Batch cultivation of Methylosinus trichosporium OB3b: II. Production of particulate methane monooxygenase

Abstract

The obligatory methanotroph, Methylosinus trichosporium OB3b, was studied to optimize the batch culture conditions for the formation of particulate methane monooxygenase (pMMO) in a nitrate minimal salts medium. The important medium components investigated were copper, carbon dioxide, and nitrate. The whole-cell specific pMMO activity decreased sharply with increasing copper concentrations in the range of 10-40 microM and remained constant upon further increases of the copper concentration to 120 microM. The cell growth rate (micro), on the other hand, decreased over the entire range (10-120 microM) of copper concentrations tested. When pMMO was produced in a bioreactor with an optimal initial copper concentration of 10 microM, M. trichosporium OB3b exhibited a much faster overall growth rate and a higher whole-cell propene epoxidation activity compared to our earlier study, in which soluble methane monooxygenase (sMMO) was produced with copper-deficient medium. The addition of external carbon dioxide to the bioreactor culture eliminated an initial lag period in the cell growth. When the standard culture medium nitrate concentration (10 mM) was depleted, the pMMO activity, but not the growth rate, decreased rapidly. The whole-cell specific pMMO activity could be maintained by subsequent supplementation of nitrate. A 4-fold higher initial culture medium nitrate concentration of 40 mM, however, resulted in slower cell growth and lower pMMO activity. These observations demonstrate that, in addition to affecting the exclusive production of pMMO, copper also has an important previously unrecognized role in enhancing the growth rate of M. trichosporium OB3b. They also indicate that for the optimal batch production of pMMO with the minimal medium under study, nitrate should be supplied intermittently during the course of cultivation until other culture medium components become growth-limiting.

Air Treatment Techniques for Abatement of Emissions from Intensive Livestock Production

“…These results confirm that the biofilter should be operated around neutral pH for optimal methanotrophic growth (Humer and Lechner, 1999a;Park et al, 1992).…”

“…In a biofilter, NH 3 is converted to nitrous and nitric acid by nitrification and H 2 S is converted to sulfuric acid. At high NH 3 and H 2 S loading rates, methanotrophs may be inhibited by high ammonium and nitrite concentrations (Bedard and Knowles, 1989;Bronson and Mosier, 1993;King and Schnell, 1994;Boeckx et al, 1996;Humer and Lechner, 1999a) or by sulfuric acid accumulation (Furusawa et al, 1984;Yang and Allen, 1994) as the pH optimum of methanotrophs lies between 6 and 8 (Park et al, 1992;Humer and Lechner, 1999a). However, at EBRT's that are common for biofiltration of methane containing air, i.e.…”

Trichloroethene degradation in a two-step system bymethylosinus trichosporium OB3b. Optimization of system performance: Use of formate and methane

“…The medium used for the two-step experiments was adapted to obtain high cell densities and consisted of a three times higher concentration of all ingredients; only the concentrations of MgSO 4 и 7H 2 O, KH 2 PO 4 , CaNO 3 , and H 2 SO 4 remained unaltered. In the two-step system the cells were grown at 30% of MAX ( MAX ‫ס‬ 0.11 h −1 ; Park et al, 1992) ( L ‫ס‬ 1 mL min −1 ; D ‫ס‬ 0.028 h −1 ) and the following gas flows were used: methane, 19 mL min −1 ; air, 5 mL min −1 , oxygen, 32 mL min −1 . Air was always included to have nitrogen present as an internal standard for GC measurements.…”

Experimental pulse technique for the study of microbial kinetics in continuous culture

“…Since the V max values of sMMO and pMMO are similar (Table 1), it was concluded that very little methane was dissolved, implying a small K s,S value resulting in a very short methane consumption period (seconds). A simulation using a literature value for the V max,S of pMMO (290 nmol min − 1 mg − 1 , Park et al, 1992) and a K s,S value of 3 vM appeared to give a good representation of the data (Fig. 4B).…”

“…trichosporium OB3b expresses different methane monooxygenases (MMOs), depending on the amount of copper available (Park et al, 1992). In the absence of copper, a soluble form (sMMO) is expressed.…”