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 bacteria that are able to degrade off-flavor compounds geosmin and 2-methylisoborneol (MIB) have continued. Strains were isolated for their ability to grow on various terpenes that have similar skeletal structure to geosmin and MIB. While many of these resemble previously isolated strains in their ability to transform MIB and geosmin, there have been some novel and important recent isolates. Products formed by dehydration of MIB by R-limonene-degrading bacterial strains Sphingomonas sp. BIR2-rlima and Pseudomonas sp. 19rlim have been characterized and shown to be further transformed by various camphor-degrading bacteria (camphor is a terpene). Previously, Tn5 mutants of strain 19-rlim were isolated, and cloning and sequencing (characterization of the deoxyribonucleic acid (DNA) involved) of mutant and neighboring genes was initiated. This sequencing is nearly completed and includes a few large blocks of genes that are likely to encode the complete metabolism of R-limonene, p-cymene, and some other related terpenes as well as the transformation of MIB. Cloning and expression of wild-type genes encoding relevant enzyme activities is ongoing. Although, constitutive (turned on all the time) mutants in the metabolic pathways of interest have not yet been isolated, methods for their isolation, including mutagenesis, enrichment, and screening have been evaluated. The genes encoding a two-component regulator of a degradative pathway involved in MIB transformation have been cloned. The encoded proteins have been expressed, partially purified, and will be used to reveal the mechanism of regulation.