remove Mennaa et al. [41] the total dissolved phosphorus and nitrogen concentrations All algal species have high removal Batista et al. [42] efficiencies for N, P, and COD High efficiency in reducing nitrogen Caporgno et al. [43] and phosphorus concentration The removal efficiencies of TN and TP Shen et al. [44] were high

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N.F. Pahazri et al.

chemical treatment which produces toxics, the final product of the phycoremediation process is biomass, which can be easily converted into value-added products such as biofuels and bio-fertilizers.[3,26] 4. Microalgae harvesting methods Microalgae harvesting is conducted by using mechanical, chemical, and biological methods. The combinations of two or more of these methods to obtain a greater separation rate is common.[46,47] The selection of the harvesting method of microalgae biomass depends on the efficiency and economic considerations, as well as the final utilization of biomass yielded. In terms of efficiency, the harvesting process should be effective for the majority of the microalgal strains and should allow the achievement of a high biomass yield.[47] The major challenge lies in the microalgal cell size, especially the one with a small size (2–0 μm) where separation from the large scale is quite difficult.[5] Economically, about 20–30% of the total cost of the production of microalgae biomass is related to the harvesting method. Therefore, the best technology is the one that has the capability of producing a large quantity of microalgae biomass with a minimal cost.[48] In this section, harvesting methods are viewed on the basis of efficiency and economy. 4.1. Mechanical harvesting Centrifugation is the most rapid, efficient, and reliable method among several mechanical harvesting methods, which is used for recovering suspended algae biomass.[49] This process depends on the principle of gravitational force that increases the sedimentation rate. The centrifugation harvesting method varies depending on the particle size. Tubular bowl centrifugation provides the most efficient method for harvesting, but its capacity is very limited. Therefore, this technique might be more preferably applied on a small laboratory scale.[20] Sim et al. [50] reported that the centrifugation method is more appropriate for smaller species of algae such as Chlorella spp. and Oocytis spp., which have higher solid contents ( > 15%). It has been reported that the efficiency of this technique might reach more than 90% at high centrifugation speeds.[51] However, this technique is not appropriate for other microalgae such as Microcytis sp., Anabaena sp., and Arthrospira sp., because these microalgae have gas vesicles which cause them to be in a vertical position and they become more difficult to harvest using sedimentation and centrifugation.[52] Another limitation of this technology is the high investment and operating costs.[53] Besides, cell wall damage due to high gravitational and shear forces might limit the application of this method. Economically, this method is not viable for harvesting at a large scale due to its practice being greatly energy intensive.[54] Many

authors in the literature have suggested to find an alternative technology with high efficiency and low cost.[55,56] The second mechanical harvesting method is the sedimentation process which depends on gravity deposition. It is suitable for various types of microalgae, because the application of sedimentation tanks is easier and inexpensive. However, the reliability of the sedimentation method is low and the biomass yield is very low without a primary flocculation and coagulation. This is due to the macroalgae density, which is a key factor to obtain a high quantity of macroalgae biomass recovered by this process. Moreover, in some larger species such as Arthrospira sp., this method might be effective.[56] In fact, sedimentation alone might be efficient to precipitate suspended solids in the wastewater. It is used as one of the treatment processes of sewage, but the main differences between suspended solids and microalgae in the wastewater is the motility, since most microalgae have active movements. Therefore, the primary flocculation and coagulation are necessary to speed up the microalgal settling by sedimentation.[47] It has been reported that the harvesting of Nannochloropsis sp. from a fermentation growth medium by sedimentation had managed to reach an efficiency of up to 99% after ferric chloride solution was added and the pH value was maintained at 8.45.[57] More discussion on the flocculation and coagulation process is provided below in detail. Flotation process is a type of harvesting method with the presence of gas bubbles, which provide the lifting force needed for particle transport and separation and it is often viewed as an ‘inverted’ sedimentation. Flotation depends on the attachment of air bubbles to solid particles. The resulting flocs float to the liquid surface and are harvested by skimming and filtration. The success of flotation depends on the nature of the suspended particles (microalgal cells in the harvesting process). Air bubbles drift up the smaller particles. This process is common in many wastewater treatment processes, and is often preceded by coagulation/flocculation.[50] Flotation process relies on the principle of low density of microalgae and self-float characteristics.[47] In contrast to sedimentation and centrifugation, flotation is more effective for microalgae because it has gas vesicles which cause the microalgae to be in a vertical position. In the flotation process, the algae move upward rather than downward like in the sedimentation process. This favours flotation as the mass cultivation of algae requires a high overflow rate. Particles with a diameter less than 500 mm can be captured by flotation.[58] Kim et al. [59] revealed that the flotation method is effective to harvest Spirulina platensis cells from a photo-bioreactor after six days of incubation period. In the study, the process was enhanced by the addition of NaCl. Table 3 illustrates the harvesting method of microalgae by using the flotation technique. Dissolved air flotation (DAF) is one of the most efficient options for sewage sludge treatment, because it seems

Environmental Technology Reviews Table 3.

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Harvesting methods of microalgae by using flotation technique.

Algae Scenedesmus quadricauda S. quadricauda Chlorella sp. Chlorella sp. S. platensis Chlorella sp. Dunaliella salina C. vulgaris, S. obliquus

Surfactant

Dosage

Harvesting percentage

SDS + Chitosan CTAB CTAB SDS + Chitosan NaCl CTAB Aluminium sulphate Ferric sulphate Ferric sulphate Saponin and chitosan

20 + 10 mg L−1 100 mg L−1 40 mg L−1 20 + 10 mg L−1 6.8 mmol l 1–3 mg L−1 150 mg L−1 150 mg L−1 75 mg L−1 20 + 5 mg L−1

95 > 90 86 85–90 80 95–99 95 98 98.7 93

to be the most economical.[50,66] In this process, the pressure of water stream, pre-saturated with air at an excess pressure, is reduced to bubbles of 10–100 μm in size.[67] This process might become more efficient if the particulate size of the algal biomass has increased via flocculant addition. Other factors which enhance the efficiency of this process are air bubbles passed into the solution, and increase in their buoyancy, making the algal particles float to the surface where a compaction zone is formed.[68] This process has more efficiency for the harvesting of microalgae biomass in comparison to settling, but the main problem associated with the DAF systems lies in the oversized bubbles that break up the flocs.[58] Dispersed air flotation (DiAF) is one of the flotation methods which generate bubbles by passing air continuously through a porous material. This process is effective to harvest microalgae biomass or algogenic organic matter (AOM).[65] It consumes less energy, but requires more expensive equipment and demands higher pressure drop for bubble generation. Electroflotation depends on the formation of fine hydrogen bubbles by electrolysis.[47] In order to improve the efficiency of DiAF, Kurniawati et al. [65] used a natural bio-surfactant saponin as the collector and chitosan as the flocculant to harvest C. vulgaris and S. obliquus. The study found that 20 mg/L of saponin was an effective collector when 5 mg/L of chitosan was added. The process managed to recover 93%, 54.4%, and 73.0% of microalgae biomass, polysaccharide, and protein, respectively. Ozonation-spread flotation is a method which relies on the principle of interaction between negatively charged microalgal cells and charged bubbles. One of the main advantages of this technique is the ability to extract lipid contents from microorganisms, as noted in C. vulgaris where the lipid contents extracted increased by 24%. This process also allows a more effective separation due to the lysis caused by several biopolymers used during the flocculation and coagulation.[47] However, one of the major threats is the toxic by-products including organic micro-pollutants and chemical compounds transformation by the ozonolysis process. It has been demonstrated that

Reference Chen et al. [60] Phoochinda and White [61] Liu et al. [62] Liu et al. [62] Kim et al. [59] Garg et al. [63] Hanotu et al. [64] Kurniawati et al. [65]

the ozonation process of potable water is associated with the generation of bromide ions which have a cancerogenic property.[3] Sort flocs depend on the size of the bubble and flux. Small bubbles have a high surface area to volume ratio and lower velocity, which lead to faster containment and tender contact with the particles. In this way, the use of micro-sized bubbles (microflotation) is a good alternative to separate fragile flocs. However, the efficiency strongly depends on the pH value and the coagulant dosage. This technique should be used preferably with a fluid oscillator, because both DAF and DiAf are not able to generate the correct bubble size in a sustainable way.[64] 4.2.

Flocculation methods for microalgae harvesting

Flocculation is a process in which the particles in a solution join together to form agglomerates called flocs, which help in settling.[68] Several studies have reported that multivalent metal ions play an important role in flocculating microalgae by pH increase. The metal ions in the microalgae medium are hydrolysed to form positive precipitates, which coagulate negative microalgal cells by sweeping flocculation and charge neutralization. At a natural pH, microalgae carry a negative charge on their surface due to the presence of functional groups on the walls of microalgal cells such as the carboxyl group.[69] The surface of microalgal cells usually receive their charge and exhibit dispersing stability from the ionization of carboxyl groups into carboxylate ions.[70] The reduction in pH values induces the carboxylate ions to accept protons, and this process makes the microalgal cells unstable and then they coagulate to form big flocs.[71] Flocculants displace the negative charge and allow connection to each other through a patch of charges, contrarily causing flocculation.[56] Flocculation improves the rate of sedimentation of microalgae by aggregating the dispersed microalgal cells into larger particles, and thus increasing the recovery of biomass.[72] Flocculation might be due to the addition of inorganic and organic flocculants. Among the inorganic flocculants, multivalent metal salts

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such as aluminium and iron are the most common. The main differences between inorganic and organic flocculants are the quality, efficiency, and the secondary products. Inorganic flocculants are used in a large quantity to achieve high efficiency and leave a lot of sludge. In contrast, organic flocculants are typically polymeric in nature, and they are effective at low concentrations.[73] Coagulant chemicals with opposite charges are used to neutralize the negatively charged suspended solids, and then they stick together to increase the stabilization process.[74] There are several factors that affect flocculation. Among the main factors are the coagulants type, ionic strength, pH, temperature, flocculant dilution, shear, and process conditions (dosing and mixing conditions) which contribute significantly to the efficiency of the flocculation process. These factors, individually and collectively, have a great influence on the type of flocculant that will produce optimum performance and optimum dose. The percentage of flocculation achieved can be markedly affected by the mixing conditions and dosing amounts. The flocculation process occurs rapidly with a relatively low polymer dose and high solid concentrations, but it is not stable and can be broken at moderate stirring rates. Moreover, by reducing the stirring shortly after polymer dosing, the floc size can be held at plateau levels, without subsequent decline.[75] Furthermore, the effective dose required to achieve high flocs depends on the type of flocculant; but before the usage of the flocculant, a jar test might be conducted to determine the effective type of coagulant and their proper dosage.[76] Temperature can affect the flocculation process by increasing water viscosity, altering coagulant solubility, and affecting the kinetics of hydrolysis reactions and particle flocculation.[77] However, the nature of the flocculant used affects strongly on charge neutralization, while temperature is the second factor.[78] Moreover, Fitzpatrick et al. [79] had investigated the effects of temperature between 6°C and 29°C on floc formation by using ferric sulphate, alum, and three polyaluminium chloride (PACI) coagulants. The jar test was conducted using a photometric dispersion analyser. The study revealed that at low temperature, the flocculation process was slow for all coagulants and was most notable at 15°C. Surendhiran and Vijay [80] revealed that the optimum temperature for harvesting N. oculata from a culture medium was 35°C. pH values of the wastewater or production medium might have an effect on the microalgae biomass harvesting by using the flocculation process. At the pH between 8 and 9, the coagulant used might consume all available alkalinity and thus increase the efficiency of the flocculation. In contrast, high doses of coagulants are required at a high pH, more than pH 11, and a high dose is required to decrease the pH value to be within the range optimal for coagulation.[77] Liu et al. [81] indicated that the flocculation method for harvesting freshwater microalgae might be induced by decreasing the pH value of the growth

medium. This method managed to achieve 90% recovery of Chlorococcum ellipsoideum, Chlorococcum nivale, and Scenedesmus sp. with a high biomass concentration ( > 1 g L−1 ). The most common coagulants used in the flocculation process are discussed below. 4.2.1. Chemical coagulant Chemical coagulation/flocculation is the main approach towards economic optimization of the microalgae harvesting processes.[82] It has been demonstrated in the literature that coagulation/flocculation processes are the most efficient methods for large-scale microalgae harvesting.[83] In chemical flocculation and coagulation, the electrolytes and synthetic polymers such as aluminium ferric cations, aluminium sulphate, and ferric chloride are typically added to increase the particle size of algal cells. Therefore, this process concentrates on the suspension 20–100 times and is used as the preliminary step for flotation harvest.[46,53] However, these practices are prohibited if the microalgae biomass will be reused for land application due to the presence of aluminium, which can cause phosphorus deficiency and increase heavy metal contents in the plant.[84] De Godos et al. [85] examined the efficiency of chemical coagulants (FeCl3 and Fe2 (SO4 )3 ) compared to polymeric flocculants (Drewfloc 447, Flocudex CS/5000, Flocusol CM/78, Chemifloc CV/300, and Chitosan) in the recovery of S. obliquus, C. sorokiniana, and Chlorococcum sp. from piggery wastewater. The study found that the highest biomass recovery was 66–98% with 150–250 mg L−1 of ferric salts and 25–50 mg L−1 of polymer flocculants. The study indicated that the efficiency of polymers in flocculation is higher than that of chemical coagulants. Granados et al. [86] found that the use of metal salts (iron chloride, iron, and aluminium sulphate) and chitosan was insufficient for harvesting Muriellopsis sp., C. vulgaris, Chlorella fusca, Scenedesmus subspicatus, and Scenedesmus sp. from freshwater, whereas polyelectrolytes were found to allow an efficient recovery of biomass with 2–25 mg per gram of microalgae biomass. Chemicals coagulants including ferric sulphate and aluminium sulphate have been tested for harvesting algae. Inorganic salts as ligands facilitated the microalgal cells to stick together and settle down.[46] These chemicals are called flocculants, although their effectiveness has a limited potential implementation. Besides, they are expensive enough to satisfy the requirement of economic viability.[87] Inorganic flocculants such as ferric chloride and aluminium sulphate and synthetic organic highpolymer flocculants such as polyacrylamide (PAA) and an amine derivative of polyethylene have been effectively used to harvest algae biomass. Vandamme et al. [88] explored the potential of inducing flocculation for harvesting C. vulgaris from freshwater, by increasing the level of pH to 11 using calcium and magnesium salts. The study

Environmental Technology Reviews showed that 0.15 mM of magnesium induced the flocculation process. This was due to the formation of magnesium hydroxide precipitate by the hydrolysis of Mg2+ in the growth medium, which coagulated the microalgal cells by charge neutralization and sweeping flocculation.[89] This study indicated that the flocculation induced by high pH might be useful for the harvesting of macroalgae biomass from freshwater. The efficiency of a combination of two coagulants was investigated. Gorin et al. [90] studied the effects of the combination of FeCl3 ·6H2 O,PAA, as well as polyethylene oxide (PEO), and flocculated biomass on the flocculation efficiency of harvesting C. vulgaris from culture media. The study showed that the efficiency of the harvesting process was 90% after 5 min of sedimentation with the addition of 50 mg/L FeCl3 and 7.5 mg L PEO, as well as the addition of 10% flocculant and 7.5 mg/L PAA. Both PAA and PEO recorded flocculation efficiency at a dosage of 0.025 and 0.015 g/L, respectively, without pH adjustment. Salama et al. [91] used acid mine drainage (AMD), rocks characterized by low pH and a high concentration of dissolved metal ions, for harvesting S. obliquus and C. vulgaris from water samples. The AMD used was in 10% of concentrations and at pH 9. The flocculation efficiency was 89% for S. obliquus and 93% for C. vulgaris. The mechanism of flocculation was investigated by using a scanning electron microscope with energy-dispersive Xray and indicated that the flocculation process depends on the floc formation, which takes place after the addition of AMD. The study concluded that the harvesting of microalgae biomass by AMD is a cost-effective method. Moreover, flocculation by metal is certainly not acceptable if the harvested biomass is to be used for aquaculture, animal feed, or organic fertilizer. It was reported that alum and acrylamide can lead to implications on human health such as involvement in the cause of Alzheimer’s disease and cancer.[92] 4.2.2. Biological flocculation Biological harvesting includes auto-flocculation and bio-flocculation.[53] Some algae are known to flocculate spontaneously without chemical addition, due to the production of extracellular polymeric substances (EPS), such as Chlorella sp. and Micractinium sp., which lead to microalgal cell agglomeration and facilitate the separation from the medium by simple gravity sedimentation.[48] Bio-flocculation might reduce the cost and energy demand for harvesting microalgae. Auto-flocculation also reduces the cost of the harvesting process of microalgae biomass. Auto-flocculation is a natural process which takes place due to the rising of pH value in wastewater as a result of dissolved carbon dioxide consumption during photosynthesis.[53] Guo et al. [93] tested the efficiency of extracellular biopolymers of S.

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obliquus in self-flocculation. The study extracted extracellular biopolymers, determined the sugar composition, and then reused it for bio-flocculation of S. obliquus and C. vulgaris. The extracellular biopolymers consisted of glucose, mannose, galatose, rhamnose, and fructose at a molar ratio of 8:5:3:2:1. The addition of 0.6 mg/L purified flocculating agent managed to achieve rapid flocculation of freely suspended cells of S. obliquus and C. vulgaris at 20–26°C and pH of 6–8. Auto-flocculation is an attractive alternative harvesting method for harvesting microalgae biomass, because it requires low cost and energy, is non-toxic to microalgae, and does not require the use of flocculants, and thus enables a simple medium reuse.[47] Bio-flocculation can also take place by secreted biopolymers such as those produced by bacteria (EPS). Microbial flocculants have been widely used for wastewater treatment due to the presence of nutrients necessary for the growth of flocculating microorganisms.[56] Furthermore, Wan et al. [94] reported that Solibacillus silvestris was effective in flocculating microalgae without the addition of chemical coagulants. However, the presence of bacteria in the production medium of microalgae might lead to microbial contamination and limit the applications of microalgae biomass as animal feeds.[47] Moreover, the potential of the microbe to produce EPS in high concentration and the ability to attach the macroalgae to form flocs are the main factors considered to achieve high efficiency of microbial flocculation.[95] Zheng et al. [96] investigated the harvesting of Chlorella protothecoides, N. oculata, Phaeodactylum tricornutum, C. vulgaris, and Botryococcus braunii by flocculation with poly (γ -glutamic acid). The study found that the efficiency of flocculation was more than 90% without damaging the cell integrity. Prochazkova et al. [97] examined modified spent yeast (MSY) prepared from a brewery by-product for harvesting C. vulgaris from different aquatic environments. The yeast cells were autolysed/hydrolysed and chemically modified with 2-chloro-N, N-dimethylethylamine hydrochloride (DEAE). The study indicated that the dose required for the harvesting process depends on the medium composition where more dosage is required in the presence of phosphorus ions and AOM was found to increase the required dosage of flocculants ranging between 0.4 and 51 mg MSY/g biomass. 4.2.3. Natural coagulant The utilization of natural coagulant produced or extracted from microorganisms, or animal or plant tissues in the harvesting of macroalgae biomass from different production media such as wastewater has received great interest during the last years. These coagulants are characterized by their biodegradability and are safe for human and environmental health such as for plants and aquatic organisms. In addition, the sludge resulting from the utilization

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Table 4.

Harvesting methods of microalgae biomass by flocculation.

Microalgae species Parachlorella, Scenedesmus, Phaeodactylum, Nannochloropsis

Coagulants

Dosage of coagulant (mg/L)

Mixing rate and time

Removal efficiency

References

5, 10, 20, 30, 40, and 60

1000 rpm (5 min) 250 rpm (30 min) Settling time 30 min

More than 90% of the biomass was removed at the optimal dose

Vandamme et al. [82]

FeCl3 and Fe2 (SO4 )3

0, 5, 25, 50, 100, 150, and 250

300 rpm (1 min) Settling time 10 min

De Godos et al. [85]

Chlorella sp.

Chitosan

0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100

10–250 rpm 5–120 min Setting time 5–10 min

Highest biomass removals (66–98%) at concentration of 150–250 mg/L Removed 99% ± 0.4% of microalgae at concentration 10 mg/L

C. vulgaris

Chitosan

30, 60, 90, and 120

50 rpm (3 min)

FeCl3 and Fe2 (SO4 )3 CGG

0.4 and 0.6 g/L 40 and 100 ppm

180 min 15 and 30 min

Cationic starch

0, 2.5, 5, 10, 20, and 40

500 rpm (5 min) 250 rpm (25 min) Settling times 0, 10, 20, and 30 min

Removal efficiency from 84% to 90% on average was achieved

10, 20, 30, 40, and 50

150 rpm (5 min) 150 rpm (5 min) 10, 20, 30, 40, and 50 rpm (15 min) Settling time 30 min

Removal efficiency more than 90% was achieved

C. sorokiniana, S. obliquus, and Chlorococcum sp.

N. oculata Chlorella sp. and Chlamydomonas sp. C. protothecoides

Chlorella sp.

M. oleifera

120 mg/L of chitosan has the highest efficiency (92 ± 0.4%) within 3 min 93.80% and 87.33% 94.5% and 92.15%

Ahmad et al. [107]

Rashid et al. [87] Surendhiran and Vijay [80] Banerjee et al. [105] Letelier-Gordo et al. [103]

Hamid et al. [92]

N.F. Pahazri et al.

Cationic starch

Environmental Technology Reviews of a natural coagulant will be more biodegradable and less voluminous, amounting to only 20–30% of alumtreated counterparts.[98] Recently, natural coagulants such as Moringa oleifera seeds and Strychnos potatorum are widely used as coagulants for the harvesting of microalgae from wastewater.[92] M. oleifera is known as a tropical plant which belongs to the family of Moringaceae. It is a tropical multipurpose tree that naturally grows in India, South Saharan Africa, and South America. In Malaysia, M. oleifera is available locally and it is inexpensive, which makes it a viable alternative in water and wastewater treatment.[92,99] Sotheeswaran et al. [100] reported that M. oleifera seeds contain proteins that have active coagulation properties. Moreover, almost every part of the plant including leaves, flowers, seeds, roots, and barks can be used as food or for medicinal and therapeutic purposes.[92] Several studies have been conducted on the performance of M. oleifera seeds as an alternative coagulant to conventional chemical coagulant in water treatment.[92,98,101] Natural polymers that do not involve the same concerns of secondary pollution might be used as flocculants. Rashid et al. [87] stated that the efficiency of the microalgae harvesting process from fresh- and seawater might be improved by adding chitosan without the contamination of microalgae biomass. The flocculation using chitosan provides high efficacy, low dose requirements, and a short settling time. Meanwhile, Farid et al. [102] showed that the use of nano-chitosan in the flocculation for harvesting Nannochloris sp. might reduce the dosage of flocculant consumption by 40% and enhance biomass recovery by 9%. However, the application of chitosan is too expensive for large-scale applications.[47] In some applications, the water generated from the harvesting process might be recycled again as the culture production of microalgae biomass. Farid et al. [102] indicated that this water managed to increase the growth of microalgae by about 7%. Vandamme et al. [82] assessed the potential of cationic starch as flocculants for harvesting Parachlorella sp. and Scenedesmus sp. using the jar test experiment. The study showed that the cationic starch to algal biomass ratios required to flocculate 80% of algal biomass were 0.1 mg L−1 for Parachlorella sp. and 0.03 mg L−1 for Scenedesmus. Letelier-Gordo et al. [103] confirmed that cationic starches are easy to use, efficient, and costeffective flocculants for harvesting microalgae without toxic by-products. Gutiérrez et al. [104] investigated the coagulation– flocculation and sedimentation of macroalgae biomass yield in wastewater using natural flocculants (Ecotan and Tanfloc). The study showed that 10 mg/L and 50 mg/L of Ecotan and Tanfloc, respectively, had recovered over 90% of biomass yield. The settling column tests revealed that both flocculants improved the microalgae settling velocity,

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performed fast, and produced efficient biomass recovery by more than 90% in 10–20 min. Moreover, the study showed that doses of 10 and 50 mg/L for Ecotan and Tanfloc, respectively, did not affect the microalgae biomass quality. Banerjee et al. [105] used cationic guar gum (CGG) that was synthesized by the introduction of quaternary amine groups onto the backbone of guar gum from N-(3-chloro-2hydroxypropyl) trimethyl ammonium chloride (CHPTAC) for the harvesting of Chlorella sp. and Chlamydomonas sp. from algal culture. CGG was investigated at different dosages until 100.0 ppm. The maximum recovery was 94.5% and 92.15% for Chlorella sp. and Chlamydomonas sp. achieved at 40 and 100 ppm within 30 and 15 min, respectively. Gutiérrez et al. [106] evaluated the efficiency of potato starch as flocculants for Chlorella sp. biomass coagulation–flocculation and sedimentation. The optimal flocculants activity was recorded at 25 mg/L with 95% of biomass recovery. Nanotechnology of flocculants was used in order to synthesize novel flocculants with a high efficiency of harvesting microalgae biomass from culture media. Vandamme et al. [5] assessed the potential of cellulose nanocrystals (CNCs) which were positively charged at a pH value between 4 and 11 for harvesting C. vulgaris from freshwater. CNCs were synthesized by grafting cationic pyridinium groups onto CNCs using two separate one-pot simultaneous esterification and nucleophilic substitution reactions. The study revealed that the flocculation efficiency reached 100% with 0.1 g CNCs g−1 biomass. The harvesting methods of microalgae biomass by flocculation processes are presented in Table 4. 5.

Harvesting process using immobilization technology

The growing harvesting challenges of macroalgae biomass due to their size which ranges between 2 and 50 μm in diameter, low density (0.5–5 g/L dry cell weight), and the characters of cell wall surface as a negative charge have led scientists to develop a more advanced technology for microalgae biomass harvesting from the production media. One of these technologies is the immobilization technique, which depends on the entrapment of microalgae in a matrix and allows continuous growth of cells within the matrix. In the immobilization process, the microalgae in the stationary growth phase are settled at the bottom of the production media. Hence, low energy is required to harvest the settled biomass and a simple filtration process might be enough to harvest a high quantity of biomass.[53] The immobilization techniques aim to improve the quality of the treated wastewater without secondary effects on the quality of the biomass yield.[108] The immobilization techniques have several advantages including a great degree of operational control, flexibility, high transparency and diffusivity, easy separation, low production

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hazards, and low polymer costs. Alginate has become the most frequently used polymer for the entrapment of microalgae.[108] The main advantage of the immobilization process is the applicability to be used in wastewater which has high salinity, metal toxicity, and pH and stress conditions that might affect the utilization of bio-flocculants such as the harmful effects of photo-inhibition. Other advantages include high biomass production, the cells are recovered in a less-destructive way, and the cost-effectiveness of the process is enhanced by reusing the regenerated biomass.[109] Immobilization technology might be an attractive alternative, since the scale-up processing represents a significant challenge associated with algal biomass recovery.[53] Nevertheless, the disadvantages of immobilization of microalgae include the destruction of macroalgae beads resulting from the availability of divalent ions and mass transfer limitation from the biofilm layer of microalgal cells, which hinder the transport of nutrients and carbon source. Hence, the applications of immobilized microalgae are limited in comparison with the usage of a free cell culture.[110] 6.

New technologies for the harvesting of microalgae biomass Most of the current harvesting method approaches, which include flocculation, floatation, centrifuge, and filtration, have limitations for an efficient and cost-effective production.[111] Therefore, in order to enhance the efficiency of microalgae harvesting methods, new technologies have been developed.[112] In this section, the newest methods used for the harvesting of biomass yield in the production media are reviewed. Electrocoagulation is a process used for water and wastewater treatment through the electrocoagulation cells, which aim to remove the pollutants that are difficult to be removed by the traditional methods such as chemical treatments.[113] Hence, it would be a suitable alternative technique for harvesting microalgae. The main advantages for this technology are it requires low energy input, and it is eco-friendly and effective. In contrast, one of the limitations lies in the need to replace the anode as well as the possible contamination of the harvested biomass due to high doses of the electrode metal ions.[114] Electrocoagulation is applied to harvest microalgae biomass by submerging two reactive electrodes (e.g. aluminium electrodes) in the microalgae suspension and then connecting it to electricity. During the electrocoagulation process, metal ions are released by electrolytic oxidation of the anode material which then act as a coagulant for the formation of microalgae flocs. During this process, oxygen and hydrogen microbubbles are also generated due to the water oxidation process. The electrocoagulation is followed by sedimentation gravity to separate the flocculated cells.[114] It was revealed that the electrocoagulation

achieved 97% of the harvested Nannochloropsis sp. from the production media.[113] Magnetic separation method is one of the latest techniques utilized for the harvesting of microalgae biomass. This technology depends on functional magnetic nanoparticles such as Fe3 O4 which provide an effective method for the recovery of macroalgae from the culture media. Magnetic separator separates the magnetic particles based on the physical capture of these particles by a magnetic field with a high process capacity.[115,116] Some of the functional magnetic particles are coated with diallyldimethylammonium chloride, chitosan, or silica and used for improving microalgae harvesting.[115,117] This technique is quick and efficient.[118] Hu et al. [115] developed a magnetic separator unit consisting of a permanent magnet drum, a separation chamber, and a scraper blade for the harvesting of Chlorella ellipsoidea cells based on the magnetic nanoparticles. The design of the magnetic separator unit exhibited more than 95% of harvesting efficiency within 40 s in each batch operation. Moreover, in the continuous operation, the harvesting efficiency relies on the liquid flow rate, where the increase in the liquid flow rate through the separation chamber is associated with the decrease in the harvesting efficiency. Therefore, in order to keep the efficiency at 95%, it is required to fix the liquid flow rate at 100 mL/min. Xu et al. [117] used the magnetic separation process for the recovery of B. braunii and C. ellipsoidea from the production media. The maximum recovery was 98%, indicating the efficiency of this process in the harvesting of different microalgae species. Ultrasound technique is a separation process of macroalgae biomass based on the ultrasound and sedimentation processes simultaneously. Ultrasound waves are used for the breakdown of the microorganism cells, but they are also used for the agglomeration process to facilitate the settling by gravity if used with high frequency and low amplitude.[112] The ultrasound method has several advantages including the absence of toxic by-products as well as the absence of mechanical failures as a result of the nature of the device, which has no freely moving parts and has the possibility of continuous operation.[119] Besides, it has the potential to be applied on an industrial scale as an alternative technology for centrifugation due to lower power consumption and high efficiency. Bosma et al. [119] used this technology for the harvest of Monodus subterraneus with 92% recovery efficiency. 7.

Microalgae milking

One of the technologies for the harvesting of microalgae biomass which has occurred recently is the milking technology. This technology is more applicable for a low productivity of algal cultures in the production of high-value compounds.[116] The main advantage of the milking process of microalgae is it keeps the algae alive,

Environmental Technology Reviews while high-value molecules are being extracted. Hence, the microalga does not require the constant need of culturing and regrowing the entire stock of algae, which have a typical timescale of a few hours to weeks.[120] Recently, this technology was used to extract carotenoids from Arthrospira platensis in a two-phase bioreactor.[116] Zhang et al. [121] studied the application of membrane dispersion for enhancing lipid milking from B. braunii. The study revealed that the solvent flow rate and the initial biomass concentration enhanced the lipid amount extracted by the solvent. The result suggested that membrane dispersion was a good choice to improve the mixing effect in the algal lipid milking process or other similar cell products extraction processes. Hejazi et al. [122] introduced a new method to continuously produce and extract carotenoids from D. salina simultaneously. The study was conducted by adding a biocompatible organic solvent (dodecane) to the bioreactor, which led to improve the production of β-carotene by the cells and extracted them in situ. The study indicated that the production of carotenoids during the microalgae growth did not need to be continuously repeated. Therefore, the milking process keeps the microalgae alive for several weeks, with constant biomass levels and increasing amounts of extracted β-carotene. Based on the studies carried out on the milking process of microalgae, it could be concluded that the milking process is a successful technology. However, the mechanism behind it is unclear, as is the influence of the organic phase on the cells.[112] Kleinegris et al. [123] investigated the effect of the organic phase on D. salina cells and its influence on the extraction mechanism and found that the D. salina cells had died due to the contact with the solvent phase, but remained viable in the water phase, which meant that the organic solvents might exert phase toxicity but did not exert molecular toxicity. 8. Drying methods of microalgae biomass The harvested biomass has to be accomplished within a few hours by the preservation process to prevent the damage in a hot climate. The specific post-harvest processing depends strongly on the desired product. Dehydration or drying of biomass is typically used to extend the shelf life of biomass, especially when the biomass is the final product.[124] The most common drying methods used for microalgae biomass are spray drying, drum drying, freezedrying, and sun-drying. Despite sun-drying being among the slower methods, it is cost and energy effective compared to the other techniques. Freeze-drying is widely used for dewatering microalgae biomass. Freeze-drying is a gentle process in which all the cell constituents are preserved without rupturing the cell wall. The concern of sun-drying of biomass yield is the high water content of algae biomass; therefore, sun-drying is not economically feasible for lowvalue products such as biofuels or protein.[55,124] The

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second concern is the applicability of solar drying in countries with temperate climates due to limited sunlight at certain times throughout the year.[110] Spray drying is the preferred method for high-value products. However, the significant impediment of this method lies in its ability to cause significant deterioration of some parts of algae such as the pigments. Moreover, these limitations can be overcome at the expense of drying appropriately to produce microalgal biomass powder to be used in food and feeds.[125] Freeze-drying or lyophilization has been widely used for drying microalgae in laboratory researches. However, freeze-drying is too expensive to be used in a large-scale commercial recovery of microalgae products. In some cases, solvent extraction of dried biomass has proven to be more effective for the recovery from the extraction of intracellular metabolites from wet biomass. Intracellular products such as oil can be difficult for the solvent extraction of wet biomass, but undisrupted cells are extracted easily if the biomass is freeze-dried.[126] Guldhe et al. [127] revealed that the lipid content of microalgal Scenedesmus sp. biomass dried by freezedrying, oven drying, and sun-drying processes was similar, which means that the drying method does not affect significantly the quality of microalgae biomass. Similarly, Balasubramanian et al. [128] stated that there was no significant effect of the drying technique on the total lipid yield after extraction, in which the biomass of Nannochloropsis sp. was dried using oven drying, freeze-drying, and sun-drying. Moreover, freeze and oven drying are energyintensive methods. 9.

Final utilization and quality of microalgae biomass

The increasing requirements for health products are associated with the utilization of algae due to the simple requirements for their growth such as CO2 and solar light. Microalgae biomass has several applications such as bioproducts, animal feeds, and biodiesel.[129] The utilization of microalgae biomass including the whole algal products or algal extracts depends on the purpose of utilization. For instance, Arthrospira sp. and Chlorella sp. are used as dietary supplements for humans and animals without any kind of processing except drying.[130] In some cases, the microalgae biomass is subjected to a further extraction process to produce specific highvalue components, especially in the pharmaceutical industries and in biofuel production. Microalgal bioproducts have more advantages than synthetic products in terms of their effectiveness to be used as infant formula, dietary supplements, and fish pigments.[131] Many secondary bioproducts are extracted from microalgae such as pigments, carotenoids, polyunsaturated fatty acids, vitamin, and antioxidants, which are formed in microalgal cells under stress environments such as a nutrient deprivation, high salinity, radiance, and temperature.[132] The most

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Table 5. Most common microalgae species used for bioproducts.

Class

Species

Cyanobacteria Spirulina/ Arthrospira sp. Synechococcus sp. Rhodophyta Bangiophytes

Chlorophyta

Cyanidophytes Porphyridiopyhtes Stylonematophytes Rhodellophytes Florideophytes Tetraselmis sp. Chlamydomonas sp. H. pluvialis Dunaliella sp. Chlorococcum sp. Scenedesmus sp. Desmodesmus sp. Chlorella sp. Parietochloris incisa

Application/ utilization/ bioproducts Dietary supplements Wastewater treatment Industrial and pharmaceutical applications Food for aquaculture animals

Source of glycerolliquid biofuel Wastewater treatment Food and supplement Chromium remediation Biofuel production Fish pigments Dietary supplements

common microalgae used in the production of bioproducts include D. salina, Haematococcus pluvialis, Arthrospira sp., and Chorella sp. (Table 5). Therefore, based on the purpose of microalgae biomass, the harvesting methods are selected. There are two main criteria which might detect the effectiveness of the biomass yield in the wastewater for further application, which are the composition of production media and the final utilization of harvested biomass yield. In terms of the production media, the harvesting process of microalgae biomass from freshwater or from any synthetic media is quite different from that of wastewater, due to the nature of wastewater which is complicated compared to the synthetic production media. Wastewaters are rich in nutrients, organic matter, and elements; these properties would definitely support the amount of microalgae biomass. Meanwhile, it would affect the harvesting process. The high content of divalent metal ions in wastewater requires a high concentration of chemical coagulant to achieve the maximum efficiency of the harvesting process. However, the final concentrations of divalent metal ions in the treated wastewater might be high, which contradicts the objective of the phycoremediation process which aims to remove metal residues from wastewater. Therefore, natural coagulants might be a better option than chemical coagulation for the harvesting of microalgae biomass. In terms of the final utilization of macroalgae biomass, the presence of pathogenic microorganisms (bacteria and

viruses) in wastewater is the main concern.[133] Microalgae biomass generated by the phycoremediation process is not pure biomass, which means that the utilization of this biomass for animal feeds or as fertilizers should undergo other treatments to reduce the risk associated with pathogenic bacteria in order to prevent their distribution to animals, plants, and then to humans as the final host. However, the microalgae biomass from wastewater can still be used as the source for biofuel and biodiesel. Moreover, the concern of heavy metals in wastewater lies in the biomass yield, which would be used for several applications such as dietary supplements. Heavy metals in the biomass yield might accumulate in humans or animals and cause several diseases. Studies confirmed that heavy metal ions have adverse effects on human health by impairing mental and neurological functions, influencing neutron-transmitter production and utilization, and altering numerous metabolic body processes. The heavy metals in the human system might induce impairment and dysfunction of blood and cardiovascular, detoxification of pathways endocrine, energy production pathways, enzymatic, gastrointestinal, immune, nervous (central and peripheral), reproductive, and urinary systems.[134] Therefore, in order to overcome the toxicity of heavy metals, wastewaters with high concentrations of heavy metals have to be subjected to a primary treatment to remove these metals. Nevertheless, meat processing wastewater has a very low concentration of heavy metals, which might play an important role as a trace element to support the microalgae biomass production. In general, the quality of microalgae biomass might be more sustainable for biodiesel production, but not for animal feeds. It was reported that one kg of wet microalgae biomass might produce 105 MJ of energy, which means that the microalgae produced in the wastewater might be used directly for biodiesel production without any further drying process.[122] Therefore, the production of microalgae in the wastewater appears to be very costly, with high contents of lipids due to the presence of high nutrient values necessary for the build-up of the microalgal cell.

10. Conclusion It can be concluded that both the composition of the production media and the final utilization of biomass yield play an important role in the selection of the harvesting method. Moreover, the natural flocculation process represents the safe method to achieve a higher quality and quantity of microalgae biomass. Milking technology is a new method which might be used to improve the quantity of biomass yields without effects on the quality.

Disclosure statement No potential conflict of interest was reported by the authors.

Environmental Technology Reviews Funding The authors gratefully acknowledge Ministry of the Higher Education of Malaysia for the research project financial support under fundamental research Grant Scheme (FRGS) [vot number 1453] and prototype research Grant Scheme (PRGS) [vot number G004].

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