Use of Microalgae for Biofuel Production in the Northeast Region of Brazil,with Emphasis on Genus Botryococcus: A Review

Abstract

The search for renewable energy sources is one of today's most pressing challenges, and biofuels have emerged as a viable solution to meet demand and reduce dependence on fossil fuels. Brazil presents the necessary conditions to stand out as a producer and exporter of biofuels worldwide, due to the country's great biodiversity and favorable climate. Among its biomass sources, microalgae stands out for presenting several advantages, such as high productivity and possibility of cultivation anywhere. This work aims to address the advantages and disadvantages of biofuel production from microalgae focused on the genus Botryococcus, known for the formation of liquid hydrocarbons and high lipid content. The species of the genus have great biotechnological potential, but are little studied, especially B. terribilis, which has diverse distribution and is generally found in alkaline reservoirs, not only in Brazil, but in other tropical climate regions.

Introduction

One of the main causes of the world's energy crisis is the progressive lack of energy resources. Fossil fuel derivatives such as oil, gas, and coal are widely used, but these sources are finite. They are also responsible for much of the greenhouse gases emissions in atmosphere, which impact health and the environment. The main natural resource, water, is seriously compromised, as increasingly lower levels of reservoirs hinder operations of hydroelectric plants. The use of alternative energy sources, such as solar, wind, geothermal and biomass derivatives, are attractive options. Biodiesel produced from microalgae is an excellent possibility for the partial or even total replacement of fossil resources.¹ In the case of biofuels produced from microalgae, countries such as Brazil, Japan, China, and the United States are leaders.²

Microalgae are single-celled and microscopic organisms that live in aquatic environments and perform photosynthesis. They reproduce rapidly and generate large amounts of oil and biomass in a short time. In addition, the oils produced by some species contain valuable compounds such as Omega 3, eicosapentaenoic acid (EPA), and carotenoids. In this sense, the biomass produced by microalgae ends up being a promising renewable alternative, given its abundance, low production cost and ability to be transformed into biofuels and other by-products.³

The production of fuel from microalgae has several advantages in relation to yield, area availability and cultivation, even in wastewater. They have high caloric value, low viscosity and density—properties that make these organisms suitable for this purpose, unlike lignocellulosic materials.^4,5 In addition to the generation of by-products, biomass can be transformed into other energy sources after processing, such as bioethanol⁶ and biohydrogen.⁷

Brazil has 12% of the world's freshwater reserves and some of the largest hydrographic basins in the world—the Amazon, Paraná and São Francisco rivers, in addition to the Aquífero Guarani water reserve in territories of Brazil, Argentina, Paraguay and Uruguay.⁸ Due to climate disparities, fresh water is distributed irregularly, and globally unequally, requiring intervention strategies such as construction of reservoirs, excessive use of groundwater, and the import and transposition of water between watersheds.⁹ The semi-arid northeast region of Brazil has the lowest water availability, but at the beginning of 2021 the volumes stored by the equivalent reservoir was 36.2%, a value 16.6% higher than that observed at the beginning of 2020.¹⁰

Sá (2016) studied the biodiversity and distribution of chlorophyta and streptophyta in 47 shallow lakes, distributed in the semiarid (RS) and non-semiarid (RNS) regions of Pernambuco. Five phytoplankton groups were identified: Cyanobacteria; Chlorophyta and Streptophyta (both, chlorophytes); Euglenophyta (euglenophyceas); Bacillariophyta (diatomaceous) and Ochrophyta, including the family Botryococcaceae.¹¹ From this observation, it is possible to make inferences about the production of biofuels in this region, reducing environmental and social impacts, stimulating local economic development and generating jobs.

Among all microalgae already identified and cataloged as biofuel producers, the genus Botryococcus Kützing 1849, stands out. This genus includes colonial chlorophytic microalgae of global distribution in lakes and dystrophic reservoirs.¹² The microalgae of this genus are known for the production of kerogen, a raw material for the formation of petroleum.¹³ Among the different species, B. braunii and B. terribilis, stand out. Although they exhibit relatively slow growth, they produce considerable lipid content.

In light of these observations, the aim of this study is to present a report on the potential of microalgae of the genus Botryococcus with a focus on species B. terribilis as a source of biomass production for the synthesis of biofuels in the Northeast region of Brazil. This paper is a systematic review of the scientific literature published between 2010 and 2021 in metasynthesis format (of non-statistical basis). The review included articles, thesis, and other revisions. Journals indexed in Google Scholar, PubMed, SciELO, Science Direct and CAPES were considered. The descriptors used in the search for the articles were microalgae, Botryococcus, Botryococcus terribilis, biofuels and biodiesel.

The Brazilian Energy Matrix: Economic Aspects

Brazil's energy matrix is self-sufficient in oil volume production, but this self-sufficiency is not guaranteed in the long term because the relationship between oil reserves and current production is about twenty years. In fact, this self-sufficiency of oil was due to the replacement of gasoline by ethanol,¹⁴ which demonstrates the value of investments in alternative energy sources such as biofuels.

Brazil is the world's second largest producer and consumer of biofuels, which together with bioelectricity, make up 18% of national energy matrix, according to the Ministry of Mines and Energy of Brazil. According to the National Energy Balance, approximately 45% of energy and 18% of fuels consumed in Brazil are currently renewable.¹⁵

In January 2008, the mixture of the biodiesel content present in fossil diesel in Brazil was of 2% (B2) throughout the national territory. With the maturing of the Brazilian market, in March 2020 this percentage was successively increased by the National Energy Policy Council (CNPE) by 12% (B12), which results in the additional need for 600 million L of biodiesel. Consistent with the projections of the Ministry of Mines and Energy of Brazil, the addition of biofuel to diesel is expected to grow by 1% per year. Thus, in 2023 the mix will reach 15%, with investments of 3 to 4 billion reais in the productive sector.¹⁶

In 2019, Brazil produced 36.0 billion L of ethanol, reaching the highest production in history. This was divided into 25.3 billion L hydrated (10% increase) and 10.7 billion L anhydrous. In the same year, 5.9 billion L of biodiesel were consumed in Brazil, which increased the national mandatory demand for biodiesel from 2.5 to 3.8 billion L between 2011 and 2020.¹⁷ In 2020, 32.6 billion L of ethanol were produced, divided into 22.6 billion L of hydrous (10.6%) and 10.0 billion L of anhydrous (6.8% reduction). Thus, the total volume of ethanol produced was 9.5% lower than in 2019, slightly higher than in 2018. This is due to social distancing measures due to the Covid-19 pandemic and the increase in sugar prices.¹⁸

National mandatory demand for biodiesel in 2021 is expected to increase to 4.1 billion L and remain positive throughout the decade, with the maintenance of B15 and an estimated surplus of 5.0 billion L of installed capacity in 2021.¹⁹

Although estimates found in the literature indicate that it is possible to obtain higher biomass productivity from algae than from oilseed plants, implying high biodiesel productivity, most of these studies are presented at the laboratory level, on a small scale. However, many government institutions have invested resources seeking to equate this process through Research, Development and Technological Innovation projects.²⁰ An example is Petrobras, which in 2018 began research for the production of biofuels from microalgae. The company forecast two to three years for pilot scale, with a “semi-commercial” scale plant planned for some time later, with the purpose of evaluating production, followed by market launch strategy.

The use of microalgae in the production of aviation biokerosene (BioQAV) is also being researched in partnership between the Federal University of Rio de Janeiro (UFRJ) and the Federal University of Viçosa (UFV), in Minas Gerais, for the production of biofuel. The biggest challenge is to optimize the process in such a way that it ensures economic viability and positive energy balance.²¹

Use of Microalgae in Biofuel Production

In addition to ethanol, microalgae biomass is an alternative to traditional raw materials.²² It has several advantages, although some disadvantages should be considered when it relates to large-scale production (Table 1).^{23

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Table 1.

Advantages and Disadvantages in the Production of Microalgae Biofuels

ADVANTAGES	DISADVANTAGES	REFERENCES
Reduction in CO₂ emissions into the atmosphere.	High cost for harvesting and isolating products.	23, 24
Higher yield and short growth cycle when compared to oilseeds.	Risk of colonization of invasive species and contamination by pathogens.	25, 26
Non-occupation of land for planting other foods. They can be grown in various places such as lakes, bioreactors, building surface, seawater, rivers, culture medium even in wastewater and effluents such as sewage.	Complexity of biomass recuperation with high energy expenditure. Growth rate and biochemical compositions affected by environmental conditions such as temperature nutrients, pH, light, salinity, and pollution, depending on the species.	25, 27, 28 25, 29, 30, 31
Obtaining by-products that can be processed. High caloric value, viscosity, and density more suitable than lignocellulosic materials.		6, 7, 32

The commercial success of microalgae requires development of strains and conditions for culture that allow rapid production of biomass with high lipid content and minimum growth of competing lines, avoiding contamination by pathogens.²⁶ Additionally, the harvesting and purification of its products are bottlenecks in obtaining biofuels. Generally, harvesting methods include chemical, physical, biological, and magnetic, such as gravity sedimentation, centrifugation, filtration and flocculation,³³ which can be used individually or in combination.³⁴

Strategies in the Use and Production of Biofuels from Microalgae

In obtaining biofuels from microalgae, it is necessary to develop economic strategies to obtain products that are competitive in terms of cost. Currently, biofuels produced by microalgae are not competitive when compared to fossil fuel production prices.³⁵ Although they can achieve high biomass yield per unit area, the growth rate and chemical compositions are significantly affected by environmental conditions.³¹

The use of low-cost medium of cultivation or reuse of medium, optimization of biomass production and collection conditions, stoichiometry to shift the balance to the target product, proper commercialization, and reduction of operational cost (mainly associated with the stages of cultivation and harvesting) are focused efforts of microalgae research.³⁶

The reuse of culture medium is a promising route to improve economic viability. The cultivation of Spirulina sp. in reused Zarrouk medium allowed the production of biomass with reduced cost and differentiated characteristics, allowing the exploration of commercially important biomolecules by performing up to four cycles.³⁷

The harvesting stage is another bottleneck that contributes to the increase in energy and financial expenditure, and it may also influence the bioactivity of the final product, which is important for pharmaceutical, cosmetic and food industries. Ramos and collaborators (2020) evaluated whether biomass collection by electroflocculation affected the toxicity of extracts of marine microalgae Isochrysis galbana and Phaeodactylum tricornutum and its antioxidant activity compared to the traditional centrifugation collection approach,³³ showing good results with the most economical method.³⁸

Even with these strategies, there are still challenges that need to be overcome to ensure stable production on a large scale, with positive energy balance in the production of biofuel in all six stages of the process, which include the selection of strains, cultivation, biomass harvesting, drying, lipid extraction and transesterification.³⁹

Recently, a microalgae biomass production technology was proposed coupled with wastewater purification.⁴⁰ This technology has high production potential due to the rapid growth rate of microalgae,⁴¹ CO₂ fixation with consequent reduction of the greenhouse effect, efficient removal of pollutants in wastewater (industrial), breweries,⁴² domestic,⁴³ and transformation of these into valuable products from biomass.⁴⁴ This technology has been widely described as efficient for nutrient removal as well as economical and sustainable, as it avoids secondary pollution. Its products have different applications, such as biofuels, bioplastics, biofertilizers, biocoal, animal feed, and aquaculture, among others.⁴⁵

Genus Botryococcus

The genus Botryococcus is formed by colonial microorganisms found in freshwater.⁴⁶ Considering the species of this genus, two stand out, B. braunii Kützing and B. terribilis J. Komákev & P. Marvan. Both have considerable lipid production, but relatively slow growth. A comparison of B. braunii and B. terribilis morphologies, distributions, lipid and biomass yields and carbon fixation rates can be verified in Table 2.^{47

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Table 2.

Morphology, Distribution, Biomass Yield and Lipid Content Produced by the Species B. braunii and B. terribilis

CHARACTERISTIC	Botryococcus braunii	Botryococcus terribilis	REFERENCE
Morphology	Dense colonies, 125.0–155.0 μm in their largest diameter, formed by sub colonies, 52.5–80.0 μm in diameter, interconnected by mucilaginous cords. Obovoid cells, 11.2–15.0 x 7.5–12.5 μm, emerging from the colony at up to 1/3 of its length. Presence of hyaline mucilaginous helmet on the apic parts in some cells. Single parietal chloroplast. Reproduction by 2 elongated autospores	Rounded, irregular colonies, formed by sub colonies, 35.7–157.0 μm diameter, interconnected by mucilaginous cords, with densely arranged cells arranged radially at the periphery of the mucilage. Colonial mucilage with irregular projections. Elongated cells, obovoid, 8.0–10.0 x 5.0–9.0μm, apexes fully covered by mucilage. Single chloroplast, parietal. Reproduction by 2(4) autospores.	47
Distribution	Reservoirs and lakes, it has worldwide distribution in the most varied types of climates, as in Lagoa do Caixão Salvador-Bahia	It presents worldwide distribution in the most varied types of climates. From Lagoa do Caixão Salvador Bahia to the Area of the Forest Steppes of Ukraine, and on Lake Naivasha in the African Rift Valley	12, 47, 48, 49, 50
Lipid yield	61.38 mg/L/day112.43 mg/L/day455 mg g⁻¹ dwt of total lipids and 59.9 mg g⁻¹ dwt of fatty acids74.7 ± 1.38 mg L⁻¹ d⁻¹ with 10% CO₂ supplementig30.2 ± 5.1 (%)	98.00 mg/L/day490mg g⁻¹ dwt of total lipid and total fatty acids 16.7mg g⁻¹dwt73.4 ± 0.92 mg L⁻¹ d⁻¹ supplementing 10% CO₂ 37.0 ± 5.3 (%)	51 52 53 54 55
Biomass productivity	87.96 (%) ^a0.235 ± 0.005 g L⁻¹ d⁻¹ when supplemented with 10% CO₂ ^b0.25 Pdwt (g L⁻¹ d⁻¹)Total concentration in dry biomass in lipids 30.1 ± 5.0 mg L⁻¹ d⁻¹ Biomass productivity was observed using wastewater 0.10–0.28 g L⁻¹ d⁻¹, and a lower value 0.13–0.22 g L⁻¹ d⁻¹ was the highest using synthetic medium	^a0.244 ± 0.004 g L⁻¹ d⁻¹ when supplemented with 10% CO₂ ^b0.20 Pdwt (g L⁻¹ d⁻¹)Total concentration in dry biomass in lipids 15.1 ± 0.1 mg L⁻¹ d⁻¹ Biomass productivity 0.10–0.18 g L⁻¹ d⁻¹ using synthetic medium.	51 54 52 55 56
Biofixation rate	496.98 mg L⁻¹ d⁻¹ in fermented cultivation0.555 ± 0.012 gL⁻¹ d⁻¹ with supplementation of 10% CO₂	0.615 ± 0.010 gL⁻¹ d⁻¹, with supplementation to 10% CO₂.	51 54

Biomass calculated at the end of the exponential phase and the initial values of biomass were B. braunii 0.084 g L⁻¹ d⁻¹ and B. terribilis 0.083 g L⁻¹ d ⁻¹; ^bPdwt: Productivity of dry biomass produced in grams per liter.

B. braunii hydrocarbon biofuel is potentially suitable for industrial production, however, it is still far from commercially viable,⁵⁷ mainly due to its relatively slow growth rate, which makes it difficult to control mass cultivation in open tanks and leads to competition with other fast-growing microalgae.⁵⁸ Furthermore, there is no clear understanding of the anabolic pathways of hydrocarbons, restricting large-scale cultivation and the application of this species as a raw material for high-yield hydrocarbons.⁵⁹

Al-Hothaly evaluated the harvest of microalgae B. braunii using Aspergillus sp. in large-scale studies, and no damage to microalgae biomass (validated by pyrolysis) was observed.⁶⁰ Some filamentous fungi can form pellets when grown in solution alone or with microalgae naturally, without any chemical inducement. These pellets will sink to the bottom of the growth medium and are therefore easily harvested. When introduced into a microalgae culture, these fungal species will trap the microalgae, forming pellets that settle at the bottom of the growth tank, as occurred with Aspergillus sp, in the harvest of B. braunii. ⁶⁰

Microalgal cell walls are composed mainly of polysaccharides and some other elements such as proteins, biopolymers, and calcified structures.⁶¹ Due to this constitution, the production of biofuels and bioproducts from these microalgae requires pretreatment increase the digestibility of the cell wall.⁶² In this sense, a non-destructive lipid extraction method that eliminates the stages of biomass harvesting and drying is suggested. This would decrease the cost of the process, since it considers the type and concentration of solvent, extraction time and agitation. It can be verified that the solvent that presented the lowest toxicity to microalgae was octane.⁶³

Although it has been observed that B. braunii has high lipid production, it is not recommended for growth in wastewater, due to the slow growth rate related to the fact that it did not consume all the available nitrogen.⁶⁴ B. terribilis also showed a low nutrient removal rate (ammoniacal nitrogen and phosphate) comparable to other evaluated species,⁶⁵ but produces fatty acids C12, C16, C18 and C20,⁶⁶ which are important in biodiesel production. B. terribilis biofuel has the highest oxidation stability, higher CN (59.50) and lower iodine values (66.90 g I₂/100 g) with cold filter obstruction point of -10.26°C,⁶⁷ and is promising for production of byproducts such as glycerol, obtained from biodiesel.⁶⁸ Such characteristics not found in other species commonly used for biodiesel production.

Genetic engineering is another strategy for improving lipid accumulation and yield to obtain C12 fatty acids with the greatest potential for biofuel production. This technique can be used to increase the yield in the production of saturated short chain fatty acids (C16–C18), which is one of the main factors necessary to obtain biofuel of optimal quality.⁶⁹

Conclusion

With current demand for new sustainable energy sources, biofuels have emerged as likely successors to fossil fuels in the global energy matrix. Accordingly, it is necessary to carry out studies and develop new technologies that focus on increasing biomass productivity and reducing costs associated with production to ensure energy efficiency and its competitiveness in the market.

Brazil has great potential to become a major global producer and exporter of biofuels due to its favorable conditions for biomass cultivation and production. The northeast region presents potentially adequate climatic conditions, with mild temperatures and sun in abundance, as well as great diversity of microalgae species from freshwater environments with potential for biofuel production. This presents a very promising socioeconomic possibility, and the development of biofuels production would generate jobs, strengthening the local economy.

Among the main sources of biomass for biofuel generation, microalgae stand out for presenting several advantages, such as not competing for space with food cultivation, higher energy conversion rate and high lipid content. The genus Botryococcus may be a viable alternative, as it produces and stores large amounts of oil and presents high adaptability to various types of adverse conditions and various culture media. Another advantage is the possibility of cultivation in open and closed systems, which enables large-scale production.

For cultivation in the Northeast, the species Botryococcus terribilis is an option that possesses the characteristics inherent to the genus that allow its adaptation to cultivation anywhere. Despite all these benefits, however, its commercial use for biomass production is not a reality yet due to lack of research and investments by industries and academia.

This work aims to stimulate the use of microalgae as an energy source in the production of biofuels, focusing on B. terribilis, due to the arguments addressed.

Footnotes

Author Disclosure Statement

No competing financial interests exist.

Funding Information

No funding information to report.

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