DAP is usually performed over a temperature range of 120-210 °C, with acid con­centration typically less than 4 wt%, and residence time from several minutes to an hour [53]. In the DAP pretreatment, the combined severity (CS) is used for an easy comparison of pretreatment conditions and for facilitation of process control, which relates to the experimental effects of temperature, residence time, and acid concen­tration [54]. Lower CS is beneficial for the hemicellulose to hydrolyze to oligomers and monomers while higher CS could bring these monomers to furfurals, which are inhibitors for the subsequent enzymatic hydrolysis [55]. In order to maximize the efficiency of pretreatments, several studies have proposed a two-step procedure for DAP of SW [45, 56]. The conditions in the first step are less severe and serve to hydrolyze the hemicelluloses resulting in a high recovery of hemicellulose-derived fermentable sugars in the pretreatment effluent. By separating the solid and liquid phases after the first step, it is possible to minimize the degradation of hemicellulosic sugars to furfural and hydroxymethylfurfural (HMF). The solid material recovered from the first step is treated again under more severe conditions which promotes the enzymatic digestibility of cellulose fibers.

The DAP offers good performance in terms of recovering hemicellulose sugars but there are also some drawbacks. The dilute acid applied in the process could cause corrosion that mandates expensive materials of construction, such as hastelloy steel and ceramic valves. The neutralization of acid before the fermentation results in the formation of solid waste. In addition, the hemicellulose sugars might be further degraded to furfural and HMF, which are strong inhibitors to microbial fermentation [57]. Furthermore, most of the reported work used materials with significant size reduction, which consumes additional energy. Previous report indicated that grinding the materials to 1 mm accounted for 33 % of the power requirement of the entire process [58]. However, this is not practical in large-scale production. In addition, the detoxification step is required in DAP when running high solids pretreatment, which adds additional cost to the process.

Two enzymes, oxaloacetase and glyoxylate oxidase that catalyze the hydrolysis of oxaloacetate and the oxidation of glyoxylate, respectively, are responsible for the biosynthesis of oxalate. An important aspect is that LiPs and MnPs are capable of decomposing oxalate in the presence of VA or Mn2+ [98,125]. The breakage of ox­alate results in the formation of carbon dioxide and formate anion radical (R-CO+-), which is further oxidized by O2 to give CO2 and superoxide (O+- or HOO+) under aerobic conditions. The active oxygen species are suggested to directly participate in the oxidation of lignin. This reaction can be observed in oxidation of phenol red and kojic acid by MnP in the presence of Mn2+ and oxalate without exogenous addition of H2O2. This suggests that oxalate maybe regarded as a passive sink for H2O2 pro­duction [98]. If the oxalate reduces the VA+- and Mn3+ ions, the mineralization rate of lignin will be affected adversely (Figs. 1.2 and 1.3). As mentioned earlier, VA+- and Mn3+ both should be reduced by phenolic and/or non-phenolic compounds of lignin for the effective degradation of lignin. For better biological treatment, it is important to conquer the excessive action of oxalate on VA+- and Mn3+.

For vascular plant [18], different organs are all composed of vascular tissue embedded in parenchyma tissue and epidermis tissue cover. For vascular tissue, the wall of vessel cell and fiber cell wall (bundle sheath cell) is rich in lignin because secondary wall is lignified. While for parenchyma tissue, parenchyma cell wall is rich in cellulose because there is only primary cell wall.

4.2.1 Tissue Level

According to Sachs’ convenient classification [18], the body of a vascular plant is composed of three tissue systems, the dermal, the vascular, and the fundamental (or ground). The dermal tissue system comprises the epidermis and the periderm. The vascular tissue system contains two kinds of conducting tissues, the phloem (food conduction) and the xylem (water conduction). The fundamental tissue system (or ground tissue system) includes parenchyma tissue, secretory tissue, collenchymas tissue, and sclerenchyma tissue.

There is cutin layer out of the dermal tissue system [19]. Cutin layer is composed of cutin and wax. Cutin is mostly polymers composed of C16 and C18 monomer. Parenchyma cell under dermal is hardened, leading to high lignin content. For vas­cular tissue system, vessel cell in xylem is thickened so that lignin content is high too. There are mainly parenchyma tissue cell in ground tissue system. For parenchyma tissue cell, there is only primary cell wall which is composed of cellulose. In terms of mass content, vascular plant is mainly composed of ground tissue system and vascular tissue system. Therefore, vascular plant could be simply fractioned into vascular tis­sue fraction which is rich in lignin and ground tissue fraction which is rich in cellulose.

Corn stalk could be fractionated into vascular tissue fraction and parenchyma tissue fraction manually. Vascular tissue fraction includes fiber cell around vascular because fiber cell is connected tightly to xylem and phloem, and both fractions are hydrolyzed with enzyme for 48 h. It is found that glucose content from parenchyma tissue is higher than that from vascular tissue by 22.5 %. Corn stalk could be frac­tionated into vascular tissue fraction and parenchyma tissue fraction with steam explosion integrated mechanical carding. For rind and leaf, the enzyme hydrolysis rates of parenchyma tissue fraction are 1.77 and 1.37 times higher than vascular tissue fraction, respectively.

Vascular tissue fraction from corn stalk is tested by pulping with ethanol self cat­alyzing. It reveals that pulp yield could reach to 57.6 % while pulp whiteness reached

65.9 % when catalyzing time is 2.0 h at 160 °C with ethanol concentration 50 % and solid-to-liquid ratio 1:10. The whiteness meets the requirement of printing paper [20].

Corn stalk has not been applied in pulping in that there is much non-fiber cell. Non-fiber cells are mainly parenchyma cell and dermal cell, and the fiber cell content in corn stalk is low. Moreover, the ratio of length to width for corn stalk fiber cell is smaller than other pulping materials. Therefore, if the whole corn stalk is applied for pulping, pulp yield is low and the quality of paper could hardly meet the requirement. If non-fiber cell could be removed and fiber cell content is high, corn stalk would become another resource for pulping.

MW technology relies on the use of electromagnetic waves to generate heat by the oscillation of molecules upon MW absorption. The electromagnetic spectrum for MWs is in between infrared radiation and radiofrequencies of 30 GHz to 300 MHz, respectively, corresponding to wavelengths of 1 cm to 1 m. Domestic and indus­trial MW systems are required to operate at either 12.2 cm (2.45 GHz) or 33.3 cm (900 MHz) in order not to interfere with the wavelength ranges being utilized for RADAR transmissions and telecommunications [5].

In MW-assisted heating, unlike the conventional methods, the heat is generated within the material, thus rapid heating occurs. As a result of this rapid heating, many MW-assisted organic reactions are accelerated, incomparable with those obtained using the conventional methods. Thus, higher yields and selectivity of target com­pounds can be obtained at shorter reaction times. In addition, many reactions not

possible using the conventional heating methods, had been reported to occur under MW heating. Some very useful information on the fundamentals of MW-enhanced chemistry, its sample preparation, and applications are well presented in the book edited by Kingston and Haswell [6]. The advantages of MW heating are briefly summarized in Table 6.1.

Other than the above-mentioned advantages of rapid, internal, and selective heat­ing, MW non-thermal effects on reaction likely occur, obtaining dramatic increase in the yield even at milder conditions. The MW non-thermal effect is defined as the system response to electromagnetic energy not attributed to temperature variation [7]. Although doubts are cast on the true existence of non-thermal effects, some evi­dences had been reported and postulates had also been made by several researchers. These were summarized in a review article published by de la Hoz et al. [8] com­paring them with the thermal effects. The review of Jacob et al. [9] on thermal and non-thermal interaction of MWs with materials attributed some interesting results on specific MW effects. Evidences on reaction rate enhancement due to some reasons other than the thermal effects such as “hotspots” or localized heating, molecular agitation, improved transport properties were discussed. They suggested that due to the interaction of MW with the materials, heating cannot be simply treated as that similar to the conventional methods as there are a lot of possible mechanisms of activation of materials that might possibly occur.

The above-mentioned thermal and non-thermal effects of MW irradiation offer enormous benefits to the pretreatment of biomass for synthesis of biofuels in­cluding energy efficiency, development of a compact process, rapid heating, and instant on-off process (instant heating-cooling process), among many other possible advantages.

The manufacture of olive oil produces large amounts of a dark colored juice called OMW that consists of a mixture of water from the olive, machinery cooling waters, fruit washings, and remainder of the fruit. Typically, OMW comprises about 15 % organic material that is composed of carbohydrates, proteins, and lipids as well as a number of other organic compounds including monoaromatic and polyaromatic molecules [62] and the toxic effects are mainly derived from its extremely high organic load and the presence of recalcitrant organic compounds such as polyphe­nols with strong antimicrobial properties. Hence valorization of OMW produced by the olive oil industry has long been an environmental concern in Mediterranean countries [61]. Beside various conventional technological treatment methods ap­plied, biovalorization of OMW to value-added chemicals is considered as the most cost-effective and environmentally compatible option [73]. Due to its composition with high carbon-to-nitrogen ratio, its use as a suitable substrate for microbial poly­mer production has been proposed [45] and applied to produce pullulan [45] and xanthan gum [74]. By studying the resource variability factors [55] and then by use of a high-producer strain and medium optimization, Lopez et al. [56] reported significant improvements in xanthan yields, reaching 7.7 g/L in 5 days. In all these studies, in order to reduce the inhibitory effect of phenols, the OMW obtained from the industry was clarified by filtration, diluted with distilled water or saline, neu­tralized, sterilized by autoclaving, and then used in microbial fermentation. OMW pretreated by this approach has also been used for the microbial production of a metal-binding EPS by Paenibacillus jamilae bioreactor cultures. Due to the high phenol biodegradation ability of Paenibacillus genus, these cultures were not only proposed for the production of an EPS that could be used as a biofilter but also for the bioremediation of OMW [62]. The main constraint associated with the use of OMW is the need for dilution in order to lower the amount of phenols which in turn limits the concentration of the used waste as culture medium [62]. On the other hand, for the в-glucan production from the fungus Botryosphaeria rhodina DABAC-P82, OMW was only clarified by centrifugation and then after steam sterilization, di­rectly applied as substrate without dilution. Due to the lack of oxidase activity, high biopolymer yields and decreases in phenol content of the culture were attributed to the adsorption action of the fungal biofilm [60]. Undiluted OMW was found to be a poor substrate for pullulan production by A. pullulans [46]. Besides EPSs, OMW has also been used as a fermentation substrate for the microbial production of other biopolymers including polyhydroxyalkanoates (PHAs) [75].

In the terms of technological economics, the more by-products are, the higher cost is apportioned, leading to low cost of the main product. That is to say, multiple products are an effective way to improve economic rationality.

The economic comparison between fractionation refining technology with multi­ple products and single technology with one product is presented in Table 4.1.

It demonstrates that if only the hydrolyzed carbohydrate of corn stalk is converted into acetone, butanol, and ethanol, products lack market competitiveness. What is worse, waste disposal would increase cost. On the contrary, if the whole stalk is frac­tionally converted into products, each product is market competitive. Moreover, there are no wastes left, which fulfills green industry requirement and realizes resource total utilization.

Compared with single pretreatment technology, the features of integrated pretreatment technology for stalk are as follows.

1. Integrated pretreatment is multiple products oriented, making best of each com­ponent for different products. Therefore, by-products increase the economic value of stalk and reduce discharge.

2. Integrated pretreatment is set up according to the heterogeneity of stalk in different levels, such as tissue level, cell level, and component level.

3. Integrated pretreatment is of wide adaptability because there are more adjustable parameters. Therefore, integrated pretreatment is also flexible and fit for various raw materials from different regions and planting manners.

4. For integrated technology, any idea and technology could be integrated includ­ing those from paper making, pinning and sheet industry, especially petroleum refining which is rational, economic, effective, clear, and operable.

5. Each technology involved in integrated pretreatment could be complement in advantages to reduce the whole pretreatment cost.

4.5 Conclusion

Stalk heterogeneous property in the level of tissue, cell, and component is ana­lyzed in terms of stalk morphology and anatomy. It is proved that different tissues, cells, and components have different conversion properties because of heterogeneous characteristics. Steam explosion integrated fractional refining technology is set up according to stalk heterogeneous intrinsic characteristics. The whole stalk is frac­tionated in the level of tissue, cell, and component and then converted into different products, respectively. Industrial demonstration proves that steam explosion inte­grated fractional refining technology is an effective way to improve the economic benefit of stalk utilization.

Acknowledgments Financial support to this study is provided by the National Basic Research Program of China (973 Project, No. 2011CB707401), the National High Technology Research and Development Program of China (863 Program, SS2012AA022502), the National Key Project of Scientific and Technical Supporting Program of China (No. 2011BAD22B02).

Although cellulose is the most abundant plant material resource, its susceptibility has been curtailed by its rigid structure. As the result, an effective pretreatment is needed to liberate the cellulose from the lignin seal and its crystalline structure so as to render it accessible for a subsequent hydrolysis step [5]. By far, most pretreatments are done through physical or chemical means.

Till date, the available pretreatment techniques include acid hydrolysis, steam explosion, ammonia fiber expansion, alkaline wet oxidation, and ozone pretreatment

[6] . Besides effective cellulose liberation, an ideal pretreatment has to minimize the formation of degradation products because of their inhibitory effects on subsequent hydrolysis and fermentation processes [7]. The presence of inhibitors will not only further complicate the ethanol production but also increase the cost of production due to entailed detoxification steps. Even though pretreatment by acid hydrolysis is probably the oldest and most studied pretreatment technique, it produces several potent inhibitors including furfural and hydroxymethyl furfural (HMF) which are by far regarded as the most toxic inhibitors present in lignocellulosic hydrolysate [8]. In fact, ammonia fiber expansion is the sole pretreatment which features promising pretreatment efficiency with no inhibitory effect in resulting hydrolysate.

Enzymes face several challenges in the degradation of macromolecular lignin [49]. As mentioned earlier, this substrate is a large heterogeneous polymer and very difficult to degrade by microbes. Indeed, lignin does not contain enzymatically hydrolysable linkages and is stereo-irregular. For lignin degradation, the enzymes or agents must be oxidative. Many extracellular enzymes involved in lignin degradation are, as mentioned earlier, LiPs (LiPs, ligninases, EC 1.11.1.14), manganese peroxidases (MnPs, Mn-dependent peroxidases, EC 1.11.1.13) and Lacs (benzenediol:oxygen oxidoreductase, EC 1.10.3.2). Further, some accessory enzymes are also involved in hydrogen peroxide production. Glyoxal oxidase (GLOX) and aryl alcohol oxidase (AAO) (EC 1.1.3.7) belong to this group. LiPs and MnPs are heme-containing glycoproteins, which require hydrogen peroxide as an oxidant [49, 98].

The major problems associated with the application of high-pressure steam for the pre-treatment of lignocellulosic materials has been the observed destruction of the xylan polysaccharide content, an incomplete disruption of the biomass lignin- carbohydrate matrix and the formation of by-products after the treatment process which inhibit any microbial and enzymatic activities utilised in subsequent down­stream conversion schemes [29]. The production of microbial inhibitors has been mainly attributed to the formation of compounds such as fufural and hydroxymethyl — furfural from the biomass pentoses and hexoses respectively and release acetate from acetyl-group from hemicelluloses during the steam pre-treatment process [13]. This sugar degradation usually occurs as a result of dehydration processes take place with the use of high steam temperatures for the biomass pre-treatment [13]. With these by-products potentially hindering the application of potential biological conversion, detoxification schemes might therefore be required to improve the use of lignocel — lulosic hydrolysates to fuels and chemicals, especially where enzymatic methods are to be applied. The use of water washing as a cheap method to aid the removal of potential inhibitory substances, as well as water soluble hemicelluloses has been discussed in the literature [30]. The use of such a method has however been reported to lead to a decrease in the overall saccharification yields obtained from the steam pre-treatment method, since the soluble sugars (i. e. generated via hemicelluloses hydrolysis) are also removed by the water washing step [9]. Other detoxification techniques, that is, the use of neutralisation and fungal treatments to aid the removal of such potential process inhibitors has also been investigated [31]. Depending on the availability of oxygen in the pre-treatment process, sugar degradation could also occur via pyrolysis (absence of oxygen) and oxidation processes resulting in the thermal decomposition of the organic matter and a partial conversion of the biomass pentoses to carboxylic acids and other by-products [13].

Commercial torrefaction technology suppliers require a strict/narrow particle size distribution as feedstock for producing fuels with a homogenous quality. Many re­actor types require a maximum chip size of 2.5 cm (1"). In particular, the reactors with a shorter residence time (in the order of seconds or a few minutes) require a more stringent particle size limitation (less than 0.5 cm), for example, Torbed and moving bed reactor. If the feedstock particles are with a larger proportion of fines, fines will block the gas to limit the heat transfer and also become difficult to fluidize or transport pneumatically.

Some reactors allow using large particles as feedstock, for example, multiple hearth reactor and rotary drum reactor. The rotary drum reactor can accommodate a mixture of large and small particles. Both groups of particle will be treated at the same temperature and reside within the same chamber at the same time. Meanwhile, the attrition of wood chips or particles occurs and generates lots of fines due to the rotational motion. However, large particles would undergo with non-homogenous reactions within a single particle and result in non-homogenous product quality. Feedstock with a wider particle size range can be used if the torrefaction temperature range is within a mild region (i. e., 260-290 °C). Besides, a pack bed of large particles are usually with lower bulk density. This limits the throughput as well as introducing a limited load capability for some reactor types including rotary drum reactor, screw reactor, and belt reactor.

A moving bed scale reactor setup for studying complex gas-solid reactions has been designed in order to obtain kinetic data for scale-up purpose. In the bench scale reactor setup, gas and solid reactants can be contacted in a co-current and counter-current manner at high temperatures. Gas and solid sampling can be per­formed through the reactor bed with their composition profiles determined at steady state. The reactor setup can be used to evaluate and corroborate model parameters accounting for intrinsic reaction rates in both simple and complex gas-solid reaction systems. The moving bed design allows experimentation over a variety of gas and solid compositions in a single experiment unlike differential bed reactors where the gas composition is usually fixed. The data obtained from the reactor can also be used for direct scale-up of designs for moving bed reactors. The recommended particle size range: 5 mm < diameter < 20 mm; length <70 mm.