الفهرس 
INTRODUCTIONOnly 14 pages are availabe for public view
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Abstract
The actinomycetes Rhodococcus erythro~olis HW1-13, Rhodococcus rhodochrous HW2-25, Rhodococcus coprophilus HW2-27, and a bacterial strain belonging to the Comamonas-, Hydrogenophaga-, Acidovorax-group were identified as heterotrophic nitrifiers. Heterotrophic nitrification activity in complex media proceeded best if the media were supplemented with 25 mM MES buffer and were adjusted to pH 6.0 (starting pH).
2. During the exponential growth phase the following intermediates/products of heterotrophic nitrification activity were excreted into the culture media: hydroxylamine, nitrite, and nitrate. Heterotrophic nitrification catalyzed by the bacterial strains tested was not a phenomenon of old or stationary cultures, but took place during the whole phase of exponential growth and must, therefore, be linked to active cell metabolism.
3. NO2- and N20-production was never detected in the experiments with intact cells. On the other hand, nitric oxide (NO) was produced by the heterotrophically nitrifying cultures of the Rhodococci in addition to NH2OH, nitrite, and nitrate.
Nitrite/nitrate production in closed cultures (for NO measurement) was lower than in parallel cultures grown under conditions, where atmospheric oxygen supply to the cultures was not limited. This may indicate that sufficient supply of molecular oxygen is a prerequisite for optimal performance of heterotrophic nitrification activity as is the case for autotrophic ammonia-oxidizers, the ammonia-monooxygenase of which depends on molecular oxygen.
4. The amount of products/intermediates of heterotrophic nitrification produced increased with increasing concentrations of peptone and meat extract in the PM-media. Meat extract was identified as a better substrate for heterotrophic nitrification than peptone at comparable concentrations. The addition of high concentrations of glucose to PM-medium resulted in a significant increase of the NO produced during heterotrophic nitrification of R. erythropolis HW1-13.
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5. Heterotrophic nitrification activity of cultures, measured as nitrite-, nitrate-, NH2OH-, and NO-production, was also observed in cultures, if defined media containing ammonium salts as the sole nitrogen source were used. The presence of an organic nitrogen source in the culture media was, therefore, not a prerequisite to catalyze heterotrophic nitrification.
6. In complex media (PM-media) and defined media containing organic nitrogen compounds the cells catalyzed parallel to heterotrophic nitrification ammonification. During the whole phase of active growth ammonia was liberated from the proteins/amino acids present in the media. Most likely, the ammonium produced from these sources was used as a substrate for nitrification, but ultimate evidence could not be given during this study.
7. The spectrum of products identified in the heterotrophically nitrifying cultures (NH2OH, nitrite, nitrate, and NO) tested indicated a comparable pathway of heterotrophic nitrification to that realized in autotrophic ammonia- and nitrite-oxidizers.
8. At a constant C/N ratio of 3:1 glutamine was identified as a better nitrogen source for heterotrophic nitrification than the other organic N-containing compounds: arginine, asparagine and urea. With glutamine the highest NO-production rates were observed in heterotrophically nitrifying cultures of R. erythropolis HW1-13.
9. Inhibitors known to inhibit autotrophic ammonia-oxidizers were tested with regard to their inhibition potential on heterotrophic nitrification performed by the bacterial strains under study. Increasing concentrations of acetylene had an increasing inhibitory effect on nitrite- and nitrate production of the cultures tested indicating that an ammonia-monooxygenase like enzyme is active also in the heterotrophic nitrifiers studied. In contrast to nitrite and nitrate production, growth remained unaffected by the acetylene concentrations used in R. erythropolis HW1-13, R. coprophilus HW2-27 and R. rhodochrous
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HW2-25. With 8-HQ, another inhibitor of ammonia-monooxygenase, no conclusive results were obtained, since in addition to nitrification activity growth was also drastically affected.
10. Hydrazin, known to inhibit hydroxylamine-oxidoreductase, but not ammonia-monooxygenase of autotrophic ammonia oxidizers, also inhibited nitrite production in cultures of R. erythropolis HW1-
13. As a consequence, a dramatic accumulation of hydroxylamine in the culture medium of cultures supplemented with hydrazin was observed, while in control cultures lacking hydrazin only small accumulation of hydroxylamine was observed. These results indicate that a hydroxylamine-oxidoreductase is present in R. erythropolis and give additional evidence that also a ammoniamonooxygenase is active.
11. Cell free extracts prepared from heterotrophically nitrifying cultures of R. erythropolis HW1-13 exhibited nitrite, nitrate, and N20 production in the presence of hydroxylamine as the substrate and cytochrome c as the electron acceptor. Furthermore, it could be demonstrated that NH2OH was very effectively converted by the extracts to nitrite and nitrate (76% recovery) in the presence of a suitable electron acceptor (cytochrome c). These results obtained with cell free extracts also strongly indicate, that a hydroxylamine-oxidoreductase is indeed present in R. erythropolis HW1-13. Since nitrate also was produced, it can be assumed from these experiments, that in addition to a HAO also a nitrite-oxidoreductase was present in the extracts of R. erythropolis HW1-13.
12. When only catalytic amounts of NH2OH (known to be effective in transforming an inactive (oxidized) ammonia-monooxygenase of N. europaea to an active (reduced state) were added to the assay mixtures, nitrite and nitrate production was also catalyzed by the extracts in the presence of cytochrome c. This results indicates that in addition to a hydroxylamine-oxidoreductase and a nitrite-oxidoreductase an ammonia-monooxygenase is most likely also present in R. erythropolis HW1-13.
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13. Extracts of R. erythropolis HW1-13 exhibiting hydroxylamineoxidoreductase-activity could be stored frozen at -18% C and in liquid nitrogen at least for 10 hours without dramatic loss in nitrite producing activity from NH2OH (hydroxylamineoxidoreductase).
14. A hydroxylamine-oxidoreductase could be enriched from extracts of R. erythropolis HW1-13 by sodium chloride precipitation (10 % saturation).
15. For the first time an activity stain (after native polyacrylamide gel electrophoresis) for a hydroxylamineoxidoreductase was successfully established, that allows to identify a hydroxylamine-oxidoreductase on gels.
16. The development of the activity stain allowed not only to identify the position/location of the hydroxylamineoxidoreductase on gels, but also was a prerequisite for the determination of the apparent molecular weight of the hydroxylamine-oxidoreductase of R. erythropolis HW1-13. The apparent molecular weight was determined to be 112.16 kD.