Degradation Of Lignin And Low Molecule Phenolic Substances By Protoplast Fusion

Lignin is widely exist in plants, and also is the second most abundant biopolymer on theearth. It is a complex，non-crystalline and three-dimensional network polymer，so is alsodifficult to be degradated and utilizated under natural conditions, which results in seriouspollution of soil, water, etc... The biodegradation of lignin not only can mitigateenvironmental pollution, but also change waste into reusing resources, so the developmentand utilization of lignin has important economic and social significance.The protoplast fusion can overcome the crop distant hybridization affinity and obstaclesthe sexual hybridization, break the boundaries of species, and reach to interspecific andintergeneric fusion. By protoplast fusion, a variety of useful genes can be tranfered from onespecies to another. we combine multiple functions into one bacterium, achieving the optimalcombination of a variety microorganisms to take good effect on the treatment of sewage. Sothis technology has good prospects to develop.The experiment fused Pseudomonas putida with Agrobacterium sp., Bacillus sp. andGordonia sp., and respectively screened out a fused strain which was best degradating lignin。Then we used these fused strains to degradate lignin, acrossing to analyse the changes of theIR、HPLC、 GC-MS and organic pollution indicators spectrum, contrasted degradating effectof seven strains. The degradating effect of parental strains was Gordonia sp.> Pseudomonasputida.> Bacillus sp.> Agrobacterium sp., fused strains was Pseudomonas putida fused withGordonia sp.> Pseudomonas putida fused with Bacillus sp.> Pseudomonas putida fused withAgrobacterium sp., and degradating effect of fused bacteria was significantly higher thanparents strains.Lignin degradation use organic pollution indicators of COD, BOD and TOC to analyse.After five days’ degradation, organic pollution indicators hadn’t too much changes. CODincreased from63-72%of the parent to76%-84%of fused strain, among which PPG reachedto83.4%. BOD increased from63-72%of the parent to74%-84%of fused strain, PPGreached to83.5%. TOC increased from64-71%of the parent to76%-83%of fused strain, thePPG reached to82.2%.The fused strain PPG degraded lignin could be80%, and was a moresuccessful fused strain.GC-MS can be used to analyse the lignin degradation products. After lignin degradation,the number of peak was increased significantly, because compounds could be more complex.Lignin degradation products included aliphatic compounds, monomer products and dimer products. In the degradation process, lignin oxidation occurred in α position, a lot of Cα-Cβbond and α-O-4bond were breaked. The phenolic hydroxyl group had been oxidized to thecarbonyl group, than the carbonyl became acid and esters. The carboxyl group on the etherlinkage which maintained the macromolecular structure of lignin was oxidized to dehydration,formed acyl group, this process was conducive to the depolymerized and the benzene ringopened. This lignin mainly had lilac structure units.To explore the pathway of lignin biodegradation, we knew that83%lignin was removed,in which60%was removed by degradating,20%was by biosorption and3%bynon-biological action （precipitation, oxidation, volatilization etc.）. In the first30minutes,biodegradation and biosorption had the same degradating ability, and then biodegradablewould play an important role.According to the basic principle of the derivative of ratio spectroscopy, we drew ratio-derivative spectra under different pH values and different concentrations of vanillin. Spectrasignal showed the value of the derivative of ratio spectroscopy at pH≤7in the230nm couldnot be effected by pH, at pH>7in the248nm could not be effected by pH. Spectra signalwas more sensitive to the change of vanillin concentration in the230nm and248nm, thevalue of the derivative of ratio spectroscopy could be used for the determination of vanillincontent in lignin solution in those two wavelengths.Low molecule phenolic substances have similar structure and functional group withlignin, so they can commonly be used as lignin model compounds. They have a double bondin α position, which is C=O double bond conjugated with a benzene ring and often be usedas representative structures of lignin. The main products of lignin degradation have4-hydroxybenzaldehyde, vanillin, syringaldehyde, syringic acid, vanillic acid and3/,5/-dimethoxy-4/-hydroxyacetophenone. We detected the contents of each low moleculephenolic substances in the lignin degradation process, and took further research on themechanism of vanillin degradation. The three intermediate products of vanillin degradationrespectively were2-methylphenol,4-hydroxy-3-methoxybenzoic acid and4-hydroxy-3-methoxybenzyl alcohol, but4-hydroxy-3-（methoxy） benzoic acid was the mainlyintermediate product. In the degradation process of low molecule phenolic substances, all thealdehydes were converted to the corresponding acids firstly, then acids did a thoroughdecomposition becoming CO₂and H₂O.