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Bioprocessing for biofuels
Available online at www.sciencedirect.com
Bioprocessing for biofuels Harvey W Blanch While engineering of new biofuels pathways into microbial hosts has received considerable attention, innovations in bioprocessing are required for commercialization of both conventional and next-generation fuels. For ethanol and butanol, reducing energy costs for product recovery remains a challenge. Fuels produced from heterologous aerobic pathways in yeast and bacteria require control of aeration and cooling at large scales. Converting lignocellulosic biomass to sugars for fuels production requires effective biomass pretreatment to increase surface area, decrystallize cellulose and facilitate enzymatic hydrolysis. Effective means to recover microalgae and extract their intracellular lipids remains a practical and economic bottleneck in algal biodiesel production. Address Department of Chemical and Biomolecular Engineering, Joint BioEnergy Institute, University of California Berkeley, Berkeley, CA 94720, United States Corresponding author: Blanch, Harvey W ([email protected])
Current Opinion in Biotechnology 2012, 23:390–395 This review comes from a themed issue on Energy biotechnology Edited by James C Liao and Joachim Messing Available online 25th October 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.10.002
Introduction Research efforts directed toward the conversion of biomass into a liquid transportation fuel have their origins in the first US energy crisis of October, 1973, a consequence of the Yom Kippur war and the OPEC oil embargo. Subsequently, the 1979 Iranian revolution and more recent concerns about the security of imported petroleum and the contribution of CO2 emissions to global warming trends have led to renewed efforts to provide an essentially CO2-neutral supply of transportation fuel. The carbohydrate content of biomass can be converted to alcohols (ethanol, butanol), fatty acid esters (biodiesel), long chain and cyclic hydrocarbons (gasoline equivalents and jet fuels), using both well-know fermentation pathways and the newly-developed methods of synthetic biology (see www.synberc.org/content/articles/whatsynthetic-biology for a description). The sources of biomass for fuels production include glucose from Current Opinion in Biotechnology 2012, 23:390–395
starch-containing crops such as corn; sucrose derived from sugar cane and sugar beets; palm and vegetable oils; and lignocellulosic biomass, including grasses such as Miscanthus and switchgrass, and woody biomass. Their use is dictated by their availability, cost, and the sustainability of production. In the US, lignocellulosic biomass, in the form of new bioenergy crops, forest and agricultural residues, have the potential to provide around 500 million dry tons of biomass at $60/ton or less in 2012 [1], and thus replace around 15% of current petroleum-based transportation fuels. In addition to biomass, algae also capture CO2 photosynthetically and produce lipids that can be transesterified to biodiesel. Marine algae, such as Nannochloropsis and Pleurochrysis, have the advantage of not competing with food crops or for fresh water resources. However, cultivation of algae at large scale poses significant economic and bioprocessing challenges, including costeffective harvesting of the algae and the recovery of the lipids, which are intracellularly located. There has also been recent interest in conversion of gaseous feedstocks such as CO and H2 (as off-gases or as syngas or as producer gas from biomass gasification) to products such as ethanol, butyrate, and acetate [2]. This is based on a biological water–gas shift reaction, which may be supplemented by a hydrogenase reaction to convert H2 to form additional reducing equivalents. Acetyl-CoA is formed from the activity of the Wood– Ljungdahl pathway, and converted to products. Transfer of sparingly-soluble gases such as CO and H2 to the aqueous phase presents a challenge in fermenter design, as high power inputs are required to obtain the required interfacial areas. These issues have been recently reviewed [3].
Fermentation of saccharides to fuels Ethanol and butanol
The microbial conversion of mono and disaccharides to ethanol and butanol is well established; the main bioprocessing challenges relate to recovery of the products and, in the case of ethanol, reducing contamination and use of antibiotics in the non-aseptic fermentations [4,5]. Energy and materials costs for dehydration of ethanol from below its azeotropic composition (95.6 wt%) to the fuel target of 99.8% have been reviewed, including pervaporation, permeation, heteroazeotropic distillation with cyclohexane, and adsorption on molecular sieves [6]. Pervaporation and membrane permeation provide the least costly routes; pervaporation has the advantage that the mixture does not need to be heated to boiling and thus low-grade www.sciencedirect.com
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heat can be used, as only permeate evaporation requires energy input. Recent advances in polyimide [7] and PDMS [8] membranes for bioalcohols separations have been detailed. Molecular sieves can remove water by adsorption. From a near azeotropic mixture, water adsorbs into the pores of the sieve, but a large amount of liquid forms in the molecular sieve regeneration stage. By operating in the vapor phase however, the sieve is not wet in adsorption or desorption, thus reducing energy consumption. Current commercial pressure-swing adsorption processes employ zeolites. Other commercial approaches include pressure-swing distillation, extractive distillation [6], and azeotropic distillation. A number of hybrid schemes that incorporate distillation and pervaporation are being developed. Advantages and disadvantages of ethanol dehydration technologies are discussed in [9]. n-Butanol and iso-butanol (a potential biofuel: www. gevo.com) have higher boiling points than water (118 and 107.9 8C, respectively), making their recovery by distillation from fermentation broth, where typical concentrations are 15–20 g/L, energy intensive. Since the early developments of extractive fermentations for butanol production using oleyl alcohol [10], there have been a number of efforts to develop non-toxic solvents for in situ extraction of butanol. Extractive fermentation relieves the solvent toxicity that typically limits butanol production, and subsequent distillation of butanol from high-boiling extractants (e.g., oleyl alcohol has a boiling point of 330–360 8C) is straightforward. An alternative is gas stripping; removal of volatile solvents by the CO2 and H2 produced during Clostridium ABE fermentation. This has also been shown to significantly enhance volumetric productivities, although high gas flow rates (3 vvm) are required for effective stripping [11], representing a high energy cost. Approaches to reduce microbial inhibition by n-butanol and iso-butanol include adaptation [12–14], incorporation of solvent pumps [15,16] or expression of chaperones [17]. These may lead to strains that can tolerate higher levels of butanol, however it is unlikely that strains will be developed that are capable of producing butanol at concentrations approaching its aqueous solubility (63 and 76 g/L for n-butanol and iso-butanol), which would permit recovery by phase separation. Thus development of fermentation processes that continuously remove butanol as it is produced would provide the most effective route to overcome butanol toxicity, improve productivity, and recover the product [18]. Hydrocarbons from the isoprenoid, polyketide, and fatty acid pathways
Advances in metabolic engineering permit the expression and regulation of heterologous pathways in a variety of hosts organisms [19]. Conversion of sugars via the isoprenoid, polyketide, and fatty acid pathways provides www.sciencedirect.com
routes to diesel, jet, and gasoline-compatible molecules [20]. The production of hydrocarbons that are more reduced than carbohydrate-based raw materials requires the oxygen in the carbohydrate to be rejected. For ethanol and butanol, this is accomplished via CO2, whereas for more reduced products, reducing equivalents need to be generated through a biological version of aqueous reforming of the sugar; these are used to reduce the sugar to form the desired product, with excess oxygen rejected as H2O. For example: C6 H12 O6 þ 6H2 O ! 6CO2 þ 12H2 C6 H12 O6 þ 4:8H2 ! 0:4C15 H24 þ 6H2 O where C15H24 is the sesquiterpene farnesene, produced via the mevalonate pathway [21]. The stoichiometric yield is thus 0.32 g farnesene/g glucose. Achieving this would require no excess reducing equivalents (as NAD(P)H2) to be formed. The inability to effectively balance reducing equivalents in existing microbial production pathways typically results in their overproduction and they must be removed aerobically in the form of water. The resulting aerobic or micro-aerobic conditions required for fermentation need to be carefully controlled, so that excess oxidation of NAD(P)H2 does not occur and reduce yields of product. A similar situation arises in the production of fatty acid ethyl esters (C12–C18 range) in Escherichia coli [22]. This presents new challenges in fermenter design, aeration, and cooling at the scales envisioned for biofuels production. Ideally, the metabolic pathway would be engineered to have pathway redox conditions balanced and thus able to operate anaerobically [23].
Lignocellulose conversion to sugars Depolymerization of cellulose and hemicellulose
A necessary step in the biological conversion of biomass to fuels is the pretreatment of biomass to make it more readily deconstructed. A number of pretreatment and chemical hydrolysis approaches have been developed over the past 80 years; these have been recently reviewed [24,25]. A promising new approach is the dissolution of biomass in certain ionic liquids, with precipitation of the cellulose by addition of water or other anti-solvents [26,27,28]. The lignin remains dissolved in the ionic liquid and can be later recovered [29]. The precipitated cellulose can be hydrolyzed by cellulases to sugars at high yields and conversions and in significantly less time than that required for diluteacid pretreated biomass [30,31]. Theoretical [32,33] and experimental [34] approaches are providing insight into the mechanisms of biomass dissolution in these novel solvents. The economics of ionic liquid pretreatment have been recently examined and shown to depend strongly on the costs of the ionic liquid, its recycle and the ionic liquid to biomass loading [35]. Routes to upgrade the value of lignin in this process significantly improve the economic viability of the process. Current Opinion in Biotechnology 2012, 23:390–395
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Ultrasound is being reexamined as a pretreatment method; cavitation induced by ultrasound increases biomass surface area, enhancing enzyme access for hydrolysis [25,36]. Recent advances in ammonia pretreatment using liquid ammonia result in production of a cellulose III polymorph, which significantly enhances the effectiveness of enzymatic depolymerization [37,38]. Obtaining a hydrolyzate solution with sufficient sugar to avoid concentration before biofuel fermentation requires a high solids loading in the hydrolysis step [39,40]. High solids loading result in high viscosities and mixing is difficult [41]. High solids loadings in alkaline pretreatment [42], with sulfuric acid, with and without bisulfite [43], are required to obtain high ethanol yields and concentrations (>4–6 w/v%) in simultaneous saccharification and fermentation approaches. The effectiveness of both pretreatment and enzymatic hydrolysis at high solids loadings requires further exploration. The energy consumption in pretreatment of woody biomass also plays a role in determining the economic effectiveness of pretreatment approaches [44]. The economic production of cellulolytic enzymes and reducing the enzyme-to-biomass ratio required for hydrolysis remain key to commercializing biomassderived fuels. Trichoderma reesei remains the most effective commercial host for cellulase production [45], although other fungal hosts may be attractive [46,47]. Protein concentrations in excess of 100 g/L are reported with T. reesei [48]. Fungal cellulase production is inducible and growth associated; lactose is an economicallyattractive inducer; the disaccharides sophorose, cellobiose, d-cellobiose-1-5-lactone, and oxidized products of cellulose hydrolysis are also inducers [49]. Bacterial cellulases suffer from low expression levels and are thus unlikely to be competitive with fungal enzymes. Efforts to improve the catalytic activity of cellulases must be viewed in the context of the ability to economically produce them at scale, particularly if glycosylation impacts activity. Cellulase costs are estimated at a minimum of $10/kg of protein [50]. Reducing both the cost and the amount of enzymes required for biomass hydrolysis is required. Typical enzyme loadings for >75% conversion of biomass to sugars are 10–15 FPU/g glucan (20– 30 mg/g glucan, depending on the method of pretreatment and the source of enzymes; 1 Filter Paper Unit (FPU) is approximately 2.1–2.2 mg enzyme mixture [51]). Thus enzyme costs represent around $1 per gallon of ethanol (for 100% yields of sugar and ethanol). Recycle of cellulase enzyme requirements has been examined as a route to these reduce costs by reducing the enzyme loading [52,53]. A three-stage hydrolysis of steam-exploded corn stover with enzyme recycle was shown to reduce hydrolysis times without adversely affecting glucose yields [54]. As cellulases adsorb irreversibly Current Opinion in Biotechnology 2012, 23:390–395
onto lignin, pretreatments that reduce or remove lignin would be desirable for enzyme recycle [55]. In this regard, pretreatment by solubilization of biomass in ionic liquids with cellulose precipitation may be attractive. The addition of a surfactant (Tween 80) to aid in enzyme release has shown to reduce enzyme loading requirements by about 60% [56,57]; the cost savings are dependent on the cost and amount of Tween 80 used. Operating modes (SSF and CBP)
Efforts to reduce capital and certain operating costs in the saccharification of pretreated biomass and fermentation of the resultant sugars have focused on conducting both steps in a single vessel. Simultaneous saccharification and fermentation (SSF), and simultaneous saccharification and co-fermentation (SSCF, where xylose is co-fermented with glucose) have been developed. The optimal temperature for fungal cellulases is 50 8C, and if coupled with ethanol production by yeast, the process must be conducted at a temperature below 50 8C to support yeast growth (Topt 30 8C; Tmax 40 8C). Fungal cellulase activity at 30 8C is 30% of that at 50 8C [58,59], reducing the potential savings of a single-vessel process. Reducing cellobiose inhibition of endoglucanase and cellobiohydrolases in SSCF processes can be accomplished by the expression of a cellobiose transporter and b-glucosidase in Saccharomyces cerevisiae strains able to ferment xylose [60,61]. This approach provides an alternative to the addition of b-glucosidase to commercial cellulase mixtures, but the temperature mismatch remains. Since early efforts to employ a single organism to simultaneously hydrolyze lignocellulosic biomass and produce ethanol or other products from the sugars released [62] (subsequently referred to as consolidated bioprocessing or CBP), development of a suitable thermophilic organism has been key. Clostridiales and Thermoanaerobiales have high rates of cellulose metabolism, with optimum cellulase activities at high temperatures (e.g., cellulases from Clostridium thermocellum show an optimum near 70 8C [63]), but typically do not produce concentrations of ethanol beyond 5 w/v%. Estimates of the economic advantages CBP might present have been based on assumptions that may be difficult to realize: cellulase production at 400 FPU/g carbohydrate (equivalent to conversion of over 90% of the carbon in the carbohydrate feedstock to protein, leaving only 10% for ethanol and biomass production) at a productivity of 400 FPU/L h, with the entire CBP process occurring in 1.5 days [64].
Algae for production of triglycerides and fatty acids Microalgae are being examined as a source of lipids (including triglycerides and fatty acids) for conversion to biodiesel. Areal yields of algal lipids can be higher than terrestrial oil crops [65] (e.g., 2650 gal/acre/yr for Pleurochrysis carterae [66] compared with palm oil at www.sciencedirect.com
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400–600 gal/acre/yr; soybean at 40 gal/acre/yr). The lipids of algae are located within the cell; the exception is Botryococcus braunii, which secretes the lipids into the cell wall, but has a low productivity. The intracellular location of the lipids requires that the algae be economically harvested and the oil recovered by breakage of the algal cell wall. These are two of the most significant issues in commercializing algal biodiesel production. Typical separation methods such as centrifugation, filtration, and chemical flocculation are expensive or introduce Al3+ as a contaminant [67]. A recent innovation is the induction of flocculation by the addition of low concentrations of organic carbon (e.g., acetate, glucose, or glycerin) to induce bacterial production of extracellular polymeric materials that attach to the microalgae and form flocs. The energy required for mixing have been detailed [67] and shown to make this a potentially attractive processing approach. Extraction of intracellular lipids from intact algae is difficult as the lipids are bound within cell membranes and cell disruption is required to maximize lipid recovery [68]. Lipids cannot be recovered effectively from algae using methods designed for oil extrusion from crops such as soybeans, due to the small size of the algae and the mechanical strength of their walls. A recent review of extraction methods indicates that mechanical methods are optimal, but these have a high specific energy consumption, which exceeds the energy content available from the extracted lipid [69]. Thus this aspect of algal lipid production requires considerable research to develop an energy-efficient method for lipid recovery. Algal bioreactors
Obtaining the high productivities required for algal biodiesel production at low cost is likely only to be possible in open raceway reactor systems [70]. Although closed photobioreactor systems offer advantages (avoidance of contamination, high algal concentrations and productivities, better control of culture conditions), only a very limited number of commercial systems have been developed. For biofuels applications, they may provide a contaminant-free inoculum for raceway systems [70]. Algal have also been considered for CO2 sequestration, provided point source of concentrated CO2 is available (e.g., a flue gas). Supply of up to 5–10% CO2 can increase algal lipid production, but due to mass transfer limitations, complete dissolution of CO2 during the time of exposure of gas to the liquid phase is difficult. This, and other aspects of microalgal biofuels production have been reviewed [71].
Acknowledgements This work was part of the DOE Joint BioEnergy Institute (http:// www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract www.sciencedirect.com
DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.
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71. Malcata FX: Microalgae and biofuels: a promising partnership? Trends in Biotechnology 2011 doi: 10.1016/j.tibtech.2011.05.005.
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Current Opinion in Biotechnology 2012, 23:390–395
Bioprocessing for biofuels Harvey W Blanch While engineering of new biofuels pathways into microbial hosts has received considerable attention, innovations in bioprocessing are required for commercialization of both conventional and next-generation fuels. For ethanol and butanol, reducing energy costs for product recovery remains a challenge. Fuels produced from heterologous aerobic pathways in yeast and bacteria require control of aeration and cooling at large scales. Converting lignocellulosic biomass to sugars for fuels production requires effective biomass pretreatment to increase surface area, decrystallize cellulose and facilitate enzymatic hydrolysis. Effective means to recover microalgae and extract their intracellular lipids remains a practical and economic bottleneck in algal biodiesel production. Address Department of Chemical and Biomolecular Engineering, Joint BioEnergy Institute, University of California Berkeley, Berkeley, CA 94720, United States Corresponding author: Blanch, Harvey W ([email protected])
Current Opinion in Biotechnology 2012, 23:390–395 This review comes from a themed issue on Energy biotechnology Edited by James C Liao and Joachim Messing Available online 25th October 2011 0958-1669/$ – see front matter # 2011 Elsevier Ltd. All rights reserved. DOI 10.1016/j.copbio.2011.10.002
Introduction Research efforts directed toward the conversion of biomass into a liquid transportation fuel have their origins in the first US energy crisis of October, 1973, a consequence of the Yom Kippur war and the OPEC oil embargo. Subsequently, the 1979 Iranian revolution and more recent concerns about the security of imported petroleum and the contribution of CO2 emissions to global warming trends have led to renewed efforts to provide an essentially CO2-neutral supply of transportation fuel. The carbohydrate content of biomass can be converted to alcohols (ethanol, butanol), fatty acid esters (biodiesel), long chain and cyclic hydrocarbons (gasoline equivalents and jet fuels), using both well-know fermentation pathways and the newly-developed methods of synthetic biology (see www.synberc.org/content/articles/whatsynthetic-biology for a description). The sources of biomass for fuels production include glucose from Current Opinion in Biotechnology 2012, 23:390–395
starch-containing crops such as corn; sucrose derived from sugar cane and sugar beets; palm and vegetable oils; and lignocellulosic biomass, including grasses such as Miscanthus and switchgrass, and woody biomass. Their use is dictated by their availability, cost, and the sustainability of production. In the US, lignocellulosic biomass, in the form of new bioenergy crops, forest and agricultural residues, have the potential to provide around 500 million dry tons of biomass at $60/ton or less in 2012 [1], and thus replace around 15% of current petroleum-based transportation fuels. In addition to biomass, algae also capture CO2 photosynthetically and produce lipids that can be transesterified to biodiesel. Marine algae, such as Nannochloropsis and Pleurochrysis, have the advantage of not competing with food crops or for fresh water resources. However, cultivation of algae at large scale poses significant economic and bioprocessing challenges, including costeffective harvesting of the algae and the recovery of the lipids, which are intracellularly located. There has also been recent interest in conversion of gaseous feedstocks such as CO and H2 (as off-gases or as syngas or as producer gas from biomass gasification) to products such as ethanol, butyrate, and acetate [2]. This is based on a biological water–gas shift reaction, which may be supplemented by a hydrogenase reaction to convert H2 to form additional reducing equivalents. Acetyl-CoA is formed from the activity of the Wood– Ljungdahl pathway, and converted to products. Transfer of sparingly-soluble gases such as CO and H2 to the aqueous phase presents a challenge in fermenter design, as high power inputs are required to obtain the required interfacial areas. These issues have been recently reviewed [3].
Fermentation of saccharides to fuels Ethanol and butanol
The microbial conversion of mono and disaccharides to ethanol and butanol is well established; the main bioprocessing challenges relate to recovery of the products and, in the case of ethanol, reducing contamination and use of antibiotics in the non-aseptic fermentations [4,5]. Energy and materials costs for dehydration of ethanol from below its azeotropic composition (95.6 wt%) to the fuel target of 99.8% have been reviewed, including pervaporation, permeation, heteroazeotropic distillation with cyclohexane, and adsorption on molecular sieves [6]. Pervaporation and membrane permeation provide the least costly routes; pervaporation has the advantage that the mixture does not need to be heated to boiling and thus low-grade www.sciencedirect.com
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heat can be used, as only permeate evaporation requires energy input. Recent advances in polyimide [7] and PDMS [8] membranes for bioalcohols separations have been detailed. Molecular sieves can remove water by adsorption. From a near azeotropic mixture, water adsorbs into the pores of the sieve, but a large amount of liquid forms in the molecular sieve regeneration stage. By operating in the vapor phase however, the sieve is not wet in adsorption or desorption, thus reducing energy consumption. Current commercial pressure-swing adsorption processes employ zeolites. Other commercial approaches include pressure-swing distillation, extractive distillation [6], and azeotropic distillation. A number of hybrid schemes that incorporate distillation and pervaporation are being developed. Advantages and disadvantages of ethanol dehydration technologies are discussed in [9]. n-Butanol and iso-butanol (a potential biofuel: www. gevo.com) have higher boiling points than water (118 and 107.9 8C, respectively), making their recovery by distillation from fermentation broth, where typical concentrations are 15–20 g/L, energy intensive. Since the early developments of extractive fermentations for butanol production using oleyl alcohol [10], there have been a number of efforts to develop non-toxic solvents for in situ extraction of butanol. Extractive fermentation relieves the solvent toxicity that typically limits butanol production, and subsequent distillation of butanol from high-boiling extractants (e.g., oleyl alcohol has a boiling point of 330–360 8C) is straightforward. An alternative is gas stripping; removal of volatile solvents by the CO2 and H2 produced during Clostridium ABE fermentation. This has also been shown to significantly enhance volumetric productivities, although high gas flow rates (3 vvm) are required for effective stripping [11], representing a high energy cost. Approaches to reduce microbial inhibition by n-butanol and iso-butanol include adaptation [12–14], incorporation of solvent pumps [15,16] or expression of chaperones [17]. These may lead to strains that can tolerate higher levels of butanol, however it is unlikely that strains will be developed that are capable of producing butanol at concentrations approaching its aqueous solubility (63 and 76 g/L for n-butanol and iso-butanol), which would permit recovery by phase separation. Thus development of fermentation processes that continuously remove butanol as it is produced would provide the most effective route to overcome butanol toxicity, improve productivity, and recover the product [18]. Hydrocarbons from the isoprenoid, polyketide, and fatty acid pathways
Advances in metabolic engineering permit the expression and regulation of heterologous pathways in a variety of hosts organisms [19]. Conversion of sugars via the isoprenoid, polyketide, and fatty acid pathways provides www.sciencedirect.com
routes to diesel, jet, and gasoline-compatible molecules [20]. The production of hydrocarbons that are more reduced than carbohydrate-based raw materials requires the oxygen in the carbohydrate to be rejected. For ethanol and butanol, this is accomplished via CO2, whereas for more reduced products, reducing equivalents need to be generated through a biological version of aqueous reforming of the sugar; these are used to reduce the sugar to form the desired product, with excess oxygen rejected as H2O. For example: C6 H12 O6 þ 6H2 O ! 6CO2 þ 12H2 C6 H12 O6 þ 4:8H2 ! 0:4C15 H24 þ 6H2 O where C15H24 is the sesquiterpene farnesene, produced via the mevalonate pathway [21]. The stoichiometric yield is thus 0.32 g farnesene/g glucose. Achieving this would require no excess reducing equivalents (as NAD(P)H2) to be formed. The inability to effectively balance reducing equivalents in existing microbial production pathways typically results in their overproduction and they must be removed aerobically in the form of water. The resulting aerobic or micro-aerobic conditions required for fermentation need to be carefully controlled, so that excess oxidation of NAD(P)H2 does not occur and reduce yields of product. A similar situation arises in the production of fatty acid ethyl esters (C12–C18 range) in Escherichia coli [22]. This presents new challenges in fermenter design, aeration, and cooling at the scales envisioned for biofuels production. Ideally, the metabolic pathway would be engineered to have pathway redox conditions balanced and thus able to operate anaerobically [23].
Lignocellulose conversion to sugars Depolymerization of cellulose and hemicellulose
A necessary step in the biological conversion of biomass to fuels is the pretreatment of biomass to make it more readily deconstructed. A number of pretreatment and chemical hydrolysis approaches have been developed over the past 80 years; these have been recently reviewed [24,25]. A promising new approach is the dissolution of biomass in certain ionic liquids, with precipitation of the cellulose by addition of water or other anti-solvents [26,27,28]. The lignin remains dissolved in the ionic liquid and can be later recovered [29]. The precipitated cellulose can be hydrolyzed by cellulases to sugars at high yields and conversions and in significantly less time than that required for diluteacid pretreated biomass [30,31]. Theoretical [32,33] and experimental [34] approaches are providing insight into the mechanisms of biomass dissolution in these novel solvents. The economics of ionic liquid pretreatment have been recently examined and shown to depend strongly on the costs of the ionic liquid, its recycle and the ionic liquid to biomass loading [35]. Routes to upgrade the value of lignin in this process significantly improve the economic viability of the process. Current Opinion in Biotechnology 2012, 23:390–395
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Ultrasound is being reexamined as a pretreatment method; cavitation induced by ultrasound increases biomass surface area, enhancing enzyme access for hydrolysis [25,36]. Recent advances in ammonia pretreatment using liquid ammonia result in production of a cellulose III polymorph, which significantly enhances the effectiveness of enzymatic depolymerization [37,38]. Obtaining a hydrolyzate solution with sufficient sugar to avoid concentration before biofuel fermentation requires a high solids loading in the hydrolysis step [39,40]. High solids loading result in high viscosities and mixing is difficult [41]. High solids loadings in alkaline pretreatment [42], with sulfuric acid, with and without bisulfite [43], are required to obtain high ethanol yields and concentrations (>4–6 w/v%) in simultaneous saccharification and fermentation approaches. The effectiveness of both pretreatment and enzymatic hydrolysis at high solids loadings requires further exploration. The energy consumption in pretreatment of woody biomass also plays a role in determining the economic effectiveness of pretreatment approaches [44]. The economic production of cellulolytic enzymes and reducing the enzyme-to-biomass ratio required for hydrolysis remain key to commercializing biomassderived fuels. Trichoderma reesei remains the most effective commercial host for cellulase production [45], although other fungal hosts may be attractive [46,47]. Protein concentrations in excess of 100 g/L are reported with T. reesei [48]. Fungal cellulase production is inducible and growth associated; lactose is an economicallyattractive inducer; the disaccharides sophorose, cellobiose, d-cellobiose-1-5-lactone, and oxidized products of cellulose hydrolysis are also inducers [49]. Bacterial cellulases suffer from low expression levels and are thus unlikely to be competitive with fungal enzymes. Efforts to improve the catalytic activity of cellulases must be viewed in the context of the ability to economically produce them at scale, particularly if glycosylation impacts activity. Cellulase costs are estimated at a minimum of $10/kg of protein [50]. Reducing both the cost and the amount of enzymes required for biomass hydrolysis is required. Typical enzyme loadings for >75% conversion of biomass to sugars are 10–15 FPU/g glucan (20– 30 mg/g glucan, depending on the method of pretreatment and the source of enzymes; 1 Filter Paper Unit (FPU) is approximately 2.1–2.2 mg enzyme mixture [51]). Thus enzyme costs represent around $1 per gallon of ethanol (for 100% yields of sugar and ethanol). Recycle of cellulase enzyme requirements has been examined as a route to these reduce costs by reducing the enzyme loading [52,53]. A three-stage hydrolysis of steam-exploded corn stover with enzyme recycle was shown to reduce hydrolysis times without adversely affecting glucose yields [54]. As cellulases adsorb irreversibly Current Opinion in Biotechnology 2012, 23:390–395
onto lignin, pretreatments that reduce or remove lignin would be desirable for enzyme recycle [55]. In this regard, pretreatment by solubilization of biomass in ionic liquids with cellulose precipitation may be attractive. The addition of a surfactant (Tween 80) to aid in enzyme release has shown to reduce enzyme loading requirements by about 60% [56,57]; the cost savings are dependent on the cost and amount of Tween 80 used. Operating modes (SSF and CBP)
Efforts to reduce capital and certain operating costs in the saccharification of pretreated biomass and fermentation of the resultant sugars have focused on conducting both steps in a single vessel. Simultaneous saccharification and fermentation (SSF), and simultaneous saccharification and co-fermentation (SSCF, where xylose is co-fermented with glucose) have been developed. The optimal temperature for fungal cellulases is 50 8C, and if coupled with ethanol production by yeast, the process must be conducted at a temperature below 50 8C to support yeast growth (Topt 30 8C; Tmax 40 8C). Fungal cellulase activity at 30 8C is 30% of that at 50 8C [58,59], reducing the potential savings of a single-vessel process. Reducing cellobiose inhibition of endoglucanase and cellobiohydrolases in SSCF processes can be accomplished by the expression of a cellobiose transporter and b-glucosidase in Saccharomyces cerevisiae strains able to ferment xylose [60,61]. This approach provides an alternative to the addition of b-glucosidase to commercial cellulase mixtures, but the temperature mismatch remains. Since early efforts to employ a single organism to simultaneously hydrolyze lignocellulosic biomass and produce ethanol or other products from the sugars released [62] (subsequently referred to as consolidated bioprocessing or CBP), development of a suitable thermophilic organism has been key. Clostridiales and Thermoanaerobiales have high rates of cellulose metabolism, with optimum cellulase activities at high temperatures (e.g., cellulases from Clostridium thermocellum show an optimum near 70 8C [63]), but typically do not produce concentrations of ethanol beyond 5 w/v%. Estimates of the economic advantages CBP might present have been based on assumptions that may be difficult to realize: cellulase production at 400 FPU/g carbohydrate (equivalent to conversion of over 90% of the carbon in the carbohydrate feedstock to protein, leaving only 10% for ethanol and biomass production) at a productivity of 400 FPU/L h, with the entire CBP process occurring in 1.5 days [64].
Algae for production of triglycerides and fatty acids Microalgae are being examined as a source of lipids (including triglycerides and fatty acids) for conversion to biodiesel. Areal yields of algal lipids can be higher than terrestrial oil crops [65] (e.g., 2650 gal/acre/yr for Pleurochrysis carterae [66] compared with palm oil at www.sciencedirect.com
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400–600 gal/acre/yr; soybean at 40 gal/acre/yr). The lipids of algae are located within the cell; the exception is Botryococcus braunii, which secretes the lipids into the cell wall, but has a low productivity. The intracellular location of the lipids requires that the algae be economically harvested and the oil recovered by breakage of the algal cell wall. These are two of the most significant issues in commercializing algal biodiesel production. Typical separation methods such as centrifugation, filtration, and chemical flocculation are expensive or introduce Al3+ as a contaminant [67]. A recent innovation is the induction of flocculation by the addition of low concentrations of organic carbon (e.g., acetate, glucose, or glycerin) to induce bacterial production of extracellular polymeric materials that attach to the microalgae and form flocs. The energy required for mixing have been detailed [67] and shown to make this a potentially attractive processing approach. Extraction of intracellular lipids from intact algae is difficult as the lipids are bound within cell membranes and cell disruption is required to maximize lipid recovery [68]. Lipids cannot be recovered effectively from algae using methods designed for oil extrusion from crops such as soybeans, due to the small size of the algae and the mechanical strength of their walls. A recent review of extraction methods indicates that mechanical methods are optimal, but these have a high specific energy consumption, which exceeds the energy content available from the extracted lipid [69]. Thus this aspect of algal lipid production requires considerable research to develop an energy-efficient method for lipid recovery. Algal bioreactors
Obtaining the high productivities required for algal biodiesel production at low cost is likely only to be possible in open raceway reactor systems [70]. Although closed photobioreactor systems offer advantages (avoidance of contamination, high algal concentrations and productivities, better control of culture conditions), only a very limited number of commercial systems have been developed. For biofuels applications, they may provide a contaminant-free inoculum for raceway systems [70]. Algal have also been considered for CO2 sequestration, provided point source of concentrated CO2 is available (e.g., a flue gas). Supply of up to 5–10% CO2 can increase algal lipid production, but due to mass transfer limitations, complete dissolution of CO2 during the time of exposure of gas to the liquid phase is difficult. This, and other aspects of microalgal biofuels production have been reviewed [71].
Acknowledgements This work was part of the DOE Joint BioEnergy Institute (http:// www.jbei.org) supported by the U.S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract www.sciencedirect.com
DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U.S. Department of Energy.
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