An integrated meta-omics approach reveals substrates involved in synergistic interactions in a bisphenol A (BPA)-degrading microbial community

Abstract

Background: Understanding microbial interactions in engineering bioprocesses is important to enhance and optimize performance outcomes and requires dissection of the multi-layer complexities of microbial communities. However, unraveling microbial interactions as well as substrates involved in complex microbial communities is a challenging task. Here, we demonstrate an integrated approach of metagenomics, metatranscriptomics, and targeted metabolite analysis to identify the substrates involved in interspecies interactions from a potential cross-feeding model community-bisphenol A (BPA)-biodegrading community, aiming to establish an identification method of microbial interactions in engineering or environmental bioprocesses.

Results: The community-level BPA-metabolic pathway was constructed using integrated metagenomics and targeted metabolite analyses. The dynamics of active functions and metabolism of major community members were identified using metagenomic and metatranscriptomic analyses in concert. Correlating the community BPA biodegradation performance to the individual bacterial activities enabled the discovery of substrates involved in a synergistic interaction of cross-feeding between BPA-degrading Sphingonomas species and intermediate users, Pseudomonas sp. and Pusillimonas sp. This proposed synergistic interaction was confirmed by the co-culture of a Sphingonomas sp. and Pseudomonas sp. isolates, which demonstrated enhanced BPA biodegradation compared to the isolate of Sphingonomas sp. alone.

Conclusion: The three types of integrated meta-omics analyses effectively revealed the metabolic capability at both community-wide and individual bacterial levels. The correlation between these two levels revealed the hidden connection between apparent overall community performance and the contributions of individual community members and their interactions in a BPA-degrading microbial community. In addition, we demonstrated that using integrated multi-omics in conjunction with culture-based confirmation approach is effective to elucidate the microbial interactions affecting the performance outcome. We foresee this approach would contribute the future application and operation of environmental bioprocesses on a knowledge-based control.

Keywords: Bacterial interactions; Biodegradation; Bisphenol A; Integrated meta-omics.

Fig. 1

Scheme of the experimental design and analytical pipeline used in this study. Targeted metabolite analysis (green lines) was used to investigate the biodegradation intermediates and dynamics, which were integrated with metagenomic annotation (blue lines) to construct the community-wide BPA-mineralizing pathways. Integrated analyses of 16S-sequencing (light blue lines), metagenomics, and metatranscriptomics (orange lines) identified functionally active populations and metabolic pathways of individual strains in the community. Correlation of individual activities and overall community-wide BPA mineralization revealed the interactions of major community populations (thick red line) which was confirmed by the genomic and metabolite analysis of bacterial isolates from the community (fine blue line and fine red line, respectively)

Fig. 2

Community-level BPA biodegradation dynamics and pathways. Biodegradation of BPA and intermediates in the enrichment culture amended with (a) 50 mg L⁻¹ BPA, (b) 40 mg L⁻¹ 1-BP, (c) 40 mg L⁻¹ 4-DM, or (d) 10 mg L⁻¹ 2-BP. (a-1) 1-BP pathway and (a-2) 2-BP metabolites detected in the enrichment culture amended 50 mg L⁻¹ BPA. * The concentrations are indicated using the secondary Y-axis. (e) Proposed BPA-mineralization pathways of the enrichment culture. Intermediates marked by orange indicate the degradation products detected by LC-MS/MS from enrichment culture. The details on detection of degradation products are summarized in Additional file 9: Table S1. Genes colored blue were deduced from metagenomic analysis. Abbreviations, BPA, bisphenol A; 1-BP, 1,2-bis(4-hydroxyphenyl)-2-propanol; 2-BP, 2,2-bis(4-hydroxyphenyl)-1-propanol; 4-DM, 4,4′-dihydroxyl-α-methylstilbene; 2,4-BP: 2,2-bis(4-hydroxyphenyl)-propanoate; 3,4-BP, 2,3-bis(4-hydroxyphenyl)-1,2-propanediol; 4-HBD, 4-hydroxybenzaldehyde; 4-HBZ, 4-hydroxybenzoate; 4-HAP, 4-hydroxy-acetophenone; 4-HPAT, 4′-hydroxyphenyl acetate; 4-HPAH, 4-hydroxyphenacyl alcohol; HQN, hydroquinone; 3,4-DHB, 3,4-dihydroxybenzoate; 4-CHS, 4-carboxy-2-hydroxymuconate semialdehyde; 2-HHD, 2-hydroxy-2-hydropyrone-4,6-dicarboxylate; 2-PD, 2-pyrone-4,6-dicarboxylate; 4-OS, 4-oxalome-saconate; 4-CHM, 4-carboxy-2-hydroxy-cis,cis-muconate; 4-CHO, 4-carboxy-4-hydroxy-2-oxoadipate; PYV, pyruvate; OLA, oxaloacetate; HMS, 4-hydroxymuconic semialdehyde; MLL, maleylacetate; β-CM, β-carboxy-muconate; γ-CL, gamma-carboxymucono-lactone; 3-OEL, 3-oxoadipate-enol-lactone; 3-ODP, 3-oxoadipate; 3-OAC, 3-oxoadipyl-CoA; SCC, succinyl-CoA. Gene names are summarized in Additional file 10: Table S6

Fig. 3

Genomes of dominant species recovered from binning analysis using bi-dimensional coverage plots on metagenomic datasets. Percentage suggests the relative abundance of 16S rRNA gene of the recovered genome in the community

Fig. 4

Differential expression of genes involved in the BPA-mineralization process and the pattern of substrate cross-feeding between BPA-degrading Sphingomonas spp. and BPA non-degraders Pseudomonas sp. and Pusillimonas sp. Specific label “S,” “1,” and “2” indicates Sph-1 and Sph-2 share the same sequence between each other, Sph-1 unique sequence and Sph-2 unique sequence, respectively

Fig. 5

Biodegradation behavior and growth of Sph-2 in axenic culture and in co-culture with Pseudomonas sp. Biodegradation of BPA by Sph-2 axenic culture (a) or Sph-2/Pseudomonas sp. co-culture (b); total organic carbon detected in Sph-2 axenic culture and co-culture with Pseudomonas sp. (c); cell growth detected in Sph-2 axenic culture and co-culture with Pseudomonas sp. (d). Error bars indicate the standard deviation of biological triplicates

Fig. 6

Proposed substrate cross-feeding between BPA-degrading Sphingomonas sp. and non-degrading Pseudomonas sp. or Pusillimonas sp. A simplified pathway presentation of major substrates found in the bulk community environment (a); Sphingomonas sp. in the community transformed BPA to 1-BP, 4-DM, and 2-BP during the initial stage of biodegradation (b); then further transformed 1-BP and 4-DM to either 4-HBD or 4-HAP; 4-HBD was quickly converted to 4-HBZ; both of 4-HBD and 4-HBZ were used by Pseudomonas sp. and Pusillimonas sp. (c); and Sphingomonas started consuming 2-BP after 4-HBD/4-HBZ depletion, while Pseudomonas coverts 4-HAP to 4-HPAT and then HQN (d); HQN was degraded by the Pusillimonas. Lines with green arrow suggests the interaction was confirmed by experiment by using isolates