Understanding the Linkage between Elevation and the Activated-Sludge Bacterial Community along a 3,600-Meter Elevation Gradient in China

Abstract

To understand the relationship between elevation and bacterial communities in wastewater treatment plants (WWTPs), bacterial communities in 21 municipal WWTPs across China, located 9 to 3,660 m above sea level (masl), were investigated by 454 pyrosequencing. A threshold for the association of elevation with bacterial community richness and evenness was observed at approximately 1,200 masl. At lower elevations, both richness and evenness were not significantly associated with elevation. At higher elevations, significant declines with increased elevations were observed for community richness and evenness. The declining evenness trend at the phylum level was reflected by distinct trends in relative abundance for individual bacterial phyla. Betaproteobacteria, Bacteroidetes, and Firmicutes displayed significant increases, while most other phyla showed declines. Spearman correlation analysis indicated that the community richness and evenness at high elevations were more correlated with elevation than with any other single environmental variable. Redundancy analysis indicated that the contribution of elevation to community composition variances increased from 3% at lower elevations to 11% at higher elevations whereas the community composition variance at higher elevations remained much more explained by operational variables (39.2%) than by elevation. The influent total phosphorus concentration, food/microorganism ratio, and treatment process were the three shared dominant contributors to the community composition variance across the whole elevation gradient, followed by effluent ammonia nitrogen and temperature at higher elevations.

FIG 1

Geographical distribution of 21 wastewater treatment plants (WWTPs) in China. Elevations of these WWTPs are represented by the sizes of symbols. (The map was created using the online software Mapping-together [Map Hui].)

FIG 2

Relative abundances of bacterial phyla and classes of Proteobacteria in 21 WWTPs. “Minor” refers to phyla with a maximum abundance of 1% in any sample. The relative abundances of bacterial phyla in each WWTP were calculated by their average percentages in the total sequences of the triplicate samples.

FIG 3

Heat map of the top 10 abundant genera in each WWTP. The relative abundances of bacterial genera in each WWTP were calculated by their average percentages in the total sequences of the triplicate samples.

FIG 4

Variance in bacterial OTU richness (a) and evenness (b) across the elevation gradient. At elevations above 1,200 masl, bacterial community richness and evenness were negatively correlated with elevation. Model choice was based on the Akaike information criteria. Taxon richness is represented by the OTU richness. Evenness represents the similarity of the population sizes of the taxa present. The two indices were calculated based on normalized sequences from across the 63 samples.

FIG 5

Spearman correlation coefficients for correlations between bacterial diversity indices and environmental variables for high-elevation WWTPs. An asterisk indicates that the variable is significantly correlated with the diversity index (P < 0.001).

FIG 6

Variance in the relative abundance of each bacterial phylum with elevation for elevations above 1,200 m. The proportions of Betaproteobacteria, Bacteroidetes, and Firmicutes in the total bacterial community increased linearly with increased elevations (regression analysis; R² > 0.97; P < 0.01). Model choice was based on the Akaike information criteria.

FIG 7

Redundancy analysis of bacterial communities and environmental variables. (a) Variation partitioning analysis was applied to calculate the contributions of wastewater characteristic parameters (W), operational parameters (O), and geographical distributions (G) to the variance of bacterial community structures. RDA ordinations corresponding to bacterial communities were used for the WWTPs at elevations of 9 to 1,200 masl (b) and 1,200 to 3,660 masl (c). E, elevation; LL, longitude and latitude; HRT, hydraulic residence time; DO, dissolved oxygen; BOD, biological oxygen demand; COD, chemical oxygen demand; TP, total phosphorus; F/M, food/microorganism ratio; C/N, COD/TN ratio.