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1993 Special Awards Winners
State Science and Engineering Fair of Florida

April 14, 16, 1993
Central Florida Fairgrounds, Orlando

animated globe FDACS Senior / FDACS Junior / FAITC Senior / FAITC Junior

Awards Given By The Florida Department of Agriculture and Consumer Services
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Senior Section

Neil A. Hattangadi
Region: Orange

Category: Botany
Awards: Plaque
Project Title: Nitrogen Fixation with Non-Legumes in Two Phases
Other Awards: First Place in Category. Special Award from Florida Academy of
Sciences-One-Year Journal Subscription to Florida Scientist. Special Award from National
Aeronautics and Space Administration (NASA)-Certificate of Outstanding Achievement.

"This is a formal letter of gratitude for the great plaque you gave at the State Science Fair. As a recipient of your special award, I can assure you that it truly promotes research in the agricultural field, one of the most crucial facets of science. I sincerely hope you can continue to distribute this wonderful award in the future."

Neil Hattangadi
First Place-Senior Botany

Project Synopsis
This is here for your information only and is not intended for reuse!

Nitrogen Fixation with Non-Legumes in Two Phases

by Neil Hattangadi

Nitrogen fixation is the reduction of molecular nitrogen into consumable ammonia through prokaryotic organisms. This first phase deals with such a genus, collectively termed rhizobia, which have hitherto functioned only in the root nodules of symbiotically related leguminous plants. The effectiveness of artificial modulation processes comprised of various physical treatments and enzymatic incubations, with cellulose and pectinate, of the roots along with Rhizobium trifolii and R. loti bacterial inoculants was tested. The "synthetic" nodules of nonleguminous Oryza sativa rice and Zea mays maize seedlings were compared with those of leguminous Trifolium repens clover and Lotus corniculatus birdsfoot-trefoil.

In Phase I, enzymatic incubation was the most effective wounding treatment, as it best dissolves pectins in the roots' cell walls allowing bacteria to enter the surface. Only rotten stone worked as a physical treatment since it is the only fine abrasive; the use of this wounding has never been documented before, from the literature surveyed by the experimenter, and represents a break-through from costly enzymes and extends the applicability of the procedure for practical agriculture. in Phase IA, barriers in symbiosis between unrelated clover and R. loti were overcome, showing the extension of cross-inoculation groups within the legume family through the elimination of the pectin proteins that bind to specific polysaccharides in the bacteria. Also, the synergistic effects of the bacterial mixture of R. loti and R. trifolii made it the best innoculant. Polyethylene glycol was found to be compulsory for non-legume nodulation, as it dissolves holes in the roots' cell walls, allowing for greater and faster bacterial uptake. After splitting the nodules, no leghemoglobin was detected through light microscopy, resulting from the non-legumes' lack of nodulin genes to stimulate rhizobial nitrogen fixation. Also, the non-legumes seemed to undergo the effects of nitrogen deprivation through chlorosis, demonstrating that no nitrogen compounds were provided for them. Thus, the nodules in non-legumes were both smaller and less numerous than those of the legumes, and were unable to function.

In Phase II, an alternative to the symbiotic nitrogen fixation for nonlegumes was found through free-living cyanobacteria. The original intent of the experimenter was to create various nitrogenase-derepressed mutants through ethyl methanesulfonate (EMS) mutagenesis of the strain Anabaena variabilis, to overcome nitrogenase repression resulting from a series of metabolic processes in ammonium assimilation; but due to the lack of resources he was forced to choose mutant strains already developed, through few of their properties in hydroponic systems were known. Based upon documented nitrite and nitrate reductase activity, a desirable nitrogenase-derepressed mutant strain was selected from about 10 mutants and obtained from the University of Florida in Gainesville. Hydroponic systems of the following variations were set up with rice and corn seedlings: 1 mg parent strain/100 mL medium, 1 mg mutant/100mL medium, 1 mg mutant/100 mL medium + 3mM (NH4)2SO4, 5mg mutant/100mL medium, 5mM (NH4)2SO4, and unamended. That mutant strain commenced nitrogenase activity at 6.444 ppm C2H4 hour -1).1 mg-1 as determined through acetylene reduction and a gas chromatograph versus 5.817 by the parent. In addition, it was found that the parent did not excrete ammonium, and when ammonium levels were detected with Nessler's reagent at the third and fourth weeks, it was due to their decomposition. The mutant did excrete NH4 and it did not show any assimilation until the concentration reached approximately 0.5mM, when 0.02umoler-glutamyl hydroxamate 15 min-1 1 mg-1 were detected, the limit of the assay procedure. The mutant strain's nitrogenase activity did decrease slightly as the NH4 levels increased, an undesirable characteristic attributed to an increase in the production of vegetative cells with a reduction in heterocysts. Since the mutant strain recorded little to no glutamine synthetase activity, it gradually reduced density to about 55% of the original, as measured by chlorophyll determination. It did show a statistically significant increase in plant biomass over the parent strain's and unamended systems, but less than that of the chemical (NH4)2SO4 supplements.

These results have a critical application in that they provide a low cost alternative to expensive, environmentally hazardous chemical fertilizers.

Junior Section

Allison Buchholz
Region: River: East
Awards: Plaque
Project Title: Flea-Free, Killing Fleas Using Nontoxic Products
Other Awards: Second Place in Category. Special Award from Osceola Regional Science and Engineering Fair-Polaroid Camera.

Awards Given By Florida Agriculture in the Classroom
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Senior Section

Nasrin Sultana

Category: Zoology
Award: $25 Cash Award and Certificate
Project Title: The Role of Juvenile Hormones During Egg Development of Mosquitoes
Other Awards: Second Place in Category

Junior Section

Alan Pendergrass
Brevard: Intracoastal

Category: Botany
Place Award: $25 Cash Award and Certificate
Project Title: An Environmentally Friendly Pesticide for Fire Ants
Other Awards: First Place in Category. Special Award from Suzdon Farms Award Fund -
$25.00 Cash Award.

"My name is Alan Pendergrass, and I am writing this letter to thank you very much for the
special award which I received from you at the SSEF. I want to thank you for your time and
efforts in coming to the Florida State Science/Engineering Fair. Again I appreciate it very
much. Thank you."
Alan Pendergrass

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