Downloaded from www.microbiologyresearch.org by IP: 54.70.40.11 On: Fri, 02 Aug 2019 23:59:26 Journal of General Microbiology (1986), 132, 1797-1803. Printed in Great Britain 1797 Autotrophic Growth and Nitrogen Fixation in Derxia gummosa By H. N. RAVI SHANKAR,’ I. R. KENNEDY2 AND P. B. NEW1* Department of Microbiology, G08, Biochemistry Building, University of Sydney, New South Wales, Australia 2006 Department of Agricultural Chemistry, University of Sydney, New South Wales, Australia 2006 (Received 4 November 1985 ; revised 19 February 1986) Initial attempts to grow N2-fixing Derxia gummosa autotrophically on H2, C02, O2 and N2 in a closed system yielded variable results. Poor growth was found to be due to rapid O2 depletion and the requirement for an agar surface. In a closed system, C2H2 reduction assays could not be carried out due to complete consumption of H2. Hence a flow-through culturing technique was developed to supply gases at a constant partial pressure and to perform C2H2 reduction assays in a continuous flow system. Hydrogenase of autotrophic D. gummosa was not inhibited by C2H2, even at 0.5 atm, and the K,,, of hydrogenase for H2 was approximately 0.15 atm. The effects of O2 and H2on C2Hz reduction were examined, using the flow-through assay system. The rate of C2H2 reduction decreased below 0.074 atm H2,suggesting that ATP and reductant supply were limiting the nitrogenase activity. INTRODUCTION Derxia gummosa, an obligate aerobe of the family Azotobacteraceae, is described as a ‘typical awkward nitrogen-fixer’ (Hill & Postgate, 1969) because its growth under N,-fixing conditions is variable and unreliable (Tchan & Jensen, 1963; Hill & Postgate, 1969). Colony variations (massive and thin types), production of tenaceous gum, an apparent need for microaerophilic conditions during N2 fixation and some unknown factors have made the study of N2 fixation by D. gummosa difficult. Autotrophic growth and N2 fixation were studied as two independent traits until the discovery of a N2-fixinghydrogen bacterium (Ooyama, 1971). Later, many H2-oxidizers were proved to fix N2 and many N2-fixerswere shown to be capable of H2 oxidation. Some of the latter were also capable of autotrophic growth (Hanus et af., 1979). Pedrosa et al. (1980) were the first to report that D. gummosa can grow autotrophically by aerobic H2-dependent C02 incorporation. In an autotrophically growing, N2-fixing D . gummosa, electrons donated by H2 will be channelled to N2 for N2 fixation, to C 0 2 for carbon fixation and to O2 as terminal electron acceptor for bioenergetic purposes. Hence the study of autotrophic N2 fixation should prove rewarding in providing information on the regulation of electron flow. We have attempted to establish the optimum gas proportions and other cultural conditions for autotrophic growth and N2 fixation by D . gummosa. Initial attempts to grow the organism autotrophically were not reproducible and growth was very scanty whenever it occurred. This was shown to result from fast O2 depletion in the sealed growth chamber. Here we describe a continuous gas flow system giving consistent autotrophic growth of D. gummosa on agar surfaces, with provision for a continuous flow-through C2H2 reduction assay. The results obtained with this system provide information on reasons for the difficulty experienced in growing D . gummosa. METHODS Organism. Derxia gummosa strain 1-15 was originally obtained from H. L. Jensen, Statens Planteavls Laboratorium, Lyngby, Denmark. Media andgrowth conditions. The medium of Tchan & Jensen (1963) for D. gummosa consisting of (g per litre of distilled water): K2HP04, 0-5; MgS04.7H20, 0.25; NaCl, 0-1; FeSO4.7H2O, 0.1; CaCl,, 0-1; Na2MoOo.2H20, 0001-3051 0 1986 SGM