The solid state ethanol fermentation by F. velutipes was performed for ammonia treated rice straw. Solid state cultivation has a large merit to decrease the ethanol production cost. But it has demerit on the saccharification of biomass. As shown in Fig. 7A, saccharification of biomass at high concentration is quite difficult. Significant amount of cellulase is necessary to obtain enough level of saccharification, and saccharification yield do not increase in proportion to the amount of cellulase if increased the amount of cellulase. Furthermore, saccharification yield will be significantly decreased under the high substrate condition. The hydrolysis rate of 30% w/v biomass was very low (less than 10%). In contrast, ethanol yield was equivalent to 80-90% of hydrolysis rate so that the merit of our process using F. velutipes was proven (Fig. 7B). In the case that enzymes were not added, ethanol production by F. velutipes was only 0.026 l/kg of dry biomass, equivalent to a theoretical ethanol recovery rate of 5.9% from total hexose. In contrast to no enzymes addition, in the case that 1 and 5 mg/ g product of enzymes were added to the fermentation, ethanol production after 15 d by F. velutipes was 0.26 and 0.34 l/kg of dry biomass, respectively. The ethanol conversion rates of 1 and 5 mg/g product enzymes addition were 61.6% and 77.8% for total hexose, respectively. The maximum weight loss was approximately 70% in the case that no enzymes were added to the fermentation, while the maximum weight loss for enzyme addition of 1 and 5 mg/ g product were approximately 90% and 96% respectively (data not shown).

These results suggest F. velutipes has favourable properties for CBP. It could be expected that development of novel bioethanol production process by using F. velutipes.

A В

Fig. 7. Saccharification and ethanol production at high biomass concentration

Hua Song

RTI International USA

1. Introduction

Along with the maturity of the production technology (i. e., fermentation) for a long history, bioethanol has become one of the most significant chemicals and energy carriers in large quantity derived from biomass. Although ethanol production from non-food resources remains challengeable for scientists, how to utilize ethanol in an efficient and economical way opens more space for all researchers both from industry and academia to play with.

Hydrogen is likely to play an important role in the energy portfolio of the future due to its high gravimetric energy density. Especially when it is used in fuel cells, it is an ideal energy carrier that can offer clean and efficient power generation. In the United States, ~95 % of hydrogen is produced using a steam reforming process [1]. Over 50% of world’s hydrogen production relies on natural gas as the feedstock [2]. As the concern for a sustainable energy strategy grows, replacing natural gas and other fossil fuels with renewable sources is gaining new urgency. In this context, producing hydrogen from bio-derived liquids such as bio-ethanol has emerged as a promising technology due to the low toxicity, ease of handling and the availability from many different renewable sources (e. g., sugar cane, switchgrass, algae) that ethanol has to offer. An added advantage of producing hydrogen from bio­derived liquids is that it is quite suitable for a distributed production strategy.

This chapter is aimed to provide a big overview of the current technologies for catalytic hydrogen production from bioethanol while focusing the discussion on the hydrogen production through steam reforming of bioethanol over non-precious metal based catalysts, more specifically, cobalt-based catalysts. By combing the work performed at the author’ laboratories, this chapter will also provide the professional insights on the future development direction of such technologies. Through the estimated economic analysis of this process simulated at industrial scale, the ways of final commercialization of the developed catalyst system specially tailored for central and distributed hydrogen production from steam reforming of bioethanol will be suggested.

Sorghum grain is a naked caryopsis composed of three major anatomical parts: pericarp, germ, and endosperm. The pericarp is composed of epicarp, mesorcarp and endocarp (cross and tube cells). Among cereals, sorghum is the only one that can contain significant amounts of starch granules in the mesocarp cells. The starch-devoid germ is rich in fat, soluble sugars and proteins (albumins and globulins) whereas the endosperm is divided into the single layered aleurone and the starchy endosperm cells positioned in the corneous and floury or chalky regions of the endosperm. The endosperm constitutes the largest fraction of the kernel and where almost all the starch is contained. Similar to maize, sorghum contains 60 to 70% of starch. The endosperm texture and hardness are highly related to the performance of the grain during several stages of ethanol production. In general terms, composition of sorghum is similar to maize with a few small but significant differences mainly in protein and fat concentrations. Sorghum for instance, has an average 1% less fat and 1.5 to 2.0% more crude protein compared to maize. Both sorghum and maize have more than 50% of this protein as prolamins named kafirins and zeins, respectively. In sorghum, approximately half of the prolamin fraction is bound. In contrast, approximately 70% of the maize prolamins are free or alcohol-soluble. There are some sorghum varieties that contain significant amounts of condensed tannins in the testa. These sorghums are classed as type III and have a lower nutritional value compared to other sorghums and maize. This is due to the presence of tannins that bind proteins and inactivate enzymes. As a result, high tannin sorghums may have reduced ethanol yields (Serna-Saldivar, 2010).

One of the most noteworthy differences between sorghum and maize is its starch granule — protein matrix interaction that negatively affects the susceptibility of both proteins and starch to enzyme hydrolyses. These structural differences affect protein digestibility and the speed of dextrins and glucose production during liquefaction and saccharification and thereafter the efficiency of yeast fermentation. Kafirins, despite the high sequence homology with zeins, tend to be less digestible especially after wet-cooking indicating the change in conformational structure attributed to formation of disulphide bonds. This is due to its high hydrophobicity which also makes possible the formation of additional protein aggregates that enhance the formation of more covalent bonds compared to zeins (Wong et al., 2009). Prolamins in the kernel are concentrated in protein bodies arranged among starch granules. The protein body composition in maize and sorghum is also similar, with alpha kafirin in the inner core surrounded by beta and gamma kafirins. The difference with maize is that during wet thermal processes the external part of protein body seems to form a net that makes difficult to access the alpha portion that is in turn more digestible than the beta and gamma counterparts. This phenomenon affects starch digestibility because in sorghum is 15 to 25% less digestible compared to maize. Taylor & Belton (2002) indicate that in sorghum, a complex rather than a simple obstruction mechanism between kafirins and starch is more likely to occur. This is the main reason why sorghum has lower susceptibility to hydrolysis and fermentation and yields less fuel ethanol compared to maize. Besides the starch-protein relationship, some other factors such as mash viscosity, amount of phenolic compounds, ratio of amylose to amylopectin and formation of amylose-lipid complex in the mash, limit the rate of enzymatic hydrolysis and fermentation efficiency during bioethanol production. For instance, starch in amylose-lipid complex cannot be converted into fermentable sugars, reducing conversion rate and final ethanol yield (Wang et al., 2008).

Based on the economic analysis, the profits of the three different modes in 5,000 tons sugarcane pressed plants are showed in table 4. There are high costs of fermentation and distillation for sugarcane directly for ethanol fermentation due to low concentration of sugarcane juice, and about 15 tons waste water need treatment. It is also uneconomic to produce fuel ethanol using concentrated juice because of high energy costs. Therefore, that sugarcane is used directly for fuel ethanol production does not reflect its best economic benefits and flexible market response capacity.

In traditional opinion, people prefer to produce sugar as possible as they can rather than use more molasses to produce ethanol. They think that it is uneconomic to produce 1 ton ethanol with nearly 2 tons sugar consumption. In fact, we can achieve the maximized economic benefits applying "the simultaneous production of sugar and ethanol " mode, in which we boil the A-syrup that have the good characteristic of low energy consumption, to produce the top-grade white sugar production. B-green syrup and second pressed juice are mixed to produce the fuel ethanol. Costs of the ethanol production can be greatly reduced.

According to the calculations, it will bring more economic benefits while employ "the simultaneous production of sugar and ethanol" mode.

6.
Conclusions

Various technologies have been identified for immediate increases in the efficiency and sustainability of current and future sugarcane ethanol. In conclusion, recycle utilization design are seems to be suitable for sugarcane bioethanol development, for example, recycling of byproducts of sugarcane in the fields reduces chemical fertilizers application rates, reducing water consumption with closure of water-processing circuits and the use of bagasse to generate electricity or to manufacture bagasse polymer composites (Xu et al., 2010), improving the energy balance of ethanol production; as well as in production and

harvesting processes. At present, we think bagasse is not preferable for directly bioethanol production due to their high bioconversion costs. Adequate developed technology is available to achieve sustainable sugarcane production and bioethanol. However, the adoption of new technologies requires a favorable economic and political environment that facilitates investments in clean technologies. Pollution problems require strict enforcement of legislation and inspection of agricultural and industrial activities.

Developing the sugarcane ethanol provides a novel option for utilization of the sugar industry, and it will be also helpful to the fuel ethanol development in China.

7. Acknowledgements

This research work was supported by Ministry of Science and Technology of China, (NCSTE-2006-JKZX-023), Guangdong Science and Technology Department (2010A010500005) and the Natural Science Foundation of Guangdong Province (10451031601006220). We specially thank Guangdong Key Laboratory of Sugarcane Improvement and Biorefinery, Guangzhou Sugarcane Industry Research Institute for supporting this research.

The glucose concentration obtained after hydrolysis from wheat straw pretreated with different levels/degrees of severity (conditions ranging from 160°C, 10 minutes to 200°C, 20 minutes) is demonstrated in Fig. 7. The pretreatment at 200°C over 20 minutes (severity factor 18000; log(R0)=4.26) achieved the highest sugar concentration, converting about 100% cellulose during the hydrolysis. Recirculation strategies with wheat straw were developed, where the sugar solution of a first hydrolysis reaction was recycled twice to fresh straw and the subsequent hydrolysis reaction. The glucose concentration was further increased by a recirculation process to fresh washed solids and subsequent hydrolysis from 30 g/l to 143 g/l

(20% solids, third hydrolysis). After fermentation with Saccharomyces cerevisiae, an ethanol concentration of 7.5%(vol) was obtained. In Fig. 8, the final glucose concentrations after recirculation processes with inhibitor-controlled wheat straw as well as bioethanol yields after fermentation are shown.

Corn stover was pretreated at 190°C for 10 minutes. Initially, 10% of the dry substance corn stover was hydrolyzed and fermented. Here, the sugar concentration was 32 g/l glucose and 10 g/l xylose yielding 1.9% bioethanol (Table 3). The dry substance was increased to 15% and 20%, yielding considerably higher sugar and bioethanol concentrations (Table 3).

Wheat straw was moistened with water before steam explosion pretreatment. Ethanol was added during pretreatment (10 minutes at 200°C) to test for a possible effect on the hydrolysis and fermentation step. The wet straw was hydrolyzed with the enzymes and fermented with yeast. Additional ethanol during the pretreatment process did not influence the sugar and bioethanol content (Table 4).

Alternatively, pellets from mixed straw were used to increase the dry substance already during the pretreatment step. It was possible to increase the glucose concentration from wet straw pellets to 60 g/l resulting in 2.5%(vol) bioethanol (from 10 % dry substance).

The obtained concentration of ethanol versus the concentration of furfuryl alcohol is shown in Fig. 6. The amount of produced ethanol increased from 10 g/L with pure glucose to 24 g/ L at the dose of 1.5 mL/L furfuryl alcohol. At higher concentrations a decrease in ethanol is observed with almost no ethanol being produced at the dose of 15 mL/L. This indicates that with the presence of small doses of furfuryl alcohol, ethanol production can increase.

To provide information about the sharp increase between measurement one and two, another experiment was conducted with the same method as the previous, except the dose of furfuryl alcohol were (0.0, 0.5, 1.0, 1.5, 2.0 and 2.5) mL/L. Samples of this experiment were analyzed by HPLC and the results are shown in table 3.

As shown in table 3, the fermentation will peak with a dose of 1.5 mL/L and at levels above that the amount of ethanol produced will decrease versus the concentration of furfuryl alcohol. It was further shown that there was plenty of leftover glucose for the yeast to ferment. Therefore in none of the cases the loss in fermentation rate was due to lack of glucose.

As shown in Table 3 there is a strong correspondence between the amount of ethanol produced and the dose of furfuryl alcohol. Furfuryl alcohol is a microbial growth inhibitor, but when added in small concentrations it will force the yeast cells to increase their metabolism to survive. This effect should be present for the norm of microbial inhibitors. Despite of the fact that furfuryl alcohol is toxic to the yeast, the stress that it causes can lead

to approximately 80% increase in ethanol production given that sufficient amounts of glucose are available. For SSF the amount of glucose needed for this effect to be visible is only present in the start of the SSF which is also where the positive feedback effect of the yeasts fermentation rate is mainly present.

To describe the effect of lacking inhibitory stress on the yeast the maximum fermentation rate with 2 g/L yeast of both 80 g/L DM and 170 g/L DM in the SSF experiments with rape straw are compared and shown in Fig. 7.

The rate of fermentation for the furfuryl alcohol experiment is calculated for the period of 5 to 19 hours, which is estimated to be the highest rate of fermentation for the experiment. As shown in Fig. 7, it seems that even though microbial inhibitors are generally restricting the fermentation process, a medium completely without inhibitors (such as the control from Fig. 8) will have a decreased fermentation rate compared to medium with a small dose of microbial inhibitors.

A series of catalyst optimization efforts have been carried out in the past several years aiming to enhance the catalytic performance during BESR. Studies on cobalt-based catalysts supported on y-A12O3, TiO2, ZrO2 supports have indicated that ethanol conversion correlates closely with metal dispersion and hence, the metallic Co sites. Among the supports studied, zirconia is shown to provide the highest metal dispersion and the highest H2 yield. H2 yields as high as 92% (5.5 mol of H2 per mole of ethanol fed) are achieved over a 10% Co/ZrO2 catalyst at 550 oC [69].

Investigation of the evolution of the Co-ZrO2 catalysts through different stages of the synthesis process showed that catalyst precursors start out with Co existing primarily in a nitrate phase and transforming into a Co3O4 phase in the fully calcined state. The reduction proceeds in two distinct steps as in Co3O4 ^ CoO and CoO ^ Co. There is an optimum in each of the synthesis parameters, which gives the highest metallic Co surface area. The maximum in metallic Co area is often determined by a series of competing processes, such as transformation from a nitrate to an oxide phase and onset of crystallinity versus reaction with the support at higher calcination temperatures, reduction to metallic state versus sintering at higher reduction temperatures. The maximum in metallic Co area was seen to coincide with the maxima in both ethanol adsorption capacity and H2 yield in the BESR reaction, suggesting a strong correlation between metallic Co sites and BESR activity [99].

Although promising activity toward hydrogen production is observed over Co/ZrO2, steady-state reaction experiments coupled with post-reaction characterization experiments showed significant deactivation of Co/ZrO2 catalysts through deposition of carbon on the surface, mostly in the form of carbon fibers, the growth of which is catalyzed by the Co particles. The addition of ceria appears to improve the catalyst stability due to its high OSC and high oxygen mobility, allowing gasification/oxidation of deposited carbon as soon as it forms. Although Co sintering is also observed, especially over the ZrO2-supported catalysts, it does not appear to be the main mode of deactivation. The high oxygen mobility of the catalyst not only suppresses carbon deposition and helps maintain the active surface area, but it also allows delivery of oxygen to close proximity of ethoxy species, promoting complete oxidation of carbon to CO2, resulting in higher hydrogen yields. Overall, oxygen accessibility of the catalyst plays a significant role on catalytic performance during BESR [100].

the effect of impregnation medium on the activity of Co/CeO2 catalysts was also systematically investigated under the environment of BESR. The significant catalytic performance improvement has been observed over ethanol impregnated Co-CeO2 catalyst, especially at lower temperature (300-400 oC), compared with its counterpart with aqueous impregnation. This promotion effect is considered to be closely related to the cobalt dispersion amelioration through cobalt particle segregation under the facilitation of surface carbon oxygenated species derived from ethanol impregnation. Moreover, even better catalytic performance is achieved using ethylene glycol as impregnation medium in our recent study, which might be closely related with the achievement of even smaller cobalt particle size due to its superior ability in preventing cobalt agglomeration probably originating from the presence of organic surface species [101].

In order to further improve the oxygen mobility within the catalyst, the effect of Ca doping on CeO2 support has been intensively studied. According to the observations obtained from the various characterization techniques employed, the introduction of calcium into the CeO2 lattice structure leads to the unit cell expansion and creation of oxygen vacancies due to lower oxidation state of Ca (2+) compared to Ce (4+), which facilitates the improvement of oxygen mobility. As a result, the catalytic performance has been significantly enhanced when Ca is present, leading to larger amount of final product formations (H2 and CO2) from BESR reaction [102].

The influence of cobalt precursor on catalytic performance was also systematically investigated. Multiple cobalt precursors including inorganic salts and organometallic compounds were used to prepare Co/ CeO2 catalysts. The steady-state reaction experiments show much higher H2 yields and fewer side products over the catalysts prepared using organometallic precursors. Among these, the catalyst prepared using cobalt acetyl acetonate has the highest H2 yield, most favorable product distribution, and best stability. The superior performance is verified by the transient data. Characterization results point to an improved dispersion on the surface. It is possible that the organic ligands surrounding Co ions provide a spatial barrier effect, keeping the particles segregated and leading to better dispersion [103].

In the interest of figuring out the impact of catalyst preparation method on its performance during BESR, in addition to conventional Incipient Wetness Impregnation (IWI) method, solvothermal, hydrothermal, colloidal crystal templating, and reverse microemulsion methods have also been employed to prepare CeO2 support and CeO2 supported Co catalysts with various morphologies. All of the novel preparation techniques led to superior behavior in ethanol steam reforming reaction compared to IWI method. Among the catalysts studied, the one prepared with the reverse microemulsion technique showed the best performance, giving higher H2 yields at much higher space velocities. The catalyst also showed good stability, with no sign of deactivation when it was kept on-line at 400 °C for 120 h. The superior performance is likely to be related to the improved cobalt dispersion, enhanced metal-support interaction and increased metal-support interphase facilitated by the reverse microemulsion technique. In addition, the hydrothermal method has also been employed to prepare the Co/CeO2 catalyst. The CeO2 particles with various shapes and size distribution have been successfully achieved in our laboratories by controlling the parameters during preparation process. The morphological effect on the catalytic performance will be evaluated in the future [104].

During cassava harvest season, fresh roots are plenty available and the price is low. Therefore, it is common to use them to make slurry by grinding and then mix with cassava chip. Alternatively, cassava roots are used as a main raw material and then cassava chips are used to adjust the solid concentration. Similar to dried chips, there are two processes for preparing cassava fresh roots for bioethanol production, namely "With fiber" and "De­fiber" process.

In "With Fiber" process (Figure 4), the roots are transferred to the root hopper, in which soil and sand are effectively removed by root peelers. The roots are then washed and subjected to the chopper and rasper. The puree of milled roots is then slurried without fiber removal and used for liquefaction. This process requires less equipment and investment cost and is recommended for batch-type fermentation (Sriroth & Piyachomkwan, 2010b). However, with the presence of cell wall materials, ground fresh roots has developed semi-solid like characteristic and should be slurried with water to reduce viscous behavior. This causes dilution of solid loading in a fermentor, yielding a low ethanol concentration in final beer. A pretreatment of ground fresh roots with appropriate cell wall degrading enzymes has been introduced to handle that inferior flowability (Martinez-Gutierrez et al., 2006; Piyachomkwan et al., 2008), allowing potential use of fresh roots with Very High Gravity (VHG), i. e. high solid loading (> 30%) process and resulting in a higher ethanol concentration (upto 14.6% w/w or 18%v/v) in beer (Thomas et al., 1996). By almost doubling the ethanol concentration in the final beer, the VHG process can not only minimize the energy consumed during the downstream distillation process, but also improve the plant capacity. This concept can be applied to improve fermentation of other feedstock as well.

Similar to wet milling process of cassava chips, in "De-fiber" process (Figure 5), the starch slurry is prepared from fresh roots by modifying a typical cassava starch production

process. After desanding and washing, roots are subjected to the chopper and rasper. The pulp is removed and starch is extracted by a series of extractors. After depulping, the starch slurry is then concentrated by a separator and subjected to a jet cooker for liquefaction. This process requires a higher investment cost and also generates high starch losses in the pulp. However, defiber process is more controllable and can be readily applied to current well — established technology of ethanol production from other materials. It is also practical for

applying in high solid loading and continuous fermentation process (Sriroth & Piyachomkwan, 2010b).

Extrusion has been widely used for the processing of cereal grains because this thermoplastic technology is continuous and saves unit operations and energy. In extrusion, the materials are subjected to heating, mixing, and shearing, resulting in physical and chemical changes during its passage through the extruder. The major advantages of extrusion include: improvement of starch digestibility and reduction of its molecular weight, production of free sugars and dextrins, changes in the native structure of both starch granules and proteins and reduced viscosity of fermentation broths. Therefore, extrusion could be an effective process to improve the bioconversion rate of sorghum starch (Zhan et al., 2006).

An innovative processing technology patented by researchers of Cornell University combines extrusion process and supercritical-fluid technology. The main difference between supercritical-fluid (SCFX) and conventional extrusion is the injection of supercritical carbon dioxide, which replaces water as blowing agent for expansion. The injection of supercritical- fluid carbon dioxide breaks the intimate bonds between starch granules and protein matrix and results in the improvement of starch availability (Zhan et al., 2006). These researchers suggested that SCFX produces molecular degradation of starch during extrusion of sorghum. This process also increased about 8% the protein digestibility, the measurable starch content, the free sugar concentration and gelatinized starch and other parameters that increased ethanol yield (+5%) and boosted fermentation efficiency compared to the non­extruded counterpart. The SCFX cooking also affected the crude fiber, chemical fraction that after microscope examination showed disruption and fissures. These authors describe the sorghum extrudates with "porous structure". Thus, this thermoplastic procedure was indeed effective as pretreatment to improve bioconversion of sorghum into ethanol.

In literature, only few papers are available on the cellulases immobilization. This is due to the fact that cellulose is not soluble and some immobilization techniques, such as enzymes entrapment, impede the interaction enzyme-substrate. Immobilization of cellulases via covalent bonds appears to be the most suitable technique. Besides the enzyme stabilization, the covalent-immobilization allows the use of supported enzymes for several cycles of reactions (Brady & Joordan, 2009; Li et al., 2007; Mateo et al., 2007; Dourado et al., 2002; Yuan et al., 1999).

In 1999, Yuan and coworkers, immobilized cellulases onto acrylamide grafted acrylonitrile copolymer membranes (PAN) by means of glutaraldehyde. They showed that the enzyme stability was increased after the immobilization process. Also, the activity of the immobilized cellulases was higher than the free cellulases at pH 3 — 5 and at temperatures above 45 °C (Yuan et al., 1999).

In 2002, cellulases from T. reesei were immobilized on Eudragit L-100 by researchers of the University of Minho (Portugal). They used the commercial mixture Celluclast® 1.5L supplied by Novozymes (Denmark). This method allowed to improve the stability of the enzymes without significant loss of its specific activity. The adsorption of cellulases on Eudragit lowered the enthalpy of denaturation, but affected only slightly the denaturation temperature (Dourado et al., 2002).

In 2006, Li and coworkers, immobilized cellulase enzymes by means of liposomes. These are phospholipid vesicle, ranging in size from 25 nm to 1pm. In this method, glutaraldehyde — activated liposome bound to the enzyme thus forming the liposome-cellulase complex. Following this step, the complex was immobilized on chitosan-gel. The immobilized enzyme by the liposome molecules showed efficiency higher by 10% compared to the enzyme immobilized in chitosan-gel without liposome. The immobilized cellulase-liposome complex showed a loss of activity of 20% with respect to the original value after six cycles of reaction. Therefore, liposome-binding cellulase appeared to prevent or limit the enzyme deactivation (Li et al., 2007).

In recent investigations, two commercial cellulase enzymes (Celluclast 1.5 and Novozym 188) were immobilized on epoxy Sepabeads® support (Resindion s. rl.). The preliminary data showed that 60% of loaded Celluclast proteins were adsorbed by the support and that more than 90% of these proteins remained stably linked even after repeated washings (Verardi et al., 2011).