The aromatic polymer lignin is well-known for resistance to microbial degradation because of its high molecular weight and presence of various biologically stable carbon-to-carbon and ether linkages. Microorganisms that degrade plant lignin via an oxidative process, are fungi , actinomycetes and to a lesser extent, bacteria. Study shows that the bacterial strains degrade the low molecular weight portion of lignin, but are probably unable to depolymerize the high molecular weight backbone of the lignin polymer because, unlike fungi which secrete extracellular enzymes called ligninases , the bacterial cells do not secrete lignin-depolymerizing enzymes (Vicuna 1988). However, bacterial lignin degradation systems consist of many unique and specific enzymes with the ability to catalyze the production of various useful compounds.
Due their productivity, bacterial enzyme systems are expected to serve as useful tools for the conversion of lignin into intermediate metabolites. There are many types of lignins, the properties and composition of which depend upon the source and method of isolation.
Recommended sites for the sample collection for the isolation of Ligninase producing Bacteria:-
1.Activated sludge samples from the effluent treatment plant for Eg: Century Pulp and Paper Mill Ltd, Lalkuan Nainital, Uttranchal (India) in sterile test tubes.
2.Decomposing tree stump, oak leaf compost, birch log, Spruce chip pile.
3.Crude Cork material from cork factory.
Here I am discussing the Method of isolation and screening of ligninase enzyme producing bacteria.The Enzymes on which i am working is :-
1.Laccase Enzyme ((benzenediol: oxygen oxidoreductase, EC 22.214.171.124) are multi-copper phenoloxidases )
2.LiP (Lignin Peroxidase)
4.MnP (manganese Peroxidase)
Media composition, for bacterial Isolation and Screening
The medium used for the isolation is: Kraft lignin -MSM (in g/l):
Trace element solution (1ml/lit, pH 7.6)
pH finally adjusted to 7.6 with NaOH (1M)
For bacterial isolation 5 g of sludge was transferred to a conical flask (250-ml) containing sterile MSM (100 ml) amended with KL (250 mg l-1) (designated as KL-MSM). The flasks were incubated at 30 C in a rotary shaking incubator at 120 rpm for 2 weeks.
When a sample exhibited decolorization, an aliquot (0.1 ml) was spread on KL-MSM agar plates and incubated at 30 C. The different colonies on plates were further purified on KL-MSM agar plates. Purity of each bacterial culture was checked with the microscope. This bacterium was stored at 4 C on nutrient agar (NA) slant and used in subsequent experiments.[Biodegradation of kraft lignin by a newly isolated bacterial strain, Aneurinibacillus aneurinilyticus from the sludge of a pulp paper mill Abhay Raj Æ Ram Chandra Æ Mohan M. Krishna Reddy Æ Hemant J. Purohit Æ Atya Kapley,Published online: 14 November 2006 Springer Science+Business Media B.V. 2006]