0026-2617/04/7304- © 2004 åÄIä “Nauka /Interperiodica” 0488 Microbiology, Vol. 73, No. 4, 2004, pp. 488–490. Translated from Mikrobiologiya, Vol. 73, No. 4, 2004, pp. 574–576. Original Russian Text Copyright © 2004 by Kolesnikov, Dedysh, Panikov. Methanotrophs are unique and ubiquitous bacteria that use methane as the carbon and energy source [1]. Boreal acidic Sphagnum bogs, in which CH 4 oxidation is driven by acidophilic methanotrophs, are one of the major sources of atmospheric methane [2]. The cur- rently recognized acidophilic methanotrophs have been assigned to two new genera and species: Methylocella palustris and Methylocapsa acidiphila [3, 4]. They pre- fer media with low salt concentrations (100–500 mg/l) and fail to grow on conventional media with a salt con- tent of 1.5–3 g/l [1]. It is worth noting that Methylocella isolates were obtained on a medium with nitrate, whereas Methylocapsa acidiphila was isolated on a nitrogen-free medium. The growth of M. acidiphila does not depend on the availability of bound nitrogen, which fact, in conjunction with acidophily, allows it to thrive in acidic low-temperature habitats with a low content of available nitrogen, such as bogs and acidic soils of the boreal zone. Our studies on the influence of nitrates, nitrites, , and other mineral salts on the methane-oxidizing activity of peat samples revealed that nitrates and chlo- rides caused maximum inhibition, whereas phosphates and sulfates were almost without effect, regardless of the cations involved [5]. The mechanism of the inhibi- tory effect of a number of mineral salts on the methane- oxidizing activity of peat and soil samples has not yet been elucidated. is known to inhibit the key enzyme of methanotrophic metabolism, methane monooxygenase, which catalyzes methane oxidation to methanol [6]. However, in our studies with peat sam- ples, we demonstrated that ammonium does not pro- duce a stronger effect on methane consumption than other cations, ä + in particular [5]. The present study on the effects of various salts on the growth of the acidophilic methanotroph Methylo- capsa acidiphila B2 was aimed at elucidating the mechanism of the inhibitory effect of minerals on methane consumption. M. acidiphila B2 (DSM 13967 T = NCIMB 13765 T ) was cultivated on a minimal nitrogen-free medium [4] NH 4 + NH 4 + at 25°C on a shaker (120 rpm) in hermetically closed 500-ml serum flasks containing 100 ml of medium. The culture purity was tested using phase-contrast micros- copy and media that promote the growth of heterotrophs [4]. Methane was introduced to a final concentration of 55 mg CH 4 -C/l by means of a syringe through a bacte- rial filter with a pore size of 0.22 μm. Mineral salts (KNO 3 , KCl, KBr, KI, LiCl, SrCl 2 , BaCl 2 , AlCl 3 , and K 2 SO 4 ) were added to the medium at concentrations of 1–10 mM. The control system was M. acidiphila B2 grown on the minimal medium without the above salts. Culture growth was estimated from the optical density of bacterial suspension samples taken at regular inter- vals. The optical density was measured with a SPEKOL 10 (Germany) spectrophotometer at 410 nm, and the results obtained were used to calculate biomass values based on carbon balance data [7]. CH 4 consumption and ëé 2 production were measured with an INFRA- LYT-4 IR (Germany) gas analyzer. The specific growth rate (μ) was determined from the dynamics of OD 410 during the exponential growth phase. The growth yield (Y = dx/ds) was estimated from the mass balance, taking into account the amount of methane consumed and CO 2 produced [7]. The table contains our data on the influence of a number of mineral salts on methane consumption, spe- cific growth rate (μ), and growth yield (Y). AlCl 3 and KI were the strongest inhibitors; they suppressed culture growth at a concentration of 1 mM. Low KNO 3 concen- trations (1 mM) stimulated methane consumption and increased the specific growth rate of M. acidiphila, but the bacterium virtually failed to grow during the first four days of cultivation if higher nitrate concentrations were used (3, 5, and 10 mM; see Fig. 1). The lag phase duration increased with an increase in the salt concen- tration. The addition of 1 mM BaCl 2 caused a lag phase of 4 days; 3 mM BaCl 2 completely inhibited growth. SrCl 2 and LiCl decreased the μ value but produced vir- tually no effect on the growth yield. The inhibition of the culture growth was enhanced by increasing KCl concentration, whereas the yield drastically decreased only at a KCl concentration of 10 mM. KBr produced an intermediate inhibitory effect in comparison to KCl Inhibition of Growth and Methane Consumption in Methylocapsa acidiphila by Mineral Salts O. M. Kolesnikov*, S. N. Dedysh*, and N. S. Panikov** *Winogradsky Institute of Microbiology, Russian Academy of Sciences, pr. 60-letiya Oktyabrya 7, k. 2, Moscow, 117312 Russia **Stevens Institute of Technology, New Jersey, USA Received February 10, 2003; in final form, March 11, 2004 SHORT COMMUNICATIONS