Engineering Duckweed as a potential biofuel crop
The problem
In 2007, global liquid fuel consumption was 86.1 million barrels per day and is growing steadily towards the projected 110.6 million barrels per day by 2035 (International Energy Outlook 2010). Growing economies such as China and India are rapidly increasing transportation fuel demands, and high energy consuming countries like the United States have not yet reached a plateau in their fuel needs. There is an undisputed need for the development of renewable energy and fuel sources which address the issues of increased need, rising levels of carbon dioxide, economic viability, and domestic energy security. There are a number of options for renewable fuel (ethanol, butanol, biodiesel, methane, hydrogen, and advanced biofuels comprised of alkanes, alkenes and/or aromatic hydrocarbon mixtures), but currently most options are heavily based on food crops. One promising option is cellulosic fuels. Cellulosic biofuels feedstock is estimated to be over 1 billion dry tons per year in the United States which could replace 30% of current demand for transportation fuels.
The process of breaking down a complex polysaccharide carbohydrate (such as starch, cellulose or hemicellulose) into monosaccharide components that can be fermented into biofuels is called saccharification. Saccharification of corn starch, alpha-linked glucose polymers, is relatively easy compared with breaking down the beta-linked glucose polymers that make up the aligned and hydrogen-bonded cellulose polymers in cellulose microfibrils. Decomposition of cellulosic biomass presents a formidable challenge that requires costly, energy intensive and environmentally detrimental biomass pretreatment steps involving high temperatures, acids and/or enzymes in order to increase the accessibility and effectiveness of cellulase enzymes. In addition, high ratios of cellulase enzyme to biomass are currently about 100 times higher than enzyme loadings used for corn starch saccharification.
In 2007, global liquid fuel consumption was 86.1 million barrels per day and is growing steadily towards the projected 110.6 million barrels per day by 2035 (International Energy Outlook 2010). Growing economies such as China and India are rapidly increasing transportation fuel demands, and high energy consuming countries like the United States have not yet reached a plateau in their fuel needs. There is an undisputed need for the development of renewable energy and fuel sources which address the issues of increased need, rising levels of carbon dioxide, economic viability, and domestic energy security. There are a number of options for renewable fuel (ethanol, butanol, biodiesel, methane, hydrogen, and advanced biofuels comprised of alkanes, alkenes and/or aromatic hydrocarbon mixtures), but currently most options are heavily based on food crops. One promising option is cellulosic fuels. Cellulosic biofuels feedstock is estimated to be over 1 billion dry tons per year in the United States which could replace 30% of current demand for transportation fuels.
The process of breaking down a complex polysaccharide carbohydrate (such as starch, cellulose or hemicellulose) into monosaccharide components that can be fermented into biofuels is called saccharification. Saccharification of corn starch, alpha-linked glucose polymers, is relatively easy compared with breaking down the beta-linked glucose polymers that make up the aligned and hydrogen-bonded cellulose polymers in cellulose microfibrils. Decomposition of cellulosic biomass presents a formidable challenge that requires costly, energy intensive and environmentally detrimental biomass pretreatment steps involving high temperatures, acids and/or enzymes in order to increase the accessibility and effectiveness of cellulase enzymes. In addition, high ratios of cellulase enzyme to biomass are currently about 100 times higher than enzyme loadings used for corn starch saccharification.
Current solution/limitations
For the enzymatic conversion, enzyme preparations (referred to as cellulases) comprised of endoglucanses, exoglucanases, xylanases and/or beta-glucosidases, are produced in separate microbial fermentations, either on-site or at commercial enzyme manufacturing plants. Typically these enzymes are produced utilizing genetically modified fungal hosts (Trichoderma reesei, Aspergillus niger, Penicillium funiculosum, Humicola insolens, etc) grown in expensive stainless steel fermentors under aerobic and aseptic conditions. Commercial enzyme manufacturers such as Genencor, Inc. and Novozymes, Inc. produce these enzymes in large scale (up to ~300,000 L) aerobic fermentation (requiring energy for fermentor and media sterilization, agitation for mixing viscous fungal suspensions, gas compression for air/oxygen sparging, fluid heating for temperature control, pumps for fluid transfer, etc), followed by downstream processing which may include host cell deactivation and/or removal, protein concentration, and stabilization/formulation.
For the enzymatic conversion, enzyme preparations (referred to as cellulases) comprised of endoglucanses, exoglucanases, xylanases and/or beta-glucosidases, are produced in separate microbial fermentations, either on-site or at commercial enzyme manufacturing plants. Typically these enzymes are produced utilizing genetically modified fungal hosts (Trichoderma reesei, Aspergillus niger, Penicillium funiculosum, Humicola insolens, etc) grown in expensive stainless steel fermentors under aerobic and aseptic conditions. Commercial enzyme manufacturers such as Genencor, Inc. and Novozymes, Inc. produce these enzymes in large scale (up to ~300,000 L) aerobic fermentation (requiring energy for fermentor and media sterilization, agitation for mixing viscous fungal suspensions, gas compression for air/oxygen sparging, fluid heating for temperature control, pumps for fluid transfer, etc), followed by downstream processing which may include host cell deactivation and/or removal, protein concentration, and stabilization/formulation.
Solution
To succeed in producing economically viable biofuels, we need non-food crops capable of producing high quantities of biomass and industrial quantities of cellulase enzymes. Duckweed is a tiny stemless aquatic plant that grows on the surface of ponds. It has a 2-3 day doubling time making it well suited as a high productivity crop for biofuels and recombinant enzyme production. It utilizes non-arable land, accumulates low levels of lignin, and has the capacity to obtain low cost nutrients through wastewater remediation, thereby reducing the fresh water demand. This scalable biomass crop is readily recovered from the liquid phase due to its size and does not require extensive biomass pretreatment for biofuel productions. Moreover, Duckweed has a high starch content of up to 45.8% dry weight, which already makes for a promising feedstock for ethanol and butanol production. Duckweed’s demonstrated field productivity (20-46 tons/ha-yr) provide sound scientific and economic grounds for utilizing it as a model for biofuels and recombinant enzymes like cellulases.
To succeed in producing economically viable biofuels, we need non-food crops capable of producing high quantities of biomass and industrial quantities of cellulase enzymes. Duckweed is a tiny stemless aquatic plant that grows on the surface of ponds. It has a 2-3 day doubling time making it well suited as a high productivity crop for biofuels and recombinant enzyme production. It utilizes non-arable land, accumulates low levels of lignin, and has the capacity to obtain low cost nutrients through wastewater remediation, thereby reducing the fresh water demand. This scalable biomass crop is readily recovered from the liquid phase due to its size and does not require extensive biomass pretreatment for biofuel productions. Moreover, Duckweed has a high starch content of up to 45.8% dry weight, which already makes for a promising feedstock for ethanol and butanol production. Duckweed’s demonstrated field productivity (20-46 tons/ha-yr) provide sound scientific and economic grounds for utilizing it as a model for biofuels and recombinant enzymes like cellulases.
Market demand
Ethanol fuel production has steadily grown from 0.175 billion gallons per year (BGPY) in 1980 to 13 BGPY in 2010 (RFA 2011 Ethanol Industry Outlook). Since 2000, the ethanol fuel production rate has been accelerated and consistently been supported with politically long-term commitments to energy independence. However, conventional ethanol (corn ethanol) will cap out at 15 BGPY in 2015 by the Renewable Fuels Standard program (EPA Proposes New Regulations for the National Renewable Fuel Standard Program for 2010 and Beyond, 2009). Under the Renewable Fuel Standard program, mandates to reach 36 billion gallons of biofuel by 2022 have been set, and 21 billion gallons of the biofuels must be derived cellulosic biomass and non-corn-based feedstocks (EPA 2009).
Production of cellulosic ethanol is currently inhibited by the high cost of enzymes. Estimates of 15-25 kilograms of cellulase per ton of biomass (Carroll and Somerville, 2009; Taylor et al., 2008) are needed to produce 84 gallons of ethanol (Himmel et al., 1997, 1999). The scale, cost, and speed required to meet production cellulase cannot be met using current industry standard methods based on fungal fermentation. Instead, various cellulase enzymes can be produced in plants for subsequent tailoring of enzyme cocktails for specific lignocellulosic feedstocks. Meeting production targets of 21 billion gallons of advanced biofuels from cellulosic sources using biological routes would create a market demand of over 3.75 million tons of cellulase enzymes in the United States alone.
Ethanol fuel production has steadily grown from 0.175 billion gallons per year (BGPY) in 1980 to 13 BGPY in 2010 (RFA 2011 Ethanol Industry Outlook). Since 2000, the ethanol fuel production rate has been accelerated and consistently been supported with politically long-term commitments to energy independence. However, conventional ethanol (corn ethanol) will cap out at 15 BGPY in 2015 by the Renewable Fuels Standard program (EPA Proposes New Regulations for the National Renewable Fuel Standard Program for 2010 and Beyond, 2009). Under the Renewable Fuel Standard program, mandates to reach 36 billion gallons of biofuel by 2022 have been set, and 21 billion gallons of the biofuels must be derived cellulosic biomass and non-corn-based feedstocks (EPA 2009).
Production of cellulosic ethanol is currently inhibited by the high cost of enzymes. Estimates of 15-25 kilograms of cellulase per ton of biomass (Carroll and Somerville, 2009; Taylor et al., 2008) are needed to produce 84 gallons of ethanol (Himmel et al., 1997, 1999). The scale, cost, and speed required to meet production cellulase cannot be met using current industry standard methods based on fungal fermentation. Instead, various cellulase enzymes can be produced in plants for subsequent tailoring of enzyme cocktails for specific lignocellulosic feedstocks. Meeting production targets of 21 billion gallons of advanced biofuels from cellulosic sources using biological routes would create a market demand of over 3.75 million tons of cellulase enzymes in the United States alone.
Ten isolates of Lemnaceae and their geographic isolation origins selected from the Rutgers Duckweed Stock Cooperative
| Species | Collection ID* | Origin |
|---|---|---|
| Landoltia punctata | 565 | Hawaii, USA |
| Landoltia punctata | 242-7776 | Victoria, Australia |
| Lemna gibba | 38-7705 | Gujarat, India |
| Lemna valdiviana | 79-8845 | Rio de Janeiro, Brazil |
| Lemna minor | 145-8627 | Sjaelland, Denmark |
| Lemna perpusilla | 275-8539 | Virginia, USA |
| Lemna trisulca | 584-8193 | Utah, USA |
| Lemna turionifera | 364-6573 | Montana, USA |
| Spirodela polyrrhiza | 20-7003 | Louisiana, USA |
| Spirodela intermedia | 457-8979 | Cundinamarca, Colombia |
Funding: E-Team grant from VentureWell, formerly known as the National Collegiate Inventors and Innovators Alliance (NCIIA) and Provost's Undergraduate Fellowship (PUF) awarded to Derrick Hicks.
