Water is an ecologically safe and abundantly available solvent in nature. Water has a relatively high critical point (374°C and 22.1 MPa) because of the strong interaction between the molecules due to strong hydrogen bond. Liquid water below the critical point is referred as subcritical water whereas water above the critical point is called supercritical water. Density and dielectric constant of the water medium play major role in solubilizing different compounds. Water at ambient conditions (25°C and

0. 1 MPa) is good solvent for electrolytes because of its high dielectric constant 78.5), whereas most organic matters are poorly soluble under these conditions.

As water is heated, the H-bonding starts weakening, allowing dissociation of water into acidic hydronium ions (H3O+) and basic hydroxide ions (OH-). Structure of water changes significantly near the critical point because of the breakage of infinite network of hydrogen bonds and water exists as separate clusters with a chain structure [52]. In fact, dielectric constant of water decreases considerably near the critical point, which causes a change in the dynamic viscosity and also increases self-diffusion coefficient of water [71].

Supercritical water has liquid-like density and gas-like transport properties, and behaves very differently than it does at room temperature. For example, it is highly nonpolar, permitting complete solubilization of most organic compounds and oxygen. The resulting single-phase mixture does not have many of the conventional transport limitations that are encountered in multiphase reactors. However, the polar species present, such as inorganic salts, are no longer soluble and start precipitating. The physicochemical properties of water, such as viscosity, ion product, density, and heat capacity, also change dramatically in the supercritical region with only a small change in the temperature or pressure, resulting in a substantial increase in the rates of chemical reactions. It is interesting to see (Fig. 7) that the dielectric behavior of 200°C water is similar to that of ambient methanol, 300°C water is similar to ambi­ent acetone, 370°C water is similar to methylene chloride, and 50°C water is similar to ambient hexane.

In addition to the unusual dielectric behavior, transport properties of water are significantly different than the ambient water as presented in Table 2.

A schematic of typical integrated BTL process is shown in Fig. 4. Some modifications of this basic process are also being investigated. For example, researchers at University of California, Riverside are examining a process illustrated in Fig. 5. The key modification is the replacement of a conventional gasification by a hydrogasification reactor [36] to produce biosyngas from biomass. In general, various

Biosyngas

EIcctfKity (to* utt m plontj

light product

Fischer-Tropsch

Diesel

Fig. 4 itic lineup of the integrated BTL plant [38]

modifications of an integrated BTL process are possible depending on the use of preferred technologies in basic five elements of an integrated BTL process.

Boerrigter et al. [43] point out that a design of an integrated BTL process depends on whether or not one takes a front-end approach or a back-end approach.

The importance and benefit to develop precipitated Fe catalyst have been discussed in Part 1 which can use CO2-containing syngas directly for hydrocarbon synthesis.

We have studied precipitated Fe catalyst since 2002 and reported some results [40-45]. It was found that promoter combination of Zn, K, and Cu can improve catalytic stability under CO2-containing syngas [42] .

In this part, the role of promoter Zn, K, and Cu on CO2 activation is investigated by CO2 temperature-programmed-desorption (TPD). A correlation is found between the characteristic of CO.-TPD and CO. selectivity based on converted CO in FT synthesis. It indicates the possibility to convert CO2 into hydrocarbons at low temperature. The possible is tested with CO2 hydrogenation.

The process described under this category aims at reducing the number of process steps (SSFR) as opposed to SHFR. In the SSFR process, after pretreatment of the cellulosic biomass, enzymes are added to the reactor and at the same time the reactor is inoculated with a butanol producing culture. Since optimum pH for hydrolytic enzymes and culture that produce butanol is the similar (5.0), these two unit opera­tions can be performed simultaneously in the same reactor. It should be noted that enzymes perform more efficiently at 45 °C while the optimum temperature for butanol producing culture is only 35°C. In spite of the different optimum temperatures for the enzymes and the culture, this process performs well. In order to make this
process more efficient, it is recommended that new hydrolytic enzymes be developed with 35°C as their optimum temperature. Since butanol is toxic to the microbial cells, the product should be removed simultaneously. Continuous removal of butanol from the fermentation broth would also prolong the reaction thus improving efficiency of the process further. A schematic diagram of the SSFR process is shown in Fig. 1b. The overall process benefits from this system as all three unit operations are performed in a single reactor. This system has been applied to butanol produc­tion from wheat straw [49, 58].

In a study, Marchal et al. [40] produced ABE in an integrated system where hydrolysis and fermentation were combined. These authors did not apply simulta­neous product removal technique to remove ABE from the system/fermentation broth. In this system wheat straw was pretreated with alkali followed by washing the straw several times with tap water. Cellulase enzyme was prepared from Trichoderma reesei Cl-847 and added to the fermentation medium which was inoc­ulated by C. acetobutylicum IFP 921. These investigations were performed in a 6 L bioreactor with 2 L medium containing 194 g (dry weight) of pretreated wheat straw, 12 g of dried corn steep liquor and 360 mL of undiluted enzyme preparation. Approximately 17.3 g/L ABE was produced from the wheat straw.

Cellulose binding modules (CBMs) are noncatalytic modules that induce surface disruption of cellulose fibers [34] as they adsorb to accessible sites on cellulose substrates to form a complex held together by specific, noncovalent, thermodynami­cally preferred bonds [2, 75]. CBMs include bacterial and fungal cellulose binding domains (CBDs), expansins, and swolenins. All CBMs share highly similar struc­ture and function [116]. Expansins are plant cell wall proteins involved in the exten­sion and loosening of the plant cell wall, promoting plant growth and expansion [29]. Swollenins, originating from the cellulose-degrading fungus Trichoderma reesei. are CBMs that share sequence homology with expansin-like proteins and effectively disrupt cellulosic materials [21].

Plant growth and biomass can be increased by bacterial CBMs transgenically expressed in the cell wall [116], where the postulated mechanism is based on sepa­ration of cellulose-biosynthesis polymerization and crystallization steps [71]. This separation may result in more flexible cellulose microfibrils that consequently increase cellulose synthesis rates in the presence of CBMs. The first observations of enhanced elongation followed in vitro addition of low concentrations of exoge­nous bacterial CBM to peach pollen tubes and Arabidopsis seedlings [71, 117]. The cellulose generated in the presence of the CBM resembled the loose ribbon­like appearance of newly formed fibrils, as opposed to the well-arranged ribbons of control fibers formed in the absence of excess CBM [117]. Accelerated cell and plant growth has also been observed in transgenic tobacco, poplar, and potato plants expressing a cell wall-targeted Clostridium cellulovorans CBM [71, 103, 114, 115].

The xyloglucan-cellulose matrix of the primary wall must be loosened for cell expansion to occur [28]. Part of the expansion or fragmentation of cellulose microfibrils is due to expansin activity [30] , often upregulated in growing tissues [23, 66, 127]. Expansins cause wall loosening in vitro [80] and are correlated with growth promotion in vivo [41]. Targeted expansin expression in transgenic Arabidopsis [22], rice [24], and poplar plants [49] resulted in enhanced growth rates. Most of the transgenic plants described above had fairly low expression levels of the nonnative bacterial CBMs or expansins. In contrast, high level expression of different CBMs resulted in retarded stem elongation and reduced mechanical prop­erties in term of extensibility vs. constant load as well as collapsed stems [86, 101]. These results corroborate the first reported attempt to apply exogenous bacterial CBM to germinated Arabidopsis seedlings. At low concentrations, CBM enhanced root elongation, whereas higher concentrations inhibited the process in a dose — dependent manner [117].

The role of CBM expression in increased plant growth rates bears profound potential in yield enhancement challenges and can be utilized through genetic engi­neering with almost all biofuel feedstocks. Ectopic CBMs and/or glycoside hydro­lase expression can change the carbon partitioning between source and sink tissues by creation of stronger sinks in cellulose synthesizing cells, leading to enhanced growth, biomass, and yield.

Although many individual catalytic domains show measurable activity against cellulose, their activities are rather low. On their own, they would be poor catalysts for large-scale biofuel production. In nature, there are two ways that reactivity is enhanced. First, multiple different types of cellulases (endocellulases, exocellu — lases, b-glucosidases, as well as hemicellulases and lignases) are coordinately expressed, producing a synergistic enhancement of activity (Fig. 4; [76]).

Second, catalytic domains are attached (either covalently or noncovalently) to cellulose-binding modules (CBMs; see [5] for a review), which localizes enzyme activity to the cellulosic substrates, allowing for multiple rounds of hydrolysis (Fig. 5). In aerobic bacteria and fungi, this is achieved in cis by direct fusion of CBM-coding and catalytic domain-coding sequences in cellulase genes (Fig. 5b).

In addition to enhancing activity by tethering to substrate surfaces, these accessory domains can directly enhance activity by providing structural integrity to the cata­lytic domain.

In anaerobic bacteria, particularly in the Clostridium genus, a strategy is used that incorporates both modes of enhancement. Although most catalytic domains in these bacteria are not covalently fused to CBMs, they are chained together nonco­valently to molecular scaffolds at the cell surface, structures referred to as “cellu- losomes” (Fig. 5c; [3, 39]). This is achieved by fusion of cellulases to domains called “dockerins.” Dockerins bind tightly to domains called “cohesions,” which are repeated in multiple copies in proteins called “scaffoldins.” By attachment of multiple scaffoldins to the cell surface, a highly dendritic structure of different catalytic domains can be produced. Because scaffoldins often contain terminal CBMs, the cellulosome directly attaches the bacterium to the cellulosic substrate, ensuring a high local concentration of complementary catalytic domains at its surface.

According to one of the various embodiments, a designer Calvin-cycle-channeled pathway is created that takes the Calvin-cycle intermediate product, 3-phospho — glycerate, and converts it into 1-hexanol by using, for example, a set of enzymes consisting of (as shown with the numerical labels 34, 35, 03-10, 07′-12′ in Fig. 7): NADPH-dependent glyceraldehyde-3-phosphate dehydrogenase 34, NAD — dependent glyceraldehyde-3-phosphate dehydrogenase 35, phosphoglycerate mutase 03, enolase 04, pyruvate kinase 05, pyruvate-ferredoxin oxidoreductase 06, thiolase 07 , 3-hydroxybutyryl-CoA dehydrogenase 08, crotonase 09, butyryl-CoA dehydrogenase 10, designer 3-ketothiolase 07′, designer 3-hydroxyacyl-CoA dehy­drogenase 08′, designer enoyl-CoA dehydratase 09′, designer 2-enoyl-CoA reductase 10′, designer acyl-CoA reductase 11′, and hexanol dehydrogenase 12′. The net result of this designer pathway in working with the Calvin cycle is photo­biological production of 1-hexanol (CH3CH2CH2CH2CH2CH2OH) from carbon dioxide (CO2) and water (H2O) using photosynthetically generated ATP and NADPH according to the following process reaction:

6CO2 + 7H2O ^ CH3CH2CH2CH2CH2CH2OH + 9O2 (9)

According to another embodiment, a designer Calvin-cycle-channeled pathway is created that takes the intermediate product, 3-phosphoglycerate, and converts it into 1-octanol by using, for example, a set of enzymes consisting of (as shown with the numerical labels 34, 35, 03-10, 07′-10′, and 07"-12" in Fig. 7): NADPH — dependent glyceraldehyde-3-phosphate dehydrogenase 34, NAD-dependent glycer — aldehyde-3-phosphate dehydrogenase 35, phosphoglycerate mutase 03, enolase 04, pyruvate kinase 05, pyruvate-ferredoxin oxidoreductase 06, thiolase 07,3-hydroxy — butyryl-CoA dehydrogenase 08, crotonase 09, butyryl-CoA dehydrogenase 10, designer 3-ketothiolase 07′, designer 3-hydroxyacyl-CoA dehydrogenase 08′, designer enoyl-CoA dehydratase 09′, designer 2-enoyl-CoA reductase 10′, designer 3-ketothiolase 07", designer 3-hydroxyacyl-CoA dehydrogenase 08", designer enoyl-CoA dehydratase 09", designer 2-enoyl-CoA reductase 10", designer acyl- CoA reductase 11", and octanol dehydrogenase 12".

These pathways represent a significant upgrade in the pathway designs with part of a previously disclosed 1-butanol production pathway (03-10). The key feature is the utilization of an NADPH-dependent glyceraldehyde-3-phosphate dehydroge­nase 34 and an NAD-dependent glyceraldehyde-3-phosphate dehydrogenase 35 as a mechanism for NADPH/NADH conversion to drive an NADH-requiring designer hydrocarbon chain elongation pathway (07′-10′) for 1-hexanol production (07′-12′ as shown in Fig. 7). The net result of this pathway in working with the Calvin cycle is photobiological production of 1-octanol (CH3CH2CH2CH2CH2CH2CH2CH2OH) from carbon dioxide (CO2) and water (H2 O) using photosynthetically generated ATP and NADPH according to the following process reaction:

8CO2 + 9H2O ^ CH3CH2CH2CH2CH2CH2CH2CH2OH + 12O2 (10)

Post reaction processing comprises ester/glycerol separation, ester washing, ester drying, alcohol recovery, and glycerol refining. These steps are very important in producing fuel-grade biodiesel and in decreasing biodiesel oil cost through alcohol recovery and glycerol refining.

For best economy and pollution prevention, alcohol must be fully recycled. Glycerol is an economically coproduct that should be fully refined [7]. As by-prod­uct, 1 mol of glycerol produces every 3 mol of methyl esters, which is equivalent to approximately 10 wt.% of the total product. Glycerol markets have reacted strongly to the increasing availability of glycerol. Although the global production of biodie­sel is still limited, the market price of glycerol has dropped rapidly [37]. Therefore, new uses for glycerol need to be developed and economical ways of the low-grade glycerol utilization should be further explored.

As Jatropha oil possesses a significant amount of fatty acids with double bonds, oxidative stability is of concern, especially when storing biodiesel over an extended period of time. The storage problem is worsening by storage conditions such as exposure to air and/or light, temperature above ambient, and the presence of extra­neous materials (contaminants) with catalytic effect on oxidation [43]. The presence of air or oxygen will hydrolyze the oil to alcohol and acid. The presence of alcohol will lead to reduction in flash point and the presence of acid will increase the total acid number. All these conditions make methyl ester relatively unstable on storage and cause damage to engine parts [23]. That is why oxidation stability is an impor­tant criterion for biodiesel production.

The stability of biodiesel is very critical. Various strategies for the improvement of biodiesel fuel quality have been suggested. Biodiesel requires antioxidant to meet storage requirements and to ensure fuel quality at all points along the distribution chain. In order to meet EN 14112 specification, around 200 ppm concentration of antioxidant is required, except for palm biodiesel, which is much higher than those required for petroleum diesel. To minimize the dosage of antioxidant, appropriate blends of Jatropha and palm biodiesel are made. Antioxidant dosage could be reduced by 80-90% if palm oil biodiesel is blended with Jatropha biodiesel at around 20-40% concentration [72].

Although there are multiple conversion pathways for microalgae biomass conver­sion (see Sect. 5.2), biodiesel production through the extraction of algal bio-oil and subsequent esterification is as arguably the optimum route to biofuels with the cur­rent technologies [38, 92, 99, 104, 134, 185]. Esterification refers to the chemical reaction between TAG and alcohols to produce the mono-esters biodiesel [190]. Bio-oils from high lipid content microalgae (Table 1) are ideal for such conversion due to their high TAG contents [92]. Algae-derived biodiesel is a technically more attractive biofuel because, if sustainably produced, will incur zero net CO[ emis­sion, with only trace amounts of sulphur released in combustion [92] , and contain

no aromatic compounds or other harmful chemicals [124]. However, microalgae — derived bio-oils contain a high degree of PUFAs, which makes biodiesel products to be susceptible to oxidation in storage and therefore limits the potential utility [38] .

Numerous research projects have been focused on improving the lipid content of microalgae species, to enhance the production process in order to make it more economically competitive [48, 112, 154, 219, 223, 229, 232]. The focus on improve­ment of the overall total lipid productivity may only achieve marginal economic gain, since algal lipids are composed of both neutral lipids (e. g. triglycerides, cholesterol) and polar lipids (e. g. phospholipids, galactolipids) that bear different desired qualities for biodiesel production. For Biodiesel Standard EN14214, the neutral lipids, i. e. FFAs and TAG content of the total lipid fraction, are the most suitable. Therefore, when TAG and FFA make up only a small percentage of the total lipid fraction [209] , the oil is unsuitable for esterification, and additional processing (e. g. pyrolysis, thermochemical liquefaction) would be necessary before it can be used [104]. Microalgal biodiesel also tends to be unsuitable for long-term storage as the constituent esters hydrolyse back to the parent carboxylic acids. The reverse process results in polymerisation, thereby affecting the fuel quality, with potential negative impacts on engine service life [209] .

Biodiesel fuels must meet stringent chemical, physical and quality requirements as specified in standards EN14214 and ASTM D6751 [68]; ASTM [10]. Table 7 benchmarks the performance characteristics of algal biodiesel against other com­mon biodiesel fuels and petroleum diesel. Data on the properties of algal biodiesel are limited; therefore, it is still not possible to compare the full range of performance indicators against active diesel EN590 [69] and the biodiesel standards (EN14214 and ASTM D6751).

From available data, it can be seen that algal biodiesel compares favourably with both diesel and biodiesel standards. The HHV of algal biodiesel is lower when compared to diesel (EN590). The kinematic viscosity of algal biodiesel is within the standards but considerably higher than diesel (EN590). This indicates poor flow properties of algal biodiesel for use in conventional diesel engines. The higher density of algal biodiesel enhances the fuels’ energy density and potentially lower logistics/transportation associated with distribution. Recorded flash point is higher than diesel (EN590), which implies it is a safer fuel for handling and trans­portation, but could result in poor ignition in conventional diesel engines. The pour point is within the prescribed range in biodiesel standards, indicating the suitability for use in cold climates.

Biodiesel can be blended with diesel (EN590) for use by diesel engines. The most common biodiesel blends from terrestrial crops are B2 (2% biodiesel and 98% diesel (EN590)), B5 (5% biodiesel and 95% diesel (EN590)) and B20 (20% biodiesel and 80% diesel (EN590)) [11] . Such biodiesel blends will be more resistance to polymerisation improving its oxidative stability [204], which is a major limitation of microalgal biodiesel. Blending of microalgal biodiesel with diesel (EN590) could potentially enable it meet the required fuel standards and is the first step towards realising the potential of microalgae as a substitute for fossil fuel. Overall, the future potential of microalgae-derived biodiesel is ultimately dependent on the ability of production systems to meet active biodiesel standards. Therefore, research should aim to produce microalgae biomass with high proportion of TAG and FFA to minimise the cost of meeting these standards.

This review has shown that each biochar evaluated has a unique set of chemical and physical properties. While one biochar may be effective at resolving one soil prob­lem, it may also have properties that are either benign or promote gross changes to another soil property. It would be beneficial, if a negative characteristic of a biochar could be turned into an advantage through blending. Blending of different biochars to produce a hybrid product with designed characteristics for a specific soil purpose

Poultry litter biochar with high pH, P, N, etc

of the hybrid biochar

Fig. 13 Blending biochars creates a multifunctional hybrid biochar is possible (Fig. 13). For instance, in acid environments, such as in mine reclamation, metals such as Al, Cu, Mn, Pb, and Zn are highly soluble [10]. These metals, in sufficient quantities, can pose a hazard to plants. A proper remediation strategy would be to reduce their solubility by raising the pH and/or by complexation with soil organic compounds. This may be achieved by applying a high temperature pyrolyzed biochar (>400°C) produced from peanut hulls, pecan shells, and switch — grass, which have alkaline pH values (Table 1). In fact, peanut hull biochar produced at both pyrolysis temperatures did raise the pH of a sandy soil (Table 2). Biochars made from other feedstocks (i. e., hardwoods, pine chips, poultry litter, etc.) may be unsuitable for application in mine spoil sites because of their inability to act as a liming agent or by potential increases in other nutrients solubility (i. e., P, Fe, etc.). The impact of biochars or blends on remediating mine reclamation sites is largely unknown, but could be a viable assignment for biochars found not to be suit­able for agricultural soil improvement.

Poultry litter biochar, although it has some difficult characteristics, can still be used as a low-grade fertilizer (Table 1). It contained the highest N-P-K ratios, is extremely alkaline, and also contained high levels of Na [74]. Problems associated with these properties could be rebalanced or diminished by blending with other bio­chars (i. e., hardwood, pine chips, etc.) to produce a hybrid biochar (Fig. 13) that has more benign characteristics (Tables 1 and 3; lower N-P-K ratios, ash contents, pH, etc.) or added to improve another soil issue (i. e., low water holding capacity, etc.). The blending ratio of other biochars can be chosen depending upon the purpose of the hybrid biochar (Fig. 13). As an example, a hybrid biochar blend could consist of a mixture of hardwoods, pine chips, and poultry litter biochar; a blend designed to improve soil water storage while also delivering C, N, P and raising the pH as well.

Blending biochars for agricultural production or commercial purposes is beyond the concept stage. Commercial companies have internet sites advertising that their designer biochars made from blended materials that have their own unique proper­ties. These companies have developed a biochar product that could be used in a number of different market sectors (greenhouse, nursery, golf courses, etc.) as a plant media, improvement in golf greens, or in site reclamation.

5 Conclusions

Biochars can be produced from diverse feedstocks and under a variety of pyrolysis conditions. Because resultant biochar properties vary, no one biochar will fit all soil improvement intentions. Each biochar has its own unique chemical and physical signature and when applied to soils may have a positive, negative, or a benign effect. To avoid creating unwanted long-lasting effects in soils, thus the concept of designer biochar was introduced and the utility of producing biochars tailored for specific soil problems was illustrated. If one biochar has unsatisfactory properties, then blends of biochars in unique proportions can be created to produce a hybrid biochar that has tailored characteristics to provide multiple benefits for specific soil prob­lems. In this review, designer biochars were shown to have a positive effect by improving soil fertility and physical properties. In the future, biochars or their hybrid blends may also be formulated to reduce N2O emissions.

Because of their costs, designer biochars may be regarded as a product for bou­tique markets; but, they would definitely improve production in agricultural fields if costs were reduced. Given the fact that biochars react in a different way in different soils, more research is needed to understand relationships between feedstock and pyrolysis conditions vs. biochar quality. Therefore, this review has suggested poten­tial protocols and guidelines for the selection of feedstock’s and pyrolysis condi­tions to produced biochars with tailored properties for selected soil problems.

Acknowledgments This publication is based on work supported by the US Department of Agriculture, Agriculture Research Service, under the ARS-GRACEnet project. Sincere gratitude is expressed to scientists, and support staff at the ARS-Florence location for their time and com­mitment on the myriad of biochar projects. Mention of specific product or vendor does not consti­tute a guarantee or warranty of the product by the U. S. Department of Agriculture or imply its approval of the exclusion of other products that may be suitable.