Diversity Manipulation of Psychrophilic Bacterial Consortia for Improved Biological Treatment of Medium-Strength Wastewater at Low Temperature

Abstract

Psychrophilic bacteria are valuable biocatalysts to develop robust bioaugmentation formulations for enhanced wastewater treatment at low temperatures or fluctuating temperature conditions. Here, using different biodiversity indices [based on species richness (SR), phylogenetic diversity (PD) and functional diversity (FD)], we studied the effects of microbial diversity of artificial bacterial consortia on the biomass gross yields (measured through OD₆₀₀) and removal efficiency of soluble chemical oxygen demand (mg sCOD removed/mg sCOD introduced) in synthetic, medium-strength wastewater. We built artificial consortia out of one to six bacterial strains isolated at 4°C through combinatorial biodiversity experiments. Increasing species richness resulted in improved sCOD removal efficiency (i.e., 0.266 ± 0.146, 0.542 ± 0.155, 0.742 ± 0.136, 0.822 ± 0.019 for mono-, tri-, penta-and hexacultures, respectively) and higher biomass gross yields (i.e., 0.065 ± 0.052, 0.132 ± 0.046, 0.173 ± 0.049, 0.216 ± 0.019 for mono-, tri-, penta,- and hexacultures, respectively). This positive relationship between biodiversity, sCOD removal and biomass gross yield was also observed when considering metabolic profiling (functional diversity) or evolutionary relationships (phylogenetic diversity). The positive effect of biodiversity on sCOD removal efficiency could be attributed to the selection of a particular, best-performing species (i.e., Pedobacter sp.) as well as complementary use of carbon resources among consortia members (i.e., complementarity effects). Among the biodiversity indices, PD diversity metrics explained higher variation in sCOD removal than SR and FD diversity metrics. For a more effective bioaugmentation, our results stress the importance of using phylogenetically diverse consortia, with an increased degradation ability, instead of single pure cultures. Moreover, PD could be used as an assembly rule to guide the composition of mixed cultures for wastewater bioaugmentation under psychrophilic conditions.

Keywords: bioaugmentation; functional diversity; phylogenetic diversity; psychrophiles; synthetic consortia; wastewater treatment.

Figure 1

Effect of species richness (SR) (A, D), Faith's phylogenetic diversity (FPD) (B, E) and dendrogram-based functional diversity (dFP) (C, F) on biomass growth (OD₆₀₀) after a 144-h incubation time (A–C) and sCOD removal efficiency (sCOD-RE) at the end of the experiment (192 h) (D–F). The initial OD₆₀₀ in all cultures was adjusted to 2.0 × 10⁷ total cells mL⁻¹ and the initial value of sCOD in SW was 540 ± 21 mg sCOD L⁻¹. Continuous lines denote simple linear regression significant at p < 0.05.

Figure 2

Soluble COD removal efficiency (sCOD-RE) as a function of Faith's phylogenetic diversity (FPD) and dendrogram-based functional diversity (dFD) for intermediate species richness levels: (A,C) SR = 3; (B,D) SR = 5. Continuous lines indicate simple linear regression significant at p < 0.05; dashed lines denote a non-significant simple linear regression (p > 0.05).

Figure 3

(A) Plot of the t-ratio for the estimation of species identity on the cell-density (black bars, 144 h of incubation) and sCOD degradation (gray bars, 192 h of incubation). The horizontal dotted lines indicate the 95% confidence interval. A, Arthrobacter sp.; PD, Pedobacter sp.; J, Janthinobacterium sp.; PS, Pseudomonas sp.; R, Rhodococcus sp.; B, Brevundimonas sp. (B) box plot comparing the sCOD degradation activity of synthetic consortia including Pedobacter sp. and synthetic consortia without Pedobacter sp. (two-sample t-test). The box plot shows the minimum and maximum values, with the box presenting the median and the quartiles.

Figure 4

Non-transgressive overyielding values associated with biomass growth (A) and sCOD removal efficiency (B) for each richness level. Transgressive overyielding values associated with biomass growth (C) and sCOD removal (D) for each richness level. Values that did not differ significantly from 0 are shown in light gray (one sample t-test). Values with different letters indicates statistically significant difference [Tukey's test (A); Dunn's test (B,C); Dunnett's T3 test (D)]. Detailed values of non-transgressive and transgressive overyielding for each bacterial consortium are presented in Figure S8. The violin plot shows the minimum and maximum values, with the dashed line representing the median and the dotted lines the quartiles.