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Cometabolic bioremediation is trending for the treatment of 1,4-dioxane (dioxane) and other emerging contaminants to meet stringent regulatory goals ( e.g. , <10 μg L −1 ) since biodegradation activities can be fueled by the supplementation of auxiliary substrates. In this study, we compared and investigated the effectiveness of two types of common auxiliary substrates, short-chain alkane gases ( e.g. , propane and butane) and primary alcohols ( e.g. , 1-propanol, 1-butanol, and ethanol), for dioxane removal in diverse environmental matrices with Azoarcus sp. DD4 as the inoculum. Physicochemical characterization at the pure culture level revealed that propane and 1-propanol are advantageous for stimulating cell growth and dioxane biodegradation by DD4. Parallel microcosm assays were conducted to assess the compatibility of DD4 bioaugmentation in diverse microbiomes recovered from five different environmental samples, including shallow and deep aquifer groundwater, contaminated river sediment, and municipal activated sludge. Propane was effective in sustaining efficient dioxane removal and the dominance of DD4 across all environmental matrices. Notably, amendment with 1-propanol promoted superior dioxane degradation in the deep aquifer groundwater, in which low pre-treatment biomass and post-treatment diversity were observed, suggesting its potential for intrinsic field applications. The combination of microbial community analysis and differential ranking identified that Ochrobactrum and several other indigenous bacteria were boosted by the inoculation of DD4, implying their commensal or mutualistic relationship. Collectively, propane and 1-propanol can be effective auxiliary substrate alternatives tailored for in situ bioaugmentation and their effectiveness is affected by the density and structure of environmental microbiomes.
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Discovery of an Inducible Toluene Monooxygenase that Co-oxidizes 1,4-Dioxane and 1,1-Dichloroethylene in Propanotrophic Azoarcus sp. DD4
Cometabolic degradation plays a prominent role in bioremediation of commingled groundwater contamination (e.g., chlorinated solvents and the solvent stabilizer 1,4-dioxane [dioxane]). In this study, we untangled the diversity and catalytic functions of multi-component monooxygenases in Azoarcus sp. DD4, a gram-negative propanotroph that is effective in degrading dioxane and 1,1-dichloroethylene (1,1-DCE). Using a combination of knockout mutagenesis and heterologous expression, a toluene monooxygenase (MO) encoded by the tmoABCDEF gene cluster was unequivocally proved as the key enzyme responsible for the cometabolism of both dioxane and 1,1-DCE. Interestingly, in addition to utilizing toluene as a primary substrate, this toluene MO can also oxidize propane into 1-propanol. Expression of this toluene MO in DD4 appears inducible by both substrates (toluene and propane) and their primary hydroxylation products (m-cresol, p-cresol, and 1-propanol). These findings coherently explain why DD4 can grow on propane and express toluene MO for active co-oxidation of dioxane and 1,1-DCE. Furthermore, upregulation of tmo transcription by 1-propanol underlines the implication potential of using 1-propanol as an alternative auxiliary substrate for DD4 bioaugmentation. The discovery of this toluene MO in DD4 and its degradation and induction versatility renders broad applications spanning from environmental remediation and water treatment to biocatalysis in green chemistry. Importance Toluene MOs have been well recognized given their robust abilities to degrade a variety of environmental pollutants. Built upon previous research efforts, this study ascertained the untapped capability of a toluene MO in DD4 for effective co-oxidation of dioxane and 1,1-DCE, two of the most prevailing yet challenging groundwater contaminants. This report also aligns the induction of a toluene MO with non-toxic and commercially accessible chemicals (e.g., propane and 1-propanol), extending its implication values in the field of environmental microbiology and beyond.
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- Award ID(s):
- 1846945
- PAR ID:
- 10183349
- Date Published:
- Journal Name:
- Applied and Environmental Microbiology
- ISSN:
- 0099-2240
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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ABSTRACT Azoarcus sp. strain DD4 can cometabolically degrade 1,4-dioxane and 1,1-dichloroethylene (1,1-DCE) when grown with propane and other substrates. The complete genome sequence of strain DD4 reveals a diverse collection of bacterial monooxygenase genes that may contribute to its versatility in degrading commingled groundwater pollutants.more » « less
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Hydroxyl radical (OH) oxidation of toluene produces ring-retaining products: cresol and benzaldehyde, and ring-opening products: bicyclic intermediate compounds and epoxides. Here, first- and later-generation OH oxidation products from cresol and benzaldehyde are identified in laboratory chamber experiments. For benzaldehyde, first-generation ring-retaining products are identified, but later-generation products are not detected. For cresol, low-volatility (saturation mass concentration, C* ∼ 3.5 × 104 − 7.7 × 10−3 µg m−3), first- and later-generation ring-retaining products are identified. Subsequent OH addition to the aromatic ring of o-cresol leads to compounds such as hydroxy, dihydroxy, and trihydroxy methyl benzoquinones and dihydroxy, trihydroxy, tetrahydroxy, and pentahydroxy toluenes. These products are detected in the gas phase by chemical ionization mass spectrometry (CIMS) and in the particle phase using offline direct analysis in real-time mass spectrometry (DART-MS). Our data suggest that the yield of trihydroxy toluene from dihydroxy toluene is substantial. While an exact yield cannot be reported as authentic standards are unavailable, we find that a yield for trihydroxy toluene from dihydroxy toluene of ∼ 0.7 (equal to the reported yield of dihydroxy toluene from o-cresol; Olariu et al., 2002) is consistent with experimental results for o-cresol oxidation under low-NO conditions. These results suggest that even though the cresol pathway accounts for only ∼ 20 % of the oxidation products of toluene, it is the source of a significant fraction (∼ 20–40 %) of toluene secondary organic aerosol (SOA) due to the formation of low-volatility products.more » « less
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Co-contamination with chlorinated compounds and 1,4-dioxane has been reported at many sites. Recently, there has been an increased interest in bioremediation because of the potential to degrade multiple contaminants concurrently. Towards improving bioremediation efficacy, the current study examined laboratory microcosms (inoculated separately with two soils) to determine the phylotypes and functional genes associated with the biodegradation of two common co-contaminants (cis-dichloroethene [cDCE] and 1,4-dioxane). The impact of amending microcosms with lactate on cDCE and 1,4-dioxane biodegradation was also investigated. The presence of either lactate or cDCE did not impact 1,4-dioxane biodegradation one of the two soils. Lactate appeared to improve the initiation of the biological removal of cDCE in microcosms inoculated with either soil. Stable isotope probing (SIP) was then used to determine which phylotypes were actively involved in carbon uptake from cDCE and 1,4-dioxane in both soil communities. The most enriched phylotypes for 13C assimilation from 1,4-dioxane included Rhodopseudomonas and Rhodanobacter. Propane monooxygenase was predicted (by PICRUSt2) to be dominant in the 1,4-dioxane amended microbial communities and propane monooxygenase gene abundance values correlated with other enriched (but less abundant) phylotypes for 13C-1,4-dioxane assimilation. The dominant enriched phylotypes for 13C assimilation from cDCE included Bacteriovorax, Pseudomonas and Sphingomonas. In the cDCE amended soil microcosms, PICRUSt2 predicted the presence of DNA encoding glutathione S-transferase (a known cDCE upregulated enzyme). Overall, the work demonstrated concurrent removal of cDCE and 1,4-dioxane by indigenous soil microbial communities and the enhancement of cDCE removal by lactate. The data generated on the phylotypes responsible for carbon uptake (as determined by SIP) could be incorporated into diagnostic molecular methods for site characterization. The results suggest concurrent biodegradation of cDCE and 1,4-dioxane should be considered for chlorinated solvent site remediation.more » « less
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Characterization of a unique polysaccharide monooxygenase from the plant pathogen Magnaporthe oryzaeBlast disease in cereal plants is caused by the fungus Magnaporthe oryzae and accounts for a significant loss in food crops. At the outset of infection, expression of a putative polysaccharide monooxygenase ( Mo PMO9A) is increased. Mo PMO9A contains a catalytic domain predicted to act on cellulose and a carbohydrate-binding domain that binds chitin. A sequence similarity network of the Mo PMO9A family AA9 showed that 220 of the 223 sequences in the Mo PMO9A-containing cluster of sequences have a conserved unannotated region with no assigned function. Expression and purification of the full length and two Mo PMO9A truncations, one containing the catalytic domain and the domain of unknown function (DUF) and one with only the catalytic domain, were carried out. In contrast to other AA9 polysaccharide monooxygenases (PMOs), Mo PMO9A is not active on cellulose but showed activity on cereal-derived m ixed (1→3, 1→4)- β -D- g lucans (MBG). Moreover, the DUF is required for activity. Mo PMO9A exhibits activity consistent with C4 oxidation of the polysaccharide and can utilize either oxygen or hydrogen peroxide as a cosubstrate. It contains a predicted 3-dimensional fold characteristic of other PMOs. The DUF is predicted to form a coiled-coil with six absolutely conserved cysteines acting as a zipper between the two α-helices. Mo PMO9A substrate specificity and domain architecture are different from previously characterized AA9 PMOs. The results, including a gene ontology analysis, support a role for Mo PMO9A in MBG degradation during plant infection. Consistent with this analysis, deletion of Mo PMO9A results in reduced pathogenicity.more » « less
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1128/AEM.01163-20
