We investigated the influences of introducing method of SiO2 and its quantity on the reactive performance of Fe catalysts. Table 2 lists results of some catalysts. The con­tents of promoter Zn, K and Cu in catalyst were arranged by uniform design. CO conversion of these catalysts is distributed in a wide range. It reflects marked influence of promoter composition on catalyst performance. CH4 selectivity is only influenced by promoter K content, and it decreases with increased K content. CO2 selectivity is not completely relied on CO conversion. For example, Z8K3C6/FS10-I and Z6K2C2/ FS15-I have similar CO conversion, but their CO2 selectivity is different.

The molar ratio of H2/CO in reactor outlet is given in Table 2 for the studied cata­lysts. All of them are higher than the H2/CO ratio in reactor inlet. Such increase of H2 content is resulted from WGS activity of Fe catalyst. It brings out H2-rich tail gas after FT synthesis reaction.

In order to improve the converting efficiency of syngas in FT synthesis and pro­duce profitable chemicals, some kinds of FT synthesis process have been projected

[65] . It needs catalysts having corresponding performance to construct a desired process. The catalysts we studied are able to meet the requirement due to their per­formance distributed in wide range.

Naturally occurring fungi such as T. reesei and Aspergillus species have the capability of secreting massive quantities of cellulases including endo-b glucanases, exo-b glucanases, and b glucosidases. The action of these three enzymes can work syner — gistically to solubilize lignocellulosic feedstocks yielding glucose that can be converted via fermentation to ethanol. Some arguments have recently been presented for developing T. reesei as a CBP host [78]. While research in this direc­tion is still preliminary, we mention it here because it represents out-of-the-box thinking that will be required to drive CBP research forward. While T. reesei and other fungal species have been shown to harbor the metabolic pathways that are necessary to produce ethanol from sugars, the levels of ethanol produced by these microorganisms are nevertheless rather low compared to the more robust fermenta­tion organisms such as Zymomonas mobilis and Saccharomyces cerevisiae [78]. Moreover, T. reesei is an obligate aerobe making ethanol production process more complicated through either strain engineering or process reconfiguration. Furthermore, its filamentous nature will make mixing within the fermentation tank challenging yet more essential than with a unicellular organism. Despite these for­midable challenges, the raw cellulolytic power of T. reesei and the innate, if not overwhelming, ability to produce ethanol make it an intriguing CBP candidate. However, much more research will need to be done to determine the feasibility of using this organism as a CBP host.

The classification above resolves whether catalytic domains of cellulases hydrolyze glycosidic bonds at the end of cellulose chains (exocellulases) or in the middle (endocellulases). An important additional distinction among endocellulases is whether they are processive (binding to a cellulase chain and then hydrolyzing at multiple sites, perhaps in sequence on the chain) or nonprocessive (binding, hydro­lyzing a single bond, and releasing). Comparison of multiple crystal structures of cellulases has revealed some of the basis of processivity, along with the basis for endo — vs. exocellulolytic activity (see [84] for a review). It should be emphasized that the type of processivity discussed above relates to individual catalytic domains, since the attachment of CBDs in cis is likely to significantly enhance processivity by tethering the enzyme to the substrate, as is the presence of multiple catalytic domains in a single polypeptide chain.

An important subdivision of the exocellulases is whether they attack the reduc­ing end or nonreducing end of the cellulose chain [2]. In many cases, exocellulases produce the disaccharide cellobiose and are appropriately designated “cellobiohy — drolases,” although many exocellulases also produce longer oligosaccharides such as cellotriose and cellotetraose.

A final mechanistic subdivision that can be made is whether a cellulase cleaves the b-glucosidic bond with retention of chirality at the C1 position (producing the b-anomer) or with inversion (producing the a-anomer). For both mechanisms, cel — lulases use a pair of acidic (asp/glu) residues in a general acid/general base scheme. Other common features include water attack at the C1 position on the nonreducing side of the glycosidic bond to be cleaved and displacement of the O4-sugar. In the inverting mechanism, the water attacks C1 directly, with one of the acidic residues acting as a general base to abstract a proton from the attacking water, and the other acting as a general acid to protonate the O4 leaving group. In the retaining mecha­nism, the general base forms a covalent adduct (an ester linkage between the side — chain carboxyl and C1), displacing the O4, with protonation from the general acid. In a second step, water is activated by the general acid (now in its deprotonated conjugate base form), which attacks the C1 and displaces the C1-asp/glu linkage.

Some of the designer enzymes discussed above, such as the alcohol dehydrogenase, pyruvate decarboxylase, phosphoglycerate mutase and enolase, are known to func­tion in the glycolytic pathway in the cytoplasm, but chloroplasts generally do not possess these enzymes to function with the Calvin cycle. Therefore, nucleic acids encoding for these enzymes need to be genetically engineered such that the enzymes expressed are inserted into the chloroplasts to create a desirable designer organism of the present invention. Depending on the genetic background of a particular host organism, some of the designer enzymes discussed above may exist at some back­ground levels in its native form in a wild-type chloroplast. For various reasons including often the lack of their controllability, however, some of the chloroplast background enzymes may or may not be sufficient to serve as a significant part of the designer ethanol-production pathway(s). Furthermore, a number of useful inducible promoters happen to function in the nuclear genome. For example, both the hydrogenase (Hydl) promoter and the nitrate reductase (Nial) promoter that can be used to control the expression of the designer ethanol-production pathways are located in the nuclear genome of C. reinhardtii, of which the genome has recently been sequenced. Therefore, it is preferred to use nuclear-genome-encodable designer genes to confer a switchable ethanol-production pathway. Consequently, nucleic acids encoding for these enzymes also need to be genetically engineered with proper sequence modification such that the enzymes are controllably expressed and are inserted into the chloroplasts to create a designer ethanol-production pathway. Figure 5 illustrates how the use of designer nuclear genes including their transcription in nucleus, translation in cytosol, and targeted insertion of designer proteins into chloroplast, can form the designer enzymes conferring the function of the ethanol — production pathway(s) for photosynthetic production of ethanol (CH3CH2OH) from carbon dioxide (CO2) and water (H2O) in a designer organism.

Additionally, it is best to express the designer ethanol-producing-pathway enzymes only into chloroplasts (at the stroma region), exactly where the action of the enzymes is needed to enable photosynthetic production of ethanol. If expressed without a chloroplast-targeted insertion mechanism, the enzymes would just stay in the cytosol and not be able to directly interact with the Calvin cycle for ethanol production. Therefore, in addition to the obvious distinctive features in pathway designs and associated approaches, another significant distinction to the prior art is that the present invention innovatively employs a chloroplast-targeted mechanism for genetic insertion of many designer ethanol-production-pathway enzymes into chloroplast to directly interact with the Calvin cycle for photobiological ethanol production.

With a chloroplast stroma-targeted mechanism, the cells will not only be able to produce ethanol but also to grow and regenerate themselves when they are returned to conditions under which the designer pathway is turned off, such as under aerobic conditions when designer hydrogenase promoter-controlled ethanol-production­pathway genes are used. Designer algae, plants, or plant cells that contain normal mitochondria should be able to use the reducing power (NADH) from organic reserves (and/or some exogenous organic substrate such as acetate or sugar) to power the cells immediately after the return to aerobic conditions. Consequently, when the designer algae, plants, or plant cells are returned to aerobic conditions after use under anaerobic conditions for photosynthetic ethanol production, the cells will stop mak­ing the ethanol-producing enzymes and start to restore the normal photoautotrophic capability by synthesizing new and functional chloroplasts. Therefore, it is possible to use such genetically engineered designer alga/plant organisms for repeated cycles of photoautotrophic growth under normal aerobic conditions and efficient production of ethanol directly from CO2 and H2O under certain specific designer ethanol-pro­ducing conditions such as under anaerobic conditions.

The targeted insertion of designer ethanol-production enzymes can be accom­plished through use of a DNA sequence that encodes for a stroma “signal” peptide. A stroma-protein signal (transit) peptide directs the transport and insertion of a newly synthesized protein into stroma. In accordance with one of the various embodiments, a specific targeting DNA sequence is preferably placed in-between the promoter and a designer ethanol-production-pathway enzyme sequence, as shown in a designer DNA construct (Fig. 6a). This targeting sequence encodes for a signal (transit) peptide that is synthesized as part of the apoprotein of an enzyme.

The transit peptide guides the insertion of an apoprotein of a designer ethanol — production-pathway enzyme into the chloroplast. After the apoprotein is inserted into the chloroplast, the transit peptide is cleaved off from the apoprotein, which then becomes an active enzyme.

A number of transit peptide sequences are suitable for use for the targeted insertion of the designer ethanol-production-pathway enzymes into chloroplast, including but not limited to the transit peptide sequences of: the hydrogenase apoproteins (such as HydAl (Hydl) and HydA2, GenBank accession number AJ308413, AF289201, AY090770), ferredoxin apoprotein (Frxl, accession numbers L10349, P07839), thioredoxin m apoprotein (Trx2, X62335), glutamine synthase apoprotein (Gs2, Q42689), Lhcll apoproteins (AB051210, AB051208, AB051205), PSII-T apoprotein (PsbT), PSII-S apoprotein (PsbS), PSII-W apoprotein (PsbW), CF0CF1 subunit-g apoprotein (AtpC), CF0CF1 subunit-Y5 apoprotein (AtpD, U41442), CFoCFj subunit-II apoprotein (AtpG), photosystem I (PSI) apoproteins (such as, of genes PsaD, PsaE, PsaF, PsaG, PsaH, and PsaK), and Rubisco SSU apoproteins (such as RbcS2, X04472). Throughout this specification, when reference is made to a transit peptide sequence, such as, for example, any of the transit peptide sequence described above, it includes their functional analogs, modified designer sequences, and combinations thereof. A “functional analog” or “modified designer sequence” in this context refers to a peptide sequence derived or modified (by, e. g., conservative substitution, moderate deletion or addition of amino acids, or modification of side chains of amino acids) based on a native transit peptide sequence, such as those identified above, that has the same function as the native transit peptide sequence, i. e., effecting targeted insertion of a desired enzyme.

In certain specific embodiments, the following transit peptide sequences are used to guide the insertion of the designer ethanol-production-pathway enzymes into the stroma region of the chloroplast: the Hyd1 transit peptide (having the amino acid sequence: msalvlkpca avsirgsscr arqvaprapl aastvrvala tleaparrlg nvacaa), the RbcS2 transit peptides (having the amino acid sequence: maaviakssv saavarpars svrp — maalkp avkaapvaap aqanq), ferredoxin transit peptide (having the amino acid sequence: mamamrs), the CF0CF1 subunit-5 transit peptide (having the amino acid sequence: mlaaksiagp rafkasavra apkagrrtvv vma), their analogs, functional derivatives, designer sequences, and combinations thereof.

In Indonesia, Jatropha oil is usually extracted by hydraulic press and screw press at 60°C heat treatment. The yield of Jatropha oil using hydraulic press method at maximum pressure of 20 tons is 47.2% and the oil extraction is done twice [84, 85].

In Tanzania presently, the Jatropha oil is obtained only mechanically with a ram press or a screw press, that is a small hand-press [89]. With the ram press method, the seeds are poured by giving a pressure on the seeds. About 5 kg of seed is needed to obtain 1 L of oil. The oil is extracted and then dripped into a container. The extraction rate of this press is quite low as the seedcake, which is left after the press­ing, still contains part of the oil.

Larger expellers and screw presses which are run by an engine can also be used. The screw which turns continuously transports the seeds from one side of the press to the other while squeezing out the oil. The extraction rate of this press is higher because more oil is extracted from the seeds; the cake residue is also much dryer. The capacity of this screw is higher than that of the ram press. For example, the Sayari oil expeller, which is used in Tanzania, has a capacity of about 20 L/h (60 kg/h) and can extract 1 L of oil from 3 kg of seeds. The larger screw expellers, like Chinese expellers, can extract about 50 L/h (150 kg/h). After the oil is expelled, it is filtered by letting it stand for some times or pouring it through a cloth [89].

Biofuels derived from microalgae are currently considered to be the most economi­cal and technically viable route for producing biofuels to compete with petroleum — based fuels [40] . This is due to a number of intrinsic advantages they could have, when compared to biofuels extracted from terrestrial bioenergy crops. These include: (1) higher annual growth rates, e. g. rates of up to 37 tonnes ha-1 per annum have been recorded, primarily due to higher photosynthetic efficiencies when compared with terrestrial plants [221]; (2) higher lipid productivity (up to 75% dry weight for some algae species), with higher proportion of triacylglycerol (TAG) that is essential for efficient biodiesel production [185]; (3) microalgae production can effect biofixation of CO2 (production utilises about 1.83 kg of CO2 per kg of dry algal biomass yield) thus making a contribution to air quality improvement [38]; (4) capability of growing in wastewater, which offers the duel potential for integrating the treatment of organic effluent with biofuel production [28]; and (5) inherent yield of valuable co-products such as proteins and polyunsaturated fatty acids (PUFAs) may be used to enhance the economics of production systems [197]. Figure 2 summarises the distinct stages in the production and processing of microalgae to biofuel.

Despite the outlined advantages, there are still a number of significant obstacles to realisation of the full potential of microalgae-derived biofuels. They include: (1) energy-intensive downstream processes including pumping and dewatering of bio­mass, and conversion processes can result in negative energy balance [ 88] ; (2) trade-off requirements in species selection and genetic enhancement for biofuel production vs. extraction of co-products [ 157 ] ; and (3) need for techniques for enabling real-time control of pH, photoinhibition, evaporation and CO. diffusion losses in cultivation systems [213].

Table 5 provides a preliminary comparison between organic solvent extraction and SCCO2 extraction for lipid extraction from microalgae. A more thorough under­standing of mass transfer mechanism and kinetic parameters involved in lipid extraction from microalgae is needed for an extensive comparison. Large use of toxic solvents and energy-intensive solvent-lipid separation represent the main dis­advantages with commercial use of organic solvent extraction, while expensive fluid compression and high installation cost of an extraction pressure vessel remain the primary obstacles for large-scale SCCO2 extraction [50]. The theoretical advan­tages of SCCO2 extraction, such as tunable selectivity for specific lipid fractions and enhanced kinetics due to the fluid intermediate physicochemical properties, still need to be verified for microalgal lipid extraction.

Well-engineered, slow-pyrolysis technology, optimized for the production of bioenergy and biochar from sustainable feedstocks, could deliver significant environmental and economical advantages to industry. The growing field of scientific research on biochar by agronomists, soil and plant scientists, is leading to a rigourous body of knowledge on the effective use of biochar for agricultural productivity gains. This work will assist in justifying a market price for biochar products, which is needed to underpin the economic feasibility of the emerging indus­try. Several companies, such as Pacific Pyrolysis in Australia, are working towards commercializing new technology for the production of biochar along with bioen­ergy from low-grade organics. Establishing one or more commercial-scale produc­tion facilities dedicated to demonstrating the technical, environmental, and economic outcomes of the business will be an essential next step for the emerging industry.

Slow-pyrolysis technology has the potential to deliver renewable energy, and biochar products while exhibiting a carbon negative greenhouse gas balance [22, 35]. The carbon sequestration achieved by the high carbon biochar product results in a net removal of carbon dioxide from the atmosphere. If organics are not used as fuel they decompose relatively quickly in the natural environment, releasing the carbon as CO2 back to the atmosphere. Production of biochar from these organics removes this material from the short-term carbon cycle, into the long-term carbon cycle. Biochar is far more stable in the environment when compared to the original organics and prevents the release of the carbon in its structures (Fig. 5).

When compared to typical bioenergy GHG balances, where all of the carbon in the fuel source (biomass organics) is released through the energy cycle as green­house gases, in pyrolysis a portion of the carbon is stabilized as the biochar product. The coproduction of biochar along with renewable energy results in a significant net removal of GHGs from the atmosphere via this pathway. It should be noted, however, that not all biochar technologies are necessarily carbon negative as carbon

Atmospheric Carbon

Bioenergy

Terrestrial Carbon

Fig. 5 Comparison of carbon balances: fossil fuel, bioenergy, and slow-pyrolysis for bioenergy and biochar
leakage and poor combustion systems can have a significant negative impact on the carbon lifecycle analysis [17]. It is essential that modeling, monitoring, and audit­ing of the system is carried out to verify carbon offsets generated.

Key pathways to GHG mitigation via production and use of biochar include:

• Renewable energy generation (displacing fossil fuel).

• Bio-sequestration (stabilizing organic carbon as biochar and storing it in terrestrial sinks).

• Stabilization of labile soil organic carbon onto biochar surfaces.

• Reduced agriculture emissions (from reduced; nitrous oxide from soil, fuel use, fertilizer use, and improved water use efficiency).

• Decreased emissions from waste biomass (including avoided methane generation from landfills and compost production).

• Increased agricultural productivity (increased biomass yields taking up more atmospheric carbon, less land area required for food production).

The sequestration of carbon via biochar and mitigation of GHGs are offset by various steps along the biochar production lifecycle. These aspects might include:

• Use of fossil fuels for harvesting, transporting, and processing.

• Fugitive emission from feedstock degradation being stored or preprocessed.

• Emissions from the processing plant, such as uncombusted syngas.

• Land use change, for example biomass requirements provide a market for more purpose-grown organics which may result in deforestation [14].

Technology considerations that should be optimized to ensure carbon negative balances are achieved:

• Energy efficiency of processes.

• Emissions control, including utilization of syngas.

• Limited distances for feedstock collection and product distribution.

• Alternate higher uses of organics is fully considered, e. g., waste organics are sourced over purpose-grown feedstocks.

The contribution biochar can make in maintaining soils for agricultural production during climate variability may prove a vital tool for adaptation.

In addition to the above chemicals, many other chemicals can be produced by selective fast pyrolysis of biomass. For example, Chen et al. [18] reported that fast pyrolysis of biomass impregnated with Na2 CO3 produced HA with high purity. Badri [10] revealed that catalytic pyrolysis of cotton with reactive dyes favored the formation of 5-hydroxymethyl-furfural (HMF) and 3-(hydroxymethyl)-furan. Lu et al. [51] found that fast pyrolysis of cellulose followed with catalytic cracking of the vapors by sulfated metal oxides could obtain high yields of furan and 5-methyl furfural. In another study, Lu et al. [52] confirmed that catalytic cracking of the biomass fast pyrolysis vapors using ZrO2 and TiO2 increased the formation of three light carbo­nyl products (acetaldehyde, acetone, and 2-butanone).

2 Conclusion

Most of the selective fast pyrolysis techniques are only in their early stage of devel­opment, and none of the techniques is commercially practical to produce specific chemicals in marketable quantities at present. Three aspects should be considered for the commercialization of the selective fast pyrolysis techniques, including (1) the technique to produce specific chemicals in high yield and purity, (2) the method to recover the target chemicals from pyrolysis liquids, and (3) the ready markets for the chemicals.

Among the above indicated chemicals, the LG, HAA, and AA can be produced without catalyst utilization, and thus, their large-scale production might be rela­tively easy to achieve through slight modification of the conventional fast pyrolysis technique. The production of other chemicals requires catalysts, which will add difficulty to their scale up. Various methods have been proposed for the chemicals recovery, and further studies should be conducted to reduce the purification cost. Finally, it is important to note that currently there are no existing markets for the LG, LGO, LAC, anhydro-oligosaccharides, and some other chemicals. Corresponding markets should be developed by manufacturers who would incorporate these chemi­cals into various products.