Cushman Bioenergy Feedstocks Arid Land

Journal of Experimental Botanydoi:10.1093/jxb/erv087

Review PaPeR

Development and use of bioenergy feedstocks for semi-arid and arid lands

John C. Cushman*,1, Sarah C. Davis2, Xiaohan Yang3 and Anne M. Borland3,4

1 Department of Biochemistry & Molecular Biology, MS330, University of Nevada, Reno, NV 89557-0330, USA2 Voinovich School of Leadership and Public Affairs and Department of Environmental and Plant Biology, Ohio University, Athens, OH 45701, USA3 Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37831-6407, USA4 School of Biology, Newcastle University, Newcastle upon Tyne, NE1 7RU, UK

* To whom correspondence should be addressed. E-mail: [email protected]

Received 30 November 2014; Revised 26 January 2015; Accepted 6 February 2015

Abstract

Global climate change is predicted to increase heat, drought, and soil-drying conditions, and thereby increase crop sensitivity to water vapour pressure deficit, resulting in productivity losses. Increasing competition between agri-cultural freshwater use and municipal or industrial uses suggest that crops with greater heat and drought durabil-ity and greater water-use efficiency will be crucial for sustainable biomass production systems in the future. Agave (Agavaceae) and Opuntia (Cactaceae) represent highly water-use efficient bioenergy crops that could diversify bio-energy feedstock supply yet preserve or expand feedstock production into semi-arid, abandoned, or degraded agri-cultural lands, and reclaim drylands. Agave and Opuntia are crassulacean acid metabolism species that can achieve high water-use efficiencies and grow in water-limited areas with insufficient precipitation to support traditional C3 or C4 bioenergy crops. Both Agave and Opuntia have the potential to produce above-ground biomass rivalling that of C3 and C4 crops under optimal growing conditions. The low lignin and high amorphous cellulose contents of Agave and Opuntia lignocellulosic biomass will be less recalcitrant to deconstruction than traditional feedstocks, as confirmed by pretreatments that improve saccharification of Agave. Refined environmental productivity indices and geographi-cal information systems modelling have provided estimates of Agave and Opuntia biomass productivity and terrestrial sequestration of atmospheric CO2; however, the accuracy of such modelling efforts can be improved through the expansion of field trials in diverse geographical settings. Lastly, life cycle analysis indicates that Agave would have productivity, life cycle energy, and greenhouse gas balances comparable or superior to those of traditional bioenergy feedstocks, but would be far more water-use efficient.

Key words: Agave, arid lands, bioenergy feedstocks, ethanol, Opuntia, renewable energy, semi-arid lands.

Introduction

Global climate change, driven by heat-trapping greenhouse gas (GHG) emissions, has resulted in a 0.7–1.0°C increase in mean global temperatures over the last 50 years, and is pre-dicted to result in an additional 1.1–4.8°C rise in long-term

global mean temperatures depending on the magnitude of future GHG emissions (Field et al., 2014; Walsh et al., 2014). Terrestrial areas are projected to warm by 1.4–1.7°C more than the oceans. Global climate change has already altered

© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For permissions, please email: [email protected]

Abbreviations: CAM, crassulacean acid metabolism; CBP, consolidated bioprocessing; EPI, environmental productivity index; GHG, greenhouse gas; GIS, geo-graphical information systems; LCA, life cycle analysis; NPP, net primary productivity; pCO2, partial pressure of carbon dioxide; PEP, phosphoenolpyruvate; PEPC, phosphoenolpyruvate carboxylase; PV, photovoltaic; RUBISCO, ribulose-1, 5-bisphosphate carboxylase/oxygenase; VPD, water vapour pressure deficit; WUE, water-use efficiency.

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precipitation patterns (Treberth, 2011) and less precipita-tion is projected for regions of North and South America, South Africa, Australia, and the Mediterranean basin (Field et al., 2014). Furthermore, heating of land areas will result in increased soil-drying because of increased surface evapora-tion and evapotranspiration from plants (Dai, 2013; Sheffield et al., 2012). Ongoing soil moisture losses, driven mainly by altered precipitation patterns, have already been documented for the southern and central US, southern South America, the Middle East, central Eurasia, northern Siberia, Mongolia, north-eastern China, and western Australia (Dorigo et al., 2012). Because drier soils cannot moderate the heating effects of the sun by evaporative cooling of the soil or vegetation, further heating occurs under drier conditions (Dai, 2013; Mueller and Seneviratne, 2012).

Drier conditions linked to increased demand for ground water pumped for agricultural irrigation, particularly in the central and western US (Brown et al., 2013), result in the depletion of aquifers (Crosbie et al., 2013; Scanlon et al., 2012). Projections of a hotter and drier future suggest a need for food, forage, and bioenergy crops with greater heat and drought durability—defined as an ability to withstand longer intervals of heat and lack of precipitation than those observed for traditional C3 or C4 crops. Furthermore, agricultural crop production systems consume large percentages (>80%) of the global freshwater supply (Hoekstra and Chapagain, 2007; Hoekstra and Mekonnen, 2012). Such consumption increas-ingly competes with the municipal and industrial needs of a growing human population that is estimated to reach 9.2 bil-lion by 2050 (United Nations, 2007). Using more water-use efficient crops or improving the water-use efficiency (WUE) of agricultural crops could lead to more sustainable biomass production systems for food, forage, fodder, fibre, and biofuel needs (Gerbens-Leenes et al., 2009).

Global climate change: effects of heat and drought on crops

The warmer and drier growing environment caused by global climate change is expected to negatively impact agricultural productivity in the long term and thus decrease certainty regarding food security (Hatfield et al., 2014; Long and Ort, 2010). Negative impacts of climate change have already been linked to decreases in global Triticum spp. (wheat) and Zea mays (maize) production (Field et al., 2014; Lobell et al., 2011). Many crops [e.g. Gossypium spp. (cotton), Helianthus annuus (sunflower), wheat, rice (Oryza sativa), Solanum lyco-persicum (tomato), and maize] are projected to exhibit declin-ing yields in the future that might not be fully offset by CO2 fertilization effects (Lee et al., 2011). A secondary negative impact of increasing temperatures will be the migration of crop production areas into northern latitudes of North America and Eurasia, where soil conditions are inferior to the high-quality soils of the American prairie or Eurasian steppe that are currently under cultivation (Easterling et al., 2007; Long and Ort, 2010). However, a positive effect of such crop migration would be to permit the expansion of production

areas for cold-sensitive bioenergy crop species within the continental US.

Higher temperatures have a strong negative effect on maize yield, primarily by increasing the plant’s sensitivity to water vapour pressure deficit (VPD), which is the gradi-ent of humidity between the water vapour-saturated interior of a leaf and the drier external air (Lobell et al., 2013; Ort and Long, 2014). Increasing VPD elevates demand for soil moisture and increases transpiration rates, thereby driving additional removal of water from the soil. Increasing temper-atures and, to a lesser extent, reduced precipitation are pro-jected to have a large negative impact on maize (and wheat) yields (Lobell et al., 2013). Crop yield is most sensitive to VPD because it is directly related to water loss from leaves via water vapour escape through stomata. Thus, although corn yields have increased in the US in recent decades, these increases have been accompanied by increased drought sensi-tivity (Lobell et al., 2014).

Increasing temperatures can also lead to stomatal closure, which in turn reduces transpiration and CO2 assimilation. Concurrently, photosynthesis is impaired by the increasing leaf temperatures caused by decreased latent heat loss (Long and Ort, 2010). The elevated atmospheric [CO2] predicted for 2050 will likely decrease evapotranspiration rates, increase WUE, and thus reduce crop water demand in soybean and maize (Bernacchi et al., 2007; Hussain et al., 2013). However, even with such improved WUE, the water needed for these crops to produce the yields required by 2050 will probably remain above that supplied by current average precipitation rates in the US corn belt (Ort and Long, 2014). Traditional bioenergy crops, such as Saccharum officinarum (sugar cane) and maize, carry large water footprints for the production of sweeteners and bioethanol (Gerbens-Leenes and Hoekstra, 2012; Gerbens-Leenes et al., 2009). More than 30% of the US maize crop is converted to ethanol (Schill, 2013; USDA, 2010). Clearly, strategies for moving towards increased reli-ance on crops that are more water-use efficient and drought durable will be necessary if current and future CO2 emissions are not curtailed.

Dryland degradation and agriculture

Approximately 50% of the global land area is considered arid, semi-arid, or dry sub-humid (Zika and Erb, 2009). Such dry-lands are vulnerable to soil and vegetation degradation from various effects, such as soil erosion, compaction, crusting, salinization, and nutrient depletion, resulting in 4–10% losses in their net primary productivity (NPP) potential each year, or approximately 2% of the global terrestrial NPP (Zika and Erb, 2009). Dryland degradation, also referred to as desertifi-cation, results in huge economic losses and directly threatens the well-being of more than 20 million human inhabitants of such areas, particularly smallholder farmers (Pimentel et al., 1995; United Nations Environment Programme, 2012). Increasing such NPP by reclaiming drylands and increasing yields on land suitable for agricultural use can be an environ-mentally favourable option (Daily, 1995). Such reclamation

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often requires direct human intervention and the implementa-tion of sustainable farming practices, can take many years to complete, and is invariably expensive (Daily, 1995; Pimentel et al., 1995). However, implementation of such reclamation efforts will be critical to increasing the NPP of such dryland regions for food, forage, fodder, fibre, and biofuel feedstock production. Reclamation efforts that utilize highly water-use efficient crop species that are heat and drought durable are likely to be most successful.

Several target species have been identified for combating desertification, including Atriplex spp., Opuntia spp. (Mulas and Mulas, 2004; Nefzaoui and Ben Salem, 2001), and Agave spp. (Schwilch et al., 2014). Opuntia spp. can also serve as drought ‘insurance’ in arid and semi-arid rangelands as reserve, seasonal, or year-round forages (Guevara et al., 2009). While these species provide important ecosystem ser-vices, such as improving soil properties and preventing soil erosion, they also have the potential to rehabilitate degraded dryland regions (Le Houérou, 1996; Rodrígueza et al., 2006) and to provide dietary resources for wildlife (Russell and Felker, 1987). Given this potential utility in the reclamation of dryland regions, these same species have also attracted attention as feedstocks for bioenergy production because of their high WUE and drought tolerance (Borland et al., 2009; Chávez-Guerrero and Hinojosa, 2010; Li et al., 2014). With the gradual depletion of fossil fuels, the demand for renewable biomass-based feedstocks as sources of lignocel-lulosic biofuels is expected to increase in the future (National Research Council, 2011). Such increased demand might be met through the cultivation of highly water-use efficient plants with the capacity for high biomass production, such as Agave spp. and Opuntia spp. If properly managed, such large-scale cultivation projects might also be linked to effective dryland reclamation efforts to provide both bioenergy and ecosystem services in the context of the development of novel ‘emerging ecosystems’ that are the result of human interven-tions (Hobbs et al., 2009).

Diversifying the bioenergy feedstock portfolio

Most biomass feedstocks currently used for biofuels are also consumed for food production (Ragauskas et al., 2006; Somerville et al., 2010). The production of crops used for both biofuels and food can be expanded, in part, by the devel-opment of drought-tolerant bioenergy feedstocks. These can be grown in an environmentally responsible manner on aban-doned agricultural lands where traditional food crops are typically not cultivated, with an estimated mean productivity of 4.3 Mg (tons) ha–1 y–1 (Campbell et al., 2008; Owen and Griffiths, 2014; Somerville et al., 2010). Such land areas are typically abandoned or considered marginal because they are unmanaged, are of low productivity because of edaphic or climatic limitations, are vulnerable to erosion or have under-gone degradation, are environmentally sensitive, or are often reserved for conservation. While the global energy production potential for such lands is less than 10% of current primary

energy demand for most developed nations, their potential productivity is greater than the energy demands of many developing nations (Campbell et al., 2008).

In addition to expanding feedstock production for food and biofuel uses, one of the major challenges for bioenergy crops is the ability to increase above-ground biomass production with-out increasing water use (Karp and Shield, 2008). Indeed, water availability is the major factor that constrains the cultivation of bioenergy crops (Dauber et al., 2012). While mainstream bioenergy crops, such as perennial grasses [e.g. maize, sugar cane, Panicum virgatum (switchgrass), Miscanthus] and short-rotation forestry trees [e.g. Populus (poplar) and Salix (willow)], have received considerable attention (Karp and Shield, 2008), relatively less consideration has been granted to more water-use efficient species. One potential opportunity for the production of biomass feedstocks on water-limited areas is to use various cultivated crassulacean acid metabolism (CAM) species from the genera Agave (Agavaceae) and Opuntia (Cactaceae, prickly pear cactus), which have growth characteristics that allow these species to operate at near-maximum productivity with relatively low water requirements (Borland et al., 2009; Borland et al., 2011). Furthermore, Agave and Opuntia are considered low-input perennial crops, similar to Miscanthus spp. and switch-grass, that exhibit lower GHG emissions and nitrogen leaching during production than does maize (Davis et al., 2012).

Although the interactions between feedstock and land use are complex, perennial lignocellulosic feedstocks could pro-vide greater ecosystem services (e.g. habitat, biodiversity), carbon sequestration, and soil and water quality improve-ment than annual grain crops (Dale et al., 2010; Dale et al., 2011). High-yielding perennial feedstocks that represent a net addition of biomass grown with low nitrogen inputs are preferable to the diversion of existing crop production into biofuels (Smith and Searchinger, 2012). The low CO2 emis-sions and fertilization requirements of Agave combined with its high WUE have stimulated considerable interest in its use as a sustainable bioenergy feedstock (Davis et al., 2011; Holtum et al., 2011; Somerville et al., 2010; Yan et al., 2011). CAM crops can serve to reduce competition for existing land resources because they can be grown in areas where precipi-tation amount or frequency is insufficient to support tradi-tional food and bioenergy crops (Davis et al., 2011; Somerville et al., 2010). Under irrigation, Agave and Opuntia require up to 5-fold less water input than C3 or C4 bioenergy feedstocks, while maintaining above-ground biomass productivity that rivals that of C3 and C4 photosynthesis crops (Borland et al., 2009; Nobel, 1996) (see below). While CAM feedstocks offer many theoretical advantages over conventional biofuel feed-stocks, CAM biomass feedstocks are not exempt from some of the criticisms made of C3 and C4 bioenergy crops. These include potentially negative environmental impacts, such as soil erosion, deforestation, loss of biodiversity, nitrogen ferti-lizer pollution of waterways, herbicide and pesticide use, water use, and the release of GHGs associated with the production and combustion of liquid fuels (Pimentel et al., 1995; Pimentel et al., 2008; Pimentel et al., 2009). Thus, a strict regulatory framework that avoids such negative environmental impacts and provides tangible societal benefits is necessary.



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CAM engenders favourable attributes for arid land bioenergy feedstocks

At a metabolic level, the desirable traits of high produc-tivity and water conservation in Agave and Opuntia can be attributed to the temporal separation of C3 and C4 car-boxylation processes that define CAM. In essence, CAM is expressed in a background of ribulose-1, 5-bisphosphate carboxylase/oxygenase (RUBISCO)-mediated CO2 fixation via the engagement of nocturnal CO2 uptake catalysed by phosphoenolpyruvate carboxylase (PEPC) and subsequent daytime decarboxylation processes (Fig. 1). The interplay and metabolic coordination of carboxylation processes are described as a four-phase model that encompasses diurnal and nocturnal metabolism (Osmond, 1978). Phase I describes the nocturnal opening of stomata when evapo-transpiration rates are low and atmospheric plus respira-tory CO2 is fixed in the cytosol by PEPC. The 3-C substrate phosphoenolpyruvate (PEP) is formed from the glycolytic breakdown of carbohydrates, and the final 4-C product malic acid is then stored in a large central vacuole. At the start of the day, phase II represents a surge in the rate of net CO2 uptake that may be mediated via both PEPC and RUBISCO. During phase III, malate exits the vacuole and is decarboxylated through the single or combined action of three enzymes (depending on plant species): nicotinamide adenine dinucleotide phosphate-malic enzyme, nicotinamide

adenine dinucleotide-malic enzyme, and PEP carboxykin-ase. In addition to the 3-C products (PEP or pyruvate), CO2 is released at a high internal partial pressure (pCO2). This is accompanied by stomatal closure and transpirational water loss is then curtailed. The high pCO2 generated via decarboxylation in phase III also suppresses photorespira-tion for much of the day. While the recovery of carbohy-drate via gluconeogenesis in phase III imposes additional energetic costs (estimated to be approximately 10%) on the CAM pathway (Winter and Smith, 1996), the production of substrate for subsequent nocturnal carboxylation and parti-tioning for growth is ensured.

During the latter part of the day (phase IV), when malate reserves have been exhausted, the stomata may re-open and atmospheric CO2 may be fixed directly by RUBISCO. Although the manifestation of the four CAM phases is highly plastic and dependent upon prevailing environmental condi-tions, A. tequilana has been shown to exhibit all four phases of CAM as described above (Nobel and Valenzuela, 1987), whereas O. ficus-indica (L.) Miller has been shown to exhibit atmospheric CO2 exchange only during phase I (Nobel and Hartsock, 1983). Overall, the CAM pathway is efficient as reflected by productivity values of certain CAM species with greater productivity than C3 species, but sometimes less effi-cient than C4 species (Nobel, 1991a).

The responses of crops to increasing atmospheric [CO2], or the so called CO2 fertilization effect, have been well studied in C3 and C4 species (Attavanich and McCarl, 2014; Sakurai et al., 2014). In comparison, data describing the physiological impacts of atmospheric [CO2] on CAM plants are limited, but it would appear that the productivity of CAM species under elevated atmospheric [CO2] may be expected to either increase or be maintained at levels comparable to those under ambi-ent [CO2], but with reduced inputs of water (Ceusters and Borland, 2010). Drennan and Nobel (2000) demonstrated an average of 1% increase in daily net CO2 uptake per 10 μmol mol–1 increase in atmospheric [CO2] (up to 750 μmol mol–1) for a range of desert CAM succulents, alongside increases in temperature and drought (Drennan and Nobel, 2000). Similarly, dry biomass production of O. ficus-indica was stim-ulated by 40% over a 1-year period of exposure to 750 μmol mol–1 [CO2] (Nobel and Israel, 1994). These results suggest that nocturnal uptake of CO2 seems to be far from saturated at the current atmospheric CO2 concentration for a number of CAM species. However, such increased biomass produc-tion might also be an indirect effect of greater soil moisture retention from reduced evaporative water losses. Future stud-ies conducted under elevated [CO2] conditions should include an evaluation of soil moisture status to explore this possi-bility. Sustained increases in productivity may be facilitated by the high succulence of CAM species such as Agave and Opuntia, which can accommodate large increases in chloren-chyma thickness and accumulation of photosynthate without feedback inhibition of photosynthesis (Nobel, 2000). Tight cell packing and low amounts of intercellular air space vol-umes are typical of succulent photosynthetic CAM leaves, cladodes, and stems, which impose significant constraints on the internal diffusive supply of CO2-. This is in contrast to the

Fig. 1. A simplified view of crassulacean acid metabolism (CAM). Stomata open at night allowing atmospheric CO2 to enter the cell and together with CO2 generated internally from respiration are converted to HCO3

– which is captured by cytosolic phosphoenolpyruvate carboxylase (PEPC). This leads to the formation of malate (MAL), which undergoes protonation and is stored in the vacuole as malic acid. Stomata are closed for all or part of the subsequent day and malic acid efflux from the vacuole and decarboxylation release CO2, which is re-fixed by plastidic ribulose-1,5-bisphosphate carboxylase/oxygenase (RUBISCO) through the Calvin–Benson cycle. Triose-P serves as substrate for the gluconeogenic pathway during which carbohydrates are regenerated, which may be used for growth and maintenance or stored as starch or other carbohydrates for subsequent nocturnal mobilization and provision of PEP. Modified from Borland and Taybi. Synchronization of metabolic processes in plants with Crassulacean acid metabolism. Journal of Experimental Botany. 2004. 55, 1255–1265, by permission of the Society of Experimental Biology.



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‘regenerative’ phase III, when internal CO2 concentrations are likely to saturate RUBISCO (Maxwell et al., 1997; Maxwell et al., 1998; Nelson and Sage, 2008). At night, the carbon iso-tope signals associated with PEPC-mediated uptake of CO2 indicated diffusion limitation in the CAM species Kalanchoë daigremontiana (Griffiths et al., 2007). Additionally, meso-phyll conductance derived during phase IV of gas exchange in the light is inversely related to the degree of leaf succu-lence in several CAM species, with internal CO2 supply at RUBISCO potentially as low as 110 μmol mol–1 (Griffiths et al., 1999). However, the relationship between leaf thick-ness or succulence and CO2 diffusion is not always intuitive. A recent comparison of CAM dynamics in the highly suc-culent leaves of both K. daigremontiana and A. tequilana reported a higher stomatal density and a more aerated chlor-enchyma in A. tequilana, which appears to be integral to the higher recorded values of instantaneous and integrated net CO2 uptake in this species compared to K. daigremontiana (Owen and Griffiths, 2013). The extent to which elevated CO2 stimulates diurnal and nocturnal CO2 uptake in CAM species by overcoming diffusional constraints needs to be established for different CAM species.

Physiological attributes of CAM

Maximum instantaneous rates of net photosynthesis for Agave and Opuntia species of 23–34 μmol CO2 m

–2 s–1 have been measured under field conditions, often at dusk during the transition between phases IV and I, but also well into the dark period (Nobel, 1991a; Nobel, García-Moya et al., 1992; Nobel and Hartsock, 1983, 1984; Nobel et al., 2002; Pimienta-Barrios et al., 1991). While these photosynthetic rates are among the highest recorded for any CAM species, they are significantly lower than the maximum rates for C3

and particularly C4 species (Table 1, modified from data pre-sented by Nobel, 1991a and Borland et al., 2009), and likely reflect the diffusive resistance to CO2 uptake that is presented by the thick, succulent leaves of CAM plants in general and Agave and Opuntia species in particular (Nelson and Sage, 2008). However, when net CO2 uptake is integrated over 24 h, which is more meaningful in terms of plant productivity, the maximal measured values for Agave and Opuntia fall well within those predicted for C3 and approach those predicted for C4 plants, with the benefit of substantial savings in water-use for the CAM species (Table 1). In addition, Agave and Opuntia species typically display high shoot to root ratios (up to 10:1) such that the entire shoot is photosynthetic in both leaf (Agave) and stem (Opuntia) (Borland et al., 2009). So, while crop water demands for some Agave and Opuntia species average only 16% of those for C3 crops and 28% for C4 crops, above-ground biomass productivities are compara-ble or may even exceed those reported for C3 and C4 species (Borland et al., 2009; Nobel, 1991a). Additional desirable attributes of candidate CAM feedstocks include tolerance of high temperatures, UV-B radiation, and drought condi-tions by virtue of their typically waxy cuticles and ability to rapidly take up and store water in above-ground succulent tissues (Borland et al., 2009). Moreover, mature roots of Agave and Opuntia display rectifier-like hydraulic conduct-ance responses to fluctuating plant water potential, which enable plants to avoid plant–soil water losses during periods of drought (Nobel, 1988).

Traditional uses and products of Agave

Agave spp. (Agavaceae) are native to the Americas but have been introduced throughout the world for commercial use, including in Australia, Brazil, Tanzania, Kenya, Madagascar,

Table 1. A comparison of physiological traits related to photosynthetic carbon gain and water use for potential CAM bioenergy feedstocks, as exemplified by Agave and Opuntia, in relation to C3 and C4 species

Physiological trait Photosynthetic pathway

CAM C3 C4

Maximum instantaneous rate net CO2 uptake (μmol m–2 s–1) 34 (Agave mapisaga)a 48c 64c

34 (A. tequilana)b

29 (A. salmiana)a

23 (Opuntia ficus-indica)e,f

Maximum integrated net 24 h CO2 uptake (mmol m–2 day–1) 1170 (A. mapisaga)c 930g–1244h 1240g–1659h

1081 (O. ficus-indica)e,f

Average instantaneous WUE (mmol CO2 mol–1 H2O)d 7 1 1.5Integrated water loss over 24 h (mol H2O m–2 day–1)i 154–167 930–1244 827–1106

aNobel et al., 1992; bPimienta-Barrios et al., 1991; cNobel, 1991a; dBorland et al., 2009; eNobel and Hartsock, 1983; fNobel and Hartsock, 1984, gCalculated for C3 and C4 on the assumption that net CO2 uptake rate increases linearly from zero at dawn to the maximum instantaneous rate of net CO2 uptake (i.e. 48 or 64 μmol m–2 s–1for C3 and C4) at noon then decreases linearly back to zero at dusk over a 12 h photoperiod. Daytime net CO2 uptake would be 1033 and 1378 mmol m–2 for C3 and C4. The integrated net 24 h CO2 uptake is obtained by subtracting CO2 respired at night from daytime net CO2 uptake with the assumption that 10% of C acquired during the day is respired at night (i.e. 103 and 139 mmol m–2 for C3 and C4). Assumptions for calculation adapted from Nobel (1991). hCalculated for C3 and C4 on the assumption that the sinusoidal variation in daily photosynthetic photon flux density can be represented by a square wave with a magnitude of 2000 μmol m–2 s–1 (maximum photosynthetic photon flux density) for 8 h per day. Daytime net CO2 uptake would be 1382 and 1843 mmol m–2 for C3 and C4. Nocturnal respired CO2 (10% of daytime CO2 uptake) is subtracted from the values of daytime net CO2 uptake. iCalculated by dividing the integrated 24 h uptake of CO2 by the values presented for average water-use efficiency.



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Mexico, China, and across the Caribbean and Mediterranean regions (Davis et al., 2011; Yan et al., 2011). The majority of the more than 200 species of Agave can be found in Mexico (Garcia-Mendoza, 2007), and historically have served as sources of food, fibre, shelter, beverages, and a wide range of artisanal specialty products (Colunga-García Marín et al., 2007; Escamilla-Treviño, 2012). Mexico is the domi-nant commercial producer of A. tequilana, which is grown for fermentation and double distillation into tequila because of its high content of water-soluble sugars (Valenzuela, 2011). However, many Agave spp. (e.g. A. angustifolia, A. esperrima, A. weberi, A. potatorum, A. salmiana) are used for the produc-tion of other beverages including the sweet drink aguamiel (honey water), nectar or syrup sweeteners, fermented pulque, and the single distillate mezcal (Escamilla-Treviño, 2012; Nobel, 2010; Núñez et al., 2011). Many countries, including Colombia, Cuba, Guatemala, Ecuador, Mexico, Nicaragua, El Salvador, and the Philippines, are major commercial pro-ducers of A. fourcroydes (henequen) and other species (FAO, 2013). A. fourcroydes and A. lechuguilla are grown for fibres used in cordage and textiles production and also for sugars necessary for alcoholic beverage production (Martinez-Torres et al., 2011; Núñez et al., 2011; Valenzuela, 2011). A. sisalana is grown commercially for fibre (sisal) in many countries, including Brazil, China, Kenya, Madagascar, Mexico, and Tanzania (FAO, 2013). Following extraction of sugars for fermentation, residual biomass from harvested leaves, waste fibre, and bagasse from juice extraction can be used for com-post, animal feed, and fibreboard construction, and as a solid, combustible fuel (Chávez-Guerrero, 2013; Chávez-Guerrero and Hinojosa, 2010; Iñiguez-Covarrubias, Díaz-Teres et al., 2001; Iñiguez-Covarrubias, Lange et al., 2001). Important by-products of the fibre industry derived from A. sisalana and A. americana include the steroidal saponins, tigogenin and hecogenin, which are extracted from waste residues after fibre extraction and used as starting materials for the synthesis of steroidal hormones (Santos and Branco, 2014). The major uses of Agave spp. are summarized graphically in Fig. 2A.

Traditional uses and products of Opuntia

Opuntia spp. (Cactaceae) originated in the Americas with the centre of diversity in Mexico (Chávez-Moreno et al., 2009; DeFelice, 2004; Griffith, 2004), but various species have been introduced worldwide, with major production in the Mediterranean basin [Algeria, Italy (Sicily), and other north-ern African nations], Argentina, Bolivia, Brazil, Chile, Israel, Mexico, South Africa, and the USA (Basile, 2001; Inglese et al., 2002; Le Houérou, 1996; Nobel, 1994). Opuntia spp. have historically been cultivated primarily for commercial fod-der and forage in semi-arid regions worldwide (Le Houérou, 1996; Nobel, 1988; Russell and Felker, 1987). However, the tender, young cladodes (nopalitos) and fruits (tunas) are also consumed by humans, primarily in Mexico, South America, south-western USA, and throughout the Mediterranean basin (Hegwood, 1994; Iñiguez-Covarrubias, Lange et al., 2001; Le Houérou, 1996; Nobel, 1994; Shedbalkar et al., 2010). The

young cladodes and fruits are also dried and sold as dietary supplements (Bensadón et al., 2010; Sáenz et al., 2002), in cosmetic formulations (Sáenz et al., 2002), and for medicinal use (de los Angeles Aguilera-Barreiro et al., 2013; Feugang et al., 2006; Stintzing and Carle, 2005). Opuntia fruits are used for human food in both the Mediterranean Basin and the Americas, and as animal fodder worldwide (Le Houérou, 1996). Fruits can be consumed fresh, canned, or frozen, or made into a variety of processed foods, including jams, jellies, sauces, marmalades, fruit sheets or rolls, powders, candies, syrups, purées, juices, vitamin water, and liquor. Extracts of the fruit can be used as a natural food colouring because of their high betalain pigment content, and as a sweetener owing to the high sugar content (>50% on a dry matter basis) of the fruit syrup (Inglese et al., 2002; Moßhammer et al., 2006; Sáenz et al., 1998; Stintzing and Carle, 2005; Stintzing et al., 2001). The fruits can also be fermented into a low-alcohol beverage called colonche (Sáenz, 2000). The major uses of Opuntia spp. are summarized graphically in Fig. 2B.

Agave biomass production

Global production of Agave peaked in the 1960s when demand for strong natural fibres was high (FAO, 2012). More than 100 M ha are estimated to be under commercial cultivation for production of Agave-based fibre (Nobel, 2010). Sisal fibres were considered ideal for making rope, and A. sisalana has been prized for its long leaves that yield similarly long, continu-ous fibres. However, global fibre production has continued to decline over the last three decades, with global production sta-bilizing at about 20 Mg y–1 (FAO, 2012). The older, outer leaves are harvested annually once the plant matures (circa 4 years). Although there is an establishment period, the crop acts as a perennial in that it can be harvested annually without replant-ing. Unlike some other perennial crops, only a portion of the above-ground canopy can be harvested in a given year, and harvesting is usually accomplished manually so that the oldest leaves can be selectively trimmed from the base of the stem.

More than 130 M ha are estimated to be in commer-cial production for Agave-based beverages (Nobel, 2010). A. tequilana, grown commercially for tequila, is harvested destructively on a 5–12 year rotation cycle (Cedeño, 1995; Núñez et al., 2011). These plants are valued for the high per-centage of soluble carbohydrates by fresh weight (16–28%), which is concentrated mostly in the main stem or piña (Núñez et al., 2011; Yan et al., 2011). The leaves are typically trimmed from the stem in the field; skilled labourers accomplish this task using a coa de jima (a specialized axe). The leaves are usually left in the field, burned, or composted. Because of increasing tequila production, there is increasing interest in utilizing these harvest residues and bagasse for biofuel pro-duction (Núñez et al., 2011; Valenzuela, 2011).

Agave plants are typically propagated asexually from bul-bils, which are offsets from a mother plant. In the tequila industry, micropropagation is now also used (Ramírez-Malagón et al., 2008; Robert et al., 2006). The plantlets are grown in culture and then transferred to a greenhouse for



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1–2 years prior to planting in the field at a typical density of 2000–4000 plants ha–1 for tequila production (Cedeño, 1995).

Biomass productivity of Agave varies considerably with agricultural practices and water inputs, with average annual above-ground dry-weight productivity values for various Agave species ranging from <1 to 34 Mg ha–1 y–1 under ambi-ent precipitation conditions (Davis et al., 2011; Escamilla-Treviño, 2012; Nobel, 1988, 1994; Somerville et al., 2010; Yan et al., 2011). Under cultivated conditions with supple-mental irrigation, average annual dry-weight productivi-ties of Agave spp. can reach as high as 38–44 Mg ha–1 y–1 in

Mexico (García-Moya et al., 2011; Nobel, 1988, 1991a, 1994, 1996; Nobel, García-Moya et al., 1992). More information is needed from field trials to model the biomass production associated with these water-wise feedstocks (Holtum and Chambers, 2010; Holtum et al., 2011; Nair et al., 2012).

Opuntia biomass production

In Mexico, about 10,000 ha y–1 of Opuntia are cultivated (Hegwood, 1994; Shedbalkar et al., 2010), resulting in the pro-duction of 600 000 Mg y–1 (fresh weight) (Sáenz et al., 2002).

Fig. 2. Uses and products derived from (A) Agave spp. and (B) Opuntia spp. Agave image © SSSCCC. Opuntia image © andylin. Mescal image © Patricia Hofmeester. Tequila image © Evageny Karandaev. Fuel pump image © Fejas. Cordage image © Lubava. Textile image © homydesign. Fiberboard image © Voyagerix. Fuel brick image © African Studio. Vial image © Nikolay Litov. Cladode image © Scisetti Alfio. Fruit image © Evikka. Jam jar image © Zern Liew. Jelly candy image © de2marco. Food coloring image © sarininka. Cosmetics image © Neomov. Dietary supplements image © PeoGeo.



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Global cultivated production of Opuntia fruit is estimated to exceed 400 000 Mg y–1 (fresh weight) (Inglese et al., 1995; Nobel, 1994). Biomass productivity of Opuntia also varies with agricul-tural practices and water inputs. Under arid (rain-fed) conditions with annual precipitation of 200–400 mm, average annual above-ground dry-weight productivity for Opuntia species varies from 3 to 15 Mg (dry matter) ha–1 y–1 (Le Houérou, 1996; Nobel, 1991b; Nobel et al., 1987). Under semi-arid (rain-fed) conditions with annual precipitation of 400–600 mm, average annual productiv-ity increases to 9–15 Mg (dry matter) ha–1 y–1 or more under extensively managed conditions (Le Houérou, 1996). A more recent estimate of biomass productivity based on geographical information systems (GIS) modelling estimated that with annual precipitation of 250 mm or 500 mm, average annual productivi-ties of 4.2 or 9.4 Mg (dry matter) ha–1 y–1, respectively, were possible in the Almeria province, Spain (Sánchez et al., 2012). To validate such GIS modelling approaches, more detailed sampling studies will be needed in the future from field trials at multiple geographical locations. However, under well-irrigated conditions, average annual dry matter productivities in the range of 40–50 Mg ha–1 y–1 have been reported under intensive culti-vation and fertilization conditions in Mexico and Chile (Garcia de Cortazar and Nobel, 1992; Garcia de Cortázar and Nobel, 1991; García-Moya et al., 2011; Nobel, 1991a, 1996; Nobel, García-Moya et al., 1992). Such biomass production rates are comparable with those of other bioenergy feedstocks, such as maize, sugarcane, switchgrass, and poplar (Borland et al., 2009; Davis et al., 2011; Somerville et al., 2010). Opuntia plants are typically propagated asexually by dividing the plant into indi-vidual cladodes and replanting them; however, propagation by seed is also possible, as fruits are produced within 1–4 years and each fruit produces large numbers of seeds (Mondragon-Jacobo and Bordelon, 1996; Nefzaoui and Ben Salem, 2001).

Agave biomass characteristics and composition

Water-soluble carbohydrates are found in high concentrations in the plant tissue of Agave spp. In the case of A. tequilana, these soluble carbohydrates are concentrated in the piña just before the flowering stage of the life cycle. Varieties like A. tequilana Weber

var. azul (blue agave) have been bred to have shortened flower-ing times of 5–6 years, but other Agave species will flower only after decades. Tissue composition thus differs among varieties and changes over the lifetime of the plants (Arrizón et al., 2010).

The sugar found in greatest abundance in agave plant tissues is fructose, which is easily fermented by yeast (e.g. Saccharomyces cerevisiae, Kluyveromyces marxianus). The total sugar content of the Agave piña can range from 12% to 28% (fresh weight) (Yan et al., 2011). Concentrations of fructans in the piña range from 36% to 73% of tissue at maturity depending on the species (Davis et al., 2011). The succulent leaves also have high amounts of extractable compounds that include mainly fructose, along with glucose and sucrose (Li et al., 2012), but also include com-pounds such as fructans, which are suspected to contribute to survival during long periods of drought, and other compounds that afford protection from pathogens. Agave leaves can represent more than 30% of the plant’s total biomass and contain 13–16% of the total reducing sugar, and the bagasse can represent 40% of the total wet weight of the milled Agave, and contain 5–20% total reducing sugar (Iñiguez-Covarrubias, Díaz-Teres et al., 2001; Iñiguez-Covarrubias, Lange et al., 2001; Valenzuela, 2011; Yan et al., 2011).

In addition to the composition of extractable sugars, recent interest in the use of Agave spp. as lignocellulosic biofuel feed-stocks has resulted in a growing number of studies examining the composition of its residual biomass (Li et al., 2012; Núñez et al., 2011; Perez-Pimienta et al., 2013). Numerous reports have estimated the structural carbohydrate composition from vari-ous Agave spp. (Table 2). Although the estimates vary consider-ably, likely due to differences among species, growth conditions and age of the plants, and analytical methods used, overall these results show that Agave spp. have relatively low lignin content (4.9–19.3%) relative to some traditional lignocellulosic feedstocks, which range from 9% to 32% lignin (Ragauskas et al., 2014). Lignin is the major cause of recalcitrance to hydrolysis compared with other plant cell wall biopolymers (Trajano et al., 2013; Zeng et al., 2014). Thus, a low lignin content can be beneficial for over-coming recalcitrance to cellulose degradation and improving sac-charification (Ragauskas et al., 2014). In addition to a low lignin content, A. americana, A. salmiana, and A. tequilana have a low crystalline cellulose content and high paracrystalline (disordered) cellulose content relative to woody biomass feedstocks such as

Table 2. Comparison of biomass composition of different Agave feedstocks

Agave ssp. (fraction) Structural component (dry weight %)

Solubles (extractives) Cellulose Hemicellulose Lignin Ash Citation

A. americana (Bagasse) 14.5 n/a n/a 8.2 7.4 Li et al., 2012A. fourcroydes (Leaf fibre) 3.6 77.6 5–7 13.1 n/a Vieira et al., 2002A. lechugulla (Leaf fibre) 2–4 79.8 3–6 15.3 n/a Vieira et al., 2002A. salmiana (Bagasse) n/a 47.3 12.8 10.1 n/a Garcia-Reyes and Rangel-Mendez, 2009A. salmiana (Bagasse) 17.9 n/a n/a 9.8 6.1 Li et al., 2012A. sisalana (Leaf fibre) n/a 77.3–84.4 6.9–10.3 7.4–11.4 n/a Vieira et al., 2002; Martin et al., 2009A. tequilana (Bagasse) 14 64.8 5.1 15.9 1.0 Iñiguez-Covarrubias, Díaz-Teres et al., 2001A. tequilana (Bagasse) n/a 68.4 15.7 4.9 n/a Mylsamy and Rajendran, 2010A. tequilana (Bagasse) 17.4 n/a n/a 11.9 6.4 Li et al., 2012A. tequilana (Bagasse) n/a n/a n/a 19.3 4.4 Perez-Pimienta et al., 2013A. tequilana (Bagasse) 29.0 26.0 22.8 13.8 6.0 Yang et al., 2015



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Pinus taeda (loblolly pine), poplar, and switchgrass, suggesting their lignocellulosic biomass will be more readily hydrolysable than that of other feedstocks (Li et al., 2012; Yang et al., 2015). Lastly, fresh Agave piñas and leaves have high water contents that range from 60% to 70% and 78% to 89%, respectively (Yan et al., 2011). Such high water content could help to reduce water inputs needed for downstream processing of lignocellulosic bio-mass during mechanical, physiochemical, or enzymatic hydrolysis pretreatment steps.

Opuntia biomass characteristics and composition

In contrast to Agave spp., the cladodes of Opuntia spp. contain relatively low amounts of water-soluble carbohy-drates. The cladodes comprise 3–7% carbohydrate (fresh weight) (Stintzing and Carle, 2005) with the major non-fibrous storage carbohydrate stored as pectin or mucilage composed mostly of arabinose, xylose, galactose, rham-nose, and galacturonic acid (Nobel, Cavelier et al., 1992). Opuntia fruit contains 6–14% soluble sugar (fresh weight) (Duru and Turker, 2005; Parish and Felker, 1997) com-prised largely of glucose and fructose (El Kossori et al., 1998). However, Opuntia spp. cladodes contain an even lower range of lignin content (0.01–16%) than estimated for Agave spp. (Table 3). In addition to a low lignin con-tent, cladode tissues of O. ficus-indica have a lower crys-talline cellulose content and a higher amorphous and paracrystalline (disordered) cellulose content than those of Agave spp., suggesting its lignocellulosic biomass would be even more readily hydrolysable into fermentable sugars than would biomass from traditional herbaceous or woody feedstocks (Yang et al., 2015). O. ficus-indica cladodes have relatively high ash content (8.5–23.7%) (Table 3) due to the accumulation of inorganic ions (e.g. Ca2+, K+, Mg+, Na+) and salts, such as Ca2+-oxalate crystals (Contreras-Padilla et al., 2011). Although the ash contents of A. tequi-lana and O. ficus-indica are higher than those present in woody biomass feedstocks, which range from 0.2% to 3% (Demirbas, 2004; Zhu and Pan, 2010), they are in the range of those found in herbaceous bioenergy feedstocks, such as Arundo, Cynara, Miscanthus, Panicum, Phalaris, Saccharum, Sorghum, Triticum, and Zea, which range from

5% to 11% (Bakker and Elbersen, 2005; Demirbas, 2004; Monti et al., 2008; Zhu and Pan, 2010). High ash contents can potentially cause corrosion and slagging problems during bioprocessing, although this might be overcome by washing the biomass with dilute acid, which can effec-tively remove inorganic constituents (Runge et al., 2013). Fresh Opuntia cladodes and fruits have very high water contents that range from 88% to 95% and 84% to 90%, respectively (Feugang et al., 2006; Stintzing and Carle, 2005; Stintzing et al., 2001). Similar to Agave, this high water content could help to reduce water inputs needed for downstream processing of Opuntia lignocellulosic biomass during mechanical, physiochemical, or enzymatic hydroly-sis treatment steps.

Biomass conversion and biofuel production

Agave spp. are currently receiving attention as potential biofuel feedstocks because of their drought tolerance, tis-sue composition, and history in commercial production (Borland et al., 2009; Davis et al., 2011; Somerville et al., 2010). Following the tequila industry as a model, it is clear that Agave carries great potential to be developed for the commercial-scale production of alcohol-based fuels like eth-anol (Davis et al., 2011; Davis et al., 2014). The theoretical maximum ethanol yield from Agave biomass (363 L Mg–1) is comparable to that of two major non-CAM biofuel crops, poplar (438 L Mg–1) and switchgrass (354 L Mg–1), on a dry matter basis (Li et al., 2012). Although the scope of etha-nol production from Opuntia has historically been far more limited than from Agave, many attempts to convert Opuntia cladodes or fruit to ethanol have been reported, dating back to the 19th century (Casas and Barbera, 2002) to the present (Sánchez et al., 2012).

Recent life cycle analysis (LCA) of Agave for bioenergy con-cluded that both energy outputs and GHG offsets are greater in an Agave bioenergy production system than in either corn or switchgrass bioenergy production systems (Yan et al., 2011). A LCA should be developed for Opuntia and the existing LCA for Agave should be improved upon to allow revised inferences to be made as new data are collected or as new LCA models are developed. Economically, however, there are substantial costs that need to be addressed before Agave can be produced

Table 3. Comparison of biomass composition of different Opuntia feedstocks.

Opuntia spp. (fraction) Structural component (dry weight %)

Solubles (extractives) Cellulose Hemicellulose Lignin Ash Citation

O. ficus-indica (Fruit pulp) 58.3b 14.2 15.5 0.01 8.5 El Kossori et al., 1998; Li et al., 2012O. ficus-indica (Fruit skin) 27.6 b 71.4 20.8 0.06 12.1 Li et al., 2012; El Kossori et al., 1998Opuntia spp. (Cladode) n/a 21.6 n/a 3.6 19.6 Malainine et al., 2003Opuntia spp.a (Cladode) n/a 6.8 9.1 11.8 22.5 Mciteka, 2008O. ficus-indica (Cladode) 17.7 n/a n/a 16 n/a Ginestra et al., 2009O. ficus-indica (Cladode) 24.3 13.5 n/a 7.9 16.8 Kuloyo, 2012O. ficus-indica (Cladode) 25 13.1 18.5 12.3 23.7 Yang et al., 2015

aMean of six different Opuntia ficus-indica varieties. bEthanol soluble fraction.



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for bioenergy in the US (Núñez et al., 2011). These costs are primarily associated with the manual labour that has been traditionally used for Agave production. Agave would be comparable to or superior to other ethanol feedstocks, such as maize, switchgrass, and sugarcane, for bioethanol produc-tion in terms of life cycle energy and GHG balances (Nair et al., 2012), but is far more water-use efficient than these tra-ditional crops (Davis et al., 2014).

The efficient conversion of lignocellulosic biomass into value-added chemicals and renewable biofuels is limited primarily by the recalcitrance of plant cell wall material to degradation (DeMartini et al., 2013). Because deconstructive pretreatments of raw lignocellulosic biomass account for a majority of the costs associated with lignocellulosic biofuel production (Ragauskas et al., 2006), the use of feedstocks like Agave and Opuntia, which both have low lignin and high dis-ordered cellulose contents, would theoretically require fewer or less-aggressive pretreatment steps than those needed for traditional lignocellulosic biomass feedstocks. Recent experi-ments have now verified the low recalcitrance of lignocellu-losic fractions of Agave leaves and piñas (Li et al., 2014).

Agave biomass conversion and biofuel production

Agave bagasse is a good feedstock for biofuels production (Caspeta et al., 2014). Conversion of lignocellulosic biomass into biofuels generally involves three steps: (i) pretreatment using chemical, heat, and mechanical methods (e.g. acid, steam, alkali, and ammonium-freeze explosion) to disrupt the lignin bonds and thereby enhance the susceptibility of lig-nocellulosic materials to enzyme activity; (ii) saccharification using enzymatic hydrolysis to release sugars from pretreated biomass; and (iii) fermentation to convert the released sugars into biofuels (Saucedo-Luna et al., 2011). Although Agave biomass is a low-recalcitrance lignocellulosic feedstock (Li et al., 2014), pretreatment is still necessary for conversion of Agave into biofuels. Pretreatment of Agave bagasse with an ionic liquid (1-ethyl-3-methylimidazolium acetate) at 160°C for 3 h was very effective in improving its digestibility, result-ing in 45.5% delignification, a highly amorphous cellulose structure, and a significant reduction in recalcitrance of bio-mass for biofuel production (Perez-Pimienta et al., 2013). Unlike A. tequilana, in which the piña serves as a reservoir for fructan accumulation, A. americana lacks a significant piña. Instead, the large compact panicle (flower stalk) of A. ameri-cana was tested as a lignocellulosic feedstock for biofuel pro-duction and various pretreatments were found to improve its enzymatic digestibility and hydrolysability (Yang and Pan, 2012).

As the second step in biofuel conversion, sugar release is critical for converting Agave biomass into biofuels. A. atro-virens leaf fibre contains 67% cellulose, 42% of which can be converted into glucose by enzymatic degradation (Medina-Morales et al., 2011). Recently, Li and colleagues systemati-cally evaluated the sugar release from four biomass samples from the leaves or hearts of three Agave species (A. americana

leaves and heart, A. salmiana leaves, A. tequilana leaves), and identified an optimized fungal enzyme mixture for increas-ing sugar release (Li et al., 2014). Some cellulose-decompos-ing bacterial isolates, such as a Bacillus strain (65S3) and a Pseudomonas strain (CDS3), were demonstrated to efficiently decompose Agave biomass for bioethanol and xylitol produc-tion, with bioethanol yields as high as 0.92 g/g (Xiong et al., 2014). Recently, consortia of cellulase-degrading bacterial isolates were tested for the decomposition of A. americana fibre: six co- and two tri-cultures were shown to generate the greatest decomposition, indicating that synergy among the enzymes produced by the different microorganisms can help overcome inefficient conversion by a single strain (Maki et al., 2014).

Various methods have been used for Agave fermentation, the final step for biofuel conversion. Using an industrial yeast strain for the fermentation of Agave bagasse can yield up to 0.25 g/g of ethanol per gram of dry Agave bagasse (Caspeta et al., 2014). A native yeast strain, Pichia caribbica UM-5, was used to ferment sugar released from enzymatic hydrolysis of A. tequilana (L.) Weber bagasse to ethanol with an efficiency of 87% (Saucedo-Luna et al., 2011). While yeast (S. cerevisiae) is widely used for alcoholic fermenta-tion, Zymomonas mobilis, a Gram-negative anaerobic bac-terium, was shown to generate higher ethanol yields (5%) from leaves of Aloe barbadensis than could S. cerevisiae (4%) (Murugan and Rajendran, 2013). The ability of Z. mobilis and related bacteria to improve the conversion efficiency of Agave bagasse into ethanol should be investigated during this optimization.

Agave is rich in inulin as a reserve carbohydrate (Suárez-González et al., 2014). Inulinase-expressing microor-ganisms, including yeast (e.g. Pichia guilliermondii), filamentous fungi (e.g. Penicillium sp. TN-88), and bacteria (e.g. Streptomyces sp. GNDU 1), can be used to transform inulin into bioethanol (Chi et al., 2009). The fructans in A. fourcroydes leaf juice can be fermented into ethanol with K. marxianus that is able to grow in the hydrolysates, giv-ing a higher ethanol yield from the enzymatic hydrolysate (Villegas-Silva et al., 2014).

As an alternative to the classic biofuel conversion approaches, which involve separate processes for sugar release and fermentation, consolidated bioprocessing (CBP) exploits microorganisms that can simultaneously convert biomass to fermentable sugars and ferment the resultant sugars to etha-nol (Xu et al., 2009). CBP represents a future direction for cost-effective production of biofuels from Agave biomass. Lastly, the bagasse and vinasse from Agave have been used to make methane fuel through the process of anaerobic diges-tion (Espinoza-Escalante et al., 2009; Hernandez-Salas et al., 2009).

Opuntia biomass conversion and biofuel production

Relative to Agave spp., fewer studies have examined the conversion of Opuntia spp. biomass to biofuel. Enzyme mixtures consisting of Cytolase, Pectinex, Rapidase, and



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Viscozyme were used to hydrolyse Opuntia cladode muci-lage to improve its extractability and release of sugars (Kim et al., 2013). Pretreatment of fresh or dried O. ficus-indica cladodes with cellulase and acid slightly improved sugar release and ethanol yields above those of non-pre-treated material; however, ethanol yields remained low at ~1.4% (w/v) (Retamal et al., 1987). Separate hydroly-sis and fermentation and simultaneous saccharification and fermentation experiments on O. ficus-indica cladodes were conducted using K. marxianus and S. cerevisiae and resulted in similar ethanol yields of ~2.6% (w/v) (Kuloyo et al., 2014). While these results represented an improve-ment over earlier attempts, the authors concluded that greater hydrolysis of the biomass was needed to reduce the viscosity of the slurry and increase the release of fer-mentable sugars to attain an economically viable ethanol yield of >4% (w/v) (Kuloyo et al., 2014).

Global growth potential, distribution, and productivity modelling studies

The development of predictive models of biomass pro-duction that facilitate a systems approach to land use and natural resource management, and identify geographical regions that CAM feedstocks might successfully exploit in a warmer and drier world will accelerate the potential of Agave and Opuntia as bioenergy feedstocks on semi-arid and arid lands. This potential for significant biomass pro-ductivity and terrestrial sequestration of atmospheric CO2 in arid and semi-arid regions was first highlighted by the environmental productivity index (EPI) developed by Park Nobel and colleagues to inform and improve agronomic practices for CAM cultivation (Nobel, 1988; Nobel and Decortazar, 1991). EPI represents a first-order approxima-tion of the combined influence of environmental factors (water, temperature, photosynthetically active radiation, and nutrients) on net CO2 uptake over 24 h. Each com-ponent index represents the fractional net CO2 uptake expected under limitations on net CO2 uptake imposed by that factor. Thus, annual productivity is predicted by averaging daily EPIs, while the component indices of EPI highlight the limitations imposed by specific environmen-tal factors on net CO2 uptake in CAM species (Nobel and Decortazar, 1991; Nobel, García-Moya et al., 1992). For O. ficus-indica, EPI was used to predict an average global dry biomass productivity of 12 Mg ha–1 y–1 (Garcia de Cortazar and Nobel, 1990).

More recently, refinements have been made to the EPI model that accommodate the water retention properties of soil, the persistence of CO2 uptake under water-deficit stress, and the estimates of CO2 accumulation across varying night-day temperature regimes (Owen and Griffiths, 2014). Using this theoretical framework for predicting environmentally responsible bioethanol production for Agave and Opuntia across contrasting landscapes and varying environmental conditions in Australia has indicated that CAM feedstocks could produce yields in excess of 5 kL ha–1 y–1 across 57,700

km2 (~ 0.7% land area of Australia) (Owen and Griffiths, 2014).

One potential constraint of the Nobel EPI for accurate prediction of biomass yield is the reliance on measure-ments of titratable plant acidity between dusk and dawn (ΔH+) as a proxy for nocturnal carbon assimilation under variable inputs for water, temperature, and photosyn-thetically active radiation. Because measurements of ΔH+ do not capture the direct daytime uptake of CO2 during phases II and IV, which can make significant contributions to daily carbon gain in many CAM species (Borland et al., 2011), potential biomass productivity may be underesti-mated. By contrast, diurnal acidity changes can occur with internal recycling of respired CO2 carboxylation at night, which might lead to an overestimation of productivity if this is used as a proxy for CO2 exchange. Other potential limitations of EPI include possible errors associated with sampling methodologies and scaling, as well as changes in plant responses that occur from the interplay of multiple conditions as opposed to in response to a single condition with other conditions held constant. To improve EPI pro-ductivity modelling, multiple regression analysis of agro-nomic-scale eddy covariance gas exchange measurements collected over a range of contrasting environmental condi-tions might be useful.

To address these perceived limitations of EPI, a sys-tems dynamic (SD) model has been designed to capture the inherent plasticity that exists both within and between CAM species in the magnitude and duration of noctur-nal and daytime gas exchange (Owen and Griffiths, 2013). Using a restricted set of measured inputs (vacuole capac-ity, stomatal and mesophyll conductance, maximum rate of PEPC activity, Michaelis–Menten constant for PEPC), the SD model predicts and delineates the four phases of CAM gas exchange according to biochemical and physi-ological parameters that rate-limit carbon uptake over the diel cycle. The SD model created by Owen and Griffiths provides a workable platform for improving predictions of the potential global productivity of CAM bioenergy feed-stocks and moving towards the targeted genetic manipula-tion of traits that would improve carbon gain and WUE in CAM crops (Borland and Yang, 2013). Integration of SD modelling of CAM biochemistry with canopy-scale gas exchange measurements collected over diverse environ-mental conditions might further improve existing produc-tivity models.

A related GIS modelling study based upon productivity and yield estimates calibrated for annual precipitation rates in the Almeria province, Spain, calculated that the total potential eth-anol production from cultivated Opuntia spp. was 0.9 kL ha–1 y–1 based on the production of approximately 500 000 Mg (dry matter) ha–1 y–1, assuming an average annual biomass produc-tivity of 5 Mg (dry matter) ha–1 y–1 (Sánchez et al., 2012). To improve the accuracy of all of the aforementioned productivity estimation approaches, larger and more detailed field studies are needed to validate Agave and Opuntia productivity estimates at multiple geographical locations with contrasting environmental conditions.



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Hybrid bioenergy and renewable energy production systems

Agave and Opuntia production typically occurs in arid and semi-arid regions with high insolation rates. This realiza-tion inspired a recent comparative LCA of the energy out-puts and GHG offsets from photovoltaic (PV) systems and Agave-to-ethanol conversion and indicated that the mean energy outputs from PV systems are more than 11-fold greater than Agave, whereas GHG offsets were more than 25-fold greater than Agave (Ravi et al., 2014). However, as the water input required for both cleaning of solar panels and dust suppression are similar to that needed for annual Agave growth, co-localization of both systems into an inte-grated solar PV–Agave system could generate a higher rate of energy return than either system alone (Ravi et al., 2014). A similar co-localization strategy could also be envisioned for Opuntia production systems (Fig. 3). However, imple-mentation of such co-production systems will have to over-come major logistical concerns, such as the possible effects of shading on plant physiological responses and productivity, and the compatibility of PV arrays with mechanical harvest-ing equipment. Furthermore, reliable yield estimates in such co-localization systems will have to be obtained from various

species or cultivars of Agave and Opuntia before realistic economic assessments can be made regarding the economic viability of such systems.

Conclusions and future directions

Agave spp. and, to a lesser extent, Opuntia spp. have been viewed as potential biofuel feedstocks because of their historical use in commercial-scale ethanol production. However, the prospects of an increasingly hotter and drier climate have led many research-ers to re-evaluate these highly water-use efficient, heat- and drought-durable CAM species for use as bioenergy feedstocks from biomass production on semi-arid and arid lands. Using these species as feedstocks would allow food and biofuel pro-duction on land not currently used for the production of C3 and C4 crops. However, to realize the potential of these low water-use bioenergy crops, up-to-date, realistic ‘ground truth’ estimates will be needed for biomass productivity, and fertilizer and water inputs. Furthermore, both the environmental impacts and eco-system services effects of such agricultural production systems on fragile semi-arid and arid lands should be monitored care-fully to ensure that negative environmental impacts are avoided and societal benefits are gained from their use. In addition, more

Fig. 3. Co-localization of solar PV arrays with (A) Agave and (B) Opuntia production systems to utilize wastewater runoff. In arid and semi-arid environments, PV arrays require periodic washing to remove dust residue to maximize electricity production. Wastewater is captured to provide irrigation for Agave and Opuntia plantations, thereby maximizing the production of both electricity and biofuel production. PV panel image © Freshpaint. Agave image © SSSCCC. Opuntia image © sarininka.



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research into the physiological impacts of increased atmospheric [CO2] on these CAM species will be needed to better understand whether such increases will likely affect their biomass productiv-ity relative to C3 and C4 species in the future.

While detailed LCA have been performed for A. tequi-lana, similar studies will also be needed for O. ficus-indica to obtain a more realistic estimate of biofuel production in terms of life cycle energy and GHG balances, as will an economic analysis of the profitability of converting Agave and Opuntia feedstocks to biofuels. For example, one of the major barriers to the economic viability of these bio-fuel feedstocks will be high production costs, such as the high cost of manual labour traditionally used for Agave and Opuntia production. This can be overcome, in part, by the development of specialized, mechanical harvesting and automated, biomass-processing equipment for both Agave and Opuntia. Furthermore, more precise estimates of crop water demand and of the amount of fuel that can be produced per unit land area are needed for these crops, particularly for Opuntia. Such estimates have been derived from modelling studies but more replicated field trials at various locations with varying levels of water and ferti-lizer inputs are needed. More efficient biomass-to-biofuel conversion strategies are also needed, particularly for the lignocellulosic fractions of biomass derived from these low water-use species. Innovative strategies are also needed to identify and to reduce the costs of hydrolytic enzymes that have been customized for the efficient degradation of these feedstocks, such as inulinase-producing microorgan-isms for Agave and pectinase-producing microorganisms for Opuntia. Testing and optimizing bacterial and fungal combinations and consortia for biopolymer degradation and saccharification, and also developing efficient CBP systems will be essential for maximizing biofuel produc-tion while reducing costs. In summary, Agave spp. and Opuntia spp. have great potential as bioenergy feedstocks and their relative importance is likely to increase as ter-restrial crop production areas become warmer and drier in the future.

AcknowledgementsThis review is based on work supported by the Department of Energy (DOE), Office of Science, Genomic Science Program under Award Number DE-SC0008834. Additional support from the Nevada Agricultural Experiment Station under projects NAES-00377 and NAES-00380 is acknowledged. The contents of this review are solely the responsibility of the authors and do not necessarily represent the official views of the DOE. The authors wish to Mary Ann Cushman for critical review and clarifying comments on the manuscript and Lori Kunder (Kunder Design Studio) for assistance with figure preparation. Oak Ridge National Laboratory is managed by UT-Battelle, LLC for the US DOE under Contract Number DE–AC05–00OR22725.

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