Solid-state 13C NMR spectroscopy has been frequently employed to characterize organic matter. The most-commonly used technique is 13C CP/MAS. In this tech­nique, the magnetization from abundant ‘H is transferred to dilute 1 3C by cross polarization and then 13C magnetization is detected [20]. Its recycle delays depend on 7]H (‘H spin-lattice relaxation time), rather than T1C (13C spin-lattice relaxation time) [15]. Usually, T1H is much shorter than Tf. Therefore, this technique has the advantages of both enhancing sensitivity and shortening recycle delays. The combi­nation of these two factors substantially reduces the measurement time. Despite its advantages, CP has inherent shortcomings which render it unreliable for biochar characterization, especially for quantitative characterization. For CP/MAS, CP efficiency is reduced for nonprotonated carbons, mobile components, or regions having short proton rotating-frame spin-lattice relaxation time (7^) [16]. Note that biochars usually contain significant nonprotonated fused-ring aromatic carbons. Fused-ring aromatic carbons are far away from protons, cannot cross polarize well with ‘H, and are thus difficult to detect by CP. Also, the biochars produced via pyrolysis contain significant radicals which shorten [8]. These two problems

cause significant signal loss, especially for fused-ring aromatics in biochars. Compared with quantitative DP, 13C CP/MAS can only detect approximately 1/3 the aromaticity that ‘ 3C DP/MAS can detect [3]. Solid-state 13C CP/MAS with either regular CP or ramp CP cannot be used to quantify chars [15]. In extreme cases, 13C CP/MAS may not even be able to provide qualitative structural information of chars.

The following operating costs may be incurred by a biochar production business:

• Management, administration, monitoring, and reporting.

• Operations staff.

• Maintenance, service agreements, and sustaining capital.

• Debt servicing.

• Insurance.

• Transport of feedstocks and products.

• Consumables.

• Energy requirements (start-up, shut-down, and sustaining).

The magnitude of the project operating costs varies greatly depending on the loca­tion of the project and the regulatory regime it is subjected to. For example, in a developed country the cost of human resources is likely to be one of, if not, the most significant contributors to operating costs. However, in a developing country these resources come at a significantly lower cost. Likewise, in developing countries the level of administration, monitoring, and reporting required to meet government requirements could also be less and therefore represent a decreased operating cost.

Furfural (FF) is a typical pyrolytic product formed from both of cellulose and hemi — cellulose. It is widely used as an organic solvent or an organic reagent for the produc­tion of medicines, resins, food additives, fuel additives, and other special chemicals. Currently, FF is industrially produced from agricultural raw materials rich in pentosan. By aqueous acid catalysis (e. g., sulfuric acid or phosphoric acid), the pentosan is firstly hydrolyzed to pentose which is then dehydrated to form FF [96].

Similar as the LGO and LAC, the formation of FF can be promoted in the acid — catalyzed pyrolysis of biomass [2, 29, 30, 50, 85, 89]. Zinc chloride (ZnCl2) is one of the promising catalysts to produce FF. It was found that fast pyrolysis of cellulose impregnated with small amounts of ZnCl2 (below 10 wt%) would generate several dehydrated products as the primary pyrolytic products, mainly the FF, LGO, LAC, and 1,4:3,6-dianhydro-a-D-glucopyranose (DGP). With the increasing of ZnCl2 impregnation (at least 15 wt% or more), the ZnCl2 catalysis would increase the FF formation, while decrease the anhydrosugars. Moreover, the secondary catalytic cracking of the primary pyrolysis vapors by ZnCl2 could promote the conversion of LGO and other anhydrosugars to FF, leaving the FF as the only predominant product (Fig. 7). Compared with the cellulose, the ZnCl2-catalzyed fast pyrolysis of xylan would obtain the FF as the only predominant product, and the FF yield would be higher than that from cellulose, indicating that biomass rich xylan would be suitable for the FF production.

In another study, microwave-assisted fast pyrolysis was applied to treat biomass mixed with various catalysts, and the results revealed that the MgCl2 exhibited high selectivity on the FF production [93]. The highest FF content was more than 80% (peak area% on the GC/MS ion chromatograms) at the 8 g MgCl2 mixed with 100 g biomass.

In addition, ZnCl2 (or sometimes MgCl2 ) is an effective chemical activation agent for the production of activated carbons from biomass. Hence, for the biomass pretreated by ZnCl2 (or MgCl2) impregnation, they can be firstly subjected to fast pyrolysis to produce FF. The solid residues which contained char and ZnCl) (or MgCl2) could be further activated to produce activated carbons, so as to achieve the coproduction of FF and activated carbons.

In general, conventional higher land plants are not very efficient in capturing solar energy. Even the fastest growing energy crops can convert solar energy to biomass at a yearly rate of no more than 1 W/m2 [133]. However, the biomass productivity of microalgae, a photosynthetic microorganism, can be 50 times greater than switch — grass [20]. Microalgae grow in marine and freshwater environments. Due to their simple cellular structure and submergence in an aqueous environment where they are in vicinity of water, CO2 , and other nutrients, microalgae are generally more efficient in converting solar energy into biomass. Microalgae can be used to pro­duce wide range of second-generation biofuels and bioactive compounds [47, 106]. They offer many potential advantages [14, 106, 133] over conventional biomass sources. Microalgae primarily comprise of varying proportion of proteins, carbohy­drates, lipids, and ash. The percentages vary depending upon the species. Table 5 presents the general composition of different microalgae. What really makes algal biomass feasible for biofuels production is the fact that many forms of algae have

very high lipid contents. The biomass that is nonlipid provides a high-value co­product such as animal feed or fertilizer that offsets the cost of converting the algae to fuels. Growing algae also removes nitrogen and phosphorus from water and con­sumes atmospheric CO2.

1.2 Characterization of Monometallic Systems

The SBET and total pore volume (obtained at 0.95 of P/P°) of calcined Co(x)/SiO2 and Fe(x)/SiO2 catalysts are shown in Table 1. As shown, the SBET decreases slightly with the Co and Fe loading in both systems. This result suggests that Co and Fe species were highly dispersed into the pores of the silica substrate and that pore blockage was almost absent.

In Table 1, the cobalt and iron particle average size estimated from TEM micro­graphs of Co(x)/SiO2 and Fe(x)/SiO2 catalysts are summarized. In general, both catalyst systems present a broadening in the metal particles size distribution upon increasing Co and Fe loading and the average particle was found to increase gradu­ally with metals loading from 37 to 52 nm and from 29 to 40 nm for Co and Fe, respectively. Also, Table 1 shows that Fe catalysts display slightly lower metal par­ticles size comparing to Co counterpart.

TPR profiles of the oxide precursors for both catalytic systems are given in Fig. 1 and show that the reduction process of the Co(x)/SiO2 catalysts occurs in two distinct stages, while for the Fe(x)/SiO2 catalysts occurs according to the three char­acteristic steps of the reduction of Fe2O3 species. For the Co(x)/SiO2 catalysts the first peak centred at 350°C is ascribed to the transformation of Co3O4 to CoO, whereas the second stage centred to 406°C represents the reduction of CoO to Co [20, 21]. The relative intensity and width of the second reduction peak increases with Co-loading in a higher extent than the first peak, suggesting a higher reduction degree of CoO to metallic Co with an increase of the average diameter of Co3 O4 particles, in agreement with results reported previously by Martinez et al. [22] . This is also in agreement with those results obtained by TEM. On the other hand, in Fig. 1b, the first peak centred at 420°C in the Fe(x)/SiO2 catalysts is related to the transformation of Fe2O3 to Fe3O4, the second peak centred to 615°C represents the reduction of Fe3O4 to FeO and the third peak centred around 720°C corresponds to

the transformation of FeO to metallic Fe [23, 24]. The intensities of the three peaks gradually increase with the Fe loading. Figure 1b also shows that the position of the maximum reduction of three peaks does not change significantly as iron loading increases. This observation suggests that iron species are homogeneously dispersed on the surface of the silica carrier.

XPS results of reduced Fe(x)/SiO2 and Co(x)/SiO2 catalysts are summarized in Table 2. XP spectra showed that all catalysts in the Fe 2p and Co 2p region display the doublet corresponding to Fe 2p3/2-Fe 2p1/2 and Co 2p3/2-Co 2p1/2 for iron and cobalt species, respectively. Table 2 summarizes the most intense peaks of each doublet. Thus, the peak at 707.3 eV represents a signal due to metallic iron (2p3/2) [25] and the peak at710.5.3 eV is due to iron oxide (2p3/2) [26]. Due to no satellite line is observed somewhere around 719.0 eV indicative of the presence of Fe3+ ions, it is inferred that the iron oxides responsible for the peak around 710.5 eV in the reduced catalysts comes from partially reduced iron oxides, such as Fe3O4 (magne­tite) species. The relative intensities of the two Fe 2p components (peaks at 707.3 and 710.5 eV) are also included in Table 2 (in parentheses). It can be seen that the fraction of metallic iron determined on the surface region of these catalysts is much lower than the fraction of Fe oxides. In addition, the fraction of reduced iron to metallic state (peak at 707.3 eV) increases upon increasing the iron loading in the catalysts. This behaviour suggests that at low Fe content, the ionic Fe species strongly interact with the SiO2 surface and therefore are difficult to reduce to the metal state under the experimental conditions of this work. On the contrary, in the catalysts with higher Fe loadings a higher proportion of tridimensional iron oxide structures are developed and therefore they can be easily reduced to zero valent

oxidation state. On the other hand, the most intense Co2p3/2 peak was fitted to two components: one at 778.0 eV belonging to metallic cobalt [27] and at 780.0 eV originated from cobalt oxide [28]. The relative intensities of the two Co 2p peaks were calculated and the values obtained are also showed in Table 2. It can be seen that the proportion of Co metallic phase increases gradually with Co-loading whereas the cobalt oxide phase follows an opposite trend. This behaviour suggests that at low Co content Co+2 interacts strongly with the SiO2 support and is not completely reduced to metallic cobalt. Conversely, at higher Co-loading, cobalt is present in the form of larger particles which are easier to reduce.

In order to examine the extent of dispersion of the active phase over the silica surface, the Fe/Si and Co/Si atomic ratio were calculated (see Table 2). The variation of the Fe/Si and Co/Si atomic surface ratio as a function of metal-loading of the catalysts is shown in Fig. 2. Clearly, the Fe phase appears as rather large crystallites (around 32 nm) up to 15% of Fe. This trend is similar to that found with Co(x)/SiO2 catalysts, where the activity increased almost linearly as Co-loading increases, reaching the maximum at about 20 wt% Co (around of 41 nm) and then levelled off. The observed deviation from linearity above 15% of Fe and 20% of Co suggests the formation of large segregated crystalline particles mainly on the external surface of the silica particles. The higher Fe/Si ratios were observed at higher Fe loading, especially for Fe(20)/SiO2 and Fe(25)/SiO2 catalysts. These results are likely due to the presence of a high density iron oxide particles and therefore keep less exposed the silica surface to incident photons. Similar behaviour was observed for Co(25)/ SiO2 and Co(30)/SiO2 catalysts.

We also measured reactive performance of precipitated Fe catalysts for CO2 hydrogenation and the results are shown in Table 3.

Comparing the results in Table 3 to Table 2 , it can be found that the reactive performances of studied catalysts depend on reactant composition. For example, catalyst Z6K4C8/FS10-I is more active than Z6K2C2/FS15-I for CO hydrogena­tion, while the former is weaker than the latter to convert reactant in the case of CO2 hydrogenation.

We had found that low K content is helpful to increase hydrocarbon yield and Fe catalyst with high Zn/K ratio shows high C2 + hydrocarbon selectivity for CO2 hydrogenation at lower reaction temperature [42]. The results of catalyst Z6K2C6/ FS15-I in Table 3 still support the above conclusions after SiO2 was introduced into precipitated Fe catalyst. Although the content of promoter K in it is the lowest among studied catalysts, more CO2 is hydrogenated into hydrocarbons rather than terminated as CO. It supports that the CO2 is able to be activated by suitable promoter(s) for hydrocarbon synthesis at lower temperature [42], too. The effect of H2/CO2 ratio on reactive performance of catalyst Z6K4C8/FS15-II is evident according to the results in Table 3. After the H2/CO2 ratio is increased to 5, more hydrocarbon is synthesized from CO2, and most liquid products are in C6-C10 range.

1.4 Conclusions

SiO2 is used commonly as structure promoter for precipitated Fe catalyst in order to enhance its mechanical strength besides to increase its specific surface area. The influences of introducing method and the content of SiO2 on the reactive perfor­mance of Fe catalyst were studied under CO + H2 and CO2 + H2, respectively.

Although the amount of effective potassium is decreased by the introduced SiO2, the correlation between promoter composition and catalyst reactivity found for SiO2-free Fe catalysts is still in effect for SiO2-added Fe catalysts. It is beneficial to improve precipitated Fe catalysts for FT synthesis with CO2-containing syngas.

2 Perspectives

In order to develop precipitated Fe catalyst active to convert CO2-containing syngas, we decompose the work into three steps. The first step is to develop Fe catalyst active to convert CO + H2. The second one is to exploit Fe catalyst propitious for CO2 hydro­genation or to activate CO2 into CO. The third step is to set up guidance on how to couple the above two kinds of catalysts according to contents of CO2 and CO in reactants in order to convert effectively CO2-containing syngas into liquid fuels.

By now, the first step is nearly completed, and several Fe catalysts are found with high CO conversion during FT synthesis reaction. Much work is being done for the second aim. We have observed the relation between the pattern of CO2 — TPD and CO2 hydrogenation activity for Fe catalysts promoted with Zn, K, and Cu. It helps us to find Fe catalysts active to hydrogenate CO2 at low temperature. However, new characteristic tool or method is needed to accelerate making up ideal Fe catalyst after SiO2 is introduced into catalyst as support or binder. Based on the catalysts selected in the first two steps, it will be promise to complete the third step and realize acquiring liquid fuels efficiently from CO2-containing syngas.

Acknowledgments This work is partially supported by the Science and Technology Department of Zhejiang Province (2009C21002), Zhejiang Provincial Natural Science Foundation of China (Y4100410) and National Ministry of Science and Technology of China (2009AA05Z435).

The basic premise of the native ethanologen CBP approach is to start with a robust ethanologen, and engineer in it the ability to produce and secrete a suite of cellulolytic enzymes. This approach generally takes advantage of already having an excellent ethanol production system with ethanol tolerance mechanisms in place, and either natural or engineered abilities to utilize the major biomass-derived sugars including xylose and arabinose. With the metabolic foundation intact, the strain development effort can focus solely on producing large amounts of cellulases and directing them to the extracellular space. However, these related processes represent distinct chal­lenges in and of themselves, and can indeed be quite complex. While most attempts at pursuing this route have expressed one to several separate cellulases, a native cellulose-degrading organism will generally produce dozens of distinct enzymes in order to achieve this task. For example, T. reesei a cellulolytic fungi with the pre­miere cellulose-degrading enzyme, CBHI [32], expresses 10 cellulose-degrading and 16 hemicellulose-degrading enzymes [12]. Optimizing the functional expres­sion of many enzymes can be complicated as it includes transcription, translation, peptide folding, and ensuring protein stability. Further complicating the case is that many enzymes, particularly of fungal origin, need post-translational modifications including glycosylation to achieve maximal activity or stability [33-35]. In addi­tion, the enzymes must be targeted either directly or indirectly to be secreted extra­cellularly. Of course, the advantage that recombinant CBP organisms have over cellulolytic organisms in nature is that they are fed a partially deconstructed sub­strate due to thermochemical pretreatment, making the task of cellulose and hemi- cellulose depolymerization less daunting, and requiring fewer enzymes. At an absolute minimum, an endoglucanase, an exoglucanase (cellobiohydrolase) and a b-glucosidase will be required to fully depolymerize cellulose [36], and very likely additional accessory enzymes will be required, making the expression of sufficient enzymes a challenging task.

There are numerous ethanologens that have been considered as candidates for conversion to a CBP organism including Escherichia coli [7, 37, 38], Klebsiella oxytoca [7, 39-41], Z. mobilis [42-47], and several yeasts including S. cerevisiae [6, 9, 48-50]. Given their strong representation in the cellulosic biofuels research landscape, we will discuss S. cerevisiae and Z. mobilis in more detail below.

For cellulases with well-characterized enzymatic properties, a classification scheme could be created that captures the reactivities described above. However, there are a large number of cellulase sequences that have been identified from DNA sequencing that have not been characterized biochemically. To include these new sequences in a classification scheme, and to represent the evolutionary relationships that connect them, a system has been developed [26] and refined [27, 28] that groups like sequences together. This information is available in a searchable database (CAZy, for carbohydrate-active enzyme database; http://www. cazy. org/) that includes structural, functional, and phylogenetic information [7]. At the time of this writing, CAZy currently subdivides the glycohydrolase sequences (EC 3.2.1.x) into 122 distinct sequence families, designated GH1-GH122 (and 956 additional nonclassified sequences).

The GH families that are tagged with relevant EC numbers for cellulose degrada­tion (EC 3.2.1.4, EC 3.2.1.21, and EC 3.2.1.91) are shown in Table 1. Although there are 21 families that contain one or more members from one of these three EC groups, the bulk of the cellulases are in GH families 5, 6, 7, 8, 9, 12, and 44, 45. GH families 5, 8, 9, 12, 44, and 45 are largely composed of endocellulases (at the exclu­sion of exocellulases), whereas GH families 6 and 7 include both endocellulases and exocellulases (Table 1); exocellulases in GH-6 act on the nonreducing end, whereas exocellulases in GH-7 act on the reducing end. There do not appear to be GH families composed exclusively of exocellulases. Nearly all b-glucosidases are in GH families 1 and 3.

Another key feature of the invention is the application of a genetic switch to control the expression of the designer ethanol-producing pathway(s), as illustrated in Fig. 2. 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 (Fig. 6a) . 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 reported 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 (Fig. 4a), and when the designer organism culture is grown and ready for photosynthetic ethanol production, anaerobic conditions will be applied to turn on the promoter and the designer genes (Fig. 4b).

A number of promoters that become active under anaerobic conditions are suit­able for use in the present invention. For example, the promoters of the hydrogenase 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 hydrogenase structural gene itself (Hyd1), the Cyc6 gene encoding the apoprotein of Cytochrome C6, 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 [ 5]. 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 (Nia1) promoter, which is inducible by growth in a medium containing nitrate and repressed in a nitrate-deficient but ammonium-containing medium [6]. Therefore, the Nia1 (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 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 [7] (accession number: DQ059999, AY456093, M98823), the promoter of CabII-1 gene (accession number M24072), the promoter of Ca1 gene (accession number P20507), and the promoter of Ca2 gene (accession number P24258). Throughout this specification, when refer­ence is made to inducible promoter, such as, for example, any of the inducible pro­moters described above, 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.

Aqueous oil extraction (AOE) is a method in which Jatropha seeds are cracked and the shells are carefully removed. Shah et al. [ 78] used Jatropha kernels for oil extraction. The suspension was prepared with powdered (obtained by using a homogenizer) Jatropha kernels in distilled water, and then was incubated at desired temperature with constant shaking at 100 rpm for specified time period. The upper oil phase was collected after a centrifugation at 10,000 x g for 20 min. Enzyme- assisted AOE was performed similar to AOE. The difference is that the prepara­tions, i. e., Protizymee, Cellulase, Pectinex Ultra SP-L, Promozyme, as well as mixture of all these enzymes, were added after the pH of the suspension was adjusted. The amounts of oil obtained were calculated as the percentage of total oil present in Jatropha kernels.

It is found that the use of ultrasonication as a pretreatment before aqueous oil extraction and aqueous enzymatic oil extraction is useful in the case of extraction of oil from the seeds of J. curcas L. [78]. The use of ultrasonication for a period of 10 min at pH 9.0 followed by AOE resulted in a yield of 67% of available oil. The maximum yield of 74% was obtained by ultrasonication for 5 min followed by aqueous enzymatic oil extraction using an alkaline protease at pH 9.0 (44 g oil/100 g Jatropha kernels was taken as 100% recovery). Use of ultrasonication can also reduce the process time from 18 to 6 h [78].

To obtain an optimized condition for an extraction using microwave is by using petroleum ether as the solvent, with the ratio of seed powder to solvent is at 1:3. It is done under a microwave power of 810 W for a total radiation time of 5 min [97]. The extraction rate was 31.49% with the oil product containing 5.22 mg/g of acid number and 8.78 meq/g of peroxide value. For the ultrasonic method, hexane was used as the solvent and the ratio of seed powder to solvent was 1:7; the soaking time applied is 18 h and the sonication is 0.5 h. The extraction rate was 37.37% contain­ing 5.91 mg/g of acid number and 8.37 meq/g of peroxide value in the final oil product [97] .

Alkyl acetates, especially methyl acetate and ethyl acetate, are important chemi­cals and suitable solvents for seed oil extraction which are assisted by Novozym 435 [83]. The results were compared to those obtained by extraction with я-hexane. Ground seeds were mixed with methyl acetate or ethyl acetate in screw-caped glass vials. And, 30% (w/w) of Novozym 435 based on theoretical oil content was added. The reactions were carried out at 50°C and 180 rpm for 6 h in a shaker which was fitted with a thermostat. After filtration, the ground seed mixture was mixed with another solvent and then extracted at the same condition for another 2 h. The two filtrates were pooled and centrifuged at 17,400 x g for 10 min; the supernatant was collected into a round bottom flask and the solvent was evaporated using a rotary evaporator. The oil content in g/100 g was 54.90% (я-Hexane), 55.92% (Methyl acetate), and 56.65% (Ethyl acetate) [83].