In general, cellulase activities measured using insoluble substrates are more likely to be relevant to large-scale biomass deconstruction than chemically modified, soluble chains. A variety of different insoluble substrates are used to measure cellulase activ­ities. These substrates differ in their biological sources and pretreatment methods, and as a result, they have different structural and physical properties (Table 2). As a result, different substrates are more appropriate for different types of enzymes.

By pretreatment with acid (typically phosphoric acid), insoluble cellulose preparations can be obtained that have decreased crystallinity, and thus are typically more susceptible to enzymatic digestion than crystalline substrates [22] . Although published protocols differ in subtle but important ways, high concentrations of phosphoric acid can produce PASC (for phosphoric acid swollen cellulose, [78]). By prehydrating Avicel prior to acid treatment, Zhang et al. prepared an amorphous cellulose of even higher reactivity, which they term regenerated amorphous cellu­lose (RAC, [82] ).

Cellulose substrates with high crystallinity include bacterial cellulose (BC), which has high DP values of 2,000-8,000, and a high 60-90% crystallinity index [37]. Microcrystalline cellulose, or “Avicel” PH, which is prepared by acid hydro­lysis of wood pulp, has a lower DP value (150-300) [37], although it retains a high level of crystallinity. Because it has a high ratio of free ends to accessible b-gluco — sidic bonds due to its lower DP, Avicel is especially suited for the measurement of exoglucanase activity from a crystalline substrate [ 82]. Finally, Whatman No. 1 filter paper, manufactured from cotton linters, is highly heterogeneous and is often used for measuring total cellulase activity.

Another key feature of the invention is the application of a genetic switch to control the expression of the designer butanol-producing pathway(s), as illustrated in Fig. 1. This switchability is accomplished through the use of an externally inducible pro­moter so that the designer transgenes are inducibly expressed under certain specific inducing conditions. Preferably, the promoter employed to control the expression of designer genes in a host is originated from the host itself or a closely related organ­ism. The activities and inducibility of a promoter in a host cell can be tested by placing the promoter in front of a reporting gene, introducing this reporter construct into the host tissue or cells by any of the known DNA delivery techniques, and assessing the expression of the reporter gene.

In a preferred embodiment, the inducible promoter used to control the expression of designer genes is a promoter that is inducible by anaerobiosis, i. e., active under anaero­bic conditions but inactive under aerobic conditions. A designer alga/plant organism can perform autotrophic photosynthesis using CO2 as the carbon source under aerobic conditions, and when the designer organism culture is grown and ready for photosyn­thetic butanol production, anaerobic conditions will be applied to turn on the promoter and the designer genes that encode a designer butanol-production pathway(s).

A number of promoters that become active under anaerobic conditions are suitable for use in the present invention. For example, the promoters of the hydro — genase genes (HydA1 (Hyd1) and HydA2, GenBank accession number: AJ308413, AF289201, AY090770) of C. reinhardtii. which is active under anaerobic condi­tions but inactive under aerobic conditions, can be used as an effective genetic switch to control the expression of the designer genes in a host alga, such as C. reinhardtii. In fact, Chlamydomonas cells contain several nuclear genes that are coordinately induced under anaerobic conditions. These include the hydroge- nase structural gene itself (Hydl), the Cyc6 gene encoding the apoprotein of Cytochrome C. . and the Cpxl gene encoding coprogen oxidase. The regulatory regions for the latter two have been well characterized, and a region of about 100 bp proves sufficient to confer regulation by anaerobiosis in synthetic gene constructs [7]. Although the above inducible algal promoters may be suitable for use in other plant hosts, especially in plants closely related to algae, the promoters of the homologous genes from these other plants, including higher plants, can be obtained and employed to control the expression of designer genes in those plants.

In another embodiment, the inducible promoter used in the present invention is an algal nitrate reductase (Nial) promoter, which is inducible by growth in a medium containing nitrate and repressed in a nitrate-deficient but ammonium-containing medium [8]. Therefore, the Nial (gene accession number AF203033) promoter can be selected for use to control the expression of the designer genes in an alga accord­ing to the concentration levels of nitrate and ammonium in a culture medium. Additional inducible promoters that can also be selected for use in the present inven­tion include, for example, the heat-shock protein promoter HSP70A [9] (accession number: DQ059999, AY456093, M98823), the promoter of CabII-1 gene (acces­sion number M24072), the promoter of Cal gene (accession number P20507), and the promoter of Ca2 gene (accession number P24258).

In the case of blue-green algae (oxyphotobacteria including cyanobacteria and oxychlorobacteria), there are also a number of inducible promoters that can be selected for use in the present invention. For example, the promoters of the anaerobic — responsive bidirectional hydrogenase hox genes of Nostoc sp. PCC 7120 (GenBank: BA000019), P. hollandica (GenBank: U88400; hoxUYH operon promoter), Synechocystis sp. strain PCC 6803 (CyanoBase: sll1220 and sll1223), S. elongatus PCC 6301 (CyanoBase: syc1235_c), A. platensis (GenBank: ABC26906), Cyanothece sp. CCY0110 (GenBank: ZP_01727419), and Synechococcus sp. PCC 7002 (GenBank: AAN03566), which are active under anaerobic conditions but inactive under aerobic conditions [ 10], can be used as an effective genetic switch to control the expression of the designer genes in a host oxyphotobacterium, such as Nostoc sp. PCC 7120, Synechocystis sp. strain PCC 6803, S. elongatus PCC 6301, Cyanothece sp. CCY0110, A. platensis, or Synechococcus sp. PCC 7002.

In another embodiment in creating switchable butanol-production designer organisms such as switchable designer oxyphotobacteria, the inducible promoter selected for use is a nitrite reductase (nirA) promoter, which is inducible by growth in a medium containing nitrate and repressed in a nitrate deficient but ammonium- containing medium [11, 12]. Therefore, the nirA promoter sequences can be selected

for use to control the expression of the designer genes in a number of oxyphotobacteria according to the concentration levels of nitrate and ammonium in a culture medium. The nirA promoter sequences that can be selected and modified for use include (but not limited to) the nirA promoters of the following oxyphotobacteria: S. elongatus PCC 6301 (GenBank: AP008231, region 355890-255950), Synechococcus sp. (GenBank: X67680.1, D16303.1, D12723.1, and D00677), Synechocystis sp. PCC 6803 (GenBank: NP_442378, BA000022, AB001339, D63999-D64006,

D90899-D90917), Anabaena sp. (GenBank: X99708.1), Nostoc sp. PCC 7120 (GenBank: BA000019.2 and AJ319648), Plectonema boryanum (GenBank: D31732.1), S. elongatus PCC 7942 (GenBank: P39661, CP000100.1), T. elongatus BP-1 (GenBank: BAC08901, NP_682139), Phormidium laminosum (GenBank: CAA79655,Q51879),M. laminosus(GenBank: ABD49353, ABD49351, ABD49349, ABD49347), Anabaena variabilis ATCC 29413 (GenBank: YP_325032), P. marinus str. MIT 9303 (GenBank: YP_001018981), Synechococcus sp. WH 8103 (GenBank: AAC17122), Synechococcus sp. WH 7805 (GenBank: ZP_01124915), and Cyanothece sp. CCY0110 (GenBank: ZP_01727861).

In yet another embodiment, an inducible promoter selected for use is the light — and heat-responsive chaperone gene groE promoter, which can be induced by heat and/or light [13]. A number of groE promoters such as the groES and groEL (chap­erones) promoters are available for use as an inducible promoter in controlling the expression of the designer butanol-production-pathway enzymes. The groE promoter sequences that can be selected and modified for use in one of the various embodi­ments include (but not limited to) the groES and/or groEL promoters of the follow­ing oxyphotobacteria: Synechocystis sp. (GenBank: D12677.1), Synechocystis sp. PCC 6803 (GenBank: BA000022.2), S. elongatus PCC 6301 (GenBank: AP008231.1), Synechococcus sp. (GenBank: M58751.1), S. elongatus PCC 7942 (GenBank: CP000100.1), Nostoc sp. PCC 7120 (GenBank: BA000019.2), A. vari­abilis ATCC 29413 (GenBank: CP000117.1), Anabaena sp. L-31 (GenBank: AF324500); T. elongatus BP-1 (CyanoBase: tll0185, tll0186), S. vulcanus (GenBank: D78139), Oscillatoria sp. NKBG091600 (GenBank: AF054630), P marinus MIT9313 (GenBank: BX572099), P. marinus str. MIT 9303 (GenBank: CP000554), P marinus str. MIT 9211 (GenBank: ZP_01006613), Synechococcus sp. WH8102 (GenBank: BX569690), Synechococcus sp. CC9605 (GenBank: CP000110), P marinus subsp. marinus str. CCMP1375 (GenBank: AE017126), and P. marinus MED4 (GenBank: BX548174).

Additional inducible promoters that can also be selected for use in the present invention include: for example, the metal (zinc)-inducible smt promoter of Synechococcus PCC 7942 [14] ; the iron-responsive idiA promoter of S. elongatus PCC 7942 [15]; the redox-responsive cyanobacterial crhR promoter [16]; the heat — shock gene hsp16.6 promoter of Synechocystis sp. PCC 6803 [17]; the small heat — shock protein (Hsp) promoter such as S. vulcanus gene hspA promoter [18]; the CO2-responsive promoters of oxyphotobacterial carbonic-anhydrase genes (GenBank: EAZ90903, EAZ90685, ZP_01624337, EAW33650, ABB 17341, AAT41924, CAO89711, ZP_00111671, YP_400464, AAC44830; and CyanoBase: all2929, PMT1568 slr0051, slr1347, and syc0167_c); the nitrate-reductase-gene (narB) promoters (such as GenBank accession numbers: BAC08907, NP_682145, AAO25121; ABI46326, YP_732075, BAB72570, NP_484656); the green/red light- responsive promoters such as the light-regulated cpcB2A2 promoter of Fremyella diplosiphon [19]; and the UV-light responsive promoters of cyanobacterial genes lexA, recA, and ruvB [20].

Furthermore, in one of the various embodiments, certain “semi-inducible” or constitutive promoters can also be selected for use in combination of an inducible promoter(s) for construction of a designer butanol-production pathway(s) as well. For example, the promoters of oxyphotobacterial Rubisco operon such as the rbcL genes (GenBank: X65960, ZP_01728542, Q3M674, BAF48766, NP_895035, 0907262A; CyanoBase: PMT1205, PMM0550, Pro0551, tll1506, SYNW1718, glr2156, alr1524, slr0009), which have certain light dependence but could be regarded almost as constitutive promoters, can also be selected for use in combina­tion of an inducible promoter(s) such as the nirA, hox, and/or groE promoters for construction of the designer butanol-production pathway(s) as well.

Throughout this specification, when reference is made to inducible promoter, such as, for example, any of the inducible promoters described earlier, it includes their analogs, functional derivatives, designer sequences, and combinations thereof. A “functional analog” or “modified designer sequence” in this context refers to a promoter sequence derived or modified (by, e. g., substitution, moderate deletion or addition or modification of nucleotides) based on a native promoter sequence, such as those identified hereinabove, that retains the function of the native promoter sequence.

Foidl et al. [26] reported a technical process for processing seed oil and production of methyl ester and ethyl ester from the oil of Jatropha seeds. The fuel properties were also determined. Production of biodiesel used two-step transesterification: alkali-alkali transesterification for methyl ester and alkali-acid transesterification for ethyl ester.

Chitra et al. [ 17] found that methyl ester yield of 98% was obtained using 20 wt.% methanol and 1.0% NaOH at 60°C. The maximum reaction time needed for a maxi­mum ester yield was 90 min. Total biodiesel of 96% was obtained from experimental studies on large-scale production (reactor capacity of 75 kg). Esterification — transesterification reaction for Jatropha biodiesel was done by Sudradjat et al. [83, 84], but methyl ester yield was not reported.

Tiwari et al. [88] and Berchmans and Hirata [14] have developed a technique to produce biodiesel from Jatropha with high FFA contents (15% FFA). They selected two-stage transesterification processes to improve methyl ester yield. The first stage involved the acid pretreatment process to reduce the FFA level of crude Jatropha seed oil to less than 1%. The second was the alkali base-catalyzed transesterification process resulting in 90% methyl ester yield. Tiwari et al. [88] found that the opti­mum combination to reduce the FFA of Jatropha oil from 14% to less than 1% was 1.43% v/v H2SO4 acid catalyst, 0.28 v/v methanol-to-oil ratio, and 88-min reaction time at a reaction temperature of 60°C. This process produced yield of biodiesel of more than 99%.

Berchmans and Hirata [14] reduced the high FFA level of Jatropha oil to less than 1% by a two-step process. The first step was carried out with 0.60 w/w metha — nol-to-oil ratio in the presence of 1% w/w H2SO4 as an acid catalyst in 1 h reaction at 50°C. The second step was transesterified using 0.24 w/w methanol to oil and 1.4% w/w NaOH to oil as alkaline catalyst to produce biodiesel at 65°C. The final yield for methyl esters was achieved ca. 90% in 2 h.

1.2 Bio-mitigation of CO2 Emission

The rationale for enhancement of strategies for mitigation of CO2 emissions related to energy production and utilisation has been outlined in a large body of environ­mental science research and the underpinning policy considerations pertaining to sustainable energy systems [83, 216]. Methods for CO2 capture and storage include the use of physiochemical absorbents, injection into deep oceans and geological formations (e. g. saline aquifers and deep ocean basalt), and biological fixation [17]. However, it could be argued that biological fixation is the only economical and environmentally sustainable long-term strategy for CO2 mitigation because the other strategies incur high cost and space requirements, and there are risks of CO2 leakage over time [183] . It has also been argued that carbon capture and storage might be the easier technique for controlling GHG, than building energy conversion systems that do not emit them. Consequently, hypotheses for “artificial trees” that capture CO2 much faster than terrestrial plants have been advanced [107].

Microalgae can be used for the biological capture and storage of CO2 as they readily consume CO2 during photosynthesis with a more efficient system than ter­restrial energy crops [15, 218]. They can be used to capture CO2 from three different sources, namely: the atmospheric CO2, emission from power plants and industrial processes employing fossil fuels, and the CO2 from soluble carbonate [218]. Capture of atmospheric CO2 is the most basic carbon mitigation strategy that can be coupled to microalgae production systems. However, due to the relatively low concentration of atmospheric CO. (ca. 360 ppm), the potential biomass yield is limited, which makes it uneconomical [ 198]. The CO2 emissions in the flue gases from power plants or industrial processes are of higher concentrations, usually 10-14% [207], and therefore offer better mitigation potential, with commensurate increase in algal biomass production. The process is practicable for both PBR and raceway pond microalgae production systems [17]. However, only a limited number of algae species are tolerant to the typical levels of SOX and NOX and the high temperatures of flue gases. A few species have the ability to assimilate CO2 from soluble carbon­ates such as Na2CO3 and NaHCO3 [218].

The selection of suitable microalgae strains has a significant effect on efficacy and cost-competitive of the CO2 bio-mitigation process. Table 5 provides experi­mental data on ranges of known characteristics of selected species that have been

studied. From the data, it can be seen that a wide range of species are capable of growing under enhanced CO2 and flue gas conditions. The data for Chlorella sp. also show that carbon utilisation efficiency of the microalgae decreases with increase in CO2 concentration in the flue gas. This would imply that, flue gas with higher CO2 concentration may necessitate a recirculation through the algae production system to attain the same level of CO2 extraction, or in order to meet a set level of residual CO2 in the exhaust.

Annual precipitation in the coastal plain region of South Carolina is high enough for crop production (1,310 mm, [86]). However, erratic rainfall with dry spells of a few days to a few weeks [16, 85] reduces production as seen in (Fig. 6) where less than 5 mm of rainfall was recorded in June 2008 in Darlington SC during the corn growing season (April to July). Low rainfall caused crop moisture stress to occur (Fig. 6, left), resulting in low corn yields (3.8-4.7 Mg ha-1). In contrast, rainfall was sufficient during the 2009 corn growing season (Fig. 6, right) and yield was double the drought year (8.4-9.3 Mg ha-1).

Low water storage [18, 79] and poorly aggregated, hard layers, that restricts root penetration to the top 25-30 cm of the soil profil e [36] limit soil water holding capacity to »22.5 mm [37, 81]. During the hot summer, evapotranspiration rates of 16.8 mm day-1 for soybeans [82] will use this in less than 2 days. Unless water is replenished with rain or irrigation, crops will stress. In contrast, a finer-textured

SOC-enriched Coxville soil series can have between 29 and 51 mm of available H2O per 300 mm of soil [79]. Under similar conditions, a soybean crop growing in the Coxville soil would have more time (1.7-3 days) before soil water is depleted.

1.1 Cellulose Fast Pyrolysis

Cellulose is the main component of lignocellulosic biomass and is predominantly located in the cell wall. It is a linear homopolysaccharide of b-D-glucopyranose units linked together by (1^4)-glycosidic bonds. Among the three major compo­nents of lignocellulosic biomass, the cellulose has received the most attention in its pyrolytic mechanism study.

Cellulose starts pyrolysis at as low as 150°C. At temperatures lower than 300°C, pyrolysis of cellulose mainly involves the reduction in degree of polymerization, the formation of free radicals, elimination of water, formation of carbonyl, carboxyl and hydroperoxide groups, and evolution of carbon monoxide and carbon dioxide, finally leaving a charred residue [31, 86]. The low temperature pyrolysis will pro­duce very low yield of organic liquid products.

At temperatures above 300°C, the pyrolysis of cellulose involves many new reactions, mainly leading to a liquid product with the yield as high as 87 wt% [72]. Generally, cellulose is firstly decomposed to form activated cellulose [15]. Afterwards, two major parallel pathways will take place, the depolymerization and the fragmentation (ring scission), as shown in Fig. 1. The depolymerization process mainly forms anhydro-oligosaccharides, levoglucosan (LG) and other monomeric anhydrosugars, furans, cyclopentanones, pyrans, and other related derivatives. The ring scission process mainly obtains hydroxyacetaldehyde (HAA), acetol (HA), other linear carbonyls, linear alcohols, esters, and other products [46, 47, 71, 79]. In a recent study, Shen and Gu [87] proposed the detailed possible routes for the pyro­lytic formation of several major products from cellulose, as shown in Fig. 2.

The use of sub — and supercritical water media also known as hydrothermal media, which can be broadly defined as water-rich phase above 200°C, offers several advantages over the other biofuels production methods [94]. Some of the major benefits are

— Ability to wet biomass

— Can use mixed feedstock or waste biomass from other process residues

— High energy and separation efficiency (since water remains in liquid phase and the phase change is avoided)

— High throughputs

— Versatility of chemistry (solid, liquid, and gaseous fuels)

— Reduced mass transfer resistance

— Improved selectivity for the desired energy products (methane, hydrogen, liquid fuel) or biochemicals (sugars, furfural, organic acids, etc.)

I

cmperature ( C)

Fig. 8 Application referenced to pressure-temperature phase diagram of water

— No need to maintain specialized microbial cultures

— Products are completely sterilized with respect to any pathogens including biotoxins, bacteria, or viruses

— Processing of postfermentation residues

Since the process is conducted in liquid/water-rich phase, the energy required for phase change of water from liquid to vapor is avoided. This provides an opportunity to reduce the requirement of process heat compared to steam-based processes. As an example, we know that 2.869 MJ/kg of energy is required to convert ambient water from 25°C to steam at 250°C and 0.1 MPa whereas only 0.976 MJ/kg of energy is required to convert ambient water from 25°C to subcritical water at 250°C and 5 MPa. This energy need for heating water to subcritical condition (0.976 MJ/kg) is equivalent to 6-8% of energy contained in dry biomass. Generally, the higher heating value of dry biomass is in the range of 16-19 MJ/kg. This also means that the energy contained in the subcritical water is insufficient to vaporize the water on decom­pression. Further, it is possible to recover much of the heat from subcritical water. The technology can be applied to produce solid (biochar), liquid (bioethanol, biocrude/bio-oil), and gaseous (methane, hydrogen) fuels from biomass depending on the processing temperature and pressure as shown in Fig. 8.

The substantial changes in the physical and chemical properties of water in the vicinity of its critical point can be utilized advantageously for converting lignocel — lulosic biomass to desired biofuels [22, 94] . In fact, reactions in subcritical and supercritical water also provide a novel medium to conduct tunable reactions for the synthesis of specialty chemicals from biomass [76] .

In the subcritical region, the ionization constant (Kw) of water increases with temperature and is about three orders of magnitude higher than that of ambient water (Fig. 7) and the dielectric constant (e) of water drops from 80 to 20 [118].

A low dielectric constant allows subcritical water to dissolve organic compounds, while a high ionization constant allows subcritical water to provide an acidic
medium for the hydrolysis reactions. These ionic reactions can be dominant because of the liquid-like properties of subcritical water. Moreover, the physical properties of water, such as viscosity, density, dielectric constant, and ionic product, can be tuned by small changes in pressure and/or temperature in subcritical region [31, 79, 103]. In the supercritical region, density of water drops down to lower value. This means that ionic product of water is much lower and ionic reactions are inhibited because of the low relative dielectric constant of water. The lower density favors free-radical reactions, which may be favorable for gasification [61].

In the front-end approach, the scale of the plant is dependent upon the scale of the gasifier. The principal idea in this approach is that FT liquid, heat and electricity are the desired products. This is generally used for the gasifier of the size 1-100 MWth

[45] . In this approach, the off-gases from FT reactor are used to generate heat and electricity in a combined cycle. The fundamental assumptions and other elements that justify this approach are:

1. The gasifier is small and air blown.

2. Once pass through FT reactor operation to avoid accumulations of inert like N2 (pres­ent at 40 vol.% in biosyngas, CO2, CH4, and other gaseous C1 to C4 FT products).

3. No adjustment of H2/CO ratio via WGS reaction and CO2 removal thus reducing the cost of gas conditioning.

4. H2 concentration is the limiting factor for the conversion of once-through FT synthesis. The unconverted H2, CO and other hydrocarbons are used to generate heat and electricity. The conversion to liquid is, therefore, low. When the purpose of the tri-generation plant is to improve the production of FT liquid, a shift step is introduced as a part of the overall system. This step allows to maximize the total yield of H2 + CO in the FT synthesis.

5. The basic gas conditioning system will include the removal of tars and BTX by OLGA unit, the removal of inorganic impurities by wet gas cleaning technique, and the removal of H2S and remaining trace impurities by ZnO and active carbon filters.

The methods to prepare precipitated Fe catalyst and to test catalyst activity were given in previous works [40, 42]. The studied catalysts are expressed as ZlKmCnl Fe. L, m, and n are the nominal mass percent of promoter Zn, K, and Cu relative to Fe2O3, respectively.

The program for CO.-TPD was to reduce 0.1 g catalyst in 95% CO/Ar at 573 K for 4 h and then purge it with He for 1 h at the same temperature. After the catalyst was cooled to 303 K in He atmosphere, it was exposed to 0.1 MPa CO2 for 1 h. The CO2-TPD was done with He as carrying gas and the catalyst was heated in 10 K min" 1 to 1,073 K. Some of the exit gas was inducted into a differentially pumped atmospheric sampling system connected to a quadrupole mass spectrom­eter (LEDA-MASS Ltd.). The measurable partial pressure range is 10-5 ~ 10-11 Torr with Faraday detector.

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The studies on butanol production employing a SSFR process were performed using wheat straw as a substrate [60] . In this process wheat straw was pretreated with dilute sulfuric acid at 121°C for 1 h followed by cooling the mixture to 45°C and adjusting pH to 5.0 with concentrated NaOH solution. During these studies a num­ber of experiments were performed with the following conclusions: (1) presence of sediments in the reactor does not inhibit fermentation; (2) agitation by gas stripping was necessary to improve mass transfer which helped wheat straw to hydrolyze to near completion and remove butanol simultaneously; and (3) hydrolysis of wheat straw to sugars using enzymes was slower than sugar utilization by the culture to produce butanol and often the culture was found deficient in sugar. This may have been due to different optimum temperatures for enzymes (45°C) and butanol fer­mentation (35°C). Although, SSFR in a batch reactor was successful, it had some problems that are listed below: (1) aseptic transfer of pretreated wheat straw to the bioreactor was difficult, introducing the possibility of contamination; (2) liquid sampling from the reactor was problematic due to the presence of significant amount of solids in the reactor; and (3) axial agitation of biomass and cell broth affects the culture negatively and hence agitation by gas stripping was considered as an option. While use of gas stripping was helpful, rate of butanol removal from the broth was low thus requiring a large amount of gas recycle. In a batch reactor where SSFR was applied a productivity of 0.31 g/L h was observed (when wheat straw was used) as compared to 0.30 g/L h when glucose was used as a substrate. In this integrated batch process 21.42 g/L total ABE was produced from 86 g/L wheat straw. This system resulted in an ABE yield of 0.37 (g ABE/g sugar released) based on 95% hydrolysis of wheat straw (Table 4).