Application Of Single Cell Raman Spectroscopy To The Elucidation Of Ammonia Oxidizing Microbial Metabolic Activities

Nitrogen cycle plays an important role in affecting ozone layer destruction,water eutrophication and greenhouse processes.Ammonium oxidation driven by microbes is one of the limited steps in nirtrogen cycling.Those microbes were firstly known as ammonia-oxidizing bacteria?AOB?.Ammonia-oxidizing archaea?AOA?,which began to be recognized as a key player in nitrogen cycling in 2005,were found to be widely distributed in differernt environments.However,the contributions of AOA and AOB to the ammonia oxidation process and their responses in different environments are still largely unrevealed.The research goal of this study are as follows:Revealing the microbial abundances of AOA and AOB associated with different particle sizes along the salinity gradient of the Pearl River Estuary and their controlling factors.Developing a new method to analyze the metabolic activity of AOA?taking Nitrosopumilus maritimus SCM1 as a model stain?,which was based on the red shifts of single cell Raman spectrum resulting from assimilating deuterium,¹³C or ¹⁵N.The results show:?1?From the freshwater area to the low salinity mixing area,the content of AOB decreased?P=0.03?.From the low salinity mixing area to the high salinity mixing area,the content of AOB continued to decrease?P=0.031?,indicating that AOB prefer to grow in low salinity environments.The abundance of particle-attached?0.7?m?AOB was 17.9±34.6 times?n=12,P=0.001?that of the free-living AOB,indicating that the particle-attached?0.7?m?AOB were higher than the free-living AOB.The salinity has no significant effect on the abundance of AOA.The abundance of A-type particle-attached?0.7?m?AOA was 4.0±3.3 times?n=12,P=0.025?and 4.1±3.4times?n=12,P=0.013?higher than those of the B-type particle-attached?2.7?m?and the free-living AOA,respectively.It is indicated AOA were more abundant on 0.7².7?m fractions.The ratio of AOA to AOB in total three particle sizes increased from 0.76times?n=4?in the fresh water zone to 1.13 times?n=3??P=0.026?in the high salinity mixing area,indicating that the total AOA of three particle sizes are more adaptable to high salinity than AOB.Pearson's correlation analysis between the copy number of AOA and AOB and the ratio of the two to environmental factors showed that salinity,pH,dissolved oxygen and temperature were the main factors that controlled the distributions of AOA and AOB in the Pearl River Estuary.?2?This study also explored the use of single-cell Raman spectroscopy to determine the metabolic activity of ammonia-oxidizing microorganisms.The Raman spectra of SCM1 could be dectected,and the main cellular components could be recognized in the spectrum,for example,lipids(1443.92 cm^-1),proteins(1655.99 cm^-1),nucleic acids(855.29 cm^-1),and C-H bond(2922.81 cm^-1).SCM1 was cultured in a gradient D₂O medium,the strong peaks between 2040 cm^-1 and 2300 cm^-1 were detected in the Raman spectra of SCM1 archaeal cells after 15 days'cultivation.By calculating the integrated area of the C-D peak and the C-H peak,we found that the ratio of C-D peak and C-H peak was positively correlated with the percentage of D₂O in the growing water,the correlation coefficient was 0.97.The C-D and C-H characteristic regions could be used as a marker to quantify the amount of deuterium that was assimilated from water,indicating that Raman-SIP?D₂O?is a promising method for rapidly determining the activity of AOA.There was no significant change in the Raman spectrum of SCM1 after in situ fluorescence hybridization?FISH?,indicating that Raman-SIP?D₂O?analysis can also be performed with formaldehyde-fixed cells for FISH analysis.The integration of Raman-SIP-FISH has far-reaching significance for the rapid assessment of the metabolic activity of AOA in different environments.