Simultaneous heterotrophic nitrification and aerobic denitrification by Chryseobacterium sp. R31 isolated from abattoir wastewater

Abstract

A heterotrophic carbon utilizing microbe (R31) capable of simultaneous nitrification and denitrification (SND) was isolated from wastewater of an Indian slaughterhouse. From an initial COD value of 583.0 mg/L, 95.54% was removed whilst, from a starting NH4 (+)-N concentration of 55.7 mg/L, 95.87% was removed after 48 h contact. The concentrations of the intermediates hydroxylamine, nitrite, and nitrate were low, thus ensuring nitrogen removal. Aerobic denitrification occurring during ammonium removal by R31 was confirmed by utilization of both nitrate and nitrite as nitrogen substrates. Glucose and succinate were superior while acetate and citrate were poor substrates for nitrogen removal. Molecular phylogenetic identification, supported by chemotaxonomic and physiological properties, assigned R31 as a close relative of Chryseobacterium haifense. The NH4 (+)-N utilization rate and growth of strain R31 were found to be higher at C/N = 10 in comparison to those achieved with C/N ratios of 5 and 20. Monod kinetic coefficients, half saturation concentration (K s ), maximum rate of substrate utilization (k), yield coefficient, (Y) and endogenous decay coefficient (K d ) indicated potential application of R31 in large-scale SND process. This is the first report on concomitant carbon oxidation, nitrification, and denitrification in the genus Chryseobacterium and the associated kinetic coefficients.

Figure 1

Unrooted phylogenetic tree obtained by the neighbor-joining (NJ) method based on 16S rRNA gene sequences depicting the position of strain Chryseobacterium sp. R31 amongst its phylogenetic neighbors. Numbers at nodes designate levels of bootstrap support (%) based on a NJ analysis of 1000 resampled datasets; only values higher than 50% are displayed. Asterisks denote branches that were obtained using the maximum parsimony and maximum likelihood algorithms. NCBI accession numbers are provided in parentheses. Bar = 0.1 nucleotide substitutions per site. The sequence of Bacillus subtilis DSM 10 (AJ276351) was applied as an outgroup.

Figure 2

Time profile of carbon oxidation and growth of Chryseobacterium sp. R31. Growth (filled diamonds) and COD removal (%) (filled squares). Error bars represent one SD (n = 9).

Figure 3

Time profile of ammonium oxidation by Chryseobacterium sp. R31. Ammonium nitrogen (filled diamonds), nitrite nitrogen (filled squares), nitrate nitrogen (filled triangles), and hydroxylamine (filled circle). Error bars represent one SD (n = 9).

Figure 4

(a) Influence of various carbon substrates on growth of Chryseobacterium sp. R31. Sodium succinate (filled diamonds), glucose (filled squares), trisodium citrate (filled triangles), and sodium acetate (filled circles). Error bars represent one SD (n = 9). (b) Influence of various carbon substrates on ammonium removal by Chryseobacterium sp. R31. Sodium succinate (filled diamonds), glucose (filled squares), trisodium citrate (filled triangles), and sodium acetate (filled circles). Error bars represent one SD (n = 9).

Figure 5

(a) Influence of various nitrogen substrates on growth of Chryseobacterium sp. R31. Ammonium chloride (filled squares), sodium nitrate (filled triangles), and sodium nitrite (filled circles). Error bars represent one SD (n = 9). (b) Influence of various nitrogen substrates on nitrogen removal by Chryseobacterium sp. R31. Ammonium chloride (filled squares), sodium nitrate (filled triangles), and sodium nitrite (filled circles). Error bars represent one SD (n = 9).

Figure 6

(a) Influence of different C/N ratios on growth of Chryseobacterium sp. R31. C/N = 5 (filled diamonds), C/N = 10 (filled squares), and C/N = 20 (filled triangles). Error bars represent one SD (n = 9). (b) Influence of different C/N ratios on ammonium removal by Chryseobacterium sp. R31. C/N = 5 (filled diamonds), C/N = 10 (filled squares), and C/N = 20 (filled triangles). Error bars represent one SD (n = 9).

Figure 7

Lineweaver-Burk plot for determination of substrate utilization kinetic constants for (a) carbon oxidation, (b) nitrification, and (c) denitrification and growth kinetic constants for (d) carbon oxidation, (e) nitrification, and (f) denitrification by Chryseobacterium sp. R31. Each data point represents the mean value of nine determinations. Error is within one SD of the mean.