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
Isolation and characterization of new bacteria that are capable of degrading the main catfish off-flavors, geosmin and 2-methylisoborneol (MIB), have continued. Geosmin and MIB are terpene-type compounds produced in catfish ponds by algae. Bacterial strains were isolated for their ability to grow on compounds having structures similar to these terpenes. While many of the newly isolated bacteria resemble previously strains in their ability to transform these compounds, some novel and important strains were identified. Of the bacteria tested, Rhodococcus wratslaviensis DLC-cam, Pseudomonas putida G1, and Rhodococcus sp. T1 were found to be the most promising by virtue of having the highest amount of transformation of geosmin or MIB. Rhodococcus wratslaviensis DLC-cam was shown to have constitutive activity against both compounds. To improve the activity of these organisms, a key enzyme in the degradation of terpene-like compounds was recently crystallized. We have collaborated with this group and used molecular modeling tools to show that MIB will also bind to the active site of this enzyme. The work suggests that degradation of MIB differs from the degradation of the enzyme’s normal substrate, but also suggests that modifications of the enzyme’s structure might improve its ability to degrade MIB. Initial tests were conducted to determine if these organisms had properties that would allow them to be used for bioremediation of off-flavors in aquaculture environments. In a preliminary round of testing, Rhodococcus wratslaviensis DLC-cam was found to not be pathogenic against fish, even at very high concentrations. All three bacterial strains were able to be grown to very high densities on inexpensive media, a condition necessary for mass production of the organism. All three strains were also able to be grown in minimal media with fish feed as the sole carbon source, a condition typical of aquaculture environments. Finally, all three strains can be frozen without significant loss in viability, which is desirable for storage and transport to a treatment area.
1. R. wratislaviensis DLC-cam (DLC) for bioremediation of off-flavors in aquaculture. Off-flavors are a persistent problem for the aquaculture industry. Work conducted by Agricultural Research Service scientists at the Southern Regional Research Center in New Orleans, LA, in collaboration with the University of Louisiana at Lafayette, has identified a strain of R. wratislaviensis that will be useful for reducing off-flavor problems in aquaculture farms. This strain has constitutive biotransformation activities for 2-methylisoborneol and geosmin, is non-pathogenic towards fish, and can be grown under conditions found in static and recirculating aquaculture systems. Once a delivery system is established, strain DLC will benefit aquaculturists with fish off-flavor problems.