Biological pre-treatment is a slow process which can last for few weeks. However, the process has a mild reaction, less energy demand, low chemical usage, low capital cost, less side reaction, and is environment friendly [59,60]. The process if compared to other pre-treatment processes can be an attractive alternative process due to its green technology. This pre-treatment employs microorganism and their enzymatic mechanism to break down the lignin and liberate cellulose and hemicellulose from the complex lignin. These selective microorganisms which produce oxidative enzyme to break the lignin are from type of fungi and bacteria.

Fungi, the wood rotten microorganism can be divided into three groups according to the morphology of wood decay which are soft-rot ascomycetes or detromycetes, white-and brown-rot basidomycetous. Degradation of the lignin and hemicelluloses by the action of white-rot fungi is an aerobic process but there are some bacteria like E. lignoluticus SCF1 and rumen microorganisms with lignin degrading capability under anaerobic condition [16, 17].

These fungi have shown positive effect on delignification process [61, 62]. White — and soft-rot fungi attack both cellulose and lignin while brown-rot fungi mainly attack cellulose and hemicellulose components in wood [62-64]. Some white-rot fungi can selectively delignify (lignin and hemicellulose) and leave enriched cellulose. De­pending on types of fungi and wood, the lignin lost, observed, can reach up to 44 % [64]. These microorganisms, associated with lignin-degrading enzyme, consist of mainly two major families of enzymes which are laccase and peroxidase. Bacteria such asactinomycetes, which is a filamentous bacteria belonging to the genus Strep — tomyces are well known degrader that can mineralize up to 15 % of lignin. Others nonfilamentous bacteria mineralize lignin less than 10 % and can degrade the low molecular weight part of lignin [64].

Laccase (benzenediol:oxygenoxidoreductase, EC 1.10.3.2) belongs to the small group of enzymes called the blue copper proteins or the blue copper oxidases that catalyze one-electron oxidations of aromatic amines and phenolic compounds such as phenolic structures of lignin [60, 65]. Laccase, widely distributed in higher plants and fungi, is especially found abundant from white-rot fungi and is also found in insects and bacteria [65]. Fungal laccases have higher redox potential than bacterial or plant laccases (up to +800 mV), and their action seems to be relevant in nature, also finding important applications in biotechonology [65]. Thus, fungal laccases are involved in the degradation of lignin and detoxification of phenols arising during lignin degradation which inhibit fermentation process. Vikineswary et al. studied the production of laccase from sago hampas and OPF parenchyma tissue, which gave higher laccase productivity compared to rubberwood sawdust, using Pycnoporus sanguineus [66].

Peroxidase family consists of ligninolytic enzymes such as lignin peroxidase (LiP), manganese peroxidase (MnP), and versatile peroxidases (VP) [60, 62]. Lignin peroxidase (LiP, diarylpropane peroxidase, EC 1.11.1.14), an extracellular lignin olyticperoxidases which is commonly associated with Phanerochaete chrysosporium

[67] . It has capability of catalyzing the depolymerization of the aromatic polymer lignin and a variety of non-phenolic lignin model compounds in the presence of H2O2 [60]. Others such as Phanerochaete sordida [68], Aspergilllusstrains, and bacteria such as Acinetobactercalcoaceticus, Streptomyces viridosporus and Streptomyces lividans [59] are also producing extracellular LiP.

MnP, hydrogen peroxidase oxidoreductase (EC 1.11.1.13), is able to oxidize Mn(II) to Mn(III) [60, 67]. It has also been reported isolated from Cunninghamella elegans [69], Schizophyllum sp., Ceriporiopsis subvermispora, Panus tigrinus, Lentinula edodes, Nematolomafrowardii, Bjerkandera adusta, Tinea versicolor, and Dichomitus squalens. VP (EC 1.11.1.16) oxidize Mn(II) and a high redoc-potential aromatic compound as MnP and LiP, respectively [68]. VP is also reported to be produced by fungi from the genera Pleurotus, Bjerkandera, and Lepista and maybe also by Panus and Trametes species [70].

The enzyme activity and lignin degradation are influenced by several factors which include the type of strain and nutrient composition (i. e., in the case of delignification by using microorganism), enzyme dosage (i. e., in the case of delignification by isolated enzyme), moisture content, pH, aeration, and temperature. These factors can be manipulated to obtain the optimum pre-treatment process. Ahmad Khushairi and Zainol screened factors affecting biological delignification process of oil palm trunk using local oyster mushroom (Pleurotus ostreatus) [61]. In their study, temperature contributed the most in delignification process followed by pH. Other studied factors were fungi-to-medium ratio, moisture content, contact time, lighting, and humidity. Interesting to note that, in the study, even though biological delignification is known to be time consuming, the contact time between 2 and 10 days are among the least important compared to others.

In another study, direct delignification with a commercial biocatalyst called lac — case was performed. Taguchi method was applied to determine the optimum lignin degradation of OPF from laccase treatment. The effects of laccase dose, pH, pre­treatment temperature, and treatment time were investigated. The experiment results of the nine trial conditions with two runs per trial condition are shown in Table 17.3 where the percentage of lignin degradation is exhibited.

Table 17.3 Lignin Trial Lignin degradation (%)

degradation (%) of laccase

treated OPF ________ RR

Results in Table 17.4 indicated that factor that influenced lignin degradation is in the following order: pH value > laccase dose > treatment time > treatment tem­perature. The optimum combination of factors and levels in lignin degradation of OPF may be predicted in general as follow: pH value of 4, laccase dose of 20 mg, treatment time for 2 h, and treatment temperature at 30 °C. However, the experiment results revealed very low percentage of lignin degradation (10-11 %) as shown in Table 17.3.

Syafwina et al. studied three white-rot fungi and found that Dichomitus squalens degraded lignin most rapidly compared to Ceriporiopsis subvermispora and Pleu- rotus ostreatuson in OPEFB [71]. After 8 weeks, weight loss of the lignin and holocellulose in beech wood reached 75.9, and 49.9 %, respectively. The fungus also delignifled EFB. After 8 weeks, weight loss of lignin and holocellulose in EFB was

25.7 % and 22.8 %, respectively. Hamisan et al. performed chemical pre-treatment and compared to microbial on OPEFB [72]. Microbial pre-treatment using Phane- rochate chrysosporium was shown to significantly removed the lignin, but it is timely (7 days) compared to chemical pre-treatment (3 h) which is fast reaction but quite harsh to the substrate. Namoolnoy et al. isolated 63 fungal of white-rot fungi; 27 of them showed high activity of laccase, MnP or LiP was selected to culture on OPFs [73]. Only seven isolates could degrade more than 50 % lignin content in OPFs within 30 days of cultivation. The efficiency in lignin degradation of selected fungal isolates seemed to be related to the ability of the fungi to produce more than one ligninolytic enzyme.