De Vos, Paul

Heylen, Kim

Sun, Yi-hua

Willems, Anne

English

Ghent University Academic Bibliography

Dissimilatory nitrate reductions in N2O producing Bacillus azotoformans LMG 9581TYihua Sun1, Paul De Vos1,2 , Kim Heylen1 , Anne Willems11 Laboratory of Microbiology, (LM-UGent) Ghent University, Belgium; 2 BCCM/LMG Culture Collection, Ghent University, Belgiumwww.lmg.ugent.beYihua.Sun@UGent.beThis work was financially supported by FWO-Flanders & Chinese Scholarship Council (CSC) • B. azotoformans has reduced N assimilation capacity, and requires organic nitrogen for assimilation.• NH4+ can be assimilated only if certain organic nitrogen is supplied.• NH4+ concentration has a clear influence on growth rate.• Low NH4+ concentration, low NO3- concentration and high C/N ratio do not trigger DNRA in denitrifier B. azotoformans in batch test.Topological model of enzymes involved indissimilatory nitrate reduction (DNR) pathwaysFig 2. Electron transport chain of denitrification and DNRA, basedon gene inventory of B. azotoformans.Fig 1. genes in green exist, genes in redare absent in the genome.• Anaerobic • Dfferent concentrations of nitrate• Different C/N ratios• Measurement of OD600, NO2-/NO3-/NH4+ , N2OTable 1 . Growth observations of B. azotoformans after 15 days aerobic incubation under different combinations of nitrogen sources.Figure 4. NO3- concentration test: 0.2 mM, 0.5 mM, 1 mM, 2 mM, 4 mM, 10 mM. n = 3.  After growth, all initial NO3- converts to N2O, no NH4+production is observed. DNRA is not observed.Figure 5. C/N-NO3- ratio test: C/N=1.5, 3, 7.5, 15, 30, 150, with 0.2 mM or1 mM NO3-. n = 3. Slower growth was obtained after 3 days incubation under high C/N ratio of 150 (P <0.05, SPSS). After 7 days, all initial NO3-converts to N2O. No NH4+ production is observed(P=0.308). DNRA is not observed .N2O is a potent greenhouse gas and a contributor to ozone layer destructions. There are three N2O producingmicrobial processes that have been studied thus far: denitrificaion, dissimilatory nitrate reduction to ammonium(DNRA), and nitrification. The former two respiratory pathways were previously thought to be mutually exclusive,but recently both pathways have been demonstrated to be functional in one organism Shewanella loihica PV-4.Soil bacteria Bacillus azotoformans LMG 9581T also possesses these two pathways, besides, its genome analysesshowed a remarkable redundancy of dissimilatory nitrogen reduction, with multiple copies of each denitrificationgene as well as nrfAH, but has reduced capacity for nitrogen assimilation, with no nas operon nor amtB gene. Wehypothesize that (i) NH4+ and NO3- could not assimilated, and (ii) DNRA, if expressed, would be used for energyconservation, not for nitrogen assimilation.Fig 3. NH4+ concentration test: 4.6 mM (a), 1mM, 0.1mM and 0mM NH4+ (b).n > = 2. Similar growth is obtained in 4 different media (P >0.05), all initialNO3- converts to N2O, growth rate is significantly different (P =2.9x10-5, SPSS),no NH4+ production is observed.A denitrification phenotype, no DNRA observed in Batch testmineral media + 100mg/L yeast extract, 30 mM sodium acetate, 10 mM NO3- , 4.6 mM NH4+ if not indicated otherwise Growth could be partially supported by NH4+ only if certain organic nitrogen existsConclusions:a '+' indicates growth observed ; '-' indicates no growth b 'YNB+' means defined yeast nitrogen base with 5g/l (NH4)2SO4 with amino acids (10mg/l L-histidine, 20mg/l L-methionine, and 20mg/l L-tryptophan)c 'YNB-' means defined yeast nitrogen base without (NH4)2SO4without amino acids 

Dissimilatory nitrate reductions in N2O producing Bacillus azotoformans LMG 9581T

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Dissimilatory nitrate reductions in N2O producing Bacillus azotoformans LMG 9581T

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