The development of insecticides resistance among many insect species and the ecological damage occasionally caused by the lack of specificity in the toxic effects of insecticides have provided the impetus to seek alternative methods of insect control. This observation led to the development of bioinsecticides based on the insecticidal action Bacillus sphaericus (Bs), Bacillus turingiensis (Bt). The discovery of biolarvicidal actions of Bacillus thuringiensis and Bacillus sphaericus opened a new perspective for insect control. In the first part of the study was initiated to determine a suitable fermentation medium formulation and optimal fermentation conditions for large scale, low cost production of Bs. Bs 2362 was tested in whey and soy flour based media. These media was reformulized form of NYSM (Nutrient Broth Yeast Extract Sporulation Medium). Soy flour based medium, SYSM, gave the promising results in terms of cell yield, sporulation frequency and toxin production. In the second part of the study, fermentation productivity anlaysis of a local isolate Bacillus thuringiensis subsp. kurstaki 81 was evaluated. In order to compare different C:N ratios (1:1, 2:1, 4:1, 8:1, 10:1 20:1 and 30:1) of YSM medium. Btk 81 were run for 72 h and cell growth, sporulation and toxin protein profile of Btk 81 were determined for each. When all the quantitative toxin data for both glucose and sucrose varying C:N ratios were compared, it was determined that the crystal protein concentrations had the highest value in sucrose based medium when C:N ratio was 10:1. Regulation by C:N ratio of crystal protein biosynthesis was investigated for improving the production of this protein by our third candidate strain Bacillus thuringiensis subsp. israelensis ONR60. The experiments were performed by using TBL medium, at three different C:N ratios, 2:1, 4:1 and 8:1 respectively. In view of the cell growth characteristics and bioassy results, TBL medium designed with 2:1 C:N ratio was chosen as the best for further steps. In addition, running time of the culture determined as 60 hours as was also determined in the previous experiment. As the last step of this study, the pre-determined optimal conditions were applied to a 30L batch type fermentor for toxin production by using Bacillus thuringiensis subsp. israelensis ONR60. Unfortunately, the toxicity was not satisfactory, being much below the level of that expected as based on the results of the laboratory scale studies.

Pine tree substrate (PTS) is a relatively new substrate for container crop production. There are no detailed studies that elucidate how storage time impacts PTS chemical, physical, and biological aspects. The objective of this research was to determine how PTS storage time influenced PTS chemical and physical properties, nitrification, and plant growth. Pine tree substrate was manufactured by hammer-milling chips of loblolly pine trees (Pinus taeda L.) through two screen sizes, 4.76 mm (PTS) and 15.9 mm amended with peat (PTSP). PTS and PTSP were amended with lime at five rates. A peat-perlite mix (PL) served as a control treatment. Prepared substrates were placed in storage bags and stored in an open shed in Blacksburg, Virginia. Subsamples were taken at 1, 42, 84, 168, 270, and 365 days. At each subsampling day, twelve 1-L containers were filled with each substrate. Six containers were left fallow and six were planted with marigold (Tagetes erecta L. "Inca Gold") seedlings. Substrate was also collected from select treatments for Most Probable Number assays to estimate density of nitrifying microorganisms, and for chemical and physical property analyses. Pour-through extracts were collected from fallow containers at 0, 2, and 4 weeks, and from marigold containers at harvest for determination of pH, electrical conductivity, ammonium-N and nitrate-N. At harvest, marigold height, width, and dry weight were measured. At least 1 kg·m-3 lime for PTS, and 2 to 4 kg·m-3 lime for PTSP were needed to maintain pH values ≥ 5.5 for 365 days. Bound acidity of unlimed PTS increased but cation exchange capacity for unlimed PTS and PTSP decreased over 365 days. Carbon to nitrogen ratio and bulk density values were unchanged over time in all treatments. There were minor changes in particle size distribution for limed PTS and unlimed and limed PTSP. Marigold growth in PTS and PTSP was ≥ PL in all limed treatments, except at day 1. Nitrite-oxidizing microorganisms were present and nitrification occurred in PTS and PTSP at all subsampling days. Pine tree substrate is relatively stable in storage, but pH decreases, and lime addition may be necessary to offset this decrease. / Ph. D.