Comparative study of aerobic and nitrate respiration in Pseudomonas stutzeri Public Deposited

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  • Some forms of bacterial respiration do not involve molecular oxygen but instead utilize other hydrogen acceptors for oxidation of the substrate. Various organic and inorganic acceptors may be used. In the present study, the inorganic radicals, nitrate and nitrite, were investigated. Preliminary evidence indicated that nitrate and oxygen are able to compete effectively as acceptors of hydrogen in respiration. It is the purpose of this dissertation to extend this observation with the hope that the degree of competition may be elucidated and information obtained regarding the conditions under which nitrate and nitrite can most effectively compete with oxygen. A strain of Pseudomonas stutzeri was used throughout the study. Experiments were conducted with the closed electrolytic respirometer flasks which could be flushed with helium gas for anaerobic studies or with 20% oxygen in helium for aerobic studies. The vessels containing a magnetic bar were set on magnetic stirrers to obtain maximum aeration of the medium. Samples of the respirometer atmosphere were assayed with a Beckman GC-2 gas chromatograph. Analyses for CO₂, NO₃⁻, NO₂⁻ and fermentation products were performed by conventional procedures. Dissolved oxygen was measured with a Precision Scientific Oxygen Analyzer. P. stutzeri has a definite requirement for some component supplied by yeast extract. No denitrifying activity is noted in the absence of yeast extract. Neither nitrate nor nitrite can be assimilated by the cell although either can be used as the sole hydrogen acceptor in respiration. Data are presented for nitrite respiration which indicate that this respiratory system may be similar to that of the oxygen system insofar as the atoms of oxygen required for oxidation of the carbon substrate. The nitrate respiring system seems to be less efficient, particularly when using the nitrate to nitrite reduction step. An [0]/C ratio of 2.0 is obtained for nitrite whereas the mean ratio for nitrate is 2.5. Excretion of fermentation products appears to be due to a sluggish acceptor system or to the complete absence of acceptor. Pyruvate, acetate and succinate are metabolized without difficulty. The cell density greatly influenced the dissolved oxygen content of the agitated medium. This in turn determined the rate at which nitrate and nitrite could be reduced in the aerobic system. Too dense a cell suspension leads to "aerobic denitrification" because the conditions of the medium per se were not aerobic. Attempts to correlate dissolved oxygen with "aerobic denitrification showed that at a D.O. as low as 0.9 ppm no denitrification occurred. With cell optical densities of 0.5 to 1.0 (0.25 to 0.5 mg dry wt.) very active stirring of the medium was required to maintain the D.O. above 1.0 ppm for a 24 hour period. Although no N₂ gas was produced, nitrate was reduced to nitrite. The reduction of oxygen uptake by high nitrate concentrations was first noted at 4000 ppm NO₃⁻-N (0.28 molar) and increased with increase of nitrate. A solution of 8000 ppm (0.57 molar) gave a lag lasting about 3 hours. Solutions of other salts at 0.57 molar (NH₄C1, KC1, KNO₃, NH₄NO₃ and K₂SO₄) also caused a reduction in oxygen uptake. The effect was greatest with the nitrate salts. A 0.57 M solution of (NH₄)₂HPO₄ had no apparent effect on oxygen consumption. Solutions (0.50 M) of KC1, K₂SO₄ and KNO₃ exerted no apparent influence on the reduction of nitrate to nitrite, but the further reduction of nitrite to N₂ (nitrite respiration) was almost completely inhibited during the 30 hour test period. This was true for both nitrite accumulated from nitrate and for nitrite added in the absence of nitrate.
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