Growth Characteristics of Different Algal Species

Abstract

Algae are large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multi-cellular forms (Singh J, Gu S, Renew Sustain Energy Rev 14:2596–2610, 2010). Productivity of these photosynthetic microorganisms which converts CO₂ into carbon-rich lipids is only a step or two away from biofuel which in turn is produced by several chemical, biochemical and thermochemical processes (Wijffels RH, Barbosa MJ, Science 329:796–799, 2010; Kirrolia A et al., Renew Sustain Energy Rev 20:642–656, 2013; Beneroso D et al., Bioresour Technol 144:240–246, 2013). Globally algal biofuel has been considered as 3^rd and 4^th generation biofuel based on its potential over 1^st and 2^nd generation crop based biofuels. Numerous scientists have discovered various applications of algal biomass apart from biofuel applications for the production of value added products to reduce its production cost towards bio-refinery approach (Rawat I et al., Appl Energy 103:444–467, 2013). Wijffels and Barbosa (Science 329:796–799, 2010) reported in Science about the broad prospect of microalgae over terrestrial crop based biofuel. In their report they mentioned how a 50-year-old concept came into focus during the oil crisis of 1970s. Since then over millions of algal species have been isolated, identified and studied towards its potential for biofuel and value added products. Table 3.1 represents microscopic view of some potential algal strains which has been studied as model organism at lab-scale and pilot-scale. Recent studies suggest that green algae are promising species bearing a substantial potential to obtain various products in a biorefinery concept (Suali E, Sarbatly R, Sustain Energy Rev 16:4316–4342, 2012). The algal oil can be transesterified to fatty acid methyl ester (FAME) and non-lipid components of algal biomass such as carbohydrates and proteins can be used for the production of bioethanol, biobutanol, neutraceuticals and animal feed (Kirrolia A et al., Renew Sustain Energy Rev 20:642–656, 2013). Moreover, the residue biomass cake can be used further to produce liquid fuel using the process of pyrolysis (Beneroso D et al., Bioresour Technol 144:240–246, 2013).

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3.1 Introduction

Algae are large and diverse group of simple, typically autotrophic organisms, ranging from unicellular to multi-cellular forms (Singh and Gu, 2010). Productivity of these photosynthetic microorganisms which converts CO₂ into carbon-rich lipids is only a step or two away from biofuel which in turn is produced by several chemical, biochemical and thermochemical processes (Wijffels and Barbosa, 2010; Kirrolia et al., 2013; Beneroso et al., 2013). Globally algal biofuel has been considered as 3rd and 4th generation biofuel based on its potential over 1st and 2nd generation crop based biofuels. Numerous scientists have discovered various applications of algal biomass apart from biofuel applications for the production of value added products to reduce its production cost towards bio-refinery approach (Rawat et al., 2013). Wijffels and Barbosa (2010) reported in Science about the broad prospect of microalgae over terrestrial crop based biofuel. In their report they mentioned how a 50-year-old concept came into focus during the oil crisis of 1970s. Since then over millions of algal species have been isolated, identified and studied towards its potential for biofuel and value added products. Table 3.1 represents microscopic view of some potential algal strains which has been studied as model organism at lab-scale and pilot-scale. Recent studies suggest that green algae are promising species bearing a substantial potential to obtain various products in a biorefinery concept (Suali and Sarbatly, 2012). The algal oil can be transesterified to fatty acid methyl ester (FAME) and non-lipid components of algal biomass such as carbohydrates and proteins can be used for the production of bioethanol, biobutanol, neutraceuticals and animal feed (Kirrolia et al., 2013). Moreover, the residue biomass cake can be used further to produce liquid fuel using the process of pyrolysis (Beneroso et al., 2013).

Table 3.1 Microscopic view of potential algae

Bearing substantial feature towards biorefinery approach, triumphing significant biomass through cost effective process has always been a pronounced challenge (Acién et al., 2012). Amongst the several challenges, isolating and identifying potential algal strains having fast growth rate, high biomass yield, and significant lipid productivity are vital. In identifying potential algal strains, growth characteristics play significant role which further depends on growth medium, growth parameters, cultivation system, CO₂ concentration, algal growth rate, biomass productivity, and many more (Roleda et al., 2013). The current chapter elaborates the growth characteristics of different algal species studied in recent time and their feasibility towards algal biorefinery approach.

3.2 Potential Algal Strains

Photosynthetic algae represent an exceptionally diverse but highly specialized group of organisms of which microalgae exists with 1 to 10 million (upper limit) species in the nature (Norton et al., 1996). However, only few have shown the potential to be grown successfully in lab-scale and pilot-scale using photobi oreactors and raceway ponds s towards production of biofuel and value added products (Wu et al., 2012). Reports suggest that green algae have high prospective towards mass cultivation due to their high growth rate and lipid content which meets the present requirement of algal biorefinery concept (Singh and Gu, 2010). Potential algal strains can be obtained from the repositories present worldwide, but indigenous isolated algal strains will have an inherent adaptability that may be the competitive edge required for mass cultivation systems. In addition, they are able to adapt quickly to the changes in the local environment and climate (Abou-Shanab et al., 2011; Rawat et al., 2013). Indigenous algal strains can be isolated from different aquatic habitats such as pond, lake, river, etc. as well as salt water or brackish water lakes and oceans. Further different microorganism isolation techniques are used to isolate pure algal strains and identified morphologically and genetically. To screen model algal strain for future experiment and mass cultivation studies, those isolate strains undergo several lab-scale experiments such as growth rate estimation, photosynthetic efficiency, nutrient recovery, biomass productivity, lipid yield, lipid profiling etc. (Singh and Gu, 2010).

In recent time, many reports have been published on identifying potential algal strains and their feasibility towards biofuel production. Abou-Shanab et al. (2011) isolated 33 numbers of microalgae strains, from which screened eight strains and studied their potential towards biodiesel production. In evidence to their characteristics study they concluded that Scenedesmous obliquus YSR01 was the most potential strain towards biodiesel production. Chaichalerm et al. (2012) isolated six microalgae strains enriched in four different growth medium s for the study of their biomass yield, lipid content, and lipid productivity in batch mode. Based on their study, Chlorococcum humicola was identified as a potential strain. In contrast to isolating potential strains which can grow in artificial culture medium, there are several strains isolated having the potential to grow in wastewater. These algal strains were proficient towards removing nitrogen and phosphorus as growth nutrients while accumulating lipids in effluents from different domestic and industrial wastewater. Sydney et al. (2011) conducted a screening experiment on 20 algal strains and found positive growth on 13 strains towards nutrient removal and lipid accumulation in secondary domestic wastewater effluents. They concluded that Botryococcus braunii showed highest nutrient removal of 79.63 % on 14 days of cultivation period. Cai et al. (2013) in their review article on “Nutrient recovery from wastewater streams by microalgae: Status and prospects” stated, Chlorella sp. has been widely studied and shown to be effective in nutrient removal and high value biomass production. It has also been claimed that some species of Chlorella are heterotrophic or mixotrophic and can consume organic forms of carbon in addition to inorganic nutrients as part of their metabolic process. This can be an advantage when using wastewater streams containing carbon residues, such as digested dairy manure (Wang et al., 2010).

3.3 Algal Growth Characteristics

Algae are photosynthetic organisms whose growth characteristics are subjected to light intensity, growth medium (nitrogen, phosphorous, potassium and micronutrients) and carbon dioxide supply (Wang et al., 2010). Since long time many scientists have developed different growth conditions and optimized several growth medium to enhance algae growth rate towards high value biomass yield. Bold’s basal medium (BBM), BG-11, TAP and artificial sea water (ASW) are the most common nutrient mediums which have been widely used as the algal (green algae and cyanobacteria) growth medium (He et al., 2013; Basu et al., 2014; Kumar et al., 2014; Bellou and Aggelis, 2012). In recent times, wastewater containing nitrogen and phosphorous as major nutrients has also been widely studied replacing lab-grade nutrient medium as low cost cultivation process coupled with wastewater treatment (Cai et al., 2013). Moreover, recent studies suggest that there are several stress effects of nutrients, light, temperature, CO₂ and pH which affect the growth rate and biochemical properties in algal biomass (Suali and Sarbatly, 2012). These stress conditions can be applied during batch mode (single/two-step) or continuous mode (Roleda et al., 2013). As algal cultivation needs to be studied towards bio-refinery approach, achieving feasible biomass productivity during mass cultivation has always been a challenge. Acién et al. (2012) in their report suggested using low cost cultivation system in addition to flue gases as carbon source and wastewater as growth medium can help achieving substantial biomass yield up to 4.86 g L⁻¹ (Basu et al., 2014).

3.3.1 Methodology to Estimate Algal Growth Rate

Determining algal growth rate is essential in identifying potential algal strains during the screening process and validates its productivity towards mass cultivation prospect. There are several methods through which algal growth rate can be estimated of which specific growth rate and biomass productivity is essential.

3.3.1.1 Spectroscopic Growth Rate Observation

Algae are photosynthetic organisms which contain different pigments. Chlorophyll ‘a’ and ‘b’ are among such pigments which is present in green algae and cyanobacterias. Spectrophotometer is used to estimate these pigments and cell concentration in the culture medium. A wide range of wavelength (550–750 nm) is considered to identify algal cell concentration which is estimated at a specific time interval and algal growth curve formed (wave length vs time).

3.3.1.2 Biomass Yield

Besides algal cell concentration, growth rate can also be determined from its biomass growth which is estimated from the total biomass achieved from a specific culture time period. To obtain algal biomass, algal culture is washed repeatedly to remove the nutrients present in it and then moisture-free biomass is obtained through centrifugation followed by drying at approx. 108 °C. There are several kinetic equations available in literatures which were usually used to determine the biomass productivity and yield.

3.3.1.3 Kinetic Equations for Growth Study

Several kinetic equations are reported for algal growth estimation and among them specific growth rate in terms of algal density is quite popular. Besides, dry algal biomass has also been considered to estimate specific growth rate of algae (Xu and Boeing, 2014). ‘μ’ was determined from a linear fit in a semi-logarithmic plot of algal density against time.

$$ \upmu =\frac{ \ln \left(\raisebox{1ex}{${W}_t$}\!\left/ \!\raisebox{-1ex}{${W}_0$}\right.\right)}{t-{t}_0} $$

(3.1)

In equation (3.1), ‘W _t’ is algal biomass concentration (g L⁻¹) at the time ‘t’, W ₀ is initial biomass concentration (g L⁻¹) and ‘t’ is the experimental time (d). Specific growth rate can also be determined from the slope of a semi-log plot of biomass concentration versus time.

3.3.2 Growth Medium and Algal Growth Characteristics

Algae are known to have different nutrient requirements not only by composition but also by concentration of the nutrients supplied which is basically composed of macro- and micro-nutrients, dissolved ions, trace metals and several vitamins (Rawat et al., 2013). Growth media for algae are grouped depending on fresh water or salt water species. There is no universal growth media recipe that works for all taxa, so researchers are forced to give great care on how growth media is composed, stored and used. In general, algal growth medium is composed of macro- and micro-nutrients. Macro-nutrients required by algae, diatoms and cyanobacteria include carbon, nitrogen, phosphorous, silicon and major ions including Na, K, Mg, Ca, Cl and SO₄ as a base media. Micro-nutrients are trace amounts of essential elements and these include iron, manganese, zinc, cobalt, copper, molybdenum and a small amount of metalloid selenium (He et al., 2013).

In the past few decades, tremendous efforts have been put into research of microalgae cultivation in wastewater s for the removal of nitrogen, phosphorus and other elements (Cai et al., 2013). This process of algal bioremediation route towards biomass production makes algal cultivation process cost effective coupled with simultaneous wastewater treatment and biomass production for biofuel and other applications. To pursue algal bioremediation photoautotrophic/photoheterotrophic/mixotrophic cultivation system were followed. Recently, untreated and treated domestic secondary effluent, municipal wastewater, brewery wastewater, thin stillage, soy whey, carpet industry wastewater and other industrial wastewater were successfully used for microalgal-based bioremediation process.

3.3.2.1 Algal Growth in Conventional Medium

Conventional lab-grade synthetic mediums are widely studied at lab-scale or small scale experiments for screening and process optimization of various algal strains (Abou-Shanab et al., 2011). During the culture process nitrate and phosphate play a central role in microalgal cell physiology and growth where the optimum concentration enhances the growth rate of microalgal cultures (Bhola et al., 2011). In addition to nutrients, CO₂ enhances algal photosynthetic efficiency leading to increase of algal growth rate (Suali and Sarbatly, 2012). In a recent study, Basu et al. (2014) reported indigenous Scenedesmus obliquus SA-1 in BG11 medium and studied its growth charac-teristics. S. obliquus showed higher biomass concentration of 4.86 g L⁻¹ which was achieved at 13.8 ± 1.5 % CO₂ supply. To study the nitrogen and phosphorous effect on algal growth they increased their concentration and achieved higher biomass concentration of 4.975 ± 0.003 g L⁻¹ which justified that nitrogen and phosphorous play important role in uplifting algal growth rate.

Algae are known to have specific nutrient requirement for their growth for which their growth characteristics varies at different growth medium. Chaichalerm et al. (2012) grew six algal strain Chlorococcum humicola, Didymocystis bicellularis, Monoraphidium contortum, Oocystis parva, Sphaerocystis sp., and Scenedesmus acutus in four different growth medium (3NBBM, BG-11, Kuhl and N-8) and the mode of algal growth effect was observed by varying nutrient compositions. Significant difference in their growth characteristic and lipid productivity with respect to its growth medium was observed for all the six strains. Among all, C. humicola had the highest biomass yield of 0.113 g L⁻¹d⁻¹ (in Kuhl medium), the highest lipid content of 45.94 % w/w (in BG-11 medium), and the highest lipid yield of 0.033 g L⁻¹d⁻¹ (in 3NBBM medium). The 3NBBM medium, which has the lowest nitrogen concentration among the four culture media, was considered the optimal culture medium for C. humicola for lipid production. This is due to the fact that nitrogen limitation does not inhibit algal growth and creates stress environment to cell which enhances synthesis of membrane and storage lipid (Cakmak et al., 2014). Some potential algal strains and their growth characteristics have been presented in Table 3.2.

Table 3.2 Growth characteristics of different algal strains grown in synthetic media at lab-scale

3.3.2.2 Algal Growth in Wastewater

Wastewater from fertilizer industry, brewery industry, untreated and treated domestic secondary effluent, municipal wastewater, etc. contain high amount of nitrogen, phosphorous and other elements which are major algal growth nutrients. Achieving high nutrient removal along with feasible biomass yield is rather difficult due to high nutrient load in wastewater which creates stress environment for algal growth. There are few algae which has inherent adaptability to sustain their growth in high nutrient load environment. Among them Chlorella sp. are more commonly studied in various types of wastewaters and achieved high nutrient removal in addition to feasible biomass yield (Cai et al., 2013). In a study by Li et al. (2011), isolated Chlorella sp. was grown in municipal wastewater where a higher growth rate of 0.677 d⁻¹ was achieved without any lag phase. During the 14 d of cultivation period, Chlorella could achieve more than 80 % of nutrient removal (ammonia, total nitrogen, total phosphate) with 0.92 g L⁻¹ of feasible biomass productivity. There is also report which claimed of achieving 9.8 g L⁻¹ and 6.3 g L⁻¹ biomass from thin stillage and soy while growing Chlorella vulgaris at mixotrophic conditions in a bioreactor. Besides high biomass yield Chlorella vulgaris could achieve high lipid yield of 43 % and 11 % (w/w) respectively (Mitra et al., 2012).

As mentioned earlier, nutritional requirement for algal growth varies from strain to strain for which different algal strains need to be screened. Sydney et al. (2011) conducted a screening experiment over 20 algal strains on secondary domestic wastewater treated effluents. Among 20 strains three strains—unknown LEM-IM 11, Botryococcus braunii and Chlorella vulgaris—have shown higher growth rate and high nutrient removal. During 2 L photobioreactor study unknown LEM-IM 11 has shown growth rate of 0.19 d⁻¹ whereas 0.11 d⁻¹ was observed in Botryococcus braunii and C. vulgaris. But greater nutrient removal was achieved by Botryococcus braunii with 79 % nitrate and 100 % phosphate in addition to higher biomass productivity of 0.68 g L⁻¹ containing over 36.14 % lipid. There are also reports where two different wastewaters were mixed in different proportion to achieve higher algal growth and nutrient removal. In one such study, Scenedesmus obliquus CCAP 276/3A reported a maximum specific growth rate of 0.074 h⁻¹, volumetric biomass productivity of 4 mg L⁻¹ h⁻¹, and net biomass generation 0.28 g L⁻¹ when grown in a culture blend of 25 % (v/v) of urban wastewater from secondary treatment added to 5 % (v/v) olive-oil mill wastewater (Hodaifa et al., 2013). Table 3.3 represents various algal strains grown in different wastewater and their biomass and lipid productivity. This suggests algal strains represent variable growth characteristics for which screening process is highly essential to select potential algal strain to achieve high biomass yield with simultaneous nutrient removal.

Table 3.3 Biomass and lipid productivity of different algal strains studied in different waste water

3.3.3 Growth Study at Mass Cultivation Prospect

Algal biofuel are globally considered as 3^rd and 4^th generation biofuel due to its significant biomass productivity containing high cellular concentration of lipids, resources and economic sustainability and overall potential advantages over other sources of biofuels (Rawat et al., 2013). Productivity of algae, microalgae in general, can be twenty times than that of oil seed crops on per hectare basis and is thus a more viable alternative towards mass cultivation prospect (Chisti, 2007). Moreover, microalgae have faster growth rates than plants and are capable to grow in highly saline waters and utilize a large fraction of solar energy making them effective solar to chemical energy converters (Huber et al., 2006). Various algal strains were studied towards mass cultivation prospect using raceway pond and outdoor photobioreactors (Singh and Gu, 2010). But feasibility over economic viability has always been a greater challenge of algal cultivation at pilot/commercial scale (Acién et al., 2012). There are many vital steps which need to be critically analyzed at each stage such as, isolation of algal strains for mass cultivation prospect which can be either freshwater or marine algae, cultures selected from various culture repositories or indigenous wild types which might be best suited for large scale production (Suali and Sarbatly, 2012). Furthermore, the screening process should identify potential strains bearing faster growth rate, high biomass yield, and significant lipid productivity. The synergistic interactions that occur between naturally grown algae and other microorganisms cannot be ignored (Rawat et al., 2013). Besides isolation and identification of potential algal strains, reactor design and biomass harvest from mass cultivation system play vital role to make mass cultivation process cost effective (Acién et al., 2012).

3.3.3.1 Raceway Pond

Low cost, low maintenance, easy setup and large volume cultivation capacity make raceway pond more commercially popular among various researchers and industrialists (Suali and Sarbatly, 2012). This is one of the oldest mass cultivation systems which are widely studied for algal biomass production. Though it is commercially popular it has several drawbacks such as wide exposure to environment always contaminates algal culture and can’t be recommended during rainy season and winter season with snowfall. Maintaining growth parameters such as temperature, pH and light intensity is also fairly possible. Besides several drawbacks, there are quite a few algal strains which have been successfully grown with a biomass productivity up to 14–50 g m⁻²d⁻¹, which can be further enhanced with supply of external CO₂ (Putt et al., 2011). In a recent study by Ranga Rao et al. (2012), Botryococcus braunii has shown high growth rate leading to biomass concentration of 1.8 g L⁻¹ in 18 days of cultivation in a raceway pond having culture capacity of 80 L. Similar biomass concentration (1.9 g L⁻¹) with specific growth rate of 0.419 d⁻¹ was achieved in 18 days of cultivation in 2000 L raceway pond for Botryococcus braunii (Ashokkumar et al., 2014). This growth rate is comparatively four times higher than that observed by Botryococcus braunii in secondary domestic wastewater treatment effluents (Sydney et al., 2011). In another experiment, fast growing marine algae Chlorella variabilis was grown in 400 L raceway pond with a culture volume of 150 L where significant specific growth rate of 0.36 day⁻¹ was observed. Cultivating the C. variabilis throughout the year, maximum biomass (1.76 ± 0.04 g L⁻¹) was achieved during summer season when high light intensity used to be observed causing higher photosynthesis and algal growth (De Bhowmick et al., 2014). Nurra et al. (2014) studied algal mass cultivation using Nannochloropsis gaditana in a 53 m³ raceway pond on a biorefinery concept where significant growth rate with biomass productivity of 19.9 g m⁻²d⁻¹ was achieved. Some of potential algal strain studied towards mass cultivation prospect using raceway pond and photobioreactor are depicted in Table 3.4.

Table 3.4 Growth characteristics of different algal strains at mass cultivation prospect

3.3.3.2 Photobioreactor

Controlled growth environment, minimal contamination and high photosynthesis leading to higher biomass yield are promising features of a photobioreactor as an algal cultivation system. Among several photobioreactor designs, stirred tank and vertical colum n photobioreactors are commonly used during lab-scale experiments, whereas high volume flat panel and tubular reactors are recommended during pilot-scale experiment (Wang et al., 2012). Due to controlled growth parameters most algal strains showed better growth rate and delivers higher biomass yield. Botryococcus braunii achieved significant growth rate when grown in photobioreactor and resulted 2.31 g L⁻¹ of biomass on 25^th day of cultivation with 20 % v/v CO₂ which was supplied in the form of flue gas (Ge et al., 2011). This is comparatively higher than the biomass obtained from raceway pond (Ranga Rao et al., 2012). Since algal growth rate is specific to strains and its potential towards mass cultivation prospect, research has been focused on cultivating potential high biomass productivity algal strains with a mass cultivation prospect.

Doucha and Lívanský (2009) cultivated fast growing Chlorella sp. in outdoor open thin-layer photobioreactor having culture volume of 2000 L where greater biomass productivity was achieved. During this maximum algal concentration of 4.3 g L⁻¹d⁻¹ was achieved whereas less growth rate was observed by Chlorella zofingiensis grown in 60 L flat plate photobioreactor. The highest specific growth rate and biomass productivity obtained was 0.994 d⁻¹ and 58.4 mg L⁻¹d⁻¹ respectively (Feng et al., 2011). Besides fresh water algal strains, sea water and brackish water algal strains have also been studied on scale-up experiments using photobioreactors. High lipid producing Nannochloropsis sp. F&M-M24 has shown significant growth rate and average biomass productivity of 0.30 g L⁻¹d⁻¹ when grown in 110 L Green Wall Panel photobioreactors under nutrient sufficient and deficient conditions containing over 60 % w/w lipid (Rodolfi et al., 2009). Similar growth rate was observed from Tetraselmis suecica of 0.35 ± 0.03 g L⁻¹d⁻¹ with optimum biomass concentration of 0.7 g L⁻¹ when grown in 40 L tubular photobioreactor (Michels et al., 2014). This suggests fresh water green algae have high potential towards mass cultivation prospect using photobioreactor.

3.4 Conclusion

Recent reports in the field of algal research suggest different algal species bear different growth characteristic which depends on their mode of cultivation in addition to medium of nutrition and CO₂ supply. Over several algal species, green algae have shown promising growth characteristics and have been widely studied. Potential green algae have shown sustainable growth in different wastewater with respect to high nutrient removal efficiency which makes algal cultivation process a cost effective process when coupled with simultaneous wastewater treatment. Fresh water algal strains have shown significant growth features towards mass cultivation prospect over saline water algal strains. However, nutritional stress can result in high lipid yield from algal biomass during mass cultivation process which has feasibility towards algal biorefinery concept.