BATCH CULTIVATION OF METHYLOSINUS-TRICHOSPORIUM OB3B .5. CHARACTERIZATION OF POLY-BETA-HYDROXYBUTYRATE PRODUCTION UNDER METHANE-DEPENDENT GROWTH-CONDITIONS

Methanotrophs have promising applications in the bioremediation of chl orinated hydrocarbons and in the production of a biopolymer, poly-beta -hydroxybutyrate (PHB). Batch bioreactor culture conditions were studi ed for the accumulation of PHB by methane-grown Methylosinus trichospo rium 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 g as chromatography (GC), after hydrochloric acid (HCl) propanolysis. A differential GC-based assay was developed for the monomer and the poly mer of beta-hydroxybutyrate utilizing 1% and 10% HCl (v/v) reaction mi xtures, respectively. During bioreactor growth in a Cu-deficient modif ied Higgins' medium, the cells accumulated PHB upon depletion of nitra te. A biomass yield of 3.2 g dry wt/L and a PHB accumulation of simila r to 10% (w/w) were reached after 140 to 160 h, without adversely affe cting the propene or TCE epoxidation specific rate given by whole cell s containing soluble methane monooxygenase (sMMO). The TCE biotransfor mation capacity (similar to 0.25 mg TCE oxidized/mg dry cell wt) of re sting cells containing similar to 10% PHB was consistently similar to 1.6-fold greater than that of cells containing only similar to 2% PHB. Higher levels (>10%) of accumulated PHB did not enhance this biotrans formation capacity further. By replacing the bioreactor inlet air + CO 2 mixture with pure O-2 at similar to 85 h of batch operation, a PHB a ccumulation of similar to 45% was achieved after 160 h, but the whole- cell sMMO activity was markedly decreased. In contrast, cells grown in a 10 mu M Cu-supplemented Higgins' nitrate minimal salts medium (part iculate MMO formation) accumulated up to 50% PHB in only 120 h, couple d with a very high biomass yield of 18 g dry cell wt/L. High PHB accum ulations above similar to 20% by both the -Cu and the +Cu grown cells resulted in a decreased ratio of the electronic cell count to the abso rbance at 660 nm, which is commonly used to monitor bacterial growth. (C) 1996 John Wiley & Sons, Inc.