Bio-Nanoparticles Mediated Transesterification of Algal Biomass

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This entry is adapted from the peer-reviewed paper 10.3390/su16010295

Immense use of fossil fuels leads to various environmental issues, including greenhouse gas emissions, reduced oil reserves, increased energy costs, global climate changes, etc. These challenges can be tackled by using alternative renewable fuels such as biodiesel. Many studies reported that biodiesel production from microalgae biomass is an environment-friendly and energy-efficient approach, with significantly improved fuel quality in terms of density, calorific value and viscosity. Biodiesel is produced using the transesterification process and the most sustainable method is utilizing enzymes for transesterification. Lipase is an enzyme with excellent catalytic activity, specificity, enantio-selectivity, compatibility and stability and hence it is applied in microalgae biodiesel production. But, difficulty in enzymatic recovery, high enzyme cost and minimal reaction rate are some of its drawbacks that have to be addressed. In this aspect, the nanotechnological approach of lipase immobilization in producing microalgae biodiesel is a promising way to increase production yield and it is due to the adsorption efficiency, economic benefit, recyclability, crystallinity, durability, stability, environmental friendliness and catalytic performance of the bio-nanoparticles used. Through increasing post-harvest biomass yield, absorption of CO₂ and photosynthesis in the photobioreactor, the use of nanoparticle immobilized lipase during the generation of biodiesel from microalgae has the potential to also remove feedstock availability constraints.

biofuel nanocatalyst algal biomass immobilized renewable sustainable lipase

1. Introduction

Biodiesel generated from microalgae is a potential source of energy ^[1] and it shows an increased cetane count, a flash point, high lubricant, combustion efficiency, reduced exhaust generation, biodegradability, nitrogen oxides, positive energy balance, prevention of a sulfur shortage, renewable energy infrastructure and domestic origin ^[2]^[3]. Using microalgae for biodiesel production is advantageous due to its ease of culturing in waste-piled areas, rivers, ponds, seas, humid wastelands and municipal and industrial waste drainage ^[4]. Its growth does not overlap or interrupt the animal and human food chains to cause potential food sustainability or safety issues ^[5]^[6]. It has the capability of fixing and sequestering carbon content derived from carbon dioxide present in air and exhausted emission ^[7]^[8]. Moreover, many species of microalgae have significant oil content; under ideal conditions, some species can have oil content as high as 75% of their dry mass. One of the potential algae genera with significant lipid content is the Scenedesmus genus. It also possesses a significant capacity to remove nutrients during wastewater treatment and a resistance to contamination. These qualities make it a desirable input for the production of biodiesel ^[1].

The most popular and affordable method for producing biodiesel using an alkali, an acid, or an enzyme as a catalyst is transesterification, where the triglycerides and primary alcohol are combined in the presence of a catalyst ^[9]. Because of high activity under reaction conditions, catalysts like KOH/NaOH are widely utilized for producing biodiesel. However, separating a catalyst can be challenging, and a significant quantity of wastewater is produced ^[10]. Using enzymes is one of the sustainable techniques and it is due to their biocompatibility, specificity, biodegradability and environmental acceptability. The enzymatic transesterification process is more advantageous with a lower energy requirement and minimal environmental adverse effects ^[11].

One of the enzymes, lipase, shows enhanced stability, specificity, enantio-selectivity, catalytic effect and region selectivity and hence it is more suitable for the enzymatic transesterification process in biodiesel production ^[12]. Additional benefits of using lipase include decreased consumption of energy during the reaction due to mild reaction conditions and low temperatures, absence of soap development in the process, minimal or no side reactions, reduced sensitivity to moisture and fatty acid (FA), ease of recovering the fuel produced, compatibility with the environment and reduced alcohol consumption during the esterification reaction ^[11].

Even while the enzymatic transesterification process and lipase provide numerous advantages for the production of biodiesel, the disadvantages should also be taken into consideration. It should be noted that carrying out enzymatic transesterification is challenging, mostly because of issues in recovery of the enzyme, sustained instability of its functioning, cost and alcohol-induced inactivation of the enzyme. Furthermore, lipases may exhibit improper enzymatic activity. Additionally, the structure and activity of lipase are altered in the aqueous phase. Also, the interfacial area, which is governed by the substrate’s moisture content, is what determines lipase’s catalytic activity ^[13]. Enzyme immobilization is developed as a solution to the drawbacks of enzymatic transesterification. It also aimed to increase reaction control, prevent contamination of the product by the enzyme, enhance catalytic stability and allow for the use of different reactor topologies. It can also be used to modify enzyme activity, selectivity and specificity, reduce enzyme inhibitions or couple with purification of enzymes ^[14]. Also, in comparison with the chemical catalysts, lipase-immobilized nanoparticles are versatile and economical in terms of cost-effectiveness and reusable nature ^[15].

The IEA World Energy Outlook 2020 reported that biodiesel production volume in 2018 reached 154 billion liters and is predicted to increase by 25% in 2024 ^[16]. A bar chart representing the production of biodiesel from 2019 to 2025 is shown in Figure 1 ^[17]. However, despite the increased biofuel production volume, they are still not enough to meet the global energy need, and also developing sustainable low-energy biofuel production technology is a need globally ^[8]^[18]. Hence, researchers have utilized nanotechnology to produce microalgae biodiesel using the enzymatic transesterification process with improved yield and quality at a reduced cost.

Figure 1. Production of biodiesel from the year 2019 to 2025, as reported by the international energy agency.

2. Microalgae Biodiesel Production

The production of biodiesel from microalgae biomass involves several processes: microalgae strain selection, microalgae cultivation, harvesting, oil extraction and transesterification ^[19]. Microalgae strains may be marine- or fresh-water-based and marine strains are preferred more due to their wide occurrence. Microalgae such as Chlorella vulgaris ^[20], Chlorella emersonii ^[21], Chlorella sorokiniana ^[22], Schizochytrium acidic limacinum ^[23], C. protothecoides ^[24], Chlorella vulgaris ESP-31 ^[22], Spirulina, Thalassiosira, Nannochloropsis, Cyclotella, Scenedesmus ^[25], etc., are employed for generating biodiesel. A few characteristics determine the selection of microalgae strains: greater capacity for producing lipids; resilience to stress in photobioreactors and open raceway ponds; competent with the wild type of strains in the event that an open pond system is employed; increased absorption of carbon dioxide; robustness; ability to tolerate limited nutrient supply; tolerance to seasonal variations in temperature; capacity to generate co-products; increased productivity; increased photosynthetic effect; and the distinctive self-flocculation feature. Among the investigated strains, Schizochytrium sp., Nannochloropsis sp. and Spirulina sp. are commonly used to generate biomass to produce biofuels, with Chlorella vulgaris as the most suitable strain and it is due to the presence of high lipid content ^[26].

Microalgae can be cultivated in open systems such as ponds, tanks and raceway ponds, closed systems and hybrid systems that utilize characteristics of both systems. All these systems have their advantages and disadvantages that can be compared based on various parameters. Open ponds have minimal capital and operating costs with low or minimum energy requirements for culture mixing. Open systems have various disadvantages, such as high contamination risk, difficulty in scaling up, more susceptibility to adverse weather conditions, lack of agitation in these systems and low biomass production yield, whereas closed cultivation systems such as photobioreactors are more efficient in terms of quality due to highly controlled conditions during operation ^[27]. However, bio-fouling and overheating along with high build-up of dissolved oxygen result in growth limitation ^[28]. In order to overcome the challenges faced in a photobioreactor, a hybrid cultivation method is developed. This method involves two cultivation stages, aiming to combine the advantages of open ponds and photobioreactor systems ^[29]. In a study, the hybrid cultivation system is developed by combining a baffled reactor with a photobioreactor to cultivate Chlorella vulgaris, Scenedesmus simris and Chlorella sorokiniana in the wastewater system and it produced a biomass with lipid content varying from 44.1 to 87.9% ^[30].

In one of the comparative studies using marine microalga Tetraselmis sp. M8, cultivation is carried out in a photobioreactor, open raceway pond and two-stage hybrid system. The two-stage hybrid system has resulted in exponential biomass in the first tank and separate synchronized lipid stimulation in nutrient-limiting conditions. The comparative account is carried out based on growth, harvesting cycles and maximum lipid accumulation. Three harvesting cycles with 2 × 10⁻⁶ cells/mL on the 28th and 29th day are possible in the photobioreactor and open raceway pond whereas six harvesting cycles with 3 × 10⁻⁶ cells/mL are achieved with two-stage hybrid systems. The growth rate (µ) is the maximum in the case of the hybrid system (0.17) as compared to the raceway pond (0.10) and photobioreactor (0.11). Thus, maximum biomass productivity and lipid accumulation have been achieved in a hybrid system (14 g/m⁻²/D⁻¹) and maximum lipid accumulation ^[31].

Product value, density and size are among the parameters taken into account when choosing the harvesting technique. The harvesting procedure accounts for 20–30% of the total production costs ^[32]. The physical harvesting method includes gravity sedimentation, centrifugation, membrane filtration and flotation approaches. However, it also has disadvantages such as a higher energy consumption and higher cost of facility implantation ^[33]. For the flotation process to work, algae cells must be trapped. Although centrifugal sedimentation is a quick method for harvesting algal biomass, it has the drawback of requiring maintenance ^[34]. Microalgae such as Spirulina and Coelastrum are ideal for filtration ^[35]. In the harvesting process, liquids are pumped out, homogenized and dewatered as well. Harvested biomass must be dried or dehydrated since it perishes quickly. Algal biomass can be dried using a variety of techniques, including drum drying, sun drying, fluidized bed drying, spray drying and freeze drying (lyophilization). Sun drying is the least expensive drying technique; however, it has drawbacks, such as a lengthy drying period. According to ^[36], freeze drying is the most costly drying process when used on a wide scale. Flocculation and coagulation are typical chemical harvesting methods. Chemicals like FeCl₃, Fe₂(SO₄)₃ or Al₂(SO₄)₃ must be added for flocculation. The disadvantages of chemical harvesting methods are sludge formation, biomass toxicity and low lipid content ^[37]. Autoflocculation and bioflocculation are good examples of biological methods of harvesting microalgae ^[38]^[39]. Compared with the above chemical and physical harvesting methods, this approach is cost-efficient and environmentally viable to generate pure harvested biomass ^[40], but the biological approach requires a high energy input ^[41].

After harvesting the algal biomass, various chemical and mechanical procedures are employed to extract oil. The methods of extraction include solvent extraction, enzymatic extraction, hot oil extraction, mechanical extraction, ultrasonic aided extraction and supercritical fluid extraction ^[42]. Although it is useful to use enzymes in the oil extraction process to obtain fractional chemicals in the downstream phase, cost is a disadvantage. The solvent extraction approach is not nearly as effective as the supercritical fluid extraction method. The supercritical fluid extraction technique that involves liquefying CO₂ under pressure yields an extract that is highly pure. The extraction of algae oil employs the stripper column approach ^[43]. About 60–70% of the oil can be extracted using the solvent extraction method, which uses cyclohexane, acetone, benzene, hexane and chloroform as the solvents. The solvent used to extract oil is very expensive. According to ^[42], the ultrasonic approach depends on the process of cavitation, which requires very little duration and extracts 76–77% of the oil. These techniques are associated with high energy consumption, significant environmental toxicity and low extraction efficiency ^[44].

Transesterification is an important process where the microalgae biomass’s glycerol-based ester is converted to fatty acid methyl ester. Triglycerides are an ester with a higher lipid content that produces a large amount of biodiesel. If there are any ketones, polar lipids or pigments in the algae oil, the transesterification process may be affected. In addition to using methanol as the alcohol, the transesterification process also makes use of a catalyst (KOH/NaOH). Biodiesel yielded from freshwater algal cultivation is around 400 kg kg⁻¹ ^[45]. The three variables that impact the transesterification process are inhibitors, humidity and triglycerides. According to ^[46], a two-step transesterification procedure is advised to prevent saponification reactions. Methanol and sulfuric acid must be used in the pretreatment procedure if the amount of FFA in the algal oil is more than 2% ^[47]. Transesterification using enzymes is more viable and specifically immobilized enzymes increase the sustainability of biodiesel productivity ^[48].

3. Effect of Nanoparticles on Microalgae Biodiesel Production

A microalgae culture system is enhanced with nanomaterials that boost microalgal growth and cause lipid accumulation ^[49]^[50]. Furthermore, the use of the nanoparticle during biodiesel production from microalgae has the capacity to eliminate the restrictions associated with the availability of feedstock by improving the post-harvest biomass yield and CO₂ absorption and enhancing photosynthesis in the photobioreactor ^[51]. The technological scheme for the production of biodiesel using bio-nanoparticles ^[35]^[52] is depicted in Figure 2 and the different types of nanoparticles used for microalgae biodiesel production ^[53]^[54]^[55]^[56] are depicted in Figure 3. In the sections that follow, the usefulness of nanotechnology in lipid accumulation, transesterification, algal culture and extraction has been evaluated.

Figure 2. Technological scheme for the production of biodiesel using bio-nanoparticles.

Figure 3. Different types of nanoparticles used for microalgae biodiesel production.

4. Enzyme Immobilization and Nanotechnology-Driven Microalgae Biodiesel Production

The process of encapsulating enzymes in supportive materials to maintain their stabilities and reusing capacities is known as enzyme immobilization ^[57]. This method helps with downstream processing and offers a complete separation of the enzymes from the reaction mixture ^[58]. Different nanomaterials have attracted a lot of attention as vehicles for enzyme immobilization ^[59]. One of the many advantages that nanoparticles offer as immobilization carriers is their significantly higher surface areas when compared to conventional materials for enzyme immobilization. The activity, stability and reusability of enzymes can be increased by using nanoimmobilized enzyme systems, which could result in a significant decrease in the expenses related to their use ^[60]. Nanoparticles are efficient in minimizing the potential steric hindrances and protein structure distortion, while increasing the contact surface area to maximize the enzyme loading capacity and mass transfer rate ^[61]. In microalgae biodiesel production using enzyme immobilization in nanoparticles, the nanoparticles overcome the challenges in biodiesel production in the absence of nanoparticles and they are shown in Figure 4.

Figure 4. Importance of nanotechnology-driven enzyme-immobilized microalgae biodiesel.

Lipases, which are the triacylglycerol acylhydrolases, function as biocatalysts that exhibit high stability and reactivity when exposed to organic solvents. They can react with a variety of substrates including esters of FAs, synthetic oils, triacylglycerides, lipids, natural oils, etc. Aspergillus, Fusarium, Mucor, Pseudomonas, Rhodotorula, Candida, Geotrichum, Penicillium and Rhizopus are among the species that are frequently listed as good producers and constitute 50% of the commercial volume of lipases ^[11]^[62]^[63]. The hydrophobicity of the nanoparticle is a characteristic that helps with both the immobilization of lipase and the synthesis of biodiesel ^[64]. Because lipases naturally bind to hydrophobic surfaces, their immobilization on nanoparticles is distinct from that of enzymes that adsorb hydrophobically. Moreover, hydrophobic triacylglycerols and fatty acid esters have a strong attraction for hydrophobic supports. It has little affinity for glycerol, which promotes the interaction of lipases with the nanoparticles and greatly inhibits glycerol adsorption ^[65]^[66].

5. Effect of Nanoparticle-Immobilized Lipase Enzyme on Biodiesel Production

Compared with lipases in the original form, the immobilized lipases showed enhanced pH tolerance, functional stability, substrate specificity and thermal stability. This ultimately increases biofuel production yield ^[50]^[67]. To date, a wide range of nanoparticles have been investigated for this purpose, including nanosilicones, nano-metal particles, carbon nanotubes, metal–organic frameworks, magnetic nanoparticles, nanoflowers and nanofibers ^[68]. The nano-immobilized lipases showed the superiority of an increased surface area, enhanced stability and eased separation, compared with the lipase immobilized using conventional supporting materials ^[69]. Furthermore, a high reactant diffusion rate can be provided to the lipase enzyme’s active areas because of the nanomaterials’ tiny pore sizes. The factors that render nanoparticles suitable for lipase immobilization in microalgae biofuel production are shown in Figure 5.

Figure 5. Factors rendering nanoparticles ideal for enzyme immobilization in microalgae biodiesel production.

Magnetic nanoparticles can offer a great substitute that is practical and affordable due to their enhanced thermostability and ease of recovery from reaction mixtures ^[70]. T. lanuginosus lipase covalently attached to aminofunctionalized magnetic nanoparticles produced 90% biodiesel conversion efficiency in one study ^[71]. A 100% success rate in turning oil into biodiesel using P. cepacia lipase immobilized on Fe₃O₄ magnetic nanoparticles is reported ^[72]. Lipases immobilized by magnetic nanoparticles are able to be detached from the reaction medium and are reusable, further reducing the biofuel production cost ^[73]. The conversion efficiency is increased by four times compared to the free lipase ^[74]. The magnetite-Fe₃O₄ and maghemite-Fe₂O₃ are widely employed for immobilization ^[75]. The magnetic support modifications are required to enhance the interaction between these nanoparticles and target lipase. In a study, amino silanes are added to the Fe₃O₄ nanoparticle to improve the lipase loading capacity ^[76]. The lipase can even be cross-linked with the incorporated amino acids in the nanoparticle using glutaraldehyde to maximize binding ^[77]. The silica-based nanoparticles are preferred more by the industry due to their biocompatible and non-toxic nature ^[78]. The silanol group can be attached on silica gel to facilitate further modifications such as the addition of functional groups such as 3–Glycidyloxypropyl trimethoxysilane to improve the immobilization efficiency ^[79]. In another study, lipase is immobilized on mesostructured onion-like silica via crosslinking using glutaraldehyde to produce biodiesel. This immobilization increased the reusability of the enzyme by 23 times ^[80]. Additionally, lipase is immobilized on silica aerogels and attained a 93% conversion rate ^[81]. Metal–organic-framework nanoparticles like zeolitic imidazolate framework-8 significantly improved the stability and reusability of lipase ^[82]^[83]. Carbon nanotubes are well-suited for the immobilization of lipase because of their higher mechanical properties ^[84]. Hybrid nanoflowers are developed to immobilize lipase for biodiesel production. The high production yield results suggested that it had economic feasibility ^[85]. Organic polymers are a good choice of supporting material for enzyme immobilization since the carboxyl, epoxy, hydroxyl or amino groups facilitate covalent attachment and modification. For example, the polyacrylamide-nanogel-encapsulated lipase has shown remarkable effectiveness in the synthesis of biodiesel. This allowed the lipase that has been rendered immobile to be placed at the oil–water interaction by the amphiphilic polymer shell of the nanogel. In addition, the polymer outer shell’s presence increased the lipase stability at high temperatures and methanol concentrations ^[86].

6. Reuse and Recovery of Lipase Nanoparticles

The two biggest advantages of using lipase nanoparticles in the biodiesel generation are the recoverability and reusability ^[87]^[88]. The lipase nanoparticles are used in repeated cycles to generate biodiesel, and the recovery process is performed at each level. Chemical techniques are often applied for lipase nanoparticle recovery. Heterogeneous catalysts facilitate the rapid and effortless recovery of both primary and secondary products. In this mode of the catalyst, the washing step is not required. The esterification process using nanoparticles has been shown to have benefits such as rapid separation and faster reactant–catalyst mixing ^[89]^[90]^[91]. Easy enzyme recovery is made possible by an external magnetic field, and prior research has demonstrated that there is multiple-time recovery without significantly lowering the catalytic effect or biodiesel yield ^[52].

7. Challenges in Large-Scale Commercialization and the Proposed Strategies to Overcome

The challenges for commercialization include both environmental and technological barriers. Environmental barriers can be the usage of land and water, emission of greenhouse gas and loss of biodiversity ^[92]. Sufficient land and water are required for scaling up of microalgae biodiesel production. The effects of algal biomass production on water stress varied significantly amongst the possible sites because of regional variations in water stress. Proper planning is necessary to increase the water efficiency of large-scale algal biodiesel production. Water stress effects become quite severe when several sites are placed in highly stressed water zones, even though choosing locations according to the yield of biomass ranking could enhance economic feasibility. Water usage can be decreased by ranking sites according to Water Use Efficiency; however, the biomass production will be reduced (~25%). On the other side, the Available Water Remaining-US hotspot avoidance method could greatly lessen the effects of water stress without negatively impacting biomass yield. In contrast to the biomass yield ranking that ignores regional water stress, avoiding locations situated within elevated water stress zones could lower the water scarcity footprint for a long-term biodiesel production aim. AWARE-US might direct the geographical planning of energy system deployment and assess the effects of microalgal biofuel systems on water stress ^[93]. Future techno-economic assessment and life cycle assessment (LCA) studies should be carried out for envisioned commercial systems in order to confirm earlier findings based on lab- or pilot-scale experiments and to more effectively demonstrate and defend the choice of a specific production process as the best option for a given location.

Even if the use of nanoparticles in microalgae culturing, harvesting and transesterification improved efficiency, there are obstacles to overcome prior to the usage of nanoparticles in the commercial strategy. Particle size, shape, size distribution and clustering of the majority of the nanoparticles from experimental study are not well defined ^[94]. Thorough research on characterizing nanoparticles well is necessary prior to large-scale implementation. For maximum productivity, consideration should be given to the selection of appropriate nanoparticles, preparation techniques and time for the chosen application. Before being used, the impact of nanocatalyst implementation on engine performance, gas emission and the quality of microalgae-biofuel combustion should be thoroughly investigated and understood. Accordingly, even while nanoparticles are sufficiently available for laboratory use, their large-scale availability may provide a problem for mass use. Another barrier is the cost-effectiveness of nanocatalysts for industrial use, which could hurt business prospects because many of them are highly costly. Thus, as the economic problem is an important factor for large-scale plant installation, a complete techno-economic study is necessary to analyze whether the nanoparticles used on microalgae biodiesel are economically feasible or not. Additionally, these applications will benefit the environment in the near future by generating value-added co-products. Even if the use of nanoparticles has been shown to be environmentally beneficial, a thorough LCA is mandatory to demonstrate the benefits to the environment. Prior to commercialization, a thorough analysis of public safety, the effect on flora and fauna and the potential for biohazards is also required ^[52].

The impacts of microalgae are not limited to natural resources but also biodiversity. Microalgae cultivation poses a threat to biodiversity and the ecosystem. Large-scale microalgae deployment necessitates effective management and control of the cultivation process ^[95]. Therefore, dependence on native plant species instead of invading species that could jeopardize biodiversity for the generation of biofuel may be a wiser decision. However, large-scale microalgae farming may result in an overabundance of nutrients in the aquatic environment, endangering biodiversity ^[92].

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