Selenate Reduction And Selenium Enrichment Of Tea By The Endophytic Herbaspirillum Sp.Strain WT00C

Herbaspirillum sp.WTOOC,isolated by our laboratory from the wild tea plant,is a tea-specific,gram-negative,endophytic bacterium.Its microbiological properties,physiological and biochemical properties,classification and identification as well as the growth-promoting of tea plants have been deeply studied,and its genome has also been sequenced.Genomic survey has found that Herbaspirillum sp.WT00C has an intact pathway for selenocompound metabolism.To confirm whether this tea-specific endophyte really has the ability to reduce selenate and explore if it can.improve selenium enrichment of tea,we paid our attention to study the selenate reduction,the optimal condition of selenate reduction,the product component of selenium reduction as well as selenium enrichment of tea by Herbaspirillum sp.WT00C.The main conclusions obtained by hard work for three years are as follows:1.Tea-endophytic Herbaspirillum sp.WT00C showed a strong selenate tolerability.When Herbaspirillum sp.WTOOC was cultured in LB medium containing selenate,its growth was severiously inhibited by rising selenate concentrations Bacterial growth was completely inhibited during 12 hours when selenate concetration in the medium approched to 200 mM.However,the bacterium began to grow after 12hours and approched to the growth level of the bacterium growing in the medium without addition of selenate at 48 hours.The detection of viable bacteria found that the bacterial cells in 12-hour cultivation were still alive,suggesting that Herbaspirillum sp.WTOOC strongly tolerated against selenate.The optimization of cultivation conditions also found that selenate reduction was more effective when the bacteriun was cultivated with selenate at 37℃ with shaking at 200 rpm.2.Tea-endophytic Herbaspirillum sp.WTOOC effectively reduced selenate to red elemental selenium.When Herbaspirillum sp.WT00C was cultured in the medium containing sodium selenate,this bacterium was able to reduce selenate effectively to form red elemental selenium,and turn bacterial colonies on the solid plate or the liquid medium into red.strong selenate tolerance.Transmission electron microscopy(TEM)and scanning electron microscopy(SEM)revealed that Herbaspirillum sp.WT00C reduced selenite(Se6+)to form elemental selenium(Se0)nanoparticles(SeNPs).Those Se-nanoparticles(<10 nm)on the surface of bacterial cells aggregated to form selenospheres,and then selenospheres grew biger and biger from less than 10 nm to more than 200 nm via fusion each other.Selenospheres eventually crystallized under the special condition to form selenoflowers with a diameter of 1μm.X-ray energy dispersive spectrometer(EDS)showed that elemental selenium mainly located in these nanoballs.X-ray diffractometry(XRD)analysis further confirmed that Herbaspirillum sp.WT00C indeed reduced selenate(Se6+)to elemental selenium(Se0).The red selenospheres were finally purified by suger density gradient centrifugation.Biochemical assays showed that selenospheres contained proteins but no carbohydrates or lipids.When bacterial cytoplasm and cytoderm were respectively cultivated with selenate under the same condition,bacterial cytoplasm reduced selenate more effective than the cytoderm.This result together with other results suggested that selenate reducation mainly occurred inside bacterial cytoplasm and formed SeNPs.SeNPs were then secreted outside of bacterial cells to form selenospheres by aggregation.Selenospheres gradually grew bigger and bigger via fusion each other,and eventually crystallized to form selenoflowers.3.Tea-endophytic Herbaspirillum sp.WT00C effectively enhanced selenium enrichment of tea.As Herbaspirillum sp.WT00C displayed strong capability of selenium reduction,we speculated that this bacterium might improve selenium enrichment of tea when it lives in tea plant.To test this hypothesis,we inoculated Herbaspirillum sp.WT00C into tea plants through two different inoculation modes,injection and soaking tea-cutting.Tea seedlings were planted in the soil containing 0-3.5 mg/kg selenate and grew under natural environment.Samples including root,stem and leaves were collected at different periods of time,and selenium content was measured.Those results obtained from either injection or soaking tea-cutting demonstrated that Herbaspirillum sp.WTOOC significantly enhanced selenium enrichment of tea.In the injection groups,selenium content increased in roots,stems and leaves with the process of time,indicating that tea plant was able to enrich a certain amount of selenium by itself.Nevertheless,the selenium content of roots and stems in experimental groups had no significant difference as compared to the control groups.In contrast,the three experimental groups showed significant enrichment of selenium in tea leaves at different time periods as compared with those for control groups,When the tea seedlings were planted in soil containing 0.622,1.5 or 3.5 mg/kg selenate,the selenium contents of tea leaves at 150 days were 1.182,11.224 and 36.236 mg/kg for three experimental groups and 0.454,4.060 and 15.154 mg/kg for the control groups respectively.When the soaking tea-cutting mode was used,the selenium contents of tea leaves at 150 days were 4.586,18.394 and 38.704 mg/kg for the experimental groups,and 1.967,7.110 and 15.228 mg/kg for the control groups.In both cases,the selenium content of tea in each experimental group was 2-fold of that for the control group.Statistical analysis found the difference between each control group and experimental group was significant(P<0.001).Our studies not only demonstrated that Herbaspirillum sp.WT00C was capable of reducing selenate but also improved that this bacterium was able to improve selenium enrichment of tea.Meanwhile,it was the first time to find that selenospheres increased their volume via fusion each other.In addition,production of tea seedlings containing Herbaspirillum sp.WTOOC through incubating bacterial cells into tea plants by soaking tea-cuttings was a simple and easy technique,which is suitable for the usage of a large area in tea plantation.Our study on enhancing selenium enrichment of tea by Herbaspirillum sp.WTOOC not only provides a novel strategy for tea farmers to produce se-enriched tea but also offers technical support.