How do the bacterial members interact with each other in apyrene-degrading consortium?
Chiho SUZUKI-MINAKUCHI
Agro-Biotechnology Research Center, Graduate School of Agricultural andLife Sciences, The University of Tokyo
Research objective
Polycyclic aromatic hydrocarbons (PAHs) are persistent environmental pollutants
comprising various fused aromatic rings with toxic, mutagenic, and carcinogenic properties.
High molecular weight PAHs containing more than three aromatic rings, such as pyrene, havehigh chemical stability, hydrophobicity, and recalcitrancy. Bioremediation, a set oftechnologies that employ bacteria to degrade target pollutants, is a powerful tool for
remediating PAH-polluted sites. In our previous work, we constructed a pyrene-degrading
bacterial consortium composed of strains isolated from mangrove sediments from Thailand1.
In this consortium, the strain Mycolicibacterium sp. PO1 contained all the genes for thecomplete mineralization of pyrene to TCA cycle intermediates. In contrast, Novosphingobium
pentaromativorans PY1 and Brucella ciceri PW1 contained the genes necessary for thedegradationofphthalateandprotocatechuate,
respectively (Fig. 1).
Aconsortium
comprising these threeFig. 1. Predicted flow of pyrene degradation metabolites among
strains PO1, PY1 and PW1 (in circles) in our model consortium.

strains was predicted to have enhanced
pyrene degradation capability, as the flow ofintermediates would be improved bysynergy between different species capable oftransforming them.
However, such
improvement was only observed when PO1
was combined with either one of the othertwo strains; the three-strain consortia
exhibited the lowest degradation rate (Fig.
2). Using these two- and three-strain

Fig. 2. Degradation of pyrene by single PO1strain and two- or three-strain consortia. Cells
were added at a concentration of 107 CFU/mL to a
medium containing pyrene as sole carbon source.

consortia, we aimed to identify factors that
improve or worsen the degradation capability of degradative consortia with varying strain

compositions.
Methods
1. Transcriptomic analysis of different bacterial consortia
RNA sequencing was performed to evaluate how the transcriptome of each strain changed
within the different consortia and affected their pyrene degradation rates. Cultures of axenic
PO1, PO1 in individual combination with strains PY1 or PW1 (two-strain consortia), and withboth strains simultaneously (three-strain consortium) were prepared at a cell concentration of~107 CFU/mL each and exposed to pyrene as single carbon source. After three days ofincubation, total RNA was extracted from the four cultures, and short-read sequencing wasperformed. For analysis, the RPKM values of each gene were calculated, and pairwise
comparisons between the different consortia were performed. RPKM values of more than
twice or less than half were considered upregulated or downregulated, respectively.
2. Analysis of metabolite flow at the single-cell level using Raman microspectroscopy
To trace the flow of metabolites during pyrene degradation within the different consortia,
Raman microspectroscopy using deuterium-labeled pyrene (pyrene-d10) was performed. This
technique enables tracking the incorporation of a deuterium-labeled substrate into thebiomolecules of individual cells when the carbon-deuterium (C-D) stretching vibration band
appears in their Raman spectrum as hydrogen is replaced by deuterium. Here, axenic culturesof strains PO1, PY1, and PW1, as well as their combinations, were incubated with pyrene-d10
as the sole carbon source. Raman spectra of individual cells in the cultures were obtained at
different time points using a Raman confocal microscope, and the number of cells in eachstrain exhibiting the C-D stretching band was quantified.
Results
1. Transcriptomic analysis of different bacterial consortia
Using the axenic PO1 culture for comparison, changes in
PO1 gene expression levels were identified in the two- andthree-strain consortia. From the previous draft genome
sequence data1, PO1 contained a total of 5574 genes. Of
these, 51 and 1445 were upregulated and downregulated,
respectively, in the three-strain consortium. In particular,
26 of the 30 pyrene-degradative genes were downregulated(Fig. 3, red dots). Downregulation of these degradative
genes may determine the delayed pyrene degradation
observed in the three-strain consortium (Fig. 2).
However, 25 of these 30 genes were also downregulated

Fig. 3. Log2 of RPKM values ofPO1 genes in the three-strain
consortium vs the axenicculture. RPKM values of twiceand half the amount are shown ingreen and blue dots, respectively,
whereaspyrene-degradative
genes are shown in red.

in the PO1+PY1 consortium, whereas none of them showed significantly changed expression
in the PO1+PW1 consortium, even though the pyrene-degradation rates of both consortia wereenhanced. This and the fact that, in the three-strain consortia, downregulated genes also
included genes related to ribosome formation, protein synthesis, and amino acid metabolism,
suggest that changes in the expression of genes other than those directly associated with pyrene
degradation exert an effect on the overall pyrene transformation capability of strain PO1.
2. Analysis of metabolite flow at the single-cell level using Raman microspectroscopy
Raman microspectroscopy results of PO1, PW1, and the PO1 + PW1 consortium aredescribed in this section. PO1 cells exhibit carotenoid resonance Raman bands at 1155 and1514 cm-1. These carotenoid Raman bands were used to distinguish between PO1 and PW1
cells in the consortium when observed under the microscope. When fed pyrene-d10, theindividual PO1 strain displayed a C-D stretching vibration band at 2160 cm-1, indicating
deuterium incorporation. This band was not observed in PW1 when incubated individuallywith the same substrate. However, in the two-strain consortium, after 9 days of incubation, theC-D band was observed in approximately 5% of the examined PW1 cells, indicating deuterium
incorporation from pyrene degradation metabolites released by PO1 strain. Currently, finetuning of the conditions to discriminate PY1 cells within the consortia using its Raman
spectrum is being performed.
Conclusion
In this study, model pyrene-degrading consortia were analyzed using transcriptomics andRaman microspectroscopy to unveil the mechanisms involved in the enhancement orhindrance of their degradation capabilities. In the case of the consortium composed of strains
PO1 and PW1, Raman spectrometric data suggested that the enhancement of its degradation
capability was determined, in part, by strengthening the flow of metabolites within theconsortium. However, the transcriptomic data of each strain combination indicated that thefactors affecting the degradation capabilities were not limited to those involved in theexpression of pyrene-degradative genes. Therefore, strain-specific metabolomic analyses oranalyses of metabolites released into the culture media are necessary. The application ofdegradative bacteria to polluted sites has frequently been hindered by the inability of strains
to sustainably display their degradation capabilities. A determining factor for this inability is
the effect of the interactions between the degrader and the coexisting strains. By elucidating
the causes of the negative effects caused by interspecies interactions, it will be possible toimprove the application of degradative strains in situ.
References
1. Wanapaisan, P. et al. (2018). Synergistic degradation of pyrene by five culturable bacteria
in a mangrove sediment-derived bacterial consortium. J. Hazard. Mater. 342: 561-570.