Functional and structural responses of a halophilic consortium to oily sludge during biodegradation.

Biotreatment of oily sludge and the involved microbial communities, particularly in saline environments, have been rarely investigated. We enriched a halophilic bacterial consortium (OS-100) from petroleum refining oily sludge, which degraded almost 86% of the aliphatic hydrocarbon (C10-C30) fraction of the oily sludge within 7 days in the presence of 100 g/L NaCl. Two halophilic hydrocarbon-degrading bacteria related to the genera Chromohalobacter and Halomonas were isolated from the OS-100 consortium. Hydrocarbon degradation by the OS-100 consortium was relatively higher compared to the isolated bacteria, indicating potential synergistic interactions among the OS-100 community members. Exclusion of FeCl2, MgCl2, CaCl2, trace elements, and vitamins from the culture medium did not significantly affect the hydrocarbon degradation efficiency of the OS-100 consortium. To the contrary, hydrocarbon biodegradation dropped from 94.1 to 54.4% and 5% when the OS-100 consortium was deprived from phosphate and nitrogen sources in the culture medium, respectively. Quantitative PCR revealed that alkB gene expression increased up to the 3rd day of incubation with 11.277-fold, consistent with the observed increments in hydrocarbon degradation. Illumina-MiSeq sequencing of 16 S rRNA gene fragments revealed that the OS-100 consortium was mainly composed of the genera Halomonas, Idiomarina, Alcanivorax and Chromohalobacter. This community structure changed depending on the culturing conditions. However, remarkable changes in the community structure were not always associated with remarkable shifts in the hydrocarbonoclastic activity and vice versa. The results show that probably synergistic interactions between community members and different subpopulations of the OS-100 consortium contributed to salinity tolerance and hydrocarbon degradation.

Mechanistic insights into sulfur source-driven physiological responses and metabolic reorganization in the fuel-biodesulfurizing Rhodococcus qingshengii IGTS8.

Comparative proteomics and untargeted metabolomics were combined to study the physiological and metabolic adaptations of Rhodococcus qingshengii IGTS8 under biodesulfurization conditions. After growth in a chemically defined medium with either dibenzothiophene (DBT) or MgSO4 as the sulfur source, many differentially produced proteins and metabolites associated with several metabolic and physiological processes were detected including the metabolism of carbohydrates, amino acids, lipids, nucleotides, vitamins, protein synthesis, transcriptional regulation, cell envelope biogenesis, and cell division. Increased production of the redox cofactor mycofactocin and associated proteins was one of the most striking adaptations under biodesulfurization conditions. While most central metabolic enzymes were less abundant in the presence of DBT, a key enzyme of the glyoxylate shunt, isocitrate lyase, was up to 26-fold more abundant. Several C1 metabolism and oligotrophy-related enzymes were significantly more abundant in the biodesulfurizing culture. R. qingshengii IGTS8 exhibited oligotrophic growth in liquid and solid media under carbon starvation. Moreover, the oligotrophic growth was faster on the solid medium in the presence of DBT compared to MgSO4 cultures. In the DBT culture, the cell envelope and phospholipids were remodeled, with lower levels of phosphatidylethanolamine and unsaturated and short-chain fatty acids being the most prominent changes. Biodesulfurization increased the biosynthesis of osmoprotectants (ectoine and mannosylglycerate) as well as glutamate and induced the stringent response. Our findings reveal highly diverse and overlapping stress responses that could protect the biodesulfurizing culture not only from the associated sulfate limitation but also from chemical, oxidative, and osmotic stress, allowing efficient resource management. IMPORTANCE Despite decades of research, a commercially viable bioprocess for fuel desulfurization has not been developed yet. This is mainly due to lack of knowledge of the physiology and metabolism of fuel-biodesulfurizing bacteria. Being a stressful condition, biodesulfurization could provoke several stress responses that are not understood. This is particularly important because a thorough understanding of the microbial stress response is essential for the development of environmentally friendly and industrially efficient microbial biocatalysts. Our comparative systems biology studies provide a mechanistic understanding of the biology of biodesulfurization, which is crucial for informed developments through the rational design of recombinant biodesulfurizers and optimization of the bioprocess conditions. Our findings enhance the understanding of the physiology, metabolism, and stress response not only in biodesulfurizing bacteria but also in rhodococci, a precious group of biotechnologically important bacteria.

Harnessing microbial phylum-specific molecular markers for assessment of environmental estrogen degradation.

Steroidal estrogens are ubiquitous contaminants that have garnered attention worldwide due to their endocrine-disrupting and carcinogenic activities at sub-nanomolar concentrations. Microbial degradation is one of the main mechanisms through which estrogens can be removed from the environment. Numerous bacteria have been isolated and identified as estrogen degraders; however, little is known about their contribution to environmental estrogen removal. Here, our global metagenomic analysis indicated that estrogen degradation genes are widely distributed among bacteria, especially among aquatic actinobacterial and proteobacterial species. Thus, by using the Rhodococcus sp. strain B50 as the model organism, we identified three actinobacteria-specific estrogen degradation genes, namely aedGHJ, by performing gene disruption experiments and metabolite profile analysis. Among these genes, the product of aedJ was discovered to mediate the conjugation of coenzyme A with a unique actinobacterial C17 estrogenic metabolite, 5-oxo-4-norestrogenic acid. However, proteobacteria were found to exclusively adopt an α-oxoacid ferredoxin oxidoreductase (i.e., the product of edcC) to degrade a proteobacterial C18 estrogenic metabolite, namely 3-oxo-4,5-seco-estrogenic acid. We employed actinobacterial aedJ and proteobacterial edcC as specific biomarkers for quantitative polymerase chain reaction (qPCR) to elucidate the potential of microbes for estrogen biodegradation in contaminated ecosystems. The results indicated that aedJ was more abundant than edcC in most environmental samples. Our results greatly expand the understanding of environmental estrogen degradation. Moreover, our study suggests that qPCR-based functional assays are a simple, cost-effective, and rapid approach for holistically evaluating estrogen biodegradation in the environment.

Biotreatment of oily sludge by a bacterial consortium: Effect of bioprocess conditions on biodegradation efficiency and bacterial community structure.

We studied the biodegradation of oily sludge generated by a petroleum plant in Bahrain by a bacterial consortium (termed as AK6) under different bioprocess conditions. Biodegradation of petroleum hydrocarbons in oily sludge (C11-C29) increased from 24% after two days to 99% after 9 days of incubation in cultures containing 5% (w/v) of oily sludge at 40°C. When the nitrogen source was excluded from the batch cultures, hydrocarbon biodegradation dropped to 45% within 7 days. The hydrocarbon biodegradation decreased also by increasing the salinity to 3% and the temperature above 40°C. AK6 tolerated up to 50% (w/v) oily sludge and degraded 60% of the dichloromethane-extractable oil fraction. Illumina-MiSeq analyses revealed that the AK6 consortium was mainly composed of Gammaproteobacteria (ca. 98% of total sequences), with most sequences belonging to Klebsiella (77.6% of total sequences), Enterobacter (16.7%) and Salmonella (5%). Prominent shifts in the bacterial composition of the consortium were observed when the temperature and initial sludge concentration increased, and the nitrogen source was excluded, favoring sequences belonging to Pseudomonas and Stenotrophomonas. The AK6 consortium is endowed with a strong oily sludge tolerance and biodegradation capability under different bioprocess conditions, where Pseudomonas spp. appear to be crucial for hydrocarbon biodegradation.

Temporal compositional shifts in an activated sludge microbiome during estrone biodegradation.

Microbial biodegradation is a key process for the removal of estrogens during wastewater treatment. At least four degradation pathways for natural estrogens have been proposed. However, major estrogen degraders and the occurrence of different estrogen biodegradation pathways in wastewater treatment plants have been rarely investigated. This study was conducted to elucidate estrone biodegradation pathway and to identify key estrone-degrading bacteria in activated sludge from a major wastewater treatment plant in Bahrain. The biodegradation experiments were performed in activated sludge microcosms supplemented with estrone. Sludge samples were retrieved at time intervals to analyze the biodegradation metabolites and the temporal shifts in the bacterial community composition. Chemical analysis revealed the biodegradation of more than 90% of the added estrone within 6 days, and the compounds 4-hydroxyestrone and pyridinestrone acid, which are typical markers of the 4,5-seco pathway of aerobic estrone biodegradation, were detected. Temporal shifts in the relative abundance of bacteria were most prominent among members of Proteobacteria and Bacteroidetes. While the alphaproteobacterial genera Novosphingobium and Sphingoaurantiacus were significantly enriched (from ≤ 6% to an average of 31%) in the estrone-amended activated sludge after 2 days of incubation, the bacteroidete Pedobacter was uniquely detected in these microcosms at day 10. The relative abundance of Polyangia (Nannocyctis) increased to an average of 10 ± 0.4% in the estrone-amended activated sludge after 4 days of incubation. Enrichment cultivation of bacteria from the activated sludge on estrone resulted in a mixed culture that was capable of degrading estrone. An estrone-degrading strain was isolated from this mixed culture and was affiliated with the known estrogen-degrading Alphaproteobacteria Sphingobium estrogenivorans. We conclude that estrone degradation in the activated sludge from the studied wastewater treatment plant proceeds via the 4,5-seco pathway and is most likely mediated by alphaproteobacterial taxa.

Biodesulfurization Induces Reprogramming of Sulfur Metabolism in Rhodococcus qingshengii IGTS8: Proteomics and Untargeted Metabolomics.

Sulfur metabolism in fuel-biodesulfurizing bacteria and the underlying physiological adaptations are not understood, which has impeded the development of a commercially viable bioprocess for fuel desulfurization. To fill these knowledge gaps, we performed comparative proteomics and untargeted metabolomics in cultures of the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 grown on either inorganic sulfate or the diesel-borne organosulfur compound dibenzothiophene as a sole sulfur source. Dibenzothiophene significantly altered the biosynthesis of many sulfur metabolism proteins and metabolites in a growth phase-dependent manner, which enabled us to reconstruct the first experimental model for sulfur metabolism in a fuel-biodesulfurizing bacterium. All key pathways related to assimilatory sulfur metabolism were represented in the sulfur proteome, including uptake of the sulfur sources, sulfur acquisition, and assimilatory sulfate reduction, in addition to biosynthesis of key sulfur-containing metabolites such as S-adenosylmethionine, coenzyme A, biotin, thiamin, molybdenum cofactor, mycothiol, and ergothioneine (low-molecular weight thiols). Fifty-two proteins exhibited significantly different abundance during at least one growth phase. Sixteen proteins were uniquely detected and 47 proteins were significantly more abundant in the dibenzothiophene culture during at least one growth phase. The sulfate-free dibenzothiophene-containing culture reacted to sulfate starvation by restricting sulfur assimilation, enforcing sulfur-sparing, and maintaining redox homeostasis. Biodesulfurization triggered alternative pathways for sulfur assimilation different from those operating in the inorganic sulfate culture. Sulfur metabolism reprogramming and metabolic switches in the dibenzothiophene culture were manifested in limiting sulfite reduction and biosynthesis of cysteine, while boosting the production of methionine via the cobalamin-independent pathway, as well as the biosynthesis of the redox buffers mycothiol and ergothioneine. The omics data underscore the key role of sulfur metabolism in shaping the biodesulfurization phenotype and highlight potential targets for improving the biodesulfurization catalytic activity via metabolic engineering. IMPORTANCE For many decades, research on biodesulfurization of fossil fuels was conducted amid a large gap in knowledge of sulfur metabolism and its regulation in fuel-biodesulfurizing bacteria, which has impeded the development of a commercially viable bioprocess. In addition, lack of understanding of biodesulfurization-associated metabolic and physiological adaptations prohibited the development of efficient biodesulfurizers. Our integrated omics-based findings reveal the assimilatory sulfur metabolism in the biodesulfurization reference strain Rhodococcus qingshengii IGTS8 and show how sulfur metabolism and oxidative stress response were remodeled and orchestrated to shape the biodesulfurization phenotype. Our findings not only explain the frequently encountered low catalytic activity of native fuel-biodesulfurizing bacteria but also uncover unprecedented potential targets in sulfur metabolism that could be exploited via metabolic engineering to boost the biodesulfurization catalytic activity, a prerequisite for commercial application.

Heavy Vacuum Gas Oil Upregulates the Rhamnosyltransferases and Quorum Sensing Cascades of Rhamnolipids Biosynthesis in Pseudomonas sp. AK6U.

We followed a comparative approach to investigate how heavy vacuum gas oil (HVGO) affects the expression of genes involved in biosurfactants biosynthesis and the composition of the rhamnolipid congeners in Pseudomonas sp. AK6U. HVGO stimulated biosurfactants production as indicated by the lower surface tension (26 mN/m) and higher yield (7.8 g/L) compared to a glucose culture (49.7 mN/m, 0.305 g/L). Quantitative real-time PCR showed that the biosurfactants production genes rhlA and rhlB were strongly upregulated in the HVGO culture during the early and late exponential growth phases. To the contrary, the rhamnose biosynthesis genes algC, rmlA and rmlC were downregulated in the HVGO culture. Genes of the quorum sensing systems which regulate biosurfactants biosynthesis exhibited a hierarchical expression profile. The lasI gene was strongly upregulated (20-fold) in the HVGO culture during the early log phase, whereas both rhlI and pqsE were upregulated during the late log phase. Rhamnolipid congener analysis using high-performance liquid chromatography-mass spectrometry revealed a much higher proportion (up to 69%) of the high-molecularweight homologue Rha-Rha-C10-C10 in the HVGO culture. The results shed light on the temporal and carbon source-mediated shifts in rhamonlipids' composition and regulation of biosynthesis which can be potentially exploited to produce different rhamnolipid formulations tailored for specific applications.

Integrated Multi-omics Investigations Reveal the Key Role of Synergistic Microbial Networks in Removing Plasticizer Di-(2-Ethylhexyl) Phthalate from Estuarine Sediments.

Di-(2-ethylhexyl) phthalate (DEHP) is the most widely used plasticizer worldwide, with an annual global production of more than 8 million tons. Because of its improper disposal, endocrine-disrupting DEHP often accumulates in estuarine sediments in industrialized countries at submillimolar levels, resulting in adverse effects on both ecosystems and human beings. The microbial degraders and biodegradation pathways of DEHP in O2-limited estuarine sediments remain elusive. Here, we employed an integrated meta-omics approach to identify the DEHP degradation pathway and major degraders in this ecosystem. Estuarine sediments were treated with DEHP or its derived metabolites, o-phthalic acid and benzoic acid. The rate of DEHP degradation in denitrifying mesocosms was two times slower than that of o-phthalic acid, suggesting that side chain hydrolysis of DEHP is the rate-limiting step of anaerobic DEHP degradation. On the basis of microbial community structures, functional gene expression, and metabolite profile analysis, we proposed that DEHP biodegradation in estuarine sediments is mainly achieved through synergistic networks between denitrifying proteobacteria. Acidovorax and Sedimenticola are the major degraders of DEHP side chains; the resulting o-phthalic acid is mainly degraded by Aestuariibacter through the UbiD-dependent benzoyl coenzyme A (benzoyl-CoA) pathway. We isolated and characterized Acidovorax sp. strain 210-6 and its extracellular hydrolase, which hydrolyzes both alkyl side chains of DEHP. Interestingly, genes encoding DEHP/mono-(2-ethylhexyl) phthalate (MEHP) hydrolase and phthaloyl-CoA decarboxylase-key enzymes for side chain hydrolysis and o-phthalic acid degradation, respectively-are flanked by transposases in these proteobacterial genomes, indicating that DEHP degradation capacity is likely transferred horizontally in microbial communities. IMPORTANCE Xenobiotic phthalate esters (PAEs) have been produced on a considerably large scale for only 70 years. The occurrence of endocrine-disrupting di-(2-ethylhexyl) phthalate (DEHP) in environments has raised public concern, and estuarine sediments are major DEHP reservoirs. Our multi-omics analyses indicated that complete DEHP degradation in O2-limited estuarine sediments depends on synergistic microbial networks between diverse denitrifying proteobacteria and uncultured candidates. Our data also suggested that the side chain hydrolysis of DEHP, rather than o-phthalic acid activation, is the rate-limiting step in DEHP biodegradation within O2-limited estuarine sediments. Therefore, deciphering the bacterial ecophysiology and related biochemical mechanisms can help facilitate the practice of bioremediation in O2-limited environments. Furthermore, the DEHP hydrolase genes of active DEHP degraders can be used as molecular markers to monitor environmental DEHP degradation. Finally, future studies on the directed evolution of identified DEHP/mono-(2-ethylhexyl) phthalate (MEHP) hydrolase would bring a more catalytically efficient DEHP/MEHP hydrolase into practice.

Diesel-born organosulfur compounds stimulate community re-structuring in a diesel-biodesulfurizing consortium.

We enriched and characterized a biodesulfurizing consortium (designated as MG1). The MG1 consortium reduced the total sulfur of diesel by 25 % and utilized each of the diesel-born compounds dibenzothiophene (DBT), benzothiophene (BT), 4-methyldibenzothiophene (4-MDBT) and 4, 6-dimethyldibenzothiophene (4, 6-DMDBT) as a sole sulfur source. MiSeq analysis revealed compositional shifts in the MG1 community according to the type of the sulfur source. A DBT-grown MG1 culture had Klebsiella, Pseudomonas, Rhodococcus and Sphingomonas as the most abundant genera. When diesel or 4, 6-DMDBT was provided as a sole sulfur source, Klebsiella and Pseudomonas spp. were the most abundant. In the BT culture, Rhodococcus spp. were the key biodesulfurizers, while Klebsiella, Pseudomonas and Sphingomonas spp. dominated the 4-MDBT-grown consortium. MG1 also utilized 2-hydroxybiphenyl (the product of the 4S biodesulfurization pathway) where Pseudomonas spp. uniquely dominated the consortium. The data improves our understanding of the sulfur source-driven structural adaptability of biodesulfurizing consortia.

Phylogenomic Classification and Biosynthetic Potential of the Fossil Fuel-Biodesulfurizing Rhodococcus Strain IGTS8.

Rhodococcus strain IGTS8 is the most extensively studied model bacterium for biodesulfurization of fossil fuels via the non-destructive sulfur-specific 4S pathway. This strain was initially assigned to Rhodococcus rhodochrous and later to Rhodococcus erythropolis thus making its taxonomic status debatable and reflecting the limited resolution of methods available at the time. In this study, phylogenomic analyses of the whole genome sequences of strain IGTS8 and closely related rhodococci showed that R. erythropolis and Rhodococcus qingshengii are very closely related species, that Rhodococcus strain IGTS8 is a R. qingshengii strain and that several strains identified as R. erythropolis should be re-classified as R. qingshengii. The genomes of strains assigned to these species contain potentially novel biosynthetic gene clusters showing that members of these taxa should be given greater importance in the search for new antimicrobials and other industrially important biomolecules. The plasmid-borne dsz operon encoding fossil fuel desulfurization enzymes was present in R. qingshengii IGTS8 and R. erythropolis XP suggesting that it might be transferable between members of these species.

Natural Attenuation Potential of Polychlorinated Biphenyl-Polluted Marine Sediments.

The marine environment in Kuwait is polluted with various hazardous chemicals of industrial origin. These include petroleum hydrocarbons, halogenated compounds and heavy metals. Bioremediation with dedicated microorganisms can be effectively applied for reclamation of the polluted marine sediments. However, information on the autochthonous microbes and their ecophysiology is largely lacking. We analyzed sediments from Shuwaikh harbor to detect polychlorinated biphenyls (PCBs) and total petroleum hydrocarbons (TPHs). Then we adopted both culture-dependent and culture-independent (PCR-DGGE) approaches to identify bacterial inhabitants of the polluted marine sediments from Shuwaikh harbor. The chemical analysis revealed spatial variation among the sampling stations in terms of total amount of PCBs, TPHs and the PCB congener fingerprints. Moreover, in all analyzed sediments, the medium-chlorine PCB congeners were more abundant than the low-chlorine and high-chlorine counterparts. PCR-DGGE showed the presence of members of the Proteobacteria, Spirochaetes, Firmicutes and Bacteroidetes in the analyzed sediments. However, Chloroflexi-related bacteria dominated the detected bacterial community. We also enriched a biphenyl-utilizing mixed culture using the W2 station sediment as an inoculum in chemically defined medium using biphenyl as a sole carbon and energy source. The enriched mixed culture consisted mainly of the Firmicute Paenibacillus spp. Sequences of genes encoding putative aromatic ring-hydroxylating dioxygenases were detected in sediments from most sampling stations and the enriched mixed culture. The results suggest the potential of bioremediation as a means for natural attenuation of Shuwaikh harbor sediments polluted with PCBs and TPHs.

Estrogen Degraders and Estrogen Degradation Pathway Identified in an Activated Sludge.

The environmental release and fate of estrogens are becoming an increasing public concern. Bacterial degradation has been considered the main process for eliminating estrogens from wastewater treatment plants. Various bacterial isolates are reportedly capable of aerobic estrogen degradation, and several estrogen degradation pathways have been proposed in proteobacteria and actinobacteria. However, the ecophysiological relevance of estrogen-degrading bacteria in the environment is unclear. In this study, we investigated the estrogen degradation pathway and corresponding degraders in activated sludge collected from the Dihua Sewage Treatment Plant, Taipei, Taiwan. Cultivation-dependent and cultivation-independent methods were used to assess estrogen biodegradation in the collected activated sludge. Estrogen metabolite profile analysis revealed the production of pyridinestrone acid and two A/B-ring cleavage products in activated sludge incubated with estrone (1 mM), which are characteristic of the 4,5-seco pathway. PCR-based functional assays detected sequences closely related to alphaproteobacterial oecC, a key gene of the 4,5-seco pathway. Metagenomic analysis suggested that Novosphingobium spp. are major estrogen degraders in estrone-amended activated sludge. Novosphingobium sp. strain SLCC, an estrone-degrading alphaproteobacterium, was isolated from the examined activated sludge. The general physiology and metabolism of this strain were characterized. Pyridinestrone acid and the A/B-ring cleavage products were detected in estrone-grown strain SLCC cultures. The production of pyridinestrone acid was also observed during the aerobic incubation of strain SLCC with 3.7 nM (1 µg/liter) estrone. This concentration is close to that detected in many natural and engineered aquatic ecosystems. The presented data suggest the ecophysiological relevance of Novosphingobium spp. in activated sludge.IMPORTANCE Estrogens, which persistently contaminate surface water worldwide, have been classified as endocrine disruptors and human carcinogens. We contribute new knowledge on the major estrogen biodegradation pathway and estrogen degraders in wastewater treatment plants. This study considerably advances the understanding of environmental estrogen biodegradation, which is instrumental for the efficient elimination of these hazardous pollutants. Moreover, this study substantially improves the understanding of microbial estrogen degradation in the environment.

Biochemical Mechanisms and Microorganisms Involved in Anaerobic Testosterone Metabolism in Estuarine Sediments.

Current knowledge on the biochemical mechanisms underlying microbial steroid metabolism in anaerobic ecosystems is extremely limited. Sulfate, nitrate, and iron [Fe (III)] are common electron acceptors for anaerobes in estuarine sediments. Here, we investigated anaerobic testosterone metabolism in anaerobic sediments collected from the estuary of Tamsui River, Taiwan. The anaerobic sediment samples were spiked with testosterone (1 mM) and individual electron acceptors (10 mM), including nitrate, Fe3+, and sulfate. The analysis of androgen metabolites indicated that testosterone biodegradation under denitrifying conditions proceeds through the 2,3-seco pathway, whereas testosterone biodegradation under iron-reducing conditions may proceed through an unidentified alternative pathway. Metagenomic analysis and PCR-based functional assays suggested that Thauera spp. were the major testosterone degraders in estuarine sediment samples incubated with testosterone and nitrate. Thauera sp. strain GDN1, a testosterone-degrading betaproteobacterium, was isolated from the denitrifying sediment sample. This strain tolerates a broad range of salinity (0-30 ppt). Although testosterone biodegradation did not occur under sulfate-reducing conditions, we observed the anaerobic biotransformation of testosterone to estrogens in some testosterone-spiked sediment samples. This is unprecedented since biotransformation of androgens to estrogens is known to occur only under oxic conditions. Our metagenomic analysis suggested that Clostridium spp. might play a role in this anaerobic biotransformation. These results expand our understanding of microbial metabolism of steroids under strictly anoxic conditions.

Simultaneous valorization and biocatalytic upgrading of heavy vacuum gas oil by the biosurfactant-producing Pseudomonas aeruginosa AK6U.

Heavy vacuum gas oil (HVGO) is a complex and viscous hydrocarbon stream that is produced as the bottom side product from the vacuum distillation units in petroleum refineries. HVGO is conventionally treated with thermochemical process, which is costly and environmentally polluting. Here, we investigate two petroleum biotechnology applications, namely valorization and bioupgrading, as green approaches for valorization and upgrading of HVGO. The Pseudomonas aeruginosa AK6U strain grew on 20% v/v of HVGO as a sole carbon and sulfur source. It produced rhamnolipid biosurfactants in a growth-associated mode with a maximum crude biosurfactants yield of 10.1 g l-1 , which reduced the surface tension of the cell-free culture supernatant to 30.6 mN m-1 within 1 week of incubation. The rarely occurring dirhamnolipid Rha-Rha-C12 -C12 dominated the congeners' profile of the biosurfactants produced from HVGO. Heavy vacuum gas oil was recovered from the cultures and abiotic controls and the maltene fraction was extracted for further analysis. Fractional distillation (SimDist) of the biotreated maltene fraction showed a relative decrease in the high-boiling heavy fuel fraction (BP 426-565 °C) concomitant with increase in the lighter distillate diesel fraction (BP 315-426 °C). Analysis of the maltene fraction revealed compositional changes. The number-average (Mn) and weight-average (Mw) molecular weights, as well as the absolute number of hydrocarbons and sulfur heterocycles were higher in the biotreated maltene fraction of HVGO. These findings suggest that HVGO can be potentially exploited as a carbon-rich substrate for production of the high-value biosurfactants by P. aeruginosa AK6U and to concomitantly improve/upgrade its chemical composition.

Biochemical Mechanisms and Catabolic Enzymes Involved in Bacterial Estrogen Degradation Pathways.

Estrogens have been classified as group 1 carcinogens by the World Health Organization and represent a significant concern given that they are found in surface waters worldwide, and long-term exposure to estrogen-contaminated water can disrupt sexual development in animals. To date, the estrogen catabolic enzymes and genes remain unknown. Using a tiered functional genomics approach, we identified three estrogen catabolic gene clusters in Sphingomonas sp. strain KC8. We identified several estrone-derived compounds, including 4-hydroxyestrone, a meta-cleavage product, and pyridinestrone acid. The yeast-based estrogen assay suggested that pyridinestrone acid exhibits negligible estrogenic activity. We characterized 17ß-estradiol dehydrogenase and 4-hydroxyestrone 4,5-dioxygenase, responsible for the 17-dehydrogenation and meta-cleavage of the estrogen A ring, respectively. The characteristic pyridinestrone acid was detected in estrone-spiked samples collected from two wastewater treatment plants and two suburban rivers in Taiwan. The results significantly expand our understanding of microbial degradation of aromatic steroids at molecular level.

Identification of Comamonas testosteroni as an androgen degrader in sewage.

Numerous studies have reported the masculinization of freshwater wildlife exposed to androgens in polluted rivers. Microbial degradation is a crucial mechanism for eliminating steroid hormones from contaminated ecosystems. The aerobic degradation of testosterone was observed in various bacterial isolates. However, the ecophysiological relevance of androgen-degrading microorganisms in the environment is unclear. Here, we investigated the biochemical mechanisms and corresponding microorganisms of androgen degradation in aerobic sewage. Sewage samples collected from the Dihua Sewage Treatment Plant (Taipei, Taiwan) were aerobically incubated with testosterone (1 mM). Androgen metabolite analysis revealed that bacteria adopt the 9, 10-seco pathway to degrade testosterone. A metagenomic analysis indicated the apparent enrichment of Comamonas spp. (mainly C. testosteroni) and Pseudomonas spp. in sewage incubated with testosterone. We used the degenerate primers derived from the meta-cleavage dioxygenase gene (tesB) of various proteobacteria to track this essential catabolic gene in the sewage. The amplified sequences showed the highest similarity (87-96%) to tesB of C. testosteroni. Using quantitative PCR, we detected a remarkable increase of the 16S rRNA and catabolic genes of C. testosteroni in the testosterone-treated sewage. Together, our data suggest that C. testosteroni, the model microorganism for aerobic testosterone degradation, plays a role in androgen biodegradation in aerobic sewage.

Biocatalytic Desulfurization Capabilities of a Mixed Culture during Non-Destructive Utilization of Recalcitrant Organosulfur Compounds.

We investigated the biodesulfurization potential of a mixed culture AK6 enriched from petroleum hydrocarbons-polluted soil with dibenzothiophene (DBT) as a sulfur source. In addition to DBT, AK6 utilized the following compounds as sulfur sources: 4-methyldibenzothiophene (4-MDBT), benzothiophene (BT), and 4,6- dimethyldibenzothiophene (4,6-DM-DBT). None of these compounds supported the growth of AK6 as the sole carbon and sulfur source. AK6 could not grow on dibenzylsulfide (DBS) as a sulfur source. The AK6 community structure changed according to the provided sulfur source. The major DGGE bands represented members of the genera Sphingobacterium, Klebsiella, Pseudomonas, Stenotrophomonas, Arthrobacter, Mycobacterium, and Rhodococcus. Sphingobacterium sp. and Pseudomonas sp. were abundant across all cultures utilizing any of the tested thiophenic S-compounds. Mycobacterium/Rhodococcus spp. were restricted to the 4-MDBT culture. The 4-MDBT culture had the highest species richness and diversity. Biodesulfurization of DBT by resting cells of AK6 produced 2-hydroxybiphenyl (2-HBP) in addition to trace amounts of phenylacetate. AK6 transformed DBT to 2-hydroxybiphenyl with a specific activity of 9 ± 0.6 µM 2-HBP g dry cell weight(-1) h(-1). PCR confirmed the presence in the AK6 community of the sulfur-specific (4S) pathway genes dszB and dszC. Mixed cultures hold a better potential than axenic ones for the development of a biodesulfurization technology.

Integrated multi-omics analyses reveal the biochemical mechanisms and phylogenetic relevance of anaerobic androgen biodegradation in the environment.

Steroid hormones, such as androgens, are common surface-water contaminants. However, literature on the ecophysiological relevance of steroid-degrading organisms in the environment, particularly in anoxic ecosystems, is extremely limited. We previously reported that Steroidobacter denitrificans anaerobically degrades androgens through the 2,3-seco pathway. In this study, the genome of Sdo. denitrificans was completely sequenced. Transcriptomic data revealed gene clusters that were distinctly expressed during anaerobic growth on testosterone. We isolated and characterized the bifunctional 1-testosterone hydratase/dehydrogenase, which is essential for anaerobic degradation of steroid A-ring. Because of apparent substrate preference of this molybdoenzyme, corresponding genes, along with the signature metabolites of the 2,3-seco pathway, were used as biomarkers to investigate androgen biodegradation in the largest sewage treatment plant in Taipei, Taiwan. Androgen metabolite analysis indicated that denitrifying bacteria in anoxic sewage use the 2,3-seco pathway to degrade androgens. Metagenomic analysis and PCR-based functional assays showed androgen degradation in anoxic sewage by Thauera spp. through the action of 1-testosterone hydratase/dehydrogenase. Our integrative 'omics' approach can be used for culture-independent investigations of the microbial degradation of structurally complex compounds where isotope-labeled substrates are not easily available.

Stimulation of rhamnolipid biosurfactants production in Pseudomonas aeruginosa AK6U by organosulfur compounds provided as sulfur sources.

A Pseudomonas aeruginosa AK6U strain produced rhamnolipid biosurfactants to variable extents when grown on MgSO4 or organosulfur compounds as sulfur sources and glucose as a carbon source. Organosulfur cultures produced much higher biosurfactants amounts compared to the MgSO4 cultures. The surface tension of the growth medium was reduced from 72 mN/m to 54 and 30 mN/m in cultures containing MgSO4 and 4,6-dimethyldibenzothiophene (4,6-DM-DBT), respectively. AK6U cultures produced different rhamnolipid congener profiles depending on the provided sulfur source. The dibenzothiophene (DBT) culture produced more diverse and a higher number of rhamnolipid congeners as compared to the DBT-sulfone and MgSO4 cultures. The number of mono-rhamnolipid congeners in the DBT culture was also higher than that detected in the DBT-sulfone and MgSO4 cultures. Di-rhamnolipids dominated the congener profiles in all the analyzed cultures. The sulfur source can have a profound impact on the quality and quantity of the produced biosurfactants.