1.1.1 Fabrication of the microchannel

Microchannel architecture typically represents a T — or Y-channel configuration. There are mainly two fabrication techniques. The first one utilizes conventional chip-manufacturing techniques of semiconductor industries. Silicon wafers are patterned by lithography step followed by etched step in order to get the desired form of the channel (Moore et al., 2005; Lee et al., 2007). The second technique allows the fabrication of microchannels by rapid prototyping using standard soft lithography procedure to build the channel in poly(dimethylsiloxane) (PDMS) (Duffy et al., 1998). PDMS is relatively inert and compatible with most solvents and electrolytes (Kjeang et al., 2008). Besides it is permeable to gases, which is essential for biofuel cells working from enzymes with oxygen as the cofactor. Typically, a pretreated microscope glass slide or a silicon wafer is coated with a thin layer of photoresist by spin-coating and exposed directly to UV light through a photomask that defines the desired channel structure. Several thick photoresist layers are sequentially laminated on the first layer
to get the desired channel depth, and then exposed to UV light. The structure is then developed by spraying an aqueous solution of sodium carbonate (1 % wt) and hardened by a final irradiation. The result is a master with a positive pattern defined by the master. The channel structure is thus obtained by pouring PDMS monomer over the master, followed by curing at 70 °C during 2 h. After cooling, the PDMS slab is peeled off from the master, and holes are punched to provide fluid access (Stephan et al., 2007).

This is a type C3 perennial herbaceous plant that grows in the cool season and has an excellent resistance to flooding. Its productivity is strongly influenced by high levels of nitrogen fertilizers, a feature that makes it very useful for the distribution of fertilizer from livestock.

1.6 Sugar cane (saccharum officinarum)

This plant only grows well in tropical and subtropical regions, which is why it is particularly common in Brazil. It has a 12-17% sugar content, 10% of which is glucose and the other 90% is saccharose. Milling can extract 95% of the total sugar content and the juice can subsequently be used to produce sugar or allowed to ferment to produce bioethanol. The bagasse (i. e. the solid residue remaining after milling) can be used as a source of energy and heat.

1.7 Sugar beet (beta vulgaris)

This plant generally grows in the cooler temperate regions, so it is abundant in Europe, North America and Asia. In the ethanol production process, the sugar beet is sliced and, while the juice is used to produce sugar or ethanol, the pulp is dried and used as animal feed or sold for pharmaceutical purposes.

1.8 Cereals

These must be ground to obtain starch, from which bioethanol is subsequently obtained. The cereals containing fewer proteins and more carbohydrates are preferable for distilling purposes because they have a higher bioethanol conversion rate. This means that the nitrogen content in the cereals can be adapted to facilitate starch accumulation instead of proteins synthesis, thereby improving both the energy yield and the quality of the fermentation process (Rosenberg et al., 2001). The principal cereals are:

1.8.1 Wheat

It grows mainly in temperate regions. The wheat treatment process is much the same as for the other cereals and it is best to use high-gravity fermentation to obtain the best performance in the fermentation process.

1.8.2 Barley

The most suitable is the so-called Winter variety, which is often underestimated as a foodstuff, despite the fact that it can tolerate drought and is highly adaptable.

Ethylene diamine grafting on the carbon surface was conducted using the electrochemical reduction. As the potential sweeps to negative direction during the first cycle, the oxidation of amino group in ethylene diamine is reduced on pyrolyzed carbon surface at potentials between -0.8 V and -1.4 V, leading to a clear irreversible anodic peak. It also shows that any peaks by oxidation and reduction do not exist after first reduction sweep even though we notice that the anodic current of electrode is decreased. This result indicates that the ethylene diamine was grafted on the surface of pyrolyzed carbon by applying potential and pyrolyzed surface is successfully functionalized by the electrochemical method.

We have introduced three types of functionalization on carbon surface in this session. In the future work, we will immobilize different biomolecules based on these functionalization methods for EBFCs device. Work on building a prototype EBFC consisting of glucose oxidase immobilized anode and a laccase immobilized cathode using C-MEMS based interdigitated electrode arrays is underway.

Perspective utilisation of digestate is connected with envisaged modifications of the technology of biogas production in agricultural biogas plants. These plants have digesters for the solid phase only or the most frequent are liquid (suspension) digesters. These are digesters without partition wall where the biomass of microorganisms is carried by the processed substrate. In reactor systems for the technological processing of waste from chemical and food technologies and from the technology of municipal and industrial waste water treatment those digesters are preferred where the biomass of functional microorganisms is fixed onto a solid carrier or onto partition walls of apparatuses. It is often granulated and is maintained in the digester as a suspended sludge cloud. These reactors may be affected by short-circuiting and therefore they are sensitive to the particle size of the processed substrate but they withstand a much higher organic load than the digesters without partition wall. Of course, the reactor is smaller, cheaper and more efficient.

Hence a perspective modification of the biogas production technology in agricultural biogas plants is gradual transition to the procedures of anaerobic digestion that are currently used in industrial plants for the treatment of organic waste water. The promising utilisation of digestate from such digesters is mainly the manufacture of solid fuels in the form of pellets that are prepared from the solid phase of agricultural waste before the proper aerobic digestion of the material for a biogas plant. The first proposal of this type is the IFBB procedure, the principle of which was explained in Chapter 1.4. The liquid phase from the preparation of processed material, which is destined for anaerobic fermentation in digesters with partition wall, could be used as a liquid or suspension fertiliser but researchers would have to solve the cheap method of nutrient concentration in this waste. The current price of Diesel fuel, machinery and human labour and low purchase prices of agricultural products do not allow the application of highly diluted fertilisers and in fact handling of water.

The problem is that a small biogas plant is only scarcely profitable. Hence economic reasons favour large-capacity plants with the volume of digesters 5 000 — 10 000 m3. In such large plants the reactors with partition wall would be unjustifiably expensive and therefore in these large-capacity facilities for biogas production it is necessary to use reactors without partition wall. The utilisation of their digestate should be based on this scheme: separation of digestate — concentration of fugate and its utilisation as a liquid mineral nitrogenous fertiliser. The solid phase of digestate should be used as an inert aeration component in compost production and as a material for the improvement and aeration of heavy-textured soils.

In any case, researchers must resolve a cheap method of nutrient concentration in fugate.

A number of different reactors are available for small to medium-sized biogas plants with the treatment of material according to IFBB that were developed on a research basis mainly in the sixties to the nineties of the twentieth century. At first, these were reactors with suspension biomass, e. g. mixing contact anaerobic reactor (ACR — AG), its innovation was a membrane anaerobic reactor system (MARS) and sequencing batch reactors (SBR). Then reactors with immobilised biomass were developed that are divided into reactors with biomass on the surface of inert material and reactors with aggregated (granulated) biomass. The former group is divided into upflow reactors and downflow reactors. Reactors with a mobile filling are the third variant.

The latter group is divided into reactors with the internal separator of biogas and biomass, reactors with the external separator of biomass and reactors with partitions.

Further development brought about biofilm reactors where the biomass of microorganisms is fixed onto a solid carrier. These reactors are considered as facilities with the highest operating stability, very resistant to the fluctuation of operating conditions. But they do not usually allow for such a high load as reactors with suspension biomass. The oldest reactor of this series was an upflow anaerobic filter (UAF) reactor from 1967, then a downflow stationary fixed film reactor (DSFF) and downflow reactor with filling in bulk followed. Great progress was made by designing an anaerobic rotating biological contactor (ARBC) and fluidized bed reactor (FBR) in the eighties of the last century. A similar type of reactor, expanded bed reactor (EBR), also designated by AAFEB (anaerobic attached film expanded bed), is suitable to be operated at low temperatures. The detention time is only several hours and the portion of residual organic impurities is practically the same as in modern aerobic systems for the treatment of organically contaminated waters.

Further advance was the development of reactors with aggregated biomass. The most important representative of this group of digesters is an upflow anaerobic sludge blanket (UASB) reactor. It is a reactor with sludge bed and internal separator of microorganism biomass. The biggest reactor of this type (5 000 m3) processes waste water from the manufacture of starch in the Netherlands, it withstands the load of 12.7 kg chemical oxygen demand (COD) per 1 m3/day, 74% of organic matter is degraded and the detention time is 33 hours only. Besides the UASB reactor these reactors belong to this group: hybrid upflow bed filter (UBF) reactor, anaerobic baffled reactor (ABR), expanded granular sludge bed (EGSB) reactor, internal circulation (IC) reactor and upflow staged sludge bed reactor (USBB), often also called biogas tower reactor (BTR), and other design models of the UASB reactor.

At the end of this chapter it is to note that modern anaerobic reactors have almost amazing outputs — unfortunately, the more perfect the reactor, the more expensive, and also their advantage over huge digesters without partition wall we have got accustomed to in biogas plants is gradually disappearing. The selection of modern anaerobic reactors is also more difficult than the selection of conventional technology of reactors without partition wall, because they are mostly rather specific to the substrate to be processed. They also have higher demands on processing, attendance and checks.

The perspective possibility of using modern anaerobic reactors for biogas production in smaller plants and the simultaneous solution to the use of the digestate solid phase as a raw material for the production of solid pelleted biofuels initiated our study of the IFBB procedure (Chap. 1.4.) for the substrate commonly used in biogas plants in the CR. The results of our experimental work are presented below:

The IFBB technological procedure is based on a high degree of cell wall maceration as a result of the axial pressure and abrasion induced with a screw press. Reulein et al. (2007) used this procedure for dehydration of various field crops; it is also known from the technologies of processing rapeseed, sugar beet and leguminous crops for the production of protein concentrates (Telek and Graham 1983, Rass 2001) and in biorefineries for the extraction of lactic acid and amino acids (Mandl et al. 2006).

The basic substrate contained 37.5% by weight of cattle slurry and 62.5% by weight of solid substrates, i. e. a mixture of chopped maize silage and grass haylage of particle size max. 40 mm mixed at a 4.75 : 1 ratio, i. e. 51.6% of silage and 10% of haylage. In total, the substrate accounted for 19.3% of dry matter. This substrate at 15°C is designated by A. A portion of this substrate was mixed with water at a weight ratio of 1 : 5, put into a thermostat with a propeller stirrer at 15°C and intensively stirred for 15 minutes. Analogically, the other portion was also mixed with water at a substrate to water ratio of 1: 5 and put into a thermostat at a temperature of 60°C with 15-minute intensive stirring again. The sample of the substrate with water 15°C was designated by B, the sample with water 60°C was designated by C. The liquid phase from substrate A was separated by centrifugation while the liquid phases from substrate B and C were separated in a laboratory screw press for the pressing of fruits and vegetables. The separated liquid phases of substrates A, B and C were diluted with water to obtain a unit volume and the analytical results were recalculated to a transfer ratio in the liquid phase in relation to the content of particular nutrients in dry matter of the original substrate mixture.

The experiments conducted in an experimental unit of anaerobic digestion and in an equipment for IFBB made it possible to determine the content of mineral nutrients in substrate A after 42-day anaerobic digestion in mesophilic conditions (40°C), in the liquid phase of substrate A after anaerobic digestion, in the liquid phase of substrate B and C after recalculation to the dry matter content and concentration corresponding to substrate A, also after the process of anaerobic digestion under the same conditions (42 days, 40°C).

The above recalculations enable to clearly show the advantages of the IFBB process in nutrient transfer from solid to liquid phase when substrate A and 5 times diluted substrates B and C are compared, but they may unfortunately evoke a distorted idea about the real

concentration of nutrients in liquid phases. It is to recall that IFBB increases the mass flow and transfer to the liquid phase but with regard to the 5-fold dilution the nutrient concentration in liquid waste for fertilization continues to decrease. This is the reason why the table below shows the original, not recalculated concentrations in the fugate of fermented substrate A and in the fermented liquid phases of the same substrate in IFBB conditions designated by B and C, which document considerable dilution of these potential mineral fertilizers.

The solid phases of substrates A, B and C after anaerobic digestion were subjected to determination of organic matter hydrolysability in sulphur acid solutions according to Rovira and Vallejo (2000, 2002) as modified by Shirata and Yokozawa (2006); we already used this method to evaluate the degradability of a substrate composed of pig slurry and sludge from a municipal waste water treatment plant (Kolar et al. 2008).

Table 8 documents that the IFBB procedure proposed by German authors for grass haylage is applicable to the typical substrate of Czech biogas plants, to a mixture of cattle slurry, maize silage and grass haylage. In agreement with German experience the observed transfer ratios are markedly higher at 60°C compared to 15°C of hydrothermal conditions but the value of transfer ratios to the liquid phase is generally lower in our experiments. We ascribe this fact to the properties of the material and also to the achieved axial force of the used press that was apparently lower even though the same perforation size of the conical part of the press (1.5 mm) and slope of the body (1 : 7.5) were used.

The results in Table 8 illustrate that the separation of the liquid and solid phase of the substrate that has not been subjected to anaerobic digestion yet by means of centrifugation only is rather imperfect from the aspect of the mass flow of components. The IFBB system (water dilution, intensive stirring at a temperature of 60°C and subsequent separation of the liquid and solid phase with a screw press) increases the transfer of organic and mineral substances into the liquid phase by about 15 — 20%, and it is also true of the saccharidic nitrogen-free extract and organic nitrogen compounds. This fact documents that the liquid phase has a higher amount of active, well-degradable organic material for anaerobic digestion, and so it is possible to expect not only the higher production of biogas but also more mineral nitrogen in the liquid after anaerobic digestion.

The high mass flow of alkaline metals and chlorine into the liquid phase, and on the contrary, the low transfer of calcium confirm the opinion of German researchers (Wachendorf et al. 2007, 2009) that the IFBB procedure largely increases the quality of biomass solid phase as a material for the production of solid fuels: the production of polychlorinated dioxins and dibenzofurans is reduced, waste gases will be less corrosive and the temperature of ash fusion will be higher.

Nitrogen compounds of the substrate dry matter account for 16.3%, i. e. these nitrogen organic compounds contain 2.6% of nitrogen in dry matter (Table 8). The content of mineral nitrogen in the substrate before anaerobic digestion was 1%. If the digestate contains 2.26% of mineral nitrogen in the same dry matter after anaerobic digestion, it is to state that during anaerobic digestion about a half of organic nitrogen mineralized and enriched the original 1% content of the substrate before the fermentation process. But the dry matter content decreased in the course of fermentation, and therefore the concentration of all nutrients in digestate apparently increased contrary to the original substrate. In our experiment the concentration of substrate dry matter decreased from 19.3% to 13.3% by weight during anaerobic digestion. The content of mineral nitrogen amounting to 3.28% at this dry matter content corresponds to 2.26% of min. N at the original dry matter content of 19.3% by weight (Table 9). The contents of the nutrients P, K, Ca and Mg in digestate dry matter after anaerobic digestion (Table 9) are apparently substantially higher than before fermentation. However, anaerobic digestion did not actually bring about any increase in the content of these nutrients, and the increased concentrations completely correspond to a reduction in dry matter content, 19.3 : 13.3 = 1.45.

It is not a new fact, but Table 9 shows how this mineral nitrogen is transferred to the liquid phase of substrates B and C compared to substrate A. Obviously, the liquid phase of substrate B has a higher amount of mineral nitrogen than that of substrate A, and so the effects of the screw press, which already before anaerobic digestion enriched the liquid substrate B with colloidal solutions (sols) of nitrogen organic compounds from the crushed cell walls of the plant material that provided further mineral nitrogen during fermentation, were significantly positive at the same temperature. It was still more evident in the liquid phase of substrate C while a conclusion can be drawn that a higher temperature contributes to a higher extraction of insoluble or partly soluble nitrogen organic compounds from which further mineral nitrogen is released after subsequent fermentation.

Table 10. Contents of mineral nutrients after anaerobic digestion (42 days, 40°C) in the liquid phase of digestate A and in liquid phases B, C with the application of IFBB in % by weight of solutions that should be used for mineral fertilization

(Sample size n = 4, reliability interval of the mean for a significance level a = 0.05)

Table 11 shows the results of hydrolytic experiments with solid phases of substrates A, B and

C. They confirm the previously observed fact in the work with the substrate consisting of a mixture of pig slurry and primary sludge from a municipal waste water treatment plant that the solid phases of wastes from anaerobic digestion cannot be efficient as mineral fertilizers because of their very low degradability (Kolar et al. 2008). The IFBB process, which enriches the liquid phases with organic, easily degradable substances and improves biogas yields during the anaerobic degradation of only liquid phases, further depletes of these substances the solid phases of substrates and impairs their quality as organic fertilizers, even though it is not the case of an increase in the resistant component but only in worse hydrolysable LP II.

Table 11. Proportions of the three pools of carbon in the solid phase of substrate A before anaerobic digestion (A1), after anaerobic digestion (A2) and in the solid phase of substrate A1 after IFBB procedure before anaerobic digestion at 15°C (B) and at 60°C (C) as determined by the acid hydrolysis method of Rovira and Vallejo (2002).

(Sample size n = 4, reliability interval of the mean for a significance level a = 0.05).

Hence it is to state:

We tested the Integrated Generation of Solid Fuel and Biogas from Biomass (IFBB) procedure proposed for ensiled grass matter from the aspect of suitability of its use for a typical substrate of new Czech biogas plants, a mixture of cattle slurry, maize silage and grass haylage. The agrochemical value of the liquid phase from a biodigester was also evaluated. We concluded that this procedure is suitable for the tested substrate and improves the agrochemical value of a fugate from biogas production. By chlorine transfer to the liquid phase it makes it possible to use the solid phase as a material for the production of solid biofuels with a reduced threat of the generation of polychlorinated dioxins and dibenzofurans during combustion. However, the concentration of mineral nutrients in the liquid phase during IFBB procedure is extremely low after anaerobic digestion as a result of dilution with water, and so its volume value is negligible.

Here research must go on.

The detailed description of the method development, particular application conditions and its use were published by Linhova et al., (2010a). The main idea of the staining is based on fact that clostridia are usually stained according to Gram as G+ after germination from spores (motile, juvenile cells) and as G — when the cells started to sporulate. The change in Gram staining response corresponds to metabolic switch from acids to solvents formation and also with an alteration in a cell membrane composition i. e. thinning of peptidoglycan layer (Beveridge, 1990). Therefore the cells of C. pasteurianum were labelled with a combination of fluorescent probes, hexidium iodide (HI) and SYTO 13 that can be considered a fluorescent alternative of Gram staining. Cells of C. pasteurianum forming mainly acids fluoresced bright orange-red as G+ bacteria and the solvent producing, sporulating cells exhibited green-yellow fluorescence as G- bacteria (see Fig. 2). The red colour of labelled young cells was a result of a fact that green fluorescence of SYTO13 was quenched by that of HI while bright green-yellow colour of sporulating and/or old cells was caused by staining only by SYTO13 when HI did not permeate across the cell wall. Jones et al., (2008) used different combination of dyes (propidium iodide and SYTO 9) for labelling C. acetobutylicum ATCC 824 during time course of batch cultivation but attained the same conclusion.

Then, flow cytometry enabling quantification of fluorescent intensities of labelled clostridial populations was used for monitoring of physiological changes during fed-batch cultivation (Linhova et al., 2010a). For flow cytometry measurement, the cells were stained only by HI and the signal of fluorescent intensity acquired in a channel FL3 (red colour) was related to forward scatter signal (FSC) which corresponded to cell size in order to gain data independent on cell size. The data measured for C. pasteurianum were compared with those for typical G+ and G — bacteria i. e. for Bacillus megatherium and Escherichia coli and there was a striking difference between the values of FL3/FSC for C. pasteurianum on one hand and those for B. megatherium and E. coli on the other hand. While the values for B. megatherium (G+) and

E. coli (G-) oscillated ±0.1 and ±0.2, respectively, in time course of 32 h in which they were sampled, the values for C. pasteurianum dropped from 3.1 to 0.8 during the cultivation. It was

also evident that acidogenic phase had a very short duration and both metabolic phases overlapped. Further experiments are necessary to assess unambiguously the acquired data, however it is tempting to hypothesize that C. pasteurianum NRRL B-598 has a different pattern of acids and solvents formation when solvents production is connected rather with exponential growth phase than the well-known solventogenic strain C. acetobutylicum ATCC 824 in which solvents production is generally assembled with stationary growth phase.

Viscosity of a bio-oil is the measure of its internal friction which resists the flow of it. Viscosity is an important fuel property that should be considered when attempting to design and select handling, processing and transportation equipment. Viscosity of bio-oil affects the operation of fuel injection equipment, particularly when the increase in the viscosity affects the fluidity of fuel at low temperatures. Again, the quality and practical application of bio-oil as fuel is closely dependent on its viscosity and the elemental compositions i. e., the lower viscosity and oxygen content is desirable (Ertas & Alma, 2010). In general, bio-oil has high viscosity as compared to crude oil and diesel fuel (Onay & Kockar, 2006; Parihar et al., 2007; Pootakham & Kumar, 2010a, b). Pootakham and Kumar (2010a) reported that the loading equipment of the petroleum product such as gasoline and diesel fuels operates between 0.9 and 1.3 m3/ min, whereas they can be operated for bio-oil at a volume flow rate of 0.6 m3/min and an operating pressure of 205 kPa (or 30 psi) for safety (Jones & Pujado, 2006). Bio-oil is more viscous than crude oil at room temperature; however its viscosity is very similar to that of crude oil in a temperature range of 35-45°C, (Bridgewater, 1999; Thamburaj, 2000; Pootakham & Kumar, 2010a, b). In order to transport the bio-oil in pipeline, the temperature of the pipeline should be maintained in the range of 35-45°C to keep the viscosity similar to that of crude oil (Pootakham & Kumar, 2010a, b). According to Thangalazhy-Gopakumar et al (2010), viscosity of bio-oil is relatively higher than that of diesel (0.011 Pa. s) and gasoline (0.006 Pa. s). In general, high viscosity fuel results in poor atomization and incomplete combustion, formation of excessive carbon deposits on the injection nozzles and the combustion chamber, and contamination of the lubricating oil with unburnt residues. The viscosity of the fuel directly influences atomization and mixing in the combustion chamber. In fuel application, the lower the viscosity, the easier it is to pump and to atomize and achieve finer droplets (Ji-Lu, 2008). Hence bio-oils in their original form are not suitable for use in modern diesel engines (Ozaktas et al., 1997). Because of their high acidity, low thermal stability, low calorific value, high viscosity, and poor lubrication characteristics limit their use as transportation fuel (Garcia-Perez et al., 2006b). Oasmaa et al (2005) stated that for engine application, the viscosity should be in the range of 10-20 cSt with a solids content of less than 0.1 wt%. As is known, bio-oils are entirely different from petroleum fuels. There is a necessity to establish fuel specifications for commercial application of bio-oils as liquid fuels. The specifications should include the most critical properties such as viscosity, lubricity, homogeneity, stability, heating value, pH, water, flash point, solids, and ash (Qiang et al., 2008).

The viscosity of bio-oil varies depending on the temperature, feedstock, water content of the oil, amount of light ends that have been collected and the extent to which the pyrolysis oil has aged (Ji-Lu, 2008). For example, bio-oil produced from P. indicus and F. mandshurica had a kinetic viscosity of 70-350 mPa s and 10-70 mPa s separately, and bio-oil produced from rice straw had a minimum kinetic viscosity about 5-10 mPa s, which is mainly due to high water content in bio-oil from rice straw (Luo et al., 2004). The presence of water has both negative and positive effects on the storage and utilization of bio-oils. The negative effects are, it lowers heating values, causes phase separation, increases ignition delay, and reduces combustion rates and adiabatic flame temperatures during the combustion process. Further, it leads to premature evaporation and subsequent injection difficulties during the preheating process. The positive effects are, it reduces viscosity, facilitates atomization, and reduces pollutant emissions during combustion (Calabria et al., 2007). Moreover, OH radicals from water can inhibit the formation of soot and can also accelerate its oxidation. According to Senso’z and Kaynar (2006), viscosity of the bio-oils is related to fatty acid chain length and number of saturated bonds. In general, the density of bio-oil is higher than that of water confirms that it contains heavy fractions (Sensoz et al., 2006). The lignin content of original feedstock has a positive influence on molecular weight and viscosity of bio-oil (Fahmi et al., 2008). Recently, Ertas and Alma (2010) compared the average molecular weight and molecular weight distribution of laurel extraction residues bio-oil (664 g mol/l and 1.52) and found they were very close to those of switchgrass bio-oil of 658 g mol/l and

1. 49, respectively (He et al., 2009a). Viscosity of bio — oil increases during storage, due to slow polymerization and condensation reactions, the increase in viscosity is enhanced by higher temperature. The presence of inhibitors (hydroquinone) can dramatically reduce the rate of increase in bio-oil viscosity, due to the suppression of thermal polymerization reactions by the inhibitors (Ji-Lu, 2008). Garcia-Perez et al (2010) observed that the increase in viscosity of bio-oils is due to the solubilization of lignin derived oligomers. The condensation reactions occur ageing increases the water content in bio-oil with time (Garcia-Perez et al., 2002). The instability may be attributed to the presence of alkali metals in the ash, which are being carried over/entrained by the char particles with the vapors. These alkali metals catalyse the polymerization reactions and thereby increase the viscosity (Diebold, 2002).

Simple methods such as addition of polar solvents, diesel or other fuels can address some of the undesired bio-oil characteristics. Polar solvents, such as methanol or ethanol, can improve the volatility and heating value and decrease the viscosity and acidity. The addition of ethanol improves the volatility, stability and heating value and decreases the viscosity, acidity and corrosivity (Ji-Lu & Yong-Ping, 2010). The blending of diesel or other fuels can rearrange the viscosity of bio-oil (Onay & Kockar, 2006). In order to improve fuel properties of bio-oils, many methods are under investigation such as emulsification, hydrotreating, and catalytic cracking, which is beyond the scope of this chapter.

2. Materials and methods

Well known catalysts for the hydrotreatment of pyrolysis oil are conventional hydrodesulfurisation catalysts such as sulfided NiMo/ Al2O3, CoMo/ Al2O3, and NiMo/ Al2O3- SiO2 (Ferrari et al., 2002; Maity et al., 2000) to cope with the harsh reaction conditions (T > 200 C, aqueous environment with organic acids). Carbon-supported CoMo, or Mo supported on TiO2, ZrO2 and TiO2-ZrO2 mixed oxides (Satterfield&Yang, 1983; Lee&Ollis, 1984) have been tested as well. The latter catalysts all require sulphur in the feed to maintain activity. Pyrolysis oils contain only limited amounts of sulphur (Furimsky, 2000) and sulphur addition would be required during processing to maintain catalytic activity. This will lead to sulfur emissions e. g. in the flue gas, which is not preferred for an environmental point of view.

Non-sulfided, non noble metal catalysts have also been tested for the catalytic hydrotreatment of pyrolysis oil, but to a far lesser extent. The hydrodeoxygenation of phenol over a Ni/SiO2 catalyst has been reported and showed low HDO activity. Horne&Williams (1996) tested ZSM-5 zeolites as the catalyst for the deoxygenation of model compounds such as anisole. Their results, though, showed that anisole is mainly converted into phenol and methyl substituted phenols, and not to the oxygen-depleted compounds. Xu, et al. (2010) tested MoNi/y-Al2O3 (reduced prior to reaction) for the mild hydrotreatment of pyrolysis oil (3 MPa and 200 oC).

Noble metal based catalysts have also been evaluated as substitutes for sulfided catalysts. Examples are Pd on zeolite carriers (Horne&Williams, 1996), Pd on mesoporous CeO2 and ZrO2 (Senol et al., 2005), and Rh on zirconia (Gutierrez et al., 2008). Carbon supports have also been explored (Wildschut et al., 2009b; Wildschut et al., 2010a;, Wildschut et al., 2010b; Venderbosch et al., 2010). We have recently reported exploratory catalyst studies on the upgrading of fast pyrolysis oil by catalytic hydrotreatment using a variety of heterogeneous noble metal catalysts (Ru/C, Ru/TiO2, Ru/Al2O3, Pt/C and Pd/C) and the results were compared to typical hydrotreatment catalysts (sulfided NiMo/ Al2O3 and CoMoAl2O3). The reactions were carried out at temperatures in the range of 250 and 350 °C and pressures between 100 and 200 bar. The Ru/C catalyst appears superior to the classical hydrotreating catalysts with respect to oil yield (up to 60 %-wt.) and deoxygenation level (up to 90 %-wt.) (Figure 2) (Wildschut et al, 2009).

Unfortunately, noble metal catalysts are very expensive and this limits their application potential. The catalytic hydrotreatment of fast pyrolysis oil using much cheaper bimetallic NiCu/8-Al2O3 catalysts with various Ni/Cu ratios (0.32 — 8.1 w/w) at a fixed total metal intake of about 20 wt% was reported recently (Ardiyanti et al., 2009). Hydrotreatment reactions of fast pyrolysis oil (batch autoclave, 1 h at 150 oC followed by 3 h at 350 oC, at 200 bar total pressure) were performed and highest catalyst activity (in terms of H2 uptake per g of Ni) was observed for the catalyst with the lowest Ni loading (5.92Ni!8.2Cu). Product oils with oxygen contents between 10 and 17 wt% were obtained and shown to have improved product properties when compared to the feed.

Recently, we have also shown the potential of homogeneous Ru catalysts for the catalytic upgrading of fast pyrolysis oil fractions (Mahfud et al., 2007a). Fractions were obtained by treatment of the pyrolysis oil with dichloromethane or water. The former approach leads to a dichloromethane layer enriched with the lignin fraction of pyrolysis oil. The dichloromethane fraction was hydrogenated in a biphasic system (dichloromethane/water) using a homogeneous water soluble Ru/tri-phenylphosphine-tris-sulphonate (Ru-TPPTS) catalyst. Analysis revealed that the oxygen content of was lowered considerably and that particularly the amounts of reactive aldehydes were reduced substantially.

The water-soluble fraction of pyrolysis oil, obtained by treatment of fast pyrolysis oil with an excess of water was hydrogenated in a biphasic system with a homogeneous Ru-catalyst (Ru/tri-phenylphosphine, Ru-TPP) dissolved in an apolar solvent (Mahfud et al., 2007b). Initial experiments were conducted with representative, water soluble model compounds like hydroxy acetaldehyde and acetol. The effects of process parameters (e. g. temperature, initial H2 pressure, and initial substrate concentration) were investigated and quantified for acetol using a kinetic model. The hydrogenation of the pyrolysis oil water-soluble fraction using the biphasic system at optimized conditions was performed and significant reductions in the amounts of reactive aldehydes such as hydroxyacetaldehyde and acetol were observed, demonstrating the potential of homogeneous Ru-catalysts to upgrade pyrolysis oils.

Durability represents the measure of shear and impact forces that a pellet could withstand during handling, storing and transportation process. The durability of pellets is usually measured following the ASABE Standard S269 (ASABE, 2007), which require about 50-100 g of pellets/ compacts. However, due to the limited number of pellets obtained during single­pellet compression test, it is not feasible to use this method. Instead, the durability of pellets can be measured by following the drop test method (Al-Widyan and Al-Jalil, 2001; Khankari et al., 1989; Sah et al., 1980; Shrivastava et al., 1989), where a single pellet is dropped from a 1.85 m height on a metal plate. The larger intact portion of the mass retained is expressed as the percentage of the initial weight.

Adapa et al. (2010) reported that the type of agricultural biomass, steam explosion pretreatment, applied pressure and screen size all had significant effect on pellet durability. Statistically, no significant correlation (R2 values) was obtained for change in durability with applied pressure and hammer mill screen sizes. In general, pellet durability increases with an increase in applied pressure and grind size, and application of pre-treatment. Similarly, Kaliyan and Morey (2009) indicated that the durability of corn stover or switchgrass briquettes was significantly affected by pressure, moisture content and preheating temperature, while particle size did not have any significant effect. Kashaninejad et al. (2011) also reported the mean durability of pellets made of giant wild rye and mixed forage increased from 63.08 to 89.26% and from 61.47 to 89.21%, respectively when the hammer mill screen size increased from 0.8 to 3.2 mm. This could be primarily due to mechanical interlocking of relatively long fibers at higher grind sizes. They also indicated that at any specific compressive load, the pellet durability of biomass grinds with 12% moisture content was significantly higher than samples with 9 and 15% and demonstrates the moisture contents above or below 12% would lead to lower quality pellets.

A variety of microbial communities are thought to have unique lignocellulolytic capabilities making them of great interest for the development of second and third generation biofuels. Among the natural degraders of lignocellulose are microbial communities that are found e. g. in the gut of wood-degrading insects such as termites, in ruminants, in compost or soil. Electron microscopy with its ability to conduct an analysis at a spatial resolution of about a nanometer allows the investigation of the interplay between the different bacterial cells that constitute a community, its association with the plant material as well as their macromolecular inventory and degradation strategies.

SEM can only be conducted on surfaces, e. g. plant part from the digestive system of a cellulose degrading animal containing the microbial community, or microbes filtered out of a microbial culture suspension. Since SEM imaging typically requires sample fixation, dehydration and critical point drying, followed by sputter coating, the fine details tend to get lost. The microscopy on those samples in a high-resolution SEM (typically, 2 — 10 kV accelerating voltage are used) can visualize the shape of microorganisms, thus sometimes enabling an identification of certain species, and show both interaction between microbes and between microbes and the surface (e. g. plant material). It can thus be a valuable first insight into the composition and functioning of a microbial community, facilitated by the fact that the technique is rather quick and does not require extensive preparations. However, the technique does not enable a more detailed analysis of the microbial community and is limited to the surface of the samples, thus possibly representing only a small fraction of the sample that might not be typical for the sample in general.

If a more in-depth analysis of the microbial community is the goal, one has to use TEM instead of additional SEM. The analysis of a microbial community with TEM can visualize a lot of interesting traits in this community, including frequent cell-cell interactions, either directly or via a variety of microstructures such as pili and flagella. Even more interesting in this case are the interactions between microbial cells and the plant biomass, which is present in the sample. In studies of such communities (Knierim et al., in preparation) we have found different strategies of cell wall attachment and biomas digestion. An unresolved challenge is the direct identification of species in TEM or SEM. There have been recent advances in this field, combining TEM with Catalized Reporter Deposition Fluorescence In-Situ Hybridization (CARD-FISH), which can identify either groups of bacteria or even certain species (Knierim et al., submitted). However, this technique is extremely tedious and requires some compromises of the TEM imaging quality in order to enable the identification via CARD-FISH.

The described electron microscopy techniques are not only applicable to the understanding of microbial communities but can also be utilized for the analysis of bacteria that have been engineered for certain capabilities. Such capabilities that are developed by synthetic biologists can be the increased production of fuels, the production of different fuels (especially those with longer carbon chains which are more valuable than ethanol) or the assessment of toxicity of the produced biofuels. When combined with tag-based labeling TEM also allows a rational monitoring of protein expression levels, cell-to cell variation and subcellular localization. Thus the application of SEM and TEM to those samples can facilitate the re-engineering of microorganisms that are needed for the production of second and third generation biofuels.

A natural limitation of TEM usually is its coverage of very small sample volumes covered by thin sections. To overcome this problem, FIB/SEM can be employed, which provides a three dimensional view of large volumes of a microbial community at a resolution comparable to electron tomography. For FIB/SEM a similar sample preparation protocol as described above for TEM can be used, but one has to ensure a high internal contrast inside the plastic blocks. We have had very good experience with tannic acid for this purpose. During

FIB/SEM imaging one must pay attention to the area of interest careful, as this technique is very time intensive and hence currently can only be applied to a limited number of volumes. Typically, volumes of 10 x 10 x 5 gm can be covered at an estimated resolution around 10-15 nm. The datasets that are produced this way easily get very large (in the several Gigabyte range) and require careful 3D reconstruction and analysis using software packages with those capabilities such as UCSF Chimera (http://www. cgl. ucsf. edu/chimera) or Amira (http://www. amira. com). We are currently drafting a manuscript on the FIB/SEM 3D analysis of the termite hindgut microbial community, where we classify and quantify constituent microbial community member according to size, shape and internal density characteristics, and map out their distribution with respect to the biomass (Knierim et al., in preparation).

An understanding of the functioning of microbial communities that are capable of lignocellulose degradation will ultimately lead to the development of better techniques for lignocellulose degradation in an industrial setting such as a biorefinery for second or third generation biofuels. We are still in the early steps of this understanding due to the high complexity and variability of these microbial communities, but if we can reduce the complexity by identifying a small set of microbes with valuable capabilities, we may be able to speed up this process. As the principles that are present in these microbial communities have been developed by evolution over billions of years, we can assume that they are very energy efficient, thus providing a maximal energy output while taking up a small amount of energy themselves — an important challenge for the design of industrial processes for the production of biofuels

6. Outlook

Improving feedstocks properties as well as optimizing each step of the deconstruction process and the fuels synthesis production step will decrease the production cost and is therefore key for replacing fossil fuels with biofuels. To accomplish such needed technological advances, one needs to resort to a variety of different biophysical techniques, typically carried out by specialists, that can quantify the effect of experimental intervention and lead to a detailed understanding of the physical, chemical and biological processes of lignocellulosic biofuel production.

7. Acknowledgements

This work was part of the DOE Joint BioEnergy Institute (http://www. jbei. org) supported by the U. S. Department of Energy, Office of Science, Office of Biological and Environmental Research, through contract DE-AC02-05CH11231 between Lawrence Berkeley National Laboratory and the U. S. Department of Energy. This work was in part supported in part by the Energy Biosciences Institute grant 007-G18.

Next, we explained about the co-generation system by which electricity and thermal energy can be generated. In the case of BT-CGS, due to the heat balance, the reaction energy in the furnace might be shortage. Thus, the additional feedstock would be necessary. In the case of Bio-H2 production system, since off-gas through PSA is available, the additional biomass material is not required.

Also, from the viewpoint of the economic condition, the case that the additional one is fed into BT would be much better in comparison to the case without any feedstock. That is, more products (i. e. electricity and/or thermal energy) can be generated. Consequently, the economic condition of BT-CGS operation would be improved by a lot of energy products. Thus, we consider BT-CGS case in which the additional feedstock is required.

For the operation of gas-engine due to the low calorific heating value of bio-gas which means the syngas of BT gasifier, although there are sometimes problems on the heating value of fuel, we executed the process design using the practice parameters which were analysed by the engine manufacturing maker.

Table 5 shows the performance of Bio-CGS. In Tables 5, the cold gas efficiency цСоЫ, the net power efficiency ЦРош, the heat recovery efficiency ^Heat and the net total efficiency ^Toid are defined as follows:

Syngas [MJ/h]

ГІ|Cdd Feedstock [MJ/h] + Char [MJ/h] + Add. Feedstock [MJ/h]

Net Power (= Power-Auxiliary)[MJ/h]

^Pav Feedstock [MJ/h] + Add. Feedstock [MJ/h]

Steam[MJ/h] + Hot water [MJ/h]

Silent Feedstock [MJ/h] + Add. Feedstock [MJ/h]