Petra Patakova, Daniel Maxa, Mojmir Rychtera, Michaela Linhova, Petr Fribert, Zlata Muzikova, Jakub Lipovsky, Leona Paulova, Milan Pospisil, Gustav Sebor and Karel Melzoch

Institute of Chemical Technology Prague Czech Republic

1. Introduction

Nowadays, with increasing hunger for liquid fuels usable in transportation, alternatives to crude oil derived fuels are being searched very intensively. In addition to bioethanol and ethyl or methyl esters of rapeseed oil that are currently used as bio-components of transportation fuels in Europe, other options are investigated and one of them is biobutanol, which can be, if produced from waste biomass or non-food agricultural products, classified as the biofuel of the second generation. Although its biotechnological production is far more complicated than bioethanol production, its advantages over bioethanol from fuel preparation point of view i. e. higher energy content, lower miscibility with water, lower vapour pressure and lower corrosivity together with an ability of the producer, Clostridium bacteria, to ferment almost all available substrates might outweigh the balance in its favour. The main intention of this chapter is to summarize briefly industrial biobutanol production history, to introduce the problematic of butanol formation by clostridia including short description of various options of fermentation arrangement and most of all to provide with complex fermentation data using little known butanol producers Clostridium pasteurianum NRRL B-592 and Clostridium beijerinckii CCM 6182. A short overview follows concerning the use of biobutanol as a fuel for internal combustion engines with regard to properties of biobutanol and its mixtures with petroleum derived fuels as well as their emission characteristics, which are illustrated based on emission measurement results obtained for three types of passenger cars.

In order to test the efficacy of the antibiotics bleocin™ (EMD Biosciences), paromomycin (MP Biomedicals), and the PPT-containing (200 g L-1) commercial herbicide Basta® (Bayer CropSciences) on D. salina CCAP 19/18, algal cells were grown in the presence of various concentrations of each selective agent (Table 3) using 100-ml stirrer flasks under the same conditions mentioned previously, with the exception of aeration. Each culture was inoculated with 10 ml of exponentially growing cells (1 x 105 cells ml-1) and the cell density was measured with a Zeiss Axiovert 100 inverted light microscope using a hemocytometer over a one week period. Additionally, cells were plated on agar to analyze viability on solid medium. Measurements were taken in duplicate and experiments were repeated two times.

Esters (i. e. methyl acetate) are ubiquitous in nature and have vast industrial and commercial applications based on different substituent groups. An important class of industrial catalytic process for esters reduction is metal-catalyzed hydrogenolysis for the production of alcohols (e. i. ethanol) (Adkins and Folkers, 1931, Adkins et al., 1933, Turek et al., 1994a, Xu and Xu, 2010) New applications for ester hydrogenolysis are being found in catalytic upgrading and conversion of renewable resources such as lignocellulosic biomass into fuels or fine chemicals (Corma et al., 2007, Huber et al., 2006). Copper-based catalysts are widely used for ester hydrogenolysis (Adkins and Folkers, 1931, Cybulski et al., 2001, Thomas et al., 1992,

Turek et al., 1994b, Turek et al., 1994a). They show very good activity and selectivity for alcohols under high temperature and hydrogen pressure (500-700K, 200-300 bar). Precious metal-based alloys (Rh-Sn for example) show promise of lessening the severity of the reaction conditions (lowering hydrogen pressure to below 100 bar), but precious metal are costly and tend to favor hydrocarbon production.

In our approach, the recovered methyl acetate is maintained in liquid phase at 20°C. It is pumped against pressure ranging from 10 to 50 atm, through a heat exchanger that vaporizes it completely at temperature ranging 150 to 425°C. Hydrogen, preheated at the same temperature range is mixed with the methyl acetate vapor at the exit of the heat exchanger. The molar ratio hydrogen to methyl acetate is from 4 to 11. The hot mixture is flown through a catalytic bed where the catalyst CuO/Cr2O3 or CuO/ZnO/Al2O3 catalyst is placed. The CuO is reduced with H2/N2 mixtures prior to adding any methyl acetate (Claus et al., 1991). Methyl acetate is converted to ethanol and methanol according to the equation:

CH3COOCH3 + 2H2 = CH3OH + CH3CH2OH -27 KJ/mol (20)

The GHSV (based on H2) of the reaction is comprised between 1,000 and 2,000 h-1 and the methyl acetate conversion reached 95%, with a slightly higher selectivity towards ethanol.

4. Conclusion

Biomass is a renewable energy source whose conversion to biofuels is an option to reduce oil dependency and reduce the carbon dioxide footprint characteristic of fossil fuels.

This chapter has shown the chemical steps needed to convert non-homogeneous biomass into bioethanol via gasification. The syngas produced is catalytically turned into biofuels (methanol and ethanol). Such approach is practices by Enerkem’s which uses non­homogeneous residual biomass including urban biomass as feedstock for the gasification. The syngas produced is cleaned prior used for catalytic synthesis of methanol. The reactor used for the methanol synthesis is a three phase reactor based on the Liquid-Phase methanol process. The yield of methanol is about 1 kgMeOH/kgcat/h and the selectivity of the reaction into methanol is about 99%. The refined methanol produced is carbonylated to produce methyl acetate. The synthesis of methyl acetate is carried in a fixed bed reactor and uses a halide component as co-catalyst. The methyl acetate is hydrogenolized to form stoichiometrically one mole of ethanol and one mole of methanol. The methanol produced at the end of the process is recycled into the carbonylation unit.

5. Acknowledgement

The authors would like to acknowledge financial support of the sponsors of the Industrial Chair in Cellulosic Ethanol: the Quebec’s government (MRNF) and its industrial partners: Greenfield Ethanol, CRB innovations and Enerkem. Additional support from NSERC’s Synergy program is also acknowledged.

The quality of fuel pellet is usually assessed based on its density and durability. High density of pellet represents higher energy per unit volume of material, while durability is the resistance of pellets to withstand various shear and impact forces applied during handling and transportation. High bulk density increases storage and transport capacity of pellets. Since feeding of boilers and gasifiers generally is volume-dependent, variations in bulk density should be avoided (Larsson et al., 2008). A bulk density of 650 kg/ m3 is stated as design value for wood pellet producers (Obernberger and Thek, 2004). Low durability of pellets results in problems like disturbance within pellet feeding systems, dust emissions, and an increased risk of fire and explosions during pellet handling and storage (Temmerman et al., 2006). Other quality factors of biomass for thermo-chemical conversion include (FAO, 2011; Rajvanshi, 1986):

• Energy content: The choice of a biomass for energy conversion will in part be decided by its heating value. The method of measurement of the biomass energy content will influence the estimate of efficiency of a given gasifier. The only realistic way of presenting fuel heating values for gasification purposes is to give lower heating values (excluding the heat of condensation of the water produced) on an ash inclusive basis and with specific reference to the actual moisture content of the fuel.

• Moisture content: High moisture contents reduce the thermal efficiency since heat is used to drive off the water and consequently this energy is not available for the reduction reactions and for converting thermal energy into chemical bound energy in the gas. Therefore, high moisture contents result in low gas heating values during thermo­chemical processes.

• Volatile matter: The amount of volatiles in the feedstock determines the necessity of special measures (either in design of the gasifier or in the layout of the gas cleanup train) in order to remove tars from the product gas in engine applications.

• Ash content and slagging characteristics: The mineral content in the biomass that remains in oxidation form after complete combustion is usually called ash. The ash content of a fuel and the ash composition have a major impact on trouble free operation of a gasifier or a burner. Slagging or clinker formation in the reactor, caused by melting and agglomeration of ashes, at the best will greatly add to the amount of labour required to operate the gasifier. If no special measures are taken, slagging can lead to excessive tar formation and/or complete blocking of the reactor.

• Reactivity: The reactivity is an important factor determining the rate of reduction of carbon dioxide to carbon monoxide in a gasifier. Reactivity depends in the first instance on the type of fuel. For example, it has been observed that fuels such as wood, charcoal and peat are far more reactive than coal.

• Size and size distribution: Low bulk density feedstock may cause flow problems in the gasifier or burner as well as an inadmissible pressure drop over the reduction zone and a high proportion of dust in the gas. Large pressure drops will lead to reduction of the gas load, resulting in low temperatures and tar production. Excessively large sizes of particles or pieces give rise to reduction in reactivity of the fuel, resulting in start-up problems and poor gas quality, and to transport problems through the equipment. A large range in size distribution of the feedstock will generally aggravate the above phenomena. Too large particle sizes can cause gas channelling problems. Fluidized bed gasifiers are normally able to handle fuels with particle diameters varying between 0.1 and 20 mm (FAO, 2007).

• Bulk density: Fuels with high bulk density are advantageous because they represent a high energy-for-volume value. Consequently, these fuels need less bunker space for a given refuelling time. Low bulk density fuels sometimes give rise to insufficient flow under gravity, resulting in low gas heating values and ultimately in burning of the char in the reduction zone. Inadequate bulk densities can be improved by briquetting or pelletizing. All of the abovementioned biomass properties could be altered by subjecting raw biomass to various processing methods and forming composites. Before choosing a gasifier, it is important to ensure that the individual biomass meets the requirements of the gasifier or that it can be treated to meet these requirements.

Measuring the percent crystallinity of the biomass samples is particularly important as it affects rate and yield of enzymatic saccharification (Hall et al. 2010). It is challenging to measure the percent crystallinity of the biomass using DSC, as cellulose decomposes even before it undergoes melting (Paul et al. 2010; Kaloustian et al. 2003; Soares et al. 1995). As enthalpy of crystallization and enthalpy of melting are required to find the percent crystallinity of the sample, it is not possible to measure percent crystallinity of the biomass based on this technique. However, the enthalpy of dehydration of cellulose and biomass samples has been correlated to percent cellulose crystallinity of the samples based on the hydrogen bonds formed by the crystalline and amorphous cellulose. A detailed method for measuring cellulose crystallinity (CC) by DSC has been reported by Bertran and Dale (1986). They have reported that DSC provides a better way of measuring CC than other traditional approaches like XRD, especially for substance with very low crystallinity index. The amount of moisture absorbed by cellulose containing substances is dependent on the amount of crystalline and amorphous cellulose present in the substance. Amorphous cellulose absorbs a high amount of moisture while pure crystalline cellulose has no absorption capacity. Hence the endothermic dehydration peak appearing in DSC thermograms can be used to estimate the percent crystallinity of the cellulose present in the substances. Using completely amorphous cellulose measured under the same experimental conditions, the percent CC of the sample can be estimated from the following equation where AH0 is the heat of dehydration for a completely amorphous cellulose sample and AHs is the heat required to dehydrate the sample.

In spite of the many advantages of using TGA and DSC analysis they cannot be used to find bond specific information about the substrate. Knowledge of the chemical composition of the substrate is required ahead of time. Analysis through this technique always requires one to correlate the original substrate to the pure components of the substrate. The changes to DP of a polymer can only be visualized but cannot be quantitatively correlated to changes in the molecular weight of the polymer. Also if the substrates decompose very close to each other, we cannot differentiate between the increase of one of the substrate or decrease in DP of the polymer degrading at a higher temperature. As these techniques affect the samples physically and chemically the sample used in this technique cannot be recovered or reused.

A few works to date present enzyme-based microfluidic BFCs with immobilized enzymes. Enzymes in solution are only stable for a few days, it is why immobilization of active enzymes is of interest in order to improve lifetime (Bullen et al., 2006) and to develop integrated microfluidic BFC designs. Besides, full selectivity of both enzymatic half-cells allows microfluidic BFC operation in a single, combined fuel and oxidant channel with mixed reactants at constant concentrations favourable for stability studies. Additionally, the close proximity of the enzymes with the electrodes reduces ohmic losses because electrons are harvest from reaction sites with lower electrical resistance.

In microfluidic BFCs described in literature, enzymes with their respective redox mediators are immobilized on electrodes surface by encapsulation in polymer film. Currently, Nafion® is commonly used as it possesses surfactant properties interesting to immobilize enzymes in micellar structures when treated by quaternary ammonium salt (Moore et al., 2004). Such matrix provides an optimal enzyme environment where the enzyme retains its activity for greater than 90 days. Based on this strategy, a microchip-based bioanode with alcohol dehydrogenase enzymes immobilized in treated Nafion® associated with an external platinum cathode delivered a power density of 5 gW cm-2 (Moore et al., 2005). The low value was attributed to the thick coating of polymer, casting by hydrodynamic flow and thus difficult to control in the microchannels.

Another polymer to entrap enzymes is poly-L-lysine. This polymer mixed with an enzyme solution can stand on electrode surface after drying in air. According to this technique, a microfluidic glucose/O2 BFC was developed (Togo et al., 2007; Togo et al., 2008). The originality of this study concerned the location of the electrodes in a single flow channel. This device has been developed to generate electric power from glucose oxidation with an anode coated by immobilized glucose deshydrogenase and a bilirubin oxidase-adsorbed O2 cathode (Togo et al., 2008). The device was featured with a specific electrode-arrangement within a microchannel to prevent dissolved O2 to react at the anode as an interfering substance. A large biocathode (10 times the anode size) was placed strategically upstream of a bioanode to pre-electrolyse O2 to protect the anode vicinity from interfering oxygen. The maximum cell current was increased by 10% with this cell configuration at pH 7. The experimental results showed the influence of the channel height that should be in the same order of the depletion layer thickness for optimal operating of the device. However, in such design, the composition of fuel and oxidant could not be adjusted independently for optimum enzymatic activity and stability.

In order to choose independently the composition of the two streams, the configuration based on Y-microfluidic single channel is required. This approach has been developed for a microfluidic glucose biofuel cell working from GOx and laccase immobilised on gold electrodes in a poly-L-lysine matrix. The immobilization process was realized by mixing the enzymes and their respective redox mediators in poly-L-lysine solution. After drying, the device was tested with a phosphate buffer pH 7 solution with 50 mM glucose for the anolyte, and with a citrate buffer pH 3 solution saturated with oxygen for the catholyte. Different redox mediators were tested for efficient electron transfer at the anode. Among the mediators hexacyanoferrate (Fe(CN)63-), ferrocene and 8-hydroxyquinoline-5-sulfonic acid hydrate (HQS), the higher power density (60 pW cm-2) was obtained at a cell voltage 0.25 V with Fe(CN)63- (Fig. 8).

Hydrolysis is governed by the law:

(C6HwOs )n + nH2O ^ nC 6H nO6 (3)

and can be mainly of two types: acid (using diluted or concentrated acids) or enzymatic. A lignocellulose biomass is more complicated to hydrolyze than pure cellulose because it contains components that are not glucose-based, such as hemicellulose and lignin.

A lignocellulose biomass undergoing acid hydrolysis mainly produces xylose, while the lignin and cellulose fractions remain unchanged. This is because xylan is more susceptible to hydrolysis in moderately acid conditions because of its amorphous structure, while cellulose demands more severe conditions because of its crystalline nature.

If hydrolysis is implemented using 1% diluted sulfuric acid, the hemicellulose is depolymerized at a lower temperature than the cellulose. This process is usually conducted in two consecutive stages.

One of the most important characteristics of this type of hydrolysis is the rate of the reactions involved, which facilitate the continuity of the process. To speed up the diffusion of the acid, the raw material is mechanically reduced to pieces a few millimeters in size. Hydrolysis with concentrated acids (10-30%), on the other hand, rapidly and completely converts cellulose into glucose and hemicellulose into xylose, with some degree of degradation. The acids most often used are sulfuric and hydrochloric acid, and hydrogen fluoride.

This type of acid hydrolysis has the great advantage of recovering the sugars very efficiently (approximately 90% of hemicellulose and cellulose are depolymerized into monomeric sugars). From an economic standpoint, this process enables a reduction in production costs by comparison with the diluted acid solution, especially if the acids are retrieved and reconcentrated. The acids and sugars in solution are separated by ion exchange so the acid is reconcentrated by passing it through a series of multiple-effect evaporators. The remaining solid fractions, which are rich in lignin, are collected and can be made into pellets for use as fuel.

So, in short, we can divide concentrated acid hydrolysis into two stages: in the first stage, the concentrated acid (70%) destroys the crystalline structure of the cellulose, breaking up the hydrogen links between the cellulose chains; in the second stage, hydrolysis induces a hydrolytic reaction in the single isolated cellulose chains.

The enzymatic hydrolysis of natural lignocellulose materials is a very slow process, because it is hindered by several structural parameters of the substrate, such as its of cellulose and hemicellulose content, and the surface area and crystallinity of the cellulose. Pretreatments are consequently needed to make the biomass more susceptible to attack by hydrolysis. For the same reason, a cocktail of enzymes has to be used that is capable of breaking the links in the polymeric chains. This cocktail is usually a mixture of various hydrolytic enzymes, including cellulase, xylanase, hemicellulase and mannoxidase. Enzymatic cellulose degradation is a complex process because it takes place in limit conditions between the solid and liquid phases, where the enzymes are the mobile components. Generally speaking, degradation is characterized by a rapid initial phase followed by a slower second phase that can continue until all the substrate has been used up. The reason for this behavior is usually assumed to be because the accessible fraction of cellulose is quick to hydrolyze, followed by the slow activation of the absorbed enzyme molecules.

Chopping up the biomass increases the surface area accessible to the enzymes and reduces the polymerization and crystallinity of the cellulose, thus enabling a smaller quantity of enzymes to be used and the production costs to be contained.

Both bacteria and fungi can produce the cellulase for the hydrolysis of lignocellulose materials. The bacteria may be aerobic or anaerobic, mesophylic or thermophylic. The bacteria most often used are Clostridium, Cellulomonas, Bacillus, Thermomonospora, Ruminococcus, Bacteriodes, Erwinia, Acetovibrio, Microbispora and Streptomyces. The enzymes are usually classified according to their reaction site, so they may be intracellular (or cell-associated) or extracellular. The main function of extracellular enzymes is to convert the substrate into an external medium by taking effect on the cell mass constituents. Conversely, intracellular enzymes need the substrate to spread through the cellular mass before it can be converted.

The most widely accepted mechanism for the enzymatic hydrolysis of cellulose involves the synergic action of the enzymes endoglucanase (or endo-1,4-P-glucanase, EG), exoglucanase (or cellobiohydrolase, CBH), and P-glucosidase. Both EG and CBH are extracellular enzymes, while P-glucosidase is intracellular. EG randomly disrupts the cellulose chains, consequently inducing their strong degradation. It takes effect by hydrolyzing the P-1,4- glucoside bonds, creating new ends in the chains. Exoglucanase breaks up the ends of the chains, thus enabling the release of soluble cellobiose or glucose. BGL hydrolyses the cellobiose into glucose, thus eliminating the inhibitory cellobiose; then BGL completes the process by catalyzing the hydrolysis of cellobiose into glucose. Most cellulase and hemicellulase producers are microorganisms such as the filamentous fungi, e. g. Trichoderma sp., which can be used in their natural form or genetically modified (Trichoderma viride, Trichoderma reesei, Trichoderma longibrachiatum). CBH I and CBH II are the main enzymes of Trichoderma reesei, while EG I and EG II are the dominant endoglucanases.

Enzymatic activity is influenced by various parameters, such as temperature (a 20-30°C increase in temperature leads to a 3- to 5-fold increment in the end products). The crucial issue of temperature lies in the risk of an unwanted denaturation when the temperature is too high (Balat et al., 2008). Enzymatic hydrolysis, with or without the addition of catalysts, has generally proved capable of a high yield of both glucose (>90%) and xylose (>80%).

The first technological challenge restraining the commercialisation of the cellulosic biomass industry is the fractionation of the lignocellulosic biomass to isolate the cellulose macromolecule. Similar processes have been applied by the pulp and paper industry for decades but there is a new objective now, the complete utilisation of the carbon-based structures of the biomass as well as a reduction of the water consumption, hence, the concept of biorefinery. Although the traditional pulping processes are being remodelled to fit with the biorefinery approach, other processes are also investigated among which are the organosolv process and steam treatments. There is a significant variety of different steam treatments which all rely on the same concept: biomass is first saturated with a solvent (usually water) with or without the utilisation of a catalyst (acid or basic depending on the targeted macromolecule). The mixture is then "cooked" by addtion of steam in a pressure — resistant vessel for a certain period after which a valve is open, and the vapor phase exits the vessel through a nozzle entraining the solids. The exiting vapor reaches very high velocities as a function of the geometry of the nozzle thus reaching a sonic velocity. A "explosion" takes place while induced by the sonic field. The water saturating the biomass in its pores rapidly expands to vapour causing cell changes which vary from simple fibrilar disaggregation to fragmentation. During cooking, water, at high pressure and temperature, has a high dissociation constant leading to the occurence of a larger quantity of hydronium ions directly formed in the saturated pores of the biomass. Hemicelluloses, which are highly ramified and relatively easy to hydrolyse (in comparison to cellulose), will be affected by the increasing concentration of ions in the solution. The reaction of water and biomass is of course a major concern when considering steam treatments and it was found that in the absence of a catalyst, the two most important factors that were related to fractionation of the biomass and the hydrolysis of hemicelluloses were temperature and cooking period. The relationship between both parameters has been related to a mathematical equation called the "severity factor". This equation, reported first by Overend and Chornet (1987) has been from this point forward a significant contribution for the homogeneization of the steam processes.

So = log Ro (2)

Where So is the severity fractor, T is the temperature (expressed in degree celcius) and the overall equation relates on the integral of the temperature curve between the start and the end of the cooking period, including the preliminary heating leading to operating temperature. Severity factors were also related to the relative hydrolysis of the hemicelluloses macromolecule and (Overend and Chornet., 1987) showed that at a severity factor of 4, hemicelluloses were completely hydrolysed and there was starting to be an impact on the cellulosic fiber. This concept, can serve as a guide for other substrates although it was shown to vary from one feedstock to another, mostly because of the varying nature and amounts of hemicelluloses found in the biomass (Lavoie et al., 2010a, 2010b, 2010c). Impact of the calculated severity factor has also been reported for other feedstocks as

residua! cotton and recycled paper (Shen et al., 2008), aspen wood (Li et al., 2005), douglas fir (Wu et al., 1999), from rice husk and straws (Gerardi et al., 1999), from yellow poplar, from peanut hulls and from sugar cane (Glasser et al., 1998). In most of the cases reported previously, the ideal severity factor for the isolation of cellulose and hydrolysis of hemicelluloses was found to be between a severity factor of 3 and 4. A non-catalytic steam process of biomass usually leads to a brown lignocellulosic fibre as depicted in Figure 2 below:

Although hydrolysis of hemicelluloses can be performed only using the natural dissociative potential of water, many researches have investigated the effects of including a catalyst on the overall outcome of the process. Both acid and basic catalyst has been considered and each will have the tendency to target one type of macromolecule more than the other. Whilst acids will have a more pronounced effect on cellulose, bases will have a more significant effect on lignin. Among the acids that were used for catalysis of steam explosion reaction, sulphuric acid is one of the most common but also one of the less expensive at an industrial level. Utilisation of the latter has been reported repetitively in literature (Lawford et al., 2003; Emmel et al., 2003; Ballesteros et al., 2001). Directly comparable to sulphuric acid, sulphur dioxide was also widely used as a catalyst for steam explosion. The latter will interact with biomass and react with water to produce in turn sulphuric acid. The main difference might be that utilisation of SO2 would allow a more homogeneous distribution of the acid catalyst in the biomass since the diffusion of the gas should be higher than the sulphuric acid molecule. Such a treatment has been effectively applied on lodgepole pine (Ewanick et al., 2007), poplar (Lu et al., 2009), aspen (De Bari et al., 2007) and eucalyptus (Ramos et al., 1999). The acid catalyst will have a direct effect on the hydrolytic potential of the mixture increasing the natural hydrolytic potential of water considerabily. As for the previously mentioned severity factor, researchers have tried to translate this phenomenon into an equation. Abatzoglou et al. (1992) were able to introduce the concentration of acid into the calculation of the severity factor as depicted below:

Where X and Xref is the acid loading (g of acid/g of dry biomass) and reference (acid loading, g of acid/g of dry biomass) respectively, A is a parameter expressing the acid catalyst role in conversion of the system, ш’ is parameter expressing the temperature role in conversion of the catalysed reaction system and tR is the reaction time. A couple of years later, Montane and co-workers (Montane et al., 1998) developed a new version of the equation which included slight modifications over the equation proposed by Abatzoglou et al. The equation is depicted below: (4)

In this equation the шо parameter express the energetic of the process respect to a reference reaction temperature, T and t remains the temperature and time whilst у defines the shape of the distribution of activation energies. The research also showed that it was possible, for a specific species, to estimate the whole conversion of the process using a single equation:

(1 — f) = exp ^-h06*1010 exp (^—733) C0674 0— J (5)

Where f is the conversion parameter and C is the catalyst concentration. Equation 5 has been developed by Montane et al. using birch as substrate for the steam explosion process. Utilisation of an acid catalyst, for similar temperatures and times, should allow a more complete hydrolysis of the hemicelluloses but should also attack the cellulose molecule which is overall sensitive to the occurence of protons. It has been mentioned and it is still widely studied that the interactions between the hydronium and the cellose macromolecule may lead to a more efficient hydrolysis to glucose when used as a pretreatment for an enzymatic treatment (Dererie et al., 2011; Khunrong et al., 2011; Zhang et al., 2009). In most of the previously mentioned situations, utilisation of the catalyst leaded to increased value for conversion following the enzymatic hydrolysis, although in some specific cases, even if the conversion to glucose was increased, the fermentation was strongly inhibited by the production of furfural-derived compounds. Dehydration of xylose to furfural is depicted in Figure 3.

Dehydration of carbohydrates is strongly induced by acid catalysts at temperature higher than 150 oC with a classical inorganic catalyst although lower operating conditions were reported for the utilisation of ionic liquids (Tao et al., 2011). Five-carbon carbohydrates will dehydrate to furfural whilst dehydration of C6 sugars will lead to 5-hydroxymethylfurfural (5-HMF) (Zhang et al., 2010). The latter will usually be less concentrated in a steam explosion process since it will require an isomerisation of the aldohexose sugars to a ketohexose form. Furthermore, under acid catalyst, 5-HMF has been reported to undego spontaneous hydrolysis to levulinic acid and formic acid which are both fermentation inhibitors. The minimal concentration at which furfural starts to inhibit fermentation has been reported to be at 2-3 g/L (Palmqvist et al., 1999) whilst as for 5-HMF, it has been reported that the concentration that causes 50% inhibition of fermentation was of 8 g/L. Base-catalysed steam explosion may also be a potential pathway since the occurence of hydroxide ions, as in the case of kraft pulping, would lead to the hydrolysis of the hemicelluloses as well as the lignin whilst allowing the isolation of cellulose. Utilisation of NaOH as a catalyst for steam explosion has been reported in literature (Zhuang et al., 1997;

Li et al., 2005), although less frequently in comparison to the acid-catalysed reaction. Another process called ammonium fiber explosion is also slightly comparable to a base — catalysed steam explosion since it will allow defibration of the feedstock in a first time, then the interaction of ammonia with water can be directly related to an hydroxide ion catalyst although part of the hydrolysis process could be related to the ammonia itself although it is highly soluble in water and it interacts in an classical acido-basic reaction to produce ammonium hydroxide. This concept was efficiently tested on rice straws (Vlasenko et al., 1997) and the process itself has been patented by Dale et al. The Feedstock Impregnation Rapid and Sequential Steam Treatment (FIRSST)

Since a non-catalytic and an acid steam treatment allowed targeting the carbohydrate-based macromolecules from the biomass and the based-catalysed reaction allowed partial depolymerisation and solubilisation of lignin, our group has developed the two step FIRSST (Feedstock Impregnation Rapid and Sequential Steam Treatment) process. The biomass is first reduced in size to a range 3 to 6 cm long. It then follows the process flow diagram depicted below (Figure 4).

Biomass is first extracted with water, solvent and/or a mixture of both to extract the secondary metabolites. Two reasons justify the preliminary extraction, first some of the compounds could have a bioactive potential thus leading to applications in cosmetics and pharmaceuticals. Secondly, the extractives could act as inhibitors for fermentation and depending what is the targeted application for the broth obtained after the first steamexplosion, it might be beneficial to remove such compounds. After extraction, biomass is rinced with a minimal amount of water to remove traces of the residual solvent or to ensure maximal removal of extracts. Typically, at the bench scale level, a 5/1 massic ratio of water/biomass is used at this point. Biomass is then impregnated with water to ensure

maxima! penetration of the aqueous medium in the biomass’ pores. Impregnation could be performed with or without pressure, either positive or negative. Saturation with water is one of the key elements for performing an efficient steam process and has to be monitored carefully. Impregnation could be performed by letting the biomass soak in water for a time period (typically up to 24h) allowing the water molecules to fill the small pores via capillarity. Whilst both positive and negative pressure might be used, utilisation of a positive pressure to ensure water penetration is by far the most efficiently scalable approach. After impregnation, excess water has to be removed, a pressure of 100 psi is sufficient both for the pressurized impregnation process as well as the following excess water removal. Once excess water is removed, the biomass is transferred in the FIRSST reactor where it is cooked for 2-4 minutes whilst monitoring the severity factor of the whole process. In a two step FIRSST process, one must ensure that the severity factor of the first process is not excessively high or the following delignification process, although efficient, will lead to excessive conversion and a lignin-carbohydrates broth after the second process. Once the first FIRSST process is completed, the biomass is once more impregnated but this time with an aqueous diluted NaOH solution (typically 1-10%). Impregnation as well as removal of excessive solution was performed at 100 psi and room temperature. The residual lignocellulosic matrix is then cooked at temperature comparable to the first process although typically in a 10 oC inferior temperature range. Once the second cooking period is completed, biomass is rinced with a 10/1 ratio water/fibre to ensure removal of the remaining sodium ions. Such process has been tested on different feedstock including energy crops (Lavoie et al., 2010a), residual forest biomass (Lavoie et al., 2010b) and different agricultural residues (Lavoie et al., 2011). When using based-catalysed steam process, the catalyst itself, as in the case of acid hydrolysis, becomes an important factor of the reaction. Increasing the concentration of the basic catalyst showed to have a direct impact on lignin removal and using the same conditions (time and temperature), it was shown that an increasing alkali concentration in the mixture allowed the production of a whiter fibre. The texture of the fibres are strongly affected by the severity of the steam processes, example of the different textures of fibres produced according to the two-step FIRSST process used on triticale straws are depicted below (Figure 5).

At this point, the two-step FIRSST process has allowed the isolation, in high yields, of all the fractions of lignocellulosic biomass whilst producing pulp with good mechanical properties. Example of mechanical properties obtained from FIRSST pulps are depicted in the Table 4 for Salix vimimalis (Lavoie et al., 2010a), for a mixture of softwood (Lavoie et al., 2010b) and for Cannabis sativa (Lavoie et al. unpublished results). Opportunity to produce quality pulp is yet another advantage of this technique which would, from the same feedstock, allow the production of many derived products including ethanol and cellulose.

For the first step of the steam explosion and as mentionned earlier, no catalyst is used since the hemicelluloses and/or protein found in the lignocellulosic matrix were shown to be directly affected by water at the operating conditions. The first step of the FIRSST process is usually performed between temperatures of 180-230 oC depending on the nature of the biomass used as a feedstock. As an example, residual forest biomass was shown to require more severe conditions in comparison to residual agricultural biomass as triticale or hemp. Cooking period is usually ranging from 2-4 minutes, but it most of the cases investigated by our team, a 2 minute cooking period was shown sufficient. The uncatalyzed steam explosion process can be related to the severity factor that is calculated from the cooking temperature and time, needless to underline the fact that similar severity could be obtained by increasing the cooking period whilst decreasing the temperature. The 2 minutes cooking time usually
excludes the heating period where biomass is heated to the operating conditions, nevertheless, this heating period is taken into consideration when calculating the severity factor. The heating period for the FIRSST process varies from 10-30 seconds and the temperature of biomass is monitored with thermocouples strategically located in the steam explosion reactor and recorded on an acquisition system allowing control and downstream calculations with regards to the conditions of operation.

One of the most important variables affecting the yield of ester is the molar ratio of alcohol to triglyceride. The stoichiometric ratio for transesterification requires three moles of alcohol and one mole of glyceride to yield three moles of fatty acid ester and one mole of glycerol. The molar ratio is associated with the type of catalyst used. For example, a reaction conduced with an acid catalyzed needed a 30:1 ratio of BuOH to soybean oil, while a alkali — catalyzed reaction required only a 6:1 ratio to achieve the same ester yield for a given reaction time (Freedman et al., 1986). Higher molar ratios result in greater ester conversion in a shorter time. During the ethanolysis of peanut oil with a molar ratio alcohol:oil of 6:1 the amount of glycerin liberated was more than did a 3:1 molar ratio (Feuge and Grose, 1949). In this point is important to consider the type of alcohol that is been used. This is because during ethanolysis, as this alcohol has chemical affinity for both glycerine and ester, the higher the molar ratio is more difficult to separate the both phases.

Gas stripping by nitrogen as a method potentially enabling both butanol preconcentration before final distillation and a way how to mitigate butanol toxicity during fermentation was studied separately from fermentation and stripping coefficient p, defined by equation (5) was chosen as main criterion for stripping efficiency:

P= (-1/PL). (dPL/dt) (5)

where PL is butanol concentration in a liquid phase.

Gas stripping of solvents from fermentation media is however only the first step towards isolation/concentration of products (ABE), further steps consist in product change of state from the gas into liquid phases. There are several ways how to carry out this change of state but there are scarcely discussed. Two of them i. e. application of low temperature (- 4 °C in condenser) and adsorption on charcoal followed by desorption by steam were tested. If low temperature was used for butanol conversion from the gas to liquid, average achieved preconcentration lay in the interval from 7 to 9. However, when the method of freezing was used then only 60% of solvents were captured in one gas cycle (probably due to insufficiency of freezing unit capacity) while at charcoal adsorption, 90% of solvents was captured. This affected stripping efficiency which was lowering gradually at freezing. On the contrary, main disadvantage of charcoal use was a gain of more diluted butanol solution (preconcentration from 2 to 4) after its displacement from charcoal by steam. Energy balance must be done for this process but it needs measurement in pilot scale.

The profound influence of solution composition on stripping efficiency is shown in Table 3, where comparison of model (water) solution of solvents with medium after fermentation is provided. In this case, the stripping was carried out directly in the bioreactor (liquid volume 3L) using aeration ring as nitrogen distributor (flow rate 2 VVM). Schemes of stripping arrangements are provided in (Fribert et al., 2010).

Table 3. Comparison of butanol stripping from model solution and cultivation medium after fermentation

Nevertheless, if summarized it can be stated that the mean rate of stripping for butanol and butanol preconcentrations achieved after application of freezing corresponded with already published values (Ezeji et al., 2003; Ezeji et al., 2005; Qureshi & Blaschek, 2001b). The mean butanol stripping rate exceeded the butanol productivity what indicated a potential successful integration of gas stripping with fermentation into one process.