Characterization and activity assay of Nitrous Oxide (N2O) reducing denitrifier isolated from rice soils

Abstract

Nitrous oxide (N2O) is one of the main greenhouse gases and rice fields are among major contributors of this ozone depleting agent. Soil denitrifiers possessing high N2O reduction activity are important for controlling N2O emission. Nitrous oxide reduction is the last step of denitrification process. The aim of this study was to isolate denitrifying bacteria from rice soils possessing high ability of N2O reduction and potential in reducing N2O emission. Soil samples were collected from 6 locations of rice fields in Bogor (West Java) and Tangerang (Banten), Indonesia. Bacteria were isolated through enrichment culture supplemented with NO3 -. Measurement of growth and N2O reduction activity were conducted by growing bacterial isolates in medium with N2O as a sole terminal electron acceptor. Physiological characterization and identification were performed using API 20NE while molecular identification was conducted based on 16S rRNA gene sequence. nosZ gene was analyzed by cloning and sequencing. N2O emission activity was carried out using rice soil slurry experiment. It was found that 10 isolates of denitrifying bacteria could grow on N2O. The bacterial growth indicated by optical density (OD) increased up to 0.12-0.47. During 5 days incubation, isolate BL1, BL2 and BLN1 reduced N2O up to 4.09, 5.41 and 3.91 μmol mL-1 bacterial cultures respectively. BL1, BL2 and BLN1 grew well in medium supplemented with 900, 1380 and 1979 μM N2O but did not grow well with 88 μM N2O in medium during 10 hours, this facts indicated that the isolates consumed N2O during their growth. Maximum growth rate (μmax) using N2O and Monod constant (Ks) of BL1, BL2 and BLN1 were 0.21 h-1 and 102.3 μM h-1, 0.23 h-1 and 213.3 μM h-1, 0.18 h-1 and 172.4 μM h-1 respectively. N2O reduction rate of BL1, BL2 and BLN1 were 0.26, 0.28 and 0.43 μmol mL-1 h-1 respectively. N2O reduction proceeded along with growth. Based on the API 20NE assay, 3 isolates showed different physiological characteristics on hydrolysis of esculine and P-nitrophenyl-β-D-galactopyranoside and also for the assimilation of potassium gluconate and trisodium citrate. From its physiological characteristics, BLN1 was identified as Ochrobactrum anthropi with 99.9% similarity, while BL1 and BL2 were not identified. Based on their 16S rRNA sequence, BL1, BL2 and BLN1 were closely related to Ochrobactrum anthropi ATCC 49188 with similarity of 99, 95 and 98% respectively. Analysis of nosZ gene did not give the expected result. BLN1 isolate reduced the dissolved N2O concentration in surface water from 32.12 to 12.94 nmol L-1 after 6 hours supplementation of 0.6 mmol NO3 -.