Johanna Dbereiner Fifty Years Dedicated To The Biological Nitrogen Fixation Research Area

Embrapa Agrobiología, BR465, Km47, 23851-970-Seropédica, Rio de Janeiro, Brazil

Johanna Dóbereiner was born in the former Czechoslovakia in 1924, immigrated to Brazil in 1950 and became a Brazilian citizen by option in 1956. She was an extremely enthusiastic person in the field of Biological Nitrogen Fixation (BNF) which began, almost incidentally, when she was employed by the Research Department of the Brazilian Ministry of Agriculture (nowadays Embrapa) to work in the soil microbiology area. Her first publication was in 1951 and referred to the effect of cover vegetables on soil microbe populations. Together with her colleagues she identified in 1958 the first rhizosphere nitrogen-fixing bacterium, named Beijerinckia fluminensis, associated with sugarcane plants grown in Rio de Janeiro State. The persistent green color of the grass plants grown on the campus around Embrapa stimulated the young scientist to discover another rhizosphere nitrogen-fixing bacterium. This bacterium was named Azotobacter paspali (1966) since its occurrence was predominantly found on the rhizoplane of Paspalum notatum cv. batatais. Other groups later confirmed this unique association and the amount of fixed nitrogen (around 30 kg N ha" year" ) was quantified using sophisticated techniques such as the 15N isotopic dilution method.

Despite her important work with grasses, her greatest contribution occurred in the legume field, when together with other Brazilian scientists between the years 1960 and 1970, she convinced the organizers of the Brazilian Soybean breeding program to select soybean varieties dependent only on the BNF process. This approach allowed Brazil to become the World's second largest soybean producer and has saved more than a billion dollars annually in nitrogen fertilizer. It has enabled Brazil to compete successfully in the international market through the production and export of soybean where BNF, biological control and zero tillage are low cost techniques commonly applied. During this period, Johanna trained many students in the legume area investigating the growth of common beans, pastures legumes and legume trees. These former students are now research scientists working in the same field contributing to the development of Brazilian Agriculture with a strong emphasis on sustainability. BNF in legume trees was a special area stimulated by her, which began with experiments on rhizobial inoculation of the legume tree called Sabiá. Later on, one of her first generation students identified new nodule types as well as new modes of infection processes induced by rhizobia in symbioses with legume trees. The group led by Avilio Franco has taken advantage of this symbiosis and, by aggregating mycorrhiza, they developed a technology to rapidly recover degraded areas, including bauxite mining, with legume trees.

The birth of the acetylene reduction technique in the 1970s allowed the detection of very low levels of nitrogenase activity and therefore renewed the interest in BNF associated with graminaceous plants. This period coincided with the energy crisis and The Green Revolution, a system that demanded high amounts of nitrogen fertilizer application and which caused an increase in pollution of the environment. In between 1970 and 1974, Johanna published in collaboration with colleagues at home and abroad many papers measuring nitrogenase activity of Paspalum notatum and other pasture grasses as well as sugarcane. With the participation of the late J. Day, she isolated from Digitaria decumbens, a micro-aerophilic nitrogen-fixing bacterium resembling Spirillum lipoferum, subsequently reclassified as Azospirillum lipoferum in collaboration with Dr Noel Krieg. This isolation was made possible by the development of a nitrogen-free semi-solid medium called NFb (Fb stands for Fábio Pedrosa) that reproduced the soil oxygen gradient. Another Azospirillum species, named A. brasilense was also identified at that time.

Two members of the original BNF grass team, my wife Vera and I, began working with her in late 1976 when the BNF work on graminaceous plants reached its peak. We learnt how to isolate nitrogen-fixing bacteria, not only the known ones but also new species that could be identified based on those "ten commandments" published during the Brazilian BNF Conference, held in Rio de Janeiro, in 1987. These "ten commandments" helped to isolate and identify new nitrogen-fixing bacteria including the diazotrophic endophytes, which colonize the interior of plant tissues. These were the species Azospirillum amazonense in 1983, using the semi-solid LGI medium (I stands for Ivo), and the endophytes Herbaspirillum seropedicae in 1986, Gluconacetobacter diazotrophicus (former Acetobacter diazotrophicus) and Herbaspirillum rubrisubalbicans in 1996. All these bacteria are known to have an important role in association with graminaceous plants, especially for some sugarcane varieties that are reported to obtain between 30 and 50% of their N from BNF. Johanna's determination was that these new bacteria should be explored as much as possible, therefore, she encouraged many scientists around the world to work with them and today many of these microrganisms are subjects of discussion at this conference.

Johanna's contribution to Science can be measured by the several international prizes that she received, including election to the Vatican Pontifical Academy of Sciences. She was always proud to attend its annual meetings. In addition, she received the title of "Honoris Causa" from the University of Florida (1975) and the Federal Rural University of Rio de Janeiro (1979). She received many other national and international prizes that are listed in her brief biography written by Avilio Franco and Robert Boddey published in Soil Biology and Biochemistry, special issue (29: ix-xi, 1997). She was the most cited female Brazilian scientist by the international community and the seventh most cited Brazilian scientist in a search prepared in 1995 by the Folha de Sao Paulo Newspaper. Johanna has contributed around 190 scientific publications (national and international) in addition to two books, several chapters, more than two hundred abstracts and was an invited speaker at over 50 international meetings. Over a fifty-year scientific career, she averaged 3.7 papers per annum.

Training was another of Johanna's strong characteristics, and today more than one hundred students are learning how to do research at Embrapa. At least three generations of scientists have already trained under her guidance and are now transferring her work philosophy and ideas on the Biological Nitrogen Fixation theme to new students in Brazil and overseas. She was a woman with a very strong personality, sincere and dedicated to her work. Despite her rigorous treatment of scientific aspects, she behaved always like a mother to all students and trainees in our center. In Brazil, she was known as the "soil doctor", although for those that had the privilege to work with her we regarded her as our "Aunt Johanna".

Johanna never agreed that personal names should be given to a bacterium. However, because of her important contribution to the area, two diazotrophs have been named in her honour: Azospirillum doebereinerae (Hartmann's group) and Gluconacetobacter johannae (Caballero-Mellado's group). For this, and much more, future scientists working in the field of biological nitrogen fixation will remember her name.

Supported by Embrapa, Pronex II, FAPERJ and PADCTII

INORGANIC NITROGEN COMPLEXES: A CANADIAN DISCOVERY

The discovery of Nitrogenpentaamminerutheniumchloride [Run(NH3)5N2]Cl2 - the first nitrogen complex by A.D. Allen and C.V. Senoff in 1965

In the early 1960s, A.D. Allen and his coworkers at the University of Toronto were studying the ammine complexes of ruthenium. They routinely prepared these complexes by the reaction between ruthenium trichloride and zinc dust in concentrated ammonia, followed by treatment with hydrochloric acid to give [Run(NH3)6]ZnCl4. Mild oxidation and treatment with hydrochloric acid then gave [Ru(NH3)5Cl]Cl2. In early 1965 they became intrigued by the report by some Russian workers of the direct preparation of [Rum(NH3)5Cl]2+ by refluxing an aqueous solution of K2[RumCl5H20] with hydrazine monohydrochloride. On repeating this preparation but using hydrazine hydrate instead of the hydrochloride, they obtained a different product in the form of a yellow solid which proved very difficult to characterize. Eventually by chemical analysis, infrared spectroscopy of the compound and its fully deuterated form, decomposition with sulfuric acid to give N2, and other experiments they came to the conclusion that it must be [Run(NH3)5N2]Cl2 and with some trepidation submitted a paper to this effect to Chemical Communications. The idea that the very unreactive N2 molecule could form a complex with a transition metal was something that at the time nobody had considered might be possible. Finally, at the end of 1966 after having some difficulty obtaining suitable crystals, an X-ray diffraction study was completed and the composition of the compound and its structure were fully confirmed. Many more nitrogen complexes were then rapidly prepared particularly when it was discovered that many could be prepared by the direct reaction of a transition metal complex with N2 under mild conditions. Today many hundreds of inorganic nitrogen complexes of almost all the transition metals are known.

References

Allen AD, Senoff CV (1965) Chem. Cornmun. 621

Allen AD, Bottomley F (1965) Accts. Chem. Res. Vol. 1, 360

The above summary was generously contributed by:

Professor R.J. Gillespie Department of Chemistry McMaster University 1280 Main Street West Hamilton, Ontario, ON L8S 4M1 Email: [email protected]

Website: http://www.chemistry.mcmaster.ca/faculty/gillespie

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