Plant Biology ISSN 1435-8603
REVIEW ARTICLE
Growing duckweed for biofuel production: a review W. Cui1 & J. J. Cheng2,3 1 School of Water Resources and Environment, China University of Geosciences (Beijing), Beijing, China 2 Shenzhen Engineering Laboratory for Algal Biofuels, School of Environment and Energy, Peking University-Shenzhen Graduate School, Shenzhen, China 3 Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC, USA
Keywords Biofuels; biogas; butanol; duckweed; ethanol; starch. Correspondence W. Cui, School of Water Resources and Environment, China University of Geosciences (Beijing), Beijing 100083, China. E-mail: [email protected] and J. J. Cheng, Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695-7625, USA. E-mail: [email protected] Editor K. Appenroth
ABSTRACT Duckweed can be utilised to produce ethanol, butanol and biogas, which are promising alternative energy sources to minimise dependence on limited crude oil and natural gas. The advantages of this aquatic plant include high rate of nutrient (nitrogen and phosphorus) uptake, high biomass yield and great potential as an alternative feedstock for the production of fuel ethanol, butanol and biogas. The objective of this article is to review the published research on growing duckweed for the production of the biofuels, especially starch enrichment in duckweed plants. There are mainly two processes affecting the accumulation of starch in duckweed biomass: photosynthesis for starch generation and metabolism-related starch consumption. The cost of stimulating photosynthesis is relatively high based on current technologies. Considerable research efforts have been made to inhibit starch degradation. Future research need in this area includes duckweed selection, optimisation of duckweed biomass production, enhancement of starch accumulation in duckweeds and use of duckweeds for production of various biofuels.
Received: 3 January 2014; Accepted: 24 April 2014 doi:10.1111/plb.12216
INTRODUCTION The world energy consumption has been increasing steadily with population growth and industrialisation processes since 1900. Fossil fuels, e.g. crude oil and natural gas, are currently the predominant energy sources. However, crude oil and natural gas are limited resources that will be depleted sometime in the near future. Although there are debates about the exact year of peak oil production, it is generally believed that it will occur before 2025, after which a decline in worldwide crude oil production will begin (Campbell 2013). Campbell & Laherrere (1998) also predicted that annual global oil production would decline from the current 25 billion barrels to approximately 5 billion barrels in 2050. An increasing demand for energy and inevitable depletion of fossil fuels has stimulated exploration for alternative energy sources. Bio-renewable energy is one of the important energy alternatives to reduce world dependence on notorious fossil-based fuels. Unlike fossil fuels, bioethanol is a renewable energy source produced through fermentation of sugars, and it has been recognised as a potential alternative renewable energy source to petroleum-derived transportation fuels. Developing bioethanol from renewable biomass would provide environmental and social benefits (Lynd et al. 1991; Wyman 1994). The production of bioethanol and its consumption as a fuel could substantially lower CO2 emissions compared with those from fossil fuels. The production of renewable biomass and its
conversion to bioethanol could also generate jobs for local communities. Ethanol has been widely used as a gasoline additive worldwide. The production of ethanol fuel has been increasing over the last 10 years, and reached a level of 85.2 billion litres in the year 2012 (Renewable Fuels Association 2013). The United States is the world’s largest producer of bioethanol fuel, accounting for nearly 47% of global bioethanol production. Brazil is the world’s largest exporter of bioethanol and second largest producer after the United States (Balat & Balat 2009). More than 95% of all cars sold in Brazil are ‘flex-fuel’ cars that can use any blend of gasoline and ethanol (Amorim et al. 2011). Using ethanol-blended fuel for automobiles can significantly reduce petroleum use and greenhouse gas emissions (Wang 1999). Ethanol is also a safer alternative to methyl tertiary butyl ether (MTBE), which was the most common additive to gasoline to provide cleaner combustion before 2000 (Uhler et al. 2001). MTBE is a toxic chemical compound and has been found to contaminate groundwater. Concern over ground- and surface water contamination caused by MTBE leakage led the US Environmental Protection Agency (EPA) to propose reducing or eliminating its use as a gasoline additive (Ahmed 2001). Bioethanol can be produced via several processes based on the properties of the feedstock, i.e. sugar, starch and cellulose platforms (Cheng 2010). Corn grain is currently the dominant feedstock for bioethanol production in the United States. However, using corn for fuel production is inevitably competing for
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limited cropland for food/feed production because corn is also an important food/feed source (Endo et al. 2008). Moreover, intensive corn production has raised environmental concerns. Corn production has high requirements for agricultural inputs, and its cultivation causes more total soil erosion than any other crop (Pimentel 2003). About 79% of ethanol in Brazil is produced from fresh sugarcane juice, and the remaining percentage from cane molasses (Wilkie et al. 2000); sugarcane molasses is also the main feedstock for ethanol production in India (Ghosh & Ghose 2003). Sugarcane grows in tropical regions and its planting area is very limited. The same dilemma of food/feed versus fuel also exists in ethanol production using other feedstocks containing abundant carbohydrates, such as sweet potato and cassava (Zhang et al. 2010; Papong & Malakul 2011). It is evident that lignocellulosic biomass is of great potential importance for ethanol production because the material is abundant in many regions of the world. However, conversion of lignocellulosic biomass to bioethanol is difficult and prohibitively expensive because of the tight structure of the biomass (Sarkar et al. 2012). Among the three platforms for bioethanol production, the starch platform is currently the most widely used in the world because of availability of the feedstock and relatively mature technology (Cheng 2010). However, almost all the current starch feedstocks (corn, rice, wheat, sweet potatoes, etc.) are important food/feed sources and need precious cropland to produce. Therefore, there is great interest in exploring novel starch crops that do not necessarily compete for cropland to make bioethanol production more sustainable. Compared to ethanol, butanol has higher energy density as a fuel (Cheng 2010). Many fuel-related physical and chemical properties (e.g. energy density, heat of vaporisation, octane test value) of butanol are very close to those of gasoline, making butanol an ideal alternative to gasoline (Cheng 2010). Because of its superior physical and chemical properties, biobutanol has attracted the attention of many bioenergy researchers. Currently, production of biobutanol is mainly through acetone– butanol–ethanol (ABE) fermentation, which is much more expensive than ethanol production. Recent research on biobutanol has focused on improvement of the ABE fermentation and butanol separation, as well as new feedstock for the fermentation (Cheng 2010). Biogas is another important bioenergy product, which is usually produced through anaerobic digestion of organic waste materials, such as municipal organic waste or wastewater and sludge, agricultural waste and wastewater, and organic industrial wastes (Cheng 2010). Original organic waste materials usually contain mainly large-molecule compounds, such as carbohydrates, proteins, lipids and celluloses. These organic compounds are hydrolysed with anaerobic bacteria to mainly smaller molecules, such as sugars, fatty acids, amino acids and peptides, as well as a small amount of acetic acid, hydrogen and carbon dioxide in hydrolysis. The sugars, fatty acids, amino acids and peptides are fermented by the anaerobic bacteria to volatile fatty acids (VFAs) such as propionic and butyric acids during acidogenesis. The volatile fatty acids are completely degraded into acetic acid, hydrogen and carbon dioxide during acetogenesis. The whole anaerobic digestion process is complete when both hydrogen and acetic acid are converted to methane during methanogenesis (Cheng 2010). Because of its mature technologies and relatively low cost, 2
biogas production has experienced tremendous growth in the world in the last few years, especially in Europe (Linke 2011). There is great interest in biogas production from different organic feedstocks, such as animal manure, organic municipal wastes and agricultural residues (Linke 2011). Duckweed is a small floating aquatic plant that usually proliferates through vegetative budding of new fronds from the leaf-like thallus (Landolt 1986). Duckweeds are monocotyledons with 37 species distributed among five genera (Landoltia, Lemna, Spirodela, Wolffia and Wolfiella). Based on duckweed species and growing conditions applied, duckweed starch contents from 3% to 75% (dry based) have been reported (Reid & Bieleski 1970; Landolt & Kandeler 1987). Studies have also shown that starch content of duckweed can be substantially increased by manipulating growing conditions, such as pH, phosphate concentration and other nutrient concentration in the medium (Tasseron-De-Jong & Veldstra 1971; McLaren & Smith 1976), which makes duckweed a promising starch source and a potential feedstock for bioethanol and other biofuel production. Duckweed grows faster than most other plants and can double its biomass every 16–24 h under appropriate environmental conditions (Peng et al. 2007). Cheng et al. (2002) reported that the growth rate of duckweed cultivated in swine lagoon liquid could reach 29 g(dry based) m 2day 1, which could be translated to 106 t(dry based)ha 1year 1 if the duckweed could grow for 365 daysyear 1. This yield is much higher than most starch crops such as corn (7.84 tha 1year 1), wheat (3.15 tha 1year 1) and barley (3.70 tha 1year 1), indicating that duckweed has great potential as an alternative starch crop and therefore for bioethanol production (USDA-NASS 2013). Duckweed is not only a great aquatic plant for biomass production, it can also be grown in wastewaters with high nutrient concentrations, making duckweed biomass production an environmentally friendly process. Actually, because of its tolerance to high nutrients and excellent nutrient uptake ability, duckweed has been extensively studied in tertiary treatment of municipal and industrial wastewaters, as well as nutrient recovery from swine wastewater (Alaerts et al. 1996; Shen et al. 2006; Xu & Shen 2011). The objective of this paper is to summarise recent studies on developing high-starch duckweeds for bioethanol and biobutanol production, as well as growing duckweed for the production of biogas. Starch accumulation in duckweeds is specifically discussed in this article. ACCUMULATION OF STARCH IN DUCKWEED As a novel feedstock with a high biomass production rate, duckweed has recently gained attention of researchers and governments for bioenergy production (Zhao et al. 2012a). Green plants generate starch through photosynthesis. Some starch is used in cell metabolism and the remainder is stored in the chloroplasts in the form of starch granules (Fig. 1). At night, without photosynthesis, some starch is degraded and exported from the chloroplasts for heterotrophic metabolism. Numerous enzymes participate in starch breakdown in plant leaves (Smith et al. 2004; Lloyd et al. 2005; Lu & Sharkey 2006). These enzymes allow phosphorolytic or hydrolytic starch degradation. Different pathways of transitory starch (starch temporarily stored in leaves that can be readily used in
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Duckweed for biofuel
Starch granule
Thylakoid
Chloroplast envelope Outer membrane Inner membrane
Stroma Ribosome Plastoglobulus
Nucleoid
Fig. 1. General structure and main components of the chloroplast of green plants.
cell metabolism) degradation in leaves are shown in Fig. 2. As shown in the figure, starch in the chloroplast exists in the form of semicrystalline granules that can be phosphorylated by glucan-water dikinase (GWD; Ritte et al. 2002) and phosphoglucan-water dikinase (PWD; K€ otting et al. 2005), which loosen the starch structure. Two classes of debranching enzymes, isoamylase (ISA) and limit dextrinase (LDA) have been identified in plants (Ishizaki et al. 1983; Doehlert & Knutson 1991; Zhu et al. 1998) and localised in chloroplasts (Okita et al. 1979; Ludwig et al. 1984; Kakefuda et al. 1986; Li et al. 1992; Zeeman et al. 1998). ISA and LDA act on the loosened starch granules to release phosphoglucans (Dinges et al. 2003; Hussain et al. 2003). Phosphate groups on phosphoglucans are cut off by phosphoglucan phosphatase (SEX4) to form linear glucans (K€ otting et al. 2005; Hejazi et al. 2010), which can be degraded through direct hydrolysis of semicrystalline granules by aamylase (AMY) and a-glucosidase (Sun et al. 1995; Frandsen & Svensson 1998; Asatsuma et al. 2005). A primary role of â-amylase (BMY) is production of maltose during hydrolytic starch degradation of branched and linear glucans (Scheidig et al. 2002; Chia et al. 2004; Lu & Sharkey 2004; Sharkey et al. 2004; Smith et al. 2004; Weise et al. 2004). BMY and disproportionating enzyme (DPE) act in complementary roles during starch degradation. DPE catalyses a wide range of reactions; it transfers one part of a glucan molecule (donor) to another (acceptor). Glucan fragments transferred can be maltosyl residues or larger molecules; maltotriose is the smallest donor species. The acceptor molecule can be a malto-oligosaccharide, a polyglucan or even glucose (Kakefuda et al. 1986; Lin et al. 1988; Takaha et al. 1993). The concerted actions of BMY and DPE result in the production of small amounts of glucose and large amounts of maltose. Both products can be exported from the chloroplast to the cytosol: maltose is transferred by a maltose transporter (MEX1; Niittylea et al. 2004), and glucose is transferred by the Glc transporter (Trethewey & ap Rees 1994). a-Glucan phosphorylase (PHS1) releases glucose-1-phosphate from the non-reducing ends of the linear chains, and has been extensively studied in leaves and frequently cited as an important enzyme in starch breakdown (Stitt & Heldt 1981; Kruger & ap Rees 1983). Experiments conducted to evaluate the contribution of phosphorylase to starch degradation indicate that this enzyme has a minor role (Zeeman et al. 2004a). Inclusion of phosphate in the incubation medium results in the production of increased amounts of phosphorylated compounds
(hexose-phosphates, 3-phosphoglycerate (3-PGA) and triosephosphates), presumably through stimulation of a-glucan phosphorylase. Most phosphorylated compounds are exported as triose-phosphates or 3-PGA via the triose-phosphate/phosphate translocator (TPT), rather than as hexose phosphates. The metabolic conclusion is that the neutral compounds released from hydrolysis of starch, i.e. glucose and maltose, are exported from the plastid to provide substrates for sucrose synthesis. It remains possible that some carbohydrate (i.e. products of phosphorolysis) may exit via the TPT, but it seems likely that this is a minor flux (Zeeman et al. 2004b). During daytime, carbon in the form of triose phosphate is exported from chloroplasts via the triose-phosphate translocator (TPT). Schleucher et al. (1998) indicated that most carbon leaves chloroplasts at night as hexoses, not as trioses, while Weise et al. (2004) found that maltose is the major form of carbon exported from chloroplasts, but only at night. Several factors, such as low temperature, nutrient starvation and exposure to inhibitory chemicals, can inhibit starch decomposition in duckweed. To increase starch generation in duckweed, one can either stimulate photosynthesis or decrease starch decomposition. However, to stimulate photosynthesis, one needs to increase the intensity of illumination and/or the concentration of carbon dioxide in the medium, which is difficult and expensive to achieve, especially outdoors. On the other hand, manipulating growing conditions (i.e. temperature, nutrients and inhibitory chemicals) to inhibit starch decomposition is relatively easy and less expensive than stimulating photosynthesis. NUTRIENT STARVATION It is generally recognised that nutrient deficiency can trigger starch accumulation in duckweed, which can be stimulated through nutrient deficiency of phosphorus (Reid & Bieleski 1970), potassium (White 1939) and/or nitrogen (Eyster 1978). In these cases, nutrient deficiency might lead to a reduced starch use in cells, resulting in starch accumulation. Other researchers reported that the response of Lemna gibba to phosphate deficiency was starch accumulation (Thorsteinsson & Tillberg 1987; Ciereszko & Barbachowska 2000). Water containing very low nutrient concentrations is a good medium in which to grow high-starch duckweed. Through a simple transfer of fresh duckweed fronds from a nutrient-rich solution to tapwater, the starch content in a Spirodela polyrhiza increased from about 20% to 45.8% (dry based) after 5 days (Cheng & Stomp 2009). Tao et al. (2013) transferred Landoltia punctata from nutrient-rich solution to distilled water and sampled it at different time points. They found that the activity of ADPglucose pyrophosphorylase, the key enzyme of starch synthesis, and starch content in this duckweed increased continuously under nutrient starvation. Comparative gene expression analysis using RNA sequencing revealed the expression profile of L. punctata under nutrient starvation, with down-regulated global metabolic status and redirected metabolic flux of fixed CO2 into the starch synthesis branch, resulting in starch accumulation (Tao et al. 2013). On the basis of nutrient starvation, the effect of temperature and the daily light integral on starch accumulation in a S. polyrhiza was studied (Cui et al. 2011a). S. polyrhiza grown at 5 °C had a starch content 114% higher than that grown at
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P
P
Chloroplast
BMY
Semicry stalline starch granule
GWD, PWD
AMY -glucosidase
P
Loosened Starch granule
P
Cytosol
BMY
Maltose
Maltose P
MEX1
P
ISA LDA Phosphoglucan P
BMY DPE Maltotriose
SEX4 Glucan
Glucose
Glucose
BMY
pGlcT
PHS1 G1P
Ru5P
G6P PGM1
Triose phosphate
Triose phosphate
G6PDH
TPT
Fig. 2. Pathways of transitory starch degradation in the plant chloroplast. The size of the arrows indicates estimated flux. Dotted lines indicate phosphorolytic starch degradation. Solid lines indicate hydrolytic starch degradation. AMY: a-amylase; BMY: b-amylase; DPE: disproportionating enzyme; G1P: glucose-1phosphate; G6P: glucose-6-phosphate; G6PDH: glucose-6-phosphate dehydrogenase; GWD: glucan-water dikinase; ISA: isoamylase; LDA: limit dextrinase; MEX1: maltose transporter; pGlcT: plastid glucose transporter; PGM1: plastidial phosphoglucomutase; PHS1: a-glucan phosphorylase; PWD: phosphoglucanwater dikinase; Ru5P: ribulose 5-phosphate; TPT: triose phosphate translocator; SEX4: phosphoglucan phosphatase (modified from Weise et al. 2004).
25 °C. With the increase in daily light integral, starch content increased at all temperatures studied (5 °C, 15 °C, 25 °C). The results showed that lower temperature and higher daily light integral favour starch accumulation in duckweed. Starch content in duckweed increases continuously during the light phase and decreases during the night. Cui et al. (2011b) determined the effect of day and night temperature on starch content of S. polyrhiza. They found that, at the same daytime temperature, lower nighttime temperature favoured starch accumulation in S. polyrhiza, while at the same night temperature varying daytime temperature had no real effect on duckweed starch accumulation. In summary, a relatively low temperature, especially at night, favours starch accumulation in duckweed. In addition to indoor laboratory experiments under controlled conditions, outdoor tests of duckweed starch accumulation under natural climate conditions have also been carried out. Xu et al. (2011) performed a pilot study cultivating highstarch duckweed for bioethanol production on a commercial swine farm in Zebulon, North Carolina, USA, which was the first report on producing high-starch duckweed in the field and converting the harvested biomass into ethanol. Farrell (2012) cultivated duckweed in a 23-ha lagoon and also found that duckweed starch content could be increased from 2% duckweed. Huang et al. (2013) studied anaerobic digestion of swine manure with duckweed at a ratio of swine manure to duckweed of 1:1 in a 4 m3 pilot-scale plug-flow baffled digester (a digester with baffles to define compartments and guide flow in the digester). The results showed that biogas yield, COD (chemical oxygen demand) conversion rate and volumetric biogas production were 0.31 lg 1 COD, 63.2% and 1.00 m3m 3day 1, respectively. In the control anaerobic digester with swine manure as sole substrate, biogas yield, COD conversion rate and volumetric biogas production were 0.28 lg 1 COD,57.1% and 0.71 m3m 3day 1, respectively. The results indicate that addition of duckweed significantly improved biogas production in the anaerobic digester. FUTURE PERSPECTIVES To improve the application of duckweeds for biofuel production, further studies are needed into duckweed selection, optimisation of biomass production, enhancement of starch accumulation and use for production of various biofuels. When duckweed is used for biogas production, it is important to select strains with high growth and biomass production rates to maximise the generation of organics. Use of duckweed for biogas production is most likely to be associated with growing duckweed for wastewater treatment, especially for nutrient removal. Thus, selection of the right duckweed strains that have a high capacity for nutrient absorption is also important. When duckweed is used for bioethanol or biobutanol production, it is critical that the duckweed has both high biomass production and high starch content. To increase starch content, enrichment of CO2 in growth media could stimulate photosynthesis and carbohydrate production. Accumulation of starch could also be achieved through the inhibition of starch consumption in cell metabolism. An understanding of starch degradation in cells is very important for further studies of starch accumulation in duckweed. As shown in Fig. 2, many enzymes are involved in transitory starch degradation. Duckweed fronds are similar to higher plant leaves, so information on starch degradation in such leaves could be important for exploring transitory starch degradation in duckweed fronds. A fundamental understanding of transitory starch degradation will be essential in developing strategies to inhibit starch degradation and thus improve starch accumulation in duckweed. ACKNOWLEDGEMENTS The authors would like to acknowledge financial support from the Fundamental Research Funds for the Central Universities (No. 53200959458) and National Natural Science Foundation of China (No. 51309206).
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Wang S.-L., Xu X.-R., Sun Y.-X. (2013) Heavy metal pollution in coastal areas of South China: a review. Marine Pollution Bulletin, 76, 7–15. Weise S.E., Weber A.P.M., Sharkey T.D. (2004) Maltose is the major form of carbon exported from the chloroplast at night. Planta, 218, 474–482. White H.L. (1939) The interaction of factors in the growth of Lemna. XIV. The interaction of potassium and light intensity in relation to growth and assimilation. Annals of Botany, 3, 619–648. Wilkie A.C., Riedesel K.J., Owens J.M. (2000) Stillage characterization and anaerobic treatment of ethanol stillage from conventional and cellulosic feedstocks. Biomass and Bioenergy, 19, 63–102. Wyman C.E. (1994) Ethanol from lignocellulosic biomass: technology, economics, and opportunities. Bioresource Technology, 50, 3–6. Xiao Y., Fang Y., Jin Y.L., Zhang G.H., Zhao H. (2013) Culturing duckweed in the field for starch accumulation. Industrial Crops and Products, 48, 183–190. Xu J., Shen G. (2011) Growing duckweed in swine wastewater for nutrient recovery and biomass production. Bioresource Technology, 102, 848–853. Xu J., Cui W., Cheng J.J., Stomp A.-M. (2011) Production of high-starch duckweed and its conversion to bioethanol. Biosystems Engineering, 110, 67–72. Zeeman S.C., Umemoto T., Lue W.-L., Au-Yeung P., Martin C., Smith A.M., Chen J. (1998) A mutant of Arabidopsis lacking a chloroplastic isoamylase accumulates both starch and phytoglycogen. The Plant Cell, 10, 1699–1711. Zeeman S.C., Smith A.M., Smith S.M. (2004a) The breakdown of starch in leaves. New Phytologist, 163, 247–261. Zeeman S.C., Thorneycroft D., Schupp N., Chapple A., Weck M., Dunstan H., Haldimann P., Bechtold N., Smith A.M., Smith S.M. (2004b) The role of plastidial a-glucan phosphorylase in starch degradation and tolerance of biotic stress in Arabidopsis leaves. Plant Physiology, 135, 849–858. Zhang L., Chen Q., Jin Y., Xue H., Guan J., Wang Z., Zhao H. (2010) Energy-saving direct ethanol production from viscosity reduction mash of sweet potato at very high gravity (VHG). Fuel Processing Technology, 91, 1845–1850. Zhao H., Appenroth K.-J., Landesman L., Salmean A.A., Lam E. (2012a) Duckweed rising at Chengdu: summary of the 1st International Conference on Duckweed Application and Research. Plant Molecular Biology, 78, 627–632. Zhao X., Elliston A., Collins S.R.A., Moates G.K., Coleman M.J., Waldron K.W. (2012b) Enzymatic saccharification of duckweed (Lemna minor) biomass without thermophysical pretreatment. Biomass and Bioenergy, 47, 354–361. Zhu Z.P., Hylton C.M., Rossner U., Smith A.M. (1998) Characterization of starch-debranching enzymes in pea embryos. Plant Physiology, 118, 581–590.
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
REVIEW ARTICLE
Growing duckweed for biofuel production: a review W. Cui1 & J. J. Cheng2,3 1 School of Water Resources and Environment, China University of Geosciences (Beijing), Beijing, China 2 Shenzhen Engineering Laboratory for Algal Biofuels, School of Environment and Energy, Peking University-Shenzhen Graduate School, Shenzhen, China 3 Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC, USA
Keywords Biofuels; biogas; butanol; duckweed; ethanol; starch. Correspondence W. Cui, School of Water Resources and Environment, China University of Geosciences (Beijing), Beijing 100083, China. E-mail: [email protected] and J. J. Cheng, Department of Biological and Agricultural Engineering, North Carolina State University, Raleigh, NC 27695-7625, USA. E-mail: [email protected] Editor K. Appenroth
ABSTRACT Duckweed can be utilised to produce ethanol, butanol and biogas, which are promising alternative energy sources to minimise dependence on limited crude oil and natural gas. The advantages of this aquatic plant include high rate of nutrient (nitrogen and phosphorus) uptake, high biomass yield and great potential as an alternative feedstock for the production of fuel ethanol, butanol and biogas. The objective of this article is to review the published research on growing duckweed for the production of the biofuels, especially starch enrichment in duckweed plants. There are mainly two processes affecting the accumulation of starch in duckweed biomass: photosynthesis for starch generation and metabolism-related starch consumption. The cost of stimulating photosynthesis is relatively high based on current technologies. Considerable research efforts have been made to inhibit starch degradation. Future research need in this area includes duckweed selection, optimisation of duckweed biomass production, enhancement of starch accumulation in duckweeds and use of duckweeds for production of various biofuels.
Received: 3 January 2014; Accepted: 24 April 2014 doi:10.1111/plb.12216
INTRODUCTION The world energy consumption has been increasing steadily with population growth and industrialisation processes since 1900. Fossil fuels, e.g. crude oil and natural gas, are currently the predominant energy sources. However, crude oil and natural gas are limited resources that will be depleted sometime in the near future. Although there are debates about the exact year of peak oil production, it is generally believed that it will occur before 2025, after which a decline in worldwide crude oil production will begin (Campbell 2013). Campbell & Laherrere (1998) also predicted that annual global oil production would decline from the current 25 billion barrels to approximately 5 billion barrels in 2050. An increasing demand for energy and inevitable depletion of fossil fuels has stimulated exploration for alternative energy sources. Bio-renewable energy is one of the important energy alternatives to reduce world dependence on notorious fossil-based fuels. Unlike fossil fuels, bioethanol is a renewable energy source produced through fermentation of sugars, and it has been recognised as a potential alternative renewable energy source to petroleum-derived transportation fuels. Developing bioethanol from renewable biomass would provide environmental and social benefits (Lynd et al. 1991; Wyman 1994). The production of bioethanol and its consumption as a fuel could substantially lower CO2 emissions compared with those from fossil fuels. The production of renewable biomass and its
conversion to bioethanol could also generate jobs for local communities. Ethanol has been widely used as a gasoline additive worldwide. The production of ethanol fuel has been increasing over the last 10 years, and reached a level of 85.2 billion litres in the year 2012 (Renewable Fuels Association 2013). The United States is the world’s largest producer of bioethanol fuel, accounting for nearly 47% of global bioethanol production. Brazil is the world’s largest exporter of bioethanol and second largest producer after the United States (Balat & Balat 2009). More than 95% of all cars sold in Brazil are ‘flex-fuel’ cars that can use any blend of gasoline and ethanol (Amorim et al. 2011). Using ethanol-blended fuel for automobiles can significantly reduce petroleum use and greenhouse gas emissions (Wang 1999). Ethanol is also a safer alternative to methyl tertiary butyl ether (MTBE), which was the most common additive to gasoline to provide cleaner combustion before 2000 (Uhler et al. 2001). MTBE is a toxic chemical compound and has been found to contaminate groundwater. Concern over ground- and surface water contamination caused by MTBE leakage led the US Environmental Protection Agency (EPA) to propose reducing or eliminating its use as a gasoline additive (Ahmed 2001). Bioethanol can be produced via several processes based on the properties of the feedstock, i.e. sugar, starch and cellulose platforms (Cheng 2010). Corn grain is currently the dominant feedstock for bioethanol production in the United States. However, using corn for fuel production is inevitably competing for
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limited cropland for food/feed production because corn is also an important food/feed source (Endo et al. 2008). Moreover, intensive corn production has raised environmental concerns. Corn production has high requirements for agricultural inputs, and its cultivation causes more total soil erosion than any other crop (Pimentel 2003). About 79% of ethanol in Brazil is produced from fresh sugarcane juice, and the remaining percentage from cane molasses (Wilkie et al. 2000); sugarcane molasses is also the main feedstock for ethanol production in India (Ghosh & Ghose 2003). Sugarcane grows in tropical regions and its planting area is very limited. The same dilemma of food/feed versus fuel also exists in ethanol production using other feedstocks containing abundant carbohydrates, such as sweet potato and cassava (Zhang et al. 2010; Papong & Malakul 2011). It is evident that lignocellulosic biomass is of great potential importance for ethanol production because the material is abundant in many regions of the world. However, conversion of lignocellulosic biomass to bioethanol is difficult and prohibitively expensive because of the tight structure of the biomass (Sarkar et al. 2012). Among the three platforms for bioethanol production, the starch platform is currently the most widely used in the world because of availability of the feedstock and relatively mature technology (Cheng 2010). However, almost all the current starch feedstocks (corn, rice, wheat, sweet potatoes, etc.) are important food/feed sources and need precious cropland to produce. Therefore, there is great interest in exploring novel starch crops that do not necessarily compete for cropland to make bioethanol production more sustainable. Compared to ethanol, butanol has higher energy density as a fuel (Cheng 2010). Many fuel-related physical and chemical properties (e.g. energy density, heat of vaporisation, octane test value) of butanol are very close to those of gasoline, making butanol an ideal alternative to gasoline (Cheng 2010). Because of its superior physical and chemical properties, biobutanol has attracted the attention of many bioenergy researchers. Currently, production of biobutanol is mainly through acetone– butanol–ethanol (ABE) fermentation, which is much more expensive than ethanol production. Recent research on biobutanol has focused on improvement of the ABE fermentation and butanol separation, as well as new feedstock for the fermentation (Cheng 2010). Biogas is another important bioenergy product, which is usually produced through anaerobic digestion of organic waste materials, such as municipal organic waste or wastewater and sludge, agricultural waste and wastewater, and organic industrial wastes (Cheng 2010). Original organic waste materials usually contain mainly large-molecule compounds, such as carbohydrates, proteins, lipids and celluloses. These organic compounds are hydrolysed with anaerobic bacteria to mainly smaller molecules, such as sugars, fatty acids, amino acids and peptides, as well as a small amount of acetic acid, hydrogen and carbon dioxide in hydrolysis. The sugars, fatty acids, amino acids and peptides are fermented by the anaerobic bacteria to volatile fatty acids (VFAs) such as propionic and butyric acids during acidogenesis. The volatile fatty acids are completely degraded into acetic acid, hydrogen and carbon dioxide during acetogenesis. The whole anaerobic digestion process is complete when both hydrogen and acetic acid are converted to methane during methanogenesis (Cheng 2010). Because of its mature technologies and relatively low cost, 2
biogas production has experienced tremendous growth in the world in the last few years, especially in Europe (Linke 2011). There is great interest in biogas production from different organic feedstocks, such as animal manure, organic municipal wastes and agricultural residues (Linke 2011). Duckweed is a small floating aquatic plant that usually proliferates through vegetative budding of new fronds from the leaf-like thallus (Landolt 1986). Duckweeds are monocotyledons with 37 species distributed among five genera (Landoltia, Lemna, Spirodela, Wolffia and Wolfiella). Based on duckweed species and growing conditions applied, duckweed starch contents from 3% to 75% (dry based) have been reported (Reid & Bieleski 1970; Landolt & Kandeler 1987). Studies have also shown that starch content of duckweed can be substantially increased by manipulating growing conditions, such as pH, phosphate concentration and other nutrient concentration in the medium (Tasseron-De-Jong & Veldstra 1971; McLaren & Smith 1976), which makes duckweed a promising starch source and a potential feedstock for bioethanol and other biofuel production. Duckweed grows faster than most other plants and can double its biomass every 16–24 h under appropriate environmental conditions (Peng et al. 2007). Cheng et al. (2002) reported that the growth rate of duckweed cultivated in swine lagoon liquid could reach 29 g(dry based) m 2day 1, which could be translated to 106 t(dry based)ha 1year 1 if the duckweed could grow for 365 daysyear 1. This yield is much higher than most starch crops such as corn (7.84 tha 1year 1), wheat (3.15 tha 1year 1) and barley (3.70 tha 1year 1), indicating that duckweed has great potential as an alternative starch crop and therefore for bioethanol production (USDA-NASS 2013). Duckweed is not only a great aquatic plant for biomass production, it can also be grown in wastewaters with high nutrient concentrations, making duckweed biomass production an environmentally friendly process. Actually, because of its tolerance to high nutrients and excellent nutrient uptake ability, duckweed has been extensively studied in tertiary treatment of municipal and industrial wastewaters, as well as nutrient recovery from swine wastewater (Alaerts et al. 1996; Shen et al. 2006; Xu & Shen 2011). The objective of this paper is to summarise recent studies on developing high-starch duckweeds for bioethanol and biobutanol production, as well as growing duckweed for the production of biogas. Starch accumulation in duckweeds is specifically discussed in this article. ACCUMULATION OF STARCH IN DUCKWEED As a novel feedstock with a high biomass production rate, duckweed has recently gained attention of researchers and governments for bioenergy production (Zhao et al. 2012a). Green plants generate starch through photosynthesis. Some starch is used in cell metabolism and the remainder is stored in the chloroplasts in the form of starch granules (Fig. 1). At night, without photosynthesis, some starch is degraded and exported from the chloroplasts for heterotrophic metabolism. Numerous enzymes participate in starch breakdown in plant leaves (Smith et al. 2004; Lloyd et al. 2005; Lu & Sharkey 2006). These enzymes allow phosphorolytic or hydrolytic starch degradation. Different pathways of transitory starch (starch temporarily stored in leaves that can be readily used in
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
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Duckweed for biofuel
Starch granule
Thylakoid
Chloroplast envelope Outer membrane Inner membrane
Stroma Ribosome Plastoglobulus
Nucleoid
Fig. 1. General structure and main components of the chloroplast of green plants.
cell metabolism) degradation in leaves are shown in Fig. 2. As shown in the figure, starch in the chloroplast exists in the form of semicrystalline granules that can be phosphorylated by glucan-water dikinase (GWD; Ritte et al. 2002) and phosphoglucan-water dikinase (PWD; K€ otting et al. 2005), which loosen the starch structure. Two classes of debranching enzymes, isoamylase (ISA) and limit dextrinase (LDA) have been identified in plants (Ishizaki et al. 1983; Doehlert & Knutson 1991; Zhu et al. 1998) and localised in chloroplasts (Okita et al. 1979; Ludwig et al. 1984; Kakefuda et al. 1986; Li et al. 1992; Zeeman et al. 1998). ISA and LDA act on the loosened starch granules to release phosphoglucans (Dinges et al. 2003; Hussain et al. 2003). Phosphate groups on phosphoglucans are cut off by phosphoglucan phosphatase (SEX4) to form linear glucans (K€ otting et al. 2005; Hejazi et al. 2010), which can be degraded through direct hydrolysis of semicrystalline granules by aamylase (AMY) and a-glucosidase (Sun et al. 1995; Frandsen & Svensson 1998; Asatsuma et al. 2005). A primary role of â-amylase (BMY) is production of maltose during hydrolytic starch degradation of branched and linear glucans (Scheidig et al. 2002; Chia et al. 2004; Lu & Sharkey 2004; Sharkey et al. 2004; Smith et al. 2004; Weise et al. 2004). BMY and disproportionating enzyme (DPE) act in complementary roles during starch degradation. DPE catalyses a wide range of reactions; it transfers one part of a glucan molecule (donor) to another (acceptor). Glucan fragments transferred can be maltosyl residues or larger molecules; maltotriose is the smallest donor species. The acceptor molecule can be a malto-oligosaccharide, a polyglucan or even glucose (Kakefuda et al. 1986; Lin et al. 1988; Takaha et al. 1993). The concerted actions of BMY and DPE result in the production of small amounts of glucose and large amounts of maltose. Both products can be exported from the chloroplast to the cytosol: maltose is transferred by a maltose transporter (MEX1; Niittylea et al. 2004), and glucose is transferred by the Glc transporter (Trethewey & ap Rees 1994). a-Glucan phosphorylase (PHS1) releases glucose-1-phosphate from the non-reducing ends of the linear chains, and has been extensively studied in leaves and frequently cited as an important enzyme in starch breakdown (Stitt & Heldt 1981; Kruger & ap Rees 1983). Experiments conducted to evaluate the contribution of phosphorylase to starch degradation indicate that this enzyme has a minor role (Zeeman et al. 2004a). Inclusion of phosphate in the incubation medium results in the production of increased amounts of phosphorylated compounds
(hexose-phosphates, 3-phosphoglycerate (3-PGA) and triosephosphates), presumably through stimulation of a-glucan phosphorylase. Most phosphorylated compounds are exported as triose-phosphates or 3-PGA via the triose-phosphate/phosphate translocator (TPT), rather than as hexose phosphates. The metabolic conclusion is that the neutral compounds released from hydrolysis of starch, i.e. glucose and maltose, are exported from the plastid to provide substrates for sucrose synthesis. It remains possible that some carbohydrate (i.e. products of phosphorolysis) may exit via the TPT, but it seems likely that this is a minor flux (Zeeman et al. 2004b). During daytime, carbon in the form of triose phosphate is exported from chloroplasts via the triose-phosphate translocator (TPT). Schleucher et al. (1998) indicated that most carbon leaves chloroplasts at night as hexoses, not as trioses, while Weise et al. (2004) found that maltose is the major form of carbon exported from chloroplasts, but only at night. Several factors, such as low temperature, nutrient starvation and exposure to inhibitory chemicals, can inhibit starch decomposition in duckweed. To increase starch generation in duckweed, one can either stimulate photosynthesis or decrease starch decomposition. However, to stimulate photosynthesis, one needs to increase the intensity of illumination and/or the concentration of carbon dioxide in the medium, which is difficult and expensive to achieve, especially outdoors. On the other hand, manipulating growing conditions (i.e. temperature, nutrients and inhibitory chemicals) to inhibit starch decomposition is relatively easy and less expensive than stimulating photosynthesis. NUTRIENT STARVATION It is generally recognised that nutrient deficiency can trigger starch accumulation in duckweed, which can be stimulated through nutrient deficiency of phosphorus (Reid & Bieleski 1970), potassium (White 1939) and/or nitrogen (Eyster 1978). In these cases, nutrient deficiency might lead to a reduced starch use in cells, resulting in starch accumulation. Other researchers reported that the response of Lemna gibba to phosphate deficiency was starch accumulation (Thorsteinsson & Tillberg 1987; Ciereszko & Barbachowska 2000). Water containing very low nutrient concentrations is a good medium in which to grow high-starch duckweed. Through a simple transfer of fresh duckweed fronds from a nutrient-rich solution to tapwater, the starch content in a Spirodela polyrhiza increased from about 20% to 45.8% (dry based) after 5 days (Cheng & Stomp 2009). Tao et al. (2013) transferred Landoltia punctata from nutrient-rich solution to distilled water and sampled it at different time points. They found that the activity of ADPglucose pyrophosphorylase, the key enzyme of starch synthesis, and starch content in this duckweed increased continuously under nutrient starvation. Comparative gene expression analysis using RNA sequencing revealed the expression profile of L. punctata under nutrient starvation, with down-regulated global metabolic status and redirected metabolic flux of fixed CO2 into the starch synthesis branch, resulting in starch accumulation (Tao et al. 2013). On the basis of nutrient starvation, the effect of temperature and the daily light integral on starch accumulation in a S. polyrhiza was studied (Cui et al. 2011a). S. polyrhiza grown at 5 °C had a starch content 114% higher than that grown at
Plant Biology © 2014 German Botanical Society and The Royal Botanical Society of the Netherlands
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Duckweed for biofuel
Cui & Cheng
P
P
Chloroplast
BMY
Semicry stalline starch granule
GWD, PWD
AMY -glucosidase
P
Loosened Starch granule
P
Cytosol
BMY
Maltose
Maltose P
MEX1
P
ISA LDA Phosphoglucan P
BMY DPE Maltotriose
SEX4 Glucan
Glucose
Glucose
BMY
pGlcT
PHS1 G1P
Ru5P
G6P PGM1
Triose phosphate
Triose phosphate
G6PDH
TPT
Fig. 2. Pathways of transitory starch degradation in the plant chloroplast. The size of the arrows indicates estimated flux. Dotted lines indicate phosphorolytic starch degradation. Solid lines indicate hydrolytic starch degradation. AMY: a-amylase; BMY: b-amylase; DPE: disproportionating enzyme; G1P: glucose-1phosphate; G6P: glucose-6-phosphate; G6PDH: glucose-6-phosphate dehydrogenase; GWD: glucan-water dikinase; ISA: isoamylase; LDA: limit dextrinase; MEX1: maltose transporter; pGlcT: plastid glucose transporter; PGM1: plastidial phosphoglucomutase; PHS1: a-glucan phosphorylase; PWD: phosphoglucanwater dikinase; Ru5P: ribulose 5-phosphate; TPT: triose phosphate translocator; SEX4: phosphoglucan phosphatase (modified from Weise et al. 2004).
25 °C. With the increase in daily light integral, starch content increased at all temperatures studied (5 °C, 15 °C, 25 °C). The results showed that lower temperature and higher daily light integral favour starch accumulation in duckweed. Starch content in duckweed increases continuously during the light phase and decreases during the night. Cui et al. (2011b) determined the effect of day and night temperature on starch content of S. polyrhiza. They found that, at the same daytime temperature, lower nighttime temperature favoured starch accumulation in S. polyrhiza, while at the same night temperature varying daytime temperature had no real effect on duckweed starch accumulation. In summary, a relatively low temperature, especially at night, favours starch accumulation in duckweed. In addition to indoor laboratory experiments under controlled conditions, outdoor tests of duckweed starch accumulation under natural climate conditions have also been carried out. Xu et al. (2011) performed a pilot study cultivating highstarch duckweed for bioethanol production on a commercial swine farm in Zebulon, North Carolina, USA, which was the first report on producing high-starch duckweed in the field and converting the harvested biomass into ethanol. Farrell (2012) cultivated duckweed in a 23-ha lagoon and also found that duckweed starch content could be increased from 2% duckweed. Huang et al. (2013) studied anaerobic digestion of swine manure with duckweed at a ratio of swine manure to duckweed of 1:1 in a 4 m3 pilot-scale plug-flow baffled digester (a digester with baffles to define compartments and guide flow in the digester). The results showed that biogas yield, COD (chemical oxygen demand) conversion rate and volumetric biogas production were 0.31 lg 1 COD, 63.2% and 1.00 m3m 3day 1, respectively. In the control anaerobic digester with swine manure as sole substrate, biogas yield, COD conversion rate and volumetric biogas production were 0.28 lg 1 COD,57.1% and 0.71 m3m 3day 1, respectively. The results indicate that addition of duckweed significantly improved biogas production in the anaerobic digester. FUTURE PERSPECTIVES To improve the application of duckweeds for biofuel production, further studies are needed into duckweed selection, optimisation of biomass production, enhancement of starch accumulation and use for production of various biofuels. When duckweed is used for biogas production, it is important to select strains with high growth and biomass production rates to maximise the generation of organics. Use of duckweed for biogas production is most likely to be associated with growing duckweed for wastewater treatment, especially for nutrient removal. Thus, selection of the right duckweed strains that have a high capacity for nutrient absorption is also important. When duckweed is used for bioethanol or biobutanol production, it is critical that the duckweed has both high biomass production and high starch content. To increase starch content, enrichment of CO2 in growth media could stimulate photosynthesis and carbohydrate production. Accumulation of starch could also be achieved through the inhibition of starch consumption in cell metabolism. An understanding of starch degradation in cells is very important for further studies of starch accumulation in duckweed. As shown in Fig. 2, many enzymes are involved in transitory starch degradation. Duckweed fronds are similar to higher plant leaves, so information on starch degradation in such leaves could be important for exploring transitory starch degradation in duckweed fronds. A fundamental understanding of transitory starch degradation will be essential in developing strategies to inhibit starch degradation and thus improve starch accumulation in duckweed. ACKNOWLEDGEMENTS The authors would like to acknowledge financial support from the Fundamental Research Funds for the Central Universities (No. 53200959458) and National Natural Science Foundation of China (No. 51309206).
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