Metagenomic analysis reveals specific BTEX degrading microorganisms of a bacterial consortium

Bacterial diversity of the sampling site and the enrichment culture

A total of 1914 OTUs were identified from 340,472 sequences for all samples. The relative abundances at the bacterial phyla level showed the dominance of Proteobacteria (41.5%) and Actinobacteria (16.8%) in the polluted soil samples (Fig. 1a). The enrichment culture was also dominated by Proteobacteria and Actinobacteria, while the relative abundances were different (35.0% and 55.5%, respectively). At class level, Actinobacteria and Alphaproteobacteria dominated the enrichment culture, accounting for 53.5% and 27.0%, respectively (Fig. 1b). At genus level, the top ten dominant genera of the enrichment culture were Rhodococcus, Azospirillum, Microbacterium, Arthrobacter, Methylobacterium-Methylorubrum, Mycobacterium, Gordonia, norank_f__JG30-KF-CM45, Sphingobium and Nocardioides (Fig. 1c).

Fig. 1figure 1

a Bacterial community composition of the polluted soil and enrichment culture. Relative abundance of taxonomic classification of the enrichment culture at a b class level and c genus level

Identification of aliphatic and aromatic hydrocarbon‑degrading coding DNA sequences

Functional analysis of the metagenome derived from the microbial enrichment culture revealed that 12 potential enzymatic classes represented by 1128 coding DNA sequences (CDSs) were involved in the degradation of aliphatic and aromatic hydrocarbons.

The enzymes considered to be responsible for the degradation of aliphatic hydrocarbons included alkane 1-monooxygenase, long-chain alkane monooxygenase, cyclohexanone monooxygenase and gluconolactonase (Fig. 2). Two hundred and eighty CDSs were detected to play a role in aliphatic hydrocarbon degradation, in which 121 CDSs belonged to the Actinomycetia and 76 CDSs to the Alphaproteobacteria. It is worth mentioning that our consortium contained all the genes involved in cyclohexane degradation, including the alkM, cpnA, chnB, chnC, gnl, chnD adh, chnE and aldH genes (Fig. 3a). These genes were assigned to seventy four genera (Table S1, despite of the unclassified genera), and seven of which contain more than 10 CDSs: Rhodococcus (25 CDSs), Mycolicibacterium (18 CDSs), Bradyrhizobium (16 CDSs), Mycobacterium (14 CDSs), Sphingopyxis (14 CDSs), Gordonia (13 CDSs) and Nocardioides (10 CDSs) (Fig. 3b).

Fig. 2figure 2

The number of sequences associated with specific hydrocarbon-degrading enzymes of the enrichment culture with a taxonomic classification at a class level

Fig. 3figure 3

a Biodegradation of cyclohexane via the Baeyer-Villiger oxidation pathway, b The number of the CDSs involved in this biodegradation pathway at a genus level. Genera that contain over three of the nine genes or over five CDSs are shown

In aerobic conditions, the first step of aromatic hydrocarbon biodegradation is an oxidation catalyzed by monooxygenases (hydroxylases) or by dioxygenases. In the enrichment culture, five hundred and thirty-seven CDSs were detected as oxygenases. Among those CDSs, one hundred and ten were catechol 2,3-dioxygenase, ninety-nine were homogentisate 1,2-dioxygenase, ninety-five were benzoate/toluate 1,2-dioxygenase, fifty-nine as phthalate 4,5-dioxygenase, thirty-seven as catechol 1,2-dioxygenase, thirty as phenol/toluene 2-monooxygenase, twenty-nine as anthranilate 1,2-dioxygenase, twenty-one as p-cumate 2,3-dioxygenase, fourteen as naphthalene 1,2-dioxygenase, fourteen as terephthalate 1,2-dioxygenase, fourteen as toluene monooxygenase, six as biphenyl 2,3-dioxygenase, five as toluene methyl-monooxygenase, four as ethylbenzene dioxygenase (Fig. 2).

Upper pathway in the degradation of BTEX

The results showed that our consortium contained the genes involved in all the steps for the conversion of benzene to catechol, toluene to either benzoate or 3-methylcatechol, and (o-, m-, p-,) xylene to (o-, m-, p-,) methylbenzoate.

The metagenome results revealed that thirty putative CDSs were involved in the upper pathway in the degradation of benzene. These CDSs were classified as phenol/toluene 2-monooxygenase, corresponding to six genes dmpKLMNOP (Fig. 4a). Taxonomic assignments indicated that eleven CDSs affiliated to the bacterial genus Novosphingobium, which contained all the six genes. The other twelve CDSs were assigned to bacterial genus Acidovorax (1 CDS), Pseudomonas (2 CDSs), Sphingomonas (1 CDS), Janibacter (1 CDS), Methyloversatilis (4 CDSs), Mycobacterium (2 CDSs) and Thauera (1 CDS) (Fig. 4c).

Fig. 4figure 4

The activation of a benzene by monooxygenases and b ethylbenzene by dioxygenases. The number of CDSs involved in the c benzene and d ethylbenzene activation at a genus level

Ethylbenzene degradation is initiated by ethylbenzene dioxygenase (etbAaAbAc) and subsequently transformed to 3-ethylcatechol (Fig. 4b). In our consortium, eight CDSs participated in the initial oxidation of ethylbenzene, three of which were assigned to Croceicoccus, Pelagerythrobacter and Sphingobium (Fig. 4d).

In the toluene degradation pathway of our consortium, the initial oxidation step was catalyzed by toluene 2-monooxygenases (tomA0A1A2A3A4A5), toluene monooxygenases (tmoABCDEF) or toluene methyl-monooxygenases (xylMA), producing o-cresol, m-cresol or benzyl alcohol, respectively (Fig. 5a). The toluene 2-monooxygenases can further transform the o-cresol to 3-methylcatechol, these genes (tomA0A1A2A3A4A5) possess the same functions as dmpKLMNOP genes and assigned to the same genera shown in Fig. 4c. Genus assignments demonstrated that 8 out of 14 of toluene monooxygenases (tmoABCDEF) belonged to Hyphomicrobium (5 CDSs) and Pseudonocardia (3 CDSs) (Fig. 5c). Meaningwhile, five CDSs that belonged to Mycolicibacterium (2 CDSs), Novosphingobium (2 CDSs) and Croceicoccus (1 CDS), were potentially involved in toluene methyl-monooxygenase (xylMA) degradation step. Benzyle alcohol is transformed to benzoate by two dehydrogenases, E1.1.1.90 and XylC, while m-cresol is metabolized to 3-methylcatechol by a phenol 2-monooxygenase (E1.14.13.7). Our results indicate that 92 CDSs were identified based on these three genes (E1.1.1.90, xylC and E1.14.13.7), which affiliate to 35 bacterial genera (despite of these unclassified genera), including Microbacterium (10 CDSs), Nocardioides (8 CDSs), Rhodococcus (6 CDSs), Mycobacterium (4 CDSs) and Sphingobium (4 CDSs), etc.

Fig. 5figure 5

The activation of a toluene and b xylene by monooxygenases. c The number of CDSs involved in the toluene and xylene activation at a genus level. Tulene 2-monooxygenase genes (tmoA0A1A2A3A4A5) belong to the same genera as dmpKLMNOP genes shown in Fig. 4c

The degradation pathway of xylene is similar to toluene. Toluene methyl-monooxygenase genes xylM and xylA initiate the degradation of ortho-, meta-, and para-xylenes (Fig. 5b). The aryl-alcohol dehydrogenase (E1.1.1.90) and benzaldehyde dehydrogenase (xylC) are involved in the subsequent oxidation of 2-methylbenzyl alcohol, 3-methylbenzyl alcohol and 4-methylbenzyl alcohol to o-methylbenzoate, m-methylbenzoate and p-methylbenzoate.

Central intermediates degradation pathways of BTEX

The degradation of toluene and xylene produces benzoate and methylbenzoate, which are further transformed by the benzoate/toluate 1,2-dioxygenase (benA-xylX, benB-xylY and benC-xylZ) and dihydroxycyclohexadiene carboxylate dehydrogenase (benD-xylL) to catechol and methylcatechol (Fig. 6a). A total of 109 CDSs played a role in the benzoate degradation pathway, which were assigned to 25 bacterial genera (despite of these unclassified genera). The majority of these CDSs were assigned to Nocardioides (12 CDSs), Rhodococcus (12 CDSs), Gordonia (9 CDSs), Marinobacter (5 CDSs), and Sphingopyxis (5 CDSs) (Fig. 6b).

Fig. 6figure 6

a The transformation of benzoate and (o-, m-, p-,) methylbenzoate to catechol and (3-, 4-) methylcatechol, respectively. b The number of CDSs involved in the degrading pathways at a genus level

In the ortho-cleavage of catechol pathway, catechol is first oxidized to cis,cis-muconate by catechol 1,2-dioxygenase (catA), then converted to 3-oxoadipate enol-lactone by muconate cycloisomerase (catB) and muconolactone D-isomerase (catC), and further metabolized to 3-oxoadipate with 3-oxoadipate enol-lactonases (pcaDL) (Fig. 7a). The metagenome data showed that our consortium contained 237 CDSs involved in this pathway, of which, 19 belonged to Rhodococcus, 13 to Delftia, 13 to Pseudonocardia, 10 to Gordonia, 9 to Bradyrhizobium, 9 to Nocardioides, 8 to Ramlibacter, and 7 to Mycobacterium (Fig. 7b). Our results demonstrated that 66 CDSs could not be assigned to any specific genus, and the other 83 CDSs were affiliated to 43 bacterial genera.

Fig. 7figure 7

a The ortho-cleavage of catechol. b The number of CDSs involved in the ortho-cleavage of catechol at a genus level

In the meta-cleavage of catechol pathway, the catechol and methylcatechol are initiated by the catechol 2,3-dioxygenase, then converted to a 4-hydroxy-2-oxoacid intermediate, which is cleaved by the aldolase to produce pyruvate, acetaldehyde or propanal. The propanal and acetaldehyde are transformed by the aldehyde dehydrogenase to propanoyl-CoA and acyl-CoA, respectively (Fig. 8a). Our results showed that the consortium contained all the genes involved in these reactions, including the catE, todF, dmpBCDH, mhpDEF, bphHIJ and praC genes, adding up to 523 CDSs. The todF gene, involved in the transformation of 3-methylcatechol to 2-hydroxy-2,4-pentadienoate, corresponding to only 1 CDS affiliated to a unclassified genus. Four hundred and eleven of these 523 CDSs affiliated to 100 specific bacterial genera, and the other 112 CDSs were assigned as unclassified genera (Table S2). Among the 100 bacterial genera, 19 of which contained more than four genes, including Rhodococcus (35 CDSs), Novosphingobium (30 CDSs), Pseudonocardia (26 CDSs), Mycolicibacterium (20 CDSs), Sphingopyxis (20 CDSs), Gordonia (19 CDSs), Nocardioides (16 CDSs), Mycobacterium (15 CDSs), Bradyrhizobium (11 CDSs), Sphingobium (10 CDSs), Sphingomonas (10 CDSs), Dietzia (8 CDSs), Aestuariivirga (7 CDSs), Azospirillum (7 CDSs), Janibacter (7 CDSs), Prauserella (7 CDSs), Methyloversatilis (6 CDSs), Nocardia (5 CDSs) and Micromonospora (4 CDSs) (Fig. 8b).

Fig. 8figure 8

a The meta-cleavage of catechol. b The number of CDSs involved in the meta-cleavage of catechol at a genus level. Genera that contain over four of the twelve genes are shown

The activation of ethylbenzene degradation resulted in the production of 3-ethylcatechol. The central metabolism of 3-ethylbenzene is initiated with a ring cleavage reaction by the 2,3-dihydroxyethylbenzene 1,2-dioxygenase (etbC). The product 2-hydroxy-6-oxo-octa-2,4-dienoate is then transformed to 2-hydroxy-2,4-pentadienoate by the hydrolase gene etbD. The etbC gene is not detected in our consortium, and the etbD gene corresponded to only 1 CDS, which assigned to the genus Sphingobium.

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