Robert H. Burris

Bio: Robert H. Burris is an academic researcher from University of Wisconsin-Madison. The author has contributed to research in topics: Nitrogenase & Hydrogenase. The author has an hindex of 43, co-authored 105 publications receiving 8463 citations.

Biological Nitrogen Fixation

Abstract: Biological nitrogen fixation (BNF) is the process of the reduction of dinitrogen from the air to ammonia carried out by a large number of species of free-living and symbiotic microbes called diazotrophs. BNF presents an inexpensive and environmentally sound, sustainable approach to crop production and constitutes one of the most important Plant Growth Promotion (PGP) scenarios. Here I will summarize various aspects of BNF, including the dinitrogen reduction catalysed reaction carried out by “nitrogenase” and the enzymes/genes involved and their regulation, the inherent “oxygen paradox” , the identification of diazotrophs, sustainable agricultural uses of BNF, symbiotic plant-diazotroph interactions and endophytic diazotrophs, data from the field, and future prospects in BNF.

In situ studies on n2 fixation using the acetylene

Abstract: The measurement of in situ N2-fixation rates on the basis of total nitrogen changes or 15N2 uptake is not entirely satisfactory-the former method is insufficiently sensitive and accurate, and the '5N method is time consuming, expensive, and requires a mass spectrometer. The discovery in 1966 by Sch6llhorn and Burris' and by Dilworth2 that the nitrogen-fixing complex (nitrogenase) reduces acetylene to ethylene2 suggested that the rate of acetylene reduction may be used as an index of the rate of N2 fixation. Subsequently, the measurement of ethylene production from acetylene'-' and the measurement of cyanide,8 isocyanide,9 and azide reduction8' 10 have been used to aid in laboratory studies of N2 fixation. However, the potential of the method for field investigations of N2 fixation generally has not been appreciated by limnologists, marine biologists, and soil scientists. In this paper, data obtained in experiments designed to test the feasibility of employing a simple method for measuring acetylene reduction as an index of N2 fixation in the field illustrate that the method is practical and extremely sensitive. Sites Studied.-Lake studies were performed in Lake Mendota, Madison, Wisconsin, either offshore or in mid-lake; algal samples also were obtained from Lakes Monona and Wingra. Soil studies were made in the Madison area, studies on nonlegumes on land surrounding Plummer Lake, Vilas County, Wisconsin, and studies on soybeans at the University of Wisconsin experimental farm, Arlington, Wisconsin. Gases.-Acetylene (purified grade) and a gas mixture of 02 (22%), CO2 (0.04%), and argon (78%, high purity) were obtained commercially (Matheson Co.). Field Method.-Experiments were carried out in 5.0-ml capacity glass serum bottles fitted with rubber serum stoppers. Lake samples (1.0 ml with or without prior concentration, depending on the experiment and the density of the algal population) were added to each bottle. Soil samples 0.78-cm in area and 1-cm deep were taken with a cork borer. Root nodules were detached immediately after the plants were dug, and 100-500 mg fresh weight of nodules was added per bottle. With lake samples, air was removed by flushing the liquid with the premixed gas phase for 1.5 min; then each bottle was stoppered and flushed for a further 1.5 min by introducing gas through a no. 22 hypodermic needle and venting it through a second needle. With soil and with root nodule samples, air was removed from the stoppered bottles through a hypodermic needle with a hand vacuum pump prior to flushing with the premixed gas phase. Evacuation and flushing were performed twice. Acetylene (0.5 ml) or 15N2 (1.0 ml) was then injected from a hypodermic syringe, and the samples were incubated in situ for the desired period. Generally, 18 samples could be prepared, gassed, and replaced in situ for incubation within 30 min of sampling the material. Reactions were terminated by the injection of 50% trichloroacetic acid (0.2 ml to lake water samples and 0.5 or 1.0 ml to root nodule and soil samples). A covering of RTV sealant (General Electric Co.) was applied to each stopper after gassing and after injection of trichloroacetic acid. Samples were returned to the laboratory and analyzed for total nitrogen, for ethylene production, or for 15N enrichment as required. Laboratory Studies.-Samples of lake algae were exposed in the laboratory using the methods described above or as detailed later. During incubation, the samples were continuously shaken at 300 in a water bath tinder a constant light intensity of 320 ft-c. Analysis.-Ethylene formation was detected by gas chromatography with a Varian-Aerograph model 600D gas chromatographic apparatus (H-flame ionization detector) fitted with a 9-ft long,

Methods for Growing Spirillum lipoferum and for Counting It in Pure Culture and in Association with Plants.

Abstract: Methods are described for growing Spirillum lipoferum in quantities sufficient to serve as inoculant in field trials of its associative N(2)-fixing ability with higher plants and as a source of cells for the preparation of nitrogenase, cytochromes, respiratory enzymes, etc A heavy inoculum of S lipoferum grown on NH(4) was transferred to a medium of minimal nitrogen content, and initial rapid growth at the expense of residual combined nitrogen was replaced later by slower growth on N(2) Conversion to N(2) fixation was prompt upon exhaustion of fixed nitrogen; growth on N(2) was most rapid at a pO(2) of 0005 to 0007 atm Numbers of S lipoferum can be estimated by diluting soil, crushed roots, or other material, and inoculating diluted samples into a stagnant semisolid medium Development of a characteristic subsurface layer of organisms and demonstration the these organisms can reduce C(2)H(2) are presumptive evidence that they are S lipoferum With most-probable-number tables the observations can be converted to numbers of S lipoferum in the samples The most-probable-number method indicated that numbers of S lipoferum may increase 100-fold or more in roots of maize removed from the plant and incubated for 24 h at 30 degrees C at a pO(2) initially adjusted to 001 atm

A nitrogen pressure of 50 atmospheres does not prevent evolution of hydrogen by nitrogenase

Abstract: The effect of a partial pressure of nitrogen of 50 atmospheres (5065 kilopascals ) on the hydrogen evolution reaction of nitrogenase has been investigated. Evolution of hydrogen was not blocked completely by 50 atmospheres of nitrogen in any of four experiments; rather, 27.3 +/- 2.4 percent of the total electron flux through nitrogenase was directed toward production of hydrogen. The ratio of hydrogen evolved to nitrogen fixed was close to 1:1, which implies that hydrogen evolution is obligatory in the fixation of molecular nitrogen by nitrogenase.

Cell biology and molecular basis of denitrification.

Abstract: Denitrification is a distinct means of energy conservation, making use of N oxides as terminal electron acceptors for cellular bioenergetics under anaerobic, microaerophilic, and occasionally aerobic conditions. The process is an essential branch of the global N cycle, reversing dinitrogen fixation, and is associated with chemolithotrophic, phototrophic, diazotrophic, or organotrophic metabolism but generally not with obligately anaerobic life. Discovered more than a century ago and believed to be exclusively a bacterial trait, denitrification has now been found in halophilic and hyperthermophilic archaea and in the mitochondria of fungi, raising evolutionarily intriguing vistas. Important advances in the biochemical characterization of denitrification and the underlying genetics have been achieved with Pseudomonas stutzeri, Pseudomonas aeruginosa, Paracoccus denitrificans, Ralstonia eutropha, and Rhodobacter sphaeroides. Pseudomonads represent one of the largest assemblies of the denitrifying bacteria within a single genus, favoring their use as model organisms. Around 50 genes are required within a single bacterium to encode the core structures of the denitrification apparatus. Much of the denitrification process of gram-negative bacteria has been found confined to the periplasm, whereas the topology and enzymology of the gram-positive bacteria are less well established. The activation and enzymatic transformation of N oxides is based on the redox chemistry of Fe, Cu, and Mo. Biochemical breakthroughs have included the X-ray structures of the two types of respiratory nitrite reductases and the isolation of the novel enzymes nitric oxide reductase and nitrous oxide reductase, as well as their structural characterization by indirect spectroscopic means. This revealed unexpected relationships among denitrification enzymes and respiratory oxygen reductases. Denitrification is intimately related to fundamental cellular processes that include primary and secondary transport, protein translocation, cytochrome c biogenesis, anaerobic gene regulation, metalloprotein assembly, and the biosynthesis of the cofactors molybdopterin and heme D1. An important class of regulators for the anaerobic expression of the denitrification apparatus are transcription factors of the greater FNR family. Nitrate and nitric oxide, in addition to being respiratory substrates, have been identified as signaling molecules for the induction of distinct N oxide-metabolizing enzymes.

Organic acids in the rhizosphere: a critical review

Abstract: Organic acids, such as malate, citrate and oxalate, have been proposed to be involved in many processes operating in the rhizosphere, including nutrient acquisition and metal detoxification, alleviation of anaerobic stress in roots, mineral weathering and pathogen attraction. A full assessment of their role in these processes, however, cannot be determined unless the exact mechanisms of plant organic acid release and the fate of these compounds in the soil are more fully understood. This review therefore includes information on organic acid levels in plants (concentrations, compartmentalisation, spatial aspects, synthesis), plant efflux (passive versus active transport, theoretical versus experimental considerations), soil reactions (soil solution concentrations, sorption) and microbial considerations (mineralization). In summary, the release of organic acids from roots can operate by multiple mechanisms in response to a number of well-defined environmental stresses (e.g., Al, P and Fe stress, anoxia): These responses, however, are highly stress- and plant-species specific. In addition, this review indicates that the sorption of organic acids to the mineral phase and mineralisation by the soil's microbial biomass are critical to determining the effectiveness of organic acids in most rhizosphere processes.

The Acetylene-Ethylene Assay for N2 Fixation: Laboratory and Field Evaluation

Abstract: The methodology, characteristics and application of the sensitive C(2)H(2)-C(2)H(4) assay for N(2) fixation by nitrogenase preparations and bacterial cultures in the laboratory and by legumes and free-living bacteria in situ is presented in this comprehensive report. This assay is based on the N(2)ase-catalyzed reduction of C(2)H(2) to C(2)H(4), gas chromatographic isolation of C(2)H(2) and C(2)H(4), and quantitative measurement with a H(2)-flame analyzer. As little as 1 mumumole C(2)H(4) can be detected, providing a sensitivity 10(3)-fold greater than is possible with (15)N analysis.A simple, rapid and effective procedure utilizing syringe-type assay chambers is described for the analysis of C(2)H(2)-reducing activity in the field. Applications to field samples included an evaluation of N(2) fixation by commercially grown soybeans based on over 2000 analyses made during the course of the growing season. Assay values reflected the degree of nodulation of soybean plants and indicated a calculated seasonal N(2) fixation rate of 30 to 33 kg N(2) fixed per acre, in good agreement with literature estimates based on Kjeldahl analyses. The assay was successfully applied to measurements of N(2) fixation by other symbionts and by free living soil microorganisms, and was also used to assess the effects of light and temperature on the N(2) fixing activity of soybeans. The validity of measuring N(2) fixation in terms of C(2)H(2) reduction was established through extensive comparisons of these activities using defined systems, including purified N(2)ase preparations and pure cultures of N(2)-fixing bacteria.With this assay it now becomes possible and practicable to conduct comprehensive surveys of N(2) fixation, to make detailed comparisons among different N(2)-fixing symbionts, and to rapidly evaluate the effects of cultural practices and environmental factors on N(2) fixation. The knowledge obtained through extensive application of this assay should provide the basis for efforts leading to the maximum agricultural exploitation of the N(2) fixation reaction.

The Ecology of Phytoplankton

Abstract: Communities of microscopic plant life, or phytoplankton, dominate the Earth's aquatic ecosystems. This important new book by Colin Reynolds covers the adaptations, physiology and population dynamics of phytoplankton communities in lakes and rivers and oceans. It provides basic information on composition, morphology and physiology of the main phyletic groups represented in marine and freshwater systems and in addition reviews recent advances in community ecology, developing an appreciation of assembly processes, co-existence and competition, disturbance and diversity. Although focussed on one group of organisms, the book develops many concepts relevant to ecology in the broadest sense, and as such will appeal to graduate students and researchers in ecology, limnology and oceanography.

The microbial nitrogen-cycling network

Abstract: Nitrogen is an essential component of all living organisms and the main nutrient limiting life on our planet By far, the largest inventory of freely accessible nitrogen is atmospheric dinitrogen, but most organisms rely on more bioavailable forms of nitrogen, such as ammonium and nitrate, for growth The availability of these substrates depends on diverse nitrogen-transforming reactions that are carried out by complex networks of metabolically versatile microorganisms In this Review, we summarize our current understanding of the microbial nitrogen-cycling network, including novel processes, their underlying biochemical pathways, the involved microorganisms, their environmental importance and industrial applications