Perspectives on engineering strategies for improving biofuel production from microalgae – A critical review Shih-Hsin Ho, Xiaoting Ye, Tomohisa Hasunuma, Jo-Shu Chang, Akihiko Kondo PII: DOI: Reference:
S0734-9750(14)00147-5 doi: 10.1016/j.biotechadv.2014.09.002 JBA 6842
To appear in:
Biotechnology Advances
Received date: Revised date: Accepted date:
11 June 2014 18 September 2014 19 September 2014
Please cite this article as: Ho Shih-Hsin, Ye Xiaoting, Hasunuma Tomohisa, Chang Jo-Shu, Kondo Akihiko, Perspectives on engineering strategies for improving biofuel production from microalgae – A critical review, Biotechnology Advances (2014), doi: 10.1016/j.biotechadv.2014.09.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A revised manuscript submitted to Biotechnology Advances
T
Perspectives on engineering strategies for improving biofuel
SC R
IP
production from microalgae – A critical review
Shih-Hsin Hoa, Xiaoting Yea, Tomohisa Hasunumaa, Jo-Shu Changc.d.e,**, and Akihiko
a
NU
Kondob,f,*
Organization of Advanced Science and Technology and bDepartment of Chemical
MA
Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan c
Department of Chemical Engineering, dResearch Center for Energy Technology and
D
Strategy and eCenter for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan
TE
f
Biomass
Engineering
Program,
RIKEN,
1-7-22
Suehiro-cho,
CE P
Yokohama, Kanagawa 230-0045, Japan
*
AC
Correspondence:
Prof. Akihiko Kondo Phone: +81-78-803-6196 Fax: +81-78-803-6196 E-mail: [email protected]
**
Co-correspondence: Prof. Jo-Shu Chang Phone: +886-6-2757575 ext. 62651 Fax: +886-6-2357146 E-mail: [email protected]
1
Tsurumi-ku,
ACCEPTED MANUSCRIPT Abstract Although
the
potential
for
biofuel
production
from
microalgae
via
photosynthesis has been intensively investigated, information on the selection of a
T
suitable operation strategy for microalgae-based biofuel production is lacking. Many
IP
published reports describe competitive strains and optimal culture conditions for use in biofuel production; however, the major impediment to further improvements is the
SC R
absence of effective engineering strategies for microalgae cultivation and biofuel production. This comprehensive review discusses recent advances in understanding the effects of major environmental stresses and the characteristics of various
bioethanol)
using
microalgae.
NU
engineering operation strategies on the production of biofuels (mainly biodiesel and The
performance
of
microalgae-based
MA
biofuel-producing systems under various environmental stresses (i.e., irradiance, temperature, pH, nitrogen depletion, and salinity) and cultivation strategies (i.e., fed-batch, semi-continuous, continuous, two-stage, and salinity-gradient) are
D
compared. The reasons for variations in performance and the underlying theories of
TE
the various production strategies are also critically discussed. The aim of this review is to provide useful information to facilitate development of innovative and feasible
CE P
operation technologies for effectively increasing the commercial viability of microalgae-based biofuel production.
Keywords: microalgae, biofuel, environmental stress, engineering operation strategy,
AC
fed-batch operation, semi-continuous operation, continuous operation, two-stage operation, salinity-gradient operation
2
ACCEPTED MANUSCRIPT Contents 1. Introduction 2. Biofuels produced using microalgae
T
2.1 Biodiesel
IP
2.2 Bioethanol
3. Environmental factors affecting microalgae-based biofuel production
SC R
3.1 Irradiance 3.2 Temperature 3.3 pH
NU
3.4 Nitrogen depletion 3.5 Salinity
production 4.1 Fed-batch processes
D
4.2 Continuous processes
MA
4. Engineering operation strategies for improving microalgae-based biofuel
TE
4.3 Semi-continuous processes 4.4 Two-stage processes
CE P
4.5 Salinity-gradient processes 4.6 Comparison of different operation strategies 5. Integrated processes involving microalgae-based biofuel production
AC
5.1 Mitigation of CO2 emissions 5.2 Wastewater treatment 5.3 Production of value-added co-products 5.4 Genetic engineering 5.5 Blocking starch synthesis to increase lipid content 5.6 Engineering fatty acid biosynthesis 5.7 Modifying carbon assimilation pathways 5.8 Genetic modification of carbohydrate metabolism 6. Conclusions References
3
ACCEPTED MANUSCRIPT 1. Introduction Changes in global climate attributed to greenhouse gas (GHG) emission from fossil fuel combustion threaten human life and entire ecosystems (Brennan and
T
Owende, 2010). Currently, about 90% of global energy demand is met by fossil fuels,
IP
which has generated several critical problems (e.g., energy demand crises and environmental damage) (Yen et al., 2013). With concern over these issues growing,
SC R
the search for highly sustainable renewable energy sources is now of paramount importance.
Biofuels, which are produced from biomass, are currently viewed as one of the
NU
most feasible energy alternatives to reduce our reliance on fossil fuels. Biofuels have several advantages over fossil fuels, including sustainability, non-toxicity,
MA
biodegradability, and extremely low CO2 emissions (Lam and Lee, 2012). Two widely used crop-based biofuels (or first-generation biofuels; i.e., biodiesel and bioethanol) are produced from lipid-based (biodiesel; rapeseed, palm, etc.) and sugar-based
D
(bioethanol; sugarcane, corn, etc.) feedstocks (Brennan and Owende, 2010, Chen et
TE
al., 2011, 2013b). However, the use of first-generation biofuels has caused considerable debate in recent years, primarily due to potential problems associated
CE P
with global food competition and ecosystem imbalance due to the requirement of an excessively large area of arable land for producing a sufficient amount of crop-based biofuels (Parmar et al., 2011). In addition, although various non-food feedstocks, such as agricultural residues and waste cooking oils, have been successfully converted into
AC
bioethanol or biodiesel, respectively (known as second-generation biofuels), the potential for non-food feedstocks to meet overall energy demands in the global market remains limited (Naik et al., 2010). Microalgae have received growing interest as a potential biofuel feedstock. Microalgae have numerous advantages in this respect, such as a high growth rate, high lipid and carbohydrate content (50-70% per unit of dry weight), low demand for fresh water and arable land, and high degree of environmental tolerance (Chisti, 2007, Lam and Lee, 2012). In addition, conversion of microalgal biomass to biofuels could effectively mitigate the environmental problems associated with CO2 emissions and water pollution, enhancing the benefits derived from microalgae-based biofuel production (Ho et al., 2011, Pittman et al., 2011). The commercial use of microalgae for sustainable biofuel faces some challenges due to low productivity and high cost associated with photosynthetic efficiency of 4
ACCEPTED MANUSCRIPT algal strain, photobioreactor design, harvesting method and extraction techniques. (Brennan and Owende, 2010, Chen et al., 2011, Pragya et al., 2013). In particular, the development of a simple and ideal operation strategy is considered the key to further
T
improving microalgae-based biofuel production processes. However, the existing
IP
literature indicates a lack of comprehensive knowledge of the processes for producing biofuels from microalgae and the best means of selecting, developing, and assessing
SC R
suitable operation strategies. Considering the potential of microalgae as a sustainable biofuel feedstock, this review provides an overview of the effect of different stressors on
microalgae
cultivation,
practical
operation
strategies,
and
future
NU
perspectives/challenges regarding the use of genetic engineering tools for improving the production of biofuels and value-added chemicals from microalgae. This review
MA
also emphasizes recent engineering advances in various operation strategies (e.g., fed-batch, semi-continuous, continuous, two-stage, and salinity-gradient) used for the efficient production of biofuels (primarily biodiesel and bioethanol) to address the
D
importance of engineering perspectives in promoting microalgae-based biofuel
TE
production to achieve the ultimate goal of commercialization.
CE P
2. Biofuels produced using microalgae Microalgae are able to accumulate large amounts of energy-rich compounds (e.g., triacylglycerol and starch) under favorable growth conditions. These energy-rich compounds can be further converted into biodiesel (triacylglycerol) and bioethanol
AC
(starch). Biofuels derived from microalgae are now recognized as ―third-generation,‖ representing an entirely new dimension in the alternative bioenergy field.
2.1 Biodiesel Biodiesel has attracted considerable attention as one of the most important new alternative energy sources because it is non-toxic, and biodegradable fuel with low CO2 emissions (Gouveia and Oliveira, 2009). Biodiesel is composed of methyl esters of long-chain fatty acids (mainly C12-C18 groups) derived from triglycerides contained in a variety of biological feedstocks, such as oleaginous crops, microalgae, and animal fat (Yen et al., 2013). Currently, most commercial biodiesel is produced from oleaginous crops, such as soybean, rapeseed, jatropha, sunflower, and palm trees (Chisti, 2007). However, competition for arable land and potable water supplies arising from crop-based 5
ACCEPTED MANUSCRIPT biodiesel production have become increasingly problematic global issues in recent years (Pragya et al., 2013). It has been reported that over 50% of the total arable land would be required for cultivating enough oleaginous crops to meet the biodiesel
T
demand for transportation use in the US, which could result in a serious global food
IP
crisis. However, only 6% of US cropland would be required if the demand were met through microalgae-based biodiesel production (Liu et al., 2008).
SC R
The primary microalgae species used for biodiesel production are from the genera Botryococcus (Ranga Rao et al., 2012), Chlorella (Chen et al., 2013a, Münkel et al., 2013, Zhou et al., 2013b), Scenedesmus (Ho et al., 2012a, Xia et al., 2013),
NU
Chlamydomonas (Nakanishi et al., 2014, Siaut et al., 2011), Dunuliella (Moheimani, 2013, Tang et al., 2011), and Nannochloropsis (Bartley et al., 2013, Bondioli et al.,
MA
2012, Wan et al., 2013). The advantages of using microalgae for biodiesel production include: high growth rate, high lipid yield, high environmental tolerance, low competition for arable land and potable water, and no seasonal limitations on
D
culturing (Chisti, 2007, Ho et al., 2010). However, the fatty acid profile of microalgal
TE
lipids is also vital, because the quality of biodiesel produced is highly dependent on the composition of the constituent fatty acids, which strongly influences the
CE P
combustion efficiency and heating power of engines (Singh and Gu, 2010, Talebi et al., 2013). Fortunately, under suitable conditions of environmental stress, the lipids produced by microalgae are mostly composed of neutral fatty acids with a low degree of unsaturation (in most cases no more than two unsaturated bonds), thus confirming
AC
the suitability and potential of microalgae-based biodiesel as a partial replacement for fossil diesel (Talebi et al., 2013).
2.2 Bioethanol Bioethanol is another major commercialized biofuel at present and is primarily produced from sugar- and starch-based food crops (e.g., corn and sugarcane) via biological fermentation processes. Bioethanol production in the US increased from 1.6 to 13.2 billion gallons between 2000 and 2010 (Chen et al., 2013b). Taking into account the increased global population and increased demand for crops as food sources, lignocellulosic materials (e.g., agricultural residues) are regarded as better alternative feedstocks for bioethanol, as their production does not result in competition for arable land and food supplies (Sims et al., 2010). However, the major obstacle to efficient and economical bioethanol production from lignocellulosic 6
ACCEPTED MANUSCRIPT biomass is the high lignin content since it requires a very difficult pretreatment process (Ho et al., 2013a). In contrast to lignocellulosic biomass, some microalgae can accumulate large
T
amounts of carbohydrates in the form of starch or cellulose during periods of extreme
IP
environmental stress (John et al., 2011, Siaut et al., 2011). Therefore, there is currently considerable interest in the use of microalgae as an alternative feedstock for
SC R
bioethanol fermentation. Microalgae species belonging to genera such as Scenedesmus (Ho et al., 2013d), Chlorella (Ho et al., 2013a), and Chlamydomonas (Kim et al., 2006) reportedly can accumulate a considerable amount of carbohydrate
NU
(>50% of total dry cell weight) as biomass. Moreover, the carbohydrates found in microalgae are primarily composed of starch and cellulose (with the absence of
MA
lignin), which are much easier to convert to simple sugars for fermentation compared with lignocellulosic biomass (Ho et al., 2013a, John et al., 2011). Therefore, even though the number of studies concerning the use of microalgae for bioethanol
D
production is relatively limited compared with studies concerning their use for
TE
biodiesel production, many researchers still believe that in the near future, microalgae-based bioethanol production will prove to be a feasible alternative to
CE P
conventional sugar-rich crops or lignocellulosic biomass feedstocks for producing bioethanol (Chen et al., 2013b, Ho et al., 2013d, John et al., 2011, Wang et al., 2011).
3. Environmental factors affecting microalgae-based biofuel production
AC
Although the growth rate and cell composition of microalgae varies with species, most reports agree that microalgae could grow faster but generate only a small amount of lipid or carbohydrate under environmental conditions favorable to their growth. As a result, lipids or carbohydrates usually only begin to accumulate in microalgal cells under conditions of physical or chemical stress, such as extremes in irradiance, temperature, pH, nutrient availability, salinity, and trace metal concentrations (Chen et al., 2013b, Hu et al. , 2008). However, those stresses often lead to the inhibition of growth resulting in lower biomass productivity and higher contamination risk (Hu et al. , 2008). To enhance the economic feasibility of using microalgae for biofuels production, a better understanding is needed regarding how to rapidly accumulate lipids and carbohydrates in microalgal cells with higher cell growth rate can be controlled by manipulation of environmental conditions during
7
ACCEPTED MANUSCRIPT cultivation. The effects of different environmental factors on the production of lipids and carbohydrates are described in detail below.
T
3.1 Irradiance
IP
Irradiance plays an important role in photosynthesis and thereby strongly influences the growth, rate of CO2 fixation, and cell composition of photoautotrophic
SC R
microalgae (Ho et al., 2012a, Hu et al., 2008). Microalgal growth is strongly affected by the level of irradiance, which can fall into one of three categories, namely, light limitation, light saturation, and light inhibition (Ho et al., 2012a). Before reaching the
NU
stage of light inhibition, the growth rate of microalgae increases with rising light intensity, although the extent of the light intensity effect varies from species to species.
MA
As depicted in Table 1, Liu et al. (2012) found that biomass productivity of Scenedesmus sp. 11-1 was increased from 255 to 452 mg L-1d-1 when the light intensity was increased from 50 to 250 μmol m−2s-1. Ho et al. (2012a) also showed the
D
biomass productivity of S. obliquus CNW-N increased nearly 3-fold with an increase
TE
in light intensity from 60 to 420 μmol m−2 s-1. Through photosynthesis, large amounts of ATP and NADPH are produced as sources
and
reduction
power
for
the
conversion
of
CO2
to
CE P
energy
glyceraldehyde-3-phosphate (G3P), which is the primary precursor for the synthesis of both triacylglycerol (TAG) and starch (Chen et al., 2013b, Lv et al., 2010). Exposure to an environmental stress results in a competition between TAG and starch
AC
synthesis; which type of energy-producing compound will accumulate in the microalgal cell appears to be species specific (Rismani-Yazdi et al., 2011). It has been reported that appropriate irradiance can result in changes in pH and the concentrations of Mg2+ and NADPH in the stroma, which may in turn regulate the fate of some key metabolites (e.g., G3P) and thus further influence the accumulation of TAG (Lv et al., 2010). It has also been demonstrated that one key enzyme of phosphoglucomutase (PGM), which is involved in starch synthesis, can be regulated by varying irradiance levels (Neuhaus and Stitt, 1990). A related study suggested that light intensity plays an important role in the accumulation of lipids and carbohydrates (Sun et al., 2014). As shown in Table 1, appropriately increasing light intensity can lead to a significant increase in the accumulation of TAG or carbohydrates in some microalgae with simultaneous enhancement of cell growth, resulting in a higher TAG or carbohydrate yield. Typically, a sufficient light supply leads to a decrease in the total polar lipid 8
ACCEPTED MANUSCRIPT content and a significant increase in the content of neutral lipids (primarily TAG) as energy-storage compounds (Khotimchenko and Yakovleva, 2005, Sukenik et al., 1989). Unfortunately, the correlation between light supply and carbohydrate synthesis
T
in microalgae remains unclear, although it has been proven that generation of the
IP
precursors for sucrose and starch synthesis can be stimulated by irradiance (Champigny, 1985).
SC R
In addition, microalgal TAG and carbohydrate profiles can be influenced by irradiance level. For Scenedesmus obliquus, for instance, exposure to sufficient light intensity (e.g., 180 μmol m−2s−1) strongly increases the percentages of palmitic
NU
(C16:0), stearic (C18:0), and oleic (C18:1) acids in total TAG as well as the percentage of glucose in total carbohydrate (Ho et al., 2012a). Another study
MA
demonstrated that the content of major fatty acids, including palmitic (C16:0), stearic (C18:0), and oleic (C18:1) in the microalga N. oleoabundans can be significantly increased by culturing cells under high-intensity light (300 μmol m−2s−1) (Sun et al.,
D
2014). Based upon those studies, it seems clear that high light intensity can trigger the
TE
production of the saturated and monounsaturated fatty acids that are the primary components of neutral lipids (Hu et al., 2008). In contrast, there is still no clear
CE P
information regarding the dependence of microalgal carbohydrate profiles on irradiance level (Ho et al., 2012a).
3.2 Temperature
AC
As with the cultivation of other microorganisms, temperature is one of the most important factors in microalgae cultivation and is highly correlated with growth rate. Typically, higher growth rate of microalgae can be achieved by increasing temperature to its optimal level (Table 1) (González-Fernández and Ballesteros, 2012, Ho et al., 2013d). Variations in temperature also play an important role in the accumulation of lipids and carbohydrates in many species of microalgae, as shown in Table 1. The lipid content of Monoraphidium sp. SB2, N. oculata, and Scenedesmus sp. LX1 decreases as the temperature is increased from 25 to 35C, 15 to 20C, and 20 to 30C, respectively (Converti et al., 2009, Wu et al., 2013, Xin et al., 2011). However, James et al. (2013) showed that the TAG content of C. reinhardtii BAF-J5 is positively correlated with cultivation temperature. Another study demonstrated that the lipid content of Desmodesmus sp. F2 remains stable over the temperature range of
9
ACCEPTED MANUSCRIPT 25-40°C, suggesting that the regulation of genes and the activity of enzymes involved in lipid biosynthesis are not sensitive to temperature changes in this range (Ho et al., 2014b). Therefore, the effect of growth temperature on the lipid content of microalgae
T
varies from species to species. In addition, as shown in Table 1, the suitable
eventually resulting in a lower lipid productivity.
IP
temperature for lipid accumulation often causes the inhibition of cell growth,
SC R
As for carbohydrate accumulation, it has been reported that the carbohydrate content of Chlorella vulgaris SO-26 increases by 20-30% when the culture temperature is decreased from 20 to 5C (Hosono et al., 1994). The enzymes involved
NU
in carbohydrate synthesis (e.g., starch synthase and sucrose synthase) are known to be influenced by culture temperature (González-Fernández and Ballesteros, 2012).
MA
However, some researchers have pointed out that the effect of temperature on carbohydrate accumulation in microalgae remains unclear (Chen et al., 2013b). In addition, temperature appears to affect the TAG profile in microalgae, as the
D
proportion of saturated fatty acids increases with increasing temperature (Hu et al.,
TE
2008, Wu et al., 2013).
CE P
3.3 pH
The pH of the culture medium is an important factor, influencing many biological processes associated with microalgal growth, metabolism, and uptake of ions (Khalil et al., 2010). In general, the optimum pH for growth is also species
AC
dependent. For instance, Khalil et al. (2010) reported that the optimal pH for the growth of D. bardawil is about 7.5, whereas that for C. ellipsoidea is about 10. In addition, as shown in Table 1, as the culture medium pH is raised, the TAG content increases in some species of microalgae, but this comes with the cost of a sharp decrease in growth rate (Breuer et al., 2013, Gardner et al., 2011, Santos et al., 2012). These data suggest that high-pH stress not only inhibits the cell cycle but also effectively triggers lipid accumulation. Moreover, Santos et al. (2012) also demonstrated that high-pH stress not only increases the lipid content but also enhances lipid quality. At a high pH (e.g., pH 10), the major fatty acids produced by N. oleoabundans include palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linoleic (C18:2), all of which are suitable for biodiesel production. In contrast, Khalil et al. (2010) found that the carbohydrate content of both D. bardawil and C. ellipsoidea is
10
ACCEPTED MANUSCRIPT highly correlated with biomass concentration, indicating that use of an appropriate pH can significantly improve both growth and carbohydrate accumulation in these species, as the maximum carbohydrate content of these two organisms occurs at pH 7.5 and
IP
T
9.0, respectively.
3.4 Nitrogen depletion
SC R
Of all the macronutrients in the culture medium, the nitrogen source is the most critical in terms of the effect on lipid and carbohydrate accumulation in microalgae (Chen et al., 2013b, Chisti, 2007). A wide variety of studies have demonstrated that
NU
microalgae tend to allocate their carbon molecules to energy-rich lipids or carbohydrates when they encounter conditions of nitrogen depletion (Hu et al., 2008,
MA
John et al., 2011, Siaut et al., 2011). Nitrogen limitation may cause a sharp increase in microalgal lipid/carbohydrate content, along with a marked drop in the protein and chlorophyll content, suggesting that microalgae can degrade chlorophyll and
D
proteins/peptides as nitrogen source while transforming their carbon skeletons into
TE
lipid and carbohydrate in response to the extreme environment (Ho et al., 2012a, Sun et al., 2014). As shown in Table 1, Su et al. (2011) reported achieving a lipid content
CE P
of 48% in the microalga N. oculata when the organism was cultured under nitrogen-depletion conditions for 4 days. Similarly, nitrogen starvation triggers significant lipid accumulation in Nannochloropsis sp. F&M-M24 (Bondioli et al., 2012). In addition, cultivation of S. obliquus CNW-N and C. vulgaris FSP-E under
AC
nitrogen-depletion conditions results in a dramatic increase in the cellular carbohydrate content, from 21 to 49% and 15 to 51%, respectively (Ho et al., 2013b, Ho et al., 2013c).
In addition to enhancing lipid/carbohydrate accumulation, the stress associated with nitrogen limitation also strongly influences the lipid (fatty acids) and carbohydrate profiles in many microalgae (Ho et al., 2012a). Several reports have shown that nitrogen depletion leads to major increases in the proportions of saturated (e.g., palmitic acid) and monounsaturated (e.g., oleic acid) fatty acids, both of which are primary substrates for biodiesel production (Siaut et al., 2011). Nitrogen-starvation stress can also trigger the accumulation of carbohydrates in some microalgae. As carbohydrates are primarily located in the cell wall (mostly as cellulose) and plastids (mostly as starch), in some species of microalgae carbohydrate
11
ACCEPTED MANUSCRIPT typically accumulates as glucose, which can be easily converted to bioethanol via microbial fermentation (Ho et al., 2013b).
T
3.5 Salinity
IP
Similar to other environmental factors, salinity also plays a vital role in the accumulation of lipids/carbohydrates in microalgae. However, high salinity also slows
SC R
the growth of some species, such as Chlorococcum sp. (Harwati et al., 2012), Dunaliella sp. (Takagi et al., 2006), and Botryococcus braunii (Zhila et al., 2011). The literature shows that lipid/carbohydrate accumulation in microalgae increases in
NU
response to an immediate salinity shock (Table 1). For instance, increasing the culture medium NaCl concentration from 0 to 2% in the 10-day cultivation of Chlorococcum
MA
sp. leads to an increase in the lipid content, from 10.3 to 29.8% (Harwati et al., 2012). Similarly, the TAG content in microalgae is enhanced significantly by increasing the NaCl concentration (Takagi et al., 2006, Zhila et al., 2011). Moreover, the starch
D
content of Chlamydomonas reinhardtii cultivated in medium containing 100 mM
TE
NaCl is 4-fold higher than that obtained with freshwater cultivation (Siaut et al., 2011).
CE P
Fatty acid composition is also affected significantly by changes in culture medium salinity (Zhila et al., 2011). In Dunaliella sp. and Botryococcus braunii, for instance, an increase in salinity significantly decreases the proportion of polyunsaturated fatty acids (e.g., linoleic acid [C18:3]) and increases the proportions
AC
of saturated and monounsaturated fatty acids, including palmitic (C16:0), stearic (C18:0), and oleic (C18:1) acids, a composition that is suitable for biodiesel production (Takagi et al., 2006, Zhila et al., 2011). In addition, it has been reported that the sucrose pathway in microalgae can be altered via addition of NaCl to the culture medium (González-Fernández and Ballesteros, 2012). However, the effect of changes in the salinity level on starch accumulation in microalgae has yet to be fully elucidated.
4. Engineering operation strategies for improving microalgae-based biofuel production Although microalgae can grow faster and produce higher yields of lipids/carbohydrates as compared with other energy crops, the high cost of cultivation systems continues to impede the commercialization of microalgae-based biofuel 12
ACCEPTED MANUSCRIPT production processes. It is thus necessary to develop more economically feasible processes in order to increase the production of lipids/carbohydrates and thereby reduce the cultivation cost (Chen et al., 2013b). A large number of studies have
production.
However,
to
date
no
commercially
viable
IP
lipid/carbohydrate
T
focused on the development of more efficient cultivation methods that will improve
microalgae-based systems for biofuel production have been developed. The ideal
SC R
process would enable microalgae to grow fast with a simultaneous increase in lipid/carbohydrate productivity, which is strongly affected by cell growth and lipid/carbohydrate content. However, simultaneous increases in both growth rate and
NU
lipid/carbohydrate production is difficult to achieve because the accumulation of energy-rich compounds (e.g., lipids and carbohydrates) in microalgae usually occurs
MA
only under conditions of environmental stress, which in turn often decreases the growth rate (Ho et al., 2013d). Therefore, an operation strategy optimized so as to achieve both the highest biomass productivity and lipid/carbohydrate content is
D
needed in order to reduce the costs of cultivation and product formation.
TE
From the perspective of engineering process, achieving high overall lipid/carbohydrate productivity of microalgae is always the key issue when evaluating
CE P
its commercialization potential (Ho et al., 2012a). Therefore, how to accurately estimate the productivity of lipids or carbohydrates of the microalgae is critically important. In most published reports, the lipid/carbohydrate productivity was calculated by directly multiplying the lipid content with the biomass productivity (Ho
AC
et al., 2012a). This calculation may not precisely reflect the true lipid/carbohydrate productivity since the biomass productivity and lipid/carbohydrate content are usually not linearly correlated (Xu et al., 2014). Therefore, as proposed by Su et al. (2011), a more reasonable way to quantify the overall lipid/carbohydrate productivity is to determine the difference between the initial and final lipid/carbohydrate concentrations over the period of cultivation time. The initial and final lipid/carbohydrate
concentration
can
be
obtained
by
multiplying
the
lipid/carbohydrate content with biomass concentration at the time of inoculation and of harvesting, respectively. Nevertheless, the value of initial lipid/carbohydrate concentration might be negligibly small, especially when the inoculum size is very small and/or the initial lipid/carbohydrate content is very low (Converti et al. 2009, Xu et al., 2014).
13
ACCEPTED MANUSCRIPT 4.1 Fed-batch processes For microalgae cultivation, the fed-batch operation process is one of the most promising strategies for enhancing growth rate and end product production due to
T
flexibility with respect to the specific nutrients supplied during cultivation (Abdollahi
IP
and Dubljevic, 2012). Recent studies have shown that the biomass concentration and overall lipid production of Nannochloris sp. and Chlorella sp. are increased by
SC R
intermittent nitrogen feeding, but their lipid content is not increased under conditions of nitrogen sufficiency (Hsieh and Wu, 2009, Takagi et al., 2000). It has also been reported that fatty acid production by Cyclotella sp. is increased by controlled silicon
NU
limitation in fed-batch cultivation (Jeffryes et al., 2013). Moreover, Cheirsilp and Torpee (2012) demonstrated that both the lipid content and lipid productivity of
MA
Chlorella sp. and Nannochloropsis sp. are significantly increased via stepwise addition of glucose, indicating that maintaining the glucose concentration at a low level during fed-batch cultivation is a potentially useful method for increasing
D
microalgae-based lipid production.
TE
However, fed-batch processes may not always be suitable for microalgae cultivation, as light may become significantly limited in the culture due to high cell
CE P
density following prolonged cultivation, especially when the microalgae are grown under phototrophic or mixotrophic conditions. Light limitation in turn leads to a reduction in biomass productivity. To overcome this problem, a fed-batch operation involving a stepwise increase in light intensity was proposed, and this strategy was
AC
shown to dramatically enhance the growth and lipid productivity of microalgae under mixotrophic conditions (Cerón-García et al., 2013, Cheirsilp and Torpee, 2012). However, although fed-batch operations coupled with stepwise increases in light intensity may be effective, they are very difficult to control, especially when microalgae are being cultured outdoors with a naturally fluctulating light intensity over time.
4.2 Continuous processes Under phototrophic or mixotrophic conditions, the increasing turbidity associated with microlagal growth usually becomes a significant growth-limiting factor due to the self-shading effect. Continuous cultivation may therefore be an attractive alternative because continuous feeding of fresh medium effectively dilutes the cell density inside the photobioreactor so that it does not become too high. 14
ACCEPTED MANUSCRIPT Biomass productivity is maintained at a relatively high level due to the continuous feeding of medium and nutrients to support growth. In addition, continuous cultivation systems are generally low cost and easy to operate. Hence, continuous
T
processes are suitable for large-scale cultivation of microalgae for industrial
IP
applications (Marchetti et al., 2012, Sforza et al., 2013).
Under steady-state conditions in continuous culture, the biomass concentration
SC R
can be controlled by adjusting the rate of dilution with culture medium, and irradiance can be continuously maintained at a specific level (Tang et al., 2012). Of course, these characteristics facilitate light penetration, resulting in significant increases in both
NU
specific growth rate and biomass productivity. In addition, as shown in Table 1, some studies have found that irradiance plays an important role in the accumulation of
MA
lipids/carbohydrates. As illustrated in Table 2, continuous operation systems have been successfully used to produce microalgae-based biofuels, as they lead to a higher lipid/carbohydrate content and increased biomass production with long-term
D
cultivation (Ho et al., 2013c, Mazzuca Sobczuk and Chisti, 2010, Tang et al., 2012).
TE
However, because the growth rate must remain constant in a steady-state continuous culture, the feeding medium should contain sufficient nutrients (and a nitrogen source)
CE P
to sustain microalgal growth, and the culture conditions should not be growth inhibiting. However, the non-limiting nutrient and relatively non-stressful environmental conditions of continuous culture systems may have a negative effect on the accumulation of lipids and carbohydrates, which is usually higher under of
nutrient
AC
conditions
starvation
or
environmental
stress.
Therefore,
the
lipid/carbohydrate content in microalgal cells cultivated in a continuous system may not reach a satisfactory level (e.g., 40-60% of total dry cell weight). This may increase the difficulty and costs of downstream processing (e.g., extraction). In addition, because microalgae usually grow fairly slowly, a relatively low dilution rate should be used in order to maintain steady-state conditions; however, this increases the possibility of microbial contamination. Also, if the system is operated outdoors, growth will become very slow at night, resulting in the possibility of cell washout. Therefore, to achieve a stable outdoor continuous culture, the dilution rate may need to be adjusted during the light and dark periods according to growth conditions. In addition, using artificial light sources (such as LED) to provide additional light intensity (especially during the dark period) (Tang et al., 2012) may be useful for
15
ACCEPTED MANUSCRIPT maintaining the stable microalgal growth necessary to make the continuous culture operation successful.
T
4.3 Semi-continuous processes
IP
The semi-continuous operation is one of the simplest and most efficient bioreactor strategies used in microalgae-based biofuel production (Ho et al., 2013c,
SC R
Hsieh and Wu, 2009). Semi-continuous processes are an ideal strategy for avoiding both a low cell division rate in the early exponential stage and light limitation in the late stationary stage because it allows for maintaining the microalgal culture under
NU
exponential growth conditions, resulting in enhanced biofuel production (Ho et al., 2013c). In addition, semi-continuous operation processes seem to be more practicable
MA
than other operation modes for long-term cultivation, making them suitable for industrial production of microalgae-derived lipids/carbohydrates (Chen et al., 2013a, Ho et al., 2013c). As illustrated in Table 2, the biomass productivity of S. obliquus
D
CNW-N can be dramatically enhanced in semi-continuous systems, resulting in a
TE
favorable carbohydrate content of 50-52% and consequently higher carbohydrate productivity (Ho et al., 2013c). Another study showed that both biomass production
CE P
and the lipid content of Chlorella sp. can be increased under optimal semi-continuous cultivation (Hsieh and Wu, 2009). Han et al. (2013) reported that semi-continuous operation with pH regulation and nitrogen depletion sharply increases lipid accumulation in C. pyrenoidosa. Lipid productivity in that system was 3.6-fold higher
AC
than in batch cultivation. In outdoor cultivation of Chlorella sp. NJ-18, use of a semi-continuous operation results in markedly improved biomass and lipid productivity (Zhou et al., 2013a). Available data suggest that the semi-continuous process is a reasonable strategy for simultaneously enhancing microalgal growth and lipid/carbohydrate accumulation. However, semi-continuous processes have rarely been utilized in outdoor large-scale operations (Zhou et al., 2013a). Therefore, whether the use of semi-continuous processes to cultivate biofuel-producing microalgae outdoors is feasible or not should be evaluated through further studies.
4.4 Two-stage processes As discussed earlier, when producing lipids/carbohydrates from microalgae for biofuels applications, one of the major problems is the conflict with respect to the 16
ACCEPTED MANUSCRIPT optimal conditions required for achieving a high lipid/carbohydrate content and those necessary for achieving a high growth rate. From an engineering perspective, this type of problem can be dealt with by applying a two-stage cultivation process, which
T
provides different conditions for growth and lipid/carbohydrate accumulation, thereby
IP
markedly enhancing productivity. Indeed, two-stage processes have already been utilized in both indoor laboratory-scale and outdoor large-scale microalgal cultivation
SC R
operations for the production of biofuel feedstock (Ho et al., 2010, San Pedro et al., 2013, Xia et al., 2013).
In the two-stage strategy, nutrient-rich medium is used in the first stage to
NU
achieve maximum biomass productivity. After a sufficient amount of microalgal biomass is produced, the culture conditions are then altered to induce stress, such as
MA
that associated with nitrogen depletion, salt addition, or unsuitable ion concentrations (Table 2), thereby stimulating lipid accumulation in the second stage. San Pedro et al. (2013) suggested that the use a continuous operation process is a reasonable option
D
for the first stage in order to enhance biomass by adjusting the dilution rate. In the
TE
second stage, various types of stresses can be induced in order to dramatically upregulate lipid/carbohydrate accumulation with fair biomass productivity, resulting
CE P
in higher lipid/carbohydrate productivity (Ho et al., 2013d, Mujtaba et al., 2012, Sun et al., 2014). Based on the successes demonstrated in the above-mentioned studies, the two-stage operation seems to be an attractive strategy for microalgae-based biofuel production. However, the feasibility of commercialized outdoor large-scale two-stage
AC
operations to produce microalgae-based biofuels is questionable due to their high energy demand (Sun et al., 2014).
4.5 Salinity-gradient processes Increasing attention has been paid in recent years to the effect of salinity-associated stress on microalgae-based biofuel production (Harwati et al., 2012, Pal et al., 2011). As shown in Table 1, cultivation under high salinity conditions may increase the lipid/carbohydrate content, but this comes at the cost of a lower growth rate. Achieving higher lipid/carbohydrate productivity thus necessitates that the growth inhibition resulting from salinity stress be alleviated. Ideally, inducing high-salinity stress to stimulate lipid accumulation in microalgae and maintain a satisfactory growth rate (e.g., by enhancing adaptation to salinity) seems to be a promising strategy for simultaneously enhancing both lipid accumulation and 17
ACCEPTED MANUSCRIPT productivity. Takagi et al. (2006) suggested that gradually increasing the salt concentration in the medium facilitates microalgal adaptation to high salinity. This concept, designated as a ―salinity-gradient‖ process, is demonstrated for the first time
T
in this review. In this process, microalgae are cultivated in a nutrient-sufficient
IP
medium to achieve maximum biomass productivity and are then switched to a salinity-gradient cultivation mode via a stepwise increase in the concentration of sea
SC R
salt in the culture medium. As shown in Table 2, the stepwise addition of salt dramatically increases the lipid content in Chlamydomonas sp. JSC4, without significant inhibition of growth (Ho et al., 2014c). To date, no reports have been
NU
published concerning the use of salinity-gradient operation strategies to enhance lipid/carbohydrate productivity. This review proposes that the salinity-gradient
MA
process has a high potential for microalgae-based biofuel production via a two-stage operation.
D
4.6 Comparison of different operation strategies
TE
Although lipid/carbohydrate content is usually strain dependent, the type of operation strategy chosen plays a key role in determining whether a process will be
CE P
commercially feasible for microalgae-based biofuel production. Comparison of the characteristics of the five main microalgae cultivation strategies (fed-batch, continuous, semi-continuous, two-stage, and Salinity-gradient) shows that the fed-batch and continuous culture systems have certain advantages, including high
AC
biomass productivity and low operational cost. However, since fed-batch and continuous culture are often operated under nutrient-sufficient conditions, the microalgal lipid/carbohydrate content obtained may not low, which may lead to higher cost of downstream processing (e.g., extraction ofr isolation of lipids or carbohydrates). In contrast, sufficiently high lipid/carbohydrate content can easily be obtained using two-stage, semi-continuous, or salinity-gradient processes because of the strong environmental stresses associated with these processes, which results in decreased biomass productivity. The two-stage operation strategy is currently the most widely used for increasing lipid/carbohydrate productivity. However, due to the high operational costs arising from the necessity of exchanging the nutrient-rich and nutrient-deficient media in order to support growth in the first stage or to trigger rapid lipid/carbohydrate accumulation in the second stage, two-stage processes can prove problematic when large-scale cultivation is needed. Xia et al. (2013) attempted to 18
ACCEPTED MANUSCRIPT enhance lipid productivity in S. obtusus XJ-15 by adding NaCl in the second stage, with the aim of inducing significant lipid accumulation. However, the sudden addition of NaCl also inhibited growth. These salt-addition operation procedures seem to be
T
much simpler than switching between nutrient-rich and nutrient-deficient media.
IP
However, the tolerance of microalgae for high salinity must be improved to avoid growth inhibition upon salt addition. The salinity-gradient process is particularly
SC R
suitable for marine microalgae-based biofuel production if biomass productivity can be maintained at a satisfactory level by incorporating a continuous operation process during the growth stage. Thus, based on their merits, it might be beneficial to
NU
appropriately combine two or three of the above-mentioned processes to achieve the goals of low production cost, high biomass productivity, and high lipid/carbohydrate
MA
yield. Notably, whether the single or combined engineering strategies are applied in lab- and large-scale, the life-cycle assessment (LCA) is necessary to ensure that biofuel production from microalgae with the proposed strategies is more sustainable
2014).
Identifying
the
best
engineering
strategy
could
accelerate
TE
al.,
D
and feasible than the conventional cultivation process (Collet et al. , 2014, Fortier et
commercialization of microalgae-based biofuel production. In addition, plenty of
CE P
contamination risks from zooplankton, bacteria, or other competitors, especially in large-scale cultivation, are also one of the most dominant factors encountered for the feasibility of microalgae commercialization (Wang et al., 2013). Thus, using effective engineering strategies for the contaminant control of microalgae culture is another
AC
vital factor that must be considered.
5. Integrated processes involving microalgae-based biofuel production In addition to optimizing the operation strategy for microalgae-based biofuel production, it would be beneficial and economically advantageous to combine the microalgae cultivation process with CO2 fixation (Ho et al., 2011), wastewater treatment (Cai et al., 2013), or processes leading to the production of a variety of valuable co-products (Yen et al., 2013).
5.1 Mitigation of CO2 emissions The earth is being threatened by the sharp rise in the level of atmospheric CO 2. Biological CO2 fixation, achieved primarily by microalgae through photosynthesis, has emerged as one of the most effective and environmentally friendly ways to reduce 19
ACCEPTED MANUSCRIPT atmospheric CO2 levels (Ho et al., 2012b). Moreover, through photosynthesis, microalgae can effectively convert solar energy into a variety of valuable end products, such as biofuels, food additives, and compounds used in cosmetics and
T
pharmaceuticals (Yen et al., 2013). For example, some microalgae, such as S.
IP
obliquus (Ho et al., 2012a), C. vulgaris (Ho et al., 2013b), and Chlamydomonas sp. (Nakanishi et al., 2014), exhibit both a high CO2 fixation rate and high
SC R
lipid/carbohydrate content. Thus, combining microalgae-based CO2 fixation with biofuel production or bio-based chemical production seems to have a high potential for reducing the level of atmospheric CO2 while simultaneously producing biofuels or
NU
valuable chemicals.
MA
5.2 Wastewater treatment
D
In addition to establishing a suitable microalgae-based biofuel production system, it is equally important to consider the recycling of inorganic nutrients present in wastewater. Several microalgal species are known to have a high potential for use in wastewater treatment, which would greatly facilitate biofuel production using
AC
CE P
TE
microalgae (Cai et al., 2013). Many related studies have shown impressive results regarding the potential for utilizing microalgae to remove carbon, nitrogen, phosphorus, sulfur, and other chemicals/heavy metals from a variety of wastewater streams. For example, Zhou et al. (2012) used Auxenochlorella protothecoides UMN280 to effectively remove 90-100% of nitrogen and phosphate from concentrated municipal wastewater in conjunction with the production of oil-rich biomass. Feng et al. (2011) achieved 96-97% removal of ammonium and total phosphorus from artificial wastewater using the microalgae C. vulgaris, obtaining a maximum lipid content of 42%. However, the above studies were primarily conducted indoors at the laboratory level. More challenges (such as microbial contamination and inconsistent wastewater composition) are encountered when using wastewater to grow microalgae in large-scale outdoor cultivation (Cai et al., 2013). 5.3 Production of value-added co-products With the rapid increase in global population beginning in the early 1950s, microalgae, which are protein and nutrient rich, have received considerable research attention due to their capacity to serve as additives for human and animal foods (Borowitzka, 1999). To enhance the economic feasibility of microalgae-based biofuel production, other components that have potential applications in producing foods, cosmetics, emulsifiers, and clinical drugs can be collected from microalgal biomass in 20
ACCEPTED MANUSCRIPT addition to lipids and carbohydrates (Yen et al., 2013). In particular, algal polysaccharides (e.g., fucoidan and carrageenans) (Kim et al., 2012), algal long-chain unsaturated fatty acids (e.g., EPA and DHA) (Hong et al., 2011), and carotenoids (e.g.,
T
lutein and astaxanthin) (Ho et al., 2014a) are gaining wide attention due to their
IP
special medical and food nutrient applications. However, most recent studies have focused on obtaining one specific product without regard to damage that may be done
SC R
to other product-containing fractions (Vanthoor-Koopmans et al., 2013). Thus, innovative separation methods that permit extraction of different compounds without damaging other potentially valuable compounds are urgently needed, and the
NU
technology to accomplish this should also be suitable for commercial-scale operations (Vanthoor-Koopmans et al., 2013). In addition, a thorough evaluation of
MA
food/medicine safety is required in processes in which microalgal cultivation is combined with flue gas CO2 fixation and wastewater treatment.
D
6. Genetic engineering
TE
DNA sequencing has become more efficient and accessible in recent years, and the number of industrially relevant algae for which genome sequence information is
CE P
available is increasing rapidly. At least 16 algal genomes have been sequenced to date, and many more sequencing projects are underway. Transformation systems have been set up for at least 15 species, including Chlamydomonas reinhardtii, Chlorella sp., Haematococcus pluvialis, Dunaliella viridis, Dunaliella salina, Nannochloropsis
AC
oculata, Thalassiosira pseudonanna, Thalassiosira weissfloggi, Phaeodactylum triconuntumtricornuntum, Porphyridium sp., Nannochloropsis sp., and Scenedesmus obliquus (Guo et al., 2013, Kilian et al., 2011, Neupert et al., 2012, Radakovits et al., 2010, Yu et al., 2011). However, although several studies have successfully displayed the improvement of microalgal lipid synthesis via genetic modifications, there are still many uncertain factors that hinder the utilization of genetically engineered microorganisms for this purpose, such as unknown biological contamination, possible ecological damage, and restrictive legislation (Mata et al., 2010). In addition, ‗omics‘ research involving a combination of genomics, transcriptomics, proteomics, and metabolomics has generated a substantial amount of data regarding algal metabolism (Guarnieri et al., 2011, Jamers et al., 2009). However, successful manipulation of algal metabolism will require a much more detailed understanding. As new algal species are identified and their genomes are sequenced, 21
ACCEPTED MANUSCRIPT strategies must be developed to quickly annotate and characterize genes and proteins involved in lipid and starch metabolism.
T
6.1 Genetic modification of carbohydrate metabolism
IP
Starch is a basic energy-rich storage compound of microalgae. Several studies have concentrated on altering catalytic and allosteric properties of ADP-glucose
SC R
pyrophosphorylase (AGPase; an essential enzyme in starch production) in crop plants in order to increase starch production (Smith, 2008). AGPase, which is the rate-limiting
enzyme
with
starch ATP to
synthesis, form
catalyzes
ADP-glucose
the and
reaction
of
pyrophosphate.
NU
glucose-1-phosphate
in
Starch-synthesizing enzymes have also been introduced into the cytosol of microalgae
MA
in an effort to increase the starch content (Smith, 2008). Strategies to decrease starch degradation in microalgae (Ritte et al., 2006) or alter secretion to export soluble sugars (Deschamps et al., 2008) have also received considerable interest recently.
D
Available data indicate that blocking starch synthesis to provide precursor
TE
intermediates for lipid accumulation is a plausible strategy for increasing lipid yield. Wang et al. (2009) reported an increase in the triacylglycerol content after deleting the
CE P
AGPase gene. Another starchless mutant of Chlorella pyrenoidosa has also been shown to have elevated polyunsaturated fatty acid content (Ramazanov and Ramazanov, 2006).
AC
6.2 Engineering fatty acid biosynthesis Several studies have explored the effect on TAG production of overexpressing the enzymes involved in lipid synthesis. Acetyl-CoA carboxylase (ACCase), which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, is the rate-limiting enzyme in fatty acid biosynthesis. Attempts to overexpress ACCase as a means of increasing lipid content in various systems have been disappointing. Dunahay et al. (1995) overexpressed native ACCase in the diatom C. cryptic, which resulted in a 2to 3-fold increase in ACCase activity; however, little increase in lipid production was observed.
Other
studies
have
attempted
to
increase
the
expression
of
3-ketoacyl-acyl-carrier protein synthetase III (KASIII), which catalyzes the initial condensation reaction in fatty acid biosynthesis. Overexpression of KASIII from spinach (Spinacia oleracea) or Cuphea hookeriana in tobacco (Nicotiana tabacum), Arabidopsis thaliana, and Brassica napus resulted in no enhancement of seed oil 22
ACCEPTED MANUSCRIPT content (Dehesh et al., 1996). These results highlight the importance of understanding the metabolic pathways and their underlying regulation. In addition to enhancing the lipid content and productivity of microalgae,
T
improving the quality of biodiesel is also desirable. Biodiesel (or jet fuel) is composed
IP
of methyl esters of fatty acids, ideally those that are relatively short in length (e.g., C10-C18) and either saturated or monounsaturated. However, most microalgae
SC R
synthesize fatty acids composed of 16-22 carbons. Efforts to engineer thioesterase (TE; which is responsible for the termination of chain elongation in fatty acid biosynthesis) to catalyze the formation of C8:0-C14:0 fatty acids in microalgae have
NU
shown promise as a strategy to alter microalgal fatty acid composition. Plant TEs were recently engineered into heterologous hosts to alter the fatty acid content for
MA
biodiesel production. Voelker and coworkers (1992) introduced a plant TE derived from Umbellularia californica into A. thaliana and achieved a 24% increase in the C12:0 fatty acid content. Introduction of the C10:0-specific TE from C. hookeriana
D
into B. napus shifted the fatty acid content to 38% C10:0 (Dehesh et al., 1996).
TE
Taken together, these results suggest that it is possible to manipulate fatty acid biosynthesis to optimize microalgae as a biodiesel feedstock. However, new strategies
CE P
must be envisioned to enhance fatty acid content and oil yield.
6.3 Modifying carbon assimilation pathways Rubisco catalyzes the CO2 fixation reaction in the Calvin-Benson-Bassham
AC
(CBB) cycle and has been postulated as the rate-limiting enzyme in CO2 fixation. In microalgae grown under phototrophic conditions, all newly produced biomass, including starches and lipids, is derived from the fixation of CO2 into ribulose-1,5-bisphosphate (RuBP) to form 3-phosphoglycerate (3PG). Several studies have shown that the activity of Rubisco is the major bottleneck for carbon flux through the Calvin cycle, especially when CO2 is not enriched in the medium or under high-light or high-temperature conditions. At present, we are far from fully understanding the details of Rubisco regulation, and whether Rubisco can be engineered so as to improve photosynthetic CO2 assimilation remains a matter of debate. Other limitations include precise regulation of Rubisco expression and inaccurate folding of the protein in heterologous organisms (Andrews and Whitney, 2003). Nevertheless, two recent papers have made some significant observations. For example, tobacco Rubisco was successfully replaced with its counterpart from 23
ACCEPTED MANUSCRIPT Rhodospirillum rubrum, which has a naturally high specificity (Whitney and Andrews, 2001). However, the CO2-assimilation rates of these engineered plants were much lower than those of the wild-type plants (Whitney and Andrews, 2001). In other work,
T
genes encoding the small subunit of Arabidopsis and sunflower Rubisco were
IP
transformed into a Rubisco gene–deficient strain of Chlamydomonas. As a result, the
SC R
in vitro CO2/O2 specificity increased by 11% (Genkov et al., 2010).
7. Conclusion
The production of biofuels using lipid-/carbohydrate-rich microalgae is a very
NU
promising alternative to conventional biofuel production approaches; however, most of the engineering processes are not as yet economically viable. To significantly
MA
improve the feasibility of microalgae-based biofuel production, engineering strategies that will effectively increase both growth and lipid/carbohydrate content must be developed and should be optimized by employing favorable environmental factors
D
and suitable operation strategies. Here, various innovative operation strategies that
TE
incorporate appropriate environmental stresses were critically reviewed. Selection of the appropriate engineering strategy for biofuel production can be accomplished by
CE P
comparing the biological characteristics of various microalgae and their responses under different conditions. In addition, more effective manipulation of microalgal metabolism and the implementation of genetic modifications will be required to further enhance the efficiency of biofuel production using microalgae. Hopefully, this
AC
review will facilitate the development of economically viable microalgae-based biofuel production processes in the coming years via cultivation process optimization as well as metabolic and genetic engineering.
Acknowledgement This work has been supported through project P07015 by the New Energy and Industrial Technology Development Organization (NEDO).
References Abdollahi J, Dubljevic S. Lipid production optimization and optimal control of heterotrophic microalgae fed-batch bioreactor. Chem Eng Sci 2012;84:619-27. Bartley ML, Boeing WJ, Corcoran AA, Holguin FO, Schaub T. Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and
24
ACCEPTED MANUSCRIPT
SC R
IP
T
invading organisms. Biomass Bioenerg 2013;54:83-8. Bondioli P, Della Bella L, Rivolta G, Chini Zittelli G, Bassi N, Rodolfi L, et al. Oil production by the marine microalgae Nannochloropsis sp. F&M-M24 and Tetraselmis suecica F&M-M33. Bioresour Technol 2012;114:567-72. Borowitzka MA. Commercial production of microalgae: ponds, tanks, tubes and fermenters. J Biotechnol 1999;70:313-21. Brennan L, Owende P. Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sustain Energ Rev 2010;14:557-77.
MA
NU
Breuer G, Lamers PP, Martens DE, Draaisma RB, Wijffels RH. Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresour Technol 2013;143:1-9. Cai T, Park SY, Li Y. Nutrient recovery from wastewater streams by microalgae: Status and prospects. Renew Sustain Energ Rev 2013;19:360-9.
D
Cerón-García M, Fernández-Sevilla J, Sánchez-Mirón A, García-Camacho F, Contreras-Gómez A, Molina-Grima E. Mixotrophic growth of Phaeodactylum tricornutum on fructose and glycerol in fed-batch and semi-continuous modes. Bioresour Technol 2013;147:569-76.
AC
CE P
TE
Champigny M. Regulation of photosynthetic carbon assimilation at the cellular level: a review. Photosynthesis Res 1985;6:273-86. Cheirsilp B, Torpee S. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: Effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour Technol 2012;110:510-6. Chen C-Y, Chang J-S, Chang H-Y, Chen T-Y, Wu J-H, Lee W-L. Enhancing microalgal oil/lipid production from Chlorella sorokiniana CY1 using deep-sea water supplemented cultivation medium. Biochem Eng J 2013a;77:74-81. Chen C-Y, Yeh K-L, Aisyah R, Lee D-J, Chang J-S. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review. Bioresour Technol 2011;102:71-81. Chen C-Y, Zhao X-Q, Yen H-W, Ho S-H, Cheng C-L, Lee D-J, et al. Microalgae-based carbohydrates for biofuel production. Biochem Eng J. 2013b;78:1-10. Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294-306. Collet P, Lardon L, Hélias A, Bricout S, Lombaert-Valot I, Perrier B, et al. Biodiesel from microalgae – Life cycle assessment and recommendations for potential improvements. Renew Energ. 2014;71:525-33. Converti A, Casazza AA, Ortiz EY, Perego P, Del Borghi M. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis
25
ACCEPTED MANUSCRIPT
SC R
IP
T
oculata and Chlorella vulgaris for biodiesel production. Chem Eng Process: Process Intensif 2009;48:1146-51. Dehesh K, Jones A, Knutzon DS, Voelker TA. Production of high levels of 8: 0 and 10: 0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana. The Plant J 1996;9:167-72. Deschamps P, Haferkamp I, d‘Hulst C, Neuhaus HE, Ball SG. The relocation of starch metabolism to chloroplasts: when, why and how. Trends Plant Sci 2008;13:574-82. Dunahay TG, Jarvis EE, Roessler PG. Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J Phycol 1995;31:1004-12.
MA
NU
Feng Y, Li C, Zhang D. Lipid production of Chlorella vulgaris cultured in artificial wastewater medium. Bioresour Technol 2011;102:101-5. Fortier M-OP, Roberts GW, Stagg-Williams SM, Sturm BSM. Life cycle assessment of bio-jet fuel from hydrothermal liquefaction of microalgae. Appl Energ. 2014;122:73-82.
D
Gardner R, Peters P, Peyton B, Cooksey KE. Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the Chlorophyta. J Appl Phycol 2011;23:1005-16. Genkov T, Meyer M, Griffiths H, Spreitzer RJ. Functional hybrid rubisco enzymes
AC
CE P
TE
with plant small subunits and algal large subunits engineered rbcS cDNA for expression in Chlamydomonas. J Biol Chem 2010;285:19833-41. González-Fernández C, Ballesteros M. Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation. Biotechnol Adv 2012;30:1655-61. Gouveia L, Oliveira AC. Microalgae as a raw material for biofuels production. J Ind Microbiol Biotechnol 2009;36:269-74. Guarnieri MT, Nag A, Smolinski SL, Darzins A, Seibert M, Pienkos PT. Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga. PLoS One 2011;6:e25851. Guedes AC, Meireles LA, Amaro HM, Malcata FX. Changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity. J Am Oil Chem Soc 2010;87:791-801. Guo S-L, Zhao X-Q, Tang Y, Wan C, Alam MA, Ho S-H, et al. Establishment of an efficient genetic transformation system in Scenedesmus obliquus. J Biotechnol 2013;163:61-8. Han F, Huang J, Li Y, Wang W, Wan M, Shen G, et al. Enhanced lipid productivity of Chlorella pyrenoidosa through the culture strategy of semi-continuous cultivation with nitrogen limitation and pH control by CO2 Bioresour Technol 2013;136:418-24. Harwati TU, Willke T, Vorlop KD. Characterization of the lipid accumulation in a
26
ACCEPTED MANUSCRIPT
SC R
IP
T
tropical freshwater microalgae Chlorococcum sp. Bioresour Technol 2012;121:54-60. Ho S-H, Chan M-C, Liu C-C, Chen C-Y, Lee W-L, Lee D-J, et al. Enhancing lutein productivity of an indigenous microalga Scenedesmus obliquus FSP-3 using light-related strategies. Bioresour Technol 2014a;152:275-82. Ho S-H, Chang J-S, Lai Y-Y, Chen C-NN. Achieving high lipid productivity of a thermotolerant microalga Desmodesmus sp. F2 by optimizing environmental factors and nutrient conditions. Bioresour Technol 2014b;156:108-16. Ho S-H, Chen C-Y, Chang J-S. Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga
MA
NU
Scenedesmus obliquus CNW-N. Bioresour Technol 2012a:244-52. Ho S-H, Chen C-Y, Lee D-J, Chang J-S. Perspectives on microalgal CO2-emission mitigation systems — A review. Biotechnol Adv 2011;29:189-98. Ho S-H, Chen W-M, Chang J-S. Scenedesmus obliquus CNW-N as a potential candidate for CO2 mitigation and biodiesel production. Bioresour Technol
D
2010;101:8725-30. Ho S-H, Huang S-W, Chen C-Y, Hasunuma T, Kondo A, Chang J-S. Bioethanol production using carbohydrate-rich microalgae biomass as feedstock. Bioresour Technol 2013a;135:191-8.
AC
CE P
TE
Ho S-H, Huang S-W, Chen C-Y, Hasunuma T, Kondo A, Chang J-S. Characterization and optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E. Bioresour Technol 2013b;135:157-65. Ho S-H, Kondo A, Hasunuma T, Chang J-S. Engineering strategies for improving the CO2 fixation and carbohydrate productivity of Scenedesmus obliquus CNW-N used for bioethanol fermentation. Bioresour Technol 2013c;143:163-71. Ho S-H, Li P-J, Liu C-C, Chang J-S. Bioprocess development on microalgae-based CO2 fixation and bioethanol production using Scenedesmus obliquus CNW-N. Bioresour Technol 2013d;145:142-9. Ho S-H, Lu W-B, Chang J-S. Photobioreactor strategies for improving the CO2 fixation efficiency of indigenous Scenedesmus obliquus CNW-N: Statistical optimization of CO2 feeding, illumination, and operation mode. Bioresour Technol 2012b;105:106-13. Ho S-H, Nakanishi A, Ye XT, Chang J-S, Hara K, Hasunuma T, et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnol Biofuels 2014c, in press. Hong W-K, Rairakhwada D, Seo P-S, Park S-Y, Hur B-K, Kim C, et al. Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101. Appl Biochem Biotechnol 2011;164:1468-80. Hosono H, Uemura I, Takumi T, Nagamune T, Yasuda T, Kishimoto M, et al. Effect of
27
ACCEPTED MANUSCRIPT
SC R
IP
T
culture temperature shift on the cellular sugar accumulation of Chlorella vulgaris SO-26. J Ferment Bioeng 1994;78:235-40. Hsieh C-H, Wu W-T. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour Technol 2009;100:3921-6. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, et al. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. The Plant J 2008;54:621-39. Jamers A, Blust R, De Coen W. Omics in algae: Paving the way for a systems biological understanding of algal stress phenomena? Aquat Toxicol 2009;92:114-21.
MA
NU
James GO, Hocart CH, Hillier W, Price GD, Djordjevic MA. Temperature modulation of fatty acid profiles for biofuel production in nitrogen deprived Chlamydomonas reinhardtii. Bioresour Technol 2013;127:441-7. Jeffryes C, Rosenberger J, Rorrer GL. Fed-batch cultivation and bioprocess modeling of Cyclotella sp. for enhanced fatty acid production by controlled silicon limitation.
D
Algal Res 2013;2:16-27. Jiang L, Luo S, Fan X, Yang Z, Guo R. Biomass and lipid production of marine microalgae using municipal wastewater and high concentration of CO2. Appl Energ 2011;88:3336-41.
AC
CE P
TE
John Andrews T, Whitney SM. Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants. Arch Biochem Biophys. 2003;414:159-69. John RP, Anisha GS, Nampoothiri KM, Pandey A. Micro and macroalgal biomass: A renewable source for bioethanol. Bioresour Technol. 2011;102:186-93. Khalil ZI, Asker MM, El-Sayed S, Kobbia IA. Effect of pH on growth and biochemical responses of Dunaliella bardawil and Chlorella ellipsoidea. World J Microbiol Biotechnol 2010;26:1225-31. Khotimchenko SV, Yakovleva IM. Lipid composition of the red alga Tichocarpus crinitus exposed to different levels of photon irradiance. Phytochem 2005;66:73-9. Kilian O, Benemann CS, Niyogi KK, Vick B. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Pro Natl Acad Sci 2011;108:21265-9. Kim M-S, Baek J-S, Yun Y-S, Jun Sim S, Park S, Kim S-C. Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: Anaerobic conversion and photosynthetic fermentation. Int J Hydrogen Energ 2006;31:812-6. Kim M, Yim JH, Kim S-Y, Kim HS, Lee WG, Kim SJ, et al. In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03. Antivir Res 2012;93:253-9.
28
ACCEPTED MANUSCRIPT
SC R
IP
T
Lam MK, Lee KT. Microalgae biofuels: A critical review of issues, problems and the way forward. Biotechnol Adv 2012;30:673-90. Liu J, Yuan C, Hu G, Li F. Effects of light Intensity on the growth and lipid accumulation of microalga Scenedesmus sp. 11-1 under nitrogen limitation. Appl Biochem Biotechnol 2012;166:2127-37. Liu ZY, Wang GC, Zhou BC. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour Technol. 2008;99:4717-22. Lv J-M, Cheng L-H, Xu X-H, Zhang L, Chen H-L. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour Technol 2010;101:6797-804.
MA
NU
M ü nkel R, Schmid ‐ Staiger U, Werner A, Hirth T. Optimization of outdoor cultivation in flat panel airlift reactors for lipid production by Chlorella vulgaris. Biotechnol Bioeng 2013;110:2882-93. Marchetti J, Bougaran G, Le Dean L, Megrier C, Lukomska E, Kaas R, et al.
D
Optimizing conditions for the continuous culture of Isochrysis affinis galbana relevant to commercial hatcheries. Aquac 2012;326:106-15. Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: A review. Renew Sustain Energ Rev. 2010;14:217-32.
AC
CE P
TE
Mazzuca Sobczuk T, Chisti Y. Potential fuel oils from the microalga Choricystis minor. J Chem Technol Biotechnol 2010;85:100-8. Moheimani NR. Long-term outdoor growth and lipid productivity of Tetraselmis suecica, Dunaliella tertiolecta and Chlorella sp (Chlorophyta) in bag photobioreactors. J Appl Phycol 2013;25:167-76. Mujtaba G, Choi W, Lee C-G, Lee K. Lipid production by Chlorella vulgaris after a shift from nutrient-rich to nitrogen starvation conditions. Bioresour Technol 2012;123:279-83. Naik SN, Goud VV, Rout PK, Dalai AK. Production of first and second generation biofuels: A comprehensive review. Renew Sustain Energ Rev 2010;14:578-97. Nakanishi A, Aikawa S, Ho S-H, Chen C-Y, Chang J-S, Hasunuma T, et al. Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. JSC4. Bioresour Technol 2014;152:247-52. Neuhaus HE, Stitt M. Control analysis of photosynthate partitioning. Planta 1990;182:445-54. Neupert J, Shao N, Lu Y, Bock R. Genetic transformation of the model green alga Chlamydomonas reinhardtii. Transgenic Plants: Springer; 2012. p. 35-47. Pal D, Khozin-Goldberg I, Cohen Z, Boussiba S. The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. Appl Microbiol Biotechnol 2011;90:1429-41.
29
ACCEPTED MANUSCRIPT
SC R
IP
T
Parmar A, Singh NK, Pandey A, Gnansounou E, Madamwar D. Cyanobacteria and microalgae: a positive prospect for biofuels. Bioresour Technol 2011;102:10163-72. Pittman JK, Dean AP, Osundeko O. The potential of sustainable algal biofuel production using wastewater resources. Bioresour Technol 2011;102:17-25. Pragya N, Pandey KK, Sahoo P. A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renew Sustain Energ Rev 2013;24:159-71. Radakovits R, Jinkerson RE, Darzins A, Posewitz MC. Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 2010;9:486-501. Ramazanov A, Ramazanov Z. Isolation and characterization of a starchless mutant of
MA
NU
Chlorella pyrenoidosa STL‐PI with a high growth rate, and high protein and polyunsaturated fatty acid content. Phycol Res 2006;54:255-9. Ranga Rao A, Ravishanka G, Sarada R. Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production. Bioresour Technol 2012;123:528-33.
D
Rismani-Yazdi H, Haznedaroglu BZ, Bibby K, Peccia J. Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta: Pathway description and gene discovery for production of next-generation biofuels. BMC Genomics 2011;12:148. Ritte G, Heydenreich M, Mahlow S, Haebel S, Kötting O, Steup M. Phosphorylation
AC
CE P
TE
of C6-and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases. FEBS Lett 2006;580:4872-6. San Pedro A, González-López CV, Acién FG, Molina-Grima E. Marine microalgae selection and culture conditions optimization for biodiesel production. Bioresour Technol 2013;134:353-61. Santos AM, Janssen M, Lamers PP, Evers WAC, Wijffels RH. Growth of oil accumulating microalga Neochloris oleoabundans under alkaline–saline conditions. Bioresour Technol 2012;104:593-9. Sforza E, Enzo M, Bertucco A. Design of microalgal biomass production in a continuous photobioreactor: an integrated experimental and modeling approach. Chem Eng Res Des 2013;92:1153-62. Siaut M, Cuiné S, Cagnon C, Fessler B, Nguyen M, Carrier P, et al. Oil accumulation in the model green alga Chlamydomonas reinhardtii: Characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol 2011;11:7. Sims RE, Mabee W, Saddler JN, Taylor M. An overview of second generation biofuel technologies. Bioresour Technol 2010;101:1570-80. Singh J, Gu S. Commercialization potential of microalgae for biofuels production. Renew Sustain Energ Rev 2010;14:2596-610. Smith AM. Prospects for increasing starch and sucrose yields for bioethanol
30
ACCEPTED MANUSCRIPT
SC R
IP
T
production. The Plant J 2008;54:546-58. Su CH, Chien LJ, Gomes J, Lin YS, Yu YK, Liou JS, et al. Factors affecting lipid accumulation by Nannochloropsis oculata in a two-stage cultivation process. J Appl Phycol 2011;23:903-8. Sukenik A, Carmeli Y, Berner T. Regulation of fatty acid composition by irradiance level in the eustigmatophyte Nannochloropsis sp. 1. J Phycol 1989;25:686-92. Sukenik A, Wahnon R. Biochemical quality of marine unicellular algae with special emphasis on lipid composition in Isochrysis galbana. Aquac 1991;97:61-72. Sun X, Cao Y, Xu H, Liu Y, Sun J, Qiao D, et al. Effect of nitrogen-starvation, light
MA
NU
intensity and iron on triacylglyceride/carbohydrate production and fatty acid profile of Neochloris oleoabundans HK-129 by a two-stage process. Bioresour Technol 2014;155:204-12. Takagi M, Karseno, Yoshida T. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J
D
Biosci Bioeng 2006;101:223-6. Takagi M, Watanabe K, Yamaberi K, Yoshida T. Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999. Appl Microbiol Biotechnol 2000;54:112-7.
AC
CE P
TE
Talebi AF, Mohtashami SK, Tabatabaei M, Tohidfar M, Bagheri A, Zeinalabedini M, et al. Fatty acids profiling: A selective criterion for screening microalgae strains for biodiesel production. Algal Res 2013;2:258-67. Tang H, Abunasser N, Garcia MED, Chen M, Simon Ng KY, Salley SO. Potential of microalgae oil from Dunaliella tertiolecta as a feedstock for biodiesel. Appl Energ 2011;88:3324-30. Tang H, Chen M, Simon Ng K, Salley SO. Continuous microalgae cultivation in a photobioreactor. Biotechnol Bioeng 2012;109:2468-74. Vanthoor-Koopmans M, Wijffels RH, Barbosa MJ, Eppink MHM. Biorefinery of microalgae for food and fuel. Bioresour Technol 2013;135:142-9. Voelker TA, Worrell AC, Anderson L, Bleibaum J, Fan C, Hawkins DJ, et al. Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants. Sci 1992;257:72-4. Wan C, Bai F-W, Zhao X-Q. Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01. Biochem Eng J 2013;78:32-8. Wang H, Zhang W, Chen L, Wang J, and Liu T. The contanmination and control of biologcal pollutants in mass cultivation of microalgae. Bioresour Technol 2013;128:745-750. Wang X, Liu X, Wang G. Two-stage Hydrolysis of Invasive Algal Feedstock for
31
ACCEPTED MANUSCRIPT
SC R
IP
T
Ethanol Fermentation. J Integr Plant Biol 2011;53:246-52. Wang ZT, Ullrich N, Joo S, Waffenschmidt S, Goodenough U. Algal lipid bodies: Stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii. Eukaryot Cell 2009;8:1856-68. Whitney SM, Andrews TJ. Plastome-encoded bacterial ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco. Proc Nat Acade Sci 2001;98:14738-43. Wu LF, Chen PC, Lee CM. The effects of nitrogen sources and temperature on cell growth and lipid accumulation of microalgae. Int Biodeterior Biodegrad
MA
NU
2013;85:506-10. Xia L, Ge H, Zhou X, Zhang D, Hu C. Photoautotrophic outdoor two-stage cultivation for oleaginous microalgae Scenedesmus obtusus XJ-15. Bioresour Technol 2013;144:261-7. Xin L, Hong-Ying H, Yu-Ping Z. Growth and lipid accumulation properties of a
D
freshwater microalga Scenedesmus sp. under different cultivation temperature. Bioresour Technol 2011;102:3098-102. Xu Y, Boeing WJ: Modeling maximum lipid productivity of microalgae: Review and
AC
CE P
TE
next step. Renew Sustain Energ Rev 2014; 32:29-39. Yen H-W, Hu IC, Chen C-Y, Ho S-H, Lee D-J, Chang J-S. Microalgae-based biorefinery – From biofuels to natural products. Bioresour Technol 2013;135:166-74. Yu W-L, Ansari W, Schoepp NG, Hannon MJ, Mayfield SP, Burkart MD. Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact 2011;10:91. Zhila N, Kalacheva G, Volova T. Effect of salinity on the biochemical composition of the alga Botryococcus braunii Kütz IPPAS H-252. J Appl Phycol 2011;23:47-52. Zhou W, Min M, Li Y, Hu B, Ma X, Cheng Y, et al. A hetero-photoautotrophic two-stage cultivation process to improve wastewater nutrient removal and enhance algal lipid accumulation. Bioresour Technol 2012;110:448-55. Zhou X, Ge H, Xia L, Zhang D, Hu C. Evaluation of oil-producing algae as potential biodiesel feedstock. Bioresour Technol 2013a;134:24-9. Zhou X, Xia L, Ge H, Zhang D, Hu C. Feasibility of biodiesel production by microalgae Chlorella sp.(FACHB-1748) under outdoor conditions. Bioresour Technol 2013b;138:131-5.
32
ACCEPTED MANUSCRIPT Figure legend Figure 1. Overview of metabolic pathways in microalgal lipid/starch biosynthesis. 3PG, 3-phosphoglyceric acid; ACCase, acetyl-CoA carboxylase; ACP, acyl carrier
3-ketoacyl-acyl-carrier
protein
synthetase
IP
KSIII,
T
protein; AGPase, ADP-glucose pyrophosphorylase; G3P, glyceraldehyde-3-phosphate; III;
Rubisco,
ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate;
AC
CE P
TE
D
MA
NU
SC R
TE, thioesterase.
33
ACCEPTED MANUSCRIPT
Effect/improvement
Irradiance
Carbohydrate
Pavlova lutheri
Irradiance
Carbohydrate
N. oleoabundans HK-129
Irradiance
TAG
TAG content increased from 19 to 25% with increase in light intensity from 50 to 200 μmol m−2s−1 Biomass concentration increased from 1.2 to 1.7 g L−1 with increase in light intensity from 50 to 200 μmol m−2s−1
(Sun et al., 2014)
Lipid and TAG content increased from 26 to 41% and 16 to 32%, respectively, with increase in light intensity from 50 to 250 μmol m−2s−1 Biomass concentration increased from 2.5 to 3.6 g L−1 with increase in light intensity from 50 to 250 μmol m−2s−1
(Liu et al., 2012)
Pavlova lutheri
Irradiance
TAG
Temperature
TAG content increased from 23 to78% of total fatty acids with increase in light intensity from 9 to 19 W m−2
Lipid content decreased from 33 to 29% with increase in temperature from 25 to 35C Biomass concentration increased with increase in temperature from 25 to 30C but then decreased with further increase in temperature to 35C Lipid content decreased from 15 to 8% with increase in temperature from 15 to 20C but then increased to 14% with further increase in temperature to25C Specific growth rate increased with increase in temperature from 15 to 20C but then decreased with further increase in temperature to 25C
AC
Monoraphidium sp. SB2
CE P
Lipid/TAG
Lipid
N. oculata
Temperature
Lipid
(Ho et al., 2012a)
Carbohydrate content increased almost 3-fold, from 2.7 to 7.0 pg cell−1 with increase in light intensity from 30 to 400 μmol m−2s−1
Irradiance
References
Scenedesmus sp. 11-1
CR
S. obliquus CNW-N
Carbohydrate content increased from 15 to 38% with increase in light intensity from 60 to 420 μmol m−2s−1 Biomass productivity increased around 3-fold with increase in light intensity from 60 to 420 μmol m−2s−1
US
IP
Target products
MA N
Stress condition
TE D
Microalgae
T
Table 1. Carbohydrate/lipid accumulation in different microalgae under various environmental stress conditions.
34
(Sukenik and Wahnon, 1991)
(Guedes et al., 2010)
(Wu et al., 2013)
(Converti et al., 2009)
ACCEPTED MANUSCRIPT
Effect/improvement
Temperature
Lipid
Chlorella vulgaris SO-26
Temperature
Carbohydrate
C. reinhardtii BAF-J5
Temperature
TAG
pH
TAG
S. obliquus UTEX 393
pH
TAG
Coelastrella sp. PC-3
pH
TAG
C. ellipsoidea
pH
Carbohydrate
Nitrogen depletion
TAG
N. oleoabundans HK-129
References
(Xin et al., 2011)
(Hosono et al., 1994)
TAG content increased from 56 to 76% with increase in temperature from 17 to 32C Growth rate increased with increase in temperature from 17 to 32C
(James et al., 2013)
TAG content increased from 13 to 35% with increase in pH from 8.1 to 10 Growth rate decreased around 2-fold with increase in pH from 8.1 to 10
(Santos et al., 2012)
CE P
N. Oleoabundans
CR
Scenedesmus sp. LX1
Lipid content decreased from 35 to 22% with increase in temperature from 20 to 30C Biomass concentration increased with increase in temperature from 10 to 25C but then decreased with further increase in temperature to 30C Carbohydrate content decreased from 70 to 50% with increase in temperature from 5 to 20C Specific growth rate increased with increase in temperature from 5 to 20C but then decreased with further increase in temperature to 25C
US
IP
Target products
MA N
Stress condition
TE D
Microalgae
T
Table 1 (Continued)
TAG content increased with increase in pH pH range of 6-8 optimal for growth 6-8
(Breuer et al., 2013)
TAG content increased with increase in pH Growth rate decreased with increase in pH
(Gardner et al., 2011)
pH values of 10 and 9 optimal for growth and carbohydrate accumulation, respectively
(Khalil et al., 2010)
TAG content increased from 8 to 26% after 3-day nitrogen depletion Biomass productivity decreased from 220 to 197 mg L−1d−1 after 3-day nitrogen depletion
(Sun et al., 2014)
AC
35
ACCEPTED MANUSCRIPT
Lipid
Nannochloropsis sp. F&M-M24
Nitrogen depletion
Lipid
S. obliquus CNW-N
Nitrogen depletion
Carbohydrate
Nitrogen depletion
Carbohydrate
Salinity
TAG
Chlorella vulgaris FSP-E
B. braunii Kutz IPPAS H-252
Lipid content increased 10 to 48% after 4-day nitrogen depletion
Lipid content increased from 39 to 69% after nitrogen depletion Biomass productivity obviously decreased after nitrogen depletion Carbohydrate content increased from 21 to 49% after 2-day nitrogen depletion Growth rate slightly decreased after 2-day nitrogen depletion Carbohydrate content increased from 15 to 51% after 2-day nitrogen depletion Growth rate slightly decreased after 2-day nitrogen depletion TAG content increased from 5 to 31% with an increase in NaCl concentration from 0 to 0.7 M Growth rate significantly decreased with an increase in NaCl concentration from 0 to 0.7 M Lipid content increased from 10 to 30% with an increase in NaCl concentration from 0 to 2.0% Biomass concentration significantly decreased around 4-fold with an increase in NaCl concentration from 0 to 2.0% TAG content increased from 40 to 57% with an increase in NaCl concentration from 0.5 to 1.0 M Growth rate similar over the salinity range of 0.5-1.0 M Starch content increased around 4-fold with an increase in NaCl concentration from 0 to 1.0 M Starch content increased around 5-fold with an increase in NaCl concentration from 0 to 1.0 M
Salinity
Lipid
D. tertiolecta ATCC 30929
Salinity
TAG
CE P
Chlorococcum sp.
AC
C. reinhardtii CC-124
Salinity
Starch TAG
IP
Nitrogen depletion
CR
N. oculata
Effect/improvement
US
Target products
MA N
Stress condition
TE D
Microalgae
T
Table 1 (Continued)
36
References (Su et al., 2011) (Bondioli et al., 2012) (Ho et al., 2013c)
(Ho et al., 2013b)
(Zhila et al., 2011)
(Harwati et al., 2012)
(Takagi et al., 2006)
(Siaut et al., 2011)
ACCEPTED MANUSCRIPT
Chlorella sp. S. obliquus CNW-N Choricystis minor D. tertiolecta S. obliquus CNW-N Chlorella sp. NJ-18 Chlorella sp. C. pyrenoidosa Nannochloropsis sp. N. gaditana
IP
CR
Growth markedly increased Lipid content rarely affected Growth markedly increased Lipid content rarely affected Growth markedly increased Lipid content rarely affected Growth decreased Lipid content increased Growth markedly increased Carbohydrate content decreased Growth markedly increased Lipid content rarely affected Growth markedly increased Lipid content rarely affected Growth significantly increased Carbohydrate content also increased Growth markedly increased Lipid content rarely affected Growth increased Lipid content also increased Growth markedly increased Lipid content rarely affected Growth increased Lipid content also significantly increased Growth increased Lipid content also increased
US
MA N
Nannochloris sp.
Fed-batch (Glucose feeding + High light) Fed-batch (Glucose feeding + High light) Fed-batch (Nitrate feeding) Fed-batch (Urea feeding) Continuous (Fresh medium feeding) Continuous (Fresh medium feeding) Continuous (Fresh medium feeding) Semi-continuous (Fresh medium replacement) Semi-continuous (Fresh medium replacement) Semi-continuous (Fresh medium replacement) Semi-continuous (Low-N medium replacement) Two-stage (Nitrate depletion + High light) Two-stage (Continuous culture + Nitrate depletion)
TE D
Nannochloropsis sp.
Effect/improvement
CE P
Chlorella sp.
Operation strategy
AC
Microalgae
T
Table 2. Comparison of carbohydrate/lipid productivity of different microalgae using various operation strategies.
37
Optimal productivity (mg L−1d−1)
Reference
112.4 (Lipid)
(Cheirsilp and Torpee, 2012)
148.3 (Lipid)
(Cheirsilp and Torpee, 2012)
22.7 (TAG)
(Takagi et al., 2000)
123.0 (Lipid)
(Hsieh and Wu, 2009)
312.3 (Carbohydrate)
(Ho et al., 2013c)
82.0 (Lipid)
(Mazzuca Sobczuk and Chisti, 2010)
10.0 (FAME)
(Tang et al., 2012)
467.6 (Carbohydrate)
(Ho et al., 2013c)
24.1 (Lipid)
(Zhou et al., 2013a)
139.0 (Lipid)
(Hsieh and Wu, 2009)
115.0 (Lipid)
(Han et al., 2013)
76.6 (Lipid)
(Jiang et al., 2011)
51.0 (Lipid)
(San Pedro et al., 2013)
ACCEPTED MANUSCRIPT
N. oleoabundans S. obtusus XJ-15 Chlamydomonas sp. JSC4
IP
Growth slightly decreased Lipid content significantly increased Growth rarely affected Carbohydrate content significantly improved Growth slightly decreased Lipid content significantly improved
Growth rarely affected TAG content increased
Growth rarely affected Lipid content significantly increased Growth rarely affected Lipid content significantly increased
US
CR
MA N
S. obliquus CNW-N
Two-stage (Nitrogen depletion) Two-stage (Nutrient depletion) Two-stage (Nutrient depletion) Two-stage (Nitrogen depletion + Light intensity + Iron concentration) Two-stage (NaCl addition) Salinity-gradient (Stepwise addition of NaCl)
TE D
S. obliquus CNW-N
Effect/improvement
CE P
C. vulgaris AG10032
Operation strategy
AC
Microalgae
T
Table 2 (Continued)
38
Optimal productivity (mg L−1d−1)
Reference
77.8 (Lipid)
(Mujtaba et al., 2012)
352.9 (Carbohydrate)
(Ho et al., 2013d)
78.7 (Lipid)
(Ho et al., 2010)
51.6 (TAG)
(Sun et al., 2014)
60.7 (Lipid)
(Xia et al., 2013)
223.2 (Lipid)
(Ho et al., 2014c)
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
Figure 1
39
S0734-9750(14)00147-5 doi: 10.1016/j.biotechadv.2014.09.002 JBA 6842
To appear in:
Biotechnology Advances
Received date: Revised date: Accepted date:
11 June 2014 18 September 2014 19 September 2014
Please cite this article as: Ho Shih-Hsin, Ye Xiaoting, Hasunuma Tomohisa, Chang Jo-Shu, Kondo Akihiko, Perspectives on engineering strategies for improving biofuel production from microalgae – A critical review, Biotechnology Advances (2014), doi: 10.1016/j.biotechadv.2014.09.002
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
ACCEPTED MANUSCRIPT A revised manuscript submitted to Biotechnology Advances
T
Perspectives on engineering strategies for improving biofuel
SC R
IP
production from microalgae – A critical review
Shih-Hsin Hoa, Xiaoting Yea, Tomohisa Hasunumaa, Jo-Shu Changc.d.e,**, and Akihiko
a
NU
Kondob,f,*
Organization of Advanced Science and Technology and bDepartment of Chemical
MA
Science and Engineering, Graduate School of Engineering, Kobe University, 1-1 Rokkodai, Nada-ku, Kobe 657-8501, Japan c
Department of Chemical Engineering, dResearch Center for Energy Technology and
D
Strategy and eCenter for Bioscience and Biotechnology, National Cheng Kung University, Tainan 701, Taiwan
TE
f
Biomass
Engineering
Program,
RIKEN,
1-7-22
Suehiro-cho,
CE P
Yokohama, Kanagawa 230-0045, Japan
*
AC
Correspondence:
Prof. Akihiko Kondo Phone: +81-78-803-6196 Fax: +81-78-803-6196 E-mail: [email protected]
**
Co-correspondence: Prof. Jo-Shu Chang Phone: +886-6-2757575 ext. 62651 Fax: +886-6-2357146 E-mail: [email protected]
1
Tsurumi-ku,
ACCEPTED MANUSCRIPT Abstract Although
the
potential
for
biofuel
production
from
microalgae
via
photosynthesis has been intensively investigated, information on the selection of a
T
suitable operation strategy for microalgae-based biofuel production is lacking. Many
IP
published reports describe competitive strains and optimal culture conditions for use in biofuel production; however, the major impediment to further improvements is the
SC R
absence of effective engineering strategies for microalgae cultivation and biofuel production. This comprehensive review discusses recent advances in understanding the effects of major environmental stresses and the characteristics of various
bioethanol)
using
microalgae.
NU
engineering operation strategies on the production of biofuels (mainly biodiesel and The
performance
of
microalgae-based
MA
biofuel-producing systems under various environmental stresses (i.e., irradiance, temperature, pH, nitrogen depletion, and salinity) and cultivation strategies (i.e., fed-batch, semi-continuous, continuous, two-stage, and salinity-gradient) are
D
compared. The reasons for variations in performance and the underlying theories of
TE
the various production strategies are also critically discussed. The aim of this review is to provide useful information to facilitate development of innovative and feasible
CE P
operation technologies for effectively increasing the commercial viability of microalgae-based biofuel production.
Keywords: microalgae, biofuel, environmental stress, engineering operation strategy,
AC
fed-batch operation, semi-continuous operation, continuous operation, two-stage operation, salinity-gradient operation
2
ACCEPTED MANUSCRIPT Contents 1. Introduction 2. Biofuels produced using microalgae
T
2.1 Biodiesel
IP
2.2 Bioethanol
3. Environmental factors affecting microalgae-based biofuel production
SC R
3.1 Irradiance 3.2 Temperature 3.3 pH
NU
3.4 Nitrogen depletion 3.5 Salinity
production 4.1 Fed-batch processes
D
4.2 Continuous processes
MA
4. Engineering operation strategies for improving microalgae-based biofuel
TE
4.3 Semi-continuous processes 4.4 Two-stage processes
CE P
4.5 Salinity-gradient processes 4.6 Comparison of different operation strategies 5. Integrated processes involving microalgae-based biofuel production
AC
5.1 Mitigation of CO2 emissions 5.2 Wastewater treatment 5.3 Production of value-added co-products 5.4 Genetic engineering 5.5 Blocking starch synthesis to increase lipid content 5.6 Engineering fatty acid biosynthesis 5.7 Modifying carbon assimilation pathways 5.8 Genetic modification of carbohydrate metabolism 6. Conclusions References
3
ACCEPTED MANUSCRIPT 1. Introduction Changes in global climate attributed to greenhouse gas (GHG) emission from fossil fuel combustion threaten human life and entire ecosystems (Brennan and
T
Owende, 2010). Currently, about 90% of global energy demand is met by fossil fuels,
IP
which has generated several critical problems (e.g., energy demand crises and environmental damage) (Yen et al., 2013). With concern over these issues growing,
SC R
the search for highly sustainable renewable energy sources is now of paramount importance.
Biofuels, which are produced from biomass, are currently viewed as one of the
NU
most feasible energy alternatives to reduce our reliance on fossil fuels. Biofuels have several advantages over fossil fuels, including sustainability, non-toxicity,
MA
biodegradability, and extremely low CO2 emissions (Lam and Lee, 2012). Two widely used crop-based biofuels (or first-generation biofuels; i.e., biodiesel and bioethanol) are produced from lipid-based (biodiesel; rapeseed, palm, etc.) and sugar-based
D
(bioethanol; sugarcane, corn, etc.) feedstocks (Brennan and Owende, 2010, Chen et
TE
al., 2011, 2013b). However, the use of first-generation biofuels has caused considerable debate in recent years, primarily due to potential problems associated
CE P
with global food competition and ecosystem imbalance due to the requirement of an excessively large area of arable land for producing a sufficient amount of crop-based biofuels (Parmar et al., 2011). In addition, although various non-food feedstocks, such as agricultural residues and waste cooking oils, have been successfully converted into
AC
bioethanol or biodiesel, respectively (known as second-generation biofuels), the potential for non-food feedstocks to meet overall energy demands in the global market remains limited (Naik et al., 2010). Microalgae have received growing interest as a potential biofuel feedstock. Microalgae have numerous advantages in this respect, such as a high growth rate, high lipid and carbohydrate content (50-70% per unit of dry weight), low demand for fresh water and arable land, and high degree of environmental tolerance (Chisti, 2007, Lam and Lee, 2012). In addition, conversion of microalgal biomass to biofuels could effectively mitigate the environmental problems associated with CO2 emissions and water pollution, enhancing the benefits derived from microalgae-based biofuel production (Ho et al., 2011, Pittman et al., 2011). The commercial use of microalgae for sustainable biofuel faces some challenges due to low productivity and high cost associated with photosynthetic efficiency of 4
ACCEPTED MANUSCRIPT algal strain, photobioreactor design, harvesting method and extraction techniques. (Brennan and Owende, 2010, Chen et al., 2011, Pragya et al., 2013). In particular, the development of a simple and ideal operation strategy is considered the key to further
T
improving microalgae-based biofuel production processes. However, the existing
IP
literature indicates a lack of comprehensive knowledge of the processes for producing biofuels from microalgae and the best means of selecting, developing, and assessing
SC R
suitable operation strategies. Considering the potential of microalgae as a sustainable biofuel feedstock, this review provides an overview of the effect of different stressors on
microalgae
cultivation,
practical
operation
strategies,
and
future
NU
perspectives/challenges regarding the use of genetic engineering tools for improving the production of biofuels and value-added chemicals from microalgae. This review
MA
also emphasizes recent engineering advances in various operation strategies (e.g., fed-batch, semi-continuous, continuous, two-stage, and salinity-gradient) used for the efficient production of biofuels (primarily biodiesel and bioethanol) to address the
D
importance of engineering perspectives in promoting microalgae-based biofuel
TE
production to achieve the ultimate goal of commercialization.
CE P
2. Biofuels produced using microalgae Microalgae are able to accumulate large amounts of energy-rich compounds (e.g., triacylglycerol and starch) under favorable growth conditions. These energy-rich compounds can be further converted into biodiesel (triacylglycerol) and bioethanol
AC
(starch). Biofuels derived from microalgae are now recognized as ―third-generation,‖ representing an entirely new dimension in the alternative bioenergy field.
2.1 Biodiesel Biodiesel has attracted considerable attention as one of the most important new alternative energy sources because it is non-toxic, and biodegradable fuel with low CO2 emissions (Gouveia and Oliveira, 2009). Biodiesel is composed of methyl esters of long-chain fatty acids (mainly C12-C18 groups) derived from triglycerides contained in a variety of biological feedstocks, such as oleaginous crops, microalgae, and animal fat (Yen et al., 2013). Currently, most commercial biodiesel is produced from oleaginous crops, such as soybean, rapeseed, jatropha, sunflower, and palm trees (Chisti, 2007). However, competition for arable land and potable water supplies arising from crop-based 5
ACCEPTED MANUSCRIPT biodiesel production have become increasingly problematic global issues in recent years (Pragya et al., 2013). It has been reported that over 50% of the total arable land would be required for cultivating enough oleaginous crops to meet the biodiesel
T
demand for transportation use in the US, which could result in a serious global food
IP
crisis. However, only 6% of US cropland would be required if the demand were met through microalgae-based biodiesel production (Liu et al., 2008).
SC R
The primary microalgae species used for biodiesel production are from the genera Botryococcus (Ranga Rao et al., 2012), Chlorella (Chen et al., 2013a, Münkel et al., 2013, Zhou et al., 2013b), Scenedesmus (Ho et al., 2012a, Xia et al., 2013),
NU
Chlamydomonas (Nakanishi et al., 2014, Siaut et al., 2011), Dunuliella (Moheimani, 2013, Tang et al., 2011), and Nannochloropsis (Bartley et al., 2013, Bondioli et al.,
MA
2012, Wan et al., 2013). The advantages of using microalgae for biodiesel production include: high growth rate, high lipid yield, high environmental tolerance, low competition for arable land and potable water, and no seasonal limitations on
D
culturing (Chisti, 2007, Ho et al., 2010). However, the fatty acid profile of microalgal
TE
lipids is also vital, because the quality of biodiesel produced is highly dependent on the composition of the constituent fatty acids, which strongly influences the
CE P
combustion efficiency and heating power of engines (Singh and Gu, 2010, Talebi et al., 2013). Fortunately, under suitable conditions of environmental stress, the lipids produced by microalgae are mostly composed of neutral fatty acids with a low degree of unsaturation (in most cases no more than two unsaturated bonds), thus confirming
AC
the suitability and potential of microalgae-based biodiesel as a partial replacement for fossil diesel (Talebi et al., 2013).
2.2 Bioethanol Bioethanol is another major commercialized biofuel at present and is primarily produced from sugar- and starch-based food crops (e.g., corn and sugarcane) via biological fermentation processes. Bioethanol production in the US increased from 1.6 to 13.2 billion gallons between 2000 and 2010 (Chen et al., 2013b). Taking into account the increased global population and increased demand for crops as food sources, lignocellulosic materials (e.g., agricultural residues) are regarded as better alternative feedstocks for bioethanol, as their production does not result in competition for arable land and food supplies (Sims et al., 2010). However, the major obstacle to efficient and economical bioethanol production from lignocellulosic 6
ACCEPTED MANUSCRIPT biomass is the high lignin content since it requires a very difficult pretreatment process (Ho et al., 2013a). In contrast to lignocellulosic biomass, some microalgae can accumulate large
T
amounts of carbohydrates in the form of starch or cellulose during periods of extreme
IP
environmental stress (John et al., 2011, Siaut et al., 2011). Therefore, there is currently considerable interest in the use of microalgae as an alternative feedstock for
SC R
bioethanol fermentation. Microalgae species belonging to genera such as Scenedesmus (Ho et al., 2013d), Chlorella (Ho et al., 2013a), and Chlamydomonas (Kim et al., 2006) reportedly can accumulate a considerable amount of carbohydrate
NU
(>50% of total dry cell weight) as biomass. Moreover, the carbohydrates found in microalgae are primarily composed of starch and cellulose (with the absence of
MA
lignin), which are much easier to convert to simple sugars for fermentation compared with lignocellulosic biomass (Ho et al., 2013a, John et al., 2011). Therefore, even though the number of studies concerning the use of microalgae for bioethanol
D
production is relatively limited compared with studies concerning their use for
TE
biodiesel production, many researchers still believe that in the near future, microalgae-based bioethanol production will prove to be a feasible alternative to
CE P
conventional sugar-rich crops or lignocellulosic biomass feedstocks for producing bioethanol (Chen et al., 2013b, Ho et al., 2013d, John et al., 2011, Wang et al., 2011).
3. Environmental factors affecting microalgae-based biofuel production
AC
Although the growth rate and cell composition of microalgae varies with species, most reports agree that microalgae could grow faster but generate only a small amount of lipid or carbohydrate under environmental conditions favorable to their growth. As a result, lipids or carbohydrates usually only begin to accumulate in microalgal cells under conditions of physical or chemical stress, such as extremes in irradiance, temperature, pH, nutrient availability, salinity, and trace metal concentrations (Chen et al., 2013b, Hu et al. , 2008). However, those stresses often lead to the inhibition of growth resulting in lower biomass productivity and higher contamination risk (Hu et al. , 2008). To enhance the economic feasibility of using microalgae for biofuels production, a better understanding is needed regarding how to rapidly accumulate lipids and carbohydrates in microalgal cells with higher cell growth rate can be controlled by manipulation of environmental conditions during
7
ACCEPTED MANUSCRIPT cultivation. The effects of different environmental factors on the production of lipids and carbohydrates are described in detail below.
T
3.1 Irradiance
IP
Irradiance plays an important role in photosynthesis and thereby strongly influences the growth, rate of CO2 fixation, and cell composition of photoautotrophic
SC R
microalgae (Ho et al., 2012a, Hu et al., 2008). Microalgal growth is strongly affected by the level of irradiance, which can fall into one of three categories, namely, light limitation, light saturation, and light inhibition (Ho et al., 2012a). Before reaching the
NU
stage of light inhibition, the growth rate of microalgae increases with rising light intensity, although the extent of the light intensity effect varies from species to species.
MA
As depicted in Table 1, Liu et al. (2012) found that biomass productivity of Scenedesmus sp. 11-1 was increased from 255 to 452 mg L-1d-1 when the light intensity was increased from 50 to 250 μmol m−2s-1. Ho et al. (2012a) also showed the
D
biomass productivity of S. obliquus CNW-N increased nearly 3-fold with an increase
TE
in light intensity from 60 to 420 μmol m−2 s-1. Through photosynthesis, large amounts of ATP and NADPH are produced as sources
and
reduction
power
for
the
conversion
of
CO2
to
CE P
energy
glyceraldehyde-3-phosphate (G3P), which is the primary precursor for the synthesis of both triacylglycerol (TAG) and starch (Chen et al., 2013b, Lv et al., 2010). Exposure to an environmental stress results in a competition between TAG and starch
AC
synthesis; which type of energy-producing compound will accumulate in the microalgal cell appears to be species specific (Rismani-Yazdi et al., 2011). It has been reported that appropriate irradiance can result in changes in pH and the concentrations of Mg2+ and NADPH in the stroma, which may in turn regulate the fate of some key metabolites (e.g., G3P) and thus further influence the accumulation of TAG (Lv et al., 2010). It has also been demonstrated that one key enzyme of phosphoglucomutase (PGM), which is involved in starch synthesis, can be regulated by varying irradiance levels (Neuhaus and Stitt, 1990). A related study suggested that light intensity plays an important role in the accumulation of lipids and carbohydrates (Sun et al., 2014). As shown in Table 1, appropriately increasing light intensity can lead to a significant increase in the accumulation of TAG or carbohydrates in some microalgae with simultaneous enhancement of cell growth, resulting in a higher TAG or carbohydrate yield. Typically, a sufficient light supply leads to a decrease in the total polar lipid 8
ACCEPTED MANUSCRIPT content and a significant increase in the content of neutral lipids (primarily TAG) as energy-storage compounds (Khotimchenko and Yakovleva, 2005, Sukenik et al., 1989). Unfortunately, the correlation between light supply and carbohydrate synthesis
T
in microalgae remains unclear, although it has been proven that generation of the
IP
precursors for sucrose and starch synthesis can be stimulated by irradiance (Champigny, 1985).
SC R
In addition, microalgal TAG and carbohydrate profiles can be influenced by irradiance level. For Scenedesmus obliquus, for instance, exposure to sufficient light intensity (e.g., 180 μmol m−2s−1) strongly increases the percentages of palmitic
NU
(C16:0), stearic (C18:0), and oleic (C18:1) acids in total TAG as well as the percentage of glucose in total carbohydrate (Ho et al., 2012a). Another study
MA
demonstrated that the content of major fatty acids, including palmitic (C16:0), stearic (C18:0), and oleic (C18:1) in the microalga N. oleoabundans can be significantly increased by culturing cells under high-intensity light (300 μmol m−2s−1) (Sun et al.,
D
2014). Based upon those studies, it seems clear that high light intensity can trigger the
TE
production of the saturated and monounsaturated fatty acids that are the primary components of neutral lipids (Hu et al., 2008). In contrast, there is still no clear
CE P
information regarding the dependence of microalgal carbohydrate profiles on irradiance level (Ho et al., 2012a).
3.2 Temperature
AC
As with the cultivation of other microorganisms, temperature is one of the most important factors in microalgae cultivation and is highly correlated with growth rate. Typically, higher growth rate of microalgae can be achieved by increasing temperature to its optimal level (Table 1) (González-Fernández and Ballesteros, 2012, Ho et al., 2013d). Variations in temperature also play an important role in the accumulation of lipids and carbohydrates in many species of microalgae, as shown in Table 1. The lipid content of Monoraphidium sp. SB2, N. oculata, and Scenedesmus sp. LX1 decreases as the temperature is increased from 25 to 35C, 15 to 20C, and 20 to 30C, respectively (Converti et al., 2009, Wu et al., 2013, Xin et al., 2011). However, James et al. (2013) showed that the TAG content of C. reinhardtii BAF-J5 is positively correlated with cultivation temperature. Another study demonstrated that the lipid content of Desmodesmus sp. F2 remains stable over the temperature range of
9
ACCEPTED MANUSCRIPT 25-40°C, suggesting that the regulation of genes and the activity of enzymes involved in lipid biosynthesis are not sensitive to temperature changes in this range (Ho et al., 2014b). Therefore, the effect of growth temperature on the lipid content of microalgae
T
varies from species to species. In addition, as shown in Table 1, the suitable
eventually resulting in a lower lipid productivity.
IP
temperature for lipid accumulation often causes the inhibition of cell growth,
SC R
As for carbohydrate accumulation, it has been reported that the carbohydrate content of Chlorella vulgaris SO-26 increases by 20-30% when the culture temperature is decreased from 20 to 5C (Hosono et al., 1994). The enzymes involved
NU
in carbohydrate synthesis (e.g., starch synthase and sucrose synthase) are known to be influenced by culture temperature (González-Fernández and Ballesteros, 2012).
MA
However, some researchers have pointed out that the effect of temperature on carbohydrate accumulation in microalgae remains unclear (Chen et al., 2013b). In addition, temperature appears to affect the TAG profile in microalgae, as the
D
proportion of saturated fatty acids increases with increasing temperature (Hu et al.,
TE
2008, Wu et al., 2013).
CE P
3.3 pH
The pH of the culture medium is an important factor, influencing many biological processes associated with microalgal growth, metabolism, and uptake of ions (Khalil et al., 2010). In general, the optimum pH for growth is also species
AC
dependent. For instance, Khalil et al. (2010) reported that the optimal pH for the growth of D. bardawil is about 7.5, whereas that for C. ellipsoidea is about 10. In addition, as shown in Table 1, as the culture medium pH is raised, the TAG content increases in some species of microalgae, but this comes with the cost of a sharp decrease in growth rate (Breuer et al., 2013, Gardner et al., 2011, Santos et al., 2012). These data suggest that high-pH stress not only inhibits the cell cycle but also effectively triggers lipid accumulation. Moreover, Santos et al. (2012) also demonstrated that high-pH stress not only increases the lipid content but also enhances lipid quality. At a high pH (e.g., pH 10), the major fatty acids produced by N. oleoabundans include palmitic (C16:0), stearic (C18:0), oleic (C18:1), and linoleic (C18:2), all of which are suitable for biodiesel production. In contrast, Khalil et al. (2010) found that the carbohydrate content of both D. bardawil and C. ellipsoidea is
10
ACCEPTED MANUSCRIPT highly correlated with biomass concentration, indicating that use of an appropriate pH can significantly improve both growth and carbohydrate accumulation in these species, as the maximum carbohydrate content of these two organisms occurs at pH 7.5 and
IP
T
9.0, respectively.
3.4 Nitrogen depletion
SC R
Of all the macronutrients in the culture medium, the nitrogen source is the most critical in terms of the effect on lipid and carbohydrate accumulation in microalgae (Chen et al., 2013b, Chisti, 2007). A wide variety of studies have demonstrated that
NU
microalgae tend to allocate their carbon molecules to energy-rich lipids or carbohydrates when they encounter conditions of nitrogen depletion (Hu et al., 2008,
MA
John et al., 2011, Siaut et al., 2011). Nitrogen limitation may cause a sharp increase in microalgal lipid/carbohydrate content, along with a marked drop in the protein and chlorophyll content, suggesting that microalgae can degrade chlorophyll and
D
proteins/peptides as nitrogen source while transforming their carbon skeletons into
TE
lipid and carbohydrate in response to the extreme environment (Ho et al., 2012a, Sun et al., 2014). As shown in Table 1, Su et al. (2011) reported achieving a lipid content
CE P
of 48% in the microalga N. oculata when the organism was cultured under nitrogen-depletion conditions for 4 days. Similarly, nitrogen starvation triggers significant lipid accumulation in Nannochloropsis sp. F&M-M24 (Bondioli et al., 2012). In addition, cultivation of S. obliquus CNW-N and C. vulgaris FSP-E under
AC
nitrogen-depletion conditions results in a dramatic increase in the cellular carbohydrate content, from 21 to 49% and 15 to 51%, respectively (Ho et al., 2013b, Ho et al., 2013c).
In addition to enhancing lipid/carbohydrate accumulation, the stress associated with nitrogen limitation also strongly influences the lipid (fatty acids) and carbohydrate profiles in many microalgae (Ho et al., 2012a). Several reports have shown that nitrogen depletion leads to major increases in the proportions of saturated (e.g., palmitic acid) and monounsaturated (e.g., oleic acid) fatty acids, both of which are primary substrates for biodiesel production (Siaut et al., 2011). Nitrogen-starvation stress can also trigger the accumulation of carbohydrates in some microalgae. As carbohydrates are primarily located in the cell wall (mostly as cellulose) and plastids (mostly as starch), in some species of microalgae carbohydrate
11
ACCEPTED MANUSCRIPT typically accumulates as glucose, which can be easily converted to bioethanol via microbial fermentation (Ho et al., 2013b).
T
3.5 Salinity
IP
Similar to other environmental factors, salinity also plays a vital role in the accumulation of lipids/carbohydrates in microalgae. However, high salinity also slows
SC R
the growth of some species, such as Chlorococcum sp. (Harwati et al., 2012), Dunaliella sp. (Takagi et al., 2006), and Botryococcus braunii (Zhila et al., 2011). The literature shows that lipid/carbohydrate accumulation in microalgae increases in
NU
response to an immediate salinity shock (Table 1). For instance, increasing the culture medium NaCl concentration from 0 to 2% in the 10-day cultivation of Chlorococcum
MA
sp. leads to an increase in the lipid content, from 10.3 to 29.8% (Harwati et al., 2012). Similarly, the TAG content in microalgae is enhanced significantly by increasing the NaCl concentration (Takagi et al., 2006, Zhila et al., 2011). Moreover, the starch
D
content of Chlamydomonas reinhardtii cultivated in medium containing 100 mM
TE
NaCl is 4-fold higher than that obtained with freshwater cultivation (Siaut et al., 2011).
CE P
Fatty acid composition is also affected significantly by changes in culture medium salinity (Zhila et al., 2011). In Dunaliella sp. and Botryococcus braunii, for instance, an increase in salinity significantly decreases the proportion of polyunsaturated fatty acids (e.g., linoleic acid [C18:3]) and increases the proportions
AC
of saturated and monounsaturated fatty acids, including palmitic (C16:0), stearic (C18:0), and oleic (C18:1) acids, a composition that is suitable for biodiesel production (Takagi et al., 2006, Zhila et al., 2011). In addition, it has been reported that the sucrose pathway in microalgae can be altered via addition of NaCl to the culture medium (González-Fernández and Ballesteros, 2012). However, the effect of changes in the salinity level on starch accumulation in microalgae has yet to be fully elucidated.
4. Engineering operation strategies for improving microalgae-based biofuel production Although microalgae can grow faster and produce higher yields of lipids/carbohydrates as compared with other energy crops, the high cost of cultivation systems continues to impede the commercialization of microalgae-based biofuel 12
ACCEPTED MANUSCRIPT production processes. It is thus necessary to develop more economically feasible processes in order to increase the production of lipids/carbohydrates and thereby reduce the cultivation cost (Chen et al., 2013b). A large number of studies have
production.
However,
to
date
no
commercially
viable
IP
lipid/carbohydrate
T
focused on the development of more efficient cultivation methods that will improve
microalgae-based systems for biofuel production have been developed. The ideal
SC R
process would enable microalgae to grow fast with a simultaneous increase in lipid/carbohydrate productivity, which is strongly affected by cell growth and lipid/carbohydrate content. However, simultaneous increases in both growth rate and
NU
lipid/carbohydrate production is difficult to achieve because the accumulation of energy-rich compounds (e.g., lipids and carbohydrates) in microalgae usually occurs
MA
only under conditions of environmental stress, which in turn often decreases the growth rate (Ho et al., 2013d). Therefore, an operation strategy optimized so as to achieve both the highest biomass productivity and lipid/carbohydrate content is
D
needed in order to reduce the costs of cultivation and product formation.
TE
From the perspective of engineering process, achieving high overall lipid/carbohydrate productivity of microalgae is always the key issue when evaluating
CE P
its commercialization potential (Ho et al., 2012a). Therefore, how to accurately estimate the productivity of lipids or carbohydrates of the microalgae is critically important. In most published reports, the lipid/carbohydrate productivity was calculated by directly multiplying the lipid content with the biomass productivity (Ho
AC
et al., 2012a). This calculation may not precisely reflect the true lipid/carbohydrate productivity since the biomass productivity and lipid/carbohydrate content are usually not linearly correlated (Xu et al., 2014). Therefore, as proposed by Su et al. (2011), a more reasonable way to quantify the overall lipid/carbohydrate productivity is to determine the difference between the initial and final lipid/carbohydrate concentrations over the period of cultivation time. The initial and final lipid/carbohydrate
concentration
can
be
obtained
by
multiplying
the
lipid/carbohydrate content with biomass concentration at the time of inoculation and of harvesting, respectively. Nevertheless, the value of initial lipid/carbohydrate concentration might be negligibly small, especially when the inoculum size is very small and/or the initial lipid/carbohydrate content is very low (Converti et al. 2009, Xu et al., 2014).
13
ACCEPTED MANUSCRIPT 4.1 Fed-batch processes For microalgae cultivation, the fed-batch operation process is one of the most promising strategies for enhancing growth rate and end product production due to
T
flexibility with respect to the specific nutrients supplied during cultivation (Abdollahi
IP
and Dubljevic, 2012). Recent studies have shown that the biomass concentration and overall lipid production of Nannochloris sp. and Chlorella sp. are increased by
SC R
intermittent nitrogen feeding, but their lipid content is not increased under conditions of nitrogen sufficiency (Hsieh and Wu, 2009, Takagi et al., 2000). It has also been reported that fatty acid production by Cyclotella sp. is increased by controlled silicon
NU
limitation in fed-batch cultivation (Jeffryes et al., 2013). Moreover, Cheirsilp and Torpee (2012) demonstrated that both the lipid content and lipid productivity of
MA
Chlorella sp. and Nannochloropsis sp. are significantly increased via stepwise addition of glucose, indicating that maintaining the glucose concentration at a low level during fed-batch cultivation is a potentially useful method for increasing
D
microalgae-based lipid production.
TE
However, fed-batch processes may not always be suitable for microalgae cultivation, as light may become significantly limited in the culture due to high cell
CE P
density following prolonged cultivation, especially when the microalgae are grown under phototrophic or mixotrophic conditions. Light limitation in turn leads to a reduction in biomass productivity. To overcome this problem, a fed-batch operation involving a stepwise increase in light intensity was proposed, and this strategy was
AC
shown to dramatically enhance the growth and lipid productivity of microalgae under mixotrophic conditions (Cerón-García et al., 2013, Cheirsilp and Torpee, 2012). However, although fed-batch operations coupled with stepwise increases in light intensity may be effective, they are very difficult to control, especially when microalgae are being cultured outdoors with a naturally fluctulating light intensity over time.
4.2 Continuous processes Under phototrophic or mixotrophic conditions, the increasing turbidity associated with microlagal growth usually becomes a significant growth-limiting factor due to the self-shading effect. Continuous cultivation may therefore be an attractive alternative because continuous feeding of fresh medium effectively dilutes the cell density inside the photobioreactor so that it does not become too high. 14
ACCEPTED MANUSCRIPT Biomass productivity is maintained at a relatively high level due to the continuous feeding of medium and nutrients to support growth. In addition, continuous cultivation systems are generally low cost and easy to operate. Hence, continuous
T
processes are suitable for large-scale cultivation of microalgae for industrial
IP
applications (Marchetti et al., 2012, Sforza et al., 2013).
Under steady-state conditions in continuous culture, the biomass concentration
SC R
can be controlled by adjusting the rate of dilution with culture medium, and irradiance can be continuously maintained at a specific level (Tang et al., 2012). Of course, these characteristics facilitate light penetration, resulting in significant increases in both
NU
specific growth rate and biomass productivity. In addition, as shown in Table 1, some studies have found that irradiance plays an important role in the accumulation of
MA
lipids/carbohydrates. As illustrated in Table 2, continuous operation systems have been successfully used to produce microalgae-based biofuels, as they lead to a higher lipid/carbohydrate content and increased biomass production with long-term
D
cultivation (Ho et al., 2013c, Mazzuca Sobczuk and Chisti, 2010, Tang et al., 2012).
TE
However, because the growth rate must remain constant in a steady-state continuous culture, the feeding medium should contain sufficient nutrients (and a nitrogen source)
CE P
to sustain microalgal growth, and the culture conditions should not be growth inhibiting. However, the non-limiting nutrient and relatively non-stressful environmental conditions of continuous culture systems may have a negative effect on the accumulation of lipids and carbohydrates, which is usually higher under of
nutrient
AC
conditions
starvation
or
environmental
stress.
Therefore,
the
lipid/carbohydrate content in microalgal cells cultivated in a continuous system may not reach a satisfactory level (e.g., 40-60% of total dry cell weight). This may increase the difficulty and costs of downstream processing (e.g., extraction). In addition, because microalgae usually grow fairly slowly, a relatively low dilution rate should be used in order to maintain steady-state conditions; however, this increases the possibility of microbial contamination. Also, if the system is operated outdoors, growth will become very slow at night, resulting in the possibility of cell washout. Therefore, to achieve a stable outdoor continuous culture, the dilution rate may need to be adjusted during the light and dark periods according to growth conditions. In addition, using artificial light sources (such as LED) to provide additional light intensity (especially during the dark period) (Tang et al., 2012) may be useful for
15
ACCEPTED MANUSCRIPT maintaining the stable microalgal growth necessary to make the continuous culture operation successful.
T
4.3 Semi-continuous processes
IP
The semi-continuous operation is one of the simplest and most efficient bioreactor strategies used in microalgae-based biofuel production (Ho et al., 2013c,
SC R
Hsieh and Wu, 2009). Semi-continuous processes are an ideal strategy for avoiding both a low cell division rate in the early exponential stage and light limitation in the late stationary stage because it allows for maintaining the microalgal culture under
NU
exponential growth conditions, resulting in enhanced biofuel production (Ho et al., 2013c). In addition, semi-continuous operation processes seem to be more practicable
MA
than other operation modes for long-term cultivation, making them suitable for industrial production of microalgae-derived lipids/carbohydrates (Chen et al., 2013a, Ho et al., 2013c). As illustrated in Table 2, the biomass productivity of S. obliquus
D
CNW-N can be dramatically enhanced in semi-continuous systems, resulting in a
TE
favorable carbohydrate content of 50-52% and consequently higher carbohydrate productivity (Ho et al., 2013c). Another study showed that both biomass production
CE P
and the lipid content of Chlorella sp. can be increased under optimal semi-continuous cultivation (Hsieh and Wu, 2009). Han et al. (2013) reported that semi-continuous operation with pH regulation and nitrogen depletion sharply increases lipid accumulation in C. pyrenoidosa. Lipid productivity in that system was 3.6-fold higher
AC
than in batch cultivation. In outdoor cultivation of Chlorella sp. NJ-18, use of a semi-continuous operation results in markedly improved biomass and lipid productivity (Zhou et al., 2013a). Available data suggest that the semi-continuous process is a reasonable strategy for simultaneously enhancing microalgal growth and lipid/carbohydrate accumulation. However, semi-continuous processes have rarely been utilized in outdoor large-scale operations (Zhou et al., 2013a). Therefore, whether the use of semi-continuous processes to cultivate biofuel-producing microalgae outdoors is feasible or not should be evaluated through further studies.
4.4 Two-stage processes As discussed earlier, when producing lipids/carbohydrates from microalgae for biofuels applications, one of the major problems is the conflict with respect to the 16
ACCEPTED MANUSCRIPT optimal conditions required for achieving a high lipid/carbohydrate content and those necessary for achieving a high growth rate. From an engineering perspective, this type of problem can be dealt with by applying a two-stage cultivation process, which
T
provides different conditions for growth and lipid/carbohydrate accumulation, thereby
IP
markedly enhancing productivity. Indeed, two-stage processes have already been utilized in both indoor laboratory-scale and outdoor large-scale microalgal cultivation
SC R
operations for the production of biofuel feedstock (Ho et al., 2010, San Pedro et al., 2013, Xia et al., 2013).
In the two-stage strategy, nutrient-rich medium is used in the first stage to
NU
achieve maximum biomass productivity. After a sufficient amount of microalgal biomass is produced, the culture conditions are then altered to induce stress, such as
MA
that associated with nitrogen depletion, salt addition, or unsuitable ion concentrations (Table 2), thereby stimulating lipid accumulation in the second stage. San Pedro et al. (2013) suggested that the use a continuous operation process is a reasonable option
D
for the first stage in order to enhance biomass by adjusting the dilution rate. In the
TE
second stage, various types of stresses can be induced in order to dramatically upregulate lipid/carbohydrate accumulation with fair biomass productivity, resulting
CE P
in higher lipid/carbohydrate productivity (Ho et al., 2013d, Mujtaba et al., 2012, Sun et al., 2014). Based on the successes demonstrated in the above-mentioned studies, the two-stage operation seems to be an attractive strategy for microalgae-based biofuel production. However, the feasibility of commercialized outdoor large-scale two-stage
AC
operations to produce microalgae-based biofuels is questionable due to their high energy demand (Sun et al., 2014).
4.5 Salinity-gradient processes Increasing attention has been paid in recent years to the effect of salinity-associated stress on microalgae-based biofuel production (Harwati et al., 2012, Pal et al., 2011). As shown in Table 1, cultivation under high salinity conditions may increase the lipid/carbohydrate content, but this comes at the cost of a lower growth rate. Achieving higher lipid/carbohydrate productivity thus necessitates that the growth inhibition resulting from salinity stress be alleviated. Ideally, inducing high-salinity stress to stimulate lipid accumulation in microalgae and maintain a satisfactory growth rate (e.g., by enhancing adaptation to salinity) seems to be a promising strategy for simultaneously enhancing both lipid accumulation and 17
ACCEPTED MANUSCRIPT productivity. Takagi et al. (2006) suggested that gradually increasing the salt concentration in the medium facilitates microalgal adaptation to high salinity. This concept, designated as a ―salinity-gradient‖ process, is demonstrated for the first time
T
in this review. In this process, microalgae are cultivated in a nutrient-sufficient
IP
medium to achieve maximum biomass productivity and are then switched to a salinity-gradient cultivation mode via a stepwise increase in the concentration of sea
SC R
salt in the culture medium. As shown in Table 2, the stepwise addition of salt dramatically increases the lipid content in Chlamydomonas sp. JSC4, without significant inhibition of growth (Ho et al., 2014c). To date, no reports have been
NU
published concerning the use of salinity-gradient operation strategies to enhance lipid/carbohydrate productivity. This review proposes that the salinity-gradient
MA
process has a high potential for microalgae-based biofuel production via a two-stage operation.
D
4.6 Comparison of different operation strategies
TE
Although lipid/carbohydrate content is usually strain dependent, the type of operation strategy chosen plays a key role in determining whether a process will be
CE P
commercially feasible for microalgae-based biofuel production. Comparison of the characteristics of the five main microalgae cultivation strategies (fed-batch, continuous, semi-continuous, two-stage, and Salinity-gradient) shows that the fed-batch and continuous culture systems have certain advantages, including high
AC
biomass productivity and low operational cost. However, since fed-batch and continuous culture are often operated under nutrient-sufficient conditions, the microalgal lipid/carbohydrate content obtained may not low, which may lead to higher cost of downstream processing (e.g., extraction ofr isolation of lipids or carbohydrates). In contrast, sufficiently high lipid/carbohydrate content can easily be obtained using two-stage, semi-continuous, or salinity-gradient processes because of the strong environmental stresses associated with these processes, which results in decreased biomass productivity. The two-stage operation strategy is currently the most widely used for increasing lipid/carbohydrate productivity. However, due to the high operational costs arising from the necessity of exchanging the nutrient-rich and nutrient-deficient media in order to support growth in the first stage or to trigger rapid lipid/carbohydrate accumulation in the second stage, two-stage processes can prove problematic when large-scale cultivation is needed. Xia et al. (2013) attempted to 18
ACCEPTED MANUSCRIPT enhance lipid productivity in S. obtusus XJ-15 by adding NaCl in the second stage, with the aim of inducing significant lipid accumulation. However, the sudden addition of NaCl also inhibited growth. These salt-addition operation procedures seem to be
T
much simpler than switching between nutrient-rich and nutrient-deficient media.
IP
However, the tolerance of microalgae for high salinity must be improved to avoid growth inhibition upon salt addition. The salinity-gradient process is particularly
SC R
suitable for marine microalgae-based biofuel production if biomass productivity can be maintained at a satisfactory level by incorporating a continuous operation process during the growth stage. Thus, based on their merits, it might be beneficial to
NU
appropriately combine two or three of the above-mentioned processes to achieve the goals of low production cost, high biomass productivity, and high lipid/carbohydrate
MA
yield. Notably, whether the single or combined engineering strategies are applied in lab- and large-scale, the life-cycle assessment (LCA) is necessary to ensure that biofuel production from microalgae with the proposed strategies is more sustainable
2014).
Identifying
the
best
engineering
strategy
could
accelerate
TE
al.,
D
and feasible than the conventional cultivation process (Collet et al. , 2014, Fortier et
commercialization of microalgae-based biofuel production. In addition, plenty of
CE P
contamination risks from zooplankton, bacteria, or other competitors, especially in large-scale cultivation, are also one of the most dominant factors encountered for the feasibility of microalgae commercialization (Wang et al., 2013). Thus, using effective engineering strategies for the contaminant control of microalgae culture is another
AC
vital factor that must be considered.
5. Integrated processes involving microalgae-based biofuel production In addition to optimizing the operation strategy for microalgae-based biofuel production, it would be beneficial and economically advantageous to combine the microalgae cultivation process with CO2 fixation (Ho et al., 2011), wastewater treatment (Cai et al., 2013), or processes leading to the production of a variety of valuable co-products (Yen et al., 2013).
5.1 Mitigation of CO2 emissions The earth is being threatened by the sharp rise in the level of atmospheric CO 2. Biological CO2 fixation, achieved primarily by microalgae through photosynthesis, has emerged as one of the most effective and environmentally friendly ways to reduce 19
ACCEPTED MANUSCRIPT atmospheric CO2 levels (Ho et al., 2012b). Moreover, through photosynthesis, microalgae can effectively convert solar energy into a variety of valuable end products, such as biofuels, food additives, and compounds used in cosmetics and
T
pharmaceuticals (Yen et al., 2013). For example, some microalgae, such as S.
IP
obliquus (Ho et al., 2012a), C. vulgaris (Ho et al., 2013b), and Chlamydomonas sp. (Nakanishi et al., 2014), exhibit both a high CO2 fixation rate and high
SC R
lipid/carbohydrate content. Thus, combining microalgae-based CO2 fixation with biofuel production or bio-based chemical production seems to have a high potential for reducing the level of atmospheric CO2 while simultaneously producing biofuels or
NU
valuable chemicals.
MA
5.2 Wastewater treatment
D
In addition to establishing a suitable microalgae-based biofuel production system, it is equally important to consider the recycling of inorganic nutrients present in wastewater. Several microalgal species are known to have a high potential for use in wastewater treatment, which would greatly facilitate biofuel production using
AC
CE P
TE
microalgae (Cai et al., 2013). Many related studies have shown impressive results regarding the potential for utilizing microalgae to remove carbon, nitrogen, phosphorus, sulfur, and other chemicals/heavy metals from a variety of wastewater streams. For example, Zhou et al. (2012) used Auxenochlorella protothecoides UMN280 to effectively remove 90-100% of nitrogen and phosphate from concentrated municipal wastewater in conjunction with the production of oil-rich biomass. Feng et al. (2011) achieved 96-97% removal of ammonium and total phosphorus from artificial wastewater using the microalgae C. vulgaris, obtaining a maximum lipid content of 42%. However, the above studies were primarily conducted indoors at the laboratory level. More challenges (such as microbial contamination and inconsistent wastewater composition) are encountered when using wastewater to grow microalgae in large-scale outdoor cultivation (Cai et al., 2013). 5.3 Production of value-added co-products With the rapid increase in global population beginning in the early 1950s, microalgae, which are protein and nutrient rich, have received considerable research attention due to their capacity to serve as additives for human and animal foods (Borowitzka, 1999). To enhance the economic feasibility of microalgae-based biofuel production, other components that have potential applications in producing foods, cosmetics, emulsifiers, and clinical drugs can be collected from microalgal biomass in 20
ACCEPTED MANUSCRIPT addition to lipids and carbohydrates (Yen et al., 2013). In particular, algal polysaccharides (e.g., fucoidan and carrageenans) (Kim et al., 2012), algal long-chain unsaturated fatty acids (e.g., EPA and DHA) (Hong et al., 2011), and carotenoids (e.g.,
T
lutein and astaxanthin) (Ho et al., 2014a) are gaining wide attention due to their
IP
special medical and food nutrient applications. However, most recent studies have focused on obtaining one specific product without regard to damage that may be done
SC R
to other product-containing fractions (Vanthoor-Koopmans et al., 2013). Thus, innovative separation methods that permit extraction of different compounds without damaging other potentially valuable compounds are urgently needed, and the
NU
technology to accomplish this should also be suitable for commercial-scale operations (Vanthoor-Koopmans et al., 2013). In addition, a thorough evaluation of
MA
food/medicine safety is required in processes in which microalgal cultivation is combined with flue gas CO2 fixation and wastewater treatment.
D
6. Genetic engineering
TE
DNA sequencing has become more efficient and accessible in recent years, and the number of industrially relevant algae for which genome sequence information is
CE P
available is increasing rapidly. At least 16 algal genomes have been sequenced to date, and many more sequencing projects are underway. Transformation systems have been set up for at least 15 species, including Chlamydomonas reinhardtii, Chlorella sp., Haematococcus pluvialis, Dunaliella viridis, Dunaliella salina, Nannochloropsis
AC
oculata, Thalassiosira pseudonanna, Thalassiosira weissfloggi, Phaeodactylum triconuntumtricornuntum, Porphyridium sp., Nannochloropsis sp., and Scenedesmus obliquus (Guo et al., 2013, Kilian et al., 2011, Neupert et al., 2012, Radakovits et al., 2010, Yu et al., 2011). However, although several studies have successfully displayed the improvement of microalgal lipid synthesis via genetic modifications, there are still many uncertain factors that hinder the utilization of genetically engineered microorganisms for this purpose, such as unknown biological contamination, possible ecological damage, and restrictive legislation (Mata et al., 2010). In addition, ‗omics‘ research involving a combination of genomics, transcriptomics, proteomics, and metabolomics has generated a substantial amount of data regarding algal metabolism (Guarnieri et al., 2011, Jamers et al., 2009). However, successful manipulation of algal metabolism will require a much more detailed understanding. As new algal species are identified and their genomes are sequenced, 21
ACCEPTED MANUSCRIPT strategies must be developed to quickly annotate and characterize genes and proteins involved in lipid and starch metabolism.
T
6.1 Genetic modification of carbohydrate metabolism
IP
Starch is a basic energy-rich storage compound of microalgae. Several studies have concentrated on altering catalytic and allosteric properties of ADP-glucose
SC R
pyrophosphorylase (AGPase; an essential enzyme in starch production) in crop plants in order to increase starch production (Smith, 2008). AGPase, which is the rate-limiting
enzyme
with
starch ATP to
synthesis, form
catalyzes
ADP-glucose
the and
reaction
of
pyrophosphate.
NU
glucose-1-phosphate
in
Starch-synthesizing enzymes have also been introduced into the cytosol of microalgae
MA
in an effort to increase the starch content (Smith, 2008). Strategies to decrease starch degradation in microalgae (Ritte et al., 2006) or alter secretion to export soluble sugars (Deschamps et al., 2008) have also received considerable interest recently.
D
Available data indicate that blocking starch synthesis to provide precursor
TE
intermediates for lipid accumulation is a plausible strategy for increasing lipid yield. Wang et al. (2009) reported an increase in the triacylglycerol content after deleting the
CE P
AGPase gene. Another starchless mutant of Chlorella pyrenoidosa has also been shown to have elevated polyunsaturated fatty acid content (Ramazanov and Ramazanov, 2006).
AC
6.2 Engineering fatty acid biosynthesis Several studies have explored the effect on TAG production of overexpressing the enzymes involved in lipid synthesis. Acetyl-CoA carboxylase (ACCase), which catalyzes the carboxylation of acetyl-CoA to form malonyl-CoA, is the rate-limiting enzyme in fatty acid biosynthesis. Attempts to overexpress ACCase as a means of increasing lipid content in various systems have been disappointing. Dunahay et al. (1995) overexpressed native ACCase in the diatom C. cryptic, which resulted in a 2to 3-fold increase in ACCase activity; however, little increase in lipid production was observed.
Other
studies
have
attempted
to
increase
the
expression
of
3-ketoacyl-acyl-carrier protein synthetase III (KASIII), which catalyzes the initial condensation reaction in fatty acid biosynthesis. Overexpression of KASIII from spinach (Spinacia oleracea) or Cuphea hookeriana in tobacco (Nicotiana tabacum), Arabidopsis thaliana, and Brassica napus resulted in no enhancement of seed oil 22
ACCEPTED MANUSCRIPT content (Dehesh et al., 1996). These results highlight the importance of understanding the metabolic pathways and their underlying regulation. In addition to enhancing the lipid content and productivity of microalgae,
T
improving the quality of biodiesel is also desirable. Biodiesel (or jet fuel) is composed
IP
of methyl esters of fatty acids, ideally those that are relatively short in length (e.g., C10-C18) and either saturated or monounsaturated. However, most microalgae
SC R
synthesize fatty acids composed of 16-22 carbons. Efforts to engineer thioesterase (TE; which is responsible for the termination of chain elongation in fatty acid biosynthesis) to catalyze the formation of C8:0-C14:0 fatty acids in microalgae have
NU
shown promise as a strategy to alter microalgal fatty acid composition. Plant TEs were recently engineered into heterologous hosts to alter the fatty acid content for
MA
biodiesel production. Voelker and coworkers (1992) introduced a plant TE derived from Umbellularia californica into A. thaliana and achieved a 24% increase in the C12:0 fatty acid content. Introduction of the C10:0-specific TE from C. hookeriana
D
into B. napus shifted the fatty acid content to 38% C10:0 (Dehesh et al., 1996).
TE
Taken together, these results suggest that it is possible to manipulate fatty acid biosynthesis to optimize microalgae as a biodiesel feedstock. However, new strategies
CE P
must be envisioned to enhance fatty acid content and oil yield.
6.3 Modifying carbon assimilation pathways Rubisco catalyzes the CO2 fixation reaction in the Calvin-Benson-Bassham
AC
(CBB) cycle and has been postulated as the rate-limiting enzyme in CO2 fixation. In microalgae grown under phototrophic conditions, all newly produced biomass, including starches and lipids, is derived from the fixation of CO2 into ribulose-1,5-bisphosphate (RuBP) to form 3-phosphoglycerate (3PG). Several studies have shown that the activity of Rubisco is the major bottleneck for carbon flux through the Calvin cycle, especially when CO2 is not enriched in the medium or under high-light or high-temperature conditions. At present, we are far from fully understanding the details of Rubisco regulation, and whether Rubisco can be engineered so as to improve photosynthetic CO2 assimilation remains a matter of debate. Other limitations include precise regulation of Rubisco expression and inaccurate folding of the protein in heterologous organisms (Andrews and Whitney, 2003). Nevertheless, two recent papers have made some significant observations. For example, tobacco Rubisco was successfully replaced with its counterpart from 23
ACCEPTED MANUSCRIPT Rhodospirillum rubrum, which has a naturally high specificity (Whitney and Andrews, 2001). However, the CO2-assimilation rates of these engineered plants were much lower than those of the wild-type plants (Whitney and Andrews, 2001). In other work,
T
genes encoding the small subunit of Arabidopsis and sunflower Rubisco were
IP
transformed into a Rubisco gene–deficient strain of Chlamydomonas. As a result, the
SC R
in vitro CO2/O2 specificity increased by 11% (Genkov et al., 2010).
7. Conclusion
The production of biofuels using lipid-/carbohydrate-rich microalgae is a very
NU
promising alternative to conventional biofuel production approaches; however, most of the engineering processes are not as yet economically viable. To significantly
MA
improve the feasibility of microalgae-based biofuel production, engineering strategies that will effectively increase both growth and lipid/carbohydrate content must be developed and should be optimized by employing favorable environmental factors
D
and suitable operation strategies. Here, various innovative operation strategies that
TE
incorporate appropriate environmental stresses were critically reviewed. Selection of the appropriate engineering strategy for biofuel production can be accomplished by
CE P
comparing the biological characteristics of various microalgae and their responses under different conditions. In addition, more effective manipulation of microalgal metabolism and the implementation of genetic modifications will be required to further enhance the efficiency of biofuel production using microalgae. Hopefully, this
AC
review will facilitate the development of economically viable microalgae-based biofuel production processes in the coming years via cultivation process optimization as well as metabolic and genetic engineering.
Acknowledgement This work has been supported through project P07015 by the New Energy and Industrial Technology Development Organization (NEDO).
References Abdollahi J, Dubljevic S. Lipid production optimization and optimal control of heterotrophic microalgae fed-batch bioreactor. Chem Eng Sci 2012;84:619-27. Bartley ML, Boeing WJ, Corcoran AA, Holguin FO, Schaub T. Effects of salinity on growth and lipid accumulation of biofuel microalga Nannochloropsis salina and
24
ACCEPTED MANUSCRIPT
SC R
IP
T
invading organisms. Biomass Bioenerg 2013;54:83-8. Bondioli P, Della Bella L, Rivolta G, Chini Zittelli G, Bassi N, Rodolfi L, et al. Oil production by the marine microalgae Nannochloropsis sp. F&M-M24 and Tetraselmis suecica F&M-M33. Bioresour Technol 2012;114:567-72. Borowitzka MA. Commercial production of microalgae: ponds, tanks, tubes and fermenters. J Biotechnol 1999;70:313-21. Brennan L, Owende P. Biofuels from microalgae—A review of technologies for production, processing, and extractions of biofuels and co-products. Renew Sustain Energ Rev 2010;14:557-77.
MA
NU
Breuer G, Lamers PP, Martens DE, Draaisma RB, Wijffels RH. Effect of light intensity, pH, and temperature on triacylglycerol (TAG) accumulation induced by nitrogen starvation in Scenedesmus obliquus. Bioresour Technol 2013;143:1-9. Cai T, Park SY, Li Y. Nutrient recovery from wastewater streams by microalgae: Status and prospects. Renew Sustain Energ Rev 2013;19:360-9.
D
Cerón-García M, Fernández-Sevilla J, Sánchez-Mirón A, García-Camacho F, Contreras-Gómez A, Molina-Grima E. Mixotrophic growth of Phaeodactylum tricornutum on fructose and glycerol in fed-batch and semi-continuous modes. Bioresour Technol 2013;147:569-76.
AC
CE P
TE
Champigny M. Regulation of photosynthetic carbon assimilation at the cellular level: a review. Photosynthesis Res 1985;6:273-86. Cheirsilp B, Torpee S. Enhanced growth and lipid production of microalgae under mixotrophic culture condition: Effect of light intensity, glucose concentration and fed-batch cultivation. Bioresour Technol 2012;110:510-6. Chen C-Y, Chang J-S, Chang H-Y, Chen T-Y, Wu J-H, Lee W-L. Enhancing microalgal oil/lipid production from Chlorella sorokiniana CY1 using deep-sea water supplemented cultivation medium. Biochem Eng J 2013a;77:74-81. Chen C-Y, Yeh K-L, Aisyah R, Lee D-J, Chang J-S. Cultivation, photobioreactor design and harvesting of microalgae for biodiesel production: A critical review. Bioresour Technol 2011;102:71-81. Chen C-Y, Zhao X-Q, Yen H-W, Ho S-H, Cheng C-L, Lee D-J, et al. Microalgae-based carbohydrates for biofuel production. Biochem Eng J. 2013b;78:1-10. Chisti Y. Biodiesel from microalgae. Biotechnol Adv 2007;25:294-306. Collet P, Lardon L, Hélias A, Bricout S, Lombaert-Valot I, Perrier B, et al. Biodiesel from microalgae – Life cycle assessment and recommendations for potential improvements. Renew Energ. 2014;71:525-33. Converti A, Casazza AA, Ortiz EY, Perego P, Del Borghi M. Effect of temperature and nitrogen concentration on the growth and lipid content of Nannochloropsis
25
ACCEPTED MANUSCRIPT
SC R
IP
T
oculata and Chlorella vulgaris for biodiesel production. Chem Eng Process: Process Intensif 2009;48:1146-51. Dehesh K, Jones A, Knutzon DS, Voelker TA. Production of high levels of 8: 0 and 10: 0 fatty acids in transgenic canola by overexpression of Ch FatB2, a thioesterase cDNA from Cuphea hookeriana. The Plant J 1996;9:167-72. Deschamps P, Haferkamp I, d‘Hulst C, Neuhaus HE, Ball SG. The relocation of starch metabolism to chloroplasts: when, why and how. Trends Plant Sci 2008;13:574-82. Dunahay TG, Jarvis EE, Roessler PG. Genetic transformation of the diatoms Cyclotella cryptica and Navicula saprophila. J Phycol 1995;31:1004-12.
MA
NU
Feng Y, Li C, Zhang D. Lipid production of Chlorella vulgaris cultured in artificial wastewater medium. Bioresour Technol 2011;102:101-5. Fortier M-OP, Roberts GW, Stagg-Williams SM, Sturm BSM. Life cycle assessment of bio-jet fuel from hydrothermal liquefaction of microalgae. Appl Energ. 2014;122:73-82.
D
Gardner R, Peters P, Peyton B, Cooksey KE. Medium pH and nitrate concentration effects on accumulation of triacylglycerol in two members of the Chlorophyta. J Appl Phycol 2011;23:1005-16. Genkov T, Meyer M, Griffiths H, Spreitzer RJ. Functional hybrid rubisco enzymes
AC
CE P
TE
with plant small subunits and algal large subunits engineered rbcS cDNA for expression in Chlamydomonas. J Biol Chem 2010;285:19833-41. González-Fernández C, Ballesteros M. Linking microalgae and cyanobacteria culture conditions and key-enzymes for carbohydrate accumulation. Biotechnol Adv 2012;30:1655-61. Gouveia L, Oliveira AC. Microalgae as a raw material for biofuels production. J Ind Microbiol Biotechnol 2009;36:269-74. Guarnieri MT, Nag A, Smolinski SL, Darzins A, Seibert M, Pienkos PT. Examination of triacylglycerol biosynthetic pathways via de novo transcriptomic and proteomic analyses in an unsequenced microalga. PLoS One 2011;6:e25851. Guedes AC, Meireles LA, Amaro HM, Malcata FX. Changes in lipid class and fatty acid composition of cultures of Pavlova lutheri, in response to light intensity. J Am Oil Chem Soc 2010;87:791-801. Guo S-L, Zhao X-Q, Tang Y, Wan C, Alam MA, Ho S-H, et al. Establishment of an efficient genetic transformation system in Scenedesmus obliquus. J Biotechnol 2013;163:61-8. Han F, Huang J, Li Y, Wang W, Wan M, Shen G, et al. Enhanced lipid productivity of Chlorella pyrenoidosa through the culture strategy of semi-continuous cultivation with nitrogen limitation and pH control by CO2 Bioresour Technol 2013;136:418-24. Harwati TU, Willke T, Vorlop KD. Characterization of the lipid accumulation in a
26
ACCEPTED MANUSCRIPT
SC R
IP
T
tropical freshwater microalgae Chlorococcum sp. Bioresour Technol 2012;121:54-60. Ho S-H, Chan M-C, Liu C-C, Chen C-Y, Lee W-L, Lee D-J, et al. Enhancing lutein productivity of an indigenous microalga Scenedesmus obliquus FSP-3 using light-related strategies. Bioresour Technol 2014a;152:275-82. Ho S-H, Chang J-S, Lai Y-Y, Chen C-NN. Achieving high lipid productivity of a thermotolerant microalga Desmodesmus sp. F2 by optimizing environmental factors and nutrient conditions. Bioresour Technol 2014b;156:108-16. Ho S-H, Chen C-Y, Chang J-S. Effect of light intensity and nitrogen starvation on CO2 fixation and lipid/carbohydrate production of an indigenous microalga
MA
NU
Scenedesmus obliquus CNW-N. Bioresour Technol 2012a:244-52. Ho S-H, Chen C-Y, Lee D-J, Chang J-S. Perspectives on microalgal CO2-emission mitigation systems — A review. Biotechnol Adv 2011;29:189-98. Ho S-H, Chen W-M, Chang J-S. Scenedesmus obliquus CNW-N as a potential candidate for CO2 mitigation and biodiesel production. Bioresour Technol
D
2010;101:8725-30. Ho S-H, Huang S-W, Chen C-Y, Hasunuma T, Kondo A, Chang J-S. Bioethanol production using carbohydrate-rich microalgae biomass as feedstock. Bioresour Technol 2013a;135:191-8.
AC
CE P
TE
Ho S-H, Huang S-W, Chen C-Y, Hasunuma T, Kondo A, Chang J-S. Characterization and optimization of carbohydrate production from an indigenous microalga Chlorella vulgaris FSP-E. Bioresour Technol 2013b;135:157-65. Ho S-H, Kondo A, Hasunuma T, Chang J-S. Engineering strategies for improving the CO2 fixation and carbohydrate productivity of Scenedesmus obliquus CNW-N used for bioethanol fermentation. Bioresour Technol 2013c;143:163-71. Ho S-H, Li P-J, Liu C-C, Chang J-S. Bioprocess development on microalgae-based CO2 fixation and bioethanol production using Scenedesmus obliquus CNW-N. Bioresour Technol 2013d;145:142-9. Ho S-H, Lu W-B, Chang J-S. Photobioreactor strategies for improving the CO2 fixation efficiency of indigenous Scenedesmus obliquus CNW-N: Statistical optimization of CO2 feeding, illumination, and operation mode. Bioresour Technol 2012b;105:106-13. Ho S-H, Nakanishi A, Ye XT, Chang J-S, Hara K, Hasunuma T, et al. Optimizing biodiesel production in marine Chlamydomonas sp. JSC4 through metabolic profiling and an innovative salinity-gradient strategy. Biotechnol Biofuels 2014c, in press. Hong W-K, Rairakhwada D, Seo P-S, Park S-Y, Hur B-K, Kim C, et al. Production of lipids containing high levels of docosahexaenoic acid by a newly isolated microalga, Aurantiochytrium sp. KRS101. Appl Biochem Biotechnol 2011;164:1468-80. Hosono H, Uemura I, Takumi T, Nagamune T, Yasuda T, Kishimoto M, et al. Effect of
27
ACCEPTED MANUSCRIPT
SC R
IP
T
culture temperature shift on the cellular sugar accumulation of Chlorella vulgaris SO-26. J Ferment Bioeng 1994;78:235-40. Hsieh C-H, Wu W-T. Cultivation of microalgae for oil production with a cultivation strategy of urea limitation. Bioresour Technol 2009;100:3921-6. Hu Q, Sommerfeld M, Jarvis E, Ghirardi M, Posewitz M, Seibert M, et al. Microalgal triacylglycerols as feedstocks for biofuel production: perspectives and advances. The Plant J 2008;54:621-39. Jamers A, Blust R, De Coen W. Omics in algae: Paving the way for a systems biological understanding of algal stress phenomena? Aquat Toxicol 2009;92:114-21.
MA
NU
James GO, Hocart CH, Hillier W, Price GD, Djordjevic MA. Temperature modulation of fatty acid profiles for biofuel production in nitrogen deprived Chlamydomonas reinhardtii. Bioresour Technol 2013;127:441-7. Jeffryes C, Rosenberger J, Rorrer GL. Fed-batch cultivation and bioprocess modeling of Cyclotella sp. for enhanced fatty acid production by controlled silicon limitation.
D
Algal Res 2013;2:16-27. Jiang L, Luo S, Fan X, Yang Z, Guo R. Biomass and lipid production of marine microalgae using municipal wastewater and high concentration of CO2. Appl Energ 2011;88:3336-41.
AC
CE P
TE
John Andrews T, Whitney SM. Manipulating ribulose bisphosphate carboxylase/oxygenase in the chloroplasts of higher plants. Arch Biochem Biophys. 2003;414:159-69. John RP, Anisha GS, Nampoothiri KM, Pandey A. Micro and macroalgal biomass: A renewable source for bioethanol. Bioresour Technol. 2011;102:186-93. Khalil ZI, Asker MM, El-Sayed S, Kobbia IA. Effect of pH on growth and biochemical responses of Dunaliella bardawil and Chlorella ellipsoidea. World J Microbiol Biotechnol 2010;26:1225-31. Khotimchenko SV, Yakovleva IM. Lipid composition of the red alga Tichocarpus crinitus exposed to different levels of photon irradiance. Phytochem 2005;66:73-9. Kilian O, Benemann CS, Niyogi KK, Vick B. High-efficiency homologous recombination in the oil-producing alga Nannochloropsis sp. Pro Natl Acad Sci 2011;108:21265-9. Kim M-S, Baek J-S, Yun Y-S, Jun Sim S, Park S, Kim S-C. Hydrogen production from Chlamydomonas reinhardtii biomass using a two-step conversion process: Anaerobic conversion and photosynthetic fermentation. Int J Hydrogen Energ 2006;31:812-6. Kim M, Yim JH, Kim S-Y, Kim HS, Lee WG, Kim SJ, et al. In vitro inhibition of influenza A virus infection by marine microalga-derived sulfated polysaccharide p-KG03. Antivir Res 2012;93:253-9.
28
ACCEPTED MANUSCRIPT
SC R
IP
T
Lam MK, Lee KT. Microalgae biofuels: A critical review of issues, problems and the way forward. Biotechnol Adv 2012;30:673-90. Liu J, Yuan C, Hu G, Li F. Effects of light Intensity on the growth and lipid accumulation of microalga Scenedesmus sp. 11-1 under nitrogen limitation. Appl Biochem Biotechnol 2012;166:2127-37. Liu ZY, Wang GC, Zhou BC. Effect of iron on growth and lipid accumulation in Chlorella vulgaris. Bioresour Technol. 2008;99:4717-22. Lv J-M, Cheng L-H, Xu X-H, Zhang L, Chen H-L. Enhanced lipid production of Chlorella vulgaris by adjustment of cultivation conditions. Bioresour Technol 2010;101:6797-804.
MA
NU
M ü nkel R, Schmid ‐ Staiger U, Werner A, Hirth T. Optimization of outdoor cultivation in flat panel airlift reactors for lipid production by Chlorella vulgaris. Biotechnol Bioeng 2013;110:2882-93. Marchetti J, Bougaran G, Le Dean L, Megrier C, Lukomska E, Kaas R, et al.
D
Optimizing conditions for the continuous culture of Isochrysis affinis galbana relevant to commercial hatcheries. Aquac 2012;326:106-15. Mata TM, Martins AA, Caetano NS. Microalgae for biodiesel production and other applications: A review. Renew Sustain Energ Rev. 2010;14:217-32.
AC
CE P
TE
Mazzuca Sobczuk T, Chisti Y. Potential fuel oils from the microalga Choricystis minor. J Chem Technol Biotechnol 2010;85:100-8. Moheimani NR. Long-term outdoor growth and lipid productivity of Tetraselmis suecica, Dunaliella tertiolecta and Chlorella sp (Chlorophyta) in bag photobioreactors. J Appl Phycol 2013;25:167-76. Mujtaba G, Choi W, Lee C-G, Lee K. Lipid production by Chlorella vulgaris after a shift from nutrient-rich to nitrogen starvation conditions. Bioresour Technol 2012;123:279-83. Naik SN, Goud VV, Rout PK, Dalai AK. Production of first and second generation biofuels: A comprehensive review. Renew Sustain Energ Rev 2010;14:578-97. Nakanishi A, Aikawa S, Ho S-H, Chen C-Y, Chang J-S, Hasunuma T, et al. Development of lipid productivities under different CO2 conditions of marine microalgae Chlamydomonas sp. JSC4. Bioresour Technol 2014;152:247-52. Neuhaus HE, Stitt M. Control analysis of photosynthate partitioning. Planta 1990;182:445-54. Neupert J, Shao N, Lu Y, Bock R. Genetic transformation of the model green alga Chlamydomonas reinhardtii. Transgenic Plants: Springer; 2012. p. 35-47. Pal D, Khozin-Goldberg I, Cohen Z, Boussiba S. The effect of light, salinity, and nitrogen availability on lipid production by Nannochloropsis sp. Appl Microbiol Biotechnol 2011;90:1429-41.
29
ACCEPTED MANUSCRIPT
SC R
IP
T
Parmar A, Singh NK, Pandey A, Gnansounou E, Madamwar D. Cyanobacteria and microalgae: a positive prospect for biofuels. Bioresour Technol 2011;102:10163-72. Pittman JK, Dean AP, Osundeko O. The potential of sustainable algal biofuel production using wastewater resources. Bioresour Technol 2011;102:17-25. Pragya N, Pandey KK, Sahoo P. A review on harvesting, oil extraction and biofuels production technologies from microalgae. Renew Sustain Energ Rev 2013;24:159-71. Radakovits R, Jinkerson RE, Darzins A, Posewitz MC. Genetic engineering of algae for enhanced biofuel production. Eukaryot Cell 2010;9:486-501. Ramazanov A, Ramazanov Z. Isolation and characterization of a starchless mutant of
MA
NU
Chlorella pyrenoidosa STL‐PI with a high growth rate, and high protein and polyunsaturated fatty acid content. Phycol Res 2006;54:255-9. Ranga Rao A, Ravishanka G, Sarada R. Cultivation of green alga Botryococcus braunii in raceway, circular ponds under outdoor conditions and its growth, hydrocarbon production. Bioresour Technol 2012;123:528-33.
D
Rismani-Yazdi H, Haznedaroglu BZ, Bibby K, Peccia J. Transcriptome sequencing and annotation of the microalgae Dunaliella tertiolecta: Pathway description and gene discovery for production of next-generation biofuels. BMC Genomics 2011;12:148. Ritte G, Heydenreich M, Mahlow S, Haebel S, Kötting O, Steup M. Phosphorylation
AC
CE P
TE
of C6-and C3-positions of glucosyl residues in starch is catalysed by distinct dikinases. FEBS Lett 2006;580:4872-6. San Pedro A, González-López CV, Acién FG, Molina-Grima E. Marine microalgae selection and culture conditions optimization for biodiesel production. Bioresour Technol 2013;134:353-61. Santos AM, Janssen M, Lamers PP, Evers WAC, Wijffels RH. Growth of oil accumulating microalga Neochloris oleoabundans under alkaline–saline conditions. Bioresour Technol 2012;104:593-9. Sforza E, Enzo M, Bertucco A. Design of microalgal biomass production in a continuous photobioreactor: an integrated experimental and modeling approach. Chem Eng Res Des 2013;92:1153-62. Siaut M, Cuiné S, Cagnon C, Fessler B, Nguyen M, Carrier P, et al. Oil accumulation in the model green alga Chlamydomonas reinhardtii: Characterization, variability between common laboratory strains and relationship with starch reserves. BMC Biotechnol 2011;11:7. Sims RE, Mabee W, Saddler JN, Taylor M. An overview of second generation biofuel technologies. Bioresour Technol 2010;101:1570-80. Singh J, Gu S. Commercialization potential of microalgae for biofuels production. Renew Sustain Energ Rev 2010;14:2596-610. Smith AM. Prospects for increasing starch and sucrose yields for bioethanol
30
ACCEPTED MANUSCRIPT
SC R
IP
T
production. The Plant J 2008;54:546-58. Su CH, Chien LJ, Gomes J, Lin YS, Yu YK, Liou JS, et al. Factors affecting lipid accumulation by Nannochloropsis oculata in a two-stage cultivation process. J Appl Phycol 2011;23:903-8. Sukenik A, Carmeli Y, Berner T. Regulation of fatty acid composition by irradiance level in the eustigmatophyte Nannochloropsis sp. 1. J Phycol 1989;25:686-92. Sukenik A, Wahnon R. Biochemical quality of marine unicellular algae with special emphasis on lipid composition in Isochrysis galbana. Aquac 1991;97:61-72. Sun X, Cao Y, Xu H, Liu Y, Sun J, Qiao D, et al. Effect of nitrogen-starvation, light
MA
NU
intensity and iron on triacylglyceride/carbohydrate production and fatty acid profile of Neochloris oleoabundans HK-129 by a two-stage process. Bioresour Technol 2014;155:204-12. Takagi M, Karseno, Yoshida T. Effect of salt concentration on intracellular accumulation of lipids and triacylglyceride in marine microalgae Dunaliella cells. J
D
Biosci Bioeng 2006;101:223-6. Takagi M, Watanabe K, Yamaberi K, Yoshida T. Limited feeding of potassium nitrate for intracellular lipid and triglyceride accumulation of Nannochloris sp. UTEX LB1999. Appl Microbiol Biotechnol 2000;54:112-7.
AC
CE P
TE
Talebi AF, Mohtashami SK, Tabatabaei M, Tohidfar M, Bagheri A, Zeinalabedini M, et al. Fatty acids profiling: A selective criterion for screening microalgae strains for biodiesel production. Algal Res 2013;2:258-67. Tang H, Abunasser N, Garcia MED, Chen M, Simon Ng KY, Salley SO. Potential of microalgae oil from Dunaliella tertiolecta as a feedstock for biodiesel. Appl Energ 2011;88:3324-30. Tang H, Chen M, Simon Ng K, Salley SO. Continuous microalgae cultivation in a photobioreactor. Biotechnol Bioeng 2012;109:2468-74. Vanthoor-Koopmans M, Wijffels RH, Barbosa MJ, Eppink MHM. Biorefinery of microalgae for food and fuel. Bioresour Technol 2013;135:142-9. Voelker TA, Worrell AC, Anderson L, Bleibaum J, Fan C, Hawkins DJ, et al. Fatty acid biosynthesis redirected to medium chains in transgenic oilseed plants. Sci 1992;257:72-4. Wan C, Bai F-W, Zhao X-Q. Effects of nitrogen concentration and media replacement on cell growth and lipid production of oleaginous marine microalga Nannochloropsis oceanica DUT01. Biochem Eng J 2013;78:32-8. Wang H, Zhang W, Chen L, Wang J, and Liu T. The contanmination and control of biologcal pollutants in mass cultivation of microalgae. Bioresour Technol 2013;128:745-750. Wang X, Liu X, Wang G. Two-stage Hydrolysis of Invasive Algal Feedstock for
31
ACCEPTED MANUSCRIPT
SC R
IP
T
Ethanol Fermentation. J Integr Plant Biol 2011;53:246-52. Wang ZT, Ullrich N, Joo S, Waffenschmidt S, Goodenough U. Algal lipid bodies: Stress induction, purification, and biochemical characterization in wild-type and starchless Chlamydomonas reinhardtii. Eukaryot Cell 2009;8:1856-68. Whitney SM, Andrews TJ. Plastome-encoded bacterial ribulose-1, 5-bisphosphate carboxylase/oxygenase (RubisCO) supports photosynthesis and growth in tobacco. Proc Nat Acade Sci 2001;98:14738-43. Wu LF, Chen PC, Lee CM. The effects of nitrogen sources and temperature on cell growth and lipid accumulation of microalgae. Int Biodeterior Biodegrad
MA
NU
2013;85:506-10. Xia L, Ge H, Zhou X, Zhang D, Hu C. Photoautotrophic outdoor two-stage cultivation for oleaginous microalgae Scenedesmus obtusus XJ-15. Bioresour Technol 2013;144:261-7. Xin L, Hong-Ying H, Yu-Ping Z. Growth and lipid accumulation properties of a
D
freshwater microalga Scenedesmus sp. under different cultivation temperature. Bioresour Technol 2011;102:3098-102. Xu Y, Boeing WJ: Modeling maximum lipid productivity of microalgae: Review and
AC
CE P
TE
next step. Renew Sustain Energ Rev 2014; 32:29-39. Yen H-W, Hu IC, Chen C-Y, Ho S-H, Lee D-J, Chang J-S. Microalgae-based biorefinery – From biofuels to natural products. Bioresour Technol 2013;135:166-74. Yu W-L, Ansari W, Schoepp NG, Hannon MJ, Mayfield SP, Burkart MD. Modifications of the metabolic pathways of lipid and triacylglycerol production in microalgae. Microb Cell Fact 2011;10:91. Zhila N, Kalacheva G, Volova T. Effect of salinity on the biochemical composition of the alga Botryococcus braunii Kütz IPPAS H-252. J Appl Phycol 2011;23:47-52. Zhou W, Min M, Li Y, Hu B, Ma X, Cheng Y, et al. A hetero-photoautotrophic two-stage cultivation process to improve wastewater nutrient removal and enhance algal lipid accumulation. Bioresour Technol 2012;110:448-55. Zhou X, Ge H, Xia L, Zhang D, Hu C. Evaluation of oil-producing algae as potential biodiesel feedstock. Bioresour Technol 2013a;134:24-9. Zhou X, Xia L, Ge H, Zhang D, Hu C. Feasibility of biodiesel production by microalgae Chlorella sp.(FACHB-1748) under outdoor conditions. Bioresour Technol 2013b;138:131-5.
32
ACCEPTED MANUSCRIPT Figure legend Figure 1. Overview of metabolic pathways in microalgal lipid/starch biosynthesis. 3PG, 3-phosphoglyceric acid; ACCase, acetyl-CoA carboxylase; ACP, acyl carrier
3-ketoacyl-acyl-carrier
protein
synthetase
IP
KSIII,
T
protein; AGPase, ADP-glucose pyrophosphorylase; G3P, glyceraldehyde-3-phosphate; III;
Rubisco,
ribulose-1,5-bisphosphate carboxylase/oxygenase; RuBP, ribulose-1,5-bisphosphate;
AC
CE P
TE
D
MA
NU
SC R
TE, thioesterase.
33
ACCEPTED MANUSCRIPT
Effect/improvement
Irradiance
Carbohydrate
Pavlova lutheri
Irradiance
Carbohydrate
N. oleoabundans HK-129
Irradiance
TAG
TAG content increased from 19 to 25% with increase in light intensity from 50 to 200 μmol m−2s−1 Biomass concentration increased from 1.2 to 1.7 g L−1 with increase in light intensity from 50 to 200 μmol m−2s−1
(Sun et al., 2014)
Lipid and TAG content increased from 26 to 41% and 16 to 32%, respectively, with increase in light intensity from 50 to 250 μmol m−2s−1 Biomass concentration increased from 2.5 to 3.6 g L−1 with increase in light intensity from 50 to 250 μmol m−2s−1
(Liu et al., 2012)
Pavlova lutheri
Irradiance
TAG
Temperature
TAG content increased from 23 to78% of total fatty acids with increase in light intensity from 9 to 19 W m−2
Lipid content decreased from 33 to 29% with increase in temperature from 25 to 35C Biomass concentration increased with increase in temperature from 25 to 30C but then decreased with further increase in temperature to 35C Lipid content decreased from 15 to 8% with increase in temperature from 15 to 20C but then increased to 14% with further increase in temperature to25C Specific growth rate increased with increase in temperature from 15 to 20C but then decreased with further increase in temperature to 25C
AC
Monoraphidium sp. SB2
CE P
Lipid/TAG
Lipid
N. oculata
Temperature
Lipid
(Ho et al., 2012a)
Carbohydrate content increased almost 3-fold, from 2.7 to 7.0 pg cell−1 with increase in light intensity from 30 to 400 μmol m−2s−1
Irradiance
References
Scenedesmus sp. 11-1
CR
S. obliquus CNW-N
Carbohydrate content increased from 15 to 38% with increase in light intensity from 60 to 420 μmol m−2s−1 Biomass productivity increased around 3-fold with increase in light intensity from 60 to 420 μmol m−2s−1
US
IP
Target products
MA N
Stress condition
TE D
Microalgae
T
Table 1. Carbohydrate/lipid accumulation in different microalgae under various environmental stress conditions.
34
(Sukenik and Wahnon, 1991)
(Guedes et al., 2010)
(Wu et al., 2013)
(Converti et al., 2009)
ACCEPTED MANUSCRIPT
Effect/improvement
Temperature
Lipid
Chlorella vulgaris SO-26
Temperature
Carbohydrate
C. reinhardtii BAF-J5
Temperature
TAG
pH
TAG
S. obliquus UTEX 393
pH
TAG
Coelastrella sp. PC-3
pH
TAG
C. ellipsoidea
pH
Carbohydrate
Nitrogen depletion
TAG
N. oleoabundans HK-129
References
(Xin et al., 2011)
(Hosono et al., 1994)
TAG content increased from 56 to 76% with increase in temperature from 17 to 32C Growth rate increased with increase in temperature from 17 to 32C
(James et al., 2013)
TAG content increased from 13 to 35% with increase in pH from 8.1 to 10 Growth rate decreased around 2-fold with increase in pH from 8.1 to 10
(Santos et al., 2012)
CE P
N. Oleoabundans
CR
Scenedesmus sp. LX1
Lipid content decreased from 35 to 22% with increase in temperature from 20 to 30C Biomass concentration increased with increase in temperature from 10 to 25C but then decreased with further increase in temperature to 30C Carbohydrate content decreased from 70 to 50% with increase in temperature from 5 to 20C Specific growth rate increased with increase in temperature from 5 to 20C but then decreased with further increase in temperature to 25C
US
IP
Target products
MA N
Stress condition
TE D
Microalgae
T
Table 1 (Continued)
TAG content increased with increase in pH pH range of 6-8 optimal for growth 6-8
(Breuer et al., 2013)
TAG content increased with increase in pH Growth rate decreased with increase in pH
(Gardner et al., 2011)
pH values of 10 and 9 optimal for growth and carbohydrate accumulation, respectively
(Khalil et al., 2010)
TAG content increased from 8 to 26% after 3-day nitrogen depletion Biomass productivity decreased from 220 to 197 mg L−1d−1 after 3-day nitrogen depletion
(Sun et al., 2014)
AC
35
ACCEPTED MANUSCRIPT
Lipid
Nannochloropsis sp. F&M-M24
Nitrogen depletion
Lipid
S. obliquus CNW-N
Nitrogen depletion
Carbohydrate
Nitrogen depletion
Carbohydrate
Salinity
TAG
Chlorella vulgaris FSP-E
B. braunii Kutz IPPAS H-252
Lipid content increased 10 to 48% after 4-day nitrogen depletion
Lipid content increased from 39 to 69% after nitrogen depletion Biomass productivity obviously decreased after nitrogen depletion Carbohydrate content increased from 21 to 49% after 2-day nitrogen depletion Growth rate slightly decreased after 2-day nitrogen depletion Carbohydrate content increased from 15 to 51% after 2-day nitrogen depletion Growth rate slightly decreased after 2-day nitrogen depletion TAG content increased from 5 to 31% with an increase in NaCl concentration from 0 to 0.7 M Growth rate significantly decreased with an increase in NaCl concentration from 0 to 0.7 M Lipid content increased from 10 to 30% with an increase in NaCl concentration from 0 to 2.0% Biomass concentration significantly decreased around 4-fold with an increase in NaCl concentration from 0 to 2.0% TAG content increased from 40 to 57% with an increase in NaCl concentration from 0.5 to 1.0 M Growth rate similar over the salinity range of 0.5-1.0 M Starch content increased around 4-fold with an increase in NaCl concentration from 0 to 1.0 M Starch content increased around 5-fold with an increase in NaCl concentration from 0 to 1.0 M
Salinity
Lipid
D. tertiolecta ATCC 30929
Salinity
TAG
CE P
Chlorococcum sp.
AC
C. reinhardtii CC-124
Salinity
Starch TAG
IP
Nitrogen depletion
CR
N. oculata
Effect/improvement
US
Target products
MA N
Stress condition
TE D
Microalgae
T
Table 1 (Continued)
36
References (Su et al., 2011) (Bondioli et al., 2012) (Ho et al., 2013c)
(Ho et al., 2013b)
(Zhila et al., 2011)
(Harwati et al., 2012)
(Takagi et al., 2006)
(Siaut et al., 2011)
ACCEPTED MANUSCRIPT
Chlorella sp. S. obliquus CNW-N Choricystis minor D. tertiolecta S. obliquus CNW-N Chlorella sp. NJ-18 Chlorella sp. C. pyrenoidosa Nannochloropsis sp. N. gaditana
IP
CR
Growth markedly increased Lipid content rarely affected Growth markedly increased Lipid content rarely affected Growth markedly increased Lipid content rarely affected Growth decreased Lipid content increased Growth markedly increased Carbohydrate content decreased Growth markedly increased Lipid content rarely affected Growth markedly increased Lipid content rarely affected Growth significantly increased Carbohydrate content also increased Growth markedly increased Lipid content rarely affected Growth increased Lipid content also increased Growth markedly increased Lipid content rarely affected Growth increased Lipid content also significantly increased Growth increased Lipid content also increased
US
MA N
Nannochloris sp.
Fed-batch (Glucose feeding + High light) Fed-batch (Glucose feeding + High light) Fed-batch (Nitrate feeding) Fed-batch (Urea feeding) Continuous (Fresh medium feeding) Continuous (Fresh medium feeding) Continuous (Fresh medium feeding) Semi-continuous (Fresh medium replacement) Semi-continuous (Fresh medium replacement) Semi-continuous (Fresh medium replacement) Semi-continuous (Low-N medium replacement) Two-stage (Nitrate depletion + High light) Two-stage (Continuous culture + Nitrate depletion)
TE D
Nannochloropsis sp.
Effect/improvement
CE P
Chlorella sp.
Operation strategy
AC
Microalgae
T
Table 2. Comparison of carbohydrate/lipid productivity of different microalgae using various operation strategies.
37
Optimal productivity (mg L−1d−1)
Reference
112.4 (Lipid)
(Cheirsilp and Torpee, 2012)
148.3 (Lipid)
(Cheirsilp and Torpee, 2012)
22.7 (TAG)
(Takagi et al., 2000)
123.0 (Lipid)
(Hsieh and Wu, 2009)
312.3 (Carbohydrate)
(Ho et al., 2013c)
82.0 (Lipid)
(Mazzuca Sobczuk and Chisti, 2010)
10.0 (FAME)
(Tang et al., 2012)
467.6 (Carbohydrate)
(Ho et al., 2013c)
24.1 (Lipid)
(Zhou et al., 2013a)
139.0 (Lipid)
(Hsieh and Wu, 2009)
115.0 (Lipid)
(Han et al., 2013)
76.6 (Lipid)
(Jiang et al., 2011)
51.0 (Lipid)
(San Pedro et al., 2013)
ACCEPTED MANUSCRIPT
N. oleoabundans S. obtusus XJ-15 Chlamydomonas sp. JSC4
IP
Growth slightly decreased Lipid content significantly increased Growth rarely affected Carbohydrate content significantly improved Growth slightly decreased Lipid content significantly improved
Growth rarely affected TAG content increased
Growth rarely affected Lipid content significantly increased Growth rarely affected Lipid content significantly increased
US
CR
MA N
S. obliquus CNW-N
Two-stage (Nitrogen depletion) Two-stage (Nutrient depletion) Two-stage (Nutrient depletion) Two-stage (Nitrogen depletion + Light intensity + Iron concentration) Two-stage (NaCl addition) Salinity-gradient (Stepwise addition of NaCl)
TE D
S. obliquus CNW-N
Effect/improvement
CE P
C. vulgaris AG10032
Operation strategy
AC
Microalgae
T
Table 2 (Continued)
38
Optimal productivity (mg L−1d−1)
Reference
77.8 (Lipid)
(Mujtaba et al., 2012)
352.9 (Carbohydrate)
(Ho et al., 2013d)
78.7 (Lipid)
(Ho et al., 2010)
51.6 (TAG)
(Sun et al., 2014)
60.7 (Lipid)
(Xia et al., 2013)
223.2 (Lipid)
(Ho et al., 2014c)
NU
SC R
IP
T
ACCEPTED MANUSCRIPT
AC
CE P
TE
D
MA
Figure 1
39
