BATCH CULTIVATION OF METHYLOSINUS-TRICHOSPORIUM OB3B .4. PRODUCTION OF HYDROGEN-DRIVEN SOLUBLE OR PARTICULATE METHANE MONOOXYGENASE ACTIVITY

Batch culture conditions were established for the formation of H-2-dri ven whore-cell soluble or particulate methane monooxygenase (sMMO or p MMO) activity in the obligate methanotroph, Methylosinus trichosporium Ob3b, to expand its potential uses in groundwater bioremediation and the production of specific chemicals. Addition of either Ni and H-2 to a nitrate-containing minimal salts growth medium or Ni and Mo to a ni trate-lacking growth medium (induces a nitrogenase that generates intr acellular H-2) markedly enhanced both the hydrogenase and the accompan ying washed-cell H-2-driven MMO activities of shake-flask cultured cel ls. For sMMO containing cells, H-2 provided in vitro reducing power fo r the oxidation of chlorinated solvents such as chloroform and trichlo roethylene. Cell cultivations under N-2-fixing conditions in a 5-L bio reactor, however, required an initial nitrate concentration of at leas t 1 to 2 mM to achieve high biomass yields (5 to 7 g of dry cell wt/L) for cells producing H-2-driven sMMO or pMMO activity. Elevation of th e initial medium nitrate concentration to 20 mM shortened the culture time for pMMO producing cells by 40%, yet still generated an equivalen t growth yield. High nitrate also shortened the culture time for sMMO containing cells by similar to 25%, but it lowered their biomass yield by 26%. Upon storage for 5 weeks at room temperature, washed resting- state cells retained 90% and 70% of their H-2-driven sMMO and pMMO act ivity, respectively. This makes their practical use quite feasible. (C ) 1995 John Wiley and Sons, Inc.