Dual C-Cl isotope fractionation offers potential to assess biodegradation of 1,2-dichloropropane and 1,2,3-trichloropropane by Dehalogenimonas cultures.

1,2-dichloropropane (1,2-DCP) and 1,2,3-trichloropropane (1,2,3-TCP) are hazardous chemicals frequently detected in groundwater near agricultural zones due to their historical use in chlorinated fumigant formulations. In this study, we show that the organohalide-respiring bacterium Dehalogenimonas alkenigignens strain BRE15 M can grow during the dihaloelimination of 1,2-DCP and 1,2,3-TCP to propene and allyl chloride, respectively. Our work also provides the first application of dual isotope approach to investigate the anaerobic reductive dechlorination of 1,2-DCP and 1,2,3-TCP. Stable carbon and chlorine isotope fractionation values for 1,2-DCP (ƐC = -13.6 ± 1.4 ‰ and ƐCl = -27.4 ± 5.2 ‰) and 1,2,3-TCP (ƐC = -3.8 ± 0.6 ‰ and ƐCl = -0.8 ± 0.5 ‰) were obtained resulting in distinct dual isotope slopes (Λ12DCP = 0.5 ± 0.1, Λ123TCP = 4 ± 2). However direct comparison of ΛC-Cl among different substrates is not possible and investigation of the C and Cl apparent kinetic isotope effects lead to the hypothesis that concerted dichloroelimination mechanism is more likely for both compounds. In fact, whole cell activity assays using cells suspensions of the Dehalogenimonas-containing culture grown with 1,2-DCP and methyl viologen as electron donor suggest that the same set of reductive dehalogenases was involved in the transformation of 1,2-DCP and 1,2,3-TCP. This study opens the door to the application of isotope techniques for evaluating biodegradation of 1,2-DCP and 1,2,3-TCP, which often co-occur in groundwaters near agricultural fields.

Environmental proteomics as a useful methodology for early-stage detection of stress in anammox engineered systems.

Anammox bacteria are widely applied worldwide for denitrification of urban wastewater. Differently, their application in the case of industrial effluents has been more limited. Those frequently present high loads of contaminants, demanding an individual evaluation of their treatability by anammox technologies. Bioreactors setting up and recovery after contaminants-derived perturbations are slow. Also, toxicity is frequently not acute but cumulative, which causes negative macroscopic effects to appear only after medium or long-term operations. All these particularities lead to relevant economic and time losses. We hypothesized that contaminants cause changes at anammox proteome level before perturbations in the engineered systems are detectable by macroscopic analyses. In this study, we explored the usefulness of short-batch tests combined with environmental proteomics for the early detection of those changes. Copper was used as a model of stressor contaminant, and anammox granules were exposed to increasing copper concentrations including previously reported IC50 values. The proteomic results revealed that specific anammox proteins involved in stress response (bacterioferritin, universal stress protein, or superoxide dismutase) were overexpressed in as short a time as 28 h at the higher copper concentrations. Consequently, EPS production was also increased, as indicated by the alginate export family protein, polysaccharide biosynthesis protein, and sulfotransferase increased expression. The described workflow can be applied to detect early-stage stress biomarkers of the negative effect of other metals, organics, or even changes in physical-chemical parameters such as pH or temperature on anammox-engineered systems. On an industrial level, it can be of great value for decision-making, especially before dealing with new effluents on facilities, deriving important economic and time savings.

Assessing labelled carbon assimilation from poly butylene adipate-co-terephthalate (PBAT) monomers during thermophilic anaerobic digestion.

PBAT (poly butylene adipate-co-terephthalate) is a widely used biodegradable plastic, but the knowledge about its metabolization in anaerobic environments is very limited. In this study, the anaerobic digester sludge from a municipal wastewater treatment plant was used as inoculum to investigate the biodegradability of PBAT monomers in thermophilic conditions. The research employs a combination of 13C-labelled monomers and proteogenomics to track the labelled carbon and identify the microorganisms involved. A total of 122 labelled peptides of interest were identified for adipic acid (AA) and 1,4-butanedio (BD). Through the time-dependent isotopic enrichment and isotopic profile distributions, Bacteroides, Ichthyobacterium, and Methanosarcina were proven to be directly involved in the metabolization of at least one monomer. This study provides a first insight into the identity and genomic potential of microorganisms responsible for biodegradability of PBAT monomers during anaerobic digestion under thermophilic conditions.

Metalloenzymes play major roles to achieve high-rate nitrogen removal in N-damo communities: Lessons from metaproteomics.

Nitrite-driven anaerobic methane oxidation (N-damo) is a promising biological process to achieve carbon-neutral wastewater treatment solutions, aligned with the sustainable development goals. Here, the enzymatic activities in a membrane bioreactor highly enriched in N-damo bacteria operated at high nitrogen removal rates were investigated. Metaproteomic analyses, with a special focus on metalloenzymes, revealed the complete enzymatic route of N-damo including their unique nitric oxide dismutases. The relative protein abundance evidenced that "Ca. Methylomirabilis lanthanidiphila" was the predominant N-damo species, attributed to the induction of its lanthanide-binding methanol dehydrogenase in the presence of cerium. Metaproteomics also disclosed the activity of the accompanying taxa in denitrification, methylotrophy and methanotrophy. The most abundant functional metalloenzymes from this community require copper, iron, and cerium as cofactors which was correlated with the metal consumptions in the bioreactor. This study highlights the usefulness of metaproteomics for evaluating the enzymatic activities in engineering systems to optimize microbial management.

Proteogenomics of the novel Dehalobacterium formicoaceticum strain EZ94 highlights a key role of methyltransferases during anaerobic dichloromethane degradation.

Dichloromethane (DCM, methylene chloride) is a toxic, high-volume industrial pollutant of long-standing. Anaerobic biodegradation is crucial for its removal from contaminated environments, yet prevailing mechanisms remain unresolved, especially concerning dehalogenation. In this study, we obtained an assembled genome of a novel DCM-degrading strain, Dehalobacterium formicoaceticum strain EZ94, from a stable DCM-degrading consortium, and we analyzed its proteome during degradation of DCM. A gene cluster recently predicted to play a major role in anaerobic DCM catabolism (the mec cassette) was found. Methyltransferases and other proteins encoded by the mec cassette were among the most abundant proteins produced, suggesting their involvement in DCM catabolism. Reductive dehalogenases were not detected. Genes and corresponding proteins for a complete Wood-Ljungdahl pathway, which could enable further metabolism of DCM carbon, were also found. Unlike for the anaerobic DCM degrader "Ca. F. warabiya," no genes for metabolism of the quaternary amines choline and glycine betaine were identified. This work provides independent and supporting evidence that mec-associated methyltransferases are key to anaerobic DCM metabolism.

Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives.

Unspecific peroxygenase (UPO) presents a wide range of biotechnological applications. This study targets the use of by-products from bioethanol synthesis to produce UPO by Agrocybe aegerita. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase in SmF using vinasse as nutrients source. Optimized UPO production of 331 U/L was achieved in 50% (v:v) vinasse with an inoculum grown for 14 days. These conditions were scaled-up to a 4 L reactor, achieving a UPO activity of 265 U/L. Fungal proteome expression was analyzed before and after UPO activity appeared by shotgun mass spectrometry proteomics. Laccase, dye-decolorizing peroxidases (DyP), lectins and proteins involved in reactive oxygen species (ROS) production and control were detected (in addition to UPO). Interestingly, the metabolism of complex sugars and nitrogen sources had a different activity at the beginning and end of the submerged fermentation.

Dual C-Br Isotope Fractionation Indicates Distinct Reductive Dehalogenation Mechanisms of 1,2-Dibromoethane in Dehalococcoides- and Dehalogenimonas-Containing Cultures.

Brominated organic compounds such as 1,2-dibromoethane (1,2-DBA) are highly toxic groundwater contaminants. Multi-element compound-specific isotope analysis bears the potential to elucidate the biodegradation pathways of 1,2-DBA in the environment, which is crucial information to assess its fate in contaminated sites. This study investigates for the first time dual C-Br isotope fractionation during in vivo biodegradation of 1,2-DBA by two anaerobic enrichment cultures containing organohalide-respiring bacteria (i.e., either Dehalococcoides or Dehalogenimonas). Different εbulkC values (-1.8 ± 0.2 and -19.2 ± 3.5‰, respectively) were obtained, whereas their respective εbulkBr values were lower and similar to each other (-1.22 ± 0.08 and -1.2 ± 0.5‰), leading to distinctly different trends (ΛC-Br = Δδ13C/Δδ81Br ≈ εbulkC/εbulkBr) in a dual C-Br isotope plot (1.4 ± 0.2 and 12 ± 4, respectively). These results suggest the occurrence of different underlying reaction mechanisms during enzymatic 1,2-DBA transformation, that is, concerted dihaloelimination and nucleophilic substitution (SN2-reaction). The strongly pathway-dependent ΛC-Br values illustrate the potential of this approach to elucidate the reaction mechanism of 1,2-DBA in the field and to select appropriate εbulkC values for quantification of biodegradation. The results of this study provide valuable information for future biodegradation studies of 1,2-DBA in contaminated sites.

Sulfamethoxazole Enhances Specific Enzymatic Activities under Aerobic Heterotrophic Conditions: A Metaproteomic Approach.

The growing concern about antibiotic-resistant microorganisms has focused on the sludge from wastewater treatment plants (WWTPs) as a potential hotspot for their development and spread. To this end, it seems relevant to analyze the changes on the microbiota as a consequence of the antibiotics that wastewater may contain. This study aims at determining whether the presence of sulfamethoxazole (SMX), even in relatively low concentrations, modifies the microbial activities and the enzymatic expression of an activated sludge under aerobic heterotrophic conditions. For that purpose, we applied a metaproteomic approach in combination with genomic and transformation product analyses. SMX was biotransformed, and the metabolite 2,4(1H,3H)-pteridinedione-SMX (PtO-SMX) from the pterin-conjugation pathway was detected at all concentrations tested. Metaproteomics showed that SMX at 50-2000 µg/L slightly affected the microbial community structure, which was confirmed by DNA metabarcoding. Interestingly, an enhanced activity of the genus Corynebacterium and specifically of five enzymes involved in its central carbon metabolism was found at increased SMX concentrations. Our results suggest a role of Corynebacterium genus on SMX risks mitigation in our bioreactors.

Assessment of aerobic biodegradation of lower-chlorinated benzenes in contaminated groundwater using field-derived microcosms and compound-specific carbon isotope fractionation.

Biodegradation of lower chlorinated benzenes (tri-, di- and monochlorobenzene) was assessed at a coastal aquifer contaminated with multiple chlorinated aromatic hydrocarbons. Field-derived microcosms, established with groundwater from the source zone and amended with a mixture of lower chlorinated benzenes, evidenced biodegradation of monochlorobenzene (MCB) and 1,4-dichlorobenzene (1,4-DCB) in aerobic microcosms, whereas the addition of lactate in anaerobic microcosms did not enhance anaerobic reductive dechlorination. Aerobic microcosms established with groundwater from the plume consumed several doses of MCB and concomitantly degraded the three isomers of dichlorobenzene with no observable inhibitory effect. In the light of these results, we assessed the applicability of compound stable isotope analysis to monitor a potential aerobic remediation treatment of MCB and 1,4-DCB in this site. The carbon isotopic fractionation factors (ε) obtained from field-derived microcosms were -0.7‰ ± 0.1 ‰ and -1.0‰ ± 0.2 ‰ for MCB and 1,4-DCB, respectively. For 1,4-DCB, the carbon isotope fractionation during aerobic biodegradation was reported for the first time. The weak carbon isotope fractionation values for the aerobic pathway would only allow tracing of in situ degradation in aquifer parts with high extent of biodegradation. However, based on the carbon isotope effects measured in this and previous studies, relatively high carbon isotope shifts (i.e., ∆δ13C > 4.0 ‰) of MCB or 1,4-DCB in contaminated groundwater would suggest that their biodegradation is controlled by anaerobic reductive dechlorination.

Bundling the removal of emerging contaminants with the production of ligninolytic enzymes from residual streams.

Enzymes offer interesting features as biological catalysts for industry: high specificity, activity under mild conditions, accessibility, and environmental friendliness. Being able to produce enzymes in large quantities and having them available in a stable and reusable form reduces the production costs of any enzyme-based process. Agricultural residues have recently demonstrated their potential as substrates to produce ligninolytic enzymes by different white rot fungi. In this study, the biotechnological production of a manganese peroxidase (MnP) by Irpex lacteus was conducted through solid-state fermentation (SSF) with wheat straw as substrate and submerged fermentation (SmF) employing wheat straw extract (WSE). The obtained enzyme cocktail also showed manganese-independent activity (MiP), related to the presence of a short MnP and a dye-decolorizing peroxidase (DyP) which was confirmed by shotgun proteomic analyses. In view of the enhanced production of ligninolytic enzymes in SmF, different parameters such as WSE concentration and nitrogen source were evaluated. The highest enzyme titers were obtained with a medium formulated with glucose and peptone (339 U/L MnP and 15 U/L MiP). The scale-up to a 30 L reactor achieved similar activities, demonstrating the feasibility of enzyme production from the residual substrate at different production scales. Degradation of five emerging pollutants was performed to demonstrate the high oxidative capacity of the enzyme. Complete removal of hormones and bisphenol A was achieved in less than 1 h, whereas almost 30% degradation of carbamazepine was achieved in 24 h, which is a significant improvement compared to previous enzymatic treatments of this compound. KEY POINTS: • Wheat straw extract is suitable for the growth of I. lacteus. • The enzyme cocktail obtained allows the degradation of emerging contaminants. • Mn-dependent and Mn-independent activities increases the catalytic potential.

Genome Sequence, Proteome Profile, and Identification of a Multiprotein Reductive Dehalogenase Complex in Dehalogenimonas alkenigignens Strain BRE15M.

Bacteria of the genus Dehalogenimonas respire with vicinally halogenated alkanes via dihaloelimination. We aimed to describe involved proteins and their supermolecular organization. Metagenomic sequencing of a Dehalogenimonas-containing culture resulted in a 1.65 Mbp draft genome of Dehalogenimonas alkenigignens strain BRE15M. It contained 31 full-length reductive dehalogenase homologous genes (rdhA), but only eight had cognate rdhB gene coding for membrane-anchoring proteins. Shotgun proteomics of cells grown with 1,2-dichloropropane as an electron acceptor identified 1152 proteins representing more than 60% of the total proteome. Ten RdhA proteins were detected, including a DcpA ortholog, which was the strongest expressed RdhA. Blue native gel electrophoresis (BNE) demonstrating maximum activity was localized in a protein complex of 146-242 kDa. Protein mass spectrometry revealed the presence of DcpA, its membrane-anchoring protein DcpB, two hydrogen uptake hydrogenase subunits (HupL and HupS), an iron-sulfur protein (HupX), and subunits of a redox protein with a molybdopterin-binding motif (OmeA and OmeB) in the complex. BNE after protein solubilization with different detergent concentrations revealed no evidence for an interaction between the putative respiratory electron input module (HupLS) and the OmeA/OmeB/HupX module. All detected RdhAs comigrated with the organohalide respiration complex. Based on genomic and proteomic analysis, we propose quinone-independent respiration in Dehalogenimonas.

Interspecies interaction and effect of co-contaminants in an anaerobic dichloromethane-degrading culture.

An anaerobic stable mixed culture dominated by bacteria belonging to the genera Dehalobacterium, Acetobacterium, Desulfovibrio, and Wolinella was used as a model to study the microbial interactions during DCM degradation. Physiological studies indicated that DCM was degraded in this mixed culture at least in a three-step process: i) fermentation of DCM to acetate and formate, ii) formate oxidation to CO2 and H2, and iii) H2/CO2 reductive acetogenesis. The 16S rRNA gene sequencing of cultures enriched with formate or H2 showed that Desulfovibrio was the dominant population followed by Acetobacterium, but sequences representing Dehalobacterium were only present in cultures amended with DCM. Nuclear magnetic resonance analyses confirmed that acetate produced from 13C-labelled DCM was marked at the methyl ([2-13C]acetate), carboxyl ([1-13C]acetate), and both ([1,2-13C]acetate) positions, which is in accordance to acetate formed by both direct DCM fermentation and H2/CO2 acetogenesis. The inhibitory effect of ten different co-contaminants frequently detected in groundwaters on DCM degradation was also investigated. Complete inhibition of DCM degradation was observed when chloroform, perfluorooctanesulfonic acid, and diuron were added at 838, 400, and 107 µM, respectively. However, the inhibited cultures recovered the DCM degradation capability when transferred to fresh medium without co-contaminants. Findings derived from this work are of significant relevance to provide a better understanding of the synergistic interactions among bacteria to accomplish DCM degradation as well as to predict the effect of co-contaminants during anaerobic DCM bioremediation in groundwater.

Molecular and carbon isotopic characterization of an anaerobic stable enrichment culture containing Dehalobacterium sp. during dichloromethane fermentation.

Biodegradation of dichloromethane (DCM) under reducing conditions is of major concern due to its widespread detection in contaminated groundwaters. Here, we report an anaerobic enrichment culture derived from a membrane bioreactor operating in an industrial wastewater treatment plant, capable of fermenting DCM and the brominated analogue dibromomethane (DBM). Comparative analysis of bacterial 16S rDNA-DGGE profiles from fresh liquid medium inoculated with single colonies picked from serial dilution-to-extinction agar vials showed that cultures degrading DCM contained a predominant band belonging to Dehalobacterium, however this band was absent in cultures unable to degrade DCM. Analysis of the microbial composition of the enrichment by bacterial 16S rRNA gene amplicon paired-end sequencing confirmed the presence of Dehalobacterium together with three additional phylotypes belonging to Acetobacterium, Desulfovibrio, and Wolinella, representing all four operational taxonomic units >99.9% of the retrieved sequences. The carbon isotopic fractionation (ε) determined for DCM degradation in this culture was -27±2‰. This value differs from the ε previously reported for the DCM-fermentative bacteria Dehalobacter (-15.5±1.5‰) but they are both significantly different from those reported for facultative methylotrophic organisms (ranging from -45 to -61‰). This significant difference in the ε allows differentiating between hydrolytic transformation of DCM via glutathione-dependent dehalogenases and fermentation pathway. CAPSULE: The carbon isotopic fractionation of dichloromethane by an enriched Dehalobacterium-containing culture has significant potential to monitor biodegradation of DCM in groundwaters.