From the mass transfer point of view, the diffusion resistance of the gas phase is larger for larger particles (e. g., wood chips). This implies that less heat is brought by saturated steam to diffuse into the inner core of a single biomass particle. A longer time is required for saturated steam to diffuse into the core of the large particle to initiate the hydrolysis. Hydrolysis usually starts to take place when the reaction temperature is higher than 160 °C, excluding the mass transfer limitation.

Saturated steam is a better heat transfer medium thanNitrogen. Its high-energy state (a higher latent heat of vaporization) allows them to reach the core of the particle with a smaller diffusion resistance. Hydrolysis is preferentially taking place inside the polymeric system instead of pyrolysis reaction using high-pressure saturated steam. In general, the development of chemical reaction kinetics models usually work with the particles with a <8 mm diameter by assuming no diffusion limitation [49, 50]. Since sawdust has a smaller particle size and a higher specific surface area than wood chip, more glycosidic bonds are accessible for cleavage by hydronium ions (H3O+) resulting in higher degree of hemicelluloses solubilisation. Hydronium ions have a typical size of 0.4 nm in wood chips [49]. Its small size allows them to penetrate into the wood pores to hydrolyze the glycosidic bonds of xylan molecules (hemicellu- loses) as well as to promote lignin solubilization and restructuring. Restructuring of lignin helps to activate and enhance the accessible lignin as a self-binding agent for durable pellet production.

Reactor Types

Steam explosion pretreatment is a commercial available process. It has been used as a pretreatment for bioethanol production. In Europe, Andritz Sprout developed a screw type horizontal continuous scale steam explosion unit. This reactor is similar to the one used by Mascoma Corporation. The feed material can be in a wide particle range in ground particles (diameter: 6 mm) or chips (diameter: 25-75 mm) form depending on the size of the hopper and the blow valve. There is an opening on the top of the horizontal chamber that can be connected to a hopper for material feeding. A steam chamber in horizontal direction is used for steam treatment of biomass. Saturated steam is supplied from the other steam generator via the nozzles from the side wall of the chamber. At the end of the chamber, there is a rapid opening blow valve controlled by a controller to release the high-pressure steam-treated biomass to the ambient condition and further conveying to the other downstream process. Discharged steam is separated by a flow discharger and recovered steam can be recycled for the pretreatment process. Both horizontal reactor and vertical reactor can be equipped with a dry discharger. The resulted material can be ready for pellet production without post-drying.

Alkaline pretreatment is one of major chemical pretreatment technologies receiving numerous studies. It employs various bases, including sodium hydroxide (NaOH) [83], calcium hydroxide (lime) [84], potassium hydroxide (KOH) [85], aqueous ammonia [86], ammonia hydroxide [87], and NaOH in combination with hydrogen peroxide or others [88-90]. Among these alkaline pretreatments, lime has received much more attentions since it is inexpensive (about 6 % cost of NaOH), has improved handling, and can be recovered easily by using carbonated wash water [91].

8.3.2.1 Process Description

In comparison with other pretreatment technologies, alkali pretreatment usually uses lower temperatures and pressures and even ambient conditions. Pretreatment time, however, is recorded in terms of hours or days which are much longer than other pretreatment processes. In the alkaline pretreatment, the residual alkali could be reused through the chemical recycle/recovery process, which may make the system more complex due to the need for chemical recovery [92, 93]. The particle size of the biomass is typically 10 mm or less [57]. A significant disadvantage of alkaline pre­treatment is the conversion of alkali into irrecoverable salts and/or the incorporation of salts into the biomass during the pretreatment reactions so that the treatment of a large amount of salts becomes a challenging issue for alkaline pretreatment [92]. The effectiveness of alkaline pretreatment varies, depending on the substrate and treatment conditions. In general, alkaline pretreatment is more effective on hard­wood, herbaceous crops, and agricultural residues with low lignin content than on softwood with high lignin content [94]. In addition, in comparison with KOH and lime, pretreatment with NaOH was found to be more efficient for the subsequent enzymatic hydrolysis [92].

In view of achieving the effective biological pretreatment, the process can be com­bined with physical and chemical treatment methods as the main drawback of biological pretreatment is loss of polysaccharide (cellulose/hemicellulose) and the longer pretreatment duration than chemical and physical pretreatment. Combina­tion of biological pretreatment with chemical/physical pretreatment can enhance the fermentable sugar conversion from biomass and can improve the performance of pretreatment as compared to sole pretreatment. It is obvious that chemical/physical pretreatment prior to biological pretreatment allows the substrate more assessable for microbes to degrade lignin. Therefore, optimization is required in order to min­imize the overall cost of the pretreatment, time, and energy and maximize the fermentable sugar yield after the enzymatic treatment. This combination can be carried out by two ways (i) chemical/physical treatment prior to biological pretreat­ment, (ii) chemical/physical treatment after biological pretreatment. The combined biological pretreatment with chemical/physical treatment and pretreatment process conditions are summarized in Tables 1.2 and 1.3, respectively.

Taniguchi et al. [142] treated rice straw with steam explosion prior to biological pretreatment using P. ostreatu and found that the pretreatment duration could be reduced from 60 days to 36 days required for obtaining 33 % net glucose yield. Yu et al. [129] reported that the treatment time could be reduced from 60 days to 18 days with considerable sugar yield, when rice straw was pretreated with H2O2 (2 %, 48h).Itoh et al. [143] reported that ethanol yield could be increased by 1.16 times when biological pretreatment was carried out prior to organosolv treatment by using C. subvermispora and saved 15 % electrical energy. Indeed, biological treatment can also be used in lignin-based oil production. For example, Fomitopsis sp. IMER2 was used in removal of amorphous region of cellulose from corn stover and resulted a significant increase in the oil yield from 32.7 % to 50.8 % in pyrolysis process. Therefore, it can be concluded that biopretreatment favors thermal decomposition of corn stover [134].

4.3.1 Fractionation Technology

As per the analysis in Sects. 4.1 and 4.2, heterogeneous characteristics of stalk lead to different conversion properties of different fractions. If corn stalk is converted into single products as a whole with linear technology, different conversion properties of each fraction could hardly be applied [1]. As a result, conversion yield is reduced and waste treatment cost increased. Therefore, it is necessary to integrate various technologies according to the intrinsic characteristics of stalk for multiple products.

Petroleum refining provided a good example for raw materials conversion into universal products. Heterogeneous raw materials are converted into homogeneous fraction at first, and then various fractions are converted into final products accord­ing to market requirement. It is an effective way for nature resource to fulfill human requirement. There are hundreds of hydrocarbon in petroleum. It would lead to high cost if only one component is applied ignoring others. The very reason for petroleum to play an important role in life and industry is the refining process invita­tion. Petroleum is split into different fractions according to a different boiling point. Pure fractions made it possible to explore technologies for further conversion.

Therefore, it would play an important role for stalk-based products to fractionate stalk and integrate various technologies.

6.5.1 Bioethanol Production

The heterogeneous nature of lignocellulosic biomass feedstock used for production of bioethanol makes the treatment process challenging. An efficient pretreatment to maximize enzymatic hydrolysis efficiency is necessary to reduce economics of the total process. Besides, there is a limitation on the use of acid and alkali in conventional high temperature and high-pressure pretreatment method due to its high-energy input. Alternative heating techniques are sought to reduce energy input at the same time increase the total process efficiency. MW pretreatment could be a good alternative because it can reduce the pretreatment time at higher temperature, and for this reason its use for bioethanol production has been extensively investigated as summarized in Table 6.2.

Binod et al. [13] have reported that MW treatment of sugarcane bagasse with 1 % NaOH at 600 W for 4 min followed by enzymatic hydrolysis gave reducing sugar yield of 0.665 g/g dry biomass. Combining MW-alkali-acid treatments with 1 %

NaOH followed by 1 % sulfuric acid resulted in an increase in reducing sugar yield to 0.83 g/g dry biomass. MW-alkali treatment at 450 W for 5 min resulted in almost 90 % of lignin removal from the bagasse. From the results, they found that combined MW-alkali-acid treatment for short duration enhanced the fermentable sugar yield.

The positive effects of the synergy between alkali and MW irradiation had also been confirmed by the works of Keshwani et al. [14] on hydrolysis of switchgrass. They reported that pretreatment using MW irradiation at lower power levels resulted in more efficient enzymatic hydrolysis. The application of MW irradiation for 10 min at 250 W to switchgrass immersed in 3 % NaOH (w/v) produced the highest yields of reducing sugars. The finding suggests that combined MW and alkali is a promising pretreatment method to enhance enzymatic hydrolysis of switchgrass.

On the contrary, instead of alkali, Balcu et al. [15] proposed the use of acid in combination with MW pretreatment. They found that elevated temperatures close to 180 °C are not necessary for better conversion of lignocellulosic biomass into sugars. They suggested the use of 0.82 % aqueous solution of sulfuric acid, getting very good yield even at low temperature of 140 °C.

Moreover, a two-stage MW pretreatment method, which includes MW-alkali pretreatment for hemicelluloses extraction and MW-glycerine pretreatment for lignin extraction of corn stalk as proposed by Zou et al. [16], seems to be more promising. They reported that MW-alkali pretreatment is suitable for hemicelluloses extraction with the following suitable treatment conditions: liquid-to-solid ratio of 20 mL/g, alkali consumption of 150 wt%, treatment time of 10 min, MW power of 116 W/g and the particle size of 40-80 mesh. Using MW and pure glycerine, lignin can be extracted at optimal treatment time of 30 min, and MW power of 66.7 W/g.

The use of glycerine as a solvent has also been investigated by Intanakul et al. [17] for an improved enzymatic hydrolysis of lignocellulosic wastes by MW pretreatment under atmospheric pressure. The benefits of using glycerine as a solvent include no high-pressure build-up even if the temperature reaches 200 °C. Their results showed that with the pretreatment, more than twice the amount of reducing sugars could be produced from enzyme saccharification compared with no pretreatment at all. Unlike the steam explosion process which requires high-pressure and subsequent pressure release, this technique provides some advantages regarding high temperature and high pressure handling.

The technique was also applied to pretreatment of green coconut fiber for bioethanol production [18]. Prior to MW irradiation for 10 min at 250W, pretreatment using alkaline hydrogen peroxide gave higher yield of reducing sugar (35.98 mg/g) and high ethanol yield (1.16 g/g) compared to alkaline sodium peroxide.

Optimization of the method as applied to pretreatment of wheat straw for ethanol production has also been investigated using various techniques such as orthogonal design (L9(34)) [19]. This optimization technique was applied by investigating the effects of four factors including the ratio of biomass to NaOH solution, pretreatment time, MW power, and the concentration of NaOH solution with three different levels on the chemical composition, cellulose/hemicellulose recoveries, and ethanol con­centration. Results showed that pretreatment with the ratio of biomass to liquid at 80g/kg, the NaOH concentration of 10kg/m3, and the MW power of 1000 W for 15 min was the optimal condition. They obtained ethanol yield of 148.93 g/kg wheat straw at this optimum condition, much higher than that from the untreated material which was only 26.78 g/kg.

The use of ionic liquids (IL) in combination with MW pretreatment was also reported [20]. In this method, MW irradiation enhances the solubility of cellulose in IL while decreasing the degree of polymerization. This results into improved cellulose hydrolysis. Results showed a 50-fold increase in the rate of enzymatic hydrolysis of cotton cellulose when MW irradiation is used in combination with IL dissolution pretreatment at 110 °C, about four times better than when only IL was used for dissolution.

Development of a continuous process, although a very challenging approach for the use of MW irradiation, could be the most economical and efficient for large — scale commercial production of bioethanol. The group of Mitani et al. [21] from Kyoto University (Japan) attempted to develop a prototype for a continuous MW pretreatment system for bioethanol production from woody biomass as shown in schematic diagram in Fig. 6.2. A ceramic pipe was set in a metal vessel, and a mixture of woody biomass, water, and solvents flows through the pipe. MW propagates in the internal space of the metal vessel, and it is absorbed by the mixture since MW can penetrate through the ceramic pipe. In the present system, the mixture flows through a metal pipe, and it is irradiated with MW at T-junction metal pipe sections. The gray arrows and white arrows in Fig. 6.2 show the mixture flow and the MW irradiation direction, respectively. MW frequency of the present pretreatment system is 2.45 GHz-band, which is the same as that of a MW oven. The diameter of the metal pipe is 75 mm.

Metal

pipe

unit

Performing MW pretreatment of a mixture consisting of 70 g of Japanese cedar sapwood chips and 770 g of solvents (ethylene glycol:phosphoric acid = 95:5), about

45.9 % of the total saccharide from woody biomass can be obtained as compared to only 43.6 % with the conventional heating. However, the energy consumption was quite higher at 552 kJ compared to only 498 kJ with the conventional heating. The amount of bioethanol that can be produced from this experiment was estimated to be 14.8 g of bioethanol corresponding to an energy of about 439 kJ.

Prof. Dr. Zhen Fang is leader and founder of biomass group, Xishuangbanna Tropical Botanical Garden, Chi­nese Academy of Sciences. He is also an adjunct full Professor of Life Sciences, University of Science and Technology of China. He is the inventor of “fast hydrol­ysis” process. He is specializing in thermal/biochemical conversion of biomass, nanocatalyst synthesis and its ap­plications, pretreatment of biomass for bioreflneries. He obtained his PhDs from China Agricultural University (Biological & Agricultural Engineering, 1991, Beijing) and McGill University (Materials Engineering, 2003, Montreal).

CO2 is a nontoxic, nonflammable, abundant, and renewable feedstock and its bio­transformation into industrially important chemicals can not only have a positive impact on the global carbon balance but also provide novel routes for the green biotechnology. As one of the oldest life forms on earth, microalgae have very high CO2 biofixation capacity, grow fast, and accumulate large quantities of lipids and carbohydrates and hence became the most promising feedstock for production of next generation biofuels like biodiesel and bioethanol [78]. Considering the fact that CO2 is a very cheap carbon source, microalgal systems should also be considered as potential resources for EPS production. However, in the literature, there are very few reports on microalgal polysaccharide production. In general, for production of value added products, the biggest advantage in using open microalgae culture is the direct use of solar energy which in turn is highly energy efficient and cheap [79]. Actually, these systems applied to phototropic and mixotrophic cultures are considered to be the most technically and economically feasible methods at commercial scale [78]. On the other hand, this advantage does not hold for EPS production where use of monocultures, closed, and controlled cultivation systems are required to reach high levels of productivity [80]. Although photobioreactors and fermenters are advanta­geous in providing optimum conditions for biomass growth and EPS production, these systems are expensive and energy intensive when compared with open systems [81].

There are various types of bioreactors that can be used for EPS production such as airlift flat plate photobioreactors (well reviewed by Zhang et al. [82]). Gener­ally, culture conditions for lipid-rich biomass production and EPS production are remarkably different. A systematic study conducted with the green colonial fresh water microalgae Botryococcus braunii strains on the effect of culture conditions on their growth, hydrocarbon and EPS production also revealed two distinct culture conditions so that cultivation in 16:8 h light dark cycle yielded higher hydrocarbons whereas continuous illumination with agitation yielded higher amounts of EPSs with

1.6 g/L—maximum yield obtained from B. braunii LB 572 strain [83]. In a study on the effect of salinity with the same strain, EPS yields of 2-3 g/L were also reported [84]. The difference in cultivation conditions could also be used for the high-level EPS production by use of a two-stage culture as reported for spirulan production by Spirulina platensis [85]. In this method, whereas the first stage focuses on rapidly increasing microalgal biomass, culture conditions in the second stage are modified to maximize the polysaccharide yield. Rhodella violacea [86] and Porphyridium cruen — tum [87] are well known as producers for viscous bioactive EPS [88] and the highest yield of 543.1 mg/L EPS production was reported for P cruentum after optimization of initial pH, light intensity, inoculation ratio, and liquid volume of shaking batch cultures [89]. By culturing P cruentum semi-continuously in flat plate photobioreac­tors, a production rate of 68.64 mg/L per day could be reached by Sun et al. [90]. Very low EPS concentrations (less than 30 mg/L) were reported for planktonic diatoms like Amphora holsatica, Navicula directa, and Melosira nummuloides [5, 91]. How­ever, these yields can be improved by further studies on optimizing the bioreactor conditions in favor of EPS production.

Another important issue for microalgal cultivation is the need for using high con­centrations of chemical fertilizers as a source for nitrogen and phosphorus. Whereas high nitrogen concentrations in the cultivation medium favors polysaccharide syn­thesis pathways and biomass formation, lipid accumulation is favored under nitrogen limited conditions where polysaccharide pathways are blocked and the photosynthet­ically fixed carbon is directed towards fatty acid synthesis [92]. Microalgae could become a favorable source for EPS production if the high expenses associated with fertilizers could be reduced by replacing them with their low cost alternatives. Be­sides the use of wastewater as an inexpensive source, the literature is very limited in such studies.

The rotary dryer is one of the most commonly used technologies for drying wood. It is effective to handle both sawdust and chips [18, 20, 25]. The most commonly used rotary dryers in industry are direct contact type, which consists of a hollow, rotational metal cylinder providing space for direct contact between the material to be dried and the drying medium, usually hot air. The heat and mass transfer between these two streams is high with a direct contact and can be further enhanced by installing a series of flights on the inner wall of the cylinder to promote contact of the two streams. In addition, uniform MC distribution of the dried product can be achieved because every piece of the solid material has an equal chance to contact with the hot air.

For the co-current rotary dryer, the wet material and the hot air enter from one end, and the dried material and the humid air exit from the other end. The wet material and the hot air enter the drum from the opposite ends and move inside the dryer in the op­posite directions. A typical drying temperature used in commercial drying of woody biomass is up to 500 °C that ensures high drying rate and energy efficiency of drying. The co-current mode is usually employed for industrial application as this ensures the dried biomass will not overheat and cause self-ignition. The operation conditions have to be well monitored to prevent fire hazard. This requires reliable measurement techniques of dryer temperature and MC in order to develop a prediction model for biomass drying.

5.2.1.1 Packed Moving Bed Dryer

For packed moving bed dryers, the wet biomass is fed from one side to a moving bed that has openings on it, allowing the drying medium (hot gas) to flow through. In order to increase energy efficiency of the drying, the drying medium is recycled by flowing back through the biomass bed in the second half of the dryer. The overall air flow rate is only half of that in the arrangement where the drying air always flows upwards. Because with the drying air reversal in the second half of the dryer, the exhaust gas has low temperature and high humidity, the corresponding equilibrium moisture content (EMC) is relatively high for the bio-originated material. This indicates that if low final MC is required, the exhaust gas temperature must be kept higher than a certain value to achieve the required dryness. On the other hand, higher exhaust air temperature results in greater heat losses [13, 16, 17].

During drying, the MC varies across the bed thickness [26]. In a co-current arrangement, the bottom layers of biomass in the first half of the dryer dry faster than that of top layers. Although the reverse flow of the drying air reduces the uneven moisture distribution, the MC gradient may not be totally eliminated because the recycling drying gas in the second half of the dryer has a lower drying temperature and higher humidity; thus, the drying rate is lower compared to the first half of the dryer.

In the case that the low final MC is required for the dried biomass, a countercurrent arrangement can be used where the hot gas is fed from the second half of the dryer, flowing upwards, and is then reversed to flow through the biomass bed in the first half of the dryer. In this improvement, the drying efficiency is higher and the required final MC can be achieved [13].

Fang Huang and Arthur J. Ragauskas

Abstract Lignocellulosic materials, such as wood, grass, and agricultural and forest residues, are potential resources for the production of bioethanol. The biochem­ical process of converting biomass to bioethanol typically consists of three main steps: pretreatment, enzymatic hydrolysis, and fermentation. During the whole pro­cess, pretreatment is probably the most crucial step since it has a large impact on the efficiency of the overall bioconversion. The aim of pretreatment is to disrupt recalcitrant structures of cellulosic biomass to make cellulose more accessible to the enzymes that convert carbohydrate polymers into fermentable sugars. Physical, physical-chemical, chemical, and biological processes have been used for pretreat­ment of lignocellulosic materials. This chapter summarizes the leading technologies in chemical pretreatment on softwood, particularly pine species, which generally show relatively higher recalcitrance than hardwood, grass, and other lignocellu — losic materials. Different chemical pretreatment techniques, including dilute acid pretreatment, alkaline hydrolysis, wet oxidation, sulfite pretreatment to overcome recalcitrance of lignocellulose (SPORL), organosolv, ionic liquids pretreatment, and ozonolysis process are intensively introduced and discussed. In this chapter, the key points are focused on the structural changes primarily in cellulose, hemicellulose, and lignin during the above leading pretreatment technologies.

Keywords Chemical pretreatment technology ■ Biofuel ■ Biorefinery ■ Softwood

8.1 Introduction

In order to cope with growing demand for energy, the depletion of fossil fuel re­sources, and environmental concerns raised by fossil fuel use, countries wishing to limit their energy dependence on petroleum exporting countries are developing

A. J. Ragauskas (H) ■ F. Huang School of Chemistry and Biochemistry,

Institute of Paper Science and Technology,

Georgia Institute of Technology, Atlanta, Georgia, 30332-0440, USA e-mail: arthur. ragauskas@ipst. gatech. edu

Z. Fang (ed.), Pretreatment Techniques for Biofuels and Biorefineries,

Green Energy and Technology,

DOI 10.1007/978-3-642-32735-3_8, © Springer-Verlag Berlin Heidelberg 2013

alternative energy sources, such as bioethanol produced from renewable biomass [1-4]. Cellulosic bioethanol is regarded as one of the most promising renewable biofuels in the transportation sector for the coming next few decades [5]. Current pro­duction of bioethanol relies on sugars that are obtained from starch-based agricultural crops by using first-generation conversion technologies [6]. Nowadays bioethanol produced from lignocellulosic biomass using second-generation technologies has become an interesting alternative, mainly because lignocellulosic raw materials do not compete with food crops or productive agricultural land, and they are also less expensive than conventional agricultural feedstocks [7, 8].

The biological process of converting biomass to bioethanol typically consists of three main steps: pretreatment, enzymatic hydrolysis, and fermentation. During the whole process, pretreatment is the most crucial step since it has a large impact on the efficiency of the overall bioconversion. In lignocellulosic biomass, cellulose and hemicellulose are densely packed together with lignin, which serves several func­tions including protection against enzymatic hydrolysis [9]. The aim of pretreatment is to disrupt recalcitrant structures of cellulosic biomass to make cellulose more ac­cessible to the enzymes that convert carbohydrate polymers into fermentable sugars (Fig. 8.1). During the pretreatment, the extent of removal of lignin and hemicellulose depends on the pretreatment conditions and severity. For example, acidic chemical pretreatment removes most of hemicellulose. The lignin is condensed when pretreat­ing temperature reaches above 170 °C. On the contrary, the ammonia fiber explosion (AFEX) pretreatment does not significantly remove hemicellulose.

Numerous pretreatment strategies have been developed to enhance the reactiv­ity of cellulose and to increase the yield of fermentable sugars. Typical goals of pretreatment include [11]:

• Production of highly digestible solids that enhances sugar yields during enzyme hydrolysis.

• Avoiding the degradation of sugars (mainly pentoses) including those derived from hemicellulose.

• Minimizing the formation of inhibitors for subsequent fermentation steps.

Among the numerous types of biomass, softwoods (SW) are generally recognized as being much more refractory than hardwoods (HW) or agricultural residues in the pretreatment process. This is, in part, due to the fact that SW have a more rigid structure and contains more lignin [12].

The goal of this paper is to review promising chemical pretreatments technologies on softwood, particularly pine species, and to discuss recent developments which have greatly aided the production of bioethanol. For each technology, a brief process description is first given with recent developments, and then the feedstocks on which these technologies are used are highlighted, followed by discussion of the technol­ogy’s advantages and disadvantages. The key points will be focused on the structural changes primarily in cellulose, hemicellulose, and lignin during the above leading pretreatment technologies.

Laccases (Lac, EC 1.10.3.2, benzendiol: oxygen oxydoreductase) belong to blue copper protein or oxidase family. Lac has been found in fungi, bacteria, and plants. The major producers of Lac are of fungi kingdom, whose diversity can be found in soil, phytopathogenic, and freshwater inhabiting ascomycetes and basidiomycetes

[104] . Lac is generally larger than peroxidases as it has a molecular weight of approx­imately 60 kDa and pi 3-6 [49]. Optimum pH for better Lac activity is found to be 3-5

[105] . Lac catalyzes four single-electron oxidations of aromatic amines and phenolic compounds such as phenolic substructure of lignin, which coincide with the reduc­tion of O2 to H2O [ 32, 98]. Indeed, it can also oxidize nonphenolic compounds under certain conditions, for example, 2,2/-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) [106], 1-hydrobenzotriazole (1-HBT) [107], and violuric acid [108]; natu­ral mediators such as 4-hydroxybenzoic acid, 4-hydroxybenzyl alcohol [109], and 3-hydroxyanthranilate [110]. Therefore, the natural mediator should be produced by organisms for the complete oxidation of lignin.

Lac is produced by almost all the white-rot fungi. Generally, it has several Lac encoding genes and secrete as multiple isoforms [49,106]. Lac contains four copper atoms of three distinct types per enzyme, and each type has a different role in the oxidation of substrate [98]. Type I copper directly involves in the reaction with the substrate. The type I copper gives a maximum absorbance at a wavelength of 610 nm, which gives to the enzyme a typical blue color. The type II copper and the two type III copper cluster are found in triangular forms. Copper II and III complexes involve in the binding, the reduction of O2 and the storage of electrons originating from the reducing substrates. The type II copper does not have absorbance in a visible range, while the type III copper has a maximum absorption at 330 nm, hence copper II and III complexes do not have any color [98]. The entire crystalline structure of Lac containing four copper atoms in the active site has been studied from T. versicolor and C. maxima [111,112]. Bourbonnaisetal. [106] reported that the white-rot fungus T. versicolor produces two laccase isozymes (I and II).

For effective biological pretreatment of lignocellulose, various white-rot fungi can be used in addition to copper ions in order to induce the secretion of Lac enzymes. In some special cases, Lac can also be induced by addition of aromatic compounds like VA and 2-5 xylidine [32]. Although Lac generally oxidizes phenolic residues of lignin, it also oxidizes non-phenolic compounds of lignin with addition of ABTS as discussed earlier. Therefore, Lac action can be induced further by addition of some special catalyst in the biological pretreatment. For some fungi such as C. subvermis — pora and Ganoderma lucidum the Lac production could be increased in the presence of lignocellulosic materials. Recently, some bacterial Lacs have also been char­acterized from Azospirillum lipoferum, Bacillus subtilis, Streptomyces lavendulae, Streptomyces cyaneus, and Marinomonas mediterranea [113].