APPLIED AND ENVIRONMENTAL MICROBIOLOGY, June 1986, P. 1239-1246 Vol. 51, No. 6 0099-2240/86/061239-08$02.00/0 Copyright ©) 1986, American Society for Microbiology Growth of Pseudomonas aeruginosa on Nitrous Oxide DENNIS A. BAZYLINSKI, CARLTON K. SOOHOO, AND THOMAS C. HOLLOCHER* Department ofBiochemistry, Brandeis University, Waltham, Massachusetts 02254 Received 16 October 1985/Accepted 24 February 1986 Three strains of Pseudomonas aeruginosa were grown anaerobically on exogenous N20 in a defined medium under conditions that assured the maintenance of highly anaerobic conditions for periods of 1 week or more. The bacteria were observed reproducibly to increase their cell density by factors of 3 to 9, but not more, depending on the initial amount of N20. Growth on N20 was cleanly blocked by acetylene. Cell yields, CO2 production, and N20 uptake all increased with initial PN2O at PN2O ' 0.1 atm. Growth curves were atypical in the sense that growth rates decreased with time. This is the first observation of growth of P. aeruginosa on N20 as the sole oxidant. N20 was shown to be an obligatory, freely diffusible intermediate during growth of strains PAO1 and P1 on nitrate. All three strains used this endogenous N20 efficiently for growth. For strains PAO1 and P1, it was confirmed that exogenous N20 had little effect on the cell yields of cultures growing with nitrate; thus, for these strains exogenous N20 neither directly inhibited growth nor was used significantly for growth. On the other hand, strain P2 grew abundantly on exogenous N20 when small and growth-limiting concentrations of nitrate or nitrite (2 to 10 mM) were included in the medium. The dramatic effect of these N-anions was realized in large part even when the exogenous N20 was introduced immediately after the quantitative conversion of anion-nitrogen to N2. No evidence was found for a factor in filter-sterilized spent medium that stimulated fresh inocula to grow abundantly on N20. It would seem that nitrate, nitrite, or a metabolic product can stimulate strain P2 (but not PAO1 or P1) to grow abundantly on exogenous N20. The phenotype of strain P2 suggests that the ability of strains PAO1 and P1 to grow on endogenous N20 may also be under control of nitrate or a metabolic product of nitrate. The metabolic defect that prevents abundant growth of strains PAO1 and P1 on exogenous N20 was not traceable to dysfunction of the respiratory proton pump, high proton permeability of the membrane, failure to form or maintain a proton motive force, or a nutritional requirement. The metabolic defect is not understood at present. Although many N2-producing denitrifying bacteria, such reducing activity (25). N20 appears not to be highly toxic to as Pseudomonas denitrificans, P. stutzeri, P. perfec- P. aeruginosa, because it can grow on nitrate under 1 atm (= tomarinus, and Paracoccus denitrificans, grow vigorously 109.29 kPa) of N20 (4, 6). on exogenous N20 (4, 6, 15) P. aeruginosa appears to be an In this report we re-examine with improved methods the exception (4-6, 25; B. A. Bryan, Ph.D. thesis, University of apparent inability of P. aeruginosa to grow on exogenous California, Davis, 1980). Growth of P. aeruginosa on N20 N20 and find that the organism can in fact grow to the extent has not been reported previously. Another possible excep- of two to three doublings. The nature of the defect that tion is Aquaspirillum magnetotacticum, a magnetic bacte- prevents abundant growth on N20 was investigated but rium, which reduces nitrate to N2 in growing cultures but remains obscure. We also observe that growth of one strain does not appear to grow on exogenous N2O (1; D. A. of P. aeruginosa on N20 can be enhanced considerably by Bazylinski and R. P. Blakemore, unpublished results). Not nitrate or nitrite. only can N2-producing denitrifiers generally grow on N2O, but their molar growth yields on nitrate, nitrite, and N20 are MATERIALS AND METHODS proportional to the oxidation number of nitrogen in these N-oxides (4, 15, 26; Bryan, Ph.D. thesis). Thus growth Bacteria and cultures. Three strains of P. aeruginosa were yields per electron are virtually identical among these N- used in this study: strain PAO1 was supplied by B. W. oxides. Moreover, the 'H+/2e- ratios measured in oxidant Holloway, Monash University, Clayton, Victoria, Australia; pulse studies of N2-producing denitrifiers are similar among strain P1 was isolated from the infected ear of a domestic dog these N-oxides (3, 7, 16, 18) and with NO as well (8); and the and characterized at the Department of Microbiology, N-oxides, including NO, support in these cells the active University of New Hampshire, Durham, from which it was transport of L-proline (8, 27), the uptake of which is driven obtained; strain P2 was isolated at Brandeis University from by the proton motive force (19). The failure of P. aeruginosa the infected ear of an adult human female. Strain P2 is an to grow on N20 is particularly puzzling, inasmuch as N20 asporogenous gram-negative rod with single polar flagella, has been shown to be an obligatory, freely diffusible inter- positive for oxidase, catalase, gelatinase (slow), and mediate in the reduction of nitrate or nitrite by nongrowing assimilation of acetamide and able to grow at 42°C but not at cells (25), and this endogenous N20 would appear to be used 4°C (2). Growth was obligately aerobic for all sugars, for growth with the same efficiency applicable to other carbohydrates, and media tested. Although it produced denitrifiers and other N-oxides (Bryan, Ph.D. thesis). In soluble fluorescent yellow-green pigment, it failed to produce addition, denitrifying P. aeruginosa has considerable N20 pyocyanin or other chloroform-soluble pigments on several media, including that of King et al. (14). These and other characteristics indicate that P2 is a strain of P. aeruginosa * Corresponding author. unable to produce pyocyanin. It is clearly distinguished from 1239 1240 BAZYLINSKI ET AL. APPL. ENVIRON. MICROBIOL. the various biotypes of P. fluorescens and P. putida (2, 12, ments, using N20 as oxidant. The most decisive control was 24). Isolation of apyocyanogenic strains of P. aeruginosa is the use of acetylene which cleanly blocks reduction of N20 not uncommon, particularly from clinical sources (12, 24). by the nitrous oxide reductase of denitrifiers but has no Two other species of N2-producing denitrifying bacteria, effect on reduction of 02, nitrate, or nitrite (29; Bryan, Ph.D. Paracoccus denitrificans ATCC 19367 and Pseudomonas thesis). In addition, P. aeruginosa produced fibrous macro- denitrificans ATCC 13867, were used as reference organisms scopic aggregates of cells when grown on 02- Cells were to exemplify performance of bacteria which can grow largely monodispersed when grown on N-oxides under abundantly on N20 at 1 atm. Strains PAO1 and P1 were used anaerobic conditions. Over the course of 1 to 3 days, cells of to show that PAO1 was not a unique phenotype. At present, strains PAO1 and P1 growing on 02 were also observed to strain P2 exhibits a unique phenotype. The bacteria were produce pigment (24). Cultures under N20 did not exhibit maintained aerobically at pH 7 and 30°C on undefined medium any of these criteria to suggest leakage of 02 during growth which contained the following (grams per liter): yeast extract, experiments. 6; Bacto-Peptone (Difco Laboratories, Detroit, Mich.), 3; Survival of cells under N20 over prolonged periods. Cul- KH2PO4, 1; K2HPO4, 1.5. tures under N20 were often incubated for up to 7 days at To obtain inocula for the anaerobic growth experiments 30°C. The viability of P. aeruginosa under such conditions described below, cells were grown semianaerobically from was checked qualitatively when incubation was ended by single colonies under nitrate-limiting conditions (10 mM plating on nutrient agar plates and by the rate at which cell KNO3) at 30°C in a succinate-salts minimal medium (4). The growth resumed following addition of nitrate or admission of suggested trace mineral solution for this medium was re- air to cultures at the end of the experiment. Cells were placed by a modified mineral solution of Wolin et al. (1, 28) judged to be viable if growth resumed without delay at the that supplied 11 trace elements commonly required for rate expected for fresh cells. By these criteria, cultures ofP. bacterial growth. The term "semianaerobically," used aeruginosa were judged to be largely if not entirely viable for above, refers to cultures which contained dissolved 02 at the periods of at least 1 week in the succinate-salts medium time of inoculation but became essentially anaerobic within under N20. a few hours due to a combination of aerobic respiration and Tests for a nutritional requirement to promote growth on N2 production. Such cultures were neither shaken nor N20. Experiments were carried out with 100 ml of the stirred during growth. Cells were judged to be competent in succinate-salts medium in 250-ml Erlenmeyer flasks. Sterile denitrification and suitable for use as inocula when N2 medium was sparged with N20 for a time sufficient to evolution was vigorous and just after the nitrate and nitrite remove dissolved 02 and then inoculated by injecting 1 ml of had become exhausted. These first-stage cultures were used a first-stage culture. A flow of N20 of 30 ml min-' was to inoculate (1.8%, vol/vol) second-stage cultures, which are maintained during the subsequent period of incubation at identical to the former, except that they were made highly 30°C (usually 48 h). Positive growth controls included me- anaerobic and sealed prior to inoculation as described be- dium supplemented with nitrate or under air and Pseudomo- low.