Study On Isolation And Diversity Of Culturable Anaerobic Bacteria In Intertidal Sediment Of Qingdao

Anaerobic bacteria are a large group of marine microorganism that lives betterwithout oxygen. Obligate anaerobic bacteria is liable to be damaged by oxygen, evento dead due to lacking superoxide dismutase (SOD), which makes them more harderto be cultured. However, as a large group of marine microorganism, anaerobicbacteria play an important role in biogeochemical cycles, bioremediation, exploitationof green energy, regulation of global climate and isolation of new drugs, etc.Intertidal falt is the largest transition zone between continent and sea andabounds in biological resource, which is severely affected by various physical andchemical factors as well as activities of human. It is characterized by a high nutrientinput from the land and open sea. This nutrient supplyment stimulates intense benthicprimary production, fuelling microbial activities in the upper sediment layers, whichmakes the lower layer an anaerobic expanse with abundant anaerobic bacteria.However, by now, researchers mainly focused on aerobic cultivation, and littleinformations about culturable anaerobic bacteria inhabiting in the areas is available.Anaerobic bacteria of the lower layer play a key role in biogeochemical cycles withmore research values.To test the easy method we designed and several media (2216E medium and4kinds of SPG media) working for various anaerobic bacteria for further studying thefunction of anaerobes in the biogeochemical cycle, diversity of culturable anaerobicbacteria in intertidal sediment of Qingdao was investigated followed by analysis ofbacterial16S rRNA gene. First of all, enrichment culture of anaerobic bacteria wascarried out with anaerobic liquid medium, followed by isolation and purification withsolid medium. A total of138bacterial isolates clustered into3phyla,15genera and32species. Bacterial isolates were mostly Gammaproteobacteria (46%), followed byDeltaproteobacteria (12%), Epsilonproteobacteria (3%), Bacteroidetes (21%) andFusobacteria (18%). At the genus level, Vibrio was the most common genus isolated, followed by Ilyobacter, Desulfovibrio, Shewanella, etc. At the species level,Ilyobacter polytropus, Desulfovibrio oceani subsp. oceani, Vibrio diabolicus, Vibrioordalii and Propionigenium maris etc. were superior in numbers. Notably, there were8isolates might be novel families or genera, whose homology is between89.38%and94.22%, one of which belonged to Epsilonproteobacteria was rarely isolated.The results of our studies indicate that for intertidal sediment which is esailybeing accessed, axenic cultivation still have the potential. Of all the isolates,facultative anaerobes account for81.25%, and aerotorelant bacteria9.37%, andobligate anaerobe9.37%, which indicates that the fast, simple and easy designedmethod was feasible. As a conclusion, the different media and enrichment strategieswere selective for specifc bacterial groups, the use of different approaches turned outto be a basic requirement for achieving high diversity. Medium2216Eenjoyed the diversitiy advantage (20of32, H4.05), especially in isolating bacteria ofVibrionales and Aeromonadales and medium SPG showed greater ability to isolatenovel stains (mean H2.06), especially to Bacteroidetes and Fusobacteria. Meanwhile,medium SPG-1showed a greater ability to isolate SRB and bacteria of-Proteobacteria, and medium SPG-4prone to isolating NRB.