Factors affecting the utilization of methanotrophic bacteria in bioremediation.

Abstract

The focus of this work was to assess the utility of sMMO-bearing (soluble methane monooxygenase) type II methanotrophs for bioremediation. These bacteria have demonstrated the highest trichloroethylene degradation rates of all organisms investigated thus far. Two problems, however, must be resolved to maximize these organisms' potential: (1) type II strains have slow growth rates, and (2) copper represses sMMO activity at low concentrations (<0.25uM Cu(II)). Selective enrichment strategies for type II strains were evaluated through two-organism, continuous-flow competition experiments between type I (Methylomonas albus BG8) and type II (Methylosinus trichosporium OB3b) methanotrophs. Type II organisms predominated under copper and nitrogen limitation because they express sMMO and nitrogenase, respectively. Secondary oxygen effects accelerated competitive success – high oxygen (> 173 uM) for copper limitation, and low oxygen (<65 uM) for nitrogen limitation. Two topics were evaluated involving sMMO repression by copper: (1) the regulation of sMMO activity, and (2) copper uptake and toxicity in type II strains. Results indicated that copper represses the expression of sMMO at the genetic level in M.trichosporium OB3b. This implies that regulatory mutants, which express sMMO at elevated copper levels, may be feasible. A colorimetric method for sMMO-bearing organisms is described which could be used for mutant screening. Experiments on Cu(II) uptake and toxicity were performed using M.trichosporium OB3b. M.trichosporium was found to have a two-component copper uptake system: (1) a constitutive component, present at low copper concentrations (<2.0 uM), and (2) an inducible component, which is expressed when copper is unavailable to the constitutive system. The inducible system is very efficient at scavenging copper. It can sequester copper even in the presence of strong copper complexing agents such as tetraethylenepentaamine (pK = 20.9). The ability to manufacture sMMO-bearing mutants may be complicated by the inducible copper uptake system. Methylosinus sp. DG1 appears to have a less efficient copper uptake system than M.trichosporium and should be studied further. Although type II organisms show tremendous talents at bioremediation, their potential cannot be maximized unless the problems associated with copper can be resolved.