HAA is formed as the most abundant linear carbonyl product from pyrolytic ring scission of holocellulose. It is the simplest aldehyde-alcohol or sugar, and can be used as an active meat-browning agent in food flavoring industry [39], or ingredient in cosmetic industry.

As been indicated above, during fast pyrolysis of biomass, the pyrolytic ring scission of holocellulose will be promoted by the small amounts of ash or alkaline cations [89], and hence, HAA can be formed with high yield in these conditions. Moreover, HAA could be further formed from the secondary cracking of the anhy — drosugar products. As a result, HAA is usually the most abundant single organic compound in crude bio-oils produced from raw biomass materials.

Similar as the LG, the difficulty for the preparation of HAA mainly lies in its recovery from pyrolysis liquids, due to its high reactivity. An efficient purification method was patented by Stradal and Underwood [90].

Although in laboratory experiments excellent results have been achieved and the technology possesses many potential benefit s over the conventional methods of processing biomass to biofuels or chemicals, there are certain issues, which need to be addressed.

— Biomass feeding at high pressure: As a “rule of thumb,” the solid loading in excess of 15-20 wt.% is considered economical on commercial point of view.

Feeding slurries at high pressure is always challenging especially for the lab scale studies since low capacity slurry pumps are rarely available. Pumping slurry at large scale is less of a problem, where progressive cavity or similar pumps are commercially available.

— Salt precipitation: Plugging of reactors caused by the precipitation of inorganic salts above supercritical temperature and low density conditions. At room tem­perature, water is an excellent solvent for most salts. On the other hand, solubility of most salts is very low (typically 1-100 ppm) in supercritical water (low density) and precipitating salts may plug the reactors even at high flow velocities [59]. However, the problem may be used as an opportunity to produce a valuable fertilizer by-product of the process, if managed properly.

— Corrosion: The halogens, such as sulfur or phosphorous, present in the organic matter are converted to the respective acids, which may cause severe corrosion on the reactor wall under harsh reaction conditions. The corrosion problem can be reduced or avoided by selecting the right material of construction and or a slightly modified reactor concept.

— Coking and deactivation of heterogeneous catalyst: Some catalyst supports degrade or oxidize in hydrothermal conditions. Decline in catalyst activity is also observed with long period of exposure of catalyst during continuous process [94].

Table 5 compiles the conversion level and selectivity under steady state at 300°C and 10 atm for the studied catalysts. It can be seen that for low promoter content (0.1 wt%), the conversion of CO increases from approximately 26% for the Co/SiO2 to values close to 32% for the promoted samples, except that promoted by Cu which exhibit a very low conversion level, close to 12%. In the series with 0.5 wt% of promoter the drop in the activity in the CoCu catalysts is even more drastic, reaching a conversion level of 8.3% and the CoZn catalyst also displays a decrease in the conversion compared with the monometallic Co/SiO2 catalyst. Even though the cobalt particle size is smaller in the promoted samples, specially in which the promoter content is higher, and the reduction degree is higher, therefore, it should be expected for an enhancement in the catalytic activity, in those samples pro­moted with Zn and Cu, the opposite behaviour was observed. This may be

interpreted as considering that in these samples the formation of spinel type oxides as a consequence of calcinations should be expected, thus reducing the extent of active sites. The presence of these mixed oxides species has already been reported for other systems. Other possibility for this behaviour is the blocking of the Co active sites by the copper itself. However, due to the small amount of promoter they were not detected by XPS.

The current fermentative production of butanol is not cost effective because of (1) a spore-forming life cycle, (2) butanol toxicity, (3) slow growth and instability of the producing strains and (4) production of other unwanted byproducts including butyrate, acetate, acetone, and ethanol [31]. In addition, no commercial microbes are available to ferment various lignocellulosic hydrolyzate mixtures into butanol. Thus, new microbes are needed for fermentative conversion of these hydrolyzates to butanol biofuel.

In the recent years, the fermentative production of butanol has been demonstrated in engineered strains of Escherichia coli [4, 30, 43] and Saccharomyces cerevisiae [71]. The entire butanol production pathway from Clostridium has been recon­structed and introduced into these model hosts. More recently, the pathway recon­struction strategy was applied to more robust and butanol tolerant species including Pseudomonasputida, Bacillus subtilis [43], Lactobacillus brevis [7], Lactobacillus buchneri [35], and Corynebacterium glutamicum [69]. Although the polycistronic expression of the butanol production pathway genes are achieved in these robust host cells, the butanol titers of these recombinant organisms are relatively low (Table 1) and has yet to exceed 19.50 g/L, a production level that can be achieved by Clostridium species [57] .

Three of the highest butanol producing strains are the engineered E. coli strains JCL187, EB4.F, and BUT2, which can produce 552, 580, and 1,200 mg/L of butanol, respectively (Table 1). Although the E. coli BUT2 was reported as producing more butanol, the cells were first grown aerobically and later resuspended for anaerobic fermentation. Furthermore, these strains suffer from butanol toxicity (very sensitive to butanol) and can be killed by the accumulation of no more than 15 g/L butanol [32]. So far, no breakthrough improvement ofbutanol production strains from ligno — cellulosic biomass hydrolyzates has been reported and yet, more research is needed for strain development.

Darby Harris, Carloalberto Petti, and Seth DeBolt

Abstract Most of the plant biomass is cell wall and therefore represents a renewable carbon source that could be exploited by humans for bioenergy and bioproducts. A thorough understanding of the type of cell wall being harvested and the molecules available will be crucial in developing the most efficient conversion processes. Herein, we review the structure, function, and biosynthesis of lignocellulosic biomass, paying particular attention to the most important bioresources present in the plant cell wall: cellulose, hemicellulose, and lignin. We also provide an update on key improvements being made to lignocellulosic biomass with respect to utilization as a second-generation biofuel and as a resource for bioproducts.

1 Terminology to Describe Cell Walls

Before we examine the details of cellulose, hemicellulose, and lignin biosynthesis we review some additional classification terms that describe the type of cell wall. Every plant cell forms a primary cell wall (PCW) early in the cell lifecycle that is continuously produced throughout the period of cell growth. The shape and morpho­genesis of plant cells are defined by the capacity of the PCW to constrain cellular turgor pressure in a directed and controlled manner thereby permitting anisotropic expansion during cell growth. All PCWs contain cellulose and a hydrated matrix consisting of hemicelluloses and pectins, with some structural proteins. Two distinctive types of PCWs, either Type I or Type II, have traditionally been described within the angiosperms based on polysaccharide composition [ 24]. However, accumulating evidence from other plant species, for example, Equisetum, suggests that PCWs are

D. Harris • C. Petti • S. DeBolt (*)

Department of Horticulture, N-318 Agricultural Science Center North, University of Kentucky, Lexington, KY 40506, USA e-mail: sdebo2@email. uky. edu

J. W. Lee (ed.), Advanced Biofuels and Bioproducts, DOI 10.1007/978-1-4614-3348-4_17, 281

© Springer Science+Business Media New York 2013

best described as falling within a continuum rather than into specific classes. For the sake of general discussion on PCWs, the traditional classification can be maintained, although keeping in mind that some plant species may be found at either extremes of a particular range.

In general, Type I PCWs are present in dicots and liliaceous monocots while Type II PCWs can be found in the cereals and other grasses. The main defining feature used in describing the differences between the two wall types is the particular class of hemicelluloses (HCs) found within these walls. HCs, as discussed below, are heterogeneous in nature with multiple classes represented in different cell types, which is contrary to cellulose, a homogenous polymer present in roughly the same configuration in all cell walls. Type I walls contain mostly the xyloglucan form of HCs embedded in a pectinaceous gel cross-linked to structural proteins [24]. Type II walls contain much less pectin and fewer proteins and their HCs are primarily glucuronoarabinoxylans (GAXs) and mixed-linkage (1,3), (1,4)-b-D-glucans embedded in an acidic polysaccharide network of highly substituted GAXs [24]. In addition to PCWs, all plants deposit a thick secondary cell wall (SCW) around certain cell types after cell growth has ceased. The SCW primarily contains cellulose, HCs, and the polyphenolic compound lignin which provides added strength, protection, and hydrophobicity to plant tissues. The SCW is also the primary component of wood cells found in trees. In typical angiosperm trees such as Populus spp., the SCW consists of three layers (S1, S2, and S3), which are collectively composed of approximately 45% cellulose, 25% HCs, and 20-25% lignin [2] . In terms of cell type, over 50% of poplar wood is composed of xylem fibers which in turn contain most of their mass in the S2 layer of the SCW, thus making this the main area of focus in attempts to modify wood properties [113]. While most herbaceous plants lack woody tissue, the SCW is generally much thicker and more energy dense than the PCW in these species. Therefore, an important overall consideration for crops being used as feedstocks for bioenergy, such as grasses and fast growing woody crops, is that a majority of their cell wall polysaccharides and lignin will be bound up in the more recalcitrant SCW tissues.

On the whole, the sequenced-based groupings do a good job clustering enzymes with like structures and activities (Table 1). However, the presence of multiple activ­ities within single GH families suggests that divergent evolution to new activities is not uncommon.[4] At the same time, structural comparisons show that a number of different GH families have the same fold ([58]; see Table 1, Fig. 3).

This is particularly true for (b/a)8-barrels (which can be decorated with addi­tional domains, Fig. 3), (a/a)6-barrels, and b-jelly rolls. It seems likely that the ubiquity of these folds reflect common, albeit more distant, evolutionary origins than are captured in the sequence families. To include structural similarity, a broader “clan” classification has been devised to group GH-families with the same fold ( [29] , also see [58]). Cellulases in different GH families with similar structures can be grouped into clans A, B, C, K, and M (Table 1). In addition, some GH families that have substantial numbers of characterized cellulases lack structural similarity to other GH families, and thus do not belong to a specific clan.

In another embodiment of the present invention, the host plant or cell is further modified to tame the Calvin cycle so that the host can directly produce liquid fuel ethanol instead of synthesizing starch, celluloses, and lignocelluloses that are often inefficient and hard for the biorefinery industry to use. According to the present invention, inactivation of starch-synthesis activity is achieved by suppressing the expression of any of the key enzymes, such as, starch synthase, glucose-1-phosphate (G-1-P) adenylyltransferase, phosphoglucomutase, and hexose-phosphate-isomerase of the starch-synthesis pathway which connects with the Calvin cycle (Fig. 4c).

Introduction of a genetically transmittable factor that can inhibit the starch-syn­thesis activity that is in competition with designer ethanol-production pathway(s) for the Calvin-cycle products can further enhance photosynthetic ethanol produc­tion. In a specific embodiment, a genetically encoded-able inhibitor (Fig. 6c) to the competitive starch-synthesis pathway is an interfering RNA (iRNA) molecule that specifically inhibits the synthesis of a starch-synthesis-pathway enzyme, for exam­ple, starch synthase, glucose-1-phosphate (G-1-P) adenylyltransferase, phosphoglu­comutase, and/or hexose-phosphate-isomerase. Figure 6d-f depicts examples of a designer iRNA gene. The DNA sequences encoding starch-synthase iRNA, glucose-

1- phosphate (G-1-P) adenylyltransferase iRNA, a phosphoglucomutase iRNA and/ or a G-P-isomerase iRNA, respectively, can be designed and synthesized based on RNA interference techniques known to those skilled in the art [ 11]. Generally speaking, an interfering RNA (iRNA) molecule is anti-sense but complementary to a normal mRNA of a particular protein (gene) so that such iRNA molecule can specifically bind with the normal mRNA of the particular gene, thus inhibiting (blocking) the translation of the gene-specific mRNA to protein [12, 13].

Supercritical Carbondioxide Extraction (SCE) is a process for the production of oil with high yields that do not use organic solvents. In this process, the oil is dissolved in CO2 and extracted from the plant material [95]. SCE method developed by Yan et al. [96] resulted in the actual extraction rate 37.45%; the final oil product con­tained 0.79 mg/g of KOH and 3.63 meq/g of peroxide value. Here, seeds of J. cur­cas were collected and powdered. The extraction pressure was 43 MPa, temperature for the extraction was 45°C, the flow rate of CO2 was 20 kg/h, and the extraction time was 80 min. Even though the cost of supercritical extraction methods was higher, the oil quality was the best and refining was not needed [96] .

In phototrophic cultures, microalgae can be grown in two distinct production systems: open pond systems and enclosed PBRs. These production units control the environmental (viz. light, temperature, pH, salinity, nutrient qualitative and quanti­tative profiles, and dissolved oxygen), biological impact factors (viz. predation, viruses, competition and growth of epiphytes) and operational factors (viz. hydrau­lic residence time, harvesting rate, gas transfer rate, mixing equipment, shear rates and light exposure) that affect the growth of microalgae [106]. Full-scale operations of both open ponds and enclosed PBRs for biofuel-directed microalgae production are still mostly in demonstration phase, hence, their long-term operation perfor­mance will be required to counter the many uncertainties that prevail [162].

3.1 Cation Exchange Capacity of Biochar Materials

The value of CEC was measured for the biochar materials made from the pelletized peanut hulls at pyrolysis temperature of 371, 402, 426, and 442°C. As shown in Fig. 3, the measurements demonstrated that the CEC value of the biochar materials is dependent on the pyrolysis temperature. Pyrolysis temperature of 402°C yielded the highest CEC value (18.2 meq/100 g) while the pyrolysis temperatures of 371, 426, and 442°C resulted in CEC values of 17.1, 16.1, and 13.9 meq/100 g, respec­tively. This experimental result indicated that it is important to control pyrolysis temperature for higher CEC value of biochar product. The optimal pyrolysis tem­perature for high-CEC biochar production is likely at around 400°C.