Three Kingdoms Wu Bamboo Corrosion Spots Microbial Isolation, Identification And Lignin Degradation Property

A large number of bamboo slips of Kingdom Wu during the Three-Kingdoms Dynasty,unearthed from Zoumalou,have been stored in Bamboo Slips Museum of Changsha,Hunan Province,China.The bamboo slips are rare cultural relic for studying Chinese history.After unearthed,the bamboo slips were severely eroded by microorganisms.Surface layer of the eroded bamboo slips became translucent film material,and it was easily to crack off because lignin and cellulose was degraded rapidly by the microorganisms.The eroded bamboo slips steeping fluid was choosen for microorganism separation and characterization.Eight bacterial strains and one fungus strain was isolated from steeping fluid of the eroded bamboo slips using bacterial and fungal medium.Based on the cultural,morphological characteristics of the strains,and sequence analysis of the nucleic acids(parts sequence of 16S rDNA of the bacterial strains and 18S rDNA and ITS of the fungi strain),the bacteria belong to seven genera(Bacillus,Acinetobacter,Staphylococcus, Pandoraea,Novosphingobium,Cupriavidus,Comamonas,respectively) of four different classes(Bacilli,α-Proteobacteria,β-Proteobacteria andγ-Proteobacteria), and the fungus strain showed highest similarity to Aspergillus.Then strains having lignin-degradation ability were screened by using the medium containing sodium lignosulphonate as sole carbon source.One fungal and six bacterial lignin-degradation strains were identified.The bacterial strains were Acinetobacter sp.B-2,Bacillus sp.B-3,Pandoraea sp.B-6,Novosphingobium sp.B-7,Cupriavidus sp.B-8,Comamonas sp.B-9 and the fungal strain was Aspergillus sp.F-1.The lignin-degrading enzyme activity was detected from all the seven strains by using plate culture with Azure-B and Remazol Brilliant Blue medium.Lignin degradation experiments were carried out by using liquid culture containing sodium lignosulphonate as sole carbon source.The degrading ability of sodium lignosulphonate for the seven strains was evaluated by using UV spectrophotometry.All of the 6 bacterial strains degraded about 20% lignin after 14 hours incubation and 33.3%lignin was degraded by fungal strain after 72 hours incubation.The results indicated that all of the 7 strains with relatively high degrading ability of lignin. The effects of 5 single culture factors on lignin degrading ability for 3 bacteria(Acinetobacter sp.B-2,Pandoraea sp.B-6 and Novosphingobium sp. B-7) were studied,and then the appropriate culture conditions of these 3 bacteria were determined.The appropriate culture conditions for Acinetobacter sp.B-2 and Novosphingobium sp.B-7 were obtained as follows: NH₄NO₃ as nitrogen source,0.01mol/L of nitrogen source concentration, pH=7.0,30℃of temperature and 120r/min of shaking speed.The appropriate culture conditions for Pandoraea sp.B-6 were similar to above strains except for NH₄H₂PO₄ as nitrogen source and 0.03mol/L of nitrogen source concentration.Under the appropriate culture conditions,the lignin degrading ability of Acinetobacter sp.B-2 was determined again by using standard curve of sodium lignin sulfonate-TC,and it degraded about 40%lignin after 4 days incubation.Moreover,Acinetobacter sp.B-2 can extracellularly secret high activities of manganese peroxidase(MnP),lignin peroxidase(LiP) and laccase (Lac).After 2 days incubation,the peak activities of MnP and LiP were 600U/L and 121.84 U/L,respectively.The peak activity of Lac was 714.28 U/L after 4 days incubation.