Desulfitobacterium contributes to the microbial transformation of 2,4,5‐T by methanogenic enrichment cultures from a Vietnamese active landfill

Summary The herbicide 2,4,5‐trichlorophenoxyacetic acid (2,4,5‐T) was a major component of Agent Orange, which was used as a defoliant in the Vietnam War. Little is known about its degradation under anoxic conditions. Established enrichment cultures using soil from an Agent Orange bioremediation plant in southern Vietnam with pyruvate as potential electron donor and carbon source were shown to degrade 2,4,5‐T via ether cleavage to 2,4,5‐trichlorophenol (2,4,5‐TCP), which was further dechlorinated to 3,4‐dichlorophenol. Pyruvate was initially fermented to hydrogen, acetate and propionate. Hydrogen was then used as the direct electron donor for ether cleavage of 2,4,5‐T and subsequent dechlorination of 2,4,5‐TCP. 16S rRNA gene amplicon sequencing indicated the presence of bacteria and archaea mainly belonging to the Firmicutes, Bacteroidetes, Spirochaetes, Chloroflexi and Euryarchaeota. Desulfitobacterium hafniense was identified as the dechlorinating bacterium. Metaproteomics of the enrichment culture indicated higher protein abundances of 60 protein groups in the presence of 2,4,5‐T. A reductive dehalogenase related to RdhA3 of D. hafniense showed the highest fold change, supporting its function in reductive dehalogenation of 2,4,5‐TCP. Despite an ether‐cleaving enzyme not being detected, the inhibition of ether cleavage but not of dechlorination, by 2‐bromoethane sulphonate, suggested that the two reactions are catalysed by different organisms.

Description of the bioactive landfill in Bien Hoa, establishment of enrichment cultures and results of 2,4,5-T degradation in the primary and secondary enrichment cultures The bioactive landfill in Bien Hoa was situated 700 meters from the Dong Nai river on the West (Dang and Nguyen, 2012). The landfill consisted of four cells (BH1-BH4) containing 3,384 m 3 of herbicide and dioxin contaminated soil. The soil contaminated with polychlorinated dibenzo-p-dioxins and herbicides was well mixed with different agents such as rice straw, hulls, biosurfactants, nutrients, electron acceptors, electron donors and vitamins and was periodically supplied with water (moisture range from 16-24%). The 2 m thick soil layer was sealed above and below with four layers of bentonite and geological protection material and 50 cm of non-contaminated soil as the top layer, which was planted with grass. Six pipes per cell allowed gas evaporation. Samples were taken from cell 1 (BH1. Collected samples were used for the preparation of primary microcosms (Dinh et al., 2015).They were prepared from soil (20 % w/v) of the sampling sites BH1.1, BH4.3 and BH4.4 in 40 ml of anoxic medium and incubated with 100 µM 2,4,5-T and a mixture of 5 mM pyruvate and 5 mM lactate as described in the Experimental Procedures.
2,4,5-T was completely removed within 2 weeks in all three enrichment cultures. The kinetics and pathways of transformation were studied in duplicate subcultures. 2,4,5-T disappeared within 10 days in all cultures. The subcultures BH1.1 and BH4.4 formed 2,4,5-TCP and 3,4-DCP as the main products and only low amounts of 3-chlorophenol and 2,5-dichlorophenol, whereas the subculture BH4.3 produced 2,5-dichlorophenol and 3-chlorophenol as the main products and only traces of 2,4,5-TCP and 3,4-DCP. This suggested that in the BH4.3 culture 2,4,5-T was first dechlorinated to 2,5dichlorophenoxyacetic acid followed by the cleavage of the ether bond, which resulted in the formation of 2,5-dichlorophenol and finally 3-chlorophenol. In contrast, in subcultures BH1.1 and BH4.4 the ether bond of 2,4,5-T was initially cleaved followed by the reductive dechlorination of the intermediate 2,4,5-TCP. The promising transformation pathway of BH4.3 leading from 2,4,5-T to 3chlorophenol changed during the further sub-cultivation to the transformation sequence described in the main text, strongly suggesting that a specific 2,4,5-T-dechlorinating organism was lost under the chosen cultivation conditions.  2,4,5-T, 2,4,5-trichlorophenoxyacetic acid; adj. p, Benjamini-Hochberg adjusted p-value from student's t-test; n.d., protein not detectable in both conditions; nondifferential, protein with a fold change that was not significant; Ident, protein was only identified but not quantifiable in at least one condition; a yeast extract; b data in bold indicate upregulation by 2,4,5-T in both conditions, in the absence and presence of yeast extract; c protein abundance >median of all proteins in this condition; d protein abundance <median of all proteins in this condition; e protein abundance <median-1SD (standard deviation) of all proteins in this condition; f adjusted p-value >0.05, raw p-value <0.05; g unique for 245T, but p>0.05, therefore regarded as non-differential.    . The concentration of acetate and hydrogen is indicated in italics and was calculated according to the reaction stoichiometry. Ethanol and CO 2 were not analysed. a) Pyruvate is fermented via the propionic acid fermentation pathway. Two moles of pyruvate are oxidized to two moles each of CO 2 and acetyl-CoA, the latter of which can be converted to two moles of acetate in an energy-conserving reaction. The formed NADH can be re-oxidized via the formation of propionic acid from a third mole of pyruvate. The detected 1.2 mM propionate suggests that 3.6 mM of the total added pyruvate (4.2 mM) were converted by this fermentation stoichiometry. b) Pyruvate (the remaining 0.6 mM) is proposed to be oxidized to acetyl-CoA by pyruvate:ferredoxin oxidoreductase or pyruvate formate lyase, thereby forming either reduced ferredoxin or formate, which both can deliver hydrogen by the action of hydrogenases or the formate hydrogen-lyase complex, respectively. c) Citrate fermentation via the citrate lyase reaction results in the formation of acetate, CO 2 , ethanol and hydrogen (Walther et al., 1977). d) Methane formation by hydrogenotrophic methanogens.
The total concentration of acetate determined after 8 days (6.5 mM, Fig. 3) exceeded only slightly the theoretical sum (6.1 mM) obtained from the fermentation of pyruvate and citrate (equations ac), supporting the proposed fermentation reactions. Additional processes such as acetogenesis or the oxidation of the proposed intermediate ethanol might have an additional limited influence on the acetate concentration. The presence of the CO dehydrogenase/acetyl CoA synthase as well of an alcohol dehydrogenase class IV in the metaproteome supports these assumptions (Table 2 and  Supporting Information, Table S2), whereas the ether cleavage of the micromolar concentrations of 2,4,5-T might contribute only negligible amounts of acetate.
Hydrogen forming (equation b, c) and consuming processes (equation d) are well balanced with only a minimal excess of hydrogen formation (0.1 mM), which might be sufficient to drive 2,4,5-T ether cleavage and dechlorination. The low level of detected hydrogen in the enrichment culture also supports the proposed metabolic routes.
*indicates that portions of the initial 4.2 mM pyruvate were assigned to reactions a and b according to the determined concentration of propionate formed in process a. l -1 yeast extract over 11 transfers (+/+YE, mean value and SD of two cultures) or 8 transfers with yeast extract, followed by two transfers without yeast extract and a third transfer with (-/+YE) or without yeast extract (-/-YE). Genera represented by more or less than 4000 reads in each sample are summarized in A and B, respectively. Genera, which were represented by less than 100 (< 0.25 % of total) reads in each sample were omitted. Fig. S6. PCA-Plot of all detected proteins of the metaproteomic analysis of four replicates and of four different conditions. noYE, without yeast extract; YE, with yeast extract; no_245T, without 2,4,5-T; 245T, with 2,4,5-T. Plots were generated with an in-house R-script using the packages gplots, ggplot2, ggbiplot, dplyr, miscTools and vegan. Fig. S7. Phylogenetic distribution of proteins. Abundances calculated from the number of proteins detected for each phylum in the mixed cultures using different cultivation conditions (four replicate cultures per condition). Cultures were supplemented with 2,4,5-trichloropenoxyacetic acid (+2,4,5-T) and without (-2,4,5-T). One set of cultures each was incubated with yeast extract (+YE) and without (-YE). P-value indicates significant difference between +2,4,5-T and -2,4,5-T. Plots were generated with an in-house R-script using the packages gplots, ggbiplot.

Fig. S8.
Log2-fold change and Benjamini-Hochberg adjusted p-values of all quantifiable and unique meta-proteins compared for +2,4,5-T and -2,4,5-T, without YE (A) and with YE (B). The logarithmic ratios of protein amounts in the mixed culture BH1.1 were plotted against Benjamini-Hochbergadjusted p-values of the t test performed from four replicates. The dotted lines indicate thresholds set for regulation (log2 fold change >1 and <-1 indicating a 2-fold higher abundance with and without 2,4,5-T) and the significance (adj. p-value <0.05). Significantly higher abundant proteins are labelled with the accession number of the meta-protein. Proteins with a fold change of ∞ or -∞ are unique to one of the conditions. A list of these proteins can be found in Table S3. Plots were generated with an in-house R-script using the packages gplots, ggplot2, ggbiplot, dplyr, miscTools and vegan.