Microbiology (1999>, 145, 1 173-1 180 Printed in Great Britain Butane metabolism by butane-grown 'Pseudomonas butanovora ' Daniel J. Arp Tel: + 1 541 737 1294. Fax: + 1 541 737 3.573. e-mail: arpdtg bcc.orst.edu Laboratory for Nitrogen The pathway of butane metabolism by butane-grown 'Pseudomonas Fixation Research, butanovora' was determined to be butane + I-butanol + butyraldehyde + Department of Botany and Plant Pathology, Oregon butyrate. Butane was initially oxidized at the terminal carbon to produce 1- State University, butanol. Up to 90% of the butane consumed was accounted for as I-butanol 2082 Cordley, Corvallis, when cells were incubated in the presence of 5 mM I-propanol (to block OR 97331, USA subsequent metabolism of I-butanol). No production of the subterminal oxidation product, 2-butano1, was detected, even in the presence of 5 mM 2- pentanol (an effective inhibitor of 2-butanol consumption). Ethane, propane and pentane, but not methane, were also oxidized. Butane-grown cells consumed I-butanol and other terminal alcohols. Secondary alcohols, including 2-butano1, were oxidized to the corresponding ketones. Butyraldehyde was further oxidized to butyrate as demonstrated by blocking butyrate metabolism with 1mM sodium valerate. Butyrate also accumulated from butane when cells were incubated with 1mM sodium valerate. The pathway intermediates (butane, I-butanol, butyraldehyde and butyrate) and 2-butanol stimulated 0, consumption by butane-grown cells. I-Butanol, butyraldehyde and butyrate supported growth of 'P. butanovora', as did 2-butanol and lactate. Keywords : butane metabolism, alkane metabolism, 'Pseudomonas butanovora ', alkane oxidation INTRODUCTION they can often carry out the hydroxylation of gaseous alkanes (Burrows et al., 1984; Colby et al., 1977). A number of bacteria have been isolated that are capable of growth on butane. Most of these bacteria are As pointed out by Ashraf et al. (1994) in a recent review members of the R h od o bac ter-No card ia-A r th r o bac ter- of the subject, the pathways for the metabolism of the Corynebacterium group of Gram-positive bacteria light n-alkanes (ethane, propane and butane) have (Ashraf et al., 1994; McLee et al., 1972; Perry, 1980). received little attention compared to those of methane However, two Gram-negative bacteria which can grow and liquid n-alkanes. The pathway of butane metab- on butane, ' Pseudomonas butanovora ' and Pseu- olism has not previously been established directly for domonas sp. strain CRL 71, have been described (Hou et any butane-oxidizing bacterium. Butane, as with other al., 1983 ; Takahashi, 1980). Butane-oxidizing bacteria alkanes, is generally assumed to be harvested by a can be considered as part of a larger group of bacteria monooxygenase, which results in hydroxylation of the which are characterized by their ability to grow on alkane (Ashraf et al., 1994; Perry, 1980). However, gaseous alkanes such as ethane and propane, but not production of 1-butanol or 2-butanol from butane had methane (Ashraf et al., 1994; Klug & Markovetz, 1971). not been directly demonstrated for any butane-grown This larger group is also dominated by Gram-positive bacterium. The subsequent metabolism of either 1- bacteria, although some Pseudomonas spp. will grow on butanol, the terminal oxidation product, or 2-butanol, C,-C, alkanes (Hou et al., 1983). Bacteria that grow on the subterminal oxidation product, would be expected one gaseous alkane will generally grow on other gaseous to require different pathways. Evidence for pathways or volatile alkanes ; for example, Mycobacterium vaccae consistent with both oxidation products has been will grow on propane (Vestal & Perry, 1969) or butane presented (Lukins & Foster, 1963; Phillips & Perry, (Phillips & Perry, 1974). Bacteria that can grow on 1974; van Ginkel et al., 1987). Terminal oxidation of methane, methanotrophs, generally do not use other butane by M. vaccae JOB5 was proposed because cells alkanes as growth substrates (Murrell, 1992) though grown on either butane or butyrate expressed isocitrate 0002-2996 0 1999 SGM 1173 D. J. ARP lyase activity, which is required to assimilate the 2- received 5 ml CO, as an overpressure. Butane (10 ml) was carbon compounds formed as a result of further added to the vial as an overpressure for growth on butane. For metabolism of butyrate (Phillips & Perry, 1974). In growth on 1-butanol, butyraldehyde or 2-butanol, appropriate contrast, cells grown on butanone did not produce volumes of each pure liquid were added directly to the sterile isocitrate lyase activity. Butanone was subsequently medium. For growth on butyrate or lactate, appropriate amounts of stock solutions (1 M) of sodium butyrate or decarboxylated to propionate and further metabolized sodium lactate were added to the medium. Cultures were by the methylmalonate-succinate pathway (Phillips & shaken at 160 oscillations min-l and maintained at 30 "C Perry, 1974). Butane-grown Nocardia TB1 produced during growth and harvested after 2 or 3 d. The limiting butyrate from n-butane while in the presence of arsenite nutrient for growth was 0, ; butane-grown cultures typically and a pathway of butane to 1-butanol to butyraldehyde reached an OD,,, of 0.6 upon exhaustion of the 0,. to butyrate was suggested (van Ginkel et al., 1987). Cells were harvested by centrifugation (10 min at 12000g; However, production of the first two proposed inter- 10 "C) and resuspended in 1 ml buffer [8 g (NH,),HPO,, 1.9 g mediates (1-butanol and butyraldehyde) was not dem- Na,HPO, .7H,O, 2 g KH,PO,, 0-5 g MgSO, .7H,O onstrated directly and the possibility of a concurrent pH 7-11. Cell suspensions were typically prepared fresh daily pathway initiated by subterminal oxidation of butane and used within 6 h. However, cell suspensions retained was not eliminated. Subterminal oxidation was in- butane consumption activity for at least 30 h when stored on dicated for propane-grown Mycobacterium smegmatis ice without agitation. Typical protein concentrations for the 422, which accumulated butanone when exposed to n- cell suspensions were 5-7 mg protein (ml suspension)-'. butane (Lukins & Foster, 1963). Measurement of cell activities. Butane consumption was measured in a 1 ml gas-tight syringe (Hamilton 1001 RN) with As with butane, the pathway of propane metabolism has the needle removed. The reaction mixture consisted of 0.7 or received limited attention. Support for both terminal 0.8 ml 0,-saturated buffer, 0.1 or 0.2 ml butane-saturated and subterminal oxidations of propane has been pre- buffer, and addition of a cell suspension (typically 0.025 ml), sented (Perry, 1980). Accumulation of acetone (from 2- other compounds (e.g. 1-propanol) as indicated, and ad- propanol) supported the conclusion that propane oxi- ditional buffer for a total volume of 1 ml. A glass bead in the dation was primarily subterminal in propane-utilizing syringe facilitated mixing of the components. Additions to the bacteria such as M. vaccae JOB5 (Lukins & Foster, syringe were made by injection through the opening into the 1963; Perry, 1980). However, consumption of the body of the syringe. No gas phase was present in the syringe. terminal oxidation product, 1-propanol, was also dem- Movement of the plunger facilitated addition and removal of onstrated for propane-grown M. vaccae JOB5 (Perry, samples without introducing a gas phase. Samples of the liquid (10 pl) were removed periodically and analysed for butane 1968). Furthermore, some propane-grown strains of content by GC as described below. In some instances, 1- Arthrobacter consumed 1-propanol (the terminal oxi- butanol was also analysed. Consumption of other alkanes dation product of propane) but not 2-propanol, while (0.14.2 pM) was measured similarly. The reactions were other strains consumed both isomers (Stephens & carried out at room temperature (20 1 "C). Dalton, 1986). Both 1-propanol and 2-propanol were Consumption and accumulation of alcohols, butyraldehyde produced by cell-free extracts of Arthrobacter sp. CRL- and butyrate were measured in 7 ml serum vials capped with 60, Pseudomonas fluorescens NRRL-B-1244 and Brevi- butyl rubber stoppers and aluminium crimp seals. The bacterium sp. NRRL B-11319 (Pate1 et al., 1983). reaction mixture consisted of the substrate with or without Current evidence indicates that both terminal and inhibitor (at the indicated concentrations), cell suspension subterminal hydroxylations of propane can occur. (10-100 pl), and buffer to a total of 1 ml. Butane (1 ml) or propane (1 ml) gas, where indicated, was added as an ' P. butanovora7grows on C,-C, n-alkanes as well as on overpressure to the headspace. Vials were shaken at 100 a number of alcohols and organic acids (Takahashi, oscillations min-l in a 20 "C water bath during the reactions. 1980). However, growth on alkenes and sugars was not Liquid samples (2-10 pl) were removed periodically and the observed. We recently demonstrated that this bacterium, concentrations of substrates and products were determined by when grown on butane, could initiate the degradation of GC. a number of chlorinated aliphatic compounds, including 0, consumption by cells in the presence of various compounds chloroform (Hamamura et al., 1997), and this degra- was measured with a Clark-style 0, electrode inserted into a dation appeared to be initiated by butane mono- 1.6 ml chamber sealed with a capillary inlet through which oxygenase. In this work, the pathway of butane metab- additions were made. The contents of the electrode chamber olism by this Gram-negative bacterium was elucidated. were stirred with a magnetic stir bar. The reaction mixture consisted of air-saturated buffer with substrates and cells added as indicated, The reactions were carried out at room METHODS temperature (20 1 "C). Cell growth and preparation of cell suspensions. Cells of Analytical techniques. Concentrations of alkanes, aldehydes, 'Pseudomonas butanovora ' (ATCC 43655) were grown as ketones and organic acids in the liquid phase of reaction previously described (Hamamura et al., 1997).