Measurements And Controlling Factors Of Key Cellular Metabolite Precursor (5-aminolevulinic Acid) In Marine Phytoplankton And Bacteria

5-aminolevulinic acid(ALA)is the first common precursor in the biosynthesis of tetrapyrroles(hemes,chlorophylls,vitamin B,2)and porphyrins in biology.ALA widely exsists in micro-organisms,plants and animals.Recently researchers measured soluble ALA in waters,body fluid or fermentation liquid.There are,however,no measurements were made for celluar ALA in phytoplankton or bacteria in the ocean.Therefore,we for the first time developed a method of measuring ALA in the cells of marine phytoplankton and bacteria,and successfully applied it in field samples taken from coastal waters and hydrothermal fluids.By conducting manipulative experiments,I determined several key factors controlling the ALA metabolism.The thesis is mainly divided into three parts:1)I established the method of measuring cellular ALA,and then applied it in field samples.I for the first time identified that particulate ALA exsists in natural waters including the Tong'an Bay and shallow water hydrothermal vents off Kueishan Tao(Taiwan,China).2)I conducted manipulative culture experiments of representative micro-organisms(Nannochloropsis oceanica and Synechococcus sp.WH7803)to compare the synthesis and dynamics of cellular ALA in eukarotes and prokaryotes.3)By adding extra organic molecules in the culture medium,I observed the dynamics of cellular ALA and metals in Nannochloropsis oceanica.My major results are shown in the following:A method by using High Performance Liquid Chromatography(HPLC)with fluorescence detection was established for measuring 5-aminolevulinic acid(ALA)in biological cells.First,I used lysis solution[5%MeOH,1mM(0.01%,pH 3.5)HCl]to break the cells;Second,I used chloroform to extract hydrophobic organics;Third,a InertSustain C18 column(150mm × 4.6mm,5?m)in HPLC was used for separating ALA[mobile phase:methanol/water/glacial acetic acid[(500/500/10 by vol)].Linear correlation was obtained with the ALA concentrations of 0-20 ?g/L,and the recovery was 98.7%-100.3%with extra addition of ALA in field samples.The method was applied to measure cellular ALA in field samples taken from Jiulong River Estuary and river watershed,Tong'an Bay,Pearl River Estuary,Minjiang River and shallow water hydrothermal vents off Kueishan Tao(Taiwan,China).The results showed that the concentrations of ALA in most samples were lower than the detection limit(0.035?g/L).ALA was identified with high concentrations in the samples taken from the Kueishan Tao(Taiwan,China)and the Tong'an Bay,which might be attributable to specific phyton species and abundance.The samples from Kueishan Tao(Taiwan,China)White Smoker were reported with the dominant by sulfur-reducing Nautilia and Thermococcus(71.9×108 cells/L),while the Yellow Smoker,sulfur-oxidizing Thiomicrospira and Eurychaeota(18×108 cells/L),respectively(Zhang et al.,2012).The highest concentration of ALA was observed in the Kueishan Tao(Taiwan,China)(0.35nmol/L).The Tong'an Bay was dominated with the red tide specie Akashiwo sanguine(69.1×104 cells/L),and the concentrations of ALA could reach as high as 39.9nmol/L.The laboratory culture experiments showed a dynamic variation of cellular ALA along with culture time.In the culture of Nannochloropsis,ALA increased rapidly in the early culture period(cell abundance:(0.6±0.1)x 106 cells/mL),with maximum value(413±11 ?mol/L)at the 2nd to 3rd day and then decreased gradually,finally tends to be stable(3 ±0.5?mol/)at the 9th to 10th day when the cell abundance was(2.8±0.2)×106 cells/mL.In the culture of Synechococcus,the concentrations of ALA was lower than the detection limit at the beginning(cell abundance:(0.7±0.05)×106 cells/mL),and during the late exponential growth(cell abundance:(3.7±0.4)×106 cells/mL),ALA increased rapidly,then reached the maximum value(32878±4.7?gmol/L).We found that the peak value of cellular ALA in Synechococcus is 100 times as high as that in Nannochloropsis.The result might be attributed to the biotype(Kipe et al.,1980;Lin D et al.,1989)as:Nannochloropsis is eukaryotic with the main pigment of chlorophylls and the C5 synthetic pathway of ALA,while Synechococcus is procaryotic with phycoerythrobilin as main photosynthetic pigment and the C4 synthetic pathway of ALA.Sasikala(1994)reported that anoxygenic photosynthetic bacteria could accumulate high levels of ALA in the medium.I conducted five experiments of culturing Nannochloropsis,as in the following:(a)f/2 medium;(b)f/2 medium+Glucose;(c)f/2 medium+Glycine;(d)f/2 medium+Glutamate;(e)f/2 medium+ALA.The concentrations of ALA all behaved dynamically in all the experiements.ALA increased rapidly at the beginning,and then decreased,finally tended to be stable at the middle exponential phase.Relative to the control,the cell abundance and the cellular ALA in the rest groups were significantly increased.The cell abundance was up to 20×106 ce11s/mL with glutamate addition.The concentrations of cellular ALA were highest(84.8mmol/L)with ALA addition.The concentrations of cellular trace elements were highest in the control,and lowest with the glutamate addition.Summarily,a protocol was established successfully for measuring cellular ALA in natural waters and algal cells.I observed that the concentrations of ALA varied largely in different waters.In addition,I observed that the medium conditions play a key role in the synthesis of ALA and absorption of trace metals.