Degradation of gaseous hydrocarbons in aerated stirred bioreactors inoculated with Rhodococcus erythropolis: Effect of the carbon source and SIFT-MS method development.

The study of microbial hydrocarbons removal is of great importance for the development of future bioremediation strategies. In this study, we evaluated the removal of a gaseous mixture containing toluene, m-xylene, ethylbenzene, cyclohexane, butane, pentane, hexane and heptane in aerated stirred bioreactors inoculated with Rhodococcus erythropolis and operated under non-sterile conditions. For the real-time measurement of hydrocarbons, a novel systematic approach was implemented using Selected-Ion Flow Tube Mass Spectrometry (SIFT-MS). The effect of the carbon source (∼9.5 ppmv) on (i) the bioreactors' performance (BR1: dosed with only cyclohexane as a single hydrocarbon versus BR2: dosed with a mixture of the 8 hydrocarbons) and (ii) the evolution of microbial communities over time were investigated. The results showed that cyclohexane reached a maximum removal efficiency (RE) of 53% ± 4% in BR1. In BR2, almost complete removal of toluene, m-xylene and ethylbenzene, being the most water-soluble and easy-to-degrade carbon sources, was observed. REs below 32% were obtained for the remaining compounds. By exposing the microbial consortium to only the five most recalcitrant hydrocarbons, REs between 45% ± 5% and 98% ± 1% were reached. In addition, we observed that airborne microorganisms populated the bioreactors and that the type of carbon source influenced the microbial communities developed. The abundance of species belonging to the genus Rhodococcus was below 10% in all bioreactors at the end of the experiments. This work provides fundamental insights to understand the complex behavior of gaseous hydrocarbon mixtures in bioreactors, along with a systematic approach for the development of SIFT-MS methods.

Environmental stressor assessment of hydrocarbonoclastic bacteria biofilms from a marine oil spill.

The environmental and economic impact of an oil spill can be significant. Biotechnologies applied during a marine oil spill involve bioaugmentation with immobilised or encapsulated indigenous hydrocarbonoclastic species selected under laboratory conditions to improve degradation rates. The environmental factors that act as stressors and impact the effectiveness of hydrocarbon removal are one of the challenges associated with these applications. Understanding how native microbes react to environmental stresses is necessary for effective bioaugmentation. Herein, Micrococcus luteus and M. yunnanensis isolated from a marine oil spill mooring system showed hydrocarbonoclastic activity on Maya crude oil in a short time by means of total petroleum hydrocarbons (TPH) at 144 h: M. luteus up to 98.79 % and M. yunnanensis 97.77 % removal. The assessment of Micrococcus biofilms at different temperature (30 °C and 50 °C), pH (5, 6, 7, 8, 9), salinity (30, 50, 60, 70, 80 g/L), and crude oil concentration (1, 5, 15, 25, 35 %) showed different response to the stressors depending on the strain. According to response surface analysis, the main effect was temperature > salinity > hydrocarbon concentration. The hydrocarbonoclastic biofilm architecture was characterised using scanning electron microscopy (SEM) and atomic force microscopy (AFM). Subtle but significant differences were observed: pili in M. luteus by SEM and the topographical differences measured by AFM Power Spectral Density (PSD) analysis, roughness was higher in M. luteus than in M. yunnanensis. In all three domains of life, the Universal Stress Protein (Usp) is crucial for stress adaptation. Herein, the uspA gene expression was analysed in Micrococcus biofilm under environmental stressors. The uspA expression increased up to 2.5-fold in M. luteus biofilms at 30 °C, and 1.3-fold at 50 °C. The highest uspA expression was recorded in M. yunnanensis biofilms at 50 °C with 2.5 and 3-fold with salinities of 50, 60, and 80 g/L at hydrocarbon concentrations of 15, 25, and 35 %. M. yunnanensis biofilms showed greater resilience than M. luteus biofilms when exposed to harsh environmental stressors. M. yunnanensis biofilms were thicker than M. luteus biofilms. Both biofilm responses to environmental stressors through uspA gene expression were consistent with the behaviours observed in the response surface analyses. The uspA gene is a suitable biomarker for assessing environmental stressors of potential microorganisms for bioremediation of marine oil spills and for biosensing the ecophysiological status of native microbiota in a marine petroleum environment.

Enhanced biodegradation of benzo[a]pyrene with Trametes versicolor stimulated by citric acid.

Polycyclic aromatic hydrocarbons (PAHs) are a class of persistent organic pollutants with carcinogenic, mutagenic and teratogenic effects. The white-rot fungi in the fungal group have significant degradation ability for high molecular weight organic pollutants. However, exogenous fungi are easily antagonized by indigenous microorganisms. Low molecular weight organic acids, a small molecular organic matter secreted by plants, can provide carbon sources for soil microorganisms. Combining organic acids with white rot fungi may improve the nutritional environment of fungi. In this study, immobilized Trametes versicolor was used to degrade benzo[a]pyrene in soil, and its effect on removing benzo[a]pyrene in soil mediated by different low molecular weight organic acids was investigated. The results showed that when the degradation was 35 days, the removal effect of the experimental group with citric acid was the best, reaching 43.7%. The degradation effect of Trametes versicolor on benzo[a]pyrene was further investigated in the liquid medium when citric acid was added, and the effects of citric acid on the biomass, extracellular protein concentration and laccase activity of Trametes versicolor were investigated by controlling different concentrations of citric acid. In general, citric acid can act as a carbon source for Trametes versicolor and promote its extracellular protein secretion and laccase activity, thereby accelerating the mineralization of benzo[a]pyrene by Trametes versicolor. Therefore, citric acid can be used as a biostimulant in the remediation of PAHs contaminated soil with Trametes versicolor.

Microbial diversity and oil biodegradation potential of northern Barents Sea sediments.

The Arctic, an essential ecosystem on Earth, is subject to pronounced anthropogenic pressures, most notable being the climate change and risks of crude oil pollution. As crucial elements of Arctic environments, benthic microbiomes are involved in climate-relevant biogeochemical cycles and hold the potential to remediate upcoming contamination. Yet, the Arctic benthic microbiomes are among the least explored biomes on the planet. Here we combined geochemical analyses, incubation experiments, and microbial community profiling to detail the biogeography and biodegradation potential of Arctic sedimentary microbiomes in the northern Barents Sea. The results revealed a predominance of bacterial and archaea phyla typically found in the deep marine biosphere, such as Chloroflexi, Atribacteria, and Bathyarcheaota. The topmost benthic communities were spatially structured by sedimentary organic carbon, lacking a clear distinction among geographic regions. With increasing sediment depth, the community structure exhibited stratigraphic variability that could be correlated to redox geochemistry of sediments. The benthic microbiomes harbored multiple taxa capable of oxidizing hydrocarbons using aerobic and anaerobic pathways. Incubation of surface sediments with crude oil led to proliferation of several genera from the so-called rare biosphere. These include Alkalimarinus and Halioglobus, previously unrecognized as hydrocarbon-degrading genera, both harboring the full genetic potential for aerobic alkane oxidation. These findings increase our understanding of the taxonomic inventory and functional potential of unstudied benthic microbiomes in the Arctic.

Draft genome sequence of the Tremellomycetes yeast Papiliotrema laurentii 5307AH, isolated from aircraft.

Papiliotrema laurentii 5307AH was isolated from an aircraft polymer-coated surface. The genome size is 19,510,785 bp with a G + C content of 56%. The genome harbors genes encoding oxygenases, cutinases, lipases, and enzymes for styrene degradation, all of which could play a critical role in survival on xenobiotic surfaces.

Synergistic preparation of a straw fiber/polylactic acid composite with high toughness and strength through interfacial compatibility enhancement and elastomer toughening.

Plant fiber-reinforced polylactic acid (PLA) composites are extensively utilized in eco-friendly packaging, sports equipment, and various other applications due to their environmental benefits and cost-effectiveness. However, PLA suffers from brittleness and poor toughness, which restricts its use in scenarios demanding high toughness. To expand the application range of plant fiber-reinforced PLA-based composites and enhance their poor toughness, this study employed a two-step process involving wheat straw fiber (WF) to improve the interfacial compatibility between WF and PLA. Additionally, four elastomeric materials-poly (butylene adipate-co-terephthalate) (PBAT), poly (butylene succinate) (PBS), polycaprolactone (PCL), and polyhydroxyalkanoate (PHA)-were incorporated to achieve a mutual reactive interface enhancement and elastomeric toughening. The results demonstrated that Fe3+/TsWF/PLA/PBS exhibited a tensile strength, elongation at break, and impact strength of 34.01 MPa, 14.23 %, and 16.2 kJ/m2, respectively. These values represented a 2.4 %, 86.7 %, and 119 % increase compared to the unmodified composites. Scanning electron microscopy analysis revealed no fiber exposure in the cross-section, indicating excellent interfacial compatibility. Furthermore, X-ray diffraction and differential scanning calorimetry tests confirmed improvements in the crystalline properties of the composites. This work introduces a novel approach for preparing fiber-reinforced PLA-based composites with exceptional toughness and strength.

Enhancing neonicotinoid removal in recirculating constructed wetlands: The impact of Fe/Mn biochar and microbial interactions.

Neonicotinoids pose significant environmental risks due to their widespread use, persistence, and challenges in elimination. This study explores the effectiveness of Fe/Mn biochar in enhancing the removal efficiency of neonicotinoids in recirculating constructed wetlands (RCWs). Results demonstrated that incorporating Fe/Mn biochar into RCWs significantly improved the removal of COD, NH4+-N, TN, TP, imidacloprid (IMI), and acetamiprid (ACE). However, the simultaneous presence of IMI and ACE in the RCWs hindered the elimination of NH4+-N, TN, and TP from wastewater. The enhanced removal of nutrients and pollutants by Fe/Mn biochar was attributed to its promotion of carbon, nitrogen, and phosphorus cycling in RCWs, along with its facilitation of the adsorption and biodegradation of IMI and ACE. Metagenomics analysis demonstrated that Fe/Mn biochar altered the structure and diversity of microbial communities in RCWs. A total of 17 biodegradation genes (BDGs) and two pesticide degradation genes (PDGs) were identified within RCWs, with Fe/Mn biochar significantly increasing the abundance of BDGs such as cytochrome P450. The potential host genera for these BDGs/PDGs were identified as Betaproteobacteria, Acidobacteria, Nitrospiraceae, Gemmatimonadetes, and Bacillus. This study offers valuable insights into how Fe/Mn biochar enhances pesticide removal and its potential application in constructed wetland systems for treating pesticide-contaminated wastewater.

A novel paraffin-based N/P controlled-release material for biostimulation of phenol biodegradation in groundwater.

To address the problem of the weak natural restoration ability of oligotrophic groundwater environments, a novel N/P controlled-release material (CRM) for biostimulation, prepared by an improved method, was developed. CRMs can encapsulate N and P (N/P) salts for sustained release in aquifers. Paraffin-based CRMs can be used to control N/P release rates by adjusting the particle size of CRMs and the mass ratio of the paraffin. The developed CRMs had a more remarkable adaptability to groundwater than other materials. Specifically, 0.4-cm CRMs released N/P stably and efficiently over a wide temperature range (7-25 â„ƒ), and the release properties of various CRMs were not affected by pH. The release of N/P followed Fickian diffusion, and a dissolution-diffusion model was established to elucidate the mechanism of the controlled release. In contrast to bare N/P, CRMs obviously enhanced the biodegradation rate of phenol and prolonged the effectiveness of supplying N/P. The degradation rate of phenol in the CRM system increased by 20.8 %. The different supply modes of N/P, CRMs and bare N/P, resulted in differences in salinity. Metagenomic analysis showed that this difference changed the proportion of various phenol-degrading genera and thus changed the abundance of genes associated with the phenol degradation pathway.

Transformation of antibiotics to non-toxic and non-bactericidal products by laccases ensure the safety of Stropharia rugosoannulata.

The substantial use of antibiotics contributes to the spread and evolution of antibiotic resistance, posing potential risks to food production systems, including mushroom production. In this study, the potential risk of antibiotics to Stropharia rugosoannulata, the third most productive straw-rotting mushroom in China, was assessed, and the underlying mechanisms were investigated. Tetracycline exposure at environmentally relevant concentrations (<500 µg/L) did not influence the growth of S. rugosoannulata mycelia, while high concentrations of tetracycline (>500 mg/L) slightly inhibited its growth. Biodegradation was identified as the main antibiotic removal mechanism in S. rugosoannulata, with a degradation rate reaching 98.31 % at 200 mg/L tetracycline. High antibiotic removal efficiency was observed with secreted proteins of S. rugosoannulata, showing removal efficiency in the order of tetracyclines > sulfadiazines > quinolones. Antibiotic degradation products lost the ability to inhibit the growth of Escherichia coli, and tetracycline degradation products could not confer a growth advantage to antibiotic-resistant strains. Two laccases, SrLAC1 and SrLAC9, responsible for antibiotic degradation were identified based on proteomic analysis. Eleven antibiotics from tetracyclines, sulfonamides, and quinolones families could be transformed by these two laccases with degradation rates of 95.54-99.95 %, 54.43-100 %, and 5.68-57.12 %, respectively. The biosafety of the antibiotic degradation products was evaluated using the Toxicity Estimation Software Tool (TEST), revealing a decreased toxicity or no toxic effect. None of the S. rugosoannulata fruiting bodies from seven provinces in China contained detectable antibiotic-resistance genes (ARGs). This study demonstrated that S. rugosoannulata can degrade antibiotics into non-toxic and non-bactericidal products that do not accelerate the spread of antibiotic resistance, ensuring the safety of S. rugosoannulata production.

Enterobacter cloacae-mediated polymer biodegradation: in-silico analysis predicts broad spectrum degradation potential by Alkane monooxygenase.

Plastic pollution poses a significant environmental challenge. In this study, the strain Enterobacter cloacae O5-E, a bacterium displaying polyethylene-degrading capabilities was isolated. Over a span of 30 days, analytical techniques including x-ray diffractometry, scanning electron microscopy, optical profilometry, hardness testing and mass spectrometric analysis were employed to examine alterations in the polymer. Results revealed an 11.48% reduction in crystallinity, a 50% decrease in hardness, and a substantial 25-fold increase in surface roughness resulting from the pits and cracks introduced in the polymer by the isolate. Additionally, the presence of degradational by-products revealed via gas chromatography ascertains the steady progression of degradation. Further, recognizing the pivotal role of alkane monooxygenase in plastic degradation, the study expanded to detect this enzyme in the isolate molecularly. Molecular docking studies were conducted to assess the enzyme's affinity with various polymers, demonstrating notable binding capability with most polymers, especially with polyurethane (- 5.47 kcal/mol). These findings highlight the biodegradation potential of Enterobacter cloacae O5-E and the crucial involvement of alkane monooxygenase in the initial steps of the degradation process, offering a promising avenue to address the global plastic pollution crisis.

Delineating acetaminophen biodegradation kinetics and metabolomics using bacterial community.

Acetaminophen [N-(4-hydroxyphenyl) acetamide, APAP] is an extensively and frequently consumed over-the-counter analgesic and antiphlogistic medication. It is being regarded as an emerging pollutant due to its continuous increment in the environment instigating inimical impacts on humans and the ecosystem. Considering its wide prevalence in the environment, there is an immense need of appropriate methods for the removal of APAP. The present study indulged screening and isolation of APAP degrading bacterial strains from pharmaceuticals-contaminated sites, followed by their molecular characterization via 16S rRNA sequencing. The phylogenetic analyses assigned the isolates to the genera Pseudomonas, Bacillus, Paracoccus, Agrobacterium, Brucella, Escherichia, and Enterobacter based on genetic relatedness. The efficacy of these strains in batch cultures tested through High-performance Liquid Chromatography (HPLC) revealed Paracoccus sp. and Enterobacter sp. as the most promising bacterial isolates degrading up to 88.96 and 85.92%, respectively of 300 mg L-1 of APAP within 8 days of incubation. Michaelis-Menten kinetics model parameters also elucidated the high degradation potential of these isolates. The major metabolites identified through FTIR and GC-MS analyses were 4-aminophenol, hydroquinone, and 3-hydroxy-2,4-hexadienedioic. Therefore, the outcomes of this comprehensive investigation will be of paramount significance in formulating strategies for the bioremediation of acetaminophen-contaminated sites through a natural augmentation process via native bacterial strains.

Enhanced removal of antibiotics and heavy metals in aquatic systems using spent mushroom substrate-derived biochar integrated with Herbaspirillum huttiense.

A novel integrated removal strategy was developed to enhance the concurrent elimination of copper (Cu), zinc (Zn), oxytetracycline (OTC), and enrofloxacin (ENR) from the aqueous environments. The underlying adsorption mechanisms of spent mushroom substrate (SMSB) and the Herbaspirillum huttiense strain (HHS1), and their efficacy in removing Cu, Zn, OTC, and ENR was also examined. Results showed that the SMSB-HHS1 composite stabilized 29.86% of Cu and 49.75% of Zn and achieved removal rates of 97.95% for OTC and 59.35% for ENR through a combination of chemisorption and biodegradation. Zinc did not affect Cu adsorption, and ENR did not impact the adsorption of OTC on SMSB. However, the co-presence of OTC and ENR modified the adsorption behaviors of both Cu and Zn. Copper and Zn enhanced the adsorption of OTC and ENR by serving as bridging agents, facilitating the interaction between the contaminants and SMSB. Conversely, OTC and ENR inhibited the adsorption process of Cu by obstructing its interaction with the SMSB and occupying the oxygen-containing functional groups. The ‒OH (3415 cm-1) and C-O-C (1059 cm-1) functional groups were identified as the principal active sites to form hydrogen bonds and interact with Cu and Zn, leading to the formation of CuP4O11 and Zn4CO3(OH)6H2O. HHS1 also enhanced antibiotic removal through biodegradation, as evidenced by the decrease of ‒C‒O and increase of ‒C = O groups. This study underscores the innovative potential of the SMSB-HHS1 composite, offering a sustainable approach to addressing multifaceted pollution challenges in the aquatic environments.

Total RNA analysis of the active microbiome on moving bed biofilm reactor carriers under incrementally increasing micropollutant concentrations.

Micropollutants are increasingly prevalent in the aquatic environment. A major part of these originates from wastewater treatment plants since traditional treatment technologies do not remove micropollutants sufficiently. Moving bed biofilm reactors (MBBRs), however, have been shown to aid in micropollutant removal when applied to conventional wastewater treatment as a polishing step. Here, we used Total RNA sequencing to investigate both the active microbial community and functional dynamics of MBBR biofilms when these were exposed to increasing micropollutant concentrations over time. Concurrently, we conducted batch culture experiments using biofilm carriers from the MBBRs to assess micropollutant degradation potential. Our study showed that biofilm eukaryotes, in particular protozoa, were negatively influenced by micropollutant exposure, in contrast to prokaryotes that increased in relative abundance. Further, we found several functional genes that were differentially expressed between the MBBR with added micropollutants and the control. These include genes involved in aromatic and xenobiotic compound degradation. Moreover, the biofilm carrier batch experiment showed vastly different alterations in benzotriazole and diclofenac degradation following the increased micropollutant concentrations in the MBBR. Ultimately, this study provides essential insights into the microbial community and functional dynamics of MBBRs and how an increased load of micropollutants influences these dynamics.

Unraveling the mechanisms of benzo[a]pyrene degradation by Pigmentiphaga kullae strain KIT-003 using a multi-omics approach.

Polycyclic aromatic hydrocarbons (PAHs), notably benzo[a]pyrene (BaP), are environmental contaminants with multiple adverse ecological implications. Numerous studies have suggested the use of BaP biodegradation using various bacterial strains to remove BaP from the environment. This study investigates the BaP biodegradation capability of Pigmentiphaga kullae strain KIT-003, isolated from the Nak-dong River (South Korea) under specific environmental conditions. The optimum conditions of biodegradation were found to be pH 7.0, 35°C, and a salinity of 0 %. GC-MS analysis suggested alternative pathways by which KIT-003 produced catechol from BaP through several intermediate metabolites, including 4-formylchrysene-5-carboxylic acid, 5,6-dihydro-5,6-dihydroxychrysene-5-carboxylic acid (isomer: 3,4-dihydro-3,4-dihydroxychrysene-4-carboxylic acid), naphthalene-1,2-dicarboxylic acid, and 2-hydroxy-1-naphthoic acid. Proteomic profiles indicated upregulation of enzymes associated with aromatic compound degradation, such as nahAc and nahB, and of those integral to the tricarboxylic acid cycle, reflecting the strain's adaptability to and degradation of BaP. Lipidomic analysis of KIT-003 demonstrated that BaP exposure induced an accumulation of glycerolipids such as diacylglycerol and triacylglycerol, indicating their crucial role in bacterial adaptation mechanisms under BaP stress. This study provides significant scientific knowledge regarding the intricate mechanisms involved in BaP degradation by microorganisms.

Stable-isotope probing combined with amplicon sequencing and metagenomics identifies key bacterial benzene degraders under microaerobic conditions.

The primary aim of the present study was to reveal the major differences between benzene-degrading bacterial communities evolve under aerobic versus microaerobic conditions and to reveal the diversity of those bacteria, which can relatively quickly degrade benzene even under microaerobic conditions. For this, parallel aerobic and microaerobic microcosms were set up by using groundwater sediment of a BTEX-contaminated site and 13C labelled benzene. The evolved total bacterial communities were first investigated by 16S rRNA gene Illumina amplicon sequencing, followed by a density gradient fractionation of DNA and a separate investigation of "heavy" and "light" DNA fractions. Results shed light on the fact that the availability of oxygen strongly determined the structure of the degrading bacterial communities. While members of the genus Pseudomonas were overwhelmingly dominant under clear aerobic conditions, they were almost completely replaced by members of genera Malikia and Azovibrio in the microaerobic microcosms. Investigation of the density resolved DNA fractions further confirmed the key role of these two latter genera in the microaerobic degradation of benzene. Moreover, analysis of a previously acquired metagenome-assembled Azovibrio genome suggested that benzene was degraded through the meta-cleavage pathway by this bacterium, with the help of a subfamily I.2.I-type catechol 2,3-dioxygenase. Overall, results of the present study implicate that under limited oxygen availability, some potentially microaerophilic bacteria play crucial role in the aerobic degradation of aromatic hydrocarbons.

Corrigendum: Plastic biodegradation by in vitro environmental microorganisms and in vivo gut microorganisms of insects.

[This corrects the article DOI: 10.3389/fmicb.2022.1001750.].

Efficient Biodegradation of Multiple Aryloxyphenoxypropionate Herbicides by Corynebacterium sp. Z-1 and the Proposed Degradation Mechanism.

Esterases are crucial for aryloxyphenoxypropionate herbicide (AOPP) biodegradation. However, the underlying molecular mechanisms of AOPP biodegradation by esterases are poorly understood. In the current work, Corynebacterium sp. Z-1 was isolated and found to degrade multiple AOPPs, including quizalofop-p-ethyl (QPE), haloxyfop-p-methyl (HPM), fenoxaprop-p-ethyl (FPE), cyhalofop-butyl (CYB), and clodinafop-propargyl (CFP). A novel esterase, QfeH, which catalyzes the cleavage of ester bonds in AOPPs to form AOPP acids, was identified from strain Z-1. The catalytic activities of QfeH toward AOPPs decreased in the following order: CFP > FPE > CYB > QPE > HPM. Molecular docking, computational analyses, and site-directed mutagenesis indicated the catalytic mechanisms of QfeH-mediated degradation of different AOPPs. Notably, the key residue S159 is essential for the activity of QfeH. Moreover, V222Y, T227M, T227A, A271R, and M275K mutants, exhibiting 2.9-5.0 times greater activity than QfeH, were constructed. This study facilitates the mechanistic understanding of AOPPs bioremediation by esterases.

Enrichment of Polycyclic Aromatic Hydrocarbon (PAH)-Degrading Strictly Anaerobic Sulfate-Reducing Cultures from Contaminated Soil and Sediment.

Sulfate-reducing bacteria (SRB) are crucial players in global biogeochemical cycling and some have been implicated in the anaerobic biodegradation of organic pollutants, including recalcitrant and hazardous polycyclic aromatic hydrocarbons (PAHs). Obtaining PAH-degrading SRB cultures for laboratories is of paramount importance in the development of the young field of anaerobic biodegradation of PAHs. SRB grow exceptionally slowly on PAH substrates and are highly sensitive to oxygen. Consequently, enrichment and maintenance of PAH-degrading SRB cultures and characterization of the biodegradation process remain a tedious and formidable task, especially for new researchers. To address these technical constraints, we have developed robust and effective protocols for obtaining and characterizing PAH-degrading SRB cultures. In this set of protocols, we describe step-by-step procedures for preparing inocula from contaminated soil or sediment, preparing anoxic medium, establishing enrichment cultures with PAHs as substrates under completely anaerobic sulfate-reducing conditions, successive culture transfers to obtain highly enriched cultures, rapid verification of the viability of SRB in slow-growing cultures, assessment of PAH degradation by extracting residuals using organic solvent and subsequent analysis by gas chromatography-mass spectrometry, and spectrophotometric determination of sulfate and sulfide in miniaturized, medium-throughput format. These protocols are expected to serve as a comprehensive manual for obtaining and characterizing PAH-degrading sulfate-reducing cultures. © 2024 The Author(s). Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Obtaining PAH-degrading strictly anaerobic sulfate-reducing enrichment cultures from contaminated soil and sediment Support Protocol 1: Operation and maintenance of an anaerobic workstation Support Protocol 2: Setup of gas purging systems for preparing anoxic solutions Support Protocol 3: Verification of viability in slow-growing SRB enrichment cultures Support Protocol 4: Extraction of genomic DNA from low-biomass cultures Basic Protocol 2: Extraction of residual PAH from liquid culture and analysis by GC-MS Basic Protocol 3: Spectrophotometric determination of sulfate concentration in SRB cultures Basic Protocol 4: Spectrophotometric determination of sulfide concentrations in SRB cultures by the methylene blue method Alternate Protocol: Spectrophotometric determination of sulfide concentrations in SRB cultures by the colloidal copper sulfide method.

Pseudomonas putida KT2440: the long journey of a soil-dweller to become a synthetic biology chassis.

Although members of the genus Pseudomonas share specific morphological, metabolic, and genomic traits, the diversity of niches and lifestyles adopted by the family members is vast. One species of the group, Pseudomonas putida, thrives as a colonizer of plant roots and frequently inhabits soils polluted with various types of chemical waste. Owing to a combination of historical contingencies and inherent qualities, a particular strain, P. putida KT2440, emerged time ago as an archetype of an environmental microorganism amenable to recombinant DNA technologies, which was also capable of catabolizing chemical pollutants. Later, the same bacterium progressed as a reliable platform for programming traits and activities in various biotechnological applications. This article summarizes the stepwise upgrading of P. putida KT2440 from being a system for fundamental studies on the biodegradation of aromatic compounds (especially when harboring the TOL plasmid pWW0) to its adoption as a chassis of choice in metabolic engineering and synthetic biology. Although there are remaining uncertainties about the taxonomic classification of KT2440, advanced genome editing capabilities allow us to tailor its genetic makeup to meet specific needs. This makes its traditional categorization somewhat less important, while also increasing the strain's overall value for contemporary industrial and environmental uses.

Unveiling a Novel Antidote for Deoxynivalenol Contamination: Isolation, Identification, Whole Genome Analysis and In Vivo Safety Evaluation of Lactobacillus rhamnosus MY-1.

Deoxynivalenol (DON) is a global contaminant found in crop residues, grains, feed, and animal and human food. Biodegradation is currently the best solution for addressing DON pollution. However, efficient detoxification bacteria or enzymes that can be applied in complex matrices are lacking. The aim of this study was to isolate a DON-detoxifying probiotic strain with a high degradation rate, a good safety profile, and a clear genetic background. One hundred and eight bacterial strains were isolated from 300 samples collected from a school farm and surrounding livestock farms. A new DON-degrading strain, Lactobacillus rhamnosus MY-1 (L. rhamnosus MY-1), with a degradation rate of 93.34% after 48 h and a comprehensive degradation method, was identified. Then, MY-1 at a concentration of 1 × 108 CFU/mL was administered to mice in a chronic intoxication experiment for 28 days. The experimental group showed significantly higher weight gain and exhibited good production performance compared to the control group. The length of the ileal villi in the experimental group was significantly longer than that in the control group. The expression of pro-inflammatory cytokines decreased, while the expression of anti-inflammatory factors increased in the experimental group. Whole-genome analysis revealed that most of the MY-1 genes were involved in carbohydrate metabolism and membrane transport, with a cluster of secondary metabolite genes encoding antimicrobial properties. In summary, this study successfully identified a Lactobacillus strain with good safety performance, high DON degradation efficiency, and a clear genetic background, providing a new approach for the treatment of DON contamination.