Batch cultivation ofMethylosinus trichosporium OB3b: V. Characterization of poly-β-hydroxybutyrate production under methane-dependent growth conditions

Methanotrophs have promising applications in the bioremediation of chlorinated hydrocarbons and in the production of a biopolymer, poly-beta-hydroxybutyrate (PHB). Batch bioreactor culture conditions were studied for the accumulation of PHB by methane-grown Methylosinus trichosporium OB3b, and to evaluate the effect of PHB on the bacterial capacity to degrade trichloroethylene (TCE), a common groundwater contaminant. The PHB content of the washed and lyophilized cells was measured by gas chromatography (GC), after hydrochloric acid (HCl) propanolysis. A differential GC-based assay was developed for the monomer and the polymer of beta-hydroxybutyrate utilizing 1% and 10% HCl (v/v) reaction mixtures, respectively. During bioreactor growth in a Cu-deficient modified Higgins' medium, the cells accumulated PHB upon depletion of nitrate. A biomass yield of 3.2 g dry wt/L and a PHB accumulation of approximately 10% (w/w) were reached after 140 to 160 h, without adversely affecting the propene or TCE epoxidation specific rate given by whole cells containing soluble methane monooxygenase (sMMO). The TCE biotransformation capacity ( approximately 0.25 mg TCE oxidized/mg dry cell wt) of resting cells containing approximately 10% PHB was consistently approximately 1.6-fold greater than that of cells containing only approximately 2% PHB. Higher levels (>10%) of accumulated PHB did not enhance this biotransformation capacity further. By replacing the bioreactor inlet air + CO(2) mixture with pure O(2) at approximately 85 h of batch operation, a PHB accumulation of approximately 45% was achieved after 160 h, but the whole-cell sMMO activity was markedly decreased. In contrast, cells grown in a 10 muM Cu-supplemented Higgins' nitrate minimal salts medium (particulate MMO formation) accumulated up to 50% PHB in only 120 h, coupled with a very high biomass yield of 18 g dry cell wt/L. High PHB accumulations above approximately 20% by both the -Cu and the +Cu grown cells resulted in a decreased ratio of the electronic cell count to the absorbance at 660 nm, which is commonly used to monitor bacterial growth.