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HYDROGENASE BACTERIAL ENZYME ACTIVATING MOLECULAR HYDROGEN ( hydrogenase-bacterial-enzyme-activating-molecular-hydrogen )

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206 M. STEPHENSON AND L. H. STICKLAND methanefromamixtureofcarbondioxideandhydrogenandsimultaneously toreducesulphatetosulphideattheexpenseofhydrogen[cf.Sohngen,1910]. This led to the conception that carbon dioxide and sulphate were acting as hydrogenacceptorsinasystemwheremolecularhydrogenwasthehydrogen donator,anditseemedlikelythatbacteriawerepresentinthemixedculture capableofactivatingmolecularhydrogen.We thereforecentrifugedamixed culture which had been grown for some 20 subcultures on formate as sole source of carbon, and tested the washed suspension in Thunberg tubes with methyleneblueandgaseoushydrogen. Forthispurposehydrogenfroma Kipp's apparatus was passed through three wash-bottles containing silver nitrate(fortheremovaloftracesofvolatilearseniccompounds),pyrogalloland water; the last bottle was connected with a 3-way tap (oblique bore) so that the Thunberg tube couldbe alternately evacuated and filedwith hydrogen. The tube was evacuatedthoroughly to remove dissolvedoxygen, thenfiled withhydrogenandagainevacuatedandfiled;finallythetapwasclosedand thetubewasplacedinabathat450andshakenfor1minutetoensuresatura- tionoftheliquidwiththegas;thereductiontimewasmeasured. Result: 1c. 1/25,000 methylene blue, 1cc. phosphate bufferPH 8-0, 1c.bacterialsuspension,inhydrogen 15 1c.1/25,000methyleneblue,1c.phosph&tebufferpH 8-0, 1c.bacterialsuspension,invacuo . ... ... ... Controlwithboiledsuspensioninhydrogen . ... ... Reduction time (mins.) > 70 >70 Having shown that our m ixed culture contained organisms capable of reducing by means of molecular hydrogen, we diluted and plated on ordinary brothagarandincubatedtheplatesanaerobically.We pickedofanumber ofcolonies,grewthecorrespondingculturesonbroth,centrifugedandwashed theorganisms,andtestedthesuspensionsasabovewithhydrogenandmethylene blue. Inthese and alsubsequent experiments 1c.of1/5000methylene blue in a total volume of 4 c. was used, and the time was taken when reduction hadproceededto75%,becausetherateofreductionappearstofalofafter thatpoint. Result: Culture No. 70 74 79 80 With hydrogen 5i >120 > 120 5j Without hydrogen > 120 > 120 > 120 > 120 Reductiontime(mins.) rA Thisshowsthatthereisaclearlymarkeddifferencebetweenorganismshaving the property of reducing by means of molecular hydrogen and those lacking thepower. Culture70wasre-platedandgavethestrainknownas111. Characteristics of organism 111. This organism is a short, motile, non- sporing, Gram-negative bacillus, growing both aerobically and anaerobically.



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