Monday, November 10, 2008

Lignin Degradation Method

LIGNIN DEGRADATION
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 1.10.3.2) are multi-copper phenoloxidases )
2.LiP (Lignin Peroxidase)
3.Xylanase
4.MnP (manganese Peroxidase)
Media composition, for bacterial Isolation and Screening
The medium used for the isolation is: Kraft lignin -MSM (in g/l):
Na2HPO4--------- 2.4g
K2HPO4----------2.0g
NH4NO3-----------0.1
MgSO4------------0.01
CaCl2------------- 0.01
glucose------------10.0
peptone------------5.0
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]

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Lignin MeasurementBio-remediation of Pulp and Paper Mill Effluent by Tannic Acid Degrading BacteriaTry to Unstable The Lignin PolymerLIGNIN PRECIPITATIONLignin Degradation ProblemLaccase Enzyme Secreting Bacteria IsolationLignin Degradation MethodLIGNIN


6 comments:

muruganantham said...

Can you pl suggest easy method for degradation of lignin in coir pith.
for eg. coir pith pH is 4-5 and if we are rising the pH to more than 9, is it possible to reduce lignin and phenol content in coir pith.Pl convey your answer thro my e-mail ID.

A.Muruganantham
muruganantham2005@gmail.com

muruganantham said...

Aslo pl tell me any suitable microbes for degradation of lignin and phenol in coir pith.

Yogendra Singh and Anita Singh said...

Mr A.Muruganantham thank you for showing your interest in http://www.ligninblack.blogspot.com . As you know that lignin is a complex compound for the microbial community. Its deterioration occur vary slow in nature. However, few microbes mostly belonging to fungi like Phanerochaete chrysosporium, Ceriporiopsis subvermispora, Trametes versicolor, Phlebia radiata, Pleurotus ostreatus and Pleurotus eryngii are reported for degradation of lignin components.
But my suggestion is to screen out the native microbes (Bactria/fungi) from the heaps of coir pith and then use it for your experiments.

abhibhawan said...

Hello sir and madam,
The media described here for isolation of lignin degrading bacteria contains glucose and lignin both. So how can we be sure that the bacteria are utilising ligin as their sole C source? Secondly the KL-MSM agar plates will also hav ethe same composition so will it not be better just to provide lignin as the sole C source in the media so that we can isolate lignin degrading bacteria

Suveera Bellary said...

Hello Sir and Madam,

Can you suggest a method used for degradation of hydrolyzed lignin from natural sources.
Also the medium that you have suggested contains peptone and glucose. But these are rich c sources and if bacteria are employed they will preferentially use glucose and peptone and then lignin, hence lignin is not the primary C source. Will bacteria alone be able to thrive on Lignin as C source.

Thanking you,
Shruti

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