Enrichment And Impact Factors Affecting The Metabolic Activity Of N-DAMO Bacteria

Nitrite-dependent anaerobic methane oxidation?N-DAMO?is a new discovered process in which methane is used as electron donor to reduce nitrite?NO₂^-?to nitrogen?N2?by some microorganisms under anaerobic conditions.The novel process provides a strategy for dealing with the biological denitrification of wastewater with low ratio of carbon to nitrogen?C/N ratio?.However,the growth of N-DAMO bacteria is very slowly and sensitive to environmental changes.Thus,it becomes a practical issue for engineering application to enrich and stimulate the metabolic activity of N-DAMO bacteria in biological wastewater treatment processes.In the present research work,an enrichment culture of N-DAMO bacteria were obtained from wetland soil.The community structure of the enrichment culture was revealed,and then the effects of major environmental factors and growth factors on the N-DAMO activity were further investigated.With wetland soil as an inoculum,two mixed cultures rich in N-DAMO bacteria were obtained after the same primary enrichment and the next intensive culturing with different strategies.Cultured for 19 hours at 30? and140 rpm with the initial NO₂^-of 1.5 mmol·L^-1 and methane partial pressure?PCH4?of 98 k Pa,both of the enrichment cultures displayed an excellent N-DAMO activety with a NO₂^-reduction rate of 0.27 mmol·g^-1·d^-1.Phylogenetic analysis based on the 16 S r RNA gene sequences showed that phylum NC10 was one of the most abundant bacteria in the two enrichment cultures with a relative abundance of 29.16% and 37.27%,respectively.Phylum NC10 has been specialy construceted for N-DAMO bacteria.To improve the metabolic activity of N-DAMO bacteria,the enrichment culture with the relative abundance of 29.16% was evaluated by major environmental factors and growth factors.The results showed that the favorable p H,temperature and NO₂^-concentration were 7.5,35? and 2.0 mmol·L^-1,respectively,for N-DAMO bacteria.The N-DAMO activity could be observably restricted by dissolved oxygen?DO?,but a NO₂^-reduction rate of 0.03 mmol·g^-1·d^-1 could be kept by the enrichment culture at DO 1.0 mg·L^-1.Though Na Cl also has a negative effect on the N-DAMO bacteria,a NO₂^-reduction rate of 0.04 mmol·g^-1·d^-1 could be obtained by the enrichment culture even with a Na Cl concentration as high as 15.0 g ·L^-1.On the contrary,the growth factors vitamin,heme and betaine all have positive effect on the N-DAMO activity.With 0.02 m L·L^-1 and 6 ?g·L^-1 and 0.5 mg·L^-1 as the correct dosage of vitamin solution,heme and betaine,the NO₂^-reduction rate in the enrichment culture reached 0.18,0.17 and 0.28 mmol·g^-1·d^-1,which are 1.32,1.22 and 1.96 times that without any supplement of growth factors,respectively.No better performance could be found with overdose of the growth factors.Reaction kinetics indicated that the NO₂^-reduction rate of N-DAMO bacteria agreed with the Monod mixing kinetics process,with a half-saturation constant KS?NO₂^-?of 0.708 mg·L^-1 and a maximum NO₂^-removal rate of 3.391 mg·L^-1·h^-1.The above findings suggested that N-DAMO bacteria have favorable adaptability to environmental conditions.It is quite possible to perform biological denitrification of wastewater with low C/N ratio by the enrichment of N-DAMO bacteria.