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CHARGE NUMBER: 1902' PROJECT TITLE: Microbial Technology PROJECT LEADER: V. S. Malik V~ PERIOD COVERED: July 1-31, 1'981 DATE OF REPORT: August 7, 1!981 A. GROWTH STUDIES WITH ATCC1 (b. Chadick) Growth curves were.performed with ATCC1 in pasteurized SEL and'TSB with 1% KN03. The rate of inoculum used was 10%. All flasks were incubated at 45, 55 and 60'C. Plate counts and denitrification were analyzed at two hour intervals for a period of twenty-four hours. The results were as expected with better growth and denitrification occurring at 45 and 55°C with less activity observed at 60°C in both the SEL and TSB. Growth of ATCC1 was also compared in nitrate broth, TSB with KN03 and pasteurized SEL. It appears that 45 and 55°C are more favorable temperatures. Growth appears equal in nitrate broth and Trypticase Soy Broth with 1% KN03. B. ISOLATION'OF ORGANISMS (D. Chadick) New isolates have been obtained'from SEL that was being actively denitriif ied. These isolates have been labeled PM 5 to 10. A11 of the isolates grow and denitrify more efficiently at 60°C versus 50°C. When plated on agar with 1% KN03 and incubated at 60°C. PM 6, 7, 8, 9 and 10 appear similar. PM 5 looks different from any of the other five. These organisms have al~so been tested with the APIE identification strips with ATCC1 as control. At 50 and 60°C, all of the isolates reacted with at least one compound on the strip in 48 hours. ATCC1 had the most activity. When the strips were incubated at 37°C, only ATCC1 had any activity after 48 hours. All of the new isolates show greater denitrification at 60'C versus 50°C. C. PLATING OF TANK H (D. Chadick) The plating of Tank H in the pilot plant is conti'.nuing. The viable cell numbers are remaining in the range of 106 to 107 bacteria per ml as judged by plating. A single ATCC1-like organism predominates as judged by the visual examination of the phenotype of the colony. D. DENITRIFICATION START-UP (V. S. Malik). The 500-liter fermentor containing pasteurized 10% SEL was inoculated with 1% inoculum of logphase cells of ATCCI. Onset of denitrification was monitored by automated control of pH. Within 16 to 22 hours, denitrification started as observed by the rise in pH. The pH was maintained constant using SCL as an acid'. The start-up process still needs perfection.

-2- C C I i E. AGAROSE GEL ELECTROPHORESIS (D. Naugle, V. S. Malik) Fragmented Bacteriophage lamda DNA was analyzed~by agarose gel electrophoresis. The DNA was separated as expected. The technique will be tested~to see if it can give a characteristic fragmentationn pattern of four specific organisms that are of importance in denitrification of tobacco extracts. F. MEDIA STUDIES AND STARTING UP OF DENITRIFICATION (P. Oglesby, E. Bravo) Two different types of media were compared--TSB media and NY media which contains Nitrate Broth, Yeast Extract, KND3, MgS04 and pH adjusted 8.4. TSB was superior at all inoculum levels. Two different types of inoculum in TSB media were also compared. The first inoculum came from Tank H, which had been stored in 55 gallon drums for several weeks. The second inoculum came from phauxostat. Inoculum from the phauxostat denitrified at a considerably higher rate than the stored inoculum. Experiments in the 14-liter fermentors were done to test denitri'fication by ATCCI as well as mixed' culture. This exper iment was not conclusive in showing which inoculum would be a better denitrifier. However, ATCCI grew faster and denitrified tobacco extracts. A denitrification start-up procedure which requires no additives is being perfected. Nine liters of tap water with one liter of SEL is steam sterilized, cooled to 50° and then inoculated with 1% inoculum (ATCC1). Denitrification starts taking place after 16 to 20 hours usiing pH as a guide of denitrification. Each fermentor was set up as a phauxostat so that when the pH increased above 7.2 SEL would be automatically added and pH would be maintained constant. G. STORAGE OF CULTURES (D. Naugle) The experiment on the storage of mixed cultures has been continued. Stored cultures were tested over a six week period. It has beenn demonstrated that cultures stored in 50% glycerol in a freezer remain viable and'maintain their ability to denitrify over this period. The cultures on slants and stabs stored at room temperature and at 4°C also remained viable during the experiment. Experimentation has shown the effect of glycerol on samples. A sample containing 50% inoculum and 50% glycerol' when plated shows less growth than a sample of 75% inoculum and 25% glycerol.'F, Further work is being done to determine the effect of the different concentrations of glycerol on long-term storage.