Microalgae are microscopic algae, typically found in freshwater and marine systems. In this article, Dr Dolly Wattal Dhar, Dr Pranita Jaiswal, Mr Sudhir Saxena, and Dr Randhir Bharti discuss the promising possibilities of using microalgae as a source of renewable energy in the form of biofuels.

Microalgae are microorganisms (prokaryotic or eukaryotic), which accumulate biomass by the process of photosynthesis using sunlight, water, and carbon dioxide. The complete cycle of microalgae development ranges from 24 hours to several days and can double every few hours during their exponential growth period.

There are 50,000 microalgae species and only around 30,000 species have been studied. For a long time, microalgae have been used as nutritional ingredients but their active cultivation has begun over the last three decades. These organisms represent exciting possibilities as promising sources of a diverse range of metabolites of immense medical and industrial significance and can convert solar energy into biomolecules including carbohydrates, proteins, lipids, and triglycerides. Their ability to grow rapidly and adaptation to extremes of environment and ecologies make them suitable models for not only understanding the metabolic and evolutionary processes, but these are also miniature factories for producing useful value-added products. Microalgae can be used to make various products which are widely used in different industries. The reported market is 5,000 tonnes per year and $1.25X109. An annual sale of microalgae is reported to be $6X109 and the productivity is 7.5X106 tonnes per year. Commercial production and harvesting of natural populations of microalgae predominantly take place in developing countries, indicating available experience, and good environmental and economical conditions, such as sunshine and low labour costs. Large-scale industrial applications require large amount of marginal, cheap but often ecologically valuable land and water sources. For poor rural communities, well-designed small-scale Integrated Food and Energy System (IFES) approaches are the most suitable, potentially reducing ecological impact while providing fuel, animal feed, human protein supplements, wastewater treatment, fertilizers, and possibly more products that generate additional income. Capital inputs have to be minimized for this group, which means that the cultivation system would most likely be the open raceway pond, constructed in an area with an easily accessible, sustainable water supply, or in situ collection.

In recent years, microalgae grown in mass culture in either open or closed bioreactors have been identified as having a realistic potential for the production of biofuel on a large scale. An approximate per cent composition of three main components, namely proteins, carbohydrates, and lipids in microalgal biomass are in the range of 6%–71%, 2%–64%, and 2%–40%, respectively. The photosynthetic machinery from algae can be exploited to provide clean and green energy by utilizing solar power—one of the main sources of clean energy. The idea of using microalgae as a source of fuel is not new, but it is now being taken seriously because of the escalating price of petroleum and, more significantly, the emerging concern about global warming that is associated with burning fossil fuels.

MICROALGAE FOR BIOENERGY

Microalgae are a large and diverse group of aquatic organisms that lack the complex cell structures found in higher plants. These have received considerable attention as a potential feedstock for biofuel production because, depending on the species and cultivation conditions, they can produce useful quantities of polysaccharides (sugars) and triglycerides (fats). Microalgae have a very short harvesting cycle (≈1–10 days depending on the process) allowing multiple or continuous harvests with significantly increased yields.

Microalgae are reported to provide diverse forms of renewable biofuels (Figure 1) including biomethane (by anaerobic digestion of the algal biomass), biodiesel (from microalgal oil), bioethanol (by fermentation of the microalgal carbohydrates), and photobiologically-produced biohydrogen.

Biodiesel production

Lipids are one of the main component of microalgae and depending upon the species and growth conditions, 2%–60% of the total cell dry matter can be lipids as membrane components, storage products, metabolites, and storehouses of energy. Various extraction methods have been reported for microalgal lipids which include traditional solvent extraction, accelerated solvent extraction, subcritical water extraction, and supercritical CO2 extraction. The conventional methods for lipid determination involve solvent extraction and gravimetric estimation. Nile Red—a lipid soluble fluorescent dye has been commonly used to identify the lipid content. Alternatively, lipophilic fluorescent dye BODIPY has also been used as a vital stain to monitor algal oil storage within viable cells. Studies have shown the use of Fourier- transform infrared spectroscopy (FTIR) as an efficient and effective tool to determine lipid contents in microalgae. The lipid content can be modified by varying growth conditions, such as nitrogen deprivation, silicon deficiency, phosphate limitation, high salinity, and heavy metal stresses. Factors, such as temperature, irradiance, and nutrient availability have been shown to affect lipid composition as well as content in algae. In addition, microalgae may assume many types of metabolisms, such as autotrophic, heterotrophic, mixotrophic, photoheterotrophic, and generally heterotrophic cultivation increases the total lipids as compared to phototrophically grown cells. Oil levels of 20%–50% are quite common and production of methyl esters or biodiesel from microalgal oil has been demonstrated and it is noteworthy to mention that the high quality oil produced by these microalgae can be converted to biofuel using existing technology. Biofuels produced from microalgae have the potential to replace a portion of fossil fuel consumption with a renewable alternative. Previous research in the early 1990s by the National Renewable Energy Laboratory showed that under controlled conditions, algae are capable of producing 40 times the amount of oil for biodiesel per unit area of land, compared to terrestrial oilseed crops. The interest in microalgae for oil production is due to the high lipid content of some species and synthesis of non-polar triacylglycerols (TAGs) which are the best substrates to produce biodiesel. A number of algal strains with the potential for making biodiesel include Botryococcus, Chlorella, Chlamydomonas, Scenedesmus, Crypthecodinium, Nannochloropsis, and Nannochloris.

Algal oil is unsaturated to a larger degree making it less appropriate for direct combustion. Triglycerides and free fatty acids can be converted into biodiesel and triglycerides production rate in algae is 45–220 times higher than the terrestrial plants. Biodiesel is the mixture of alkylene ethers of fatty acids obtained as a result of inter-esterification of lipids. This reaction is a multiphase process during which triglycerides are first converted into diglycerides and later into monoglycerides which are transformed into glycerine and ethyl ethers of fatty acids (biodiesel). The homogeneous alkaline catalysis (sodium and potassium hydroxides) is typically used for the industrial production of biodiesel. Large amount of microalgal oil was efficiently extracted from Chlorella protothecoides using n-hexane and converted to biodiesel by acidic transesterification. Chloroform-based transesterification has been reported to lead to a high FAME content. Supercritical CO2 extraction and thermochemical liquefaction have also been utilized for the production of biodiesel. Microalgal oils differ from most vegetable oils in being quite rich in polyunsaturated fatty acids and the lipids contain mainly unsaturated fatty acids; palmitoleic acid, oleic acid, linoleic acid, linolenic acid, and small amounts of saturated fatty acids, namely palmitic and stearic acid.

MICROALGAE FOR POLY UNSATURATED FATTY ACIDS (PUFAS)

Higher plants and animals are known to have no fermentation responsible for the synthesis of unsaturated fatty acids with more than 18 carbon atoms. Therefore, these compounds must be introduced while feeding. Well known PUFAs include omega-3 fatty acids in fish oil, but their consumption has decreased because they may accumulate toxins. Fish oil has limitations to be used as food additive because of its unpleasant smell and taste, and low oxidation ability. PUFAs are known to play an important role in reducing the cardiovascular diseases and obesity in cellular and tissue metabolism including the membrane’s fluidity, electron and oxygen transport, as well as adaptation ability. The use of fish oil is sometimes restricted because it contains such mixtures of unsaturated fatty acids that are structurally nonstandard. The production of eicosapentaenoic and docosahexaenoic acids from Crypthecodinium microalgae is prospective. OmegaTech (US) Company has shown that inexpensive oil (commercially known as DHA Gold) can be isolated from Schizochytrium. This oil is used as diet additive, non- alcoholic beverages, medioprophylactic diet, and animal fodder. This product is also introduced in medioprophylactic diet of pregnant women and patients with cardiovascular disease. Important PUFAs sourced from algae are reported in literature. PUFAs derived from microalgae, for example, docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), α-linoleic acid (ALA), and arachidonic acid (AA) are known to be essential for various larvae. Comparative proportion of important PUFAs in 46 strains of microalgae has been reported.

Microalgae For Bioethanol

With the rise in the demand of fossil fuels and realization of urgent need for their replacement, ethanol has become one of the most common biofuels worldwide as it reduces levels of lead, sulphur, carbon monoxide, and particulate matter. In many countries, it has been established/ considered for replacement of fossil fuels. Ethanol is generally produced mainly from alcoholic fermentation of sugar and starch (sugarcane, corn), utilizing fermentation of biomass sources, varying from agricultural crops (mainly sugarcane) to organic wastes. Use of agricultural crops poses huge competition for agricultural land for food. Another promising route for the production of ethanol is using lignocellulosic material, which is cheap, easily available, and renewable. However, its recalcitrant nature limits the commercial viability. Microalgae offer a perfect solution to the problem with simple cellular structure, carbohydrate-rich biomass, and no competition for agricultural land. They fix atmospheric CO2 to complex carbohydrates utilizing solar energy. These carbohydrates can be converted to ethanol via fermentation. Many cyanobacteria have been reported to possess the ability to secrete fermentation products. However, it is not the primary source of energy for majority of microalgae/cyanobacteria. Therefore, in order to increase the ethanol production, suitable genetic modification in the strain/s had been reported. Hundredfold higher ethanol production has been reported by cyanobacteria under salt stress conditions (1.24 M NaCl) as compared with the low salt conditions (0.24 M NaCl). Therefore, microalgae with high carbohydrate content are the potential candidates for bioethanol production.

Microalgae For Methane

Anaerobic digestion of organic matter produces biogas mainly consisting of methane (CH4) and carbon dioxide. Organic matter contained in microalgal biomass can be transformed to CH4 under anaerobic conditions. Lipid extracted cells from microalgae can be good material for conversion to CH4, thereby, improving the total energy recovery. However, the methanogenic activity of microalgal biomass, in general, is less desirable as compared to fossil fuels or other organic wastes. High protein content in microalgae lowers the C/N ratio, which in turn affects the digestion efficiency. Co-digestion of microalgal biomass with products containing a high C/N ratio was proposed to be the possible solution to this limitation. Efforts have been made to link the CH4 production with the natural ability of microalgae to mitigate contaminants from the environment. Studies have shown that a two-step system can be a viable approach wherein microalgae either produce nutrients (used by methanogenic bacteria) for methane production or can improve the biogas quality by removing CO2.

Microalgae For Hydrogen

Hydrogen offers a potential alternative source of energy for the future in place of fossil fuels. It is a clean carbon- free fuel which ultimately oxidizes to water and has the potential to reduce the dependence on hydrocarbon- based fuels. The hydrogen production ability of microalgae is linked with the photosynthesis with water as direct source of electrons. Hydrogen production potential has been reported in many cyanobacterial strains. The mechanism of H2 production in non-heterocystous cyanobacteria is similar to microalgae; heterocystous cyanobacteria have specialized cells for nitrogen fixation called heterocysts, which lack photosystem II (PS II), thus, maintaining low oxygen pressure due to absence of photolysis of water. Such conditions are required for nitrogenase and bidirectional hydrogenase activity. Efforts are being made to identify cyanobacterial strains with specific H2 metabolism, optimizing cultural  and environmental conditions, metabolic engineering, and genetic manipulation for enhancing hydrogen production.

Cultivation strategies

There are two main alternatives for cultivating photoautotrophic microalgae: A typical raceway pond system and a photobioreactor. Open ponds systems can be excavated and used unlined or lined with impermeable materials or they can be built up with walls. Unlined ponds can be used which will reduce the costs, but they suffer from silt suspension, percolation, heavy contamination, and their use is limited to a few algal species and particular soil and environmental conditions. Raceway ponds are open and outdoor ponds that are made up of circulating loop channels are typically shallow and unlined. Paddle wheels are used to circulate the suspended algae throughout the channels. Production
in the ponds usually takes 6–8 weeks to mature and typically yields only 0.1–0.2 g/l algae. Photobioreactors can be located indoors and provided with artificial light or natural light or outdoors under natural sunlight. Photobioreactors have higher efficiency and biomass concentration, shorter harvest time, reduce contamination risks, and allow greater selection of algal species for cultivation and higher surface-to-volume ratio than open ponds.

Once the algal culture reaches maturity, the biomass is harvested from the culture medium and harvesting can be one of the more contaminating processes in the production of algae-based products. Three systemic components of the harvesting process are biomass recovery, dewatering, and drying. The cost of harvesting can be a significant proportion of the total algal production cost ranging from 20% to 30%. The technically simplest option is the use of settling ponds which are filled with fully grown algae culture and drained at the end of that day, leaving a concentrated biomass at the bottom, which can be stored for further processing. Other techniques may include flocculation (chemical flocculation, bio/electro-flocculation), dissolved air floatation, centrifugation, filtration, decantation and vacuuming, dewatering, and drying.

Conclusions

Microalgae find use with less extraction/ processing and find application mostly as dried powder. Biodiesel production from microalgae provides technical and economic feasibility that also has the potential for CO2 sequestration and is therefore, likely to find wide acceptance. Capital input, immature technology, knowledge required for construction, operation, and maintenance and the need for quality control are significant barriers to algae-based systems. Although productivity and sustainability are potentially much higher for integrated systems, the time and effort needed to create a viable algae-based concept seems to be significantly higher. In addition, the remaining biomass can be utilized in the area of other biofuel products, such as biomethane and fermentation products. The role of cyanobacteria in the area of hydrogen gas is also reported, however, the economic viability of the process is not very lucrative.

Dr Dolly Wattal Dhar, Dr Pranita Jaiswal,

Mr Sudhir Saxena, and Dr Randhir Bharti, Centre for Conservation and Utilisation of Blue Green Algae, Division of Microbiology,ICAR-Indian agricultural Research Institute, New Delhi.