Front. Energy Res. Frontiers in Energy Research Front. Energy Res. 2296-598X Frontiers Media S.A. 10.3389/fenrg.2014.00026 Energy Research Mini Review Assessment of Environmental Stresses for Enhanced Microalgal Biofuel Production – An Overview Cheng Dan 1 * He Qingfang 1 * 1Department of Applied Science, University of Arkansas at Little Rock, Little Rock, AR, USA

Edited by: Qiang Wang, Chinese Academy of Sciences, China

Reviewed by: Anoop Singh, Denmark Technical University, Denmark; Umakanta Jena, Desert Research Institute, USA

 *Correspondence: Dan Cheng and Qingfang He, Department of Applied Science, University of Arkansas at Little Rock, 2801 South University Avenue, Little Rock, AR 72204, USA e-mail: dxcheng@ualr.edu; qfhe@ualr.edu

This article was submitted to Bioenergy and Biofuels, a section of the journal Frontiers in Energy Research.

 22 05 2014 07 07 2014 2014 2 26 10 04 2014 23 06 2014 Copyright © 2014 Cheng and He. 2014

This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) or licensor are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

Microalgal biofuels are currently considered to be the most promising alternative to future renewable energy source. Microalgae have great potential to produce various biofuels, including biodiesel, bioethanol, biomethane, and biohydrogen. Cultivation of biofuel-producing microalgae demands favorable environmental conditions, such as suitable light, temperature, nutrients, salinity, and pH. However, these conditions are not always compatible with the conditions beneficial to biofuel production, because biofuel-related compounds (such as lipids and carbohydrates) tend to accumulate under environmental-stress conditions of light, temperature, nutrient, and salt. This paper presents a brief overview of the effects of environmental conditions on production of microalgal biomass and biofuel, with specific emphasis on how to utilize environmental stresses to improve biofuel productivity. The potential avenues of reaping the benefits of enhanced biofuel production by environmental stresses while maintaining high yields of biomass production have been discussed.

 microalgae biofuel environment stress biomass Introduction

The rapid increase in global energy demand is driving the efforts to develop renewable energy sources. Biofuels are considered to be one of the most viable alternative sources of energy, as they are renewable, sustainable, and environment-friendly. The production of biofuels from microalgae has captured considerable interest in recent years (Schenk et al., 2008; Wijffels and Barbosa, 2010). In this review, microalgae are defined as unicellular and simple multicellular photosynthetic microorganisms, which include eukaryotic microalgae and cyanobacteria. Several attractive characters make microalgae the most hopeful feedstock for biofuel generation (Hu et al., 2008; Li et al., 2008b):

rapid growth, which provides high biomass productivity and reduces the time for development of such biofuel-producing systems;

high amount of lipids, which can be used to produce biodiesel (Chisti, 2007; Rodolfi et al., 2009; Scott et al., 2010);

the tolerance to marginal lands, which avoids competing with agricultural lands (Costa and de Morais, 2011; Day et al., 2011; Quintana et al., 2011);

greenhouse gas (carbon dioxide) sequestration capacity, which can mitigate global warming impacts (Ono and Cuello, 2007; Packer, 2009);

the ability to utilize nutrients (such as nitrogen and phosphorus) from polluted municipal, industrial, and agricultural wastewater, which provides economical and environmental benefits of wastewater bioremediation (Hall et al., 1995; Mulbry et al., 2008; de Godos et al., 2009; Markou and Georgakakis, 2011; Rawat et al., 2011);

the potential of producing various valuable co-products for commercial application (Radmer and Parker, 1994; Olaizola, 2003; Gavrilescu and Chisti, 2005; Singh et al., 2005; Walker et al., 2005; Spolaore et al., 2006; Raja et al., 2008), which also improves the economics of microalgal biofuel production.

Microalgae are able to produce diverse forms of biofuels including: microalgal lipid-derived biodiesel (Schenk et al., 2008; Scott et al., 2010), bioethanol by fermentation of carbohydrate (Deng and Coema, 1999; Dismukes et al., 2008), biomethane through anaerobic digestion (Sialve et al., 2009; Alzate et al., 2012), and biohydrogen from photosynthesis or fermentation (Benemann, 2000; Kapdan and Kargi, 2006; Hemschemeier et al., 2009). Moreover, the whole microalgae can be converted into bio-oil (via hydrothermal liquefaction and pyrolysis), hydrochar (via hydrothermal carbonization), and syngas (via gasification) (Miao et al., 2004; Amin, 2009; Heilmann et al., 2010; Jena and Das, 2011; Jena et al., 2011; Markou et al., 2012a; Broch et al., 2014).

Although microalgal biofuels hold great promise, considerable challenges exist for their commercialization. Further research efforts required to make microalgal biofuels cost-effective and sustainable include: selecting and bioengineering microalgal strains for the best biofuel producers; optimizing culturing conditions for microalgal biomass and biofuel production; developing bioreactors suitable for large-scale microalgae cultivation; improving efficiency of microalgal biomass harvesting and downstream processing, and reducing production costs and energy consumption (Greenwell et al., 2010; Scott et al., 2010; Gong and Jiang, 2011; Nigam and Singh, 2011; Singh et al., 2011a,b, 2012).

Cultivation of microalgae for biofuel production is influenced by numerous environmental factors, including physical factors, such as light and temperature and chemical factors, such as nutrients, salinity, and pH (Hu, 2004; Guschina and Harwood, 2006; Hu et al., 2008; Singh and Dhar, 2011). These environmental factors not only affect the accumulation of biomass but also influence the biochemical composition of cell, and thus the biofuel productivity.

This review highlights environmental conditions required for optimized microalgae cultivation as well as stressed conditions applied for improved microalgal biofuel production.

 Environmental Factors Affecting Microalgal Biomass Accumulation

The microalgal biomass production can be highly influenced by light intensity, temperature, nutrient availability, salinity, and pH (Table 1; Figure 1), as these conditions affect profoundly photosynthesis and the biosynthesis and accumulation of biomolecules such as lipids, carbohydrates, and biohydrogen.

Impact of environmental stresses on microalgal biomass accumulation and biochemical composition for biofuels.

Microalgae Biomass productivity (mg l−1 d−1)
 Biochemical content (% of dry cell weight)
 Environmental stresses Reference
Before stress After stress Before stress After stress
Arthrospira (Spirulina) platensis 193 87 Carbohydrate 11 67 Phosphorous limitation Markou et al. (2012b)
Nannochloropsis sp. 633 457 8 11 High carbon dioxide Hu and Gao (2003)
Scenedesmus obiquus CNW-N 441 841 16 38 High light Ho et al. (2012)
841 732 38 52 Nitrogen limitation Ho et al. (2012)
Spirulina sp. – – 14 21 High temperature Ogbonda et al. (2007)
Tetraselmis subcordiformis – – <10 32 Nitrogen limitation Ji et al. (2011)
Chaetoceros muelleri 70 – Lipid 19 36 Silicon limitation Griffiths and Harrison (2009)
Chlorella vulgaris 138 133 6 15 Nitrogen limitation Converti et al. (2009)
Cyclotella cryptica – – 18 38 Silicon limitation Griffiths and Harrison (2009)
Dunaliella tertiolecta ATCC 30929 – – 60 70 High salinity Takagi et al. (2006)
Nannochloropsis oculata 127 73 8 14 High temperature Converti et al. (2009)
127 103 8 16 Nitrogen limitation Converti et al. (2009)
Nannochloropsis sp. 633 457 7 9 High carbon dioxide Hu and Gao (2003)
Nannochloropsis sp. F&M-M24 360 300 32 60 Nitrogen limitation Rodolfi et al. (2009)
Navicula saprophila – – 24 49 Silicon limitation Griffiths and Harrison (2009)
Neochloris oleoabundans 630 400 16 34 Nitrogen limitation Li et al. (2008a)
Scenedesmus obiquus CNW-N 841 732 12 22 Nitrogen limitation Ho et al. (2012)
Scenedesmus sp. LX1 37–64 27 23–28 53 Phosphorous limitation Xin et al. (2010)

A model for environmental control of microalgal biomass accumulation and biofuel production is shown. Environmental factors, such as light, temperature, nutrient, salinity, and pH affect microalgal biomass accumulation. The biomass-derived lipid and carbohydrate can be transformed to diverse biofuels, including biodiesel, biomethane, bioethanol, and biohydrogen. The content and composition of lipid and carbohydrate in microalgae cells can be influenced by several environmental stresses, such as light stress, temperature stress, nutrient stress, and salt stress.

 Light

Light is the basic energy source for photoautotrophic organisms, and the intensity of light is one of the key parameters affecting photosynthetic activity (Falkowski and Owens, 1980; Richardson et al., 1983; Post et al., 1985). The growth rate of the microalgae culture increases with increased light intensities, until saturating light (generally around 200–400 μE) is reached (Radakovits et al., 2010). Oversaturating light can lead to the formation of reactive oxygen species (ROS), which is harmful for microalgae cells (photoinhibition), and thereby, decrease the biomass productivity (Stanier and Cohenbazire, 1977; Goldman, 1979; Richmond, 2000).

 Temperature

Although microalgae are able to survive at a variety of temperatures, optimal temperature for growth is limited to a narrow range (20–30°C) (Singh et al., 2012). Generally, in optimal temperature range, rise in temperature leads to improved microalgal biomass production. Temperatures above the optimal range cause growth declines, in severe conditions, even kill microalgae cells. However, low temperatures seem to reduce the biomass loss caused by respiration during dark periods (Weissman and Goebel, 1985; Raven and Geider, 2006; Chisti, 2007). Therefore, high biomass accumulation can be achieved by increasing the temperature to optimal in the morning (to enhance productivity during the day) and decreasing the temperature at night (to avoid biomass loss) (Hu, 2004).

 Nutrients

Nutrients supplied to microalgal cultures include macronutrients (such as nitrogen, phosphorus, carbon, and sulfur) and micronutrients (such as iron, zinc, copper, and cobalt). Nutrient limitation may cause morphological and physiological changes of microalgae cells, and therefore decrease the growth rates and biomass production. Nitrogen and phosphorus are the most important nutrients required for microalgae growth, and the ratio of nitrogen to phosphorus (N:P) can directly control the nutrient limitation status (Rhee, 1978; Wijffels and Barbosa, 2010). Nitrogen is involved in the biosynthesis nucleus acids, proteins, and photosynthetic pigments. Nitrogen limitation decreases the synthesis of photosynthetic proteins and pigments, therefore affects the yield of microalgal biomass (Berges et al., 1996; Saha et al., 2003; Li et al., 2008a). Phosphorus is essential in a variety of cellular metabolic processes, and phosphorus limitation affects the growth and development of microalgae (Geider and La Roche, 2002; Litchman et al., 2003; Hu, 2004). Carbon dioxide is necessary for photosynthesis, but it will become harmful if in excess. In addition, the carbon metabolic mode (autotrophic, mixotrophic, or heterotrophic) also affects the growth rates of microalgae (Chojnacka and Marquez-Rocha, 2004). Sulfur is essential for photosynthesis, protein synthesis, and lipid metabolism. It has been reported that sulfur limitation can limit cell division of microalgae (Yildiz et al., 1994; Ariño et al., 1995). Iron plays a key role in photosynthetic electron transport chain, and its limitation leads to defects in photosynthesis (van Oijen et al., 2004; Liu et al., 2008). However, excessive iron may result in oxidative damage to the cells (Fenton, 1894; Choudhary et al., 2007).

 Other environmental factors

Environmental factors, such as salinity and pH are also important for microalgae biomass accumulation. Different microalgae species can tolerate different ranges of salt concentrations (Kirst, 1989). Excess salinity inhibits photosynthesis, thus reduces the yield of biomass (Vonshak and Richmond, 1981; Gilmour et al., 1984; Kirst, 1989; Endo et al., 1995; Cho et al., 2007; Rao et al., 2007). The pH range optimal for microalgal growth is narrow (for most microalgae, is between 8.2 and 8.7) and strain-specific (Pedersen and Hansen, 2003; Havlik et al., 2013). The pH in microalgal cultures rise steadily during the day as carbon dioxide is consumed through photosynthesis. It has been found that pH also impacts the availability and absorption of nutrients such as iron and carbon (Coleman and Colman, 1981; Lee and Pirt, 1984; Wu et al., 2012).

 Environmental Stresses Affecting Microalgal Biofuel Productivity

The composition of microalgal biomass (such as biofuel-related lipids and carbohydrates) varies with the environmental conditions. Numerous studies have described utilizing environmental stresses (e.g., light stress, temperature stress, nutrient stress, and salt stress) to improve microalgal biofuel production (Table 1; Figure 1).

Light stress

Microalgae grown under different light conditions exhibit remarkable changes in their chemical composition. Typically, the amount of poly unsaturated fatty acids (structural lipids) increases under low light conditions, whereas high light promotes the accumulation of saturated and mono-unsaturated fatty acids (storage lipids) (Spoehr and Milner, 1949; Orcutt and Patterson, 1974; Sukenik et al., 1989, 1993; Walsh et al., 1997; Khotimchenko and Yakovleva, 2005). Because saturated and mono-unsaturated fatty acids are preferred sources for biodiesel, it may be feasible to use relatively high light intensities to improve biodiesel yield. It has also been reported that the content of carbohydrate in Scenedesmus obliquus CNW-N increased from 16.3 to 22.4% after exposure to high light (Ho et al., 2012).

 Temperature stress

Temperature can affect the lipid content in microalgae. Several microalgae, such as Ochromonas danica and Nannochloropsis oculata, have been found to increase their lipid content (37 and 89% increase, respectively) with increasing temperature (Aaronson, 1973; Converti et al., 2009). Besides, the composition of lipid can also be altered by temperature. Similar to the impact of light on lipid composition, low temperatures tend to increase the degree of unsaturation in fatty acid, whereas high temperatures improve the saturation of fatty acids (Sato et al., 1979; Wada and Murata, 1990; Renaud et al., 2002; Liu et al., 2005). Therefore, a suitably high temperature seems to promote the production of high quantity (high total lipid yield) and high quality (high saturation degrees of fatty acids) biodiesels. Temperature also influences the level of carbohydrates in microalgae, for example, the carbohydrate content in Spirulina sp. increased by 50% when the temperature was increased from 25 to 40°C (Ogbonda et al., 2007).

 Nutrient stress

When grown under nutrient-stress conditions, microalgae change their metabolic strategies and biochemical composition. Consequently, the improved production of desired biofuels can be achieved by manipulating nutrient conditions.

Nitrogen

Nitrogen is critical for protein biosynthesis. However, under nitrogen-limiting conditions, most of the carbon fixed in photosynthesis is used to synthesize lipids or carbohydrates, instead of proteins. Nitrogen is considered to be the most important nutrient affecting lipid metabolism in microalgae. It has been reported that a variety of microalgae species increase the accumulation of lipids after nitrogen deprivation. For instance, lipid content in Neochloris oleoabundans and Nannochloropsis sp. F&M-M24 increased about twofold and onefold, respectively, after nitrogen deprivation (Li et al., 2008a; Rodolfi et al., 2009). Nitrogen limitation also leads to the enhanced biosynthesis of carbohydrates in several microalgal species, such as a fourfold increase in carbohydrate content in Tetraselmis subcordiformis, and a 29% increase in carbohydrate content in S. obliquus CNW-N (Ji et al., 2011; Ho et al., 2012). Additionally, many cyanobacteria can produce hydrogen as a byproduct of nitrogen fixation when grown under nitrogen-limiting conditions (Das and Veziroglu, 2001; Dutta et al., 2005; Abed et al., 2009).

 Phosphorous

Phosphorous is involved in many cellular metabolic processes. It has been found that phosphorous limitation results in increased accumulation of lipids in microalgal cells, for example, Scenedesmus sp. LX1 accumulated up to 53% lipid under phosphorus-limiting conditions, whereas it only contained 25–28% lipid under phosphorus-replete conditions (Xin et al., 2010). In addition, Markou et al. (2012b) have shown that the carbohydrate content in Arthrospira (Spirulina) platensis increased from 11 to 67% after transfer to phosphorous-limiting medium.

 Sulfur

Sulfur is one of the most significant nutrients that affect the biohydrogen production in microalgae. Sulfur limitation causes anaerobic environment inside microalgae cells, thus induces the activity of hydrogenase and the release of hydrogen (Dutta et al., 2005; Esquível et al., 2011). Therefore, sulfur-limiting conditions have been applied to increase hydrogen productivity in many microalgae species, such as Gloeocapsa alpicola, Synechocystis sp. PCC 6803, Chlamydomonas reinhardtii, Chlamydomonas noctigama and Chlamydomonas euyale (Antal and Lindblad, 2005; Laurinavichene et al., 2006; Skjånes et al., 2008). It has been also found that total fatty acid content in C. reinhardtii doubled after exposure to sulfur limitation (Matthew et al., 2009). Furthermore, Brányiková et al. (2011) reported that Chlorella vulgaris cells synthesized 50% more starch under sulfur-limiting conditions than under sulfur-replete conditions.

 Carbon

Carbon is thought to influence the activity of nitrogenase and therefore the nitrogenase-dependent hydrogen production (Dutta et al., 2005). Moreover, different amounts and sources of carbon have been shown to affect both the content and the composition of lipids in microalgae cells. It has been reported that high concentration of carbon dioxide induced the accumulation of saturated fatty acids, whereas low concentration of carbon dioxide facilitated the production of unsaturated fatty acids (Tsuzuki et al., 1990; Riebesell et al., 2000; Hu and Gao, 2003). Certain microalgae are able to use organic carbon instead of carbon dioxide as the carbon source for heterotrophic growth. It was found that heterotrophically grown Chlorella cells synthesized about 280% more lipids and 45% more carbohydrates than did autotrophically grown cells (Miao and Wu, 2006).

 Trace mineral nutrients

Trace mineral elements may affect the accumulation of lipids and carbohydrates in numerous microalgae. It has been found that the content of glucose in Agmenellum quadruplicatum increased from 5 to 45% in response to iron limitation (Hardie et al., 1983), whereas excess iron caused up to sevenfold increase in lipid content in C. vulgaris (Liu et al., 2008). It has also been reported that silicon limitation resulted in increased lipid content in many diatom species, such as 89, 110, and 104% increase in lipid content in Chaetoceros muelleri, Cyclotella cryptica, and Navicula saprophila, respectively (Griffiths and Harrison, 2009). Additionally, trace metals, such as iron, nickel, magnesium, molybdenum, and zinc are important for nitrogenase-catalyzed hydrogen production (Horner et al., 2002; Lin and Lay, 2005; Carrieri et al., 2008).

 Salt stress

Salt has been shown to play an important role in the production of various biofuels. Carrieri et al. (2010) found that high salt concentration increased ethanol production by 121-fold compared to low salt concentration in the cyanobacterium A. (Spirulina) maxima. It is also known that many microalgae produce low molecular weight carbohydrates in response to salt stresses (Warr et al., 1985; Stal and Reed, 1987; Page-Sharp et al., 1998; Rao et al., 2007). Besides, salt stress is able to influence lipid content and composition in microalgae cells. It has been observed that elevated salinity increased lipid content from 60 to 70% in Dunaliella tertiolecta ATCC 30929 (Takagi et al., 2006). In addition, high salinity tends to induce the saturation of fatty acid, thus increase the productivity of biodiesels (Xu and Beardall, 1997; Chen et al., 2008).

 Combination of multiple stress factors

Since the impacts of different environmental stresses on biofuel production are additive, combined application of multiple stress factors might obtain better effect on improving the yield of desired biofuel products than the application of single stress factor. Recently, more and more research has been focused on the cumulative effect of multiple environmental stresses on microalgal biofuel production. Pal et al. (2011) found that the productivity of total lipids reached to the maximum when employed high light stress and high salinity stress simultaneously to Nannochloropsis sp. cultures. Sun et al. (2014) used nitrogen starvation in conjunction with high light to achieve the maximal triacylglyceride and carbohydrate production in N. oleoabundans HK-129.

 Perspectives – A Balance between Biomass Accumulation and Biofuel Production

Although applying environmental stress that can increase the production of microalgal biofuels, this is generally at the expense of decreased biomass yield. Consequently, the tradeoff between biomass accumulation and biofuel productivity is important for satisfactory biofuel production by microalgae.

In order to achieve optimum conditions for microalgal biofuel production, a two-phased cultivation method was proposed (Benemann and Oswald, 1996). In this method, the microalgae are grown under normal conditions in the first phase for biomass accumulation, followed by culturing under environmental-stress conditions in the second phase for desired biofuel production. By doing this, the microalgae can produce maximum biofuels without obvious biomass reduction. In the research performed by Rodolfi et al. (2009), a nutrient-replete first phase and a nitrogen-limiting second phase were used to increase both lipid content and areal lipid productivity in Nannochloropsis sp. F&M-M24. Dragon and co-workers used nitrogen- and iron-sufficient medium in the first phase to achieve high cell growth, then introduced nitrogen- and iron-limitation in the second phase to boost starch accumulation in C. vulgaris (Dragone et al., 2011).

Another balanced approach is using stress-tolerant microalgae strains for biofuel production. Many genes have been reported to be involved in stress responses and adaptation. It is likely that genetic manipulation of these genes might confer stress tolerance characteristics to microalgae. For example, glutathione peroxidase is an antioxidant enzyme, which plays an important role in protecting cells against oxidative damage. It has been reported that overexpression of glutathione peroxidase led to improved tolerance to high light stress, low temperature stress, and high salinity stress in transgenic plants (Takeda et al., 2003; Yoshimura et al., 2004). Another example is pfsR (photosynthesis, Fe homeostasis, and stress–response regulator) in the cyanobacterium Synechocystis sp. PCC 6803. Inactivation of pfsR resulted in stronger iron buffering capacity, and hence, improved resistance to iron limitation in Synechocystis sp. PCC 6803 (Jantaro et al., 2006). The photoinhibition caused by high light is always an important consideration when culturing microalgae, thus intensive efforts have been devoted to increase the tolerance of microalgae to high light-radiation. It has been shown that the Chlamydomonas mutants with reduced light-harvesting pigment or with truncated antenna size exhibited increased tolerance to high light and improved biomass productivity (Nakajima et al., 2001; Polle et al., 2002). The application of stress-tolerant strains not only makes the environmental-stress conditions suitable for both biomass accumulation and biofuels production but also prevents contamination, which reduces biomass production. The stress-tolerant microalgae strains, especially the strains thriving under reduced or limited nutrient conditions, such as the pfsR mutant, can outcompete other species, thus keeping desired microalgal culture in relatively pure conditions.

Growing microalgae under optimized stress conditions (such as a proper combination of nutrient limitation and light stress) can reduce the cultivation cost, maximize the accumulation of biofuel materials, and avoid contamination by competing out unwanted organisms, therefore offers a sustainable strategy for improving microalgal biofuel production. A comprehensive life-cycle assessment of the production processes (Singh and Olsen, 2011; Singh et al., 2011c, 2013) and appropriate policy supports, such as increasing funding for environmental-stress research and pilot studies, encouraging the utilization of environmental factors for sustainable biofuel production, promoting the development of high-efficient and cost-effective microalgae cultivation methods, and offering economic incentives (for example, tax exemptions) for microalgal biofuels to attract industry interest, will advance the key technologies in harnessing environmental stresses to promote biofuel production at commercial scale.

 Conflict of Interest Statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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