Biotechnology Letters 675: 675–679, 2002. © 2002 Kluwer Academic Publishers. Printed in the Netherlands. 675 The absolute requirement for carbon dioxide for aerobic methane oxidation by a methanotrophic-heterotrophic soil community of bacteria Victor Acha 1,∗ , Joel Alba 2 & Frederic Thalasso 2 1 Petroleum Biotechnology Program, Instituto Mexicano del Petr´ oleo (IMP), Eje Central L´ azaro C´ ardenas 152, 07730 Mexico City, D.F., Mexico 2 Biotechnology & Bioengineering Department, CINVESTAV, Av. I.P.N. 2508, 07360 Mexico City, D.F., Mexico ∗ Author for correspondence (Fax: +52-5557477002; E-mail: vacha@imp.mx) Received 15 January 2002; Revisions requested 18 January 2002; Revisions received 22 February 2002; Accepted 25 February 2002 Key words: carbon dioxide, methane oxidising bacteria, methanotrophs Abstract An aerobic methanotrophic-heterotrophic soil community has been characterised when growing with different partial pressures of CO 2 . The methanotrophic population using methane as carbon source reached 3 × 10 7 cfu ml -1 with one of the major methanotrophs being of type II which uses the serine pathway for C assimilation. Optimal methanotrophic activity required the addition of CO 2 , and in the absence of CO 2 no methane oxidisers grew. Partial pressures of CO 2 from 1.6 to 11.6 kPa gave optimal cell growth and production of soluble organic compounds. Bio- mass yield, soluble organics and CO 2 production were 0.36, 0.15, and 0.48 mg mg -1 methane uptake, respectively, with CO 2 at 11.6 kPa. The results presented here may have important implications for the use of methane-oxidising bacteria in bioremedial applications. Introduction Methanotrophic bacteria (methanotrophs) are Gram- negative aerobes that use methane as sole carbon and energy source (Hanson & Hanson 1996). By virtue of their non-specific methane monooxygenases (MMO), they have potential industrial and environ- mental applications for the co-metabolism in soil and groundwater of pollutants such as tetrachloroethylene, trichloroethylene (Alvarez-Cohen & McCarty 1991, Kang et al. 2001), ammonia compounds (Sullivan et al. 1998, Ren et al. 2000), or for methanol biosyn- thesis (Yu et al. 1998). The oxidation of methane is initiated by the MMO using O 2 to form methanol which, in turn, is oxidised to CO 2 via formaldehyde and formate by different de- hydrogenases (Hanson & Hanson 1996) (Figure 1). Furthermore, the cell material is synthesised either from formaldehyde by the serine pathway, in which 2 mol formaldehyde and 1 mol CO 2 are utilised, or by the ribulose monophosphate (RUMP) pathway for the assimilation of 3 mol formaldehyde (Figure 1). Since 1970 several reports have appeared about the establishment of pure cultures of methane-oxidising bacteria and applications of these bacteria in bioreme- diation processes. While many operational conditions for methane oxidation by methanotrophs (i.e., nutrient composition, temperature, pH, and oxygen tension) were studied, to our knowledge the importance of CO 2 for cell growth has not been adequately addressed. This paper deals with the characterisation and optimisation of the culture medium for aerobic methane oxidation by a methanotrophic-heterotrophic soil community under the influence of different CO 2 partial pressures. Materials and methods Growth medium For the growth of the aerobic methanotrophic- heterotrophic soil community, a nitrogen mineral salts (NMS) medium (Whittenbury & Dalton 1981) was