Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes - PubMed (original) (raw)

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes

Yi Yang et al. ISME J. 2017 Dec.

Abstract

Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55±0.64 × 108 cells were measured per μmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1

Figure 1

Community structure of PCE-fed and VC-fed grape pomace enrichment cultures as revealed by 16S rRNA gene amplicon sequencing. The relative abundances of operational taxonomic units (OTUs) representing bacteria are shown at the genus level. Rare groups representing <1% of total bacterial community were categorized as ‘Others’. OTUs representing the phyla Bacteroidetes and Firmicutes could not be classified at the genus level. WPS-2, WWE1, vadinCA02 and HA73 represent uncultured bacterial groups. The full colour version of this figure is available at ISME Journal online.

Figure 2

Figure 2

VC-to-ethene reductive dechlorination in the enrichment culture harboring strain GP (triangles, VC; squares, methane; open circles, ethene; solid bar, Dhgm 16S rRNA gene copies). Data points are averages of duplicate cultures and the error bars show one standard deviation. If no error bars are shown, the standard deviations were too small to be illustrated.

Figure 3

Figure 3

Principal component analysis of taxonomic profiles at the phylum level (a) and functional profiles (b) of metagenomes from the dechlorinating consortia ANAS, Donna ll, KB-1 and GP. Included in the analysis were the metagenome data sets from two non-dechlorinating microbial communities (AMD and Antarctica). For the functional comparison (b), the metagenomic sequences were classified into SEED categories and the distribution of SEED categories was compared. The full colour version of this figure is available at ISME Journal online.

Figure 4

Figure 4

Midpoint rooted maximum likelihood phylogenetic tree showing the relationship of ‘Candidatus Dehalogenimonas etheniformans’ to other members of the Chloroflexi based on concatenated 5S-16S-23S rRNA genes. The closest relatives of ‘Candidatus Dehalogenimonas etheniformans’ were Dhgm formicexedens strain NST-14T and Dhgm alkenigignens strain IP3-3. The accession numbers for each genome are listed in Supplementary Table 8. The scale bar indicates 0.01 nucleotide substitution per site.

Figure 5

Figure 5

Relative RDase A protein abundances based on normalized spectral counts of peptides detected in cultures grownwith TCE, cDCE, 1,1-DCE and VC as electron acceptors.The numbers indicate the normalized spectral counts. The full colour version of this figure is available at ISME Journal online.

Figure 6

Figure 6

Phylogenetic relationships of 528 RDase A protein sequences. The analysis included 355 sequences reported by Hug et al. (Hug and Edwards, 2013) plus the RDase sequences of Dhc strains CG1, CG4, CG5, SG1, and Dhgm strains WBC-2 and GP (Supplementary Information). Shaded in green and blue are clusters comprising known PCB and VC RDases, respectively. Putative RDases of strain GP are shown in red font and the stars highlight RDases prokka_02004 (blue), prokka_01300 (black), prokka_01297 (green) and prokka_00862 (red). The scale bar indicates 0.5 amino acid substitution per site. The full colour version of this figure is available at ISME Journal online.

Figure 7

Figure 7

Distribution of Dhgm and Dhc in 1173 groundwater samples collected from 111 chlorinated solvent-impacted sites. Boxes represent the upper (75th) and lower (25th) quartiles and whiskers depict the non-outlier range. Outliers have Dhgm/Dhc ratios greater than the upper quartile Dhgm/Dhc ratio plus 1.5 times the difference between upper and lower quartile Dhgm/Dhc ratios, or less than the lower quartile Dhgm/Dhc ratio minus 1.5 times the difference between upper and lower quartile Dhgm/Dhc ratios. Dhgm/Dhc ratios greater than the upper quartile plus 2 times the difference of the quartiles or less than the lower quartile minus 2 times the difference between the quartiles were defined as extremes. Box plots were created using Statistica v12.0 (StatSoft, Inc., Tulsa, OK, USA). The full colour version of this figure is available at ISME Journal online.

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