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Biological removal of metaldehyde from drinking water: from
38 Illumina MiSeq platform.To identify candidates for proteins involved in metaldehyde degradation, the predicted proteomes of the newly degrading strains were compared in order to find shared sequences using the Blast Score Ratio approach [9]. Additional gene candidates in other strains were identified by homology to these proteins. Random mutagenesis and heterologous expression in E. coli were used to confirm the degrading activity of these candidate proteins [10]. Third generation Oxford Nanopore sequencing was performed to obtain the reference genomes for model degraders A.
calcoaceticus E1 and Sphingobium CMET-H.
To identify the best candidate strains for drinking water purification, bench-scale assays for metaldehyde removal were undertaken at an environmentally-relevant starting concentration of 2.0 µg·L-1, first in pure culture in a defined medium (PBS) and subsequently in minimally-treated water either with clean sand or operational sand with a biofilm (Schmutzdecke from a full-scale SSF).
Metaldehyde was quantified using ultra high-performance liquid chromatography coupled to a triple quadrupole mass spectrometer (LC-MS/MS). The best strains were selected for bioaugmentation assays of SSFs at pilot-scale. SSFs were constructed in six columns with 22.8 L total internal volume each. Schmutzdecke biofilm was permitted to form, and filters were ripened for ~3 weeks. Replicate SSFs were prepared as follows: non-bioaugmented control systems not exposed to metaldehyde, bioaugmented test systems exposed to metaldehyde, non-bioaugmented control systems exposed to metaldehyde. A. calcoaceticus was used to inoculate the SSFs progressively at 1x, 2x and 3x concentrations, while Sphingobium CMET-H was inoculated at a 2x concentration. These systems were operated using continuous flow for a period of 72 d. Metaldehyde was quantified from the influent and effluent by LC-MS/MS. DNA was extracted from the Schmutzdecke and analysed to obtain the composition of the bacterial and archaeal community throughout time using 16S rRNA amplicon analysis. The abundance of the newly identified mahS and mahY degrading genes was used to monitor the degrading strain populations via qPCR in the Schmutzdecke and in the lower SSF levels.
Results and discussion
Six new metaldehyde-degrading bacterial strains were isolated from soil. These corresponded to the taxa Acinetobacter bohemicus, Acinetobacter lwoffii, Pseudomonas vancouverensis, Caballeronia jiangsuensis, Rhodococcus globerulus and Sphingobium sp. Comparative genomic analysis between these strains, a previously isolated A. calcoaceticus E1 [6] and closely-related non-degrading strains (A. calcoaceticus RUH2202) pointed out a highly-conserved gene cluster shared between degraders and absent in non-degrading strains presumptively responsible for metaldehyde degradation in most of the isolates (Table 1.1). These genes share low homology with other genes reported in nucleotide databases and were named mahX and mahY. Other gene with higher homology in databases in the cluster were identified as an aldehyde dehydrogenase (aldH) and a transposase (tnpA). Random mutagenesis confirmed that A. calcoaceticus E1 mutants that lose the ability to degrade metaldehyde had in common mutations in the mahX gene. Heterologous expression of mahX in E. coli DH5α granted this strain the ability to degrade metaldehyde, confirming this gene as the initial determinant in the shared metaldehyde-degrading pathway. A protein sharing a 57% similarity with the protein encoded by the mahX gene was found in the isolated degrading strain from the genus Sphingobium. Heterologous expression in E. coli was used once again to confirm its involvement in metaldehyde degradation and was subsequently denominated mahS.
Third generation sequencing was used to obtain a reference genome for the model degraders Acinetobacter calcoaceticus E1 and Sphingobium CMET-H. For both strains it was determined that the degrading genes were located in plasmids. In A. calcoaceticus IS91 and IS6-family insertion sequences were found surrounding the degrading gene cluster. In Sphingobium CMET-H the plasmid was found to be conjugative. Bioinformatic analysis of the sequences revealed that horizontal gene
39 transfer had taken place and had driven dissemination on the metaldehyde-degrading trait among different taxa.
The library of degraders was first screened in bench-scale assays for metaldehyde removal at micropollutant concentrations in progressively more challenging conditions, including a mixed microbial community with multiple carbon sources. The best performing strains, A. calcoaceticus E1 and Sphingobium CMET-H, showed removal rates of 0.0012 µg·h-1·107 cells-1 and 0.019 µg·h-1·107 cells-1 at this scale. These candidates were then used as inocula for bioaugmentation of pilot-scale SSFs. Here, removal of metaldehyde by A. calcoaceticus E1, was insufficient to achieve compliant water regardless testing increasing cell concentrations (Figure 1.1). Quantification of metaldehyde-degrading genes indicated that aggregation and inadequate distribution of the inoculum in the filters were the likely causes of this outcome. Conversely, bioaugmentation with Sphingobium CMET-H enabled sufficient metaldehyde removal to achieve compliance (volumetric removal: 0.57 µg·L-1·h-1), with undetectable levels in treated water for at least 14 d until reactor decommissioning.
Bioaugmentation did not affect the background SSF microbial community, and filter function was maintained throughout the trial. Here it has been shown for the first time that bioaugmentation is an efficient strategy to remove the adsorption-resistant pesticide metaldehyde from a real water matrix in upscaled systems.
Table 1.1 Predicted proteins shared between metaldehyde-degrading strains, absent from non-degrading strains and their respective BSR values when compared against the reference genome (A. calcoaceticus E1) [11].
______MahX____ ______MahY_____ _______AldH_____ ______TnpA_____
aa length BSR
1 aa length BSR
1 aa length BSR
1 aa length BSR
1
A. calcoaceticus E1 314 1.000 149 1.000 231 1.000 503 1.000
A. calcoaceticus
RUH2202 NP2 - NP2 - 495 0.441 NP2 -
A. bohemicus JMET- C 314 1.000 149 1.000 231 1.000 503 1.000
A. lwoffii SMET-C 314 0.994 149 0.993 231 1.000 387 0.997
P. vancouverensis
SMET-B 314 0.975 149 0.993 39 1.000 327 0.997
C. jiangsuensis SNO-D 314 0.984 149 0.993 88 1.000 262 1.000
1BSR: Blast-Score Ratio
2NP: not present
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Figure 1.1 Percentages of metaldehyde removal calculated as 100 – {([metaldehyde]outlet – [metaldehyde]inlet)/
[metaldehyde]inlet}*100 in SSFs with metaldehyde addition (3 to 6). Arrows indicate the inoculation events of bioaugmentation agents. Dotted lines indicate 95% metaldehyde removal.
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Presenting Author
Dr. V. Castro-Gutiérrez Cranfield University
Is the presenting author an IWA Young Water Professional? Y/N (i.e. an IWA member under 35 years of age) N
Bio: Víctor Castro-Gutiérrez is a Postdoctoral Research Fellow at Cranfield University Water Science Institute. His research is focused on xenobiotic-degrading microorganisms, including their isolation, characterization of their ecology, and biological strategies for contaminant elimination. He utilises cutting edge but ground-truthed tools to understand the fate of xenobiotics and degrading organisms in environmental compartments and engineered systems.
He has a Ph.D. in Biology (University of York), an M.Sc. in Bacteriology (University of Costa Rica) and a B.Sc. in Microbiology (University of Costa Rica). His scientific publication record includes 13 journal papers focused on bioremediation, six of them as a first author, and four book chapters. Significant contributions from these publications include the first identification and description of the genes involved in microbial degradation of the pesticide metaldehyde, as well as the study of the microbial populations present in on-farm pesticide biopurification systems. He has also worked on wastewater-based epidemiology for SARS-CoV-2 and other pathogens.
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