Marine macroalgae: an untapped resource for producing fuels and chemicals

Review

Marine macroalgae: an untapped resource for producing fuels and chemicals Na Wei1, Josh Quarterman2, and Yong-Su Jin1,2 1 2

Institute for Genomic Biology, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA Department of Food Science and Human Nutrition, University of Illinois at Urbana–Champaign, Urbana, IL 61801, USA

As world energy demand continues to rise and fossil fuel resources are depleted, marine macroalgae (i.e., seaweed) is receiving increasing attention as an attractive renewable source for producing fuels and chemicals. Marine plant biomass has many advantages over terrestrial plant biomass as a feedstock. Recent breakthroughs in converting diverse carbohydrates from seaweed biomass into liquid biofuels (e.g., bioethanol) through metabolic engineering have demonstrated potential for seaweed biomass as a promising, although relatively unexplored, source for biofuels. This review focuses on up-to-date progress in fermentation of sugars from seaweed biomass using either natural or engineered microbial cells, and also provides a comprehensive overview of seaweed properties, cultivation and harvesting methods, and major steps in the bioconversion of seaweed biomass to biofuels. Biofuels to meet increasing energy demands and societal needs Since the time of the Industrial Revolution, the ever-increasing demand for energy has been met primarily by fossil fuel resources such as coal, oil, and natural gas. In 2009, the U.S. Energy Information Administration estimated that 86% of the 483 quadrillion BTU (British thermal unit) of total primary energy consumption in the world was derived from these fossil fuel derivatives [1]. However, as rapid growth of low- and medium-income economies and moderate increases in world population are expected to increase consumption of gas, coal, and oil by 26% over the next 20 years [2], there is growing concern about meeting the vast energy demands of the future with limited, nonrenewable resources. In addition, there is a heightened awareness of environmental concerns and economic challenges associated with overutilization of fossil fuel resources, including greenhouse gas emissions, global warming, receding of glaciers, rising sea levels, loss of biodiversity [3], increasing crude oil prices [4], and energy insecurity. All of these factors have emphasized the need for alternative, sustainable, efficient, cost-effective, and cleaner-burning energy sources to meet present and future demands [5]. Corresponding author: Jin, Y-S. ([email protected]) Keywords: macroalgae; biofuel; metabolic engineering.

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Biofuels provide an excellent alternative to traditional fossil fuel-derived energy sources, as they can be produced from abundant supplies of renewable biomass. During growth of the biomass feedstocks for biofuel production, the plants utilize sunlight and carbon dioxide in the atmosphere for synthesis of organic molecules, particularly carbohydrates and lipids. Subsequently, these biomolecules can be used for conversion to various fuels and chemicals through chemical or biochemical catalysis. In general, the overall life-cycle analysis for biofuels showed an improvement over traditional fossil fuel resources as the greenhouse gas emitted during combustion is mitigated by the inorganic carbon fixed from atmospheric carbon dioxide during growth of the biomass feedstock [6]. Also, biofuels such as ethanol are less toxic, are biodegradable, and generate fewer pollutants than petroleum fuels [7]. The utilization of renewable sources for production of biofuels shows definite progress toward limiting greenhouse gas levels, improving air quality, achieving energy independence and security, and finding renewable energy resources that are geographically more distributed than fossil fuels [5]. Although there are numerous efforts on developing core technologies (production, harvest, storage, depolymerization, and bio/chemical conversion) for producing biofuels from terrestrial plant biomass, production of biofuels from marine plant biomass received less attention. As such, this review focuses on using macroalgae (seaweed) as a potential feedstock for the production of liquid biofuels (especially bioethanol) and examines up-to-date efforts and progress in demonstrating fermentation of sugars from seaweed biomass using either natural microorganisms or engineered microbial cells. Benefits of producing biofuels from seaweeds Challenges in biofuel production from terrestrial biomass Currently, the two most abundant and feasible biofuels for large-scale production are ethanol from corn or sugarcane and biodiesel from oil crops such as soy or oil palm [8]. These food crops are attractive and widely used as raw materials for biofuel synthesis because of well-established farming practices and simple, cheap processes for release of starches, sugars, or oils. However, the status quo of using food crops for biofuel production is only a partial solution and cannot satisfy the full demand for renewable fuels. For

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Review example, in order to achieve the 2020 federal mandate for renewable fuel in the United States with corn ethanol, approximately 100% of the domestic corn crop currently available would be required [9]. In recent years, the increased demand on food crops for fuel applications has resulted in concern about food scarcity, higher prices of food commodities, and pollution of agricultural land [7]. The revised Renewable Fuel Standard (RFS2) thus caps corn ethanol at 15 billion gal/year (see http://www.epa.gov/ otaq/fuels/renewablefuels/regulations.htm), which necessitates the use of alternative renewable feedstocks. One plausible alternative that has been a focus of extensive research is the use of terrestrial non-food lignocellulosic biomass such as agricultural residues, wood waste, or energy grasses as raw materials for biofuel production. These biomass sources are advantageous because of low cost, minimal land use change, and avoidance of the competition between food and fuel. However, current chemical and biological technologies have yet to overcome the significant obstacles to releasing sugars from recalcitrant lignocellulose and efficiently converting hexoses and pentoses to target fuel molecules with high yields and productivities [9]. Marine algae as an alternative source In the context of the previously mentioned challenges, marine algae (including macroalgae and microalgae) are an attractive renewable source for biofuel production with many advantages over biomass from food or cellulosic materials. Algae include a wide variety of photosynthetic organisms living in many diverse environments and present in all existing ecosystems on Earth [5]. Under normal conditions, autotrophic algae use sunlight and fix inorganic carbon from the atmosphere for assimilation in the form of carbohydrates and lipids, which can be exploited for biofuel production [7]. The marine algae have many advantages for renewable energy applications. First, marine algae have relatively high photon conversion efficiency and can therefore rapidly synthesize biomass through assimilating abundant resources in nature such as sunlight, carbon dioxide, and inorganic nutrients [10]. Therefore, production yields of algae per unit area are significantly higher than those for terrestrial biomass [11,12]. Also, as marine algae have a higher rate of carbon dioxide fixation compared to terrestrial biomass, they may have greater potential for carbon dioxide remediation [12,13]. Second, marine algae lack hemicellulose and lignin, which are essential for structural support in most terrestrial plants [7], and thus can be depolymerized relatively easily as compared to lignocellulosic biomass [14]. Finally, marine algae do not require arable land and can be grown in a variety of marine environments including fresh water, salt water, or municipal waste water. Algae’s ability to grow in salt water or waste water is critical for sustainable biofuel production to avoid competition with food crops that require fresh water and cultivable land [7]. Seaweed cultivation and biofuel production Seaweed production Currently, the macroalgae industry is primarily focused on food products for human consumption, which account for

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83–90% of the global value of seaweed. Algal hydrocolloids extracted from macroalgae, such as alginate, agar, and carrageenan, account for most of the remaining value [15,16]. The vast majority of the macroalgae for these products is produced by aquaculture, which included some 3.1 million dry metric tons of annual global production in 2006 as compared to only 22 000 dry metric tons by harvesting of wild algae stocks. The top ten countries harvesting wild stocks are more geographically distributed throughout the world, but the production from aquaculture is focused on Asia with China accounting for 72% of global annual production [15]. The five genera–Laminaria, Undaria, Porphyra, Euchema, and Gracilaria–represent 76% of the total tonnage of macroalgae production by aquaculture [15]. Pretreatment and hydrolysis of seaweed biomass After cultivation and harvesting of seaweed (Box 1), the biomass must be pretreated for most biofuel applications. The first step of pretreatment is to remove foreign objects and debris such as stones, sand, snails, or other litter that may be caught in the biomass either manually or by washing [17]. In many cases, chopping or milling is then required to increase the surface area/volume ratio and improve the efficiency of hydrolysis [15]. Finally, the biomass should be dewatered to 20–30% to increase shelf life and reduce transportation costs in situations where it must be stored for long periods or transported over long distances before further processing [15,17]. After pretreatment, hydrolysis of the seaweed biomass is necessary to release the sugars locked up in the structural polysaccharides for the subsequent fermentation step. Seaweed composition and sugars released by hydrolysis are summarized (see Table I in Box 2). The basic options for hydrolysis of seaweed include dilute acid hydrolysis and/ or enzymatic hydrolysis. One study found that seaweed carbohydrates from all three classes of macroalgae (brown, red, and green) can be effectively hydrolyzed to monosaccharides by dilute H2SO4 treatment at high temperature [18]. The four key factors for optimal saccharification during sulfuric acid hydrolysis were identified as (i) reaction temperature, (ii) reaction time, (iii) acid concentration, and (iv) seaweed concentration. Another study reported that sodium chlorite pretreatment of Ceylon moss (Gelidium amansii) can greatly improve the efficiency of enzymatic hydrolysis with cellulase, xylanase, and bglucosidase and thus increase glucose yield from 5% in non-pretreated samples to 70% in treated samples [19]. A third study demonstrated the effectiveness of combining acid hydrolysis and enzymatic hydrolysis for saccharification of seaweed, which resulted in a maximum sugar yield of 0.566 g/g and 0.376 g/g for G. amansii and Laminaria japonica, respectively [20]. Liquid biofuels from seaweeds Liquid biofuels such as ethanol and butanol are produced from algae by bioconversion of sugars from the plant biomass using microbial cells such as yeast or bacteria. Being rich in carbohydrates, marine macroalgae (seaweed) may be used to obtain fermentable sugars for producing liquid biofuels (Boxes 2 and 3). The overall process of 71

Review

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Box 1. Seaweed cultivation and harvesting Cultivation methods In general, seaweed can either be cultivated vegetatively or by a separate reproductive cycle [42]. For vegetative cultivation, small algal pieces are grown in a suitable aquatic environment with appropriate temperature, light, salt content, nutrients, and water movement. When the seaweed has reached a mature stage, it is harvested by either leaving a small piece that will grow again or removing the whole plant and cutting small pieces as seed for further cultivation [42]. Although this strategy is quite simple and costeffective, some of the brown macroalgae such as Laminaria cannot be cultivated by the vegetative method but rather require a reproductive cycle with alternation of generations. Cultivation by a separate reproductive cycle is much more expensive as seed production and raising of young seedlings must be conducted in land-based facilities under carefully controlled conditions [42]. There are at least three options for macroalgal farming sites: offshore farms, nearshore coastal farms, and land-based ponds [15]. Offshore farming of L. hyperborea has been tested in the North Sea with success, but the cost of such a technologically advanced system is still very high [16,43]. Nearshore farms are currently in use for culturing macroalgae in some countries such as China and Japan. However, government regulations and environmental concerns about aquaculture in coastal waters have prevented this option in the United States and Europe [15]. Land-based pond systems for cultivation of macroalgae have many advantages over farming on open marine water such as ease of nutrient applications and avoidance of bad weather, disease, and predation. Also, land-based systems can be integrated with the aquaculture of other species such as fish to provide waste materials as a cheap supply of nutrients for the macroalgae. Despite these advantages, the application of open pond systems for providing biomass to a biofuels marketplace would require reduction in pond construction costs and technology for considerable scale-up from current practice [15]. Harvesting Manual harvesting of seaweed has been used for centuries and is still common for some species grown on the shore in the intertidal zone. The recent demand for larger quantities of seaweed for hydrocolloid

biofuel production from seaweed is illustrated (see Figure I in Box 1). Generally, the seaweed biomass can be generated either by cultivation and harvesting or by collecting wild drift seaweed. Then, seaweed is processed by dewatering for the benefit of storage and transportation and to remove any impurities for the downstream bioconversion process. The processed biomass can be used for oil production, liquid renewable fuel (e.g., ethanol or butanol) production by fermentation, and/or methane production through anaerobic digestion (which is the most established process among these options). For bioethanol production, the preprocessed seaweed is sent for hydrolysis to release sugars, which then can be converted to ethanol or other advanced biofuels by microbial fermentation. Instead of covering all the potential fuel production options, this review will specifically focus on the bioconversion of macroalgae (seaweed) for production of liquid biofuels (especially bioethanol). Recent advances in metabolic engineering have permitted the overproduction of certain target compounds by engineering of cells and their metabolic pathways. New approaches to biology are being shaped by the genomics revolution with unprecedented ability to transfer genes, modulate gene expression, and engineer proteins [21]. In the future, we believe that more and more concepts from these fields will be used to overcome obstacles for economic biofuel production from seaweed. 72

extraction has led to the development of mechanical harvesting systems [17]. The type of mechanized harvesting that is used depends on the type of macroalgae and the method of cultivation. For example, Macrocystis and other attached seaweed that tend to stand upright may be well suited for mowing with rotating blades. Floating seaweeds (e.g., Sargassum) or low- growing attached forms (e.g., Gracilaria) may require other forms of harvesting such as suction or dredging with a cutter. Some species of Laminaria are grown on rings and must be harvested by transporting the growth structure with attached seaweed to shore [15]. Improved mechanization for rapid and efficient harvesting of seaweed will be another important prerequisite to supply ample feedstock for biofuel production (Figure I).

[(Box_1)TD$FIG]

Seaweed culvaon Harvest or collecon Wild seaweed

Anaerobic digeson

Feedstock processing (cleaning, dewatering, crushing, slurrying, etc)

Solid waste

Bio-gas

Wastewater and debris

Pretreatment and hydrolysis/ saccharificaon

Fermentaon Value-added byproducts Disllaon and dehydraon

Ethanol TRENDS in Biotechnology

Figure I. Major steps for bioethanol and/or biogas production from seaweed biomass.

Production of biofuels from seaweed using unengineered microbes Hydrolysis of seaweeds converts the storage carbohydrate into simple fermentable sugars, which can be easily used to produce ethanol by some natural microorganisms (Table 1). As an example, Saccharomyces cerevisiae, the most widely used yeast for ethanol fermentation, was presented in previous studies for bioethanol production from seaweed biomass [20,22]. One study used the enzyme laminarinase to hydrolyze laminarin in Saccharina latissima to glucose, which then can be fermented easily to ethanol by S. cerevisiae [22]. Seaweed slurry pretreatments were performed on stirring blocks at pH 6, 23 8C for 30 min, and after fermentation by S. cerevisiae produced 0.45% v/v ethanol within 40 h [22], which is less than 40% of the theoretical yield. Another study [20] reported that 7.0–9.8 g/l ethanol was produced from 50 g/l sugar in dilute-acid pretreated biomass of brown algae L. japonica by simultaneous saccharification and fermentation with S. cerevisiae. The ethanol yields were relatively low because S. cerevisiae could only consume glucose in the hydrolysates but not mannitol, which was determined to be 81% of the total sugars in the study. Mannitol cannot be easily fermented, and only a few organisms can utilize it. Mannitol needs to be converted to fructose-6P before being further metabolized.

Review

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Box 2. Distribution, diversity, and composition of seaweeds Based on the presence or lack of phytopigments other than chlorophyll, marine macroalgae can be classified into three major classes: brown algae (Phaeophyceae), red algae (Rhodophyceae), and green algae (Chlorophyceae) [44]. Brown macroalgae Brown macroalgae include almost 1800 species of multicellular algae with a characteristic olive-green to dark brown color derived from an abundance of fucoxanthin, a yellow-brown pigment that masks the green color of chlorophyll. This group includes the largest and most complex of the macroalgae, the kelp (Laminaria), which may reach lengths of 100 m and grow as much as 50 cm/day (see http://dtc.pima.edu/nschmidt/gallery/algae/algae_index.html). Kelp are found at depths below the low tide level in temperate and polar regions and are farmed extensively in Asia as food products, especially in China, Japan, and South Korea [22]. The composition of brown macroalgae such as Laminaria includes up to 55% dry weight of the carbohydrates laminarin and mannitol [22] (for structures, see Figure I in Box 3). Laminarin can be easily hydrolyzed by laminarase (endo-1,3(4)-b-glucanase) to release glucose monomers [22]. Mannitol is a sugar alcohol that can be readily converted to fructose by mannitol dehydrogenase for bioconversion into ethanol [25]. Brown macroalgae also contain alginate and cellulose (see Figure I in Box 3), which are two structural polysaccharides abundant in the cell wall for mechanical strength to prevent ripping during currents and tidal fluctuations (see http://www.cornishseaweedresources.org/index.htm). Although the sugars from laminarin and mannitol can be easily extracted from milled seaweed for bioconversion, the full potential of biofuel production from brown macroalgae has not yet been realized because

industrial microbes are not able to metabolize the alginate component [14,26]. Red macroalgae Red macroalgae include almost 6000 species of algae having a characteristic red or pink color from the pigments phycocyanin and phycoerythrin, which allow growth in relatively deep waters (see http:// www.cornishseaweedresources.org/index.htm). Red algae are found in the intertidal and subtidal zones of the sea at depths up to 40 m or occasionally as deep as 250 m (see http://www.seaweed.ie/index.html). The composition of red macroalgae varies from species to species but generally consists of cellulose, glucan, and galactan. The cell wall of red seaweed is constructed with cellulose and two kinds of long-chain structural polysaccharides that are valued for their gel-forming abilities – agar and carrageenan (see Figure I in Box 3). Agar can be readily hydrolyzed to release the galactose subunits. Carageenans can be classified as lambda (l), kappa (k), or iota (i) based on their gel-forming ability and are used for thickening foods such as yogurt, ice cream, and pudding [44]. Green macroalgae Green macroalgae include an estimated 1500 species, of which only about 15% are marine and the remainder live in freshwater or terrestrial environments. Because of its need for more light for photosynthesis, green seaweeds live mostly in the shallowest waters, including the intertidal pools that fill and drain with the tides. They are common in bays or estuaries where salt water and fresh water mix together (see http://nature.ca/explore/di-ef/isap_ts_e.cfm). In most cases, the composition of green macroalgae includes starch (Box 3) for food reserves with cellulose (Box 3) and pectin as the main structural polysaccharide in the cell wall [45].

Table I. Seaweed composition and sugars released by hydrolysis (% w/w dry biomass) for a variety of species Seaweed

Class

Gelidium amansii Gelidium amansii Gelidium amansii Laminaria japonica Laminaria japonica Sargassum fulvellum Ulva lactuca Ulva pertusa

Red

Carbohydrate composition Agar, Carrageenan, Cellulose

Brown

Laminarin, Mannitol, Alginate, Fucoidan, Cellulose

Green

Starch, Cellulose

Total carbohydrates (%) 75.2 77.2 83.6 51.9 59.5 39.6 54.3 65.2

Three possible pathways include (i) mannitol ! fructose ! fructose-6P, (ii) mannitol ! mannitol-1P ! fructose-6P [23], and (iii) mannitol ! mannose ! mannose-6P ! fructose-6P. All of these pathways include redox reactions [24], which reduce NAD(P)+ to NAD(P)H and may lead to excess NAD(P)H accumulation under anaerobic conditions. Zymobacter palmae was found to be capable of growing in a synthetic mannitol medium under oxygenlimited conditions and producing ethanol with a yield of 0.38 g/g mannitol [25]. Also, fermentation of mannitol from Laminaria hyperborea hydrolysate extract by Z. palmae was demonstrated, but laminarin was not utilized probably because of the lack of b-(1!3)-glucanase [25]. Another study found that the yeast Pichia angophorae was able to ferment both mannitol and laminarin simultaneously and produced ethanol from brown seaweed extracts [26]. The maximum ethanol yield was 0.43 g ethanol/g substrate at pH 4.5 and 5.9 mmol O2/l/h. Another microorganism Brettanomyces custersii, was suggested to be more suitable for galactose fermentation

Lipid (%) 0.6 1.1 0.9 1.8 1.5 1.4 6.2 2.6

Protein (%) 18.5 13.1 12.2 14.8 8.1 13.0 20.6 7.0

Ash (%) 5.7 8.6 3.3 31.5 30.9 46.0 18.9 25.2

Sugars released by hydrolysis (%) 34.6 56.6 67.5 37.6 34 9.6 19.4 59.6

Sugar composition Glucose, Galactose Glucose, Mannitol Glucose

Refs [46] [20] [18] [20] [18] [20] [20] [18]

than S. cerevisiae in the presence of mixed sugars [27]. A mutant B. custersii strain KCTC 18154P was reported to produce ethanol from hydrolysates of red seaweed G. amansii. Dilute sulfuric acid hydrolysis was applied to G. amansii biomass, resulting in a hydrolysate solution containing galactose as the major sugar and glucose as the minor sugar, and some sugar degradation products such as 5-hydroxymethyl-2-furaldehyde (5-HMF) and organic acids. Fermentation by B. custersii produced 11.8 g/l ethanol from 90 g/l sugar in a batch reactor (YEtOH = 0.13 g/g), and 27.6 g/l ethanol from 72.2 g/l sugar in a continuous reactor (YEtOH = 0.38 g/g) [27]. In addition to ethanol, n-butanol was produced using seaweed hydrolysates. Clostridium beijerinckii and Clostridium saccharoperbutylacetonicum, which are known as butanol-producing bacteria, were used to ferment sulfuric acid-treated hydrolysates of Ulva lactuca, a species of green macroalgae. The hydrolysate had a sugar composition of 27% glucose, 57% arabinose, and 16% xylose. The butanol concentration reached about 4 g butanol/l out of 73

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Box 3. Polysaccharides abundant in seaweed biomass Information on major polysaccharides present in seaweed biomass is introduced below.  Laminarin is the main storage polysaccharide isolated from brown macroalgae, consisting mostly of linear b-1,3-linked glucose units with small amounts of b-1,6-linkages (Figure I).  Alginate is a linear polymer consisting of 1,4-linked b-D-mannuronic acid (M) and a-L-guluronic acid (G) in varying sequences [44] (Figure I).  Cellulose is the structural component of the primary cell wall of green plants and many forms of algae, and it is a polysaccharide consisting of a linear chain of several hundred to more than 10 000 b-1,4-linked D-glucose units.  Starch is a storage polysaccharide found in green macroalgae that may be a linear or branched molecule, consisting of D-glucose units joined by a-1,4 and a-1,6 linkages.  Fucoidan is a heterogeneous polysaccharide in brown macroalgae, consisting primarily of 1,2-linked a-L-fucose-4-sulfate units with very small amounts of D-xylose, D-galactose, D-mannose, and uronic acid.  Carageenan is a sulfated polysaccharide molecule consisting of alternate units of b-D-galactose and a-D-galactose (Figure I) [44].  Agar is a linear polymer of altering 3-linked b-D-galactopyranosyl and 4-linked 3,6-anhydro-a-L-galactopyranosyl subunits and is commonly used as a gelling compound in food products and in culture mediums for research or industry [44].

15.2 g sugar/l in the hydrolysate, and the pilot study recovered 0.29 g butanol/g sugar [28]. Metabolic engineering for producing biofuels from red seaweed Although there are natural microorganisms able to utilize seaweed sugar extracts, economically feasible biofuel production from seaweed requires more efficient conversion of mixed sugars in seaweed hydrolysates by robust strains. Thus, metabolic engineering has been applied to improve fermentation of sugars that can be recovered from seaweeds. Galactose is a major sugar compound in the hydrolysate of red marine algae, composing up to 23% in the hydrolysate from red seaweed Ceylon moss [29]. Therefore, research effort has been made to enhance galactose fermentation to ethanol by engineering S. cerevisiae. The wild type yeast S. cerevisiae is capable of galactose fermentation, but there are two major issues that limit the ethanol yield and productivity. First, ethanol production rate and yield from galactose are considerably lower than from

[(Figure_I)TD$G] Cellulose

Laminarin

1,4

1,6 1,3

Starch

Alginate

1,6

1,4

1,4

Fucoidan

Agar

1,2 1,4

S

S

S

S

S Carrageenan

Key:

S

1,3

= Glucose

= Mannuronic acid

= Galactose

= Guluronic acid

= 3,6-anhydrogalactose

= Fucose

= Sulfate

1,4

1,3

S

S

S

= α-linkage = β-linkage

TRENDS in Biotechnology

Figure I. Structural information about polysaccharides abundant in seaweed biomass.

glucose [30–32]. Second, the presence of glucose represses utilization of galactose because of stringent transcriptional repression of GAL genes coding for enzymes for galactose metabolism [33–36]. These two issues lead to a diauxic consumption of glucose and galactose in red seaweed hydrolysates, which significantly reduces overall ethanol productivity [33,35]. Therefore, metabolic engineering strategies to achieve more efficient galactose fermentation not only focused on improving galactose metabolism itself but also on cofermentation of galactose and other carbohydrate from red seaweed [37]. Galactose fermentation by S. cerevisiae can be improved by engineering of gene regulatory network controlling expression levels of galactose metabolic enzymes or target metabolic genes. Orthogonal approaches have been applied in recent studies. First, improved galactose utilization was achieved by overexpressing a positive regulator GAL4 and deleting three negative regulators (GAL6, GAL80, and MIG1) of the galactose assimilating pathway in yeast [30]. This approach increased specific galactose uptake

Table 1. Microorganisms that convert seaweed biomass to ethanol/butanol Microorganisms Natural strains Saccharomyces cerevisiae Zymobacter palmae Pichia angophorae Brettanomyces custersii Clostridium beijerinckii, Clostridium saccharoperbutylacetonicum Engineered strains Saccharomyces cerevisiae (engineered for improved galactose fermentation) Escherichia coli (engineered for alginate metabolism) Escherichia coli KO11 74

Seaweed source

Major sugars utilized

Refs

Saccharina latissima Laminaria japonica Laminaria hyperborea Laminaria hyperborea Gelidium amansii Ulva lactuca

Glucose Glucose Mannitol Mannitol, laminarin Galactose, glucose Glucose, arabinose, xylose

[22] [20] [26] [25] [27] [28]

Red seaweed (e.g., Ceylon moss) Saccharina japonica

Galactose, or simultaneous cofermentation of galactose and cellobiose Glucose, mannitol and alginate

[13,30,32,37]

Laminaria japonica

Glucose, mannitol

[20]

[14]

Metabolic engineering for producing biofuels from brown seaweed Major sugars from brown seaweeds include glucan (laminarin or cellulose), mannitol, and alginates. However, natural microorganisms as introduced in the earlier section cannot utilize these various sugars concurrently, because they lack the ability to use alginate, and thus ethanol production from brown seaweed can hardly reach its maximum level [15]. An ethanogenic Escherichia coli strain KO11was developed by integrating Zymomonas mobilis ethanol production genes into the pflB gene and was able to ferment a mixed sugar solution containing glucose, galactose, xylose, L-arabinose, and mannitol with an ethanol yield of 0.35–0.37 g/g total sugar [20]. The strain was also used to convert glucose and mannitol to ethanol in simultaneous saccharification and fermentation of HCl acidtreated brown algae L. japonica, which resulted in an ethanol yield of 0.4 g/g of sugars [20]. A recent breakthrough in the efficient use of brown seaweed biomass arose from an engineered E. coli platform capable of alginate degradation and metabolism. Here, ethanol was produced through cofermentation of glucose, mannitol, and alginate from brown seaweed [14]. Fermentation of alginate to ethanol requires surplus reducing equivalents (NADH or NADPH), which can serve as a counterbalance to the reducing equivalents generated from oxidative mannitol metabolism, and by coupling the

(a)

Glactan (∼60%)

Pretreatment and hydrolysis

Galactose GAL2

Cellulose (∼20%)

Engineered S. cerevisiae GAL1

Cellobiose

GAL7

CDT1 (NCU00801) β-glucosidase (NCU00130)

GAL5

Glucose

Glycolysis

Ethanol

(c)

C

[Ethanol]

[Cellobiose & Galactose]

(b)

[Ethanol]

rate by 19% (to 3.57 mmol gal/g cell h) and enhanced specific ethanol production rate by 153% (to 2.71 mmol ethanol/g cell h). Second, overexpression of a key metabolic gene PGM2 coding for phosphoglucomutase increased galactose uptake rate by 70% compared to the parental strain [32]. For another example, overexpression of a truncated TUP1 coding for a transcription repressor was shown to remarkably increase galactose consumption rate and ethanol productivity [13]. In addition, the lag time between glucose and galactose fermentation was significantly shortened by overexpression of the truncated TUP. These approaches have led to progress in improving galactose fermentation by S. cerevisiae. However, efficient fermentation of mixed sugars in hydrolysates from seaweed remains a challenge, because glucose repression on galactose fermentation delays the utilization of the latter. To circumvent glucose repression, a cellobiose (a dimer of glucose) utilization pathway was expressed in S. cerevisiae by expressing genes coding for cellodextrin transporter (cdt-1) and intracellular b-glucosidase (gh1-1) from Neurospora crassa [37]. The strategy used in the study is illustrated in Figure 1. Simultaneous cofermentation of cellobiose and galactose was achieved because glucose was generated from cellobiose by b-glucosidase intracellularly and did not repress galactose metabolism (Figure 1c). By this approach, ethanol productivity during cofermentation of cellobiose (40 g/l) and galactose (40 g/l) increased significantly (0.72 g ethanol/l h) as compared to individual sugar fermentation (0.37 g ethanol/l h for cellobiose, 0.61 g ethanol/l h for galactose) or two-stage sequential fermentation of glucose and galactose (0.58 g ethanol/l h). This also reduces the enzyme cost because there is no need for addition of b-glucosidase.

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[(Figure_1)TD$IG]

[Glucose & Galactose]

Review

Time

Time (d)

Pretreatment and hydrolysis

Alginate Glucose

NAD+

NADP+ NADPH 6-phosphogluconolactone Glucose-6P NADH

Mannitol

Fructose-6P

EDP

Glycolysis

NADH

Introcellular Oligoalginate

6-P-gluconate

Fructose-1,6-biP DihydroxyGlyceraldehyde-3P acetone-P NAD+

Oligoalginate

2-keto-3-deoxy6-P-Gluconate

DEH NADH NAD+ 2-keto-3-deoxy -gluconate

Engineered pathway for alginate metabolism

P-enol-pyruvate

TCA

Pyruvate

Acetaldehyde Ethanol NADH NAD+

Acetyl-CoA

TRENDS in Biotechnology

Figure 1. (a) Metabolic engineering strategies for the simultaneous fermentation of hexose and pentose sugars derived from red seaweed hydrolysates. (b) Glucose inhibits the uptake and the metabolism of galactose, which results in a two stage fermentation of the mixture. (c) Heterologous expressions of a cellobiose transporter and intracellular b-glucosidase enable engineered Saccharomyces cerevisiae to simultaneously coferment glucose and galactose, resulting in efficient ethanol production. (d) Metabolic pathway for the simultaneous fermentation of sugars derived from brown seaweed hydrolysates by the engineered Escherichia coli platform reported in [14]. Cofactors involved redox reactions is illustrated. Alginate catabolism pathway and mannitol catabolism complement each other in terms of cofactor usage, enabling ethanol fermentation from the mixed sugars. EDP, Entner–Doudoroff pathway; TCA, tricarboxylic acid cycle; DEH, 4-deoxy-L-erythro-5-hexoseulose uronic acid.

redox-complementary pathways ethanol fermentation from all sugars in brown algae was realized in this study [14] (Figure 1d). A secretable alginate lysase (Aly) was 75

Review engineered in E. coli-enabled depolymerization of alginate into oligomers without thermal and chemical pretreatment or enzymatic saccharification. Then, a 36-kbp DNA fragment responsible for alginate degradation, transport, and metabolism from Vibria splendidus was integrated into the genome of the engineered E. coli. The pathway was improved by expression of auxiliary genes for alginate degradation, and also ethanol production phenotype was enhanced by heterologous expression of a homoethanol pathway. The constructed E. coli platform fermented dry milled brown macroalgae Saccharina japonica directly for ethanol production and reached a yield of 0.41 g ethanol/g total sugars including alginate, mannitol, and glucan, which is more than 80% of the maximum theoretical yield. Byproduct utilization for producing value-added products As production of biofuels mainly uses the carbohydrate fraction of seaweed biomass, utilization of other components such as plant protein, alginates (if not used), and phenolic compounds need to be considered to enhance economic value of seaweed fuel production process. Moreover, fermentation of hydrolysates from seaweed biomass produces not only ethanol, but also many byproducts, such as glycerol, organic acids (e.g., acetate, succinate), biomass protein, and other minor products. Biofuel industry using seaweed will be more economical when the byproducts are put into good use, just like the petroleum industry where many products besides gasoline are profitable. For example, the fermentation byproduct glycerol has various applications in manufacturing of food, pharmaceutical and personal care products, and other value-added chemicals. Organic acids are high-demand chemical feedstock for producing deicing salts, food additives, and so on. Moreover, because seaweed biomass does not contain lignin, residuals after fermentation can be used as animal feel or feed supplement. Furthermore, using macroalgae biomass

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residue from saccharification and fermentation to produce methane via anaerobic digestion is possible [29,38], and this strategy will have obvious benefits in that both products are valuable for two categories of energy supply – bioethanol as a transport fuel and methane for electricity generation. Challenges and potential environmental impacts Although seaweed has several favorable traits as a renewable feedstock, challenges and potential impacts on ecosystems need to be considered. First, polymers in seaweed biomass are generally made up of mixed sugars, and some are not found in terrestrial biomass (Box 3). Thus, although depolymerization of seaweed polysaccharides is relatively easy, efficient conversion of the unusual sugars to biofuels is challenging, and ongoing efforts are being made to develop strategies for this problem. Second, the application of seaweed biofuels will require increasing seaweed farming (expansion of farming area or increased intensity of current seaweed farming area), and thus special attention should be paid to the potential impacts on marine and coastal environments. Potential impacts include alteration of natural habitats, change of hydrology (e.g., sedimentation and water movement), nutrient depletion, decrease of biodiversity (e.g., removal of mangrove or sea grass), degradation of water quality, and disturbance of coral reefs [39]. Meanwhile, environmental impacts of seaweed farming may be minor in some cases [40] and may even have beneficial effects on increasing populations of fish and invertebrates in the area [41]. A balance must be attained between seaweed biofuel production and its environmental cost. Concluding remarks and future perspectives Advanced metabolic engineering approaches based on systems and synthetic biology offer new opportunities to improve bioconversion of sugars derived from plant biomass to biofuels and value added chemicals. Marine plant

Box 4. Current status and efforts in seaweed biofuel development Compared to terrestrial biomass, marine macroalgae has received less attention so far; however, with the urgent challenges in the pursuit of cost-effective renewable fuels as well as concerns over terrestrial biofuel crops, increasing effort and investment have been put in developing seaweed biofuels.  In Europe, the UK and Irish joint project Sustainable Fuels from Marine Biomass (the BioMara Project; see http://www.biomara.org/) aims to ‘demonstrate the feasibility and viability of producing third generation biofuels from marine biomass’. The seaweed cultivation and harvest process has been successfully established in Scotland, and the project is investigating ethanol production and/or methane generation from seaweed. Statoil and Bio-Architecture Lab (BAL) also aim to commercialize the production of ethanol and co-products from seaweed in Norway and in Europe.  In the United States, the DOE’s Advanced Research Projects Agency-Energy (ARPA-E) is supporting the development of process to convert seaweed biomass into isobutanol (see http://arpa-e. energy.gov/ProgramsProjects/OtherProjects/BiomassEnergy/ MacroAlgaeButanol.aspx), which is undertaken by DuPont and BAL under Technology Investment Agreement. The program focuses on improving seaweed aquaculture, converting seaweed biomass to fermentable sugars, isobutanol production from sugars, and economic and environmental optimization of the production process. Furthermore, BAL is leading a project supported by the 76

Chilean Economic Development Corporation to develop renewable fuels from seaweed in Chile.  In Asia, a project lead by the Mitsubishi Research Institute in Japan plans to start demonstration of ethanol production with waste seaweed in 2012, to develop cultivation technologies by 2016, and set up a production process by 2020. South Korea National Energy Ministry has started a 10-year project with the aim of producing nearly 400 million gallons a year of ethanol by 2020. The Philippine government also has invested more than $US5 million in building up an ethanol plant with seaweed bioethanol technology from South Korea.  There are several biotechnology companies that are in the process of commercializing bioethanol production from seaweed biomass, including Butamax, Seaweed Energy Solutions, Green Gold Algae, and Seaweed Sciences, Inc. Because seaweed cultivation is well established in some Asian countries and has been grown at commercial scale for food products for decades, collaborative effort that can bring together the advanced knowledge in converting seaweed biomass to biofuels and the established large-scale cultivation and harvesting technologies will be beneficial in promoting seaweed biofuel development. For example, Novozymes is collaborating with India’s Sea6 Energy to develop a seaweed bioethanol process, with the former focusing on the development of bioconversion process and the latter sharing its knowledge of offshore seaweed cultivation.

Review biomass such as seaweed has several advantages over terrestrial plant biomass and can be a promising feedstock for biofuel production because of its wide geographic distribution. Efforts are now underway to use seaweed biomass for liquid biofuel production (Box 4). As illustrated in this review, metabolic engineering is evidently essential to the development of technologies to convert seaweed biomass to various biofuels, because complex and diverse carbohydrate composition of seaweed requires fermenting microorganisms to be able to metabolize mixed sugars. Although bioethanol production from seaweed has been reported in several research studies, large-scale production at low cost still faces numerous challenges. In order to realize converting algae biomass into biofuels and valueadded chemicals, technologies allowing economically feasible design of each step, including seaweed cultivation, harvesting and transporting, pretreatment and hydrolysis, fermentation with high yields and productivity, and costeffective utilization of byproducts and biomass residues, are needed. At the same time, potential impacts of largescale cultivation of seaweeds on marine ecosystems need to be carefully assessed. Acknowledgment This work was supported by funding from the Energy Biosciences Institute.

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