Hydrogen Isotope Fractionation In Lipid Biosynthesis By The Piezophilic Bacterium Sporosarcina Sp. DSK25 Under Different Under Different Growth Pressure

Biogeochemical methods have been playing a vital role in studying marine organic matter cycles,transportation,and reconstructing paleooceanographic environment.As the largest carbon pool in the earth,marine has notably adjusted the global climate,controlled marine substance and energy transformation,affected the lives of human being.The connection between piezophilic bacteria and marine biogeochemical cycles was poorly studied,although microorganism,especially piezophilic microorganisms showed great potency.Therefore,our study is aim at determining the fractionation of hydrogen isotope during biosynthesis of piezophilic bacteria under various growth pressures and among distinct fatty acids,thus we are able to explore the relationship between fractionation signature and marine depth qualitatively and quantitatively.The results of this study are supposed to provide a scientific basis on studying marine organic matter cycles,transportation,and tracing organic matter sources.In addition,our study on hydrogen isotope fractionation in Sporosarcina sp.Strain DSK25 will fill the gap in biogeochemical researches of gram-positive bacteria with respect to that of gram-negative bacteria.We studied a gram-positive bacterium Sporosarcina sp.Strain DSK25 which was isolated from marine sediments at a depth of 6500 meters(40'06.8"N,144'11.0"E)in Japan Trench.Bacterium was cultured in custom-built high pressure vessels and suitable pouches under 0.1,10,20,30,40,50 MPa.Compounds were separated using solid phase extraction depending on the polarity of themselves.For example,neutral lipids,lipids and hydrocarbon would be separated with chloroform,methanol and hexane dripping through the solid phase extraction column which was filled with samples previously.Lipids were analyzed using Gas-Chromatography-Mass Spectrometer(GC-MS).Hydrogen isotope fractionation was determined using MAT253,the experiments can be done by these procedures.we used the custom-built portable high pressure vessels which possessed a superior pressure maintaining capacity.The lipids extraction and methylation was based on the well-studied procedures so as to get the reliable result of analyzation.Taking together,our rigorous experiments and scientific theory make sure that the successive analyzation are reliable and reasonable.Compounds analyzation of Sporosarcina sp.Strain DSK25 using GC-MS showed that anteiso-branched fatty acids a15,monounsaturated fatty acids C18:1 and saturated fatty acids C16:0 changed remarkably when growth pressure rose.a15 rose sharply after30 MPa,C18:1 increased from 0.1 MPa to 20 MPa,and decreased after 20 MPa,while C16:0 dropped from 0.1 MPa to 20 MPa and kept smooth after 20 MPa.i15 and 17br almost stayed constant when growth pressures changed,though 17br went up slightly after 30 MPa.Consider that AI index calculated from a15 and i15 was correlated with growth pressure after 30 MPa,thus,the increase of a15 may be a mechanism that DSK25 used for adapting the high hydrostatic pressure.Specifically,increasing a15 and decreasing C16:0 can reduce the density of carbon skeleton,thus increase the membrane fluidity.Ascendant monounsaturated fatty acids may attribute to the adaption to lower pressure(below 20 MPa)of DSK25.Hydrogen isotope fractionation of DSK25 decreased with the lifted pressure,in other words,hydrogen isotope was getting much heavier.R~2 values of hydrogen isotope fractionation of a15,17br,i15 relative to pressure were 0.952,0.994 and 0.790,respectively.Therefore,our results deciphered the connection between lipids hydrogen isotope fractionation and pressure,this phenomenon was supposed to be ascribed to the hydrogen tunneling.High pressure enhanced the tunneling effects so that more H from NADPH flew to fatty acids,as a result,hydrogen fractionation between NADPH and fatty acids weakened,in other words,hydrogen isotope turned heavy.Hydrogen isotope fractionations of ABFA,MUFA and SFA were-214.61‰,-66.8041‰,-160.303‰,respectively.The significant difference of these three kinds of fatty acids may be ascribed to the isotope effects itself because they experienced the same processes except the different primer sources and some specific enzymes,thus,thermodynamic isotopic factor may play an important role in hydrogen isotope fractionation of different kinds of fatty acids.In conclusion,our study revealed the tight relationship between hydrogen isotope fractionation of Sporosarcina sp.Strain DSK25 in lipids biosynthesis and marine biogeochemical cycles,in the meantime,the results indicated that hydrogen isotope fractionation may be controlled by biosynthesis process and the intrinsic isotope effects.We reached our aim and answered the scientific questions proposed in this study on biogeochemistry.Therefore,our study has essential significance on exploring marine biogeochemical cycles,vertical distribution of organic matter related to hydrostatic pressure,the source,transportation,settlement of organic matter.