ENVIRONMENTAL, SCIENCES AND PRACTICES

Introduction

Currently, fossil fuels are one of the main factors of various environmental problems, this resource obtained from the natural decomposition of organic matter is depleting day by day, causing the prices of oil reserves to rise, making it difficult to access them. A viable option to avoid these problems of depletion is the alternative of elaborating new fuels by means of other compounds. 

Alternative methods and compounds for the production of fuels mainly involve reducing the environmental impact and being able to prevent the scarcity of fossil fuel reserves. Microalgae is one of the fundamental alternatives to the production of biodiesel as well as for obtaining other energy products such as bioethanol, biomethane, and biohydrogen (Heredia et al. 2019) The present work, aims to analyze the importance of obtaining biofuels through the microalgae Chlorella vulgaris as raw material, in order to give a better ideology to the different methods and sources for obtaining renewable fuels that do not affect the environment.

In addition, they can be used in areas unsuitable for traditional cultivation and achieve high growth rates (0.5 to 1.2 d^-1). However, many microalgae cultivation technologies involve the use of uncontaminated water, fertilizers, and CO₂ injection for growth, which raises the cost of algal biomass production and reduces its attractiveness as a technology. To minimize these drawbacks, one cultivation alternative is to use municipal, agricultural, and livestock wastewater, where microalgae can grow by taking advantage of the nutrients in these types of discharges. This integration allows: 

1. Treat wastewater.
2. Obtain a high-quality effluent.
3. Generate useful algal biomass to produce biodiesel or other types of biofuels.

Therefore, algae are photoautotrophic organisms and present a rapid growth in a short time, which allows them to be cultivated in this type of water; since the absorption of nutrients by microalgae uses a high content of nitrogen, silicon, phosphate, and sulphate from human or animal waste; in addition, they can retain carbon dioxide (CO₂) from industrial sources. Thus, these reasons support the research on microalgae Chlorella vulgaris since obtaining them is not an obstacle.

In a study carried out in 2006 by the Mexican Ministry of Energy (SENER), it is mentioned that the production of biodiesel on a commercial scale can be feasible in the medium term if comprehensive actions are carried out, including technical, economic, and environmental aspects with the agricultural and agro-industrial sector, combining efforts in research and technological development. They allude that Mexico requires 10 industrial plants with a capacity of 100,000 tons/year each, just to replace 5% of petroleum diesel used each year and recommend that the production and processing be carried out with technologies designed and built in the country itself. They also argue that the use of biodiesel would reduce hydrocarbon emissions by 45%, 47% of CO₂, and up to 66% of particulate emissions into the environment (Escalante 2019).

In this sense, long-chain fatty acids obtained from renewable biomass (vegetable oils, animal fats, and microalgae oils) represent the main raw material for the production of liquid biodiesel obtained in the form of alkyl esters of short-chain alcohols such as ethanol and methanol. The most commonly used processes for obtaining biodiesel are pyrolysis and transesterification; however, in the first case, the method is expensive and offers low yields, while the second is presented as the most viable method for obtaining biodiesel. This process of converting oils to biodiesel is necessary because vegetable oils or oils extracted from microalgae have high viscosity and low volatility, causing incomplete combustion and the disposal of carbon deposits (Escalante 2019).

Large-scale cultivation of microalgae seeks to obtain significant amounts of a valuable product; therefore, the productivity of the system must be maximized. Understanding the factors that determine optimal growth is fundamental (Robles et al 2019): 

• Culture medium. 
• Gas exchange. 
• pH.
• Temperature
• Lighting regime. 

Temperature is a very important environmental factor due to its great influence on the development of microalgae. The effect of temperature on biochemical composition mainly affects two different mechanisms: the temperature dependence rate of chemical and biochemical reactions, and the temperature dependence for photosynthetic carbon fixation in various types of macromolecules, such as proteins, carbohydrates, and lipids.

 This type of microalgae has an impact especially on the ecology since through them is a very important advantage from the energy and ecological point of view, since they have a minimum level of emission of harmful gases such as carbon dioxide (CO₂), the main compound of the greenhouse effect (Gonzalez 2015). In addition to reducing other amounts of pollutants, thus defining biodiesel as a biodegradable product, which from this can also be obtained vegetable oils such as soybean, palm, sunflower, and others.

Method

Microalgae Chlorella vulgaris

Microalgae are microscopic photosynthetic and unicellular microorganisms; they are classified into prokaryotes and eukaryotes that can grow autotrophically or heterotrophically. They are divided into different groups depending on their taxonomy. Chlorella vulgaris is a unicellular algae with green pigmentation and spherical shape; considered as an option for the production of biodiesel and bioethanol because of its high content of lipids and oils; however, they have the ability to produce biomass quickly compared to other energy crops. These organisms, like plants, are able to use CO₂ and sunlight to generate complex biomolecules that are necessary for their survival. In general, they are photoautotrophic organisms, that is, they obtain energy from light coming from the sun and develop from inorganic matter. However, some species are able to grow using organic matter as a source of energy or carbon. The selection of microalgae is the first step in the development of a production process, they must have the appropriate characteristics for the specific cultivation conditions in order to achieve a certain product (Table 1) (Escalante 2019).

Table 1

Lipid percentage and biomass and lipid productivity of different microalgae

Bioreactor

Chlorella vulgaris microalgae are a type of algal cells from a genus of green algae of the Chlorococcales class. To carry out the production of biodiesel and bioethanol, open and closed systems are used by means of photobioreactors (PBR), where different containers and configurations can be used. Closed systems are those that are found as tubular (Figure 1a) and column (Figure 1b) PBR. However, they have a high cost in their construction and operation, but they have the advantage of controlling and improving the culture conditions, besides reducing the risks of contamination by other microorganisms; and providing a higher biomass productivity and increasing the efficiency in the use of light. 

Figure 1. a) Tubular Photobioreactors, b) Column Photobioreactors.

While open systems are more commonly used for commercial production of biomass from microwaves because of their speed and ease of maintenance. These types of systems operate at a low cost; however, their disadvantage lies in water losses due to evaporation, limited light penetration, high production periods, limited control of growing conditions, and limited CO₂ transfer due to its low concentration in the air.

However, there is an alternative for biofuel production by Chlorella vulgaris, where even 2 L plastic bottles can be used. As well as glass PBR of larger volume, (5, 10, 20) L, for extensive processes in the creation of biofuels (Figure 2).

Figure 2. General Operation Methodology for the Production of Biodiesel and Bioethanol.

Aeration Process

Aeration is of great importance for the production of microalgae, in this process all nutrients are homogenized. Therefore, a well distributed mixing must be carried out, and sedimentation of the algae cells must be avoided as excessive agitation can cause hydrodynamic stress and as a consequence a decrease in the growth rate. This water treatment consists of requiring an oxygen source, commonly known as aerobic biological water purification (Plata, Kafarov and Moreno 2009).

For the realization of biofuels such as bioethanol and biodiesel, any kind of electric light is needed for the motivation of algae reproduction, so the process is shaped by rotameters that control the aeration without affecting their growth. Therefore, the mixing is done by injecting air while stirring by bubbling, where by gravity the cells descend and by the injection of air rise. The mixing is continuous during the cultivation process, ensuring the homogeneity of the cells and nutrients that are inside the culture; in such a way that the gradients of light, nutrients, and temperature are eliminated.

Culture Medium

There are several factors that influence the cultivation process of the microalgae Chlorella vulgaris, so it is important to know and identify the optimal conditions both individually and as a whole that have tolerance microalgal strains. Therefore, to achieve an actively growing microalgae culture, it is necessary to have a viable inoculum, a minimum supply of nutrients, and adequate chemical and physical conditions. These conditions are:

Illumination is divided into two important components: irradiance and photoperiod. The former refers to the flux of light in which the microalgae are exposed to, while the latter term indicates the number of hours in which the microalgae are subjected to irradiance. Algae adapt to changes in light by varying the chlorophyll content of their cells, so that algae adapted to low light levels have a faster response to changes in light intensity because they have more chlorophyll than those adapted to high light intensities (Plata, Kafarov and Moreno 2009). Photosynthetic organisms only use the fraction of the sunlight spectrum that is photosynthetically active, i.e., between 350 and 700 nm. This photosynthetically active fraction accounts for 40% of the total radiation from the Sun. Most natural plant ecosystems have an efficiency of about 1% in terms of conversion of light energy into biomass. Light-biomass conversion efficiencies between 1 and 4 % have been demonstrated for microalgae in open systems and even higher in closed photobioreactors (González et al 2019).

Another influential factor is the temperature. During the cultivation process, three types of temperature are considered. The minimum temperature is the one that is below the optimum; therefore, it is not possible for growth to occur as well as the maximum which is above and is around 35ºC. While the optimum temperature is between 16 and 27ºC; however, this varies depending on the type of microalgae. Nevertheless, temperature changes can cause alterations in metabolic pathways, affecting the growth and development of crops as it dissociates the carbon molecules of the medium, making them available for photosynthesis.

Also, microalgae need a pH for their growth, the pH range for most microalgae cultures is between 7 and 9. An optimal pH in the culture is usually maintained by aeration with CO₂-enriched air. In the case of high cell density cultures, the addition of carbon dioxide corrects an increase in pH, which can reach a limit value of 9 for microalgae growth. A high or low reduction of pH decreases microalgae growth by the breakdown of many cellular processes. The optimum range can be between 8.2 to 8.7. The pH can be controlled by addition of CO₂. There is also an increase in pH with age or time of culture, and the photosynthetic process of CO₂ fixation causes a gradual increase in pH in the medium due to accumulation of OH- (Gonzalez et al 2019).

Therefore, the microalgae will be in a culture preservative for reproduction of more cells (Figure 3a), and by means of cells take inocula from 500 mL of solution with constant agitation for 5 days in order to determine certain amounts of cells (Santos, Gonzalez and Martin 2014). At the end of the process, the Chlorella vulgaris undergoes transesterification and fermentation by aeration until it becomes biodiesel and bioethanol, substances used by society as fuel in cars, collection in household items among infinite actions of human daily life (Figure 3b).

Figure 3. a) Growth Diagram of Microorganisms, b) Growth and Substrate Decrease.

Transesterification or alcoholysis

Transesterification or alcoholysis is the chemical reaction that occurs between oils and an alcohol (commonly methanol or ethanol) to produce glycerol and alkyl fatty acid esters, which are known as biodiesel. The main factors influencing the process are the alcohol: triglyceride molar ratio, the type of catalyst (alkali, acid, lipases), the temperature, the reaction time, and the content of water and free fatty acids in the feedstock. Currently, most biodiesel is produced by alkaline transesterification because of its speed and moderate operating conditions (Gonzalez 2015).

The use of a catalyst is required to improve the conversion, which can be acidic or basic, homogeneous or heterogeneous. Homogeneous catalysis has so far been the most widely implemented industrially. H2SO4 is most frequently used in acid catalysis; however, in addition to the difficulties arising from corrosion of the equipment involved in the process, high molar ratios are needed to achieve significant conversions [Robles et al. 2019, Conde et al. 2015]; therefore, basic catalysts are preferred. Among them, the most commonly used is NaOH at levels ranging from 0.75 to 1.5% w/w based on oil weight. However, in the particular case of ethanolysis, the recommended levels range from 0.4 to 0.8% (Figure 4), (Robles et al. 2019).

Figure 4. Chemical reaction of the transesterification process.

Fermentation

Microalgae are rich in carbohydrates, by this means they are used as carbon sources to obtain one of the biofuels such as bioethanol. By fermenting the biomass these algal organisms can be converted favorably with biomasses obtained from food crops such as sugar cane or corn. Bioethanol from microalgae can be obtained by two technologies: fermentation, which involves the conversion of biomass materials containing sugars into ethanol by yeasts; and gasification, which involves converting the biomass into a synthesis gas, which is then converted into ethanol by catalysis. A simplified diagram of the bioethanol production process, with fermentation and distillation as key steps, is shown in Fig. 5.

Figure 5. Diagram of the bioethanol production process.

Results

Currently, the economic and environmental factors are of high indexes; therefore, an alternative fuel of low environmental impact without affecting the economy is sought. Fuels such as biodiesel and bioethanol from microalgae, are emerging as substitutes for fossil fuels since they have advantages mainly due to their biodegradability and minimal toxicity. In addition, during their combustion, they produce components of lower emissions such as sulfates, aromatic compounds, and carbon dioxide.

Chlorella vulgaris strains were analyzed to obtain biodiesel and bioethanol in the Biotechnology laboratories of the Universidad Autónoma del Carmen (UNACAR), using an ecological PBR operation system. The bubbling column was replicated using PET bottles with a volume of 3 L, in addition to a bottle with a volume of 1 L for the distilled water used to hydrate the air by means of agitation and avoid evaporation of the culture medium (Figure 6a). In the same way, two other bottles with 20% chlorinated water were used to avoid possible contamination to the outside.

The function of the system in general had a plastic hose to a blower and at the other end a rotameter at the bottom. While at the top of the rotameter was connected another plastic hose 1 m long and its end was introduced into one of the holes of the bottle with distilled water; then a segment of plastic hose 0.7 m long was used and introduced into the second hole of the bottle of distilled water with a depth of 3 cm inside the container, the other end of the 0.7 m hose was inserted into the PBR with the culture medium; another 0.7 m long hose was taken and inserted through one of the holes in the lid of the PBR to capture the expelled air and transferred to a chlorine solution contained in a second bottle. 

The cultures were kept in acclimatization for 30 days in 250 ml Erlenmeyer flasks with a constant illumination of cold white, fluorescent light lamps that helped the motivation of algae reproduction (Figure 6d), in such a way that the process is conformed by rotameters that control the aeration without affecting their growth (Figure 6c). Enriched culture media were prepared at 90 mg L-1 (C90): 4 L of fresh medium were prepared, 3 mL of salts were added, 3 mL of trace metals (f/2 medium of Guillard and Ryther) per L of solution; the medium was sterilized in autoclave at 120ºC and 30 atm, it was left to cool to add 3 mL of vitamins per liter of water.

 During the process the exhaust air is bubbled in chlorinated water in order to avoid contaminating the exterior (Figure 6b). Once the biomass is obtained, the product is recovered for oil extraction. When the oil is extracted, a certain percentage of solvent is added to perform a transesterification process in order to obtain biodiesel, where a chemical reaction occurs between vegetable oil and alcohols, mainly influenced by the alcohol molar ratio factor such as triglycerides generated by microalgae; once the substance is catalyzed, alkyl esters of fatty acids and glycerol are obtained. While by a fermentation process, bioethanol is obtained as a result with the help of yeast as sugars from the biomass of microalgae. The world's population must solve problems related to energy shortages, hence the importance of exploring new sources of renewable energy. Thus, algal biomass could satisfy about 25% of the world's energy needs, also providing other biotechnological products (Santos, González and Martín 2014).

Figure 6. a) Mode of operation of a PBR for the culture system; b) Chlorella vulgaris transesterification method; c) Rotameter and Reactor equipment; d) Chlorella vulgaris cultures.

Discussion and conclusions

The analyses obtained from the microalgae Chlorella vulgaris, examined in the UNACAR facilities, are identified as one of the best microorganism producers of triglycerides mainly used to obtain biodiesel and bioethanol. However, nowadays, more research is necessary to determine the best cultivation method and, with it, to obtain a higher balanced yield of biomass and lipids, as a base to obtain these bi-fuels, as well as to improve the technology in order to carry out an ideal harvesting process, which is the point of higher cost in relation to the cultivation processes of these microorganisms.

At the industrial level, large-scale microalgae cultivation has proven to be effective and efficient. Therefore, the dissemination of this technology is of a scientific scope and fundamental to continue its development. However, there are certain limitations, such as the laws that allow the use of wastewater for cultivation. Another influencing factor is land use since it is of utmost importance to determine the location of the crop plant.

Microalgae are the main source of renewable energy as in human and animal nutrition. There are several extraction methods used in microalgae such as physical, mechanical, and chemical. However, the chemical extraction method is one of the fundamental processes for microalgae, since a higher yield is obtained due to the presence of organic solvents that offer a higher extraction of lipids present in the microalgae Chlorella vulgaris.

Unlike fossil fuels, fuels obtained from microalgae, or also defined as biofuels, have a high capacity to capture greenhouse gases, the main problem that currently affects the environment. Carbon dioxide (CO₂) is one of the components that affects the ozone layer, and that most of the items or products used by humans produce it; therefore, microalgae are excellent biomitigators of CO₂ and wastewater treatment. 

In the case of biodiesel and bioethanol production, it has been of great help in reducing greenhouse gas emissions produced during the combustion of fossil fuels. In addition to avoiding the use of crops traditionally used for human consumption as raw material. In spite of the current adversities to acquire this type of oil, the development of a well elaborated transesterification or fermentation methodology allows the obtaining of oil from Chlorella Vulgaris to be encouraging.

In Mexico, this type of productions to obtain biodiesel and bioethanol from microalgae is of great importance if they are considered a good development of technology and energy. Therefore, in the future, these factors will give satisfactory answers to the ecology and potentially sustainable to the requirement of liquid fuels produced, increasing the economy in our country.

Acknowledgements

This research is supported by the Faculty of Chemistry of the Universidad Autónoma del Carmen (UNACAR), within the research framework. I also thank the project advisors, Dr. Asteria Narváez García and Dr. Juan Carlos Robles Heredia, for their support, resources, and mainly for giving me the necessary knowledge to carry out the research.

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