Isolation and identification of fungalstrains incrude lignite

Due to the limited oil and gas resources worldwide, coal will become the most important energy material. With the consumption of 2007,in terms of volume, coal reserves will be maintained for 146 years, while oil will be exhausted within 50 years and natural gas will be within 63 years. After the oil and gas reserves are exhausted, coal will monopolize the entire fossil energy market. Obviously, coal is use, especially the brown coal, which accounts for 47.3% of the world’s coal reserves, will become an important research theme.

There are abundant lignite resources in Kazakhstan and the lignite with low calorific value and high ash content is piled up as rubbish causing a serious waste of resources as well as environmental pollution. Research into biological processes for the utilization of fossil energy has received more and more attention in recent years. Microbial treatment has been considered as an economically effective and environmentally safe way of processing coal via degradation of the macromolecular network into simpler, low molecular mass products [1-3]. The coal solubilization processes that have been studied in greater detail include the basidiomycetes fungi involved in wood rotting processes. Despite the advances in the understanding of the microbial transformation of coal, it is still not possible to develop efficient and economically viable biotechnological processes to produce humic substances by solubilizing LRC with fungi. Humic acids are polyelectrolytic macromolecules which play an important role in global carbon and nitrogen cycling and in the regulation of the mobility and fate of plant nutrients and environmental contaminants [4-6]. Lignite, a low-grade coal, has a lignin-like structure and contains humic acid and water-soluble humic material including fulvic acids. It is well known that humic acids have widespread utilization in agriculture, for example, they have been used as organic fertilizer and as a feed additive.

The present work was intended to isolate fungi with high-efficiency solubilising-coal activity from low rankcoal.

Lignite. Lignite sample for biosolubilization was obtained from the “Oikaragai” coal deposit (Almaty province, Kazakhstan). The samples were collected at around 15 cm depth beneath the surface, manually pulverized (particle size around <1 mm), and stored at 4°C .

Fungi strains and cultivation medium

These strains were selected because of theirability to grow in a (AMSC5) growth medium with powdered LRC at 5% as the sole source of carbon. Its composition(mg/L) was: NH4NO3- 2.50, KH2PO4- 1.75, MgSO4 -0.75, K2HPO4- 0.75, NaCl - 0.25, ZnSO4- 0.088, FeCl3- 0.08,CuSO4- 0.016, MnCl2- 0.014, MoO3- 0.007, Co (NO3)2- 0.005;noble agar 15 g.L^-1, powdered LRC 5% (p/v)[7].

Fungal ITS sequence and Phylogenetic analysis

The taxonomy of strain ES11 was evaluated by phylogenetic analysis of the ITS internal region[8]. Universal primers ITS1 (TCC GTA GGT GAA CCT GCG G) and ITS4 (TCC TCC GCT TAT TGA TAT GC) targeting the 3 and 5 end of the IT sequence were used for PCR.Chromatograms were analysed and edited using Bio-Edit version 5.0.9 (Hall TA., 1999: 95–98) and the Genbank database wassearched for sequence similarities using NCBI BLAST. It is generally believed that the similarity is more than 97% for the same type, and the above 95%is the same genus, using the software Clustal W calibration to multiplex it for comparison, apply MEGA5. 0 software, using neighbouring methods(Neighbour-Joining) build a phylogenetic tree.

Molecular identification of culturable fungi Performing ITS rDNA on the purified 4 representative fungi PCR reaction, the average length of the amplified gene fragment is about 500 bp, logs its ITS rDNA sequencing results to GenBank Database Blast comparison with existing sequences on NCBI Comparative analysis of similarity found that 3 strains and existing patterns in the database The ITS rDNA gene sequence similarity of the strain was over 97%. Molecular identification results showed 4 strains of fungi belong to Aspergillus sp.

Tab. 1 ITS rDNA sequences results of 4 culturable isolates

PCR amplification of purified RKs (RK1, RK2, RK5, RK6) fungal strains genomic DNA using ITS primers resulted in the expected product. BLACT analysis of amplicon sequence showed high homology values with the sequence from [99%] RK1, RK3 and RK4: Neosartoryafischeri (EU926976), RK2: Aspergillus sp. (KP890480), RK5: Aspergillus fumigatus (GU566217), RK6: Aspergillus sp. (KX009135). it provide an relevant information for the screening of lignite depolymerizing fungi as the biofactories of biohumic substances for further studies.

References

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