A pyridine-degrading bacterial strain was isolated from the oxic zone of spent shale column. The microorganism was an aerobic and pleomorphic coryneform bacterium with LL-diaminopimelic acid in the cell wall. On the basis of its phylogenetic and chemotaxonomic characteristics, the strain was identified as Nocardioides sp. OS4. The pyridine was completely degraded and the growth yield was 0.30g cellㆍg$pyridine^{-1}$. This strain also degraded some compounds of the basic fraction of retort water and various other aromatic compounds. It was found that the total organic carbon in retort water could be decreased remarkably by inoculation of the strain. These results imply that microorganisms have significant role in the removal of organic pollutants by spent shale column and this strain could be used at the retort water-contaminated sites.
The pathway of the pyridine degradation was investigated in the aerobic Nocardioides sp. OS4 and denitrifying Azoarcus evansii pF6. Hydroxy-intermediates apparently were not involved in pyridine metabolism by these strains. Pyridine induction of NAD-linked glutaric dialdehyde dehydrogenase and isocitrate lyase suggest that Azoarcus evansii pF6 used the N-C2 ring cleavage pathway to degrade pyridine. In case of Nocardioides sp. OS4, pyridine didn't induced the glutarate-dialdehyde dehydrogenase, isocitratase, NAD-linked succinate-semialdehyde dehydrogenase, and formamidase. The results suggest that that the system for the metabolism of pyridine by Nocardioides sp. OS4 may not use the C2-C3 or N-C2 ring cleavage.
In order to elucidate tolerance of the cells, fatty acid composition of the cells grown in the presence of pyridine was examined. When pyridine was used as a sole inhibitory substrate, the fatty acid composition of Nocardioides sp. OS4 was quite different from that in other readily available substrates. When compared with the fatty acid composition in a complex medium, the proportion of isopalmitic acid drastically decreased. Concomitantly, this decrease of the branched-chain fatty acid was accompanied by an increase of the straight-chain, especially long-chain, fatty acids. However, since the fatty acid composition was not sensitively affected by the change of the pyridine concentration, the fatty acid composition could not be used in the monitoring the tolerance of cells in the range of the pyridine concentration used.
Freely suspended and Ca-alginate-immobilized cells of Nocardioides sp. OS4 were used for degradation of pyridine. When the immobilized cells were used for pyridine degradation, neither specific pyridine degradation rate nor tolerance against pyridine was improved. However, a high volumetric pyridine degradation rate in the range of $0.082-0.129 gㆍl^{-1}ㆍhr^{-1}$ could be achieved by the immobilized cells because of a high cell concentration. The effect of the presence of supplementary carbon sources on the growth and pyridine-degrading activity of freely suspended and calcium alginate-immobilized Nocardioides sp. OS4 was investigated. Although the supplementary carbon sources could be degraded simultaneously with pyridine, Nocardioides sp. OS4 exhibited a preference of pyridine over supplementary carbon sources. In the semi-continuous immobilized cell culture, immobilized cells also exhibited the preference of pyridine over supplementary carbon sources and did not switch the substrate preference throughout the culture. Further, when the immobilized cells were reused in degrading pyridine with a concentration of $2-4 gㆍl^{-1}$, hey did not lose their pyridine degrading activity for 2 weeks. Taken together, the use of immobilized Nocardioides sp. OS4 for degradation of pyridine is quite feasible because of the preference of pyridine over supplementary carbon sources, high volumetric pyridine degradation rate, and reusability of immobilized cells.
In order to improve the processes of biodegradation of pyridine, new denitrifying bacteria which could degrade pyridine in both aerobic and anaerobic conditions were isolated and studied. These isolates appeared to be closely related to the species of Azoarcus evansii according to the 16S rRNA sequence analysis. Strain pF6 could degrade pyridine in the presence of nitrate, nitrite, or nitrous oxide as electron acceptors. Degradation of pyridine and denitrification were not sensitively affected by the redox potential which gradually decreased from 150 to -200 mV. In a batch culture with the nitrate concentration higher than 6 mM, nitrite transiently accumulated in the denitrification significantly inhibited cell growth and pyridine degradation. Growth yield to pyridine was slightly decreased in the denitrifying conditions, compared with that in the aerobic degradation of pyridine. Furthermore, when the pyridine concentration used was above $1 gㆍl^{-1}$, specific growth rate in the denitrifying conditions was higher than that in the aerobic conditions. Considering these characteristics, a newly isolated denitrifying bacterium, strain pF6, has advantages in the field application, compared to strictly aerobic bacteria.
