Category Archives: Biotechnological Applications of Microalgae

Lipid Identification and Extraction Techniques

Desikan Ramesh

Department of Farm Machinery

Agricultural Engineering College and Research Institute Tamil Nadu Agricultural University Coimbatore, Tamil Nadu, India


7.1 Lipid Quantification……………………………………………………………………………………. 89

7.2 Lipid Profiles……………………………………………………………………………………………….. 90

7.2.1 Identification of Algae Lipid Profiles…………………………………………….. 90

7.2.2 Suitability of Algae Lipid for Biodiesel Production………………………… 90

7.3 Oil Extraction………………………………………………………………………………………………. 92

7.3.1 Mechanical Extraction………………………………………………………………….. 92 Oil Expeller………………………………………………………………………. 93

7.3.2 Chemical Extraction……………………………………………………………………… 93 Solvent Extraction………………………………………………………….. 93 Supercritical Fluid Extraction (SFE)………………………………… 94

7.4 Conclusion………………………………………………………………………………………………….. 95

References…………………………………………………………………………………………………………… 96


Due to their high oil content, microalgae have attracted substantial research atten­tion for biodiesel production and furthermore, algae have the capability to replace conventional biodiesel feedstocks. Algal strains collected from diverse aquatic environments require the evaluation of various important parameters such as oil content, lipid composition, growth rate, and metabolic efficiency under different conditions. One can decide whether the selected algal strain is suitable/unsuitable for biodiesel production based on the preliminary lipid analysis (both lipid yield and lipid composition).

Microalgal strains have the potential to produce up to 50% lipid by dry cell weight, depending on the species and specific growth conditions (Chisti, 2007). The neutral lipids present in microalgae are primarily in the form of triacylglycerols (TAGs). TAGs can be converted to fatty acid methyl esters (FAMEs) via transesterification. Recovery of the accumulated algae lipids from algae paste is generally carried out after rupturing the cells to free the lipids. Different cell disruption techniques are used to rupture the algae cells, including autoclaving, microwave, sonication, osmotic shock, and bead beating. Lee et al. (2010) evaluated five different cell disruption techniques for enhancing lipid extraction efficiency. They reported that the micro­wave oven method is an efficient method for extracting lipids from microalgae. Because of its simplicity and cost effectiveness, solvent extraction is widely used by researchers (Letellier and Budzinski, 1999). For laboratory-scale studies, lipid con­tent and composition can be determined using well-established techniques. The most commonly used method for lipid extraction is the Bligh and Dyer method, or some variation thereof. Methods for simultaneous extraction and transesterification of algal biomass to extract the algal lipids are also available (Belarbi et al., 2000; Lewis et al., 2000).

Production Process

Phycocyanin is employed as a colorant to a greater degree compared to phycoer — ythrin, which is incorporated more frequently in fluorescent applications. This is evident in their production yields (Table 10.4), where C-phycocyanin yields are reported to be as high as 46%, which is consistent with its broad application profile. C-phycocyanin (PC) is the source of blue coloring and is commercially produced from Spirulina, Porphyridium, and Rhodella (Milledge, 2011).

The majority of the commercial production of PC occurs in outdoor, photo­autotrophic open raceway ponds predominantly in subtropical locations around the Pacific Ocean, specifically with Spirulina platensis (Spolaore et al., 2006; Eriksen, 2008). The range of commercial applications drives the production of high-purity phycobiliproteins—through extraction from the phycobilisomes fol­lowed by purification. The extraction process is particularly difficult because of the rigid cellular wall and the small size of the cell. Therefore, physical or chemical cell disruption is necessary to increase the bioavailability and assimilation of phyco­biliproteins from the cells (Molina-Grima, 2003; Sekar and Chandramohan, 2008). There are a number of extraction methods available to aid in the cell disruption process, of which include sonication with sand (mainly small-particle silica), French press, tissue grinding (with or without liquid nitrogen), homogenization, and causing osmotic shock with use of dilute phosphate buffer. Upon comparing all the extrac­tion methods tested, freezing and thawing of cells with liquid nitrogen, followed by grinding with a mortar and pestle (with an abrasive material) and homogenization at 10,000 rpm yielded almost 20% phycocyanin from Spirulina dry biomass (Sekar and Chandramohan, 2008).

There exists a range of patents detailing various cultivation and harvesting systems, extraction methods, and purification and production processes for phyco — biliproteins. Purification of phycoerythrin includes distilled water leaching, staged precipitation with ammonium sulfate, and ion-exchange chromatography (Sekar and Chandramohan, 2008). Good-quality algal pigments, specifically with respect to color tone and thermal stability, were patented for use as colorants in food. Such pigments were obtained by evaporating an aqueous solution containing trehalose and algal pigments to dryness (Sekar and Chandramohan, 2008). Consistently and efficiently cultivating large amounts of algae throughout the year without being affected by conditions of the culturing site can be challenging. Thus, methods have been patented to proliferate the growth of algae by irradiating the culture with monochromatic light at a wavelength of 600 nm. Cultivation of cyanobacteria under a magnetic field for the production of phycobiliproteins was patented for Spirulina and Colarina. This involves charging the algae in a test tube, by placing the test tube between the N — and S-poles of a magnet, such that both poles oppose each other on both sides of the tube. For the production of phycobiliproteins, this is done under constant irradiation with a fluorescent lamp with an illuminance of 800 to 8,000 lux at 24°C for 480 h (Sekar and Chandramohan, 2008).

The utilization of urea-type or amino-type water-soluble nitrogen compounds, together with other required nutrients, has also been patented as a cultivation method to increase phycocyanin yields (Sekar and Chandramohan, 2008).


It is easy to find algae, but finding algae suitable for biotechnology is difficult. Currently, insufficient attention is paid to the selection of algal strains that could be cultivated inexpensively by growing them in wastewater and under ambient condi­tions of light and temperature. It is necessary for entrepreneurs of microalgal bio­technology to invest in selecting algal strains and optimizing their cultivation. The choice of commercial algal strains is of paramount importance and merits rigor­ous investigation. Local species are well adapted to local environmental conditions, and their utility contributes to more successful cultivation than nonnative species; for example, a consortium of Actinastrum, Chlorella, Chlorococcum, Closterium, Euglena, Golenkinia, Micractinium, Nitzschia, Scenedesmus, and Spirogyra, and two unidentified species concentrated from local ponds grew well at a dairy farm in municipal wastewater and yielded 2.8 g m-2 lipid day-1, which would be equivalent to 11,000 L ha-1y-1 (Pitman et al., 2011). Microalgal cultivation in wastewaters is cost effective in producing algal biomass for biofuel, and it also helps in the removal of nutrients (Craggs et al., 2011).

To date, few native species have been studied for their growth and photosyn­thetic efficiencies; with extremophiles, this is seldom the case. For example, pho­tosynthetic rates of the extremophiles Chlamydomonas plethora and Nitzschia frustule, isolated from a semi-arid climate, approached their theoretical maxima corresponding to 22.8 and 18.1 mg C mg chl a-1 h-1 and high photosynthetic effi­ciencies (Subba Rao et al., 2005). Based on their specific growth rates at 10°C, 15°C, 25°C, and 30°C and threshold (I0) and saturation (S) values of irradiance and saturation irradiance for growth, Kaeriyama et al. (2011) demonstrated the existence of physiological races in Skeletonema species isolated from Dokai Bay, Japan. Cultures of microalgae from tropical, subtropical, and semi-arid climates that may have unique physiological characteristics should be studied in detail. Of note, a marine diatom, Navicula sp. strain JPCC DA0580, and a marine green alga, Chlorella sp. strain NKG400014, isolated in Japanese ocean waters (Matsumoto et al., 2009) had a cell composition that yielded energy of 15.9 ± 0.2 MJ kg-1 and

26.9 ± 0.6 MJ kg-1, respectively, which is equivalent to coal energy. Also of inter­est is the Strain B32 Dunaliella isolated from the Bay of Bengal, which yielded a maximum 0.68 pg carotene cell-1 while strain I3 yielded 17.54 pg carotene cell-1 (Keerthi et al., in press).

Extremophile algae stressed by high temperatures, light, salinity, and nutrients seem to have physiologically adapted to their harsh environmental conditions even under high irradiation, as evidenced by a chlorophycean microalga in the storage pools of nuclear reactors (Rivasseau et al., 2010). Because of their resilience, cultur­ing these algae under ambient environmental conditions reduces the dependency on seasons for cultivation and the need to shut off operations during extreme climatic conditions. This will be cost-effective and enhance their utility in biotechnology. The thermo-acidophilic red alga Galderia sulphuraria isolated from environments with pH 0 to 4 pH and temperatures up to 56°C can survive both autotrophically and heterotrophically (Weber et al., 2004). This alga has a repertoire of metabolic enzymes with high potential for biotechnology. Its tolerance for high concentra­tions of cadmium, mercury, aluminum, and nickel supports its potential for biore­mediation. The desert crusts seem to support extremophile members of five green algal classes; these unicellular algae growing under selective pressures of the desert appear to have high desiccation and photophysiology tolerance (Cardon et al., 2008). The extremophile cyanobacteria, mostly Microcoleus sp. living in the desert crust, are remarkably resistant to photo-inhibition, in contrast to Synechocystis sp. strain PCC 6803, and, within minutes of rehydration, recover their photosynthetic activ­ity (Harel et al., 2004). Comparison of the extremophile Chlamydomonas rauden — sis Ettl UWO 241 isolated from an ice-covered Antarctic lake with its mesophilic counterpart C. raudensis Ettl. SAG 49.72 (SAG) isolated from a meadow pool in the Czech Republic, showed different abilities for acclimation (Pocock et al., 2011). The UWO 241 strain, unlike the other, relied on a redox sensing and signaling system for growth that bestows better success under stressful environmental conditions.

Nannochloris sp., isolated from the Great Salt Plains National Wildlife Refuge, grew in salinities from 0 to 150 PSU (practical salinity unit) and temperatures up to 45°C; growth and photosynthesis saturation were at 500 mol photons m-2s-1. Although the division rates in this alga were equal, in cells acclimated to low or high salinity and temperature, the former had a higher photosynthetic performance (Pmax) than the latter (Major and Henley, 2008).

The extremophile Coccomyxa acidophila (pH < 2.5) accumulated more lutein (3.55 mg g-1) when grown in urea (Casal et al., 2011). In another extremophile, Chlamydomonas acidophila (pH 2-3.5), stringent limitation of phosphate resulted in higher total fatty acid levels and lower percentages of polyunsaturated fatty acids (Spijkeman and Wacker, 2011). C. acidophila cultures grown on urea as a carbon source yielded high biomass levels (~20 g dry biomass m-2d-1) compared to ~14 g dry biomass m-2d-1 grown mixotrophically utilizing glucose as a carbon source (Cauresma et al., 2011). Mixotrophic growth of C. acidophila on glucose resulted in better accumulation of carotene and lutein (10 g kg-1 DW), the highest recorded for a microalga (Cauresma et al., 2011). In Dunaliella salina living under high light and salt stress, carotenogenesis shifted to higher salinity and increased substantially under nutrient-limiting conditions (Coesel et al., 2008); nutrient availability seems to control carotenogenesis and messenger-RNA levels. The extremophile (photopsychrophile) Chlorella sp. Strain BI isolated from Antarctica is unique in retaining the ability for dynamic short-term adjustment of light energy distribution between Photosystem II and Photosystem I, and can grow as a heterotroph in the dark (Morgan-Kiss et al., 2008).


Along with light intensity, temperature is one of the most difficult parameters to optimize in large-scale outdoor culture systems. Fluctuations in temperature, both daily and seasonally, can lead to significant decreases in productivity. The optimal growth temperature for microalgae is species specific, but often in the region of 20°C to 30°C (Chisti, 2008). Many algal species can tolerate temperatures of up to 15°C lower than their optimum, with reduced growth rates, but a temperature of only a few degrees higher than optimal can lead to cell death (Mata et al., 2010). The net effi­ciency of photosynthesis declines at high temperature as the rate of respiration rises significantly, while the increased flux through the Calvin cycle is moderate. This effect is worsened by the fact that CO2 becomes less soluble at elevated temperatures, more rapidly than O2 (Pulz, 2001).

Low seasonal, morning, and evening temperatures can lead to significant losses in productivity, although low nighttime temperatures are potentially advantageous due to a reduction in the respiration rate. As much as 25% of the biomass produced during daylight hours can be lost at night due to respiration (Chisti, 2007). Cool nighttime temperatures can minimize this loss.

Closed reactor systems almost always require some form of temperature control. They often suffer from overheating during hot days when temperatures inside the reactor can reach in excess of 50°C. Heat exchangers or evaporative water-cooling systems may be employed to counteract this (Mata et al., 2010). The culture system can also be placed inside a greenhouse, or contacted with water to minimize tem­perature fluctuations (Chisti, 2007). Closed PBRs are sometimes floated, either whole or just the solar collector, in a temperature-modulating water bath. Double­walled reactors with part of the liquid volume used for heating and cooling have been devised (Ugwu et al., 2008), although all such modifications add to the cost of production.

There is a relationship between temperature and light availability. Exposure to a rapid increase in light intensity when the temperature is below optimum (as occurs in the early morning in outdoor cultures) can lead to photo-inhibitory stress as cells are too cold to process incoming photons, thereby reducing photosynthetic efficiency for a good part of the morning (Vonshak, 1997). Low temperatures are therefore particularly suboptimal in the early morning, and any efforts to employ heat reactors should be concentrated just before dawn.


As microalgae possess a simple cellular structure, their capability to efficiently convert solar energy into chemical energy is high. The production of oil per unit area of land from selected microalgae is around 30 times greater than that of ter­restrial plants. The scenario thus looks promising for the production of biodiesel from the microalgal oil. There are various steps involved—from the stage of cul­tivation of microalgae to the final stage of production of bio-oil or biodiesel. The intermediate steps include harvesting, dewatering, concentration, and extraction of microalgal oil. The composition of fatty acids and other constituents present in plant and animal oils varies considerably from microalgal oil. In addition to triglycerides and free fatty acids, microalgal oil contains hydrocarbons, sterols, wax and sterol esters, and free alcohols that cannot be saponified. The major components in microalgae include carbohydrates, proteins, and lipids. In gen­eral, the lipid content of microalgal biomass increases when they are deprived of certain nutrients (nitrogen and silicon). However, the deprivation of nitrogen and silicon does not necessarily favor all species viz. Euglena, Nannochloropsis strains where cell division has been found to be blocked. There are certain species (such as Escherichia coli and Saccharomyces cerevisiae) that can be converted to oleaginous species (microbes that can accumulate more than 20% of their cellular dry weight in lipid) by genetic engineering. Although there are several species of microalgae, only a few have been explored with respect to their potential for high biomass yield and lipid content. The FAME content in the biodiesel should have a minimum value of 96.5%, as per the recommendation of EN 14103 (Sarin et al., 2009). However, biodiesel synthesized from only a few of the microalgal species has fulfilled the minimum criteria of ester content in biodiesel as specified by the EN. The reason for this may be attributed to the presence of unsaponifiable constit­uents in the microalgae. Microalgal species not fulfilling the minimum specified criteria of ester content limits their suitability for biodiesel production. However, microalgal oil can be converted to bio-oil by pyrolysis or thermochemical catalytic liquefaction. The bio-oil can be further upgraded by chemical or physical means. While the chemical upgradation includes processes such as catalytic esterifica­tion, catalytic hydroprocessing, and catalytic cracking, the physical upgradation can be done by char removal, hot vapor filtration, liquid filtration, or solvent addi­tion (Xiong et al., 2011). The present status for the production of biodiesel and bio-oil from microalgae is cost intensive. However, the major advantage that the microalgae provide is their growth in aquatic environments and their noncompeti­tiveness with terrestrial plants. India and many other countries have a vast coastal area where microalgae can be grown, cultured, and harvested. The other important benefit of microalgae is the suitability of some of the species in wastewater. Thus, microalgae cultured with wastewater will have the dual benefit: of the production of oil and the treatment/disposal of wastewater. Numerous strains of microalgae are available in nature, and several of these species may be explored for their feasibility to be cultured as oleaginous species. The future seems bright for micro­algae to provide a future alternative to the other fuels.


The oil extracted from microalgae consists of polar lipids and neutral lipids. The neutral lipids consist of triglycerides, free fatty acids, hydrocarbons, sterols, wax and sterol esters, and free alcohols. Because only triglycerides and free fatty acids are saponifiable, they must be separated from the others so as to convert them to biodiesel by esterification or transesterification. The synthesis of biodiesel/bio-oil from microalgae involves several steps, including selection of an appropriate species among a large diversity of species of microalgae (around 300,000). For the production of bio-oil, a thermochemical method is adopted for the preparation of fuel from microalgae through pyrolysis, direct combustion, or thermochemical l iquefaction, wherein the organic compound is thermally decomposed at high temperature in the absence of oxygen. A high yield of bio-oil (97.05%) was obtained through liquefaction of Dunaliella tertiolecta. Thermochemical catalytic liquefaction has an advantage over pyrolysis or direct liquefaction, in that a low nitrogen content is present. A high oxygen content has been observed, which requires deoxygenation of the bio-oil. Other compounds are also formed along with bio-oil, such as bio-char, gases, and ash, all of which lower the calorific value of the fuel. The amount of biodiesel obtained from microalgal oil can be enormous The fatty acid composition of the feedstock has been found to play a significant role in the composition of the biodiesel. A trade-off between the oxidation stability and low-temperature proper­ties of biodiesel has been observed and hence a balance between the two must be maintained.


Bhaskar Singh is grateful to the Council of Scientific and Industrial Research (CSIR) New Delhi, India for the award of Research Associateship.


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The urgent need for substantive net reductions in CO2 emissions into the atmosphere can be addressed via biological CO2 mitigation (Ramanan et al., 2009a, b; Fulke et al., 2010; Shekh et al., 2012; Yadav et al., 2012), coupled with a transition to value-added products (VAPs) such as biofuels (Fulke et al., 2010; Kumar et al., 2010). Microalgae can fix CO2 from the atmosphere, from flue gases, or directly as soluble carbonates by the process of photosynthesis using solar energy (Wang et al., 2008). Concurrently, biomass is produced with 10 to 15 times greater efficiency than terrestrial plants, which has application in carbon credit programs (Lam and Lee, 2011). Microalgal cells con­tain approximately 45% to 65% carbon, wherein 1 kg dry biomass is produced by fixing approximately 1.8 kg CO2 (Chisti, 2007). CO2 from the external atmosphere (air/ extracellular surroundings of microalgae) can be dissolved as bicarbonates and made available to microalgae for uptake and intracellular conversion to CO2 by intracellular carbonic anhydrases. CO2 is then made available to Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) for its fixation into energy compounds (Kaplan et al., 1991). Microalgae may provide a better tool for simultaneous CO2 sequestra­tion and biofuel generation. Current CO2 levels (0.0387% (v/v)) in the atmosphere are inefficient in supporting the high microalgal growth rates and biomass productivities needed for full-scale biofuel production (Kumar et al., 2010). Flue gases from various industries typically contain CO2 in the concentration range around 15% (v/v), which will provide sufficient amounts of CO2 for large-scale microalgae biomass production (Kumar et al., 2010). Owing to the cost of upstream separation of CO2 gas, direct utili­zation of power plant flue gas would be advantageous in microalgal biofuel production systems. Flue gases that contain CO2 concentrations ranging from 5% to 15% (v/v) have been scrubbed for direct use in microalgal culture systems for biomass growth (Kumar et al., 2010). This approach is believed to be pragmatic, more eco-friendly, and technologically feasible for bio-mitigation of CO2 as compared to physicochemical adsorbents or deep-ocean injections. This is a win-win scenario wherein combating air pollution through microalgal cultivation is possible while simultaneous microalgal biomass generation can be exploited to produce biofuel and other VAPs.

A comparative evaluation of CO2 sequestration potential of various microalgal species is presented in Table 11.1. Some microalgal species such as Chlorella, Scenedesmus, and Botryococcus are among the microalgae that have been studied for CO2 consump­tion and are promising for bio-mitigation of CO2 (Griffith and Harrison, 2009; Fulke et al, 2010). Scenedesmus obliquus was found to tolerate high CO2 concentrations (up to 12% v/v) with optimal removal efficiency of 67%, when grown at pilot scale using industrial flue gas as a carbon source (Li et al., 2011). Biomass generation through CO2 sequestration and exploitation of biomass for biodiesel precursor formation has been studied by Fulke et al. (2010). Chlorella sp. was found to have biomass productivity of 0.322 g L-1d-1 with lipid productivity of 0.161 g d-1 at 3% CO2 as feed gas.

The presence of FAMEs (fatty acid methyl esters) suitable for biodiesel (e. g., palmitic acid (C 16:0), docosapentaenoic acid (C 22:5), and docosahexaenoic acid (C 22:6)) have been confirmed. The calcite produced was characterized by Fourier transform infrared (FTIR) spectroscopy, scanning electron microscopy (SEM), and x-ray dif­fraction (XRD) (Fulke et al., 2010). The ability to tolerate CO2 concentration during growth is confined to the individual specie’s characteristics. However, when exposed, the CO2 concentration in the gaseous phase does not provide a true reflection of the actual concentration of CO2 in the flue gas to which the microalgal specie is exposed during dynamic liquid suspension. It depends on the alkalinity (pH) and the CO2 concentration gradient created by the resistance to mass transfer (Kumar et al., 2010).


The microalgal reactors described above differ in features such as surface-to-volume ratio, freedom to adjust orientation and inclination, efficiency of mixing and gas supply (related to hydrodynamics and mass transfer), ease of maintenance, tempera­ture regulation, and construction materials. Table 5.4 presents a comparison of these design features in six major types of reactor. No reactor design is able to effectively control all these parameters simultaneously; therefore, any choice will be a compro­mise between the advantages and disadvantages of each system (Table 5.5).

5.4.1 The Open versus Closed System Debate

The relative merits of closed and open systems have been extensively debated in the microalgal literature (Pulz, 2001; Carvalho et al., 2006; Grobbelaar, 2009; Mata et al., 2010). There is no doubt that open ponds are the primary systems used in large-scale, outdoor microalgal cultures, but their commercial use has been limited to species that can be maintained using an extreme cultivation environment

Подпись: Microalgal Cultivation Reactor Systems


Comparison of Main Design Features of Various Reactor Types

Reactor Type


Vertical Column or Airlift

Horizontal Tubular

Helical Tubular

Flat Plate

Stirred Tank






Largely uniform

Largely uniform

Gas transfer



Low to high along length of tube

Low to high along length of tube


Low to high

Hydrodynamic stress



Low to high depending on pumping system

Low to high depending on pumping system



Light harvesting

Fair, depending

Good at low volume; poor

Excellent at narrow

Excellent at narrow

Excellent at


efficiency (area:volume ratio)

on depth

at high volume





Temperature control







Species control









Easily achievable

Potentially achievable

Potentially achievable





Land area required







Source: From Borowitzka (1999); Carvalho et al. (2006).



Advantages and Disadvantages of Different Reactor Types


Reactor Type




Relatively economical Low energy input Easy to clean and maintain No O2 build-up Large production capacity



Little control of culture conditions Poor mixing Light limitation Readily contaminated Limited growing period Poor productivity Large land area required Limited CO2 mass transfer with large CO2 losses to atmosphere if pond sparged

Temperature determined by climate


Vertical column PBR Excellent mixing

High mass transfer rates Low shear stress Easy to clean and sterilize Reduced photo-inhibition Low cost

Low land area requirement


Low angle to incident sunlight Size limited by area:volume ratio Large mixing energy due to gas compression


Gradients of pH, O2, and CO2 along tubes

Wall growth: fouling in tubes difficult to clean

Large land area required Significant water losses if evaporative cooling is used Possible hydrodynamic stress


Large illuminated surface area Good biomass productivity Relatively cheap construction materials


Tubular PBR



Подпись: Flat-plate PBR

Large illuminated surface area Short light path Good biomass productivity Easier to clean Lower O2 build-up

Scale-up requires many modules — material intensive Temperature control critical in thin reactors

Source: From Borowitzka (1999); Pulz (2001); Chisti (2007); Ugwu et al. (2008); Brennan and Owende (2010); Mata et al. (2010).

(Lee, 2001). To expand the product range, there is significant interest in the design of closed reactors, particularly in the production of high-value, low-volume prod­ucts requiring a high degree of sterility. The essence of the debate is presented in Table 5.6 through a comparison of the key parameters of open and closed reactors.

Despite their higher cost and technical complexity, closed systems promise great improvements in enhancing control over process parameters. The challenge appears to


Comparison of Key Design Features and Process Parameters of Open and Closed Systems

Подпись: Species Control Choice of species Main criteria for species selection Contamination risk Sterility Light Availability Light utilization efficiency Area-to-volume ratio Process Control Mixing Gas transfer CO2 loss O2 build-up Overheating problems Temperature control Hydrodynamic stress Evaporative water loss Weather dependence Cultivation period Productivity Biomass concentration Biomass productivity Reproducibility of production Cost Capital cost Most costly operating parameters Energy input required Harvesting efficiency
Подпись: Flexible Shear resistant Temperature tolerant O2 resistant Reduced Achievable High High (20-200 m-1) Uniform Fair/high Depends on pH, alkalinity, gas recycling High High Easier, but cooling more often necessary High Depends on cooling and sparging design Less Extended High (>2 g L-1) High Possible within certain tolerances High Temperature and oxygen control High Higher due to high biomass concentration
Подпись: Restricted Growth competition Tolerance of range of conditions
Подпись: High None Low Low (5-10 m-1) Poor Poor High
Подпись: Low Low Harder, but cooling not as necessary due to large volume Low Surface evaporation
Подпись: High Limited
Подпись: Low (<1 g L-1) Low Variable but consistent over time
Подпись: Low Mixing Low Low due to low biomass concentration

Open Systems Closed Systems

Source: From Pulz (2001); Carvalho et al. (2006); Grobbelaar (2009); Mata et al. (2010).

lie in enhancing productivity sufficiently that it outweighs the additional cost of closed reactors. Another alternative is to attempt to design PBRs that are cheap to build in terms of construction materials, as well as efficient in terms of light distribution, mixing, gas sparging, etc, which makes them cheap to operate by lowering energy requirements.

A major but rarely recognized concern, particularly for energy products such as biofuels, is the energy balance of the production system. For a process to be econom­ically viable and sustainable, the energy generated when the product is used must be greater than that involved in its manufacture. The energy inputs in microalgal reactors are particularly focused on the mixing and gas pressurization, as well as the embodied energy in reactor materials; therefore, open systems have a more favorable energy balance than closed systems (Richardson, 2011).


In the production of algal energy products, the aim is the biological conversion of sunlight to a more convenient, portable, storable, and accessible form of fuel. In the case of biodiesel production, this entails the production of algal lipids. Lipid productivity is dependent on both biomass productivity and lipid content (Griffiths and Harrison, 2009), which is determined by both the species used and the culture conditions provided by the reactor.

Most large-scale commercial algal production systems to date have been for food, feed, neutraceutical, or fine chemical production. As biofuel is a bulk commodity prod­uct, production must be on a grand scale, and costs must be extremely low. Sterility, particularly microbial contamination, is perhaps less of a concern for energy produc­tion than it would be for a product such as a neutraceutical or fine chemical for human consumption. A particular consideration with an energy product is that the energy balance must be positive; that is, the energy recovered from the product must exceed the energy input required for production. LCA (life cycle assessment) studies to date suggest that biofuel production in closed reactors is unable to achieve a net energy ratio (energy out/energy into process) of above one (Lardon et al., 2009; Richardson, 2011).

It is generally considered that closed PBRs alone will be incapable of cost-effectively producing microalgal biomass on the scale required for biofuels (Greenwell et al., 2010). While productivities will inevitably be lower in open raceways, it is envisaged that open systems, due to their lower cost, simplicity of operation, and ability to scale to large volumes, will form the basis of microalgal production for biofuels (Sheehan et al., 1998). The lipids necessary for biodiesel production are often produced under nutrient stress conditions. Therefore, it is likely that a two-phase system using closed reactors to generate contamination-free inoculum with a high biomass concentration for a second product-generating stage in open systems could be advantageous.


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9.4.1 Cost of Algal Biomass and Algal Oil

Currently, commercial processes for the production of microalgae only exist for the production of specialty products such as health supplements, carotenoids, and specific aquaculture feeds. Furthermore, most of the algal species used in these processes are extremophilic algae. Data on the cost of large-scale algal processes are largely based on pre-implementation costing of these. Typical costs estimated are presented in Table 9.4.

From Table 9.4, the disparity in costing that arises from an immature technology position is clear. This was also noted in a comparison of environmental analyses. As a general trend, the algal biomass cost from the raceway system lies in the range of US$0.23 to $0.60 kg-1 DW, with the exception of the estimate of Norsker et al. (2011), for which the biomass was recovered by centrifugation, noted as a major capital and energy cost. The production of algal biomass from photobioreactors was characterized by a greater variation from US$0.42 to $3.04 kg-1 DW. Here it is evident that the two lowest values are based on the same calculations (Chisti, 2007), while the majority of the values (four of the eight available) lie in the range US$3.18 to $9.54. Refinement in this costing is required. Factors impacting the cost­ing are discussed in Section 9.4.2.

These costs are not competitive with the cost of crude oil (US$0.48 to $0.71 L-1; US$76 to $113 bbl-1). Van Harmelen and Oonk (2006) estimated that cost of pro­duction with current technology exceeded potential earnings from algal oil as sole product by 1.7- to 3.9-fold, depending on the assumptions and process decisions made. In costing a production facility for algal oil in South Colorado, Richardson et al. (2012b) indicated that a 60% reduction in CAPEX (capital expenditure) and 90% reduction in OPEX (operating expenditure) are required for a cost-effective pond system with algal oil as the sole product. These reductions increase to 80% on CAPEX and 90% on OPEX using a photobioreactor system. Norsker et al. (2011) translated their costs based on algal biomass to an energy-based cost, yielding val­ues in the range of US$32.60 to $295.50 GJ-1. It is noted that the lower value of US$32 GJ-1 estimated for a high light intensity environment is similar to the cost of delivered electricity.

These costings raise clearly two key considerations:

1. The necessity to operate the algal oil process as a biorefinery, achieving value from multiple products for the same growth costs

2. The need to interrogate component process costs to identify key targets for cost savings and technology development

Подпись: Analysis of Microalgal Biorefineries for Bioenergy 129


Cost Estimates for the Production of Algal Biomass and Algal Oil on a Large Scale





Cost of

Cost of Algal

(g DW m_2d_’)


Lipid (L) or




Biodiesel (D)



(US$ per kg

(US$ per




(g L-1)]




Assumptions wrt Process

Benemann and




0.43 (D)


Oswald, 1996 Benemann and




0.26 (D)


Oswald, 1996 Molina Grima et al..





Van Harmelen and






Oonk, 2006 Chisti, 2007




1.41 (D)


Chisti, 2007




1.81 (D)


Norsker et al„ 2011


Eindhoven, the

[0.3 g L-4


Raceway ponds, as wet paste.



Paddlewheel circulating liquid at 0.25 m s-1

Norsker et al„ 2011

Eindhoven, the

[2.1 g L1]


Flat-plate photobioreactor, as


wet paste; aeration at 1 vvm;

polyethylene film panels of 1-year lifetime







Подпись: Biotechnological Applications of Microalgaeassumed; circulation at 0.5 m s-1; assume free supply of C02 from flue gas, nutrients from wastewater, and photosynthetic efficiency increased to 60%

Jorquera et al., 2010 Nannochloropsis

Jorquera et al., 2010 Nannochloropsis

Williams and USA 18-37

Laurens, 2010

Подпись: South Colorado, USA South Colorado, USA Richardson et al., 2012

Richardson et al., 2012

Подпись: Raceway system using Chisti (2008) and a lipid content of 29.6% Flat-plate reactor system using Chisti (2008) and a lipid content of 29.6% Horizontal tubular system using Chisti (2008) and a lipid content of 29.6% 0.79-3.08 (D) Based on costings prepared for 0.227 0.419 9.54

15-50 0.36-0.65

US Dept, of Energy’s Office of Energy Efficiency and Renewable Energy using a hybrid reactor system consisting of a front-end PBR “nursery” and “grow-on” raceway stage

3.36 (L) Open ponds

Подпись: Analysis of Microalgal Biorefineries for Bioenergy8.34 (L) Photobioreactors

The effect of process variables and process selection on the costing is discussed in Section 9.4.2, while the potential contribution of products other than biodiesel is discussed in Section 9.4.3.


The term phycoremediation was coined by John (2000) to refer to the remediation of water carried out by algae. Microalgae have high efficacy in wastewater treatment and can offer possible solutions for environmental problems (Lau et al., 1994; Craggs et al., 1997; Korner and Vermaat, 1998; Harun et al., 2010). Microalgae are eukary­otic, autotrophic microorganisms that can adapt to almost any aquatic environment (including wastewater) and produce biomass rich in various nutrients and minerals. Microalgae vary greatly in protein (10% to 53%), carbohydrate (10% to 16%), lipid (15% to 55%), and mineral (5%) constituents (Xu et al., 2006).

Phycoremediation of wastewater (domestic or industry) refers to any large-scale utilization of (desirable) microalgae for the removal of pollutants or biotransfor­mation of hazardous or harmful organic chemical compounds to nonhazardous end-products, xenobiotics, and removal of pathogens from wastewater. Biomass consumes considerable amounts of nutrients from freely available sources, such as wastewater rich in organic nutrients, inorganic chemicals, and CO2 from waste and exhaust streams (Olguin, 2003), that can accelerate the microalgal biomass propagation (45% to 60% microalgae by dry weight), nucleic acids, and phos­pholipids. Nutrient removal can be further increased by ammonia stripping or phosphorus precipitation due to the increase in the pH associated with photosyn­thesis (Laliberte et al., 1994; Oswald, 2003; Hanumantha Rao et al., 2011; Rawat et al., 2011).

Phycoremediation as a biological tertiary treatment, performed typically to treat secondary municipal wastewater, has been the focus of research during the past few decades (Oswald and Gotaas, 1957). High-rate algal ponds (HRAPs) for wastewa­ter treatment are very effective, in that HRAP-cultivated microalgal cultures can assimilate huge amount of nutrients, resulting in a reduction in BOD and chemi­cal oxygen demand (COD). Microalgae are regarded as the most versatile solution among biological wastewater treatment processes. Domestic wastewater contains the majority of nutrients such as nitrogen and phosphorous that directly and indirectly support microalgal productivity and maintain the biomass at levels high enough to achieve nutrient removal efficiently in wastewater systems. The application of micro­algae in wastewater treatment for reducing odor, coloring, nitrate, nitrite, phosphate, ammonia, TDS, TSS, BOD, and increasing pH and heavy metal absorption has been performed over the past few years. Effluent-treated microalgal biomass can be used for various purposes (Munoz and Guieysse, 2006). Recently, Kumar et al. (2011) studied high-rate algal pilot plant cultivated Chlorella vulgaris in confectionery effluent wastewater treatment, wherein harvested biomass was used for enzymatic and nonenzymatic antioxidant potential studies. However, the enriched microalgal biomass needs to be harvested at low cost using a cost-effective nutrient removal system. These are still in the infancy stage.

The application and advantages of phycoremediation include (Olguin, 2003)

1. Nutrient removal from both municipal and industrial wastewater or effluent enriched with high organic matter

2. Nutrient and xenobiotic compound removal with the aid of algae-based biosorbents

3. Efficient treatment of acidic and heavy-metal wastewater

4. Increasing oxygenation of the atmosphere

5. CO2 sequestration

6. Improving effluent quality

7. Transformation and degradation of xenobiotics

8. Biosensing of toxic compounds by algae

Enumeration of Microalgal Cells

Taurai Mutanda and Faizal Bux

Institute for Water and Wastewater Technology Durban University of Technology Durban, South Africa


4.1 Introduction……………………………………………………………………………………………….. 45

4.2 Sampling and Culturing Microalgae…………………………………………………………… 46

4.3 Microalgal Preservation……………………………………………………………………………… 46

4.4 Enumeration Methods……………………………………………………………………………….. 47

4.4.1 Spectrophotometric Analysis………………………………………………………….. 47

4.4.2 Gravimetric Analysis………………………………………………………………………. 47

4.4.3 Counting Chambers……………………………………………………………………….. 48

4.4.4 Flow Cytometry……………………………………………………………………………… 48

4.5 Conclusion………………………………………………………………………………………………… 48

Acknowledgments………………………………………………………………………………………………. 50

References………………………………………………………………………………………………………….. 50


The estimation of a microalgal population size is no easy task due to the micro­scopic size of the cells. Consequently, it is impossible to physically count them with the naked eye. The size of microalgae falls within the size of other microbes (e. g., bacteria) and, as a result, most of the methods used for microbial cell counting are also applicable to microalgae. In general, conventional microbiological proto­cols are available and are sufficient for cell enumeration despite the proliferation of modern and advanced techniques. It is recommended that the researcher choose a method after assessing the costs involved because some of the latest methods require very sophisticated and expensive equipment.

According to Caron et al. (2003), “the identification and enumeration of micro — organismal species in natural aquatic assemblages is an essential prerequisite for ecological studies of these populations.” Effective ecological studies of populations of colonial freshwater phytoplankton species are hampered by a lack of methods for cell enumeration (Box, 1981). Closely related microalgal groups must be accurately distinguished, and this is very crucial when these species pose health and environ­mental risks (Caron et al., 2003).

Microalgal cell population size is important when studying growth kinetics. It is also important to know the amounts of biochemical constituents such as pigments (e. g., chlorophyll-a). Microalgal cells can be enumerated directly using techniques such as light absorption and/or indirectly via surrogate determination of dry or wet biomass and/or measurement of cell components such as organic nitrogen, phosphorus, etc. (Guillard and Sieracki, 2005).

However, despite their common usage, direct methods and gravimetric measure­ments of microalgal cell dry weights are tedious, time consuming, and prone to errors that may exceed ±10% (Elnabarawy and Welter, 1984). Techniques for the estimation of the abundance of microalgal cells in natural microalgal samples are improving at a fast pace with the advent of electronic counting methods. Despite the progress made in the development of these advanced and sophisticated techniques, they still have major drawbacks, such as cost and the requirement of highly skilled personnel to operate the equipment. This chapter describes some of the popular methods that are available for the enumeration of microalgal cultures. The methods that are widely used are spectrophotometry, dry weight determinations, light micros­copy, haemacytometry, and flow cytometry.