Diversity Of Cold-adapted Denitrifying Bacteria Isolated From Arctic And Analysis Of Their Denitrifying Ability

Today,with the rapid developments of the economy and society,the quality of life of residents improved by a wide margin,meanwhile,it has also increased the discharge of domestic sewage in industry and agriculture.It is extremely urgent to treat sewage effectively,which contains harmful substances to the human health and ecolgical safety.The conventional biological treatment technology of sewage is difficult to meet emission standards at low temperatures,due to the mesophile microbial mechanism affecting the efficiency of sewage discharge.Therefore,it is very important to find high-efficiency denitrifying bacterias to be used in effective treatment of discharge at low temperatures.A total of 59 isolates with valid 16S rRNA gene sequences were obtained from the 26 Arctic Ocean sediments by denitrifying screening medium.The phylogenetic analysis of the 16S rRNA gene indicated that these isolates belonged to 3 phyla,4classes,8 orders,13 families,16 genera and 23 species,among which,?-Proteobacteria accounted for the largest proportion?57.63%?.One isolate?CC2-DN-5?,having the highest sequence similarity 96.44%with the model strain Halocynthiibacter arcticus PAMC 20958,might be novel species.The analysis results of physicochemical properties showed that most strains can grow using various carbon sources including arabinose and glucose,and can produce various extracellular hydrolases such as nitrate reductase,?-glucosidase and protease.The growth temperature and NaCl tolerance range of most representative strains were 0–30°C and 0–6.0%,and the optimum temperature and the NaCl concentration were 10°C or 20°C and 1.5%or 4.5%,respectively.The stains can grow well at low temperatures and wide NaCl concentrations,indicating that they had strong adaptability to the environments and good application prospects.Nitrite reductase gene?nirS?,the key enzyme in the denitrification,was cloned from three isolates of genus Pseudomonas?P13-DN-7,E24-DN-52 and C22-DN-54?and one isolate of genus Marinobacter?P12-DN-1?by the degenerate primers,indicating that these four isolates might be the true denitrifying bacteria.Considering the temperature and NaCl tolerance range and the growth patterns in the denitrifying screening medium,six isolates?CC2-DN-6,E24-DN-51,C22-DN-54,CC05-DN-57,B09-DN-60 and B10-DN-64?were screened to study the nitrogen removal ability in the nitrifying and denitrifying medium.The nitrifying and denitrifying test showed C22-DN-54 had better nitrogen removal ability.The content of NH₄⁺-N?initial concentration was 60 mg/L?in nitrifying test was decreased over 70%within 48 h by C22-DN-54;The removal efficacy of NO₃^?-N?initial concentration was40 mg/L?by C22-DN-54 was almost 100%in denitrifying test within 48 h.Marinobacter sp.Arc7-DN-1 uses NO₃^?or NH₄⁺as the sole nitrogen source to grow at low temperatures.The strain can grow at a wide range of temperatures?0–30??and NaCl concentration?1.5–9.0%?.The genome of Marinobacter sp.Arc7-DN-1 has one circular chromosome of 4,300,456 bp?57.64 mol%G+C content?,consisting of 3811 coding genes,including 50 tRNAs and three rRNA operons as16S-23S-5S rRNA.On the basis of the KEGG analysis,strain Marinobacter sp.Arc7-DN-1 encodes 43 proteins related to nitrogen metabolism,including a complete denitrifying pathway?nitrate reductase,nitrite reductase?NO formation?,NO reductase,and N₂O reductase?,a nitrate reduction pathway?nitrate reductase and nitrite oxidoreductase?,and other nitrogen metabolism related enzymes,such as nitronate monooxygenase and glutamine synthetase.On the basis of good denitrification ability,the combined microflora consisting of strain E24-DN-51?Paracoccus sp.?and C22-DN-54?Pseudomonas sp.?,was selected to study the effects of C/N,temperature and NaCl concentration on the degradation efficacy.The results showed that,the denitrification performance of the microflora increased with the increase of C/N in the range from 1 to 16 of C/N.The degradation efficacy was better in the range from 7 to 16 of C/N and was best at C/N=16,and the removal rates of NH₄⁺-N and NO₃^?-N were almost 100%at 96 h,The degradation efficiency of the microflora increases gradually with increasing temperature ranging from 5°C to 25°C.Within 96 hours,the combined microflora can degrade NH₄⁺-N and NO₃^?-N completely,and the degradation rate of COD was also close to 100%in the temperature ranging from 10°C to 25°C.The combined microflora showed the higher removal efficacy at the NaCl concentrations ranging from 0 to 4.5%,and the optimum NaCl concentration was 1.5%.This study will provide new strain resources for the biological treatment of low temperature sewage in China,and lay a certain foundation for the biological treatment of sewage at low temperatures in practice.