1a. Objectives (from AD-416):
The proposed research seeks to develop a means to eliminate off-flavor metabolites without regard for the producing species using bioremediation. To accomplish this goal, we will pursue the following objectives: 1) Physiological characterization and improvement of existing microbial strains for the bioremediation of the off-flavor chemicals, 2-methylisoborneol (MIB) and geosmin, and discovery of new bacterial strains. 2) Development and testing of characterized bacteria for bioremediation under field conditions.
1b. Approach (from AD-416):
Identification and characterization of degradative enzymes and genes encoding them, identification and characterization of regulatory mechanisms, and construct/select, isolate, and characterize constitutive (unregulated) mutants, and isolation and characterization of new bacterial strains having different or improved properties. Pre-characterization of selected strains for their ability to succeed in aquaculture, development, and use of methods for cultivation and application of large quantities of bacteria, for treatment of aquaculture sites and determination of bioremediation effects in aquaculture systems.
3. Progress Report:
A modest amount of progress was made on the first objective during the year; however, this effort has been slowed by critical vacancies and the redirection of scientists that occurred as part of the reorganization of personnel from terminated projects. The sole scientist working on these objectives was moved to an alternative project, and two scientists from terminated projects were redirected to the project at various times during the year. In addition, with these personnel changes, it was mandated that the work be refocused toward the improved utilization of catfish by-products; hence, work on the off-flavor objectives has stopped. During the early part of the year, work continued on three bacterial stains, Rhodococcus wratslaviensis DLC-cam (DLC), Pseudomonas putida G1(G1) and Rhodococcus sp. T1 (T1), which were found to be the most promising for the transformation of the two most important catfish off-flavor compounds, geosmin and 2-methylisoborneol (MIB). Strains DLC and T1 were grown under conditions to induce the production of proteins associated with MIB degradation. From these cultures, soluble proteins were recovered and separated to look for increased levels of individual proteins that might contribute to the degradation of the off-flavors compounds. No obviously induced proteins were apparent, indicating that the inducible activities are either derived from existing enzymes or from induced enzymes that accumulate to very low levels. Additionally, the enzyme responsible for transforming for loss of off-flavor MIB from strain G1 was cloned into a bacterial expression system, then over-expressed and purified to apparent homogeneity. The enzyme called p450-cam was also cloned into a vector to provide the reduction and oxidation system necessary for the enzyme's MIB transformation activity as a fusion protein. This enzyme complex was also purified to apparent homogeneity. These cloned enzymes were to be characterized for their ability to bind and metabolize geosmin and MIB.