Contents TITLE PAGE 1 TABLE OF CONTENTS 3 LIST OF FIGURES 5 LIST OF TABLES 6 LIST OF EQUATIONS 7 Abstract 8 1.0. Introduction 9 2.0. Microalgae harvesting method 10 2.1. Common harvesting technology 10 2.1.1. Centrifugation 10 2.1.2. Sedimentation 11 2.1.3. Flocculation 11 2.1.4. Flotation 13 2.1.5. Filtration 14 2.2. New Emerging Microalgae Biomass Harvesting Techniques 15 2.2.1. Flocculation using magnetic microparticles 16 2.2.2. Flocculation by natural biopolymer 17 2.2.3. Electrical approach 18 3.0. Extraction and Analysis of Lipid from Microalgae Biomass 20 3.1. Lipid extraction 21 3.1.1. Mechanical extraction 21 3.1.2. Chemical/solvent extraction 23 3.1.3. New emerging green solvents systems and process intensification techniques for lipids extraction from microalgae 25 4.0. Heterogeneous transesterification catalysts 29 4.1. Solid Bases Transesterification 33 4.2. Solid Acids Transesterification 35 4.3. Heterogeneous transesterification of algae oil 36 5.0. Reactors 44 5.1. Influence of reactor design and operating conditions 44 6.0. Conclusions 51 References 54

The dwindling rate of our fossil fuel reserves and general believe of major contribution of CO2 emissions which is linked to the climate change due to the burning of such carbon sources in engines either for locomotion or power generation have geared both the academic and industrial research towards the new routes for renewable and sustainable fuels. However, microalgae as one of the third-generation biomass feedstock has recently been proven to be one of the best option considered employable for biodiesel production. But one of the crucial challenges not yet explicitly attended to is the method of harvesting, lipids extraction and heterogenous catalyst for transesterification reaction for oil conversion to biodiesel. Herein. we reported several techniques for microalgae biomass harvesting, both the conventional and new emerging ones, as this helps in building ideas for improvement in the field. We also present a critical review on the work done on areas of lipid extraction from microalgae biomass and its conversion to biodiesel through heterogenous catalysis. it covers the progress made in this fields from the last decade, available systems for heterogenous catalysis, mechanism of the reactions and optimal process conditions. Lastly, we discuss on the reactors employed in the transesterification, effects of reactors design and way forward.

In our world, today, the demand for energy is on the increase. Fossil fuel reserves are running out. The persistent fluctuation and increase in price of fossil fuels and its adverse effect on the ecosystem through the emission of greenhouse gases makes it imperative that we seek alternative, sustainable and more environmental friendly energy source. The demand for safe alternative sources of energy such as biofuel is more pressing than ever before. Top on the list of such sustainable renewable energy is the feedstock energy source. A good source of feedstock energy is the Microalgae commonly referred to as the third-generation feedstock (Patrícya et al., 2014; Wawrik & Harriman, 2010). Various bio-products, such as biofuel and bio-hydrogen can be manufactured from Microalgae. Microalgae also have higher biomass yield and lower carbon footprint requirement compared to other plants (Besson & Guiraud, 2013; Farooq et al., 2015).

In making microalgae based biofuel production commercially available and economically viable, the challenge of what method to use in harvesting microalgae and lipid extraction must be taken into consideration. Most harvesting methods can be capital intensive, uneconomical, and produce some level of environmental pollution. Harvesting microalgae can require as high as 20-30% of total biomass production budget (Grima et al., 2003; Mata et al., 2010; Verma et al., 2010) or 50% of the cost of producing biofuel (Muradov et al., 2015).

Also, it has been proposed that world usage of biodiesel could increase to two or three times in most part of the globe by year 2020 and numerous influencing factors have not been fully addressed. Bio-oils or oils as the origin implies contains a key compound called triglyceride esters

which when react with any monohydric alcohol (i.e. methanol) would form a group of compounds called mono-alkyl esters i.e. biodiesel. However, scientist around the world suggested the use of lower monohydricalcohols (i.e. CH3OH to C3H8O), without any explicit justification of which gives the best performance and requirements in terms of its viscosity as specified by ASTM or related international agencies. Similarly, finding the best catalyst and optimized operating reaction conditions is of a great challenge to biofuel industries. Homogenous catalysis offers faster biodiesel production with even moderate reaction conditions, but faced with recovery or separation problem after the transesterification process.

These above highlighted challenges have triggered scientists at both industries and academics to seek alternative means, while emphasizing on the feedstock flexibility, and green catalytic systems. Lately, both microalgae and microalgae emerged as a better option for the biodiesel feedstock. Also, some green catalyst discussed herein emerged as a better candidate heterogenous catalysis for the transesterification process. We therefore present a critical review in these areas and lastly, the reactors involved.

Show et al., (2015) inferred in their work that, when considering a preferred harvesting procedure two important issues must first be determined; the attributes of the microalgae considered and the condition of their growth. The efficiency of any harvesting method chosen will depend largely on the specie of microalgae, size of the microalgae, its morphology and composition of the medium employed. Some harvesting techniques commonly used are centrifugation, flocculation, filtration and sedimentation.

Centrifugation is the application of a centrifugal force of higher intensity than the gravitational force to increase the rate of separation a suspension. Common centrifugation methods include Solid-bowl decanter, nozzle-type centrifuge, Hydro-cyclone, and solid ejecting disc (Milledge & Heaven, 2013). The challenge with the methods above despite their efficiency at harvesting majority of the microalgae cell types are high energy, capital and operational cost required to carry them out (requirement) (Grima et al., 2003; Milledge & Heaven, 2013; Yuan et al., 2009).Centrifugation can harvest averagely between 12-25% of microalgae biomass with an energy consumption of 50-75kW (Milledge & Heaven, 2013). The only justification for this high cost and large amount of energy is that sufficient biofuel of over 90% of the microalgae biomass must be harvested.

Sedimentation is the process by which solids are separated from liquids by capitalizing on differences in the density of the solids to obtain an effluent of clear liquid (Milledge & Heaven, 2013). Most wastewater treatment facilities use sedimentation for sludge treatment. Sedimentation is the most cost effective and least complicated method for harvesting microalgae biomass, especially heavy microalgae suspensions. Two difficulty with using sedimentation for microalgae biomass harvesting is that for solids with little difference in their densities the process can be excruciatingly slow and the dry solids concentration of microalgae biomass that can be harvested is about 0.5-3% (Grima et al., 2003; Milledge & Heaven, 2013; Yuan et al., 2009).

Golueke & Oswald, (1965) reported that using alum as a coagulant in a flocculation-sedimentation process an of 85% microalgae biomass harvest was achieved.

Flocculation seldom used alone but in conjunction with other methods (Brennan et al., 2010) such as coagulation-flocculation and flotation-flocculation. Flocculation through aggregation improves particle size of microalgae suspension and speeds up rate of suspension settling (Mata et al., 2010; Milledge & Heaven, 2013). Auto-flocculation, physical flocculation, bio-flocculation and physio-chemical flocculation are four types of flocculation commonly in use.

Auto-flocculation usually cuts the flow of carbon dioxide (CO2) to the microalgae system when the pH of the culture is above 9, therefore the microalgae flocculate on its own (Vandamme et al., 2013). Although auto-flocculation can be a slow, unreliable process and also requires the presence of calcium and magnesium ions many researchers have published results of up to 90% microalgae recovery harvest (Baya et al., 2016; Gerardo et al., 2015; Milledge & Heaven, 2013; Ras et al., 2011).

Bio-flocculation is a technique in which microorganism are used in the treatment of wastewater. Such microorganisms include fungi and bacteria (Gerardo et al., 2015; Vandamme et al., 2013). Bio-flocculation is a technique yet to be fully comprehended but it is well documented that it improves the abilities of microalgae to form in suspension (Salim et al., 2011; Zhou et al., 2013). Zhou et al., (2013) reported almost 100% success harvesting microalgae cells of Chlorella vulgaris UMN235 by employing palletization-assisted bio-flocculaton. Their further recommended in their study that adding 20g/L glucose and of spores in BG-11 medium is desirable for palletization. Two locally isolated fungi being used as bio-flocculants are; Aspergillus sp. UMN F01 and Aspergillus sp. UMN F02. More so, a bio-flocculant from Bacillus licheniformis CGMCC 2876 was discovered to be an excellent harvester with 96% efficiency. This level of efficiency results from reduction in the negative charge of Desmodesmus sp. to about zero surface charge (Ndikubwimana et al., 2015). From the findings of the studies above, it can be clearly seen that bio-flocculation in conjunction with other techniques such as flotation and electrical approach can be used to solve the challenge of high quantity of bio-flocculants and time required to efficiently harvest high amount of microalgae using bio-flocculation.

Microalgae have low densities; this characteristic can be explored during harvesting using flotation method (Gerardo et al., 2015; Show et al., 2015). Air bubbles enhance the movement of microalgae particles upwards. Microalgae cells become hydrophobic when surfactant or coagulants are added into the system. Addition of surfactant or coagulants expands the mass transfer between the air and microalgae particles improving particles separation (Gerardo et al., 2015; Uduman et al., 2010). Some readily available surfactants in use are aluminum sulfate (Al2(SO4)3), iron (III) sulfate (Fe2(SO4)3), cetyltrimethylammonium bromide (CTAB), chitosan, and iron (III) chloride (FeCl3). High efficiency harvesting rates between 70 and 99% of microalgae biomass by flotation has been reported in some studies (Aulenbach et al., 2010; Barrut et al., 2012; Coward et al., 2014; Show et al., 2015). Flotation harvesting method requires low initial equipment cost and shorter period compared to others (Gerardo et al., 2015; Show et al., 2015).

The choice of surfactants and its effect on reusability of culture and biofuel manufacturing has only been reported by few researchers as of today. Recent studies have shown an increase in the production of biomass and support for the growth of C. vulgaris – an outstanding discovery using iron (III) chloride (FeCl3) (Farooq et al., 2015). The challenge with this process is that ferric acid which is a residue of the iron (III) chloride has a negative impact on the oxidative stability of biodiesel, upon completion of the harvesting process it has to be separated from the biomass.

It is worthy of mention that studies by Farooq et al., (2015); Kim et al., (2011); Kim et al., (2013) have implied that cytotoxicity from residual alum from the alum used as surfactant inhibits the growth of microalgae. Hence the effect of surfactants need to be properly studied and understood before harvesting microalgae to be used for production of biofuel and culture recyclability.

Other flotation techniques have been reported. Some of these are; dispersed air, micro-flotation, foam flotation, dissolved air, vacuum gas, electro-flotation, and ozone flotation. A comparison of these techniques is displayed in Table 1.

*NR – Not reported

Filtration is the separation of a solid-liquid mixture of microalgae using a semi-permeable membrane with small pores that allow the passage of the liquid but retains the solid microalgae (Gerardo et al., 2015; Show et al., 2015). Microalgae with low density such as Chlamydomonas sp., Chlorella sp. and Scenedesmus sp. can easy have their biomass harvested using filtration method (Gerardo et al., 2015; Rickman et al., 2012; Show et al., 2015). One problem with filtration however is that clogging and fouling brought by settled cells that can reduce the solid content due to low volumes of liquid that is able to pass through the filter used (Huang et al., 2012; Show et al., 2015).

Two filtration setups are common, which are; the dead-end and the tangential flow. Dead-end setup made up of cartridge filtration, horizontal filter press, belt filter and vacuum drum filter are carried out in batch modes. These methods can harvest 5-37% mean solid content. Cross-flow filtration which is an alternative name for tangential flow filtration was created to overcome the challenge of fouling and decrease the accumulation of cake layer such that the filtration time is accelerated (Gerardo et al., 2015). Shear movement (Morineau-Thomas et al., 2002; Nurra et al., 2014), back-flushing (Baerdemaeker et al., 2013), supplementation of coagulant (Hwang et al., 2013), and alteration of membrane surface (Baerdemaeker et al., 2013) have been reported by many studies as ways of minimizing membrane fouling. Table 2 is a summary of the outcome obtained from using each filtration method in microalgae biomass harvesting.

*NR – Not reported

Many scientific studies have been carried out to enhance microalgae biomass harvesting, which include reducing the energy requirement and operational cost.

Recent approaches being developed to harvest microalgae biomass include flocculation employing magnetic microparticles (Seo et al., 2015; Vergini et al., 2015), flocculation using natural biopolymer (Banerjee et al., 2014; Rahul et al., 2015), sedimentation involving polymers (Zheng et al., 2015), magnetic membrane filtration (Bilad et al., 2013) as well as electrical approaches which includes electro-coagulation-filtration (ECF) (Gao et al., 2010) and electrochemical harvesting (ECH) (Misra et al., 2015).