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Microbial Biotechnology Jun 2024Alkanes, single carbon methane to long-chain hydrocarbons (e.g. hexadecane and tetradecane), are important carbon sources to anaerobic microbial communities. In anoxic... (Review)
Review
Alkanes, single carbon methane to long-chain hydrocarbons (e.g. hexadecane and tetradecane), are important carbon sources to anaerobic microbial communities. In anoxic environments, archaea are known to utilize and produce methane via the methyl-coenzyme M reductase enzyme (MCR). Recent explorations of new environments, like deep sea sediments, that have coupled metagenomics and cultivation experiments revealed divergent MCRs, also referred to as alkyl-coenzyme M reductases (ACRs) in archaea, with similar mechanisms as the C utilizing canonical MCR mechanism. These ACR enzymes have been shown to activate other alkanes such as ethane, propane and butane for subsequent degradation. The reversibility of canonical MCRs suggests that these non-methane-activating homologues (ACRs) might have similar reversibility, perhaps mediated by undiscovered lineages that produce alkanes under certain conditions. The discovery of these alternative alkane utilization pathways holds significant promise for a breadth of potential biotechnological applications in bioremediation, energy production and climate change mitigation.
Topics: Anaerobiosis; Archaea; Methane; Hydrocarbons; Oxidoreductases; Alkanes; Metabolic Networks and Pathways; Biodegradation, Environmental
PubMed: 38888492
DOI: 10.1111/1751-7915.14508 -
Journal of Visualized Experiments : JoVE May 2024A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or...
A key virulence mechanism for many Gram-negative pathogens is the type III secretion system (T3SS), a needle-like appendage that translocates cytotoxic or immunomodulatory effector proteins into host cells. The T3SS is a target for antimicrobial discovery campaigns since it is accessible extracellularly and largely absent from non-pathogenic bacteria. Recent studies demonstrated that the T3SS of Yersinia and Salmonella are regulated by factors responsive to iron and oxygen, which are important niche-specific signals encountered during mammalian infection. Described here is a method for iron starvation of Yersinia pseudotuberculosis, with subsequent optional supplementation of inorganic iron. To assess the impact of oxygen availability, this iron starvation process is demonstrated under both aerobic and anaerobic conditions. Finally, incubating the cultures at the mammalian host temperature of 37 °C induces T3SS expression and allows quantification of Yersinia T3SS activity by visualizing effector proteins released into the supernatant. The steps detailed here offer an advantage over the use of iron chelators in the absence of iron starvation, which is insufficient for inducing robust iron starvation, presumably due to efficient Yersinia iron uptake and scavenging systems. Likewise, acid-washing laboratory glassware is detailed to ensure the removal of residual iron, which is essential for inducing robust iron starvation. Additionally, using a chelating agent is described to remove residual iron from media, and culturing the bacteria for several generations in the absence of iron to deplete bacterial iron stores. By incorporating standard protocols of trichloroacetic acid-induced protein precipitation, SDS-PAGE, and silver staining, this procedure demonstrates accessible ways to measure T3SS activity. While this procedure is optimized for Y. pseudotuberculosis, it offers a framework for studies in pathogens with similar robust iron uptake systems. In the age of antibiotic resistance, these methods can be expanded to assess the efficacy of antimicrobial compounds targeting the T3SS under host-relevant conditions.
Topics: Yersinia pseudotuberculosis; Iron; Type III Secretion Systems; Anaerobiosis
PubMed: 38884494
DOI: 10.3791/66642 -
Journal of Hazardous Materials Aug 2024Nanofiltration (NF) is a promising technology in the treatment of microelectronic wastewater. However, the treatment of concentrate derived from NF system remains a...
Nanofiltration (NF) is a promising technology in the treatment of microelectronic wastewater. However, the treatment of concentrate derived from NF system remains a substantial technical challenge, impeding the achievement of the zero liquid discharge (ZLD) goal in microelectronic wastewater industries. Herein, a ZLD system, coupling a two-stage NF technology with anaerobic biotechnology was proposed for the treatment of tetramethylammonium hydroxide (TMAH)-contained microelectronic wastewater. The two-stage NF system exhibited favorable efficacy in the removal of conductivity (96 %), total organic carbon (TOC, 90 %), and TMAH (96 %) from microelectronic wastewater. The membrane fouling of this system was dominated by organic fouling, with the second stage NF membrane experiencing a more serious fouling compared to the first stage membrane. The anaerobic biotechnology achieved a near-complete removal of TMAH and an 80 % reduction in TOC for the first stage NF concentrate. Methyloversatilis was the key genus involved in the anaerobic treatment of the microelectronic wastewater concentrate. Specific genes, including dmd-tmd, mtbA, mttB and mttC were identified as significant players in mediating the dehydrogenase and methyl transfer pathways during the process of TMAH biodegradation. This study highlights the potential of anaerobic biodegradation to achieve ZLD in the treatment of TMAH-contained microelectronic wastewater by NF system.
Topics: Wastewater; Quaternary Ammonium Compounds; Anaerobiosis; Filtration; Biodegradation, Environmental; Waste Disposal, Fluid; Membranes, Artificial; Water Purification; Water Pollutants, Chemical; Bioreactors; Electronic Waste; Nanotechnology
PubMed: 38880045
DOI: 10.1016/j.jhazmat.2024.134924 -
Journal of Hazardous Materials Aug 2024Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an important methane (CH) consumption and nitrogen (N) removal pathway in estuarine and coastal...
Nitrite/nitrate-dependent anaerobic methane oxidation (n-DAMO) is an important methane (CH) consumption and nitrogen (N) removal pathway in estuarine and coastal wetlands. Antibiotic contamination is known to affect microbially mediated processes; however, its influences on n-DAMO and the underlying molecular mechanisms remain poorly understood. In the present study, using CH tracer method combined with molecular techniques, we investigated the responses of n-DAMO microbial abundance, activity, and the associated microbial community composition to sulfamethazine (SMT, a sulfonamide antibiotic, with exposure concentrations of 0.05, 0.5, 5, 20, 50, and 100 µg L). Results showed that the effect of SMT exposure on n-DAMO activity was dose-dependent. Exposure to SMT at concentrations of up to 5 µg L inhibited the potential n-DAMO rates (the average rates of nitrite- and nitrate-DAMO decreased by 92.9 % and 79.2 % relative to the control, respectively). In contrast, n-DAMO rates tended to be promoted by SMT when its concentration increased to 20-100 µg L (the average rates of nitrite- and nitrate-DAMO increased by 724.1 % and 630.1 % relative to the low-doses, respectively). Notably, low-doses of SMT suppressed nitrite-DAMO to a greater extent than nitrate-DAMO, indicating that nitrite-DAMO was more sensitive to SMT than nitrate-DAMO. Molecular analyses suggest that the increased n-DAMO activity under high-doses SMT exposure may be driven by changes in microbial communities, especially because of the promotion of methanogens that provide more CH to n-DAMO microbes. Moreover, the abundances of n-DAMO microbes at high SMT exposure (20 and 50 µg L) were significantly higher than that at low SMT exposure (0.05-5 µg L). These results advance our understanding of the ecological effects of SMT on carbon (C) and N interactions in estuarine and coastal wetlands.
Topics: Wetlands; Methane; Sulfamethazine; Anaerobiosis; Oxidation-Reduction; Denitrification; Water Pollutants, Chemical; Anti-Bacterial Agents; Estuaries; Bacteria; Nitrites; Nitrates
PubMed: 38878438
DOI: 10.1016/j.jhazmat.2024.134893 -
Journal of Hazardous Materials Aug 2024Minimization of antibiotic resistance genes (ARGs) and potential pathogenic antibiotic-resistant bacteria (PARB) during anaerobic digestion (AD) is significantly...
Minimization of antibiotic resistance genes (ARGs) and potential pathogenic antibiotic-resistant bacteria (PARB) during anaerobic digestion (AD) is significantly impacted by temperature. However, knowledge on how ARGs and PARB respond to temperature transition from thermophilic to mesophilic is limited. Here, we combined metagenomic-based with culture-based approaches and revealed the risks of antimicrobial resistance and pathogenicity during transition from 55 °C to 35 °C for AD, with strategies of sharp (ST, one-step by 20 °C/d) and mild (MT, step-wise by 1 °C/d). Results indicated a lower decrease in methane production with MT (by 38.9%) than ST (by 88.8%). Phenotypic assays characterized a significant propagation of multi-resistant lactose-fermenting Enterobacteriaceae and indicator pathogens after both transitions, especially via ST. Further genomic evidence indicated a significant increase of ARGs (29.4-fold), virulence factor genes (1.8-fold) and PARB (65.3-fold) after ST, while slight enrichment via MT. Bacterial succession and enhanced horizontal transfer mediated by mobile genetic elements promoted ARG propagation in AD during transition, which was synchronously exacerbated through horizontal transfer mechanisms mediated by cellular physiological responses (oxidative stress, membrane permeability, bacterial conjugation and transformation) and co-selection mechanisms of biomethanation metabolic functions (acidogenesis and acetogenesis). This study reveals temperature-dependent resistome and pathogenicity development in AD, facilitating microbial risk control.
Topics: Anaerobiosis; Drug Resistance, Bacterial; Temperature; Methane; Bacteria; Anti-Bacterial Agents; Drug Resistance, Microbial; Virulence Factors; Gene Transfer, Horizontal; Enterobacteriaceae; Genes, Bacterial
PubMed: 38878435
DOI: 10.1016/j.jhazmat.2024.134886 -
Journal of Hazardous Materials Aug 2024Poly(butylene adipate-co-terephthalate) (PBAT) is a biodegradable plastic that is difficult to degrade under both mesophilic and thermophilic anaerobic conditions. In...
Poly(butylene adipate-co-terephthalate) (PBAT) is a biodegradable plastic that is difficult to degrade under both mesophilic and thermophilic anaerobic conditions. In this study, the impact of the thermo-alkaline pretreatment (48 h, 70 °C, 1 % w/v NaOH) on the anaerobic degradation (AD) of PBAT, poly(lactic acid) (PLA) and PBAT/PLA blended plastics was investigated. Under mesophilic conditions, pretreatment only improved the methane yield of PBAT/PLA/starch plastic (100 days, 51 and 34 NmL/g VS for the treated and original plastics, respectively). Under thermophilic conditions, the pretreatment increased the methanogenic rate of PLA, PBAT and PBAT/PLA/starch plastic at the beginning stage (22 days, 35 and 79 NmL/g VS for original and treated PBAT, respectively), but did not change the methane yield at the end of the incubation (100 days, 91 NmL/g VS for original and treated PBAT). The reduction in the molecular weight and the formation of pore structures on the plastic surface accelerated the utilization of plastics by microorganisms. Furthermore, the pretreated plastics tend to form microplastics (MPs) with size predominantly below 500 µm (>90 %). The numbers of MPs dynamically changed with the degradation time. Several genera of bacteria showed specific degradation of biodegradable plastics under thermophilic conditions, including Desulfitibacter, Coprothermobacter, Tepidimicrobium, c_ D8A-2 and Thermacetogenium. The results suggest that more attention should be paid to the problem of MPs arising from the thermo-alkaline pretreatment.
Topics: Polyesters; Anaerobiosis; Methane; Plastics; Biodegradation, Environmental; Sodium Hydroxide; Temperature
PubMed: 38870853
DOI: 10.1016/j.jhazmat.2024.134882 -
Chemosphere Aug 2024Lignin, a major component of plant biomass, remains underutilized for renewable biofuels due to its complex and heterogeneous structure. Although investigations into...
Lignin, a major component of plant biomass, remains underutilized for renewable biofuels due to its complex and heterogeneous structure. Although investigations into depolymerizing lignin using fungi are well-established, studies of microbial pathways that enable anaerobic lignin breakdown linked with methanogenesis are limited. Through an enrichment cultivation approach with inoculation of freshwater sediment, we enriched a microbial community capable of producing methane during anaerobic lignin degradation. We reconstructed the near-complete population genomes of key lignin degraders and methanogens using metagenome-assembled genomes finally selected in this study (MAGs; 92 bacterial and 4 archaeal MAGs affiliated into 45 and 2 taxonomic groups, respectively). This study provides genetic evidence of microbial interdependence in conversion of lignin to methane in a syntrophic community. Metagenomic analysis revealed metabolic linkages, with lignin-hydrolyzing and/or fermentative bacteria such as the genera Alkalibaculum and Propionispora transforming lignin breakdown products into compounds such as acetate to feed methanogens (two archaeal MAGs classified into the genus Methanosarcina or UBA6 of the family Methanomassiliicoccaceae). Understanding the synergistic relationships between microbes that convert lignin could inform strategies for producing renewable bioenergy and treating aromatic-contaminated environments through anaerobic biodegradation processes. Overall, this study offers fundamental insights into complex community-level anaerobic lignin metabolism, highlighting hitherto unknown players, interactions, and pathways in this biotechnologically valuable process.
Topics: Lignin; Anaerobiosis; Biodegradation, Environmental; Biofuels; Archaea; Bacteria; Methane; Microbiota; Metagenome
PubMed: 38866340
DOI: 10.1016/j.chemosphere.2024.142588 -
Biochimica Et Biophysica Acta.... Jun 2024The effectivity of utilization of exogenous sterols in the yeast Saccharomyces cerevisiae exposed to hypoxic stress is dependent on the sterol structure. The highly...
The effectivity of utilization of exogenous sterols in the yeast Saccharomyces cerevisiae exposed to hypoxic stress is dependent on the sterol structure. The highly imported sterols include animal cholesterol or plant sitosterol, while ergosterol, typical of yeasts, is imported to a lesser extent. An elevated utilization of non-yeast sterols is associated with their high esterification and relocalization to lipid droplets (LDs). Here we present data showing that LDs and sterol esterification play a critical role in the regulation of the accumulation of non-yeast sterols in membranes. Failure to form LDs during anaerobic growth in media supplemented with cholesterol or sitosterol resulted in an extremely long lag phase, in contrast to normal growth in media with ergosterol or plant stigmasterol. Moreover, in hem1∆, which mimics anaerobiosis, neither cholesterol nor sitosterol supported the growth in an LD-less background. The incorporation of non-ergosterol sterols into the membranes affected fundamental membrane characteristics such as relative membrane potential, permeability, tolerance to osmotic stress and the formation of membrane domains. Our findings reveal that LDs assume an important role in scenarios wherein cells are dependent on the utilization of exogenous lipids, particularly under anoxia. Given the diverse lipid structures present in yeast niches, LDs fulfil a protective role, mitigating the risk of excessive accumulation of potentially toxic steroids and fatty acids in the membranes. Finally, we present a novel function for sterols in a model eukaryotic cell - alleviation of the lipotoxicity of unsaturated fatty acids.
PubMed: 38866087
DOI: 10.1016/j.bbalip.2024.159523 -
Proceedings of the National Academy of... Jun 2024Nitrous oxide (NO), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources...
Nitrous oxide (NO), a potent greenhouse gas, can be generated by multiple biological and abiotic processes in diverse contexts. Accurately tracking the dominant sources of NO has the potential to improve our understanding of NO fluxes from soils as well as inform the diagnosis of human infections. Isotopic "Site Preference" (SP) values have been used toward this end, as bacterial and fungal nitric oxide reductases (NORs) produce NO with different isotopic fingerprints, spanning a large range. Here, we show that flavohemoglobin (Fhp), a hitherto biogeochemically neglected yet widely distributed detoxifying bacterial NO reductase, imparts a distinct SP value onto NO under anoxic conditions (~+10‰) that correlates with typical environmental NO SP measurements. Using as a model organism, we generated strains that only contained Fhp or the dissimilatory NOR, finding that in vivo NO SP values imparted by these enzymes differ by over 10‰. Depending on the cellular physiological state, the ratio of Fhp:NOR varies significantly in wild-type cells and controls the net NO SP biosignature: When cells grow anaerobically under denitrifying conditions, NOR dominates; when cells experience rapid, increased nitric oxide concentrations under anoxic conditions but are not growing, Fhp dominates. Other bacteria that only make Fhp generate similar NO SP biosignatures to those measured from our Fhp-only strain. Fhp homologs in sequenced bacterial genomes currently exceed NOR homologs by nearly a factor of four. Accordingly, we suggest a different framework to guide the attribution of NO biological sources in nature and disease.
Topics: Nitrous Oxide; Oxidoreductases; Pseudomonas aeruginosa; Anaerobiosis; Bacterial Proteins; Nitric Oxide
PubMed: 38865268
DOI: 10.1073/pnas.2319960121 -
World Journal of Microbiology &... Jun 2024Anaerobic digestion (AD) emerges as a pivotal technique in climate change mitigation, transforming organic materials into biogas, a renewable energy form. This process...
Anaerobic digestion (AD) emerges as a pivotal technique in climate change mitigation, transforming organic materials into biogas, a renewable energy form. This process significantly impacts energy production and waste management, influencing greenhouse gas emissions. Traditional research has largely focused on anaerobic bacteria and methanogens for methane production. However, the potential of anaerobic lignocellulolytic fungi for degrading lignocellulosic biomass remains less explored. In this study, buffalo rumen inocula were enriched and acclimatized to improve lignocellulolytic hydrolysis activity. Two consortia were established: the anaerobic fungi consortium (AFC), selectively enriched for fungi, and the anaerobic lignocellulolytic microbial consortium (ALMC). The consortia were utilized to create five distinct microbial cocktails-AF0, AF20, AF50, AF80, and AF100. These cocktails were formulated based on varying of AFC and ALMC by weights (w/w). Methane production from each cocktail of lignocellulosic biomasses (cassava pulp and oil palm residues) was evaluated. The highest methane yields of CP, EFB, and MFB were obtained at 337, 215, and 54 mL/g VS, respectively. Cocktails containing a mix of anaerobic fungi, hydrolytic bacteria (Sphingobacterium sp.), syntrophic bacteria (Sphaerochaeta sp.), and hydrogenotrophic methanogens produced 2.1-2.6 times higher methane in cassava pulp and 1.1-1.2 times in oil palm empty fruit bunch compared to AF0. All cocktails effectively produced methane from oil palm empty fruit bunch due to its lipid content. However, methane production ceased after 3 days when oil palm mesocarp fiber was used, due to long-chain fatty acid accumulation. Anaerobic fungi consortia showed effective lignocellulosic and starchy biomass degradation without inhibition due to organic acid accumulation. These findings underscore the potential of tailored microbial cocktails for enhancing methane production from diverse lignocellulosic substrates.
Topics: Methane; Anaerobiosis; Lignin; Fungi; Microbial Consortia; Animals; Biomass; Rumen; Biofuels; Hydrolysis; Fermentation; Bacteria; Industrial Waste; Agriculture
PubMed: 38862848
DOI: 10.1007/s11274-024-04050-7