FACTORS AFFECTING THE GROWTH OF VARIOUS MICROALGAL SPECIES
The diverseness of microalgae is colossal; however, only few species have been relatively known and investigated by humans. Lately, microalgae are garnering great consideration because of the reality that they could serve as a feedstock for either biofuel or nutraceutical production. They have the capability of storing desired products and adapting themselves when there is a change in the environmental conditions (pH, temperature, light, carbon dioxide, salinity and nutrients). The current review focuses on how the environmental conditions affect the growth of various species of microalgae. In addition, the effect of mixing on the growth of microalgae as well as screening and selection of microalgal species are also discussed.
Keywords: Microalgae, Biofuel, Nutraceuticals, Environmental conditions.
The shortage of food, energy, water, fuel and impacts caused due to climate change poses a serious threat to the human society. These issues directly affect the economic stability of the society. According to Food and Agricultural Organization, the number of malnourished people is presumed to be over 850 million, globally (Stephens, Ross et al., 2013). In order to meet the food and fuel needs of the growing population, feedstocks obtained from natural sources play a crucial role (Ruiz, Olivieri et al., 2016). Algaculture promises to be one such method that is very economic and may satisfy the requirements of the population in a sustainable way (Starckx, 2012). Humans require food for their survival and fearing the scarcity of food resources in the future, industries were incorporated in 1950’s to check the feasibility of microalgal technology in manufacturing nutriment for humans (Burlew, 1953). The first development of this technology took place in 1960’s in Taiwan and Japan, where Chlorella was used as a supplement in human food. The biomass obtained from Chlorella was also sold globally, either in the form of tablets or powder. Another development was the usage of Spirulina as food supplement and for extraction of phycocyanin. Certain algae were also investigated for their medicinal properties. The microalgae Dunaliella and Haematococcus were found to be rich in antioxidants and were sold as products to enhance human nutrition.
Microalgae, especially diatoms and flagellates established a niche in aquaculture industry by serving as feedstock for animals and fishes. The application of microalgae in cosmetics were at a preliminary stage, hence the development of this industry was comparatively limited to a smaller scale (Slocombe and Benemann, 2016). An estimate indicated that the retail value of products acquired from microalgae was about US$ 5 – 6.5 billion and this was generated by health and food sector (US$ 1.25 – 2.5 billion), aquaculture (700 million) and through DHA production (US$ 1.5 billion) (Carlsson, 2007). Thus, in the last few decades production of microalgae has expanded commercially producing products of relatively high value and volume. Until today, DHA (Docosahexenoic Acid) obtained from microalgae Crypthecodinium cohnii, has been leading the sales as the largest sold microalgal product. Biomass productivity is an important aspect when analyzing microalgae and it is dependent on the gross photosynthetic activity which in succession relies on the prevailing environmental conditions (Slocombe and Benemann, 2016). Microalgae would therefore acquire the status of becoming the potential feedstock for products which are sustainable, ranging from food to fuels in the years to come (Chen, Wang et al., 2016). However, toxins produced by microalgae pose a serious threat to public health and World Health Organization recommends countries to monitor it closely. It also affects the economy of industry and therefore, World Health Organization has made it a requirement (Cardozo, Guaratini et al., 2007). This paper provides a critical overview of how environmental conditions like pH, temperature, light, carbon dioxide, salinity and other factors like mixing and selection and screening of microalgal strains affect the growth and the biomass productivity of algal species.
The temperature around the globe is increasing rapidly due to human-mediated changes. It is projected that the global average temperature of the sea-surface would increase by 1.4-5.8°C by the end of this century (Tait and Schiel, 2013). Temperature plays a crucial role in the growth of algae and it has become a necessity to control temperature in experiments involving algae (Raven and Geider, 1988). Temperature also affects the gross photosynthetic activity of microalgae. Depending on the prevailing temperature conditions, microalgal strains should be selected as this enhances the growth of the strain under study (Slocombe and Benemann, 2016). The uptake of nutrients and cellular chemical composition of microalgae is also influenced by the changes in temperature. In certain cases, temperature stress has the ability to restrict nutrient interactions (Chen, Pan et al., 2012). In most of the cases, there is an increase in the growth of the microalgae with increase in temperature until it reaches an optimum, and then decreases with any further increase in temperature (Cassidy, 2011). Temperatures under 16°C and over 35°C are considered to be fatal for microalgal growth (Pachiappan, Prasath et al., 2015).
According to a study, the optimum temperature for growth of Chlorella vulgaris was reported to be between 25°C to 30°C (Chinnasamy, Ramakrishnan et al., 2009). A study on unidentified Chlorella sp. and Chaetoceros calcitrans at temperatures of 20°C, 25°C and 30°C revealed that the highest growth rate of the species was achieved at 25°C and 30°C respectively. There was no significant change detected in the growth of culture at other temperatures (Adenan, Yusoff et al., 2013). An investigation was carried out on the growth rate of 4 species of microalgae (Phaeodactylum tricormutum, Tetraselmis gracilis, Chaetoceros sp.and Minutocellus polymorphus) at temperatures ranging from 11°C to 36°C. The study revealed that the growth rate of Phaeodactylum tricormutum was highest at 16°C to 26°C; Tetraselmis gracilis showed maximum growth at 11°C to 16°C; Chaetoceros sp.and Minutocellus polymorphus showed highest growth at 31°C (Sigaud and Aidar, 1993). A study by Ha Le Thi Loc revealed that the most conducive temperature was 28°C for growth of Tetraselmis sp.and it attained the highest cell density of 196 x 104cells/ml on day 18. The suitable temperature range for the growth of this micro-alga was from 22°C to 31°C. The growth of the algae began to fall rapidly at 34°C after the first few days of culturing, indicating that high temperature was not suitable for its growth (Ha, 2000). Chlorella Zofingiensis thrived at an ambient temperature of 28°C (Travieso Córdoba, Domínguez Bocanegra et al., 2008). Kessler studied the growth rate versus optimal temperatures for 14 different strains of Chlorella sp. and revealed that they grew successfully between 26°C to 36°C (Kessler, 1985). The optimal temperature for the growth of Scenedesmus almeriensis was 35°C and was competent of withstanding 48°C after which cell death occurred (Sánchez, Fernández-Sevilla et al., 2008). An analysis found that Scenedesmus sp. LX1 could grow within a temperature range of 10°C to 30°C (Xin, Hong-Ying et al., 2011). A study reported that the growth of three strains of Dunaliella salina isolated from 60 saline soil samples exhibited the highest growth at 22°C (Wu, Duangmanee et al., 2016). It was found that Dunaliella was able to withstand within a temperature range of 0°C to 45°C. Dunaliella antarctica was able to flourish at subzero temperatures. Though, Dunaliella flourished at temperatures above 40°C, it culminated in decreasing the microalgal growth but sequentially there was an increase in the carotenoid content. Hence, the optimal growth of Dunaliella sp.was 32°C with a broad optimum range between 25°C to 35°C (Hosseini Tafreshi and Shariati, 2009). Nannochloropsis salina grew well at an optimal temperature of 26°C with no growth detected above 35°C (Van Wagenen, Miller et al., 2012).
A study disclosed that Nannochloropsis oculata grew well at a temperature of 20°C and there was a gradual decrease in growth as the temperature increased (Converti, Casazza et al., 2009). An investigational study on Nannochloropsis ocenaica exhibited that the growth of the species was highest at 20°C and was incapable to growat high temperatures of 40°C to 50°C (Rai and Rajashekhar, 2014). Experiments conducted on Nannochloropsis gaditana showed that the highest cell growth was obtained at a temperature of 25°C (Al-Adali, Ahmed et al., 2012). A study on the microalgae Tetraselmis subcordiformis cultured at 15, 20, 25, 30 and 35°C indicated that it grew best at 20°C (Wei, Huang et al., 2015). Haematococcus pluvalis cultivated under different temperature conditions of 20°C, 23.5°C, 27°C and 30.5°C revealed that the culture growth rate and biomass productivity increased at 30.5°C (Giannelli, Yamada et al., 2015). The growth of the unicellular micro-alga Isochrysis galbana was studied under laboratory conditions at different temperatures of 15, 17, 22, 27, 33 and 35°C. The optimal temperature for obtaining maximum growth was 27°C. Temperatures more than 32°C or less than 19°C decreased the growth of the microalgae remarkably (Kaplan, Cohen et al., 1986). Growth responses for Pithophora oedogonia and Spirogyra sp. at different temperatures were determined. The results indicated that Pithophora oedogonia had a maximum growth rate at 35°C and exhibited inhibited growth at 15°C, indicating that the species was warm stenothermal. Similarly, Spirogyra exhibited maximum growth at 25°C and showed moderate inhibition at 15°C and 35°C, suggesting that this species was eurythermal over the temperature range employed (O’Neal and Lembi, 1995). Therefore, it is predicted that the optimum temperature for the growth of the majority of microalgal species is between 15°C to 35°C although some exceptions exist.
Light impacts the growth of microalgae under any one of the three different conditions namely light limitation, light saturation and light inhibition. When the condition is light limiting, the growth of algae increases with increase in light intensity. At light saturation, the photosynthetic activity decreases as the photon absorption exceeds the amount of electron turnover, thereby inhibiting photosynthesis. When the light intensity is further increased, an irreversible damage occurs to the photosynthetic apparatus, and this process is termed as photo-inhibition (Chang, Show et al., 2017). The duration and intensity of light, therefore, directly affects the growth and photosynthesis of microalgae. A discovery disclosed that microalgae tend to flourish under either blue (λ~420-470nm) or red light (λ~660nm). It was also observed that red to far-red light accelerated the growth of microalgal cells (Schulze, Barreira et al., 2014). Microalgae require both light and dark periods for photosynthesis. It requires light for photochemical phase for synthesizing Adenosine triphosphate, Nicotinamide adenine dinucleotide phosphate oxidase and darkness for biochemical phase. It is in the biochemical phase that the microalgae build up molecules for growth. Investigations disclosed that increase in the duration of light intensity directly increases the growth of microalgae (Al-Qasmi, Raut et al., 2012).
The light intensities for Chlorella vulgaris was regulated at 3960lux, 7920lux and 11920lux with no control over pH. The light/dark period was 12h/12h. It was observed that the maximum growth of cells was observed under 3960lux (Gong, Feng et al., 2014). The effect of light illumination was experimented on Scenedesmus obliquus for intensities varying from 10µmol m-2 s-1 to 1000µmol m-2 s-1. The highest growth was detected at 150µmol m-2 s-1 after which the increase in light intensity did not improve the growth rate of microalgae indicating that the point of saturation for photosynthesis was reached (Sforza, Gris et al., 2014). Scenedesmus almeriensis had greater resistance to higher irradiances showing no signs of photo inhibition even at the maximum irradiance of 1625µE m-2 s-1. The biomass productivities were the highest (0.66g/L day) at this light intensity (Sánchez, Fernández-Sevilla et al., 2008). Dunaliella salina CCAP 19/30 had the ability to modify their photo systems to achieve maximum photosynthesis even when they were exposed to higher light intensities. When the light intensity was increased above 1000 µmol photons m-2 s-1, cells displayed photo damage, however, the growth rate increased with increased light intensity (Xu, Ibrahim et al., 2016). Dunaliella bardawil DCCBC 15 and Dunaliella salina CCAP 19/18 were inspected for their growth at light intensities of 50, 100 and 150µmol photons m-2s-1 and the results showed that the optimal growth of Dunaliella was obtained at 50 µmol photons m-2s-1 light intensity and the growth rate decreased with increasing light intensity (Vo and Tran, 2014). Nannochloropsis salina was exposed to varying intensities of 5, 25, 50, 100, 250 and 850µmol m-2 s-1. The growth rate increased with light intensity and the highest growth rate was achieved at 250µmol m-2 s-1; however, photon conversion efficiency decreased for light efficiencies above 55µmol m-2 s-1 (Van Wagenen, Miller et al., 2012). The growth rate of Nannochloropsis ocenaica increased exponentially when exposed to light intensities in the range of 34-80µmol photons m-2s-1 and reached a maximum at 80µmol photons m-2 s-1 (Sandnes, Källqvist et al., 2005). Therefore, light illumination has a major influence on algae cultivation and light use efficiency must be optimal in order to achieve maximum productivity. In fact, sunlight provides the light required supporting metabolism, but if present in excess it leads to oxidative stress and photo inhibition thereby reducing photosynthetic efficiency (Sforza, Simionato et al., 2012). A study on the growth of Odontella aurita under two light intensities of 150µmol photons m-2s-1 and 300µmol photons m-2s-1 revealed that the micro-alga was able to grow under 150µmol photons m-2s-1, however, the alga grew faster at early stages under high light (300µmol photons m-2s-1). This was due to the low cell density at early stages which enabled the cells to receive additional amount of irradiance under high light conditions (Xia, Wan et al., 2013). The biomass concentration of Neochloris oleoabudans HK-129 increased from 1.2g/L to 1.7g/L when the light intensity was increased from 50 to 200µmol m-2 s-1 (Sun, Cao et al., 2014).
The growth of Chlamydomonas reinhardtii increased when the light intensities were varied from 60 – 300µE m-2s-1. However, there was little difference in growth between 200 and 300µE m-2s-1 and it was concluded that the light intensity of 200µE m-2s-1 was conducive for the growth of the species (Pyo Kim, Duk Kang et al., 2006). A study on the growth of Isochrysis sp.under different illuminations levels (100%, 73.6%, 57.6%, 29%, and 0%) of natural sunlight revealed that the maximum growth rate was attained under optimal illumination exposure of 73.6% and light-dark cycle of 43.66-28.28 s/height of column. This revealed that optimum photosynthetic process; cell growth rate and carbon fixation in microalgae could be achieved by altering both dark and light regimes. Direct exposure of the microalgae to sunlight could potentially damage the cells while unavailability of light could impact the growth of the microalgae in a negative way (Harun, Yahya et al., 2014). The growth of four microalgal strains namely Chlorella vulgaris, Pseudokirchneriella subcapitata, Synechocystis salina and Microcystis aeruginosa were studied under various light irradiances (36, 60, 120 and 180µE m-2s-1)and light:dark ratio(10:14, 14:10 and 24:0). The results observed showed that highest growth rate and biomass productivity for all the species under study was achieved at an irradiance of 180µE m-2s-1 and light:dark ratio of 24:0 (Gonçalves, Simões et al., 2014). Therefore, the optimal light illumination required for the growth of microalgal species is considered to be in between 50µmol m-2s-1 to 200µmol m-2s-1 except Scenedesmus almeriensis which has the capability to withstand higher irradiances. Altering the light-dark cycles could also positively or negatively impact the growth of microalgae to a certain extent.
The pH is believed to be the one of the underlying parameter that controls the cell metabolism and formation of biomass in microalgae. The growth of majority of the microalgal species flourished at neutral pH and all strains of microalgae seem to have a limited optimal range of pH (Lutzu, 2012). According to the physiology of microalgae, it is observed that either tilacoid or chloroplast carry out the vital functions at specific pH. The media pH also influences the process of photosynthesis in microalgae. Extremes of pH, that is, high as well as low pH reduce the rate of photosynthesis. At high pH, the trend of absorption of the trace metals and nutrients might get altered. Similarly at low pH, enzyme inhibition occurs in the photosynthetic process and media contamination by micro-organisms becomes unavoidable (Bakuei, Amini et al., 2015). The medium pH has an affiliation to the carbon dioxide concentration, that is, the pH increases steadily as carbon dioxide is consumed. The pH also influences the availability of nutrients such as iron and organic acids (Lutzu, 2012). Hence, pH is considered to be a major environmental factor that is regulated by carbonate equilibrium both in oceans and inland waters. The pH in oceans is 8 ± 0.5, however, it fluctuates from <2 to 12 in lakes and rivers (Weisse and Stadler, 2006). Algae were found to survive at both alkaline and acidic pH (Ying, Zimmerman et al., 2014). The effect of pH on Chlorella vulgaris species revealed that the microalgae exhibited reduced growth at acidic (3.0 – 6.2) and alkaline (8.3 – 9.0) pH. However, optimal growth was achieved when the pH was between 7.5 and 8.0 (Rachlin and Grosso 1991). The optimum pH for the growth of Spirulina platensis was observed to be in between pH 7.0 to pH 9.0. The maximum growth rate for the microalgae was observed at pH 8.0, followed by pH 9.0 and then pH 7.0 suggesting that moderate alkalinity was necessary for the ideal growth of the microalgae (Fagiri, Salleh et al., 2013). Scenedesmus almeriensis grew effectively at a pH of 8.0 with decrease in growth at higher pH and exhibited tolerance to neutral pH (Sánchez, Fernández et al., 2008). Scenedesmus obliquus grew well in neutral as well as in weakly alkaline conditions and the maximum growth was observed at a pH of 8.0 (Yang, Li et al., 2016).
The growth of Scenedesmus sp. (ADIITEC-II and GUBIOTJT116) at various pH levels ranging from 5.0 to 9.0 showed that the maximum specific growth rate and biomass productivity for the species was achieved at a pH of 7.0. In collation, acidic conditions (pH 5.0 and pH 6.0) did not alter the cell density and demonstrated lower biomass productivity (Difusa, Talukdar et al., 2015). The initial pH for Scenedesmus sp. strain R-16 was varied from pH 3.0 to pH 12.0 and it was observed that the alga had strong tolerance to pH and grew well in between pH of 4.0 to 11.0. At pH 3.0 and pH 12.0, the algal cells exhibited poor growth. The micro-alga exhibited the highest biomass productivity at a pH of 7.0 (Ren, Liu et al., 2013). Dunaliella salina exhibited maximum growth at pH of 9.18 when compared to pH between 6.75 and 7.2 (Abu-Rezq, Al-Hooti et al., 2010). The effect of pH on the growth of Dunaliella bardawil and Chlorella ellipsoidea over a wide range of pH (pH 4.0 to pH 11.0) showed that the ideal pH for the growth of the species was 7.5 and 10.0 respectively. The growth of Dunaliella bardawil and Chlorella ellipsoidea was completely retarded over a pH of 10.0 which was disclosed the fact that no carbon was accessible for the algae because carbonate ion was considered vital source of inorganic carbon (Khalil, Asker et al., 2010). The optimum growth of Nannochloropsis salina was observed at a pH of 7.5 to 8.0; however the micro-alga had the capability to grow over a wide range of pH(5.0 to 10.5) (Boussiba, Vonshak et al., 1987). Another study on the growth of Nannochloropsis salina at six different pH levels (pH 5, 6, 7, 8, 9 and 10) revealed that highest growth rate was achieved at a pH of 8 and 9. The ideal pH for growth of the microalgae was found to be pH 7.7 (Bartley, Boeing et al., 2014). The optimum pH for growth of Nannochloropsis oculata was validated using Response Surface Methodology and was found to be 8.4 (Spolaore, Joannis‐Cassan et al., 2006). Influence of media pH on Chlorococcum sp. revealed that the maximum growth of the microalgae was observed at a pH of 8.0 and the growth rate was 0.066 h-1 (Zhang, Ng et al., 1997). According to a study, the ideal pH for the growth of Tetraselmis sp. was observed to be at a pH of 8.5 when exposed to different levels of pH (5.5, 7.5, 8.5 and 9.5) (Khatoon, Rahman et al., 2014). A series of experiments to investigate the effect of pH on the growth of Nannochloris eucaryotum revealed that the maximum growth of 9.85±0.54×10-4 h-1 was achieved when the pH was controlled at 6.60±0.67 (Lutzu, 2012). A study on the growth of Chlamydomonas applanata within a pH range of 1.4 to 8.4 showed that no growth was observed from pH 1.4 to 3.4. Optimum growth was obtained for pH ranging from 5.4 to 8.4, while the maximum growth was observed at pH 7.4 (Visviki and Santikul, 2000).The growth response of Chlamydomonas acidophila was examined at pH ranging from 3.4 to 8.4 with an increment by 1point. Analysis of variance showed that growth was maximum at pH 7.4 with no growth observed at pH 1.4 and 2.4 (Visviki and Palladino, 2001). Euglena mutabilis exhibited the highest growth between pH 3.4 and pH 5.4 and it was able to survive over a wide range of pH (pH 1.4 to pH 7.9) (Dach, 1943). When the pH was 0.9, there was reduced growth and within 24 hours all the microalgal cells were dead. At pH 1.4 and pH 1.9 some cells were alive for 12 and 13 days respectively. It can be inferred that from the above discussion that the pH for growth of most of the algal species is 7.0 to 9.0, with the optimum pH range being between 8.2 to 8.7, although some species which dwell in both acidic and basic environments are also existent (Blinová, Bartošová et al., 2015).
Each strain of microalgae displays differences in adjusting to salinity. Salt stress affects cell growth and lipid formation. It was noted that as salinity increases, the expression of lipids increased but resulted in dwindling cell growth. Since, the two important traits that the researchers look for before selecting a microalgal strain for study is the ability of the algae to produce high biomass and lipid, considerable importance is given to microalgae which flourish in saline environment (Asulabh, Supriya et al., 2012). Marine microalgae are exceptionally tolerant to alterations in salinity when compared to freshwater species (Blinová, Bartošová et al., 2015). Spirulina platensis exposed to different concentrations of sodium chloride ranging from 0.1 M to 0.4 M revealed that the growth of micro-alga was higher at lower concentrations of sodium chloride (0.1 M and 0.2 M) and the growth reduced at higher concentrations (0.3 M and 0.4 M) (Sujatha and Nagarajan, 2014). Chlorella sp.were exposed to different salinities namely 0g/L, 30g/L, 35g/L and 40g/L of BG11 medium with deficient amount of sodium nitrate. The salinity was adjusted using sodium chloride. It was reported that as the salinity increased the growth of the microalgae decreased. The biomass concentration was high at 0g/L (Andrulevičiūtė, Skorupskaitė et al., 2011). The effect of salinity on growth of Scenedesmus almeriensis was carried out with different salinities (brackish water, sea water and fresh water). It was found that high number of cells was found in fresh water indicating that lower the salinity higher the growth of micro-alga (Suyono, Haryadi et al., 2015). However, Scenedesmus almeriensis showed higher tolerance to medium salt concentrations of 0.1 M sodium chloride and also showed higher biomass productivities at 0.1 M sodium chloride when compared to productivities observed in fresh water media (Benavente-Valdes, Aguilar et al., 2016).
The measurement of effect of salinity on growth of Scenedesmus obliquus was conducted at various concentrations of sodium chloride (0.05, 0.3, 0.6, 1.0, 2.0 and 3.0 M). The growth of Scenedesmus obliquus was inhibited at sodium chloride concentrations above 0.6 M. There was reduced growth at 0.3 M. At 0.05 M NaCl. The highest growth of the microalgae was observed at 0.05 M NaCl and it was equivalent to the growth that was obtained in fresh water. The results, thus, suggested that low to median salinities, that is, 0 M to 0.05 M were appropriate for the promotion of growth rate of Scenedesmus obliquus (Kaewkannetra, Enmak et al., 2012). Dunaliella bardawil was exposed to salinity levels ranging from 1M to 3M. The results revealed that the maximum growth rate was observed at lowest salinity of 1 M (Gomez, Barriga et al., 2003). A decrease in cell growth of Dunaliella tertiolecta ATCC 30929 was observed when the concentration of sodium chloride was increased from 1.0 M to 2.0 M. Hence, sodium chloride concentration of less than 1.0 M was considered to be appropriate for achieving high cell concentration (Takagi, Karseno et al., 2006). Dunaliella salina being a marine micro-alga has the ability to tolerate high salinity. Dunaliella salina CCAP 19/18 was inspected for its growth under different salinities (1 M, 1.5 M and 2.0 M). The ideal growth for Dunaliella was obtained at 1.5 M and 2.0 M salinities (Vo and Tran, 2014). Nannochloropsis salina was exposed to different salinity levels of 10, 22, 34, 46 and 58 PSU. Being a marine micro-alga, it exhibited highest growth rate at 22PSU and highest biomass accumulation at salinities of 22 PSU and 34 PSU. Nannochloropsis salina exhibited no growth above salinity of 58 PSU and below 10 PSU (Bartley, Boeing et al., 2013). The effect of salinity on growth of Nannochloropsis oculata CS 179 was carried out at various salinities (15%, 25%, 35%, 45% and 55%). The results depicted that the highest biomass was obtained at a salinity of 25% species (Gu, Lin et al., 2012). The marine micro-alga Tetraselmis suecica was capable of tolerating wider range of salt concentrations. The cultures were grown at 48 different salinity conditions from 0% to 35%. The ideal growth was achieved between 25% and 35% with a maximum cellular density of 1.3×106 cells/ml (Fabregas, Abalde et al., 1984). Four species of microalgae Desmodesmus armatus, Mesotaenium sp., Scenedesmus quadricauda and Tetraedron sp., were cultured at 2,8,11 and 18 ppt salinity for determining their growth at these salinity levels. Desmodesmus armatus showed maximum tolerance to salinity growing actively at 18 ppt while Mesotaenium sp., was less halotolerant with the growth rate decreasing from 11 ppt. Therefore, the ideal salinity level for the growth of Mesotaenium sp., was observed to be from 2 ppt to 8 ppt. Both Scenedesmus quadricauda and Tetraedron sp., grew well at salinity level of 2 ppt and 8 ppt (Von Alvensleben, Magnusson et al., 2016). The growth of Schizochytrium limacinum OUC88 at various salinities (0, 0.9, 1.8, 2.7 and 3.6% w/v) was analyzed. The strain performed better and the biomass remained steady with salinity at 1.8, 2.7 and 3.6% w/v. When there was a decrease in salinity from 0.9% to 0%, there was significant reduction in the biomass productivity (Zhu, Zhang et al., 2007). The growth of Botryococcus braunii under various salinities (17 mM, 34 mM, 51 mM, 68 mM and 85mM) revealed that though the micro-alga was able to grow at all salinity levels, however, the maximum growth rate was observed at the lowest salinity level of 17 mM (Rao, Dayananda et al., 2007). A study on the effect of salinity on three microalgal strains, Crypthecodinium cohnii ATCC 30556, Crypthecodinium cohnii ATCC 50051 and Crypthecodinium cohnii RJH revealed that C.cohnii ATCC 30556 had its maximum growth rate of 0.090 h-1 at a sodium chloride concentration of 9.0 g/L whereas C.cohnii ATCC 50051 and C.cohnii RJH had their maximum growth rates of 0.049 h-1 and 0.067 h-1respectively at a sodium chloride concentration of 5.0 g/L. When an optimum salinity had reached, the growth rate decreased with increasing salinity. Almost no growth was observed, when the medium did not contain sodium chloride, and at extremely high sodium chloride concentrations, growth was inhibited, and the cells were elongated. The elongation of the cells was attributed to the increase in external ionic concentrations that tend to inhibit cell growth (Jiang and Chen, 1999). Therefore, it can be seen that salinity is one of the important factor affecting growth especially for salt water microalgal species (Gu, Lin et al., 2012). Hence, salinities of 20-24 g/L have been considered to be optimal except for some strains of marine microalgae (Blinová, Bartošová et al., 2015).
Today about 85% of the world’s energy demand is satisfied by burning fossil fuels. The levels of carbon dioxide in air have risen from 260 ppm to 380 ppm lately. A number of suggestions has been made and studied to minimize such emissions in a sustainable way (Minillo, Godoy et al., 2013). The sum of fossil fuels being ignited is directly proportional to the increase of carbon dioxide in air. The increasing concentration of carbon dioxide in the air is considered to be one of the main causes for global warming. Therefore, fixing carbon dioxide biologically can be considerably used to solve this problem (Salih, 2011), or the elimination of carbon dioxide from the point source could also be investigated (Li, Luo et al., 2012). Capturing carbon and sequestering it is considered to be a safer technology to reduce the environmental carbon dioxide. Microalgae can fix carbon dioxide with efficiencies greater than that of terrestrial plants. The selection of microalgal species is important for attaining biological carbon dioxide systems which work and the microalgal species selected depends on the strategy involved for carbon sequestration. The amount of carbon dioxide in air plays a major role in the growth of microalgae, that is, higher the concentration of carbon dioxide the better the growth (Khairy, Shaltout et al., 2014; Salih, 2011). A study on the effect of carbon dioxide concentrations (Control, 280μatm, 385μatm, 550μatm, 750μatm and 1050μatm) on the growth of Chlorella gracilis showed that there was an increase in the cell number when the carbon dioxide concentration was 385μatm. There was a decrease in growth observed at 550μatm as the micro-alga was not tolerant above this limit (Khairy, Shaltout et al., 2014).
A study on Chlorella vulgaris ARC1 examined under different carbon dioxide concentrations (0.036% to 20%). The results obtained showed that Chlorella vulgaris had the ability to sequester 38.4mg of CO2 L/day at elevated carbon dioxide concentration of 6% thereby increasing the growth of biomass (Chinnasamy, Ramakrishnan et al., 2009). An investigation on the growth rate of three species Chlamydomonas reinhardtii, Chlorella pyrenoidosa and Scenedesmus obliquus disclosed that as the concentration of carbon dioxide was increased the growth of microalgae also increased but attained saturation at 30, 100 and 60µM of carbon dioxide respectively (Yang and Gao, 2003). Scenedesmus obliquus showed increased biomass (2.3g/L) at 15% carbon dioxide concentration (Singh and Singh, 2014). An experiment to study the consequence of carbon dioxide concentration on biomass of microalgae was organized from May to November on a lake in Denmark. Three enclosures with free carbon dioxide equivalent to 10 times atmospheric equilibrium and three enclosures at atmospheric equilibrium were taken. The biomass of the algae under study was significantly higher in the carbon dioxide enriched enclosures than in enclosures at atmospheric equilibrium (Andersen and Andersen, 2006). The microalgal strain Botryococcus braunii LB-572 was exposed to various concentrations of carbon dioxide (Control, 0.5%, 1% and 2%, v/v) and the growth pattern was studied. The results revealed that at 2% v/v of carbon dioxide, the growth of the microalgal strain flourished, however the micro-alga also exhibited growth at other concentrations (Ranga Rao, Sarada et al., 2007). A study on Dunaliella salina disclosed that there was no alterations in growth for change in concentrations of carbon dioxide from <230 ppm to 5100 ppm, thus showing that carbon dioxide had no effect on the growth of Dunaliella salina (King, Jenkins et al., 2015). Nannochloropsis oculata exhibited decreased growth at elevated carbon dioxide concentrations. The culture of Nannochloropsis oculata NCTU-3 was investigated for its growth at various carbon dioxide concentrations (2%, 5%, 10% and 15% v/v). Microalgae exhibited reduced growth at 5%, 10% and 15% v/v of carbon dioxide. However, the growth of microalgae was enhanced when aerated with 2% v/v of carbon dioxide concentration (Chiu, Kao et al., 2009). Spirulina platensis was exposed to carbon dioxide concentrations of 0%, 0.5%, 1% and 2% v/v to study their growth pattern. The pH decreased with increase in percentage carbon dioxide concentration. The results revealed that the alga grew well at carbon dioxide concentrations of up to 1% v/v though the difference in growth was insignificantly small when compared with 2% v/v carbon dioxide concentration. The productivity of the microalgae was increased to 60% when it was exposed to 1% of carbon dioxide (Ravelonandro, Ratianarivo et al., 2011). The effect of carbon dioxide concentration on Chlorocuccum littorale at concentrations of 5%, 20%, 35% and 50% respectively revealed that the growth decreased with increasing carbon dioxide concentration (Ota, Kato et al., 2009). It is seen that carbon dioxide does not alter the microalgal growth for all strains discussed except for some strains.
The growth of the algae is directly proportional to the uptake rate of the most limiting nutrient and is described by the Michaelis-Mentis equation as given below.
where µ is the growth rate, µmax is the maximal growth rate, S is the concentration of the limiting nutrient, and K the concentration which leads to half-maximal growth rate, called the half-saturation constant (Titman, 1976). Nitrogen is considered to be a building block for proteins and nucleic acids whereas phosphorus forms a part of phospholipids. If these macronutrients are limited then it tends to shift the metabolic pathways (Juneja, Ceballos et al., 2013). Redfield had stated that when the ratio of N/P exceeds 16, then phosphorus was considered to be the limiting factor and nitrogen content needs to be controlled to optimize the growing condition of microalgae. The requirement of optimum level of phosphorus was considered to be conducive for growth of microalgae. If the total phosphorus content was less than 0.045mg/L or greater than 1.65mg/L, then growth was prohibited. If the total phosphorus content equals 0.02mg/L the growth of microalgae was enhanced (Ren, 2014). A study on Chlorella vulgaris and Nannochloropsis oculata disclosed that if the supply of nitrogen was decreased, the lipid synthesis had increased but no effect on the growth pattern of microalgae was observed (Paes, Faria et al., 2016). Dunaliella sp. was able to accumulate high amount of carotenoids and astaxanthin when deprived of nitrogen. It has been observed that phosphorus was the primary limiting nutrient for microalgae. In Scenedesmus sp., it was observed that if phosphorus was the limiting factor, the lipid content would increase from 23% to 53% (Juneja, Ceballos et al., 2013). For Scenedesmus species LX1, nitrogen and phosphorus limitation increased the lipid content but the growth was low (Xin, Hong-ying et al., 2010). If the nitrogen content in the medium was reduced to 75% for Nannochloropsis salina, an increase in lipid content from 34.6% to 59.3% would be observed with a significant reduction in growth (Fakhry and El Maghraby, 2015). Trace metals are present in algal cells in extremely small quantities. Iron, manganese, cobalt, zinc, nickel and copper are some of the important trace metals required by algae for their metabolic functions (Bruland, Donut et al., 1991). Therefore, from the above discussion it can be inferred that the nitrogen and phosphorus starvation has a significant effect on the growth of microalgae either positively or negatively depending on the species.
Illumination and temperature of microalgae cultures are dependent on each other. A strict control of both is difficult and expensive due to refrigeration needs. Mixing is the most practical way to ensure even distribution of light to all the cells, thereby improving the light regime (Rocha, Garcia et al., 2003). Shading is a common problem that occurs in microalgae and it inhibits the growth by prohibiting the microalgae from absorbing light. Therefore, it is important that proper mixing is provided to the microalgae and this can be done by gas mixing when photo bioreactors are used for cultivation of microalgae (Ren, 2014). The effect of mixing on Spirulina platensis in three ways; mixing with a magnetic agitator inside the column, recirculating through a pump and bubbling air into the column, showed that the growth of the micro-alga remained the same before and after mixing (Ravelonandro, Ratianarivo et al., 2011). However, more researches have to be carried out to get a better understanding of this aspect.
Selection and screening of Microalgal strains
Isolation is a necessary process to obtain pure cultures and is generally the first step towards the selection of microalgal strains. The traditional isolation techniques include the use of micropipette for isolation under a microscope or cell dilution followed by cultivation in liquid media or agar. Single cell isolation by a micropipette is a technique which is followed widely today because it can be used for a wide range of samples and cost effective. An automated single cell isolation technique used today is flow cytometry. A bioinformatics approach could also be done to discover new algal isolates. The steps involved in obtaining data for phylogenetic analysis include primers design, DNA/RNA extraction, PCR amplification and Sequencing (Duong, Li et al., 2012). After isolation of algal strains, first level of screening is done using lipophilic fluorescent dye Nile Red as it has a stable fluorescence intensity that is unaffected by the broadest range of conditions (Cirulis, Strasser et al., 2012). Strains that pass the first level of screening are subjected to a second level of screening to determine their growth in media other than the initial isolation medium. For fresh water strains, a C medium, BG11 medium and BBM were used as these media have different concentration of nutrients. For marine strains, it is normally F/2 medium. The best growth medium for the algal strain is selected and then subjected to third level of screening. This third level screen evaluated the growth potential of the strains under conditions of sufficient CO2, constant pH and no O2 accumulation, in contrast to the CO2 limiting and uncontrolled pH, and potentially O2 inhibitory conditions present in the second level of screening. Strains that grow potentially well under both conditions are scaled-up. Therefore, strain bioprospecting is considered to be the first step in making microalgal technologies commercially successful as the selection and screening of microalgal strains play an important role in the biomass productivities.
Microalgae are considered to be a valuable bioresource and are recently receiving a lot of attention. For the past four to five decades, significant progress has been made in understanding the growth of microalgae. The present review underlines the different environmental conditions that affect the growth of a number of microalgal species and also discusses how mixing and screeening and selection of microalgal strains prove crucial for making microalgal technologies a reality in the future. The optimal conditions of temperature, light, pH, salinity and carbon dioxide is discussed inorder to understand better the growth of microalgae. Another aspect, that is, screening and selection of microalgal species is also discussed as this affects the biomass productivities of microalgae to a certain extent. Based on the literature reviewed, it is clear that temperature and light are the most important parameters that influence microalgal growth. Change in pH, salinity and carbon dioxide are also important, but this solely depends on the species under study. The microalgal species discussed in this review provide a potential for future studies as these species are considered to be of great interest among researchers. Evidence in this review suggest that microalgal growth is directly proportional to the envrionmental conditions in which the algae are grown. Therefore, it becomes important to maintain optimum conditions for microalgae to grow as it directly influences the biomass productivity and in turn the amount of value added products obtained from them. Bioprospecting in habitats which are yet to be explored may provide an avenue for identifying microalgae with novel properties and usefulness. Future studies, should be focussed on optimizing the environmental conditions, so that maximum growth can be obtained. Importance of mixing of microalgal culture should be given emphasis and the number of researches carried out in this regard should be increased. Therefore, continued research for creating novel and innovative technologies for harvesting microalgae are required to make the exploitation of microalgae economically viable, sustainable and competitive.
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