BOD is naturally capable of catalyzing the oxidation of bilirubin into biliverdin and to simultaneously reduce dioxygen (Shimizu et al., 1999). BOD is very similar to laccase. Performances of BOD electrodes are greatly related to the amino-acids sequence around T1 site of the enzyme (Li et al., 2004). It is clearly reported that the most efficient BOD enzyme comes from Myrothecium verrucaria. Redox potential of its T1 site is included between 650 and 750 mV vs. NHE, and the enzyme is thermally stable up to 60 °C (Mano et al., 2002b). It is thus possible to use it at physiological temperature without denaturing the protein. To build efficient BOD electrodes intended in working at physiological pH value, it is judicious to use positively charged mediator molecules since the isoelectric point of BOD is close to pH = 4. Actually, during oxygen reduction reaction, the use of an osmium based redox polymer has lead to performances such as 880 pA cm-2 at 0.3 V vs. Ag/ AgCl (physiological conditions) at a scan rate of 1 mV s-1 (Mano et al., 2002a). Additionally, the redox osmium based hydrogel conferred a very favorable environment to stabilize BOD since 95% of the initial activity of a BOD electrode can be preserved after three weeks storage (Mano et al., 2002a). This remarkable stability probably results in auspicious electrostatic interactions between the swelling matrix and the enzyme. Performances of BOD electrodes are furthermore unaffected in the presence of chloride ions. In fact BOD remains active for chloride concentrations lower than 1 M (Mano et al., 2002a). This property is of major interest for the development of implantable microscale glucose/O2 biofuel cells using BOD as cathode catalysts. The major encountered problem with BOD electrodes is the relative lack of stability of the enzyme in physiological serum. Cupric centers of BOD are indeed capable of binding with one urea oxidation product, oxidation reaction catalyzed by the enzyme (Kang et al., 2004). This phenomenon can nevertheless be limited by spreading a Nafion® film on the catalyst (Kang et al., 2004). It is moreover reported that chemically modified Nafion® is capable of constituting a favorable environment to stabilize BOD (Topcagic & Minteer, 2006). Consequently, it seems of interest to immobilize BOD in Nafion® films. A promising technique for the development of efficient BOD electrodes has already been reported in literature (Habrioux et al., 2010). It consists in firstly adsorbing BOD/ABTS2- (2,2-azinobis-3-ethylbenzothiazoline-5-sulfonic acid) complex on a carbon powder, Vulcan XC 72 R in order to increase both enzyme loading, the stability of the protein and the quality of the percolating network in the whole thickness of the polymer film. Actually, to realize the electrochemical reaction, a triple contact point (between the catalytic system, the electrolyte and the electronic conductor) is required. Once the catalytic

system is adsorbed, a buffered Nafion® solution is added. The whole system is then immobilized onto a solid carbon electrode (Fig. 3).

Fig. 3. Method used for the preparation of BOD cathodes according to the process described in Ref. (Habrioux et al., 2010)

Previous studies have shown the interest lying in the use of ABTS2- as redox mediator in combination with multicopper oxidases. One of them was carried out by Karnicka et al. who have shown that wiring laccase to glassy carbon through a ABTS2-/carbon nanotube system was a very efficient pathway to reduce molecular oxygen into water (Karnicka et al., 2008). The combination of ABTS2- with BOD is also known to exhibit a high electrochemical activity towards oxygen reduction reaction (Tsujimura et al., 2001). These observations are confirmed by electrochemical studies performed on electrodes previously described (Fig. 3).

Curves of Fig.4 clearly show the interest of such electrodes that exhibit a catalytic current from potentials as high as -50 mV vs. O2/H2O (0.536 V vs. SCE). Furthermore the half-wave potential is only 100 mV lower than the reversible redox potential of O2/H2O. This value is in good agreement with that reported by Tsujimura et al. (0.49 V vs. Ag/AgCl/KCl(sat.) at pH = 7.0) (Tsujimura et al., 2001). Let’s notice that the half-wave potential value is very close to the redox potential of T1 site of BOD (0.46 V vs. SCE). This has already been explained by the fact that the reaction between ABTS2- and BOD is an uphill one (Tsujimura et al., 2001).

Fig. 5. Electrochemical activity of BOD/ABTS2-/Nafion® electrode: dependence of the current value at 0.2 V vs. SCE with oxygen concentration

The current linearly increases with the oxygen concentration from low values to around 35%. This linearity suggests that the reaction is of a first order with oxygen concentration thereby, the Koutecky-Levich plots can be considered. Assuming that the rate determining step is an enzymatic intramolecular electron transfer step, it is possible to express the current density of a BOD/ ABTS2-/Nafion® electrode working in an air saturated solution as follows (Schmidt et al., 1999):

(2)

In Eq.2, represents the diffusion limiting current density expressed by Levich equation:

j“ = 0.2nFD23v _16C^/q (3)

In Eq.3, n is the number of electrons exchanged, D the diffusion coefficient, C0 is the oxygen concentration, Q is the rotation rate, F the Faraday constant and nis the kinematic viscosity. Then, jifilm corresponds to the limitation due to oxygen diffusion in the catalytic film and jLads is the limiting current density due to oxygen adsorption on the catalytic site. Since these two last contributions to the total current density do not depend on Q, it is impossible to separate them. They will be described according to Eq.4.

1 — JL _/L

ads film

jL jL jL

In Eq.2, n is the overpotential (n = E-Eeq), j0 the exchange current density, a the transfer coefficient, R = 8.31 J mol-1 K-1, F=96500 C mol-1 and T the temperature. 0 and 0c are the

covering rates of the active sites of the enzyme at E and Eeq, respectively. We will assume that Q » Qc for all potential values. From Eq.2, when Q^<x>, the limit of 1/j can be expressed as follows:

In Eq.5, when n^®, 1/jk^1/jL. It is thus possible to determine jL value by extrapolating and reporting the 1/jk values as a function of the potential value E. Transforming Eq.5 (Grolleau et al., 2008), it becomes as follows:

Under these experimental conditions, calculated values for both Tafel slope and exchange current density are respectively of 69 mV / decade and 25 pA cm-2. The high value obtained for j0 confirms the ability of BOD/ ABTS2-/Nafion® system to activate molecular oxygen in a physiological type medium. Moreover, it also certifies that the oxygen reduction reaction starts at very high potentials. The reference catalyst classically used to reduce molecular oxygen is platinum. It can be noticed that under similar conditions, the exchange current density is only of 5 pA cm-2 when we used platinum nanoparticles as catalyst. This clearly shows the great interest lying in these electrodes to reduce oxygen in glucose/O2 biofuel cells. Nowadays, the ma—or problem encountered with these electrodes is the lack of stability of the redox mediator (ABTS2-) (Tsujimura et al., 2001).

1.1 Advantages of microfluidics

An alternative approach towards the miniaturization of energy conversion devices is the use of microfabrication techniques. Microchemical systems have inherent advantages over macrosystems, including increased rates of mass transfer, low amount of reagents, increased safety as a result of smaller volumes, and coupling of multiple microreactors. Microfluidic techniques are ideal for miniaturization of devices featured with typical scale of channels of submillimeter in height and with laminar flow. Application of microfluidics to fuel cells has been developed rapidly since the years 2000 (Ferrigno et al., 2002; Choban et al., 2004). In such devices, all functions and components related to fluid delivery and removal, reactions sites and electrodes structures are confined to a microfluidic channel. In the channel, as illustrated in Fig. 2, the flow of streams of fuel (colored pink) and oxidant (colored blue) is kept near-parallel, which ensures minimal diffusional mixing between the streams. The only way that molecules in opposite streams can mix is by molecular diffusion across the interface of the two fluid streams. The lack of convective mixing promotes laminar flow of fluids.

The electrochemical reactions take place at the anode and cathode located within the respective streams, without needing a membrane to minimize the ohmic drop, what maximises the current density. Protons diffuse through the liquid-liquid interface created by the contacting streams of fuel and oxidant. The cathode and the anode are connected to an external circuit. The technique to force the fluid through microchannels is the pressure driven flow, in which the fluid is pumped through the device via positive displacement pumps, such as syringe pumps.

As summarized by authors (Luo et al., 2005; Gervais et al., 2006; Sun et al., 2007), the limiting factors in laminar flow-based microfluidic fuel cells that influence the performances are (i) cross-diffusional mixing of fuel and oxidant at the interface between the two streams, and (ii) the formation of depletion boundary layers at the surface of the electrodes as the result of the reaction of fuel and oxidant. Interesting papers have presented theoretical and experimental works to describe how to prevent or reduce these phenomena by concentring research efforts on designs, electronic and ionic conductivity, and electron-transfer kinetics in microfluidic fuel cells (Lee et al., 2007). The role of flow rate, microchannel geometry, and location of electrodes within microfluidic systems was also studied (Choban et al., 2005; Sun et al., 2007; Amatore et al., 2007; Chen et al. 2007).

Similarly to microfluidic fuel cells, advanced microfabrication techniques can be applied to build components of microfluidic enzymatic BFCs. The number of devices presented to date is limited. The devices have been developed based on both laminar flow within a microchannel and biological enzyme strategies. Indeed, the advantage of the co-laminar flow is to choose the composition of the two oxidant and fuel streams independently for optimum enzymatic activity and stability to improve reaction rates and current density (Zebda et al., 2009a).

This is a perennial herbaceous plant that grows mainly in the United States. Its ethanol yield per hectare is the same as for wheat. It responds to nitrogen fertilizers and can sequester the carbon in the soil. It is a highly versatile plant, capable of adapting easily to lean soils and marginal farmland (Heaton et al., 2004). Like maize, it is a type C4 plant, i. e. it makes an alternative use of CO2 fixation (a process forming part of photosynthesis). Most of the genotypes of Panicum virgatum have short underground stems, or rhizomes, that enable them with time to form a grassy carpet. Single hybrids of Panicum virgatum have shown a marked potential for increasing their energy yield (Bouton, 2007), but genetic engineering methods on this plant are still in a developmental stage and for the time being only their tetraploid and octaploid forms are known; we also now know that similar cell types (isotypes) reproduce easily.

1.3 Sweet sorghum (sorghum bicolor L)

The grains obtained from this plant are rich in starch and the stems have a high saccharose content, while the leaves and bagasse have a high lignocellulose content. The plant can be grown in both temperate and tropical countries, and it tolerates drought, flooding and alkalinity. Sorghum is considered an excellent raw material because the methods for growing and transporting it are well established. Ethanol can be obtained from it by exploiting both its starch and its sugar content. Research is currently underway on the use of hybrid or genetically modified species, although those obtained so far are weaker and need to be further refined and tested as concerns energy conversion efficiency (Rooney et al., 2007).

There are several factors regulating the lifetime of biofuel cells, which has always been a concern for their practical application. In most cases, the stability of enzyme determines the lifetime of biofuel cells. Immobilization of enzyme through covalent bonding on solid surface has attracted great attention for applications in catalytic processes. Therefore, in our research, covalent attachment of enzyme on supports was studied to promote rigidification of enzyme structure of the immobilized enzyme.

We have studied three different types of covalent surface functionalization for enzyme immobilization in EBFCs — (1) Direct amination; (2) Diazonium grafting; (3) Diamine grafting. In all these methods, the functional groups are realized on the pyrolyzed carbon surface.

2.2 Direct amination on pyrolyzed carbon

Direct amination was conducted by functionalizing its surface with ultraviolet (UV) irradiation under ammonia gas (Yang et, al., 2009). Quantified amino groups on the carbon surface were estimated by X-ray photoelectron spectroscopy in Fig. 3. It is found out that the amino

Fig. 3. X-ray photoelectron spectra of pyrolyzed carbon surface. (a) High-resolution scan of the C 1s peak and (b) high-resolution scan of the N 1s peak are compared before and after direct amination.

groups exist at surface by amination processes due to the high density of carbon. Ammonia gas forms as C-NH2 on carbon substrate because C-H bonds react easily with ammonia gas and undergo photochemical reaction on exposure to UV irradiation although steric limitations will limit the amine group coverage on the surface. The results showed that the amino groups were successfully formed on pyrolyzed carbon surface by direct amination.

In the past industrial applications, batch fermentation was a usual way how to produce biobutanol due to arrangement simplicity and attaining maximum biobutanol concentration, given by the used strain and cultivation medium, at the end of fermentation. Fed-batch fermentation can be regarded as modification of the batch process offering slight productivity increase by reduction of lag growth phase. However, taking into account possible industrial scale of the process, the preferential process arrangement is continuous ABE fermentation due to a lack of so called "dead" operation times. Nevertheless, its accomplishment in single bioreactor e. g. as chemostat is not usually easy because of biphasic process character when butanol production is not connected with growth directly (see Fig. 1). Theoretically, clostridial culture behaviour under chemostat cultivation conditions should follow an oscillation curve when acidogenesis is coupled with cell multiplication and decrease of substrate concentration. On the contrary, solventogenesis is coupled with decrease of specific growth rate due to sporulation what leads to cells wash-out and increase of substrate concentration in the medium. These two states should cycle regularly (Clarke et al., 1988) but in practice, irregular cycling with various depths of individual amplitudes is more probable as demonstrated several times (S. M. Lee et al., 2008). Moreover, chemostat cultivation conditions induce selection pressure on the microbial culture favouring non- sporulating, quickly multiplying cells what may cause culture degeneration i. e. the loss of the culture ability to produce solvents (Ezeji et al., 2005).

However, there are other options, tested in laboratory scale, how to arrange continuous ABE fermentation like multi-stage process splitting clostridial life cycle into at least two vessels, where first smaller bioreactor serves mainly for cells multiplication under higher dilution rate and in the second bigger bioreactor, actual solventogenesis takes place (Bahl et al., 1982). In addition, battery of bioreactors working in batch, fed-batch or semi-continuous regime ensuring continuous butanol output can also be considered continuous fermentation (Ni & Sun, 2009; Zverlov et al., 2006).

ABE fermentation in any regime can be combined with cells immobilization performed by different methods — entrapment in alginate (Largier et al., 1985), use of membrane bioreactor (Pierrot et al., 1986) or cells adsorption on porous material (S. Y. Lee et al., 2008; Napoli et al., 2010). Recently, final report of the US DOE grant (Ramey & Yang, 2004) has revealed a novel approach toward ABE fermentation. The principle of this solution is two step butanol production employing two microorganisms; at first Clostridium tyrobutyricum produces mainly butyric acid which is consumed by second microorganism Clostridium acetobutylicum and utilized for butanol production. The authors claimed they reached 50% yield of butyric acid in the first phase and 84% yield of butanol from butyrate. However, a pilot and a production plant planned for year 2005 have not been realized, yet. Nevertheless, this way of butanol production is still under research in U. S.A. (Hanno et al., 2010), focusing mainly on solventogenic clostridia that are capable of butyrate utilization for butanol production. One of the main constraints of biotechnological butanol production is its low final concentration in fermented cultivation media caused by its severe toxicity toward producing cells. Average butanol concentration, stable reached in Germiston plant in South Africa, was 13 g. L-1 (Westhuizen et al., 1982). Although higher butanol concentration (about 20 g. L-1) can be attained using e. g. mutant strain C. beijerinckii BA101 (Qureshi & Blaschek, 2001a) cost of distillation separation is still high. Therefore efficient preconcentration methods applied either after the fermentation or more often during the fermentation are being searched now. Moreover, if such separation method is integrated with fermentation process it will increase amount of utilized substrate by alleviating product toxicity. Preferential separation methods in this context seem to be gas stripping (Ezeji et al., 2003), adsorption on zeolites or pervaporation (Oudshoorn et al., 2009).

Despite others’ success with genomic PCR of Dunaliella-specific nucleotide sequences, our efforts to obtain the actin and rbcS2 promoters and nitA 3′-UTR were unsuccessful. Although the products from these PCR attempts appeared to be the correct fragment length, upon sequencing, it became clear that these were not the targets that we set out to amplify. BLAST analysis of some sequences recovered showed relevant homology to genes from Dunaliella viridis and Arabidopsis lyrata, but not the targeted promoters specific to D. salina.

3.3 Discussion

The inefficacy D. salina promoter and 3′-UTR amplification greatly inhibited our efforts to develop and test genetic transformation techniques with this alga. The failed attempts to amplify the actin, rbcS2, and nitA regulatory elements raises some concern for the accuracy of the sequences deposited in GenBank. Another possible cause for this lack of success might come from the strain of D. salina used as a source for these sequences. All of the prior work with these promoters and 3′-UTR has been done using the UTEX 1644 D. salina strain. It is conceivable that the UTEX 1664 sample supplied to us was either misidentified or contaminated. Additionally, the published sequence information might not have actually come from UTEX 1644, as claimed. From our observations, the CCAP 19/18 strain was consistently able to produce P-carotene when cells accumulated and dried on the inner surface of the culture flasks, unlike UTEX 1644. Although, the only way to know the identity of each strain for certain would be to perform genomic analysis of the 18S rRNA. This technique has been established for many species of Dunaliella, including both the UTEX 1664 and CCAP 19/18 strains (Olmos et al., 2000; Polle et al., 2008).

Due to our inability to construct Dunaliella-specific expression vectors, the attempts to genetically transform D. salina were limited to the use of the C. reinhardtii bleomycin- resistance plasmid, pSP124. There is evidence that genetic regulatory sequences from D. salina demonstrate activity in C. reinhardtii (Walker et al., 2004); thus, it is possible that the same is true of C. reinhardtii promoters for use in D. salina. Unfortunately, after numerous trials of electroporation and microparticle bombardment, no viable transformants were recovered after selection on bleocin plates.

It was surmised that the force of impact imposed by gold microparticles would be too much for D. salina, which lacks a cell wall; however, electroporation should have been more accommodating. Testing both high and low voltage (4 and 1 kV cm-1) electroporation conditions as well as high and low cell densities (4 x 107 or 1 x 106 cells ml-1) for transformation proved unacceptable for even transient expression of the ble gene. We did find that, with both methods of transformation, control samples remained viable after the procedure, so at least the electrical pulse itself was not killing the cells. Without endogenous D. salina promoters, we are unable to determine whether the absence of transgene expression was a result of improper transformation conditions or inactive promoters. Notwithstanding the pitfalls encountered during the molecular work with D. salina, experiments pertaining to antibiotic and herbicidal tolerance yielded results that will complement the microbiological understanding of D. salina and aid with future genetic manipulation of the organism.

3.4 Conclusions

Our approach to genetic transformation of a Dunaliella salina will hopefully set the stage for future efforts toward genetic engineering of this organism and, perhaps, act as a template for genetic bioprospecting with other novel algal species. Based on our dosage response experiments, we were able to narrow down the already short list of selective agents applicable to D. salina to the antibiotic bleomycin and the herbicide phosphinothricin and quantify the minimum inhibitory concentrations in both solid and liquid medium.

Limited by insufficient sequence information, we were unable to construct the proposed D. salina transformation vectors and transformation with existing Chlamydomonas vectors proved to be unsuccessful. Dunaliella salina is known to be a delicate organism due to its lack of a cell wall; thus, established transformation techniques may be too forceful for this organism. It is also reasonable to believe that gene silencing is an issue in D. salina and, in addition to optimizing transformation protocols suitable for this alga, molecular methods for promoting stable transgene integration and expression are of great interest to continued work in with Dunaliella species.

Although some accomplishments have been made in the area of D. salina molecular biology, genetic work with this alga warrants additional investigation. In addition to the chloroplast and mitochondrial genomes of D. salina CCAP 19/18, it is anticipated that the recent release of the nuclear genome will greatly encourage further genetic and metabolic engineering of this organism.

In the same way that computers are the coupling of software and hardware, microalgal cultivation systems rely on both the algal organism being grown and the vessel used to amass the cells. While one piece of software can potentially be run on various hardware devices, the two are often developed together and designed accordingly; the same is true with algal culture systems. Whether the growth environment is a raceway pond or a photobioreactor, there exist innumerable prospective algal species that could be cultivated. As the field of microalgal biotechnology moves more toward engineered algae and high- performance PBRs, the unique qualities of the organism will be paired with bioreactor design considerations. Just as the computing power of microchips is always increasing and new versions of operating systems are ever more frequently available, it is expected that algal species that are selected or engineered for high productivity will constantly demand more of their cultivation systems and vice versa.

Ultrasonic waves are energy application of sound waves which is vibrated more than 20,000 per second. In another words, it can be defined as the sound waves beyond human hearing limit. Human hear can not hear sound waves with more high-pitched sound waves of an average of 10-12 kHz. Ultrasonic or ultrasound signals are in the order of 20 kHz — 100 kHz and above the limit of human hearing. Ultrasonic waves were used as the first for medical research and detectors in the 1930s and 1940s (Newman& Rozycki, 1998). Idea of the use of ultrasound, especially in the industry since the 1980s began to develop rapidly, and today a wide range of applications using ultrasonic waves appeared. At present, ultrasonic waves are used in areas such as Atomization: Water sprays for dust suppression and humidifiers, low velocity spray coating, spray drying nozzles. Cleaning and cleaning of engineering items, small electronic items and jeweler using aqueous based solvents. Cleaning and disinfection of medical instruments and food processing equipment. Processing: Dispersion of pigments and powders in liquid media and emulsification. Extraction: Essential oil, flavonoid, resin, Crystallization and Filtration (Cintas et al., 2010; Mason et al., 1996; Mason, 2000).

Ultrasonic irradiation has three effects according to the investigators. First one is rapid movement of fluids caused by a variation of sonic pressure. It causes solvent compression and rarefaction cycles (Mason, 1999). The second and the most important one is cavitation. If a large negative pressure gradient is applied to the liquid, the liquid will break down and cavities (cavitation bubbles) will be created. At high ultrasonic intensities, a small cavity may grow rapidly through inertial effects. So, bubbles grow and collapse violently. The formation and collapse of micro bubbles are responsible for most of the significant chemical effects (Kumar et al., 2010a). Cavitation is considered as a major factor which influences on reaction speed. Cavity collapse increases mass transfer by disrupting the interfacial boundary layers known as the liquid jet effect. The last effect of ultrasound is acoustic streaming mixing. Ultrasound has been used to accelerate the rates of numerous chemical reactions, and the rate enhancements, mediated by cavitations, are believed to be originated from the build-up of high local pressures (up to 1000 atm) and temperatures (up to 5000 K), as well as increased catalytic surface areas and improve mass transfer (Yu et al., 2010). Low frequency ultrasonic irradiation is widely used for biodiesel production in recent years. In transesterification reaction, mixing is important factor for increasing biodiesel yield. Oil and methanol are not miscible completely in biodiesel processing. Ultrasonic mixing is an effective mixing method to achieve a better mixing and enchancing liquid-liquid mass transfer (Ji et al., 2006). Vigorous mixing increases the contact area between oil and alcohol phases with producing smaller droplets than conventional stirring (Mikkola & Salmi, 2001; Stavarache et al., 2006). Cavitation effects increase mass and heat transfer in the medium and hence increase the reaction rate and yields (Adewuyi, 2001). Ultrasonic cavitation also provides the necessary activation energy for initiating transesterification reaction.

Ultrasonic waves are produced with the power converter (transducer) which is piezoelectric material. Sound waves are converted to ultrasonic waves vibrating at high frequency with quartz crystal oscillator. If ultrasound waves are used in chemical reactions and processes it is called as sonochemistry. Industrial sonochemial reactors were designed more than 40 years ago by Sarocco and Arzono (Cintas et al., 2010). They showed that reactor geometry affected enormously the reaction kinetics. Later many rectors have been developed by researchers for different chemical reactions. For conventional biodiesel production, batch and continuous reactors have been developed in industry. Ultrasonic cleaning bath, ultrasonic probe which are usually operated at a fixed frequency are mainly used as ultrasonic apparatus. Frequency is dependent on particular type of transducer which is 20 kHz for probes and 40 kHz for bath. Figure 5 shows schematic diagram of biodiesel production via ultrasound assisted method.

Ultrasonic processing of biodiesel involves the following steps: 1. Mixing vegetable oil is with the alcohol (methanol or ethanol) and catalyst, 2. Heating the mixture, 3. The heated mixture is being sonicated inline, 4. Glycerin separation by using centrifuge. Alternative reactors have also been developed to lower energy consumption. Cintas et al., (2010) designed a flow reactor constituted by three transducers and showed that considerable energy saving could be achieved by large-scale multiple transducer sonochemical reactors operating in a continuous mode.

The factors affecting ultrasound assisted biodiesel production are: — Effect of catalyst type on ultrasound assisted biodiesel production, — Effect of alcohol type on ultrasound assisted biodiesel production, — Effect of ultrasonic power on biodiesel processing, — Frequency effect on ultrasonic assisted biodiesel production.

Effect of catalyst type on ultrasound assisted biodiesel production: In ultrasonic assisted biodiesel studies homogen (alkaline, acid), heterogen and enzyme catalyst were studied with many edible and nonedible oils under ultrasonic irradiation. Transesterification reactions have been studied with KOH catalyst for corn oil (Stavarache et al., 2007a; Lee et al., 2011), grape (Stavarache et al., 2007a), canola (Stavarache et al., 2007a; Thanh et al., 2010a; Lee et al., 2011), palm (Stavarache et al., 2007a), tung (Hanh et al., 2011), beef tallow (Teixeira et al.,2009), coconut (Kumar et al., 2010), soybean (Ji et al., 2006; Mahamuni & Adewuyi, 2009;Thanh et al., 2010a; Lee et al., 2011), triolein (Hanh et al., 2008; Hanh et al., 2009b), fish oil (Armenta et al.,2007),neat vegetable oil (Stavarache et al., 2005), waste cooking oil (Thanh et al., 2010b; Hingu et al.,2010).These studies were presented in Table 6 (one step transesterification), and Table 7 (two-step esterification). Generally KOH was preferred for transesterification reactions instead of NaOH. Soybean (Ji et al., 2006), neat vegetable oil (Stavarache et al., 2005), jatropha curcas L. (Deng et al., 2010) (in the second transesterification step) and triolein (Hanh et al., 2009b) were transesterified with NaOH. KOH and NaOH were used for ultrasound assisted transesterification of neat vegetable oil. They used 0.5%, 1% and 1.5 % alkali catalyst amount, 6:1 molar ratio methanol to oil and room temperature. The researchers reported that there were no great differences in the time to complete conversion between two types of catalyst (Stavarache et al., 2005).. 98% and 96% yields were achieved with 0.5 % NaOH and KOH catalyst, respectively. They also reported that when KOH was used, high yields were obtained even for 1.5% catalyst concentration. Potassium soap is softer, more soluble in water and does not make as much foam as sodium soap. The washing of esters when using potassium hydroxide is easier and the yields of isolated product are higher. In alkali catalyzed ultrasonic transesterification for biodiesel production (Tables 6 and 7), 0.3-1.5 % alkali catalyzed amounts were used. Apart from that, Cintas et al., (2010) developed a new ultrasonic flow reactor to scale up biodiesel from soybean oil in presence of (Na or K methoxide). Na and K methoxide, are alkaline metal alkoxides (as CH3ONa for the methanolysis) are the most active catalysts because of stronger hydroxide group. In their reacton mixture of oil (1.6 L), methanol and sodium methoxide 30% in methanol (wt/wt ratio 80:19.5:0.5, respectively) was fully transesterified at about 45°C in 1 h (21.5 kHz, 600 W, flow rate 55 mL/min).

Heterogen catalysts were tried by researchers in a few studies (Ye et a!., 2007; Salamatinia, 2010; Mootabadi et al., 2010;Kumar et al., 2010b). As it is known, ultrasound increase mixing of oil and alcohol with catalyst phases, as well as increase catalytic surface area. Catalyst can be broken into smaller particles by ultrasonic irradiation to create new sites of the subsequent reaction. Thus, solid catalyst is expected to last longer in the ultrasonic-assisted process (Mootabadi et al., 2010). Single component alkaline earth metal oxides (BaO, SrO, CaO) having lower solubility in alcohol catalyzed palm transesterification processes with methanol (Mootabadi et al., 2010). The catalytic activities of the three catalysts were correlated well with their basic strengths and found as the sequence of CaO < SrO < BaO. BaO catalyst achieved 95.2% of biodiesel yield within 60 min in the ultrasonic-assisted process while SrO catalyst generally demonstrated slightly lower result. CaO showed the lowest yield with 77.3%yield under optimum conditions. Although high activity of BaO as catalyst, this activity dropped severely in the BaO reusability test, especially under ultrasonic condition (compared to mechanical stirring). In another study, aluminum isopropoxide or titanium isopropoxide as heterogeneous transesterification catalysis are employed to produce nanoemulsions with large interfacial area for easy catalyst separation and enhanced reaction rate (Ye et al., 2007). These catalysts are produced by partial polymerization and metal alkoxides are connected by metal-oxygen bonds. Alkoxide parts in the polymer matrix catalyst gives the catalyst amphiphilic properties that help form and stabilize alcohol/ triglycerides nanoemulsion (Ye et al., 2007). The study showed that titanium isopropoxide also showed good catalytic activity and considerable amphiphilic properties in forming nanoemulsions. With aluminum isopropoxide or titanium isopropoxide, transparent alcohol/oil emulsions can be formed in less than four minutes and can significantly enhance the transesterification reaction rate. The micelle size was observed to be as low as 5.1 nm.

High acidity oils (Jatropha curcas L, waste frying oil) can be transesterified by two-step processes. In the first step, free fatty acids are converted to esters by direct esterification with acid catalyst. Eq. 1 shows esterification of fatty acids. In the second step, basic catalyst was used to esterify triglycerides as it was shown in Figure 2.

RCOOH +CH3OH RCOOCH3 + H2O (1)

In production of biodiesel from Jatropha curcas L. oil (non edible oil) Deng et al., (2011) used a two-step process. The first step pretreatment (acid-esterification) of Jatropha oil was performed at 318 K an ultrasonic reactor for 1.5 h in their first study (Deng, et al., 2010). After reaction, the acid value of Jatropha oil was reduced to 0.7 mg KOH/g and 93.3% esterification rate was achieved. The second step, a base-catalyzed transesterification was performed with nano sized Mg/ Al oxides under different conditions. At the optimized condition, (Table 6) 95.2% biodiesel yield was achieved, and the Jatropha oil biodiesel properties were found to be close to those of the German standard. It was reported that the catalyst could be reused for 8 times.

Although it is known that ultrasonic mixing has a significant effect on enzymatic transesterification there are a little study about using of lipases as enzyme catalyst. It has been reported that enzyme activity of Novozym 435 enhanced by ultrasound irradiation (Sinisterra, 1992; Lin & Liu, 1995). Novozym 435 (Candida antarctica lipase B immobilized on polyacrylic resin) was used in biodiesel production from soybean oil and methanol with a low frequency ultrasonic (40 kHz) waves to see enzyme activity and compare their overall effects under two different conditions — ultrasonic irradiation and vibration (Yu et al., 2010). They investigated effects of reaction conditions, such as ultrasonic power, water content, organic solvents, ratio of solvent/oil, and ratio of methanol/oil, enzyme dosage and temperature on the activity of Novozym 435. Novozym 435 activity significantly increased by ultrasonic irradiation compared with vibration and reaction rate was further increased under the condition of ultrasonic irradiation with vibration (UIV). Yu et al (2010) indicated that 96% yield of fatty acid methyl ester (FAME) could be achieved in 4 h under the optimum conditions: 50% of ultrasonic power, 50 rpm vibration, water content of 0.5%, tert — amyl alcohol/oil volume ratio of 1:1, methanol/oil molar ratio of 6:1, 6% Novozym 435 and 40 °C. Since the lipase enzyme is expensive catalyst it is important to reuse the catalyst in biodiesel industrial productions. The researchers also pointed out that Novozym 435 was not deactivated under UIV, only 4 % enzyme activity slightly decreased after five cycles. Effect of alcohol type on ultrasound assisted biodiesel production: Methanol was mostly used in transesterification reaction under ultrasonic irradiation with oils shown in Tables 6 and 7. High conversion and yields were obtained with methanol and ethanol using. Stavarache et al., (2007a) used methanol in transesterification of commercial edible oil, corn, grapeseed, canola and palm oil. Excellent yields (99%) were obtained for all type oils in 20 minutes with 6:1 methanol to oil molar ratio at 36 °C. As it is shown in Figure 6, triglycerides are converted to di and monoglycerides to produce biodiesel to produce biodiesel and glycerin. They also examined the transesterification reaction mechanism under low frequency (40 kHz) ultrasonically driven esterification.

They have reported that the major part of the transesterification took place in the first 3-10 minutes of reaction if not faster and the rate- determining reaction switches from diglyceride (DG) ^ monoglyceride (MG) (classical mechanic agitation) to MG + ROH^Gly + ME (ultrasonically driven transesterification). In another study, the conversion of FAME greater than 99.4 % was achieved after about 15 minutes at 40 °C with ultrasonic agitation for 6:1 methanol: oil molar ratio (Calucci et al., 2005). They have also concluded that hydrolysis rate constants of DG and TG are three to five times higher than those of mechanical agitation. Ji et al., (2006) used ultrasonic transesterification process for soybean oil transesterification with methanol and reported 99% yield at 10 min reaction time with 6:1 methanol to oil molar ratio at 45°C. Oleic acid, triolein, coconut were esterified with ethanol and 90% conversion, about 99% yield and >92% yields were achieved respectively (Hanh et al., 2009a; Hanh et al., 2009b; Kumar et al., 2010a). Table 8 shows the some biodiesel yield and conversion with various monoalcohols and comparing of the alcohols.

Stravarache et al., (2005) studied effects of alcohol type on transesterification of neat vegetable oil under ultrasonic and mechanical stirring. The results of transesterfication with primary, secondary and tertiary alcohols after 60 min of reaction were presented in Table 8.

a Reaction conditions for neat vegetable oil: 0.5% (wt/ wt) NaOH, 6:1 alcohol to oil molar ratio, 40 KHz, b Reaction conditions for triolein: 25 min, 25 °C, 0.1% (wt/ wt) KOH, 6:1 alcohol to triolein molar ratio,

40 KHz,

c Reaction conditions for soybean oil: 2h, 1.5% (wt/ wt) KOH, 6:1 alcohol to oil molar ratio, 40 KHz

Table 8. The influence of alcohol on the ultrasound assisted transesterification of different oils for biodiesel production

N — chain alcohols (methanol, ethanol, n — propanol, and n-butanol) showed the high yields between 88-98% in 10-20 min reaction time. The yields of biodiesel in ultrasound activation were higher than mechanical stirring since ultrasound produce less soap. By using ultrasound the reaction time was found much shorter than mechanical stirring. The secondary alcohols showed some conversion while transesterification reaction took place under stirring. Tertiary-butanol had no conversion with both type of procedure. Hanh et al., (2009b) produced biodiesel with triolein and various alcohols (methanol, ethanol, propanol, butanol, hexanol, octanol and decanol). The productions were performed at molar ratio 6:1 (alcohol: triolein) and 25°C in the presence of base catalysts (NaOH and KOH) under ultrasonic irradiation (40 kHz) and mechanical stirring (1800 rot/min) conditions. The rate

of ester formation depended on alcohol types; as the alcohol carbon number increased, reaction rate decreased. The secondary alcohols such as 2-propanol, 2-butanol, 2-hexanol, and 2-octanol showed 3% conversion, suggesting that the steric hindrance strongly affected the transesterification of triolein. N-propanol showed approximately 93% conversion under ultrasonic irradiation, while 75% conversion was obtained under mechanical stirring. Soybean oil was transesterifed with methanol, ethanol, n-butanol, and iso-propanol over 2 h reaction period with 1.5 % KOH as the catalyst and a 6:1 molar ratio of alcohol/oil at 60°C (Colucci et al., 2005). The similar results obtained with methanol, ethanol and n-butanol compared to other studies.

Effect of ultrasonic power on biodiesel processing: The effect of ultrasonic power on the biodiesel formation has been reported (Mahamuni& Adewuyi, 2009; Hingu et al., 2010; Lee et al.,2011). Biodiesel yield increased with increasing ultrasonic power in all the studies. Nahamuni& Adewuyi (2009) studied this effect for three different frequencies and various powers (181, 90, 181 W at 1300 kHz, 104, 139 ,68 W at 611 kHz, 181, 117, 81, 49 W at 581 kHz). The reactions were carried out for 60-180 minutes. The reaction rate increased with increasing ultrasound power at any given frequency and biodiesel yield was obtained above 90%. At start of the reaction, reaction rate is very low because of low interfacial area available for the reaction. As time increased the reaction rate increased. This increase is due to the amount and size of the emulsion formation varies because of ultrasonic cavitation. Ultrasonic cavitation produces finer and stable emulsion and following this higher mass transfer and hence, higher biodiesel formation. When the ultrasonic power increases acoustic amplitude increases. So, cavitation bubble will collapse each other violently resulting in high velocity and micromixing at the phase boundary between two immiscible phases. Ultrasonication can result in mean droplet sizes much lower than those generated by conventional agitation, and can be a more powerful tool in breaking methanol into small droplets (Wu et al., 2007). The emulsion droplet size of methanol/soybean oil dispersions for ultrasonic and mechanical stirring was investigated and was shown that emulsion droplet size in ultrasonic mixing 2.4 times lower than that of conventional agitation. The mean droplet sizes were 148 and 146 nm with ultrasonic energy at 50 and 70 W, respectively. However, the droplet size was about 340 nm with impeller at 1000 rpm.

Higher power levels usually gives lower conversions because of cushioning effect and hence lower cavitational activity (Ji et al., 2006; Hingu et al., 2010; Lee et al., 2011). Hingu et al. (2010) observed that while the biodiesel conversion was obtained around 66% at 150 W power 89% of conversion was obtained when the power dissipation was increased to 200 W. But further increase in power from 200 W to 250 W resulted in lower FAME conversion. FAME conversion rate also depends on the emulsification degree of reaction system (Ji et al., 2006). These authors also noted that the order of affecting factors on FAME yield was substrate molar ratio > temperature > pulse frequency > ultrasonic power.

Ultrasound pulse (few seconds on followed by second off) effects the biodiesel conversion (Hingu et al., 2010; Ji et al., 2006). Higher conversion can be obtained when higher pulse is applied to system. For example, while biodiesel conversion was obtained for the pulse 2 s ON and 2 s OFF, the conversion were 65.5% for 5 s ON and 1 s OF (Hingu et al., 2010). For a pulse duration as 1 min ON and 5 s OFF, conversion of 89.5% was obtained because of better emulsification of the methanol and oil layers. The effect of horn position on biodiesel production was investigated by same researchers. They kept the reaction parameters constant such as 6:1, methanol to waste cooking oil molar ratio, 1% catalyst concentration, 45°C temperature, 200W power ad 1 min ON and 5 s OFF pulse. Cavitation intensity depends on some parameters physicochemical properties namely viscosity, surface tension and density. Cavitation is generated due to the presence of horn in oil or methanol. According to the horn position various results can be observed. Hingu et al. (2010) applied there different positions: in the oil phase, at the interface and in methanol. While maximum conversion was achieved as 89.5% when the horn was dipped in methanol rich layer, the lowest conversion was obtained as 8.5% when the horn is dipped in the oil phase. 58.5% conversion was observed when the horn is located at the interface of two phases. Maximum ester conversion was obtained since methanol contributed cavitating conditions significantly.

Frequency effect on ultrasonic assisted biodiesel production: The effect of ultrasonic frequency was studied on the yield of transesterification reaction of vegetable oils and shortchain alcohols (Stavarache et al., 2005). NaOH or KOH were used as base catalysts. It was observed that the reaction time gets shorter (the reaction fastens) as the ultrasonicirradiation increases but the yield slightly decreases. At 40 KHz, the reaction time was shorter than 28 KHz, but the yield was obtained higher when studied at 28 kHz.. This is because of the higher formation of soap at 40 KHz and higher quantity of soap makes the purification process harder. The more soap is formed, more esters gets trapped in the soap micelles and the yield of the reaction decreases at 40 KHz as a result.

General comparison of ultrasound irradiation with conventional stirring: Ultrasonic assisted transesterification of oil presents some advantages compared to conventional stirring methods such as; reducing reaction time, increase the chemical reaction speed and decrease molar ratio and methanol, increase yield and conversion. Ultrasound irradiation reduce the reaction time compared to conventional stirring operation (Stavarache et al., 2005; Ji et al., 2006; Hanh, et al., 2008; Mootabadi, et al., 2010; Hingu et al., 2010; Lee et al., 2011). Stavarache et al. (2005) studied transesterification of vegetable oil with short-chain alcohols, in the presence of NaOH, by means of low frequency ultrasound (28 and 40 kHz). By using ultrasounds the reaction time was found much shorter (10-40 min) than for mechanical stirring. The optimal conditions for triolein methanolysis was methanol/ triolein molar ratio of 6/1, KOH concentration of 1 wt% and irradiation time of 30 min. But the optimal conditions for the conventional stirring method were found to be as were methanol/triolein molar ratio of 6/1, KOH concentration of 1 wt% and 4 h (Hanh et al.., 2008). In transesterification of waste cooking oil with methanol 89.5% conversion was obtained in 40 minutes whereas conventional stirring resulted in 57.5% conversion (Hingu et al., 2010). Palm oil was esterified with 95% yield in 60 minutes compared to 2-4 h with conventional magnetic stirring under optimal conditions. Ultrasonic irradiation method enabled to reduce the reaction time by 30 min or more comparing to conventional heating method in production of biodiesel from various vegetable oils. Also this method improved conversion rate (Hanh et al., 2007; Lee et al., 2011). In transesterification reaction, mixing is important factor for increasing biodiesel yield. Ultrasonic effect induces an effective emulsification and mass transfer compared to conventional stirring thus reaction rate increase (Hanh et al., 2009; Hingu et al., 2010). Comparison of yield and conversion of vegetable oilwith various alcohols was presented in Table 8 and also was explained in the effect of alcohol type on ultrasound assisted biodiesel production section.

Ultrasound assisted method has a similar effect as microwave assisted method that both of them reduce the separation time from 5 to 10 hours to less than 60 minutes compared to conventional transesterification method (Kumar et al., 2010). Also, during production of biodiesel via acid or base catalyst, ultrasound irradiation provides a fast and easy route (Yu et al., 2010) and the purity of glycerin increases.

The production of biodiesel from non-edible vegetable oil and waste cooking oil using ultrasonication allows under ambient operating conditions (Kumar et al., 2010a; Hingu et al., 2010). Also, biodiesel production works from vegetable oils given in Table 6 illustrates the applicability of ultrasonic irradiation under atmospheric and ambient conditions. The transesterification reaction with methanol is usually performed at 60°C with classical stirring. Roomtemperature is hardly competitive in terms of energy consumption. Room temperature is hardly competitive in terms of energy consumption. The production of biodiesel with ultrasound is effective and time and energy saving and economically functional method (Ji et al., 2006; Kumar et al., 2010a; Hanh et al., 2011). Power ultrasonic method required approximately a half of the energy that was consumed by the mechanical stirring method (Ji et al., 2006). Special mixing devices can be used to increase mass transfer. It was reported that sonochemical reactors consume only about one third the energy required for a specialty mixer for same conversion (Lifka & Ondruschka, 2004). All these results clearly indicate that ultrasonic method inexpensive, simple and efficient and would be promising to the conventional stirring method.

As seen from the Table 9, the length of the alcohol chain affects the yield of the reaction, as the frequency of the ultrasonic irradiation affects the reaction time. In longer alcohol chains, the yield of the reaction is higher. The longer alcohol chains increases the solubility (miscibility) of alcohol into the oil. 40 kHz of ultrasonic irradiation is preferable if faster reaction is needed but it has to be taken into account that the yield decreases as the reaction fastens because of the higher formation of soap in faster reactions. In conclusion, miscibility of oil and alcohol is better under the control of ultrasonic waves. This effect increases the surface area and higher yields of isolated methyl esters can be achieved. The mass transfer is better so that the soap formation is lower resulting as better and easier isolation of methyl esters. Power of the ultrasonic irradiation makes the reaction faster, as the yield slightly decreases under higher frequencies (40 kHz).

5. Conclusion

Due to the growing energy necessity and environmental problems the studies focused on renewable alternative energy sources. Biodiesel is one of the important renewable energy sources used in many countries in the world as an alternative diesel fuel. Biodiesel is generally produced transesterfication reaction of vegetable and animal oils with catalyst under conventional stirring with batch and continuous processes. Because of the economical causes, choosing efficient transesterification method for biodiesel production has become important in recent years. In this context, the researchers have been investigating different new processes such as supercritical, microwave assisted and ultrasound assisted process to avoid inefficient processes. It is found that these methods have several distinctions compared to conventional methods. Homogenous catalyst (sulfuric acid, sodium hydroxide, potassium hydroxide, sodium and potassium metoxide etc.), heterogeneous catalyst (ZnO, SiO, MgO, BaO, SrO etc.) and enzymatic catalyst (lipase) are also easily being used in microwave and ultrasonic assisted processes. However, supercrital transesterfication reaction of vegetable oils is a noncatalytic reaction and higher yields can be obtained with compared to conventional methods. New methods for biodiesel production offer more advantages but these methods have also some negative effects. For example, energy consumption, excess amount alcohol usage are the disadvantages of supercritical process. Microwave synthesis is still in lab-scale synthesis and it is not viable in large scale for industrial production due to penetration depth of microwave radiation into the absorbing materials. The safety aspect is another drawback of microwave reactors for industry. Ultrasonic biodiesel production could be advantageous for small producers, but in large scale processing maybe challenging because of necessity of many ultrasound probes. Although there are some disadvantages of novel methods in biodiesel production, these methods give several important advantages for the transesterification of oils such as: reducing reaction time and reaction temperature, unwanted by-products; and increasing ester yields, conversion easier compared to conventional method. In conclusion, these methods with their important advantages can be more preferred than conventional method anymore.

1.2 Compression test

A compression apparatus having a close fit plunger die assembly can be used to make a single compact in one stroke of the plunger from ground straw samples (Adapa et al., 2006 and 2010a; Mani et al., 2004). The compression test should be performed to study the effect of independent variables such as biomass, treatment, grind size, and moisture content on pellet density and durability. In order to simulate frictional heating during commercial pelleting operation, the compression die should be maintained at pre-heat temperatures of 75 to 100oC (Adapa et al., 2006; Kaliyan and Morey, 2009; Mani et al., 2006). Different levels of pre-set compressive forces can be applied using the Instron testing machine. Typical pre­set loads in the range of 31.0 to 150.0 MPa are applied to make pellets. Figure 1 represents the photographs of pellets made from barley, canola, oat and wheat straw grinds from hammer mill screen sizes of 3.2, 1.6 and 0.8 mm (Adapa et al., 2010a).

Plant cell walls are highly complex networks made of carbohydrates, lignins and some proteins. Apart from the complexity of individual plant cell walls, the walls can be very diverse in composition and organization, between different groups of plants, between different species and even within same plant, organ, tissue and cell type. Cell walls are also dynamic in nature and their ultrastructure alters with growth and differentiation (Carpita and Gibeaut, 1993; Niklas, 2004; Popper, 2008; Sarkar et al., 2009). Molecular resolution imaging of plant cell walls is needed to obtain a detailed structural knowledge of cell wall organization, which in turn is needed for rational engineering of cell walls for improving biofuel production from biomass. Electron microscopy allows ultrastructural analysis at molecular resolution, whereas optical microscopy techniques are typically limited by the diffraction limit of the optical microscope and the signal-to-noise ratios encountered in autofluorescent specimens.

Transmission electron microscopy (TEM) allows an in-depth analysis of cellular ultrastructure and has been used to study plant cell walls since 1940s yielding the first high — resolution ultrastructural insights (Preston et al., 1948). For TEM analysis samples need to be thin for the electron beam to penetrate (ideally ~ 100-300 nm) and must be examined a vacuum, resulting in the necessity for resin embedding, followed by ultrathin sectioning. The necessary chemical sample preparation steps employed in a typical conventional protocol results in limited preservation: chemical fixation, heavy metal postfixation and staining, as well as organic solvent dehydration, can lead to fixation staining and dehydration artifacts, such as the denaturation, aggregation and extraction of biological material as well as uneven or preferential staining. Moreover, even modern sample preparation protocols including specific staining techniques are predominantly optimized for cell membrane lipids, nucleic acids, and proteins, but not for carbohydrates and lignin. Some histochemical staining methods are used to stain cell wall components such as, (1) negative staining with uranyl acetate for cellulose; (2) PATCO (Periodic acid — Thiocarbohydrazide — Silver Proteinate) method for hemicelluloses; (3) ruthenium red for pectins, (4) potassium permanganate for lignins (Krishnamurthy, 1999). However, these stains are often not highly specific and stain multiple cell wall components to various degrees. Distinguishing between cell wall components accurately at high resolutions is difficult by sole differential staining.

Most of the early high-resolution TEM imaging of plant cell walls was done on samples prepared by metal shadowing and surface replication after freeze-fracturing or freeze­etching (Preston et al., 1948, McCann et al., 1990). While in principle no chemical fixatives, no dehydrating agents and no stains are used in this method, thus potentially retaining the samples closer to their native state, the images although potentially of high resolution are restricted to topological structural information in two-dimensions. Moreover, no chemical information can be obtained from this method as the imaging is done on the metal replica and not the biological sample. In recent years, sophisticated cryo-methods have been developed to minimize or completely overcome the limitations of conventional TEM sample preparation methods. Sophisticated cryo-methods such as high pressure freezing, followed by freeze-substitution and resin embedding typically display a superior quality of sample preservation in a much closer to native state (McDonald, 1999; McDonald & Muller — Reichert, 2002, McDonald & Auer 2006). High-pressure freezing followed by vitreous sectioning and cryo-TEM imaging offers preservation of biological samples closest to their native state (Al-Amoudi et al., 2004a, 2004b). These samples not only provide high- resolution structural information, but can also provide chemical information by specific staining or by immunolabeling with target-specific gold-conjugated antibodies. Several monoclonal antibodies and carbohydrate-binding modules (CBM) are being developed against different cell wall components in many laboratories around the world (Knox, 2008; Pattathil et al., 2010), which can be used with TEM analysis to localize the various chemical components of plant cell walls with high precision. All TEM sample preparation methods mentioned above are usually time-consuming and labor intensive, although automatic microwave tissue processors are now commercially available for rapid chemical fixation, dehydration, resin embedding and polymerization.

Apart from sample preparation issues, conventional TEM imaging runs into a few other problems. Due to a small field of view in a TEM, only small representative areas of any sample can be imaged at a time. The sections used in TEM are also very thin (under a micron), which makes imaging larger cellular structures in their entirety an almost impossible task. Use of high-resolution wide-field imaging (montaging) and imaging serial sections can help in covering relatively larger sample areas. Aligning serial sections is specimen-dependant due to the characteristics of each section. Individual sections in a series may have differences in scaling and/or may have non-linear deformation because of sectioning, folding, drying, specimen tilt, and optical distortions of the microscope (Stevens and Trogadis, 1984). A more complicated limitation of conventional TEM is that the images obtained are 2D projections of a 3D volume, which means multiple molecular layers of the sample contribute to the same layer of the image. Such images can be difficult to interpret if the structures of interest are only a few nanometers in dimension and are very closely packed. Plant cell walls are a good example of this imaging problem. They are made up of a tightly packed network of cellulose microfibrils, each microfibril being ~3 nm in diameter (Ohad & Danon, 1964; Frey-Wyssling, 1968; Heyn, 1969; Somerville et al., 2004; Ding and Himmel, 2006). The cellulose microfibrils are tightly surrounded by hemicelluloses such as xyloglucans or arabinoxylans that form hydrogen bonds with the cellulose microfibrils and form cross-links between two neighboring cellulose microfibrils. The matrix space in between cellulose and hemicelluloses is crowded with complex nano-scale molecules of pectins and/or lignins (McCann et. al., 1990; Carpita and Gibeaut, 1993; Somerville et al., 2004). It is extremely challenging to resolve the ultrastructure of plant cell walls in situ by most imaging techniques available currently, including conventional TEM. Atomic force microscopy (AFM) has been successfully used to image plant cell wall ultrastructure at high resolutions (Ding and Himmel, 2006) but the information available is two-dimensional and only topological.

Electron tomography overcomes some of the limitations faced by conventional TEM and AFM. In this method, several hundred two-dimensional TEM images of the same sample are collected by rotation of the sample along the central axis in small increments, which leads to a 3D volume of data, which can be visualized, segmented out to develop realistic models and quantitatively analyzed in 3D. Individual layers within the 3D volume can be separately visualized and analyzed in different planes using sophisticated image analysis software. Different types of algorithms for automated segmentation are being developed, though the currently available algorithms are only reliable for relatively simple image sets. Since individual components of the plant cell walls are typically spaced close to the resolution limit of the data set, manual segmentation is still the most reliable method for analyzing plant cell wall tomograms. EM tomography data of plant cell walls can be used to measure dimensions, orientations and spacing of the different cell wall components. This method paired with biochemical analysis methods like Raman imaging and immunolabeling has the potential to develop precise, comprehensive 3D ultrastructural cell wall model(s) at molecular resolution.

EM tomography has been applied to study the 3D organization of the cellulose microfibrils in the S2 layer of the secondary cell wall in Pinus wood tissue (Xu et al., 2007, 2011), however the samples used in this study had been harshly chemically treated. Cryo electron tomography of vitrified sections of plant tissue can provide preservation closest to their native state. The processing of tomographic datasets (reconstruction, filtering and segmentation) of plant cell wall cryo-sections and faithful model development however, is highly challenging, as the images obtained by cryo-tomography are extremely low in contrast. Furthermore, since plant cell walls contain densely packed nano-scale components, cryo-tomography is not suitable for high-throughput imaging of plant cell walls. Instead high pressure freezing, followed by freeze-substitution and resin embedding is a way to go and will be the key to obtain high-throughput realistic cell wall models. EM tomography of high-pressure frozen, freeze-substituted, resin embedded samples has been used to study at unprecedented resolution the dramatic structural changes during cytokinesis and the assembly process of cell plates during the final stage of cell division, (Segui-Simarro et al., 2008).

Based on the basic experiment on the pyrolysis and the reforming, we have developed the simulator by which the gaseous yields and/or the energy efficiency through BT process can be estimated. Here, we compared the practice data through the demo-plant with the result of the simulator.

In general, there would be somewhat deviation between the practice data and the estimated one due to the simulator. That is, it would be extremely significant to identify the deviation from the viewpoint of the reliable plant operation.

The calculation logic of the simulator which we developed is as follows (Dowaki et al. 2007):

1. The reaction temperature in each furnace (pyrolyzer and reformer) and the steam feeding rate are fixed.

2. Based on the gaseous yields in the pyrolysis and/or the reforming reactions, which were analysed by gas chromatograph (GC-8A, Shimadzu), the gaseous components in

each furnace were estimated due to the following two equilibrium reactions. In our experiments, Shincarbon-ST was used as a measure column, and the six kinds of gases which can be measured include H2, CO, CO2 CH4, C2H4 and C2H6, respectively.

Note that we considered the approach temperature difference between the theoretical one and the reaction one on these equilibrium reactions.

Here, in the gasification process after the pyrolysis reaction, it is usually thought that the following two reactions take place.

CH4 + H2O о CO + 3H2 (1)

CO + H2O о CO2 + H2 (2)

Using our experimental apparatus, the approach temperature differences between theoretical temperature and the actual temperature was measured. This temperature difference is known as the approach temperature.

In Eqs. (1) and (2), the equilibrium constant of shift reaction and that of methanation reaction are represented as follows:

Where, AT, R and p are the approach temperature [K], gas constant [J/molK] and the partial pressure of i-component [Pa], respectively.

Next, we compared the measurement data in Izumo plant (1t/d) with the estimated results using the simulator (see Table 1). Here, the tasks of Run 11 and Run 11.2 in which the decomposed reactions are assumed to be completed in the pyrolyzer are described. Both tasks were executed on March, 2009 (Kameyama et al. 2010).

In this verification, we focused on the molar fractions of H2, CO, CH4 and CO2, and found the average reaction temperature so that the total deviation on molar fraction between the measurement data and the estimated one due to Eqs. (3) and (4) is a minimum. That is, we investigated if the gaseous components based on the temperature which was measured in the plant corresponded to the estimated ones due to the simulator. The reason why we verified the gaseous components using the temperature as a variable is as follows; in the demo-plant, we did not know the temperature profile on the vertical and/or horizontal directions precisely since the sampling point of the temperature in reformer is one position. Thus, assumed that the estimated temperature based on the measured gaseous components would represent the average one of reformer, we made sure that the process simulator would be more suitable.

Next, based on the process simulator which has a precision to some extent, we describe the example due to the biomass feedstock of waste Japanese cedar. With regard to the gasification performance, since gaseous yields and concentrations are dependent upon the kind of materials, the operating temperature, and the inner pressure, they were examined using the gasifier apparatus which has a reformer and a pyrolyzer.

Here, Table 2 shows the ultimate analysis of the waste Japanese cedar.

^ Dry-Base

Through the tests, the syngas components and the equilibrium constants were obtained. For instance, Fig.3 illustrates the gaseous yields on the pyrolysis at 550 °C with variation of S/C =0.14 to 0.98, and the reforming reaction at S/C=1.0 with variation of 800 to 950 °C, respectively. Here, a steam carbon ratio is defined as the following equation.

Note that the gaseous components are modified at 20% moisture content. Also, the approach temperature for each reaction is shown as Table 3.

Fig. 3. Gaseous yields of pyrolysis (a) and reforming reaction (b).

Based on the above experimental results, we estimated the following material balance: C31.078H43.495 O23.677 N0.069 + 31.078#2O ^

24.052H2 + 11.682CO + 2.435CH4 + 9.442CO2 + 23.228H 2O (6)

+2.92 X 10 N2 + 4.40 X 10 NH3 + C2.686H0.482O0.343N0.004

C2 686H0482O0343N0004 is the chemical component of char, and its heating value was 32.0 MJ/kg. In our simulator, the energy performance would be solved so that the input and the output on heat and materials would be balanced.

Next, using ф=9.5mm ball, we measured the temperature profiles at the surface of ball and the center of it. In the phase of absorption of heat, the ball was kept at each designed temperature between 200 and 950 °С. At the time, there was difference between the surface temperature and the center one, and the temperature differences were measured. Inversely, in the phase of heat radiation, the ball was heated up to 1,000 °С in the furnace, and it was put in a room temperature. Simultaneously, the temperature differences were measured. Note that these temperature profiles are time series data.

As a result, the thermal conductivities can be obtained. Also, since the thermal circulation time has to be the same as the reacting time on a pyrolysis and a steam reforming reaction, the optimal size of the ball is decided. Thus, the adequate auxiliary power for the circulation of HC would be obtained. Due to this result, we can estimate the suitable residence time in each reactor for the temperature profile which would be led by the simulator. Based on the above concept, we could estimate the syngas through BT process (Dowaki et al., 2008a, Dowaki, 2011a).