Isolation And Identification Of Hydrogen-producing Photosynthetic Bacteria And Hydrogenase Phylogenetic Analysis

The energy crisis in the70s of last century forcing humans began to look for new energy.Hydrogen was thought as one of the most development potential “future energy”, and in manycountries, the scientists invest much capital in explore various hydrogen productionapproaches and the application of hydrogen. Microbial hydrogen production has attractedmuch attention and become the popular issues in numerous hydrogen production approaches.In these hydrogen-producing microbes, photosynthetic bacteria hydrogena-producing becomethe research hot because of it’s advantages such as high efficiency using the substrate, thewide range of spectrum utilization, the high utilization efficiency of light energy and dose notproduce oxygen and so on.In the present study, total28strains of photosynthetic bacteria were identificated fromnear area of Yangling. The capacity of hydrogen production of these photosynthetic bacteriaand the evolution of hydrogenases of several bacteria groups were investigated. The mainresults obtained here are as follows:1. Isolation and identification of photosynthetic bacteria: Collecting sludge fromthe Weihe River, artificial lake, industrial wastewater, sanitary sewage around Yangling, weget more than40strains of photosynthetic bacterias after preliminary and recurrence isolation.Using16SrDNA sequencing, Gram staining, colonial morphology and bacterial morphologyobserving, determination of growth curve and test specific absorption peak of photosyntheticbacteria, total28strains of bacteria grouped into three broad categories: Rhodopseudomonaspalustris, Rhodobacter sphaeroides, Rhodobacter capsulatus were identificated. And we haveFreeze-drying preserves these bateria.2. Determination of hydrogen production capacity: we measured the preliminaryhydrogen production of these strains using simple hydrogen detection system and found thatthey have different ability of hydrogen producing, in which Rhodobacter sp. CQ3, Rhodopseudomonas palustris G09, Rhodopseudomonas palustris LYJY2, Rhodopseudomonaspalustris E, Rhodobacter sphaeroides AH7and Rhodobacter capsulatus WGSW，can producehydrogen up to30mL in1L culture medium during24h. Among these bacteria,Rhodopseudomonas palustris E, an efficient hydrogen-producing strain, can produce as manyas46mL hydrogen in1L culture medium during24h.3. Phylogentic analysis of photosynthetic bacteria hydrogenase: In photosyntheticbacterias, hydrogenase is the key enzyme to regulate the capacity of hydrogen production andcatalyze the reversible reaction of hydrogen oxidation, therefore hydrogenase play animportant role in energy metabolism. We downloaded37hydrogenase protein sequences ofPhotosynthetic bacteria, Cyanobacteria, Green alga from NCBI database in this paper. Takingadvantages of bioinformatics methods, we studed amino acid sequence, properties of theprotein, transmembrane structure, subcellular localization and evolutionary classification. Theresults obtained here indicated that a conserved site GC island exist in [NiFe]-hydrogenasesmall subunit, and that the membrane bound state of [NiFe]-hydrogenase small subunit isanchoring on mitochondrial membrane. In spite of the [FeFe]-hydrogenases are smaller, buttheir sequences are highly conserved and all are unstable proteins. Through the evolution treeanalysis which build by MEGA5.10, we considered that [NiFe]-hydrogenase,[FeFe]-hydrogenase in photosynthetic bacteria, blue-green algae, green alga are evolved fromthe hydrogenase of Rhodospirillum centenum SW, and then these two containing metal atomsenzymes undergo respective evolution.