Lignin, (Villas-Boas et al, 2002). Thermo chemical methods –

Lignin,
cellulose and hemicellulose are three main components of lignocellulosic
material of plant (Prasongsuk S. et al, 2009). After cellulose, lignin is an
abundant aromatic polymer synthesized by plant. It is associated with
cellulose, hemicellulose and other polysaccharides in the cell wall (Ayyappa
and Wensheng, 2016). When lignin bounds to cellulose and hemicellulose,
it reduces the degradation of lignocellulosic materials by forming a barrier.
In paper and biofuel production, this barrier also complicates the use of plant
polysaccharides among other processes (Hofrichter, 2002 and Majumdar et al,
2014 Moreover, due to the rapid development of the agricultural industries, the
lignocellulosic residues are being produced about 40 to 50 million tons per
year worldwide (Villas-Boas et al, 2002). Thermo chemical methods – chemical
oxidation, gasification, pyrolysis – can be used to de-polymerzied lignin
(Pandey et al, 2011). However, these processes are difficult, unsafe, require
specific conditions to occur and waste lot of energy (Ward and Singh, 2002). In pulp industries, lignin is a materials
used for produce paper. After that, lignin will be discarded to streams or
rivers. However, all of the lignin is not removed efficiently from wood in the
pulp processes. In addition, to make white paper, the crude pulp is treated
with chlorine or chlorine dioxide. Then, lignin is chlorinated. Therefore, it
caused water pollution. Lignin also causes air pollution by combination with
sulfur chemicals to form of obnoxious odors. Therefore, finding out the
microorganisms used for lignin biodegradation or bioremediation is necessary.
(Abdelaal and Ahmed, 2015).

In
recent years, biological treatments by using microorganisms and enzymes have
the advantage in lignin pretreatment process due to its less costly and more
efficient than previous mentioned methods. Furthermore, biological pretreatment
process has no hazardous impact on the environment as much as physical and
chemical process. (Agbor et al, 2011 and Alvira et al, 2010). In biological
treatment systems, a wide variety of microorganisms involved in lignin
biodegradation including fungi and bacteria (Modi et al, 1998; Nagarathnamma et
al, 1999; and Perestelo et al, 1996). To date, most of the research works are
focused on fungi. Among them, white rot fungi and others such as Phanerochaete
chrysosporium, Streptomyces viridosporus, Pleurotus eryngii have capable of
degrading lignin. (Martin A, 1977). Nonetheless, white rot fungi have powerful
lignin – degrading enzymes systems, but they are also have some limitations in
practical treatment. (Hatakka, 1994). In industrial applications, the
limitations of fungi is that their filaments cause the structural hindrance.
Therefore it is not practicable. Moreover, the culture conditions for fungi
such as temperature, pH, and level of oxygen are not compatible with industrial
processing conditions. Finally, lignin is degraded very slowly by fungi, but
the fungi require of long lag period and additional food sources such as
glucose and nitrogen to support their growth. (Crawford and Murahlidhara,
2004). In addition, ligninolytic enzymes produced by fungi are fungi have low stability under high pH
condition, high substrate conditions, and high temperature. (Wang et al, 2013;
and Abdul et al, 2013). Actually, the temperature of process of
de-lignification is often at high temperature up to 70oC. Thus,
bacterial enzymes are proven that they can overcome the limitations of the
fungal enzymes. (Thambirajah et al, 1995). Because the bacteria have ability to
adapt the environment and biochemical versatility, they are a worthy
bio-resource for studied lignin – biodegradation (Daniel and Nilson, 1998; Maki
et al., 2009). There is a lot of bacterial strains have been established as
lignin – degrading microorganisms such as Pseudomonas aeruginosa, Serretia
marcescens, Xanthomonas (Kalyani D. C. et al, 2008). The sources for isolation
of lignin – degrading bacteria are from soils such as compost soil, rhizosphere
soil, from cow dung, from decomposing plant, and also from termite gut (Bholay
et al, 2012). Bacteria are tend producing extracellular enzyme such as
peroxidases and laccases. Moreover, the using of bacteria in bioremediation and
biodegradation has many advantages like short generation time, easy to be
cultured, separating of lignin from cellulose and hemicellulose better
(Abdelaal and Ahmed, 2015).

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          There are three enzymes involved in lignin biodegradation, including
lignin peroxidase (LiP), manganese peroxidases (MnP), and laccase (Lac) (Glenn
et al., 1983; Hatakka and Uusi-Rauva, 1983; Glenn and Gold, 1985). Lignin
– peroxidase or Ligninase is belonged to the group of ligninolytic enzymes. It
is an extracellular enzymes. Lignin – peroxidase (LiP) is able to degrade
lignin or lignin related aromatic compounds by oxidizing both phenolic and non
– phenolic compounds such as aromatic amines, aromatic ethers, and polycyclic
aromatic hydrocarbons (Breen and Singleton, 1999; Martínez et al., 2009).
Lignin – peroxidases have potential practical applications in many fields,
including chemical industry, fuel industry, agricultural industry, paper
industry, textile industry (Maciel et al. 2010).  Lignin – peroxidases have been proven that
they are useful in the removal of colored from industrial effluents,
bio-bleaching and useful in treating hazardous waste (Alam et al. 2009). Manganese
– peroxidase (MnP) is an extracellular ligninolytic enzyme. Manganese –
peroxidase is considered as a key enzyme in lignin biodegradation. (Silva and Souza et al 2014).
Manganese – peroxidase can oxidize the phenolic compounds by catalyzing the
oxidation of Mn2+ to Mn3+. In industry, manganese –
peroxidase can be used for remediation of azo-dyes, or can be used for
converting sugar into biofuels. Both lignin – peroxidases and manganese –
peroxidases are dependent on hydrogen peroxide. Therefore, the presence of
hydrogen peroxide is necessary for both of these enzymes activity (Archibal,
1992).

          Giant
Asia pond turtle is an omnivorous. However, it feeds lots of aquatic plants.
The giant Asia pond turtle lives in freshwater habitats, including rivers,
streams, lakes, marshes and swamps. This turtle can be found in Southeast Asia
such as Vietnam, Cambodia Malaysia, Myanmar, and Thailand. The reason for
choosing giant Asia pond turtle as the object for this project is this type of
turtle feed mainly on plant even though it can consume small fish for food. On
the other hand, many types of turtle in Vietnam such as Malayemys subtrijuga, Platysternum megacephalum, Cuora trifasciata,… are fed
mainly on marine animals. Just some types of turtle in Vietnam can consume
plant as food: Cyclemys pulchristriata, Manouria impressa, Mauremys annamensis, etc.
However, many of them are in the IUCN Red list. Finally, giant Asia pond turtle
is breeding for food or commercial products in Vietnam, so it is easier for
collecting sample.

Until
now, most of the treatments for lignin degradation are using fungi. Therefore,
the aim of this study is isolation and identification of bacteria that produce
lignin – peroxidase and manganese – peroxidase. Moreover, this study is
considered to be one of the first studies on isolation and using of bacteria as
a treatment of lignin. Finally, the object of this study (Heosemys grandis) is the new object promising to bring some new
discoveries to the science.