Abstract
Exposure history and adaptation of the inoculum to chemicals have been shown to influence the outcome of ready biodegradability tests. However, there is a lack of information about the mechanisms involved in microbial adaptation and the implication thereof for the tests. In the present study, we investigated the impact of a long-term exposure to N-methylpiperazine (NMP) and 4-chloroaniline (4CA) of an activated sludge microbial community using chemostat systems. The objective was to characterize the influence of adaptation to the chemicals on an enhanced biodegradation testing, following the OECD 310 guideline. Cultures were used to inoculate the enhanced biodegradability tests, in batch, before and after exposure to each chemical independently in chemostat culture. Composition and diversity of the microbial communities were characterised by 16s rRNA gene amplicon sequencing. Using freshly sampled activated sludge, NMP was not degraded within the 28 d frame of the test while 4CA was completely eliminated. However, after one month of exposure, the community exposed to NMP was adapted and could completely degrade it. This result was in complete contrast with that from the culture exposed for 3 months to 4CA. Long term incubation in the chemostat system led to a progressive loss of the initial biodegradation capacity of the community, as a consequence of the loss of key degrading microorganisms. This study highlights the potential of chemostat systems to induce adaptation to a specific chemical, ultimately resulting in its biodegradation. At the same time, one should be critical of these observations as the dynamics of a microbial community are difficult to maintain in chemostat, as the loss of 4CA biodegradation capacity demonstrates.
Original language | English |
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Article number | 125102 |
Journal | Chemosphere |
Volume | 242 |
DOIs | |
Publication status | Published - 1 Mar 2020 |
Funding
This work was funded by the European Chemical Industry Council (Cefic) Long-range Initiative (LRI project ECO29 ). The authors would like to acknowledge the useful advice and discussions of the CEFIC LRi ECO29 research liaison team. The authors would also like to thank Paul Eijk (Cancer Center Amsterdam, VUmc, Amsterdam) for operating the Illumina MiSeq. The authors declare no competing financial interest. Appendix A Forward primers Primer name Adapter i5 pad-forward link-V3f V3-16sf v3.SA501 AATGATAGGCGACCACCGAGATCTACAC ATCGTACG TATGGTAATT GG CCTACGGGNGGCWGCAG V3.SA501F AATGATACGGCGACCACCGAGATCTACACATGCGTACGTATGGTAATTGGCCTACGGNGGCWGCAG V3.SA502 AATGATACGGCGACCACCGAGATCTACAC ACTATCTG TATGGTAATT GG CCTACGGGNGGCWGCAG V3.SA502F AATGATACGGCGACCACCGAGATCTACACACTATCTGTATGGTAATTGGCCTACGGGNGGCWGCAG v3.SA503 AATGATACGGCGACCACCGAGATCTACAC TAGCGAGT TATGGTAATT GG CCTACGGGNGGCWGCAG v3.SA503F AATGATACGGCGACCACCGAGATCTACACTAGCGAGTTATGGTAATTGGCCTACGGGNGGCWGCAG v3.SA504 AATGATACGGCGACCACCGAGATCTACAC CTGCGTGT TATGGTAATT GG CCTACGGGNGGCWGCAG v3.SA504F AATGATACGGCGACCACCGAGATCTACACCTGCGTGTTATGGTAATTGGCCTACGGGNGGCWGCAG v3.SA505 AATGATACGGCGACCACCGAGATCTACAC TCATCGAG TATGGTAATT GG CCTACGGGNGGCWGCAG v3.SA505F AATGATACGGCGACCACCGAGATCTACACTCATCGAGTATGGTAATTGGCCTACGGGNGGCWGCAG v3.SA506 AATGATACGGCGACCACCGAGATCTACAC CGTGAGTG TATGGTAATT GG CCTACGGGNGGCWGCAG v3.SA506F AATGATACGGCGACCACCGAGATCTACACCGTGAGTGTATGGTAATTGGCCTACGGGNGGCWGCAG v3.SA507 AATGATACGGCGACCACCGAGATCTACAC GGATATCT TATGGTAATT GG CCTACGGGNGGCWGCAG v3.SA507F AATGATACGGCGACCACCGAGATCTACACGGATATCTTATGGTAATTGGCCTACGGGNGGCWGCAG v3.SA508 AATGATACGGCGACCACCGAGATCTACAC GACACCGT TATGGTAATT GG CCTACGGGNGGCWGCAG v3.SA508F AATGATACGGCGACCACCGAGATCTACACGACACCGTTATGGTAATTGGCCTACGGGNGGCWGCAG Reverse primers Primer name Adapter i7 i7-reverse-complement pad-reverse Link-V4r V4–16Sr v4.SA701 CAAGCAGAAGACGGCATACGAGAT AACTCTCG CGAGAGTT AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA701R CAAGCAGAAGACGGCATACGAGATAACTCTCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT V4.Sa702 CAAGCAGAAGACGGCATACGAGAT ACTATGTC GACATAGT AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT V4.Sa702R CAAGCAGAAGACGGCATACGAGATACTATGTCAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA703 CAAGCAGAAGACGGCATACGAGAT AGTAGCGT ACGCTACT AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA703R CAAGCAGAAGACGGCATACGAGATAGTAGCGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA704 CAAGCAGAAGACGGCATACGAGAT CAGTGAGT ACTCACTG AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA704R CAAGCAGAAGACGGCATACGAGATCAGTGAGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA705 CAAGCAGAAGACGGCATACGAGAT CGTACTCA TGAGTACG AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA705R CAAGCAGAAGACGGCATACGAGATCGTACTCAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA706 CAAGCAGAAGACGGCATACGAGAT CTACGCAG CTGCGTAG AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA706R CAAGCAGAAGACGGCATACGAGATCTACGCAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA707 CAAGCAGAAGACGGCATACGAGAT GGAGACTA TAGTCTCC AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA707R CAAGCAGAAGACGGCATACGAGATGGAGACTAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA708 CAAGCAGAAGACGGCATACGAGAT GTCGCTCG CGAGCGAC AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA708R CAAGCAGAAGACGGCATACGAGATGTCGCTCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA709 CAAGCAGAAGACGGCATACGAGAT GTCGTAGT ACTACGAC AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA709R CAAGCAGAAGACGGCATACGAGATGTCGTAGTAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA710 CAAGCAGAAGACGGCATACGAGAT TAGCAGAC GTCTGCTA AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA710R CAAGCAGAAGACGGCATACGAGATTAGCAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA711 CAAGCAGAAGACGGCATACGAGAT TCATAGAC GTCTATGA AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA711R CAAGCAGAAGACGGCATACGAGATTCATAGACAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SA712 CAAGCAGAAGACGGCATACGAGAT TCGCTATA TATAGCGA AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SA712R CAAGCAGAAGACGGCATACGAGATTCGCTATAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SB701 CAAGCAGAAGACGGCATACGAGAT AAGTCGAG CTCGACTT AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SB701R CAAGCAGAAGACGGCATACGAGATAAGTCGAGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SB702 CAAGCAGAAGACGGCATACGAGAT ATACTTCG CGAAGTAT AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SB702R CAAGCAGAAGACGGCATACGAGATATACTTCGAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SB703 CAAGCAGAAGACGGCATACGAGAT AGCTGCTA TAGCAGCT AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SB703R CAAGCAGAAGACGGCATACGAGATAGCTGCTAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT v4.SB704 CAAGCAGAAGACGGCATACGAGAT CATAGAGA TCTCTATG AGTCAGTCAG CC GGACTACHVGGGTWTCTAAT v4.SB704R CAAGCAGAAGACGGCATACGAGATCATAGAGAAGTCAGTCAGCCGGACTACHVGGGTWTCTAAT Sequencing primers V3F_seqprim F TATGGTAATTGGCCTACGGGNGGCWGCAG V4F_seqprim R AGTCAGTCAGCCGGACTACHVGGGTWTCTAAT V4P7_index ATTAGAWACCCBDGTAGTCCGGCTGACTGACT Appendix B Estimated time to reach 10%, 50% and 90% of degradation (DT10, DT50 and DT90) of N-methylpiperazine by each inoculum. Degradation times were estimated using a three-parameters log-logistic model. Estimated degradation time (d) DT10 DT50 DT90 Inoculum Activated sludge 17.92 ± 12.01 1 month exposed to NMP 1.74 ± 0.42 4.25 ± 0.58 10.34 ± 2.98 3 months exposed to NMP 6.94 ± 0.37 7.47 ± 0.26 8.04 ± 0.22 9 months exposed to NMP 6.11 ± 1.68 6.36 ± 1.74 6.63 ± 1.81 Appendix C Results from PERMANOVA Adonis test Permutation: free Number of permutations: 999 Terms added sequentially (first to last) Df SumsOfSqs Location 2 2.7766 Residuals 63 21.1139 Total 65 23.8904 MeanSqs F. Model Location 1.38828 4.1424 Residuals 0.33514 Total R2 Pr (>F) Location 0.11622 0.001 *** Residuals 0.88378 Total 1.00000 --- Signif. codes: 0 ‘***’ 0.001 ‘**’ 0.01 ‘*’ 0.05 ‘.’ 0.1 ‘ ’ 1 Permutation test for homogeneity of multivariate dispersions Permutation: free Number of permutations: 999 Response: Distances Df Sum Sq Mean Sq F N. Perm Pr (>F) Groups 2 0.00105 0.0005275 0.0618 999 0.954 Residuals 63 0.53772 0.0085353 Appendix D Results from differential analysis in chemostat blank. Image 1 A) Differential analysis representing each OTU whose abundance significantly (p 0 were enriched in comparison to the original inoculum while a Log2FoldChange0. Inocula represents the time of incubation in the chemostat used in biodegradation testing (0, 1 month, 3 months, 9 months).
Funders | Funder number |
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Lupus Research Institute | |
European Chemical Industry Council | ECO29 |
Keywords
- 4-chloroaniline
- Biodegradability testing
- Continuous culture
- Microbial community
- N-methylpiperazine
- Organic pollutants