Anaerobic Chamber Research Articles

Fate of arsenic in contaminated coastal soil induced by rising temperature and seawater intrusion

Temperature rising and seawater intrusion are expected to influence the hydrologic regime and redox conditions in coastal soil, and the fate and mechanisms of biogeochemical cycling of Arsenic (As) in the specific environment are poorly understood. This work was carried out in an anaerobic operating chamber by adding sulfate to simulate seawater intrusion under various temperature. Results demonstrated the microbial community diversity was influenced by temperature and the highest Shannon and lowest Simpson index were found at 28 °C. Firmicutes was the dominant bacteria, accounting for 81.16%–93.99%. Desulfosporosinus, with the proportion increasing with temperature, showed a significantly positive correlation with S2− for sulfate addition treatments. Actually, transformation of As was meditated by the concentration and valence of sulfur and iron in soil. The dissimilatory reduction of arsenic-bearing Fe oxides occurring in the initial stage, is suspected to be the primary driver of As release. Then, concentration of As declined in aqueous phase due to the reduction of sulfate, and the proportion of residual speciation of As in solid phase increased with temperature, ranging from 6.78% to 27.70%. The results displayed the reducing condition due to seawater intrusion and temperature change could regulate the release and sequestration of As in the coastal soil.

Fluid depth-dependent biofilm characteristics and their influence on sulfate-reducing bacteria-induced corrosion of carbon steel

This study investigates the response of biofilm characteristics to variations in fluid depth and their influence on the corrosion behavior of carbon steel (C1020) under low-flow fluid conditions, utilizing Desulfovibrio vulgaris. The experiments were conducted in an anaerobic chamber at 30 °C, utilizing modified Baar's medium as the testing medium. The findings reveal that fluid depth significantly impacts biofilm-corrosion product composite formation, with deeper depths promoting thicker and more heterogeneous biofilm-corrosion product layer compared to shallower depths, where a thinner and more uniform biofilm-corrosion product layer is observed. Moreover, the characteristics of initially attached biofilms was verified as the primary factor affecting subsequent corrosion behavior during prolonged exposure. Corrosion analysis reveals that greater fluid depth leads to increased weight loss (91 ± 13.2 mg/cm2) and deeper pit depths (540 ± 69 μm), surpassing those observed in shallower test media (21 ± 2.3 mg/cm2 and 105 ± 17 μm) after 28 days of exposure. The corrosion products within the biofilm were predominantly FeS and Fe3(PO4)2·8H2O. A direct relationship was observed between the thickness of this biofilm-corrosion product layer and the progression of pit depth, suggesting a strong correlation between carbon steel corrosion and biofilm development in limited fluid depths (e.g., 5–15 mm). Furthermore, a significant association between the deepest pits (average) and the number of sessile cells within the biofilm underscores the pivotal role of sessile cell numbers in carbon steel corrosion.

Removal of cadmium and zinc by calcium polysulfide in acidic groundwater: Injection ratio and precipitation mechanism

In this study, we conducted lab-scale experiments to assess the applicability of calcium polysulfide (CPS) for the removal of cadmium (Cd2+) and zinc (Zn2+) from acidic groundwater, with an emphasis on the injection ratio and removal mechanism. For the experiments, CPS was used to treat Cd2⁺ and Zn2⁺ contaminated solution. Solutions were purged with nitrogen gas, and CPS was injected in an anaerobic chamber. After 18 h, solutions were filtered, and heavy metal concentrations were measured using ICP-MS and ICP-OES. Total polysulfide (Sx2⁻) concentration in CPS was determined by converting it to bisulfide (HS⁻) at pH 8.20 and quantifying via UV–Vis spectrophotometry. Precipitates were analyzed using XRD and SEM-EDS after centrifugation. The findings revealed that more than 99.5% of the heavy metals were removed when CPS/Cd2+ (w/w) = 1.45 and CPS/Zn2+ (w/w) = 2.50, determined as the injection ratios for maximum efficiency. These ratios were applicable when Cd2+ and Zn2+ coexisted. From the XRD and SEM-EDS analyses, it was clarified that Cd2+ and Zn2+ precipitated in sulfide forms, consistently showing the preferential precipitation of Cd2+ because of the lower solubility of cadmium sulfide compared to zinc sulfide. In addition, the concentration of Sx2− in the 29% CPS solution was determined to be approximately 2.442 M. Finally, by comparing the injected Sx2− concentration with the concentration of heavy metals removed accordingly, it was concluded that CPS and heavy metals react with a 1:1 M ratio. Based on the above results and precise quantification method, our study suggests that CPS can be effectively utilized to address heavy metal contamination issues and may serve as a valuable tool for the remediation and management of contaminated groundwater globally.

Enhanced removal of PPCPs and antibiotic resistance genes in saline wastewater using a bioelectrochemical-constructed wetland system

Pharmaceuticals and personal care products (PPCPs) are insufficiently degraded in saline wastewater treatment processes and are found at high concentrations and detection frequencies in aquatic environments. In this study, the wetland plant Thalia dealbata was selected using a screening plant experiment to ensure good salt tolerance and high efficiency in removing PPCPs. An electric integrated vertical-flow constructed wetland (E-VFCW) was developed to improve the removal of PPCPs and reduce the abundance of antibiotic resistance genes (ARGs). The removal efficiency of ofloxacin, enrofloxacin, and diclofenac in the system with anaerobic cathodic and aerobic anodic chambers is higher than that of the control system (41.84 ± 2.88%, 47.29 ± 3.01%, 53.29 ± 2.54%) by approximately 20.31%, 16.04%, and 35.25%. The removal efficiency of ibuprofen in the system with the aerobic anodic and anaerobic cathodic chamber was 28.51% higher than that of the control system (72.41 ± 3.06%) and promotes the reduction of ARGs. Electrical stimulation can increase the activity of plant enzymes, increasing their adaptability to stress caused by PPCPs, and PPCPs are transferred to plants. Species related to PPCPs biodegradation (Geobacter, Lactococcus, Hydrogenophaga, and Nitrospira) were enriched in the anodic and cathodic chambers of the system. This study provides an essential reference for the removal of PPCPs in saline-constructed wetlands.

Performance of pharmaceutical products removal in a bioelectrochemical system at low temperatures and changes in microbial communities and antibiotic resistance genes.

Biological methods do not effectively remove pharmaceutical products (PPs) and antibiotic resistance genes (ARGs) from wastewater at low temperatures, leading to environmental pollution. Therefore, anaerobic-aerobic-coupled upflow bioelectrochemical reactors (AO-UBERs) were designed to improve the removal of PPs at low temperatures (10 ± 2 °C). The result shows that diclofenac (DIC) and ibuprofen (IBU) removals in the system with aerobic anodic and anaerobic cathodic chambers were 91.7% and 94.7%, higher than that in the control system (12.2 ± 1.5%, 36.5 ± 5.9%), and aerobic zone favors DIC and IBU removal; fluoroquinolone antibiotics (FQs) removals in the system with aerobic cathodic and anaerobic anodic chambers were 17.5-22.4% higher than that in the control system (9.1-22.4%), and anaerobic zone favors FQs removal. Analysis of microbial community structure and ARGs showed that different electrotrophic microbes (Flavobacterium, Acinetobacter, and Delftia) with cold-resistant ability to degrade PPs were enriched in different electrode combinations, and the aerobic cathodic chambers could remove certain ARGs. These results showed that AO-UBERs under intermittent electrical stimulation mode are an alternative method for the effective removal of PPs and ARGs at low temperatures.

Exploring the potential of graphite material in an unplanted electroactive wetland for the remediation of synthetic wastewater containing azo dye.

The current study was conducted to understand the sole role of graphite as a substrate material in a dual-chambered baffled electroactive wetland (EW) in the treatment of Methyl red dye-containing wastewater. The results obtained were compared with conventional gravel-based unplanted dual-chambered constructed wetlands (CW) at a lab scale. The highest dye decolorisation and COD removal efficiency achieved was 92.88 ± 1.6% and 95.78 ± 4.1%, respectively, in the electro-active wetland. Dissolved oxygen (DO) and pH conditions were appropriately maintained in both the microcosms because of separated aerobic and anaerobic chambers. UV-vis and gas chromatography-mass spectroscopy analysis revealed the production of by-products like 4-amino benzoic and N- N dimethyl phenyl-diamine of MR in microcosms and revealed further mineralisation of by-products in the aerobic zone of electroactive-wetland. Higher root growth of Cicer aerietinum and Vigna radiata was observed in the presence of effluents of baffled electroactive wetlands compared to constructed wetland, indicating a decrease in phytotoxicity. Metagenomic analysis revealed the abundance of potential microbes for MR and organic matter removal from phylum Proteobacteria, Firmicutes, Bacteroidetes, and Euryarchaeota. A batch adsorption study revealed a higher adsorption capability of graphite material in comparison to gravel. Hence, this study demonstrated that graphite is an appropriate substrate in electroactive wetland in facilitating microbial attachments and enhancing dye degradation, in addition to exhibiting superior adsorption quality.

Iron-retrofitted anaerobic baffled reactor system for rural wastewater treatment: Stable performance of nutrients removal with phosphorus recovery and minimal sludge production

An iron-retrofitted anaerobic baffled reactor (ABR) system was developed for the effective treatment of rural wastewater with reduced maintenance demand and aeration costs. Average removal efficiencies of chemical oxygen demand, total nitrogen and total phosphorus of 99.4%, 62.7% and 92.6% were achieved respectively, when the ABR system was operating at steady state. With effective bioreduction of FeIII in the anaerobic chambers, phosphorus was immobilized in the sludge as vivianite, the sole phosphorus-carrying mineral. The FeIII in the recirculated sludge induced Feammox in the ABR reactor, contributing 14.8% to total nitrogen removal. Biophase separation and enrichment of microorganisms associated with iron and nitrogen transformations were observed in the system after Fe dosing, which enhanced the removal of pollutants. The coupling of Feammox and vivianite crystallization to remove nitrogen and phosphorus in an iron-retrofitted ABR would appear to be a promising technology for rural wastewater treatment.

Intermittent electrical stimulation removes mixed antibiotics and associated antibiotic resistance genes at low temperatures

Biotechnology has limited effectiveness in terms of removing mixed antibiotics at low temperatures, leading to ecological risks arising from the presence of antibiotics in environmental waters. In this study, the removal of tetracycline (TCs) and sulfonamide (SAs) from antibiotic wastewater was improved by the intermittent electrical stimulation of anaerobic-aerobic-coupled upflow bioelectrochemical reactors (AO-UBERs) at low temperatures. The removal effects of oxytetracycline and tetracycline were 48.6 ± 3.5 % and 71.5 ± 2.9 %, respectively. Under 0.9V, the removal rates of oxytetracycline, tetracycline, and trimethoprim were significantly increased in both the aerobic-cathodic and anaerobic anodic chambers, with a more obvious increase at low temperatures. Compared with the blank control group, the removal efficiency of oxytetracycline, trimethoprim and tetracycline in the electric group was increased by 11.8 ± 2.5 %, 27.8 ± 10.5 % and 11.2 ± 5.8 %. The anaerobic chamber contributed more to the removal of TCs and trimethoprim than the aerobic chamber. Furthermore, electrical stimulation selectively enriched electroactive bacteria (Methylophage and Pleuromonas), drug-resistant bacteria (Proteobacteria), and nitrifying bacteria associated with biodegradation. The abundance of antibiotic-resistance genes is related to the distribution of potential hosts and mobile genetic elements (sul1), and electrical stimulation induces the enrichment of both. This suggests that while potentially effective for treating TCs- and SAs-containing wastewater at low temperatures, AO-UBERs may lead to the accumulation of antibiotic-resistance genes.

Efficiency of Photosynthetic Microbial Fuel Cells (pMFC) Depending on the Type of Microorganisms Inhabiting the Cathode Chamber

Photosynthetic microbial fuel cells (pMFCs) are hybrid systems that enable simultaneous wastewater treatment under anaerobic conditions and the generation of electricity by utilizing the potential difference in the anaerobic anode chamber and the oxygenated cathode chamber. Dairy wastewater with a concentration of 2000 mg COD/L was treated in the anode of a batch pMFC. In the cathode chamber, Chlorella vulgaris or Arthrospira platensis was cultivated in synthetic medium, and next in diluted effluent from the anode chamber. The highest power density of 91 mW/m2 was generated by the pMFC with the cultivation of Arthrospira platensis. Higher values of dissolved oxygen remained during the dark phase in the cathodic medium with Arthrospira platensis cultivation than with Chlorella vulgaris. This depletion of oxygen significantly decreased voltage generation, which during the light phase increased again to the maximum values. The COD removal achieved in the anodic chamber was 87%. The efficiency of nitrogen removal in the cathode chamber during the cultivation of Arthrospira platensis and Chlorella vulgaris was about 78% and 69%, respectively. The efficiency of phosphorus removal in the cathode chamber with the cultivation of Arthrospira plantensis and Chlorella vulgaris was 58% and 43%, respectively. This study has shown that the introduction of Arthrospira platensis into the cathode chamber is more effective than that of Chlorella vulgaris.

A perfusion host-microbe bioreactor (HMB) system that captures dynamic interactions of secreted metabolites between epithelial cells cocultured with a human gut anaerobe.

The human microbiota impacts a variety of diseases and responses to therapeutics. Due to a lack of robust in vitro models, detailed mechanistic explanations of host-microbiota interactions cannot often be recapitulated. We describe the design and development of a novel, versatile and modular in vitro system that enables indirect coculture of human epithelial cells with anaerobic bacteria for the characterization of host-microbe secreted metabolite interactions. This system was designed to compartmentalize anaerobes and human cells in separate chambers conducive to each organism's requisite cell growth conditions. Using perfusion, fluidic mixing, and automated sample collection, the cells continuously received fresh media, while in contact with their corresponding compartments conditioned supernatant. Supernatants from each chamber were collected in a cell-free time-resolved fashion. The system sustained low oxygen conditions in the anaerobic chamber, while also supporting the growth of a representative anaerobe (Bacteroides thetaiotaomicron) and a human colonic epithelial cell line (Caco-2) in the aerobic chamber. Caco-2 global gene expression changes in response to coculture with B. thetaiotaomicron was characterized using RNA sequencing. Extensive, targeted metabolomics analysis of over 150 central carbon metabolites was performed on the serially collected supernatants. We observed broad metabolite changes in host-microbe coculture, compared to respective mono-culture controls. These effects were dependent both on sampling time and the compartment probed (apical vs. basolateral). Coculturing resulted in the depletion of several important metabolites, including guanine, uridine 5'-monophosphate, asparagine, and thiamine. Additionally, while Caco-2 cells cultured alone predominantly affected the basolateral metabolite milieu, increased abundance of 2,3-dihydroxyisovalerate and thymine on the basolateral side, occurred when the cells were cocultured with B. thetaiotaomicron. Thus, our system can capture the dynamic, competitive and cooperative processes between host cells and gut microbes.

A new model of ischemia/reperfusion injury using precision-cut liver slices

Background: Primary cells isolated from the liver are widely used to investigate hepatic pathophysiology. However, these cells do not represent cell-to-cell interactions, three dimensional architecture, and zonal structure in the liver. Furthermore, characterization of cellular and molecular events during ischemia/reperfusion (I/R) is challenging in liver biopsies. Thus, a new model of I/R injury resembling the native liver is needed. Methods: We generated precision-cut liver slices (PCLS) from mouse and human livers using a Krumdieck automatic tissue slicer (Alabama R & D, Munford, AL). Thickness of individual PLCL was 200 microns. To simulate anoxia, nutrient depletion, and tissue acidosis during ischemia, PCLS were incubated in Krebs-Ringer-N-(2-Hydroxyethyl)piperazine-N-(2-ethanesulfonic acid) (HEPES) solution at pH 6.2 in the anaerobic chamber (Coy Laboratory, Grass Lake, MI) for 30 min. To simulate normoxia, nutrient repletion, and restoration to normal pH during reperfusion, ischemic PCLS were aerobically incubated in Dulbecco's modified eagle medium (DMEM) at pH 7.4 for up to 4 h. Changes in tissue viability were assessed with the release of lactate dehydrogenase (LDH) into the extracellular medium. Some PCLS were labeled with green fluorescent rhodamine-123 (Rd-123, mitochondrial membrane potential indicator) and red fluorescent propidium iodide (PI, necrosis marker). Confocal images of PCLS were collected after I/R. Results: LDH release assay revealed that while PCLS from chow diet (CD)-fed lean mice tolerated I/R injury well, those from high fat diet (HFD)-fed steatotic mice were susceptible to I/R injury. Confocal microscopy showed substantial mitochondrial depolarization (loss of Rd-123) and necrosis (nuclear labeling of PI) in the HFD group, which was, however, minimally observed in the CD group. Of importance, mitochondrial dysfunction and cell death after reperfusion occurred predominantly in hepatocytes. Heightened I/R injury in steatotic livers was also confirmed in primary hepatocytes isolated from either CD- or HFD-fed mice. To compare the similarities and differences between mouse and human livers, PCLS were generated from discarded human livers deemed unsuitable for transplant and subjected to 30 min of in vitro ischemia. The loss of mitochondrial membrane potential and hepatocyte death progressively increased after I/R in discarded human livers. At 1 h after reperfusion, diffusive fluorescence of Rd-123 and nuclear labeling of PI became evident, indicative of mitochondrial dysfunction and tissue injury after I/R. SUMMARY: LDH assay and confocal analysis of PCLS demonstrated heightened susceptibility of steatotic livers to I/R injury. In contrast to PCLS from lean livers, those from steatotic livers rapidly developed mitochondrial depolarization and onset of necrosis, cardinal features of I/R injury, even after short ischemia. The early phase of reperfusion injury in steatotic livers occurred predominantly in parenchymal cells. CONCLUSION: In vitro I/R with PCLS is a model suitable for hepatic I/R injury. This work (J-S K) was funded by the National Institutes of Health (DK079878), Mid-America Transplant Foundation (07201903), Foundation for Barnes-Jewish Hospital (4776, 5153), and Washington University Department of Surgery Pilot Grant. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Abstract 3090: Oral Administration Of Akkermansia Muciniphila Ameliorates Venous Wall Fibrosis: Therapeutic Implication Of A Novel Probiotic Therapy Against Post-thrombotic Syndrome

Introduction: Post-thrombotic syndrome (PTS) is the most common sequelae of deep vein thrombosis (DVT), manifested as venous insufficiency in up to 50% of patients. Unfortunately, none of the existing therapies, including anticoagulants and thrombolytics, have shown definitive effects for PTS, presenting a vital unmet clinical need. Akkermansia Muciniphila (AKK) , a gut commensal anaerobic bacterium, has been recognized with pleiotropic benefits in a wide range of diseases including cardiometabolic disorders. However, its therapeutic and mechanistic implications in thromboembolic diseases and the subsequent PTS remain unknown. We hypothesize that probiotic therapy with live AKK after DVT formation could ameliorate venous wall fibrosis, indicative of PTS. Methods: In male specific-pathogen free c57b6/j mice (10-12 weeks), DVT was experimentally created via partial ligation of inferior vena cava (IVC) as previously established, leading to progressive venous fibrosis. For the probiotic treatment, AKK (strain BAA-835) was cultured using BHI broth in an anaerobic Coy chamber, followed by daily oral gavage to mice beginning from day 1 post-DVT induction. At day 14 post-procedure, IVC was collected for Masson Trichrome and immunofluorescent staining, followed by quantitation of venous collagen deposition/fibrosis and phosphorylation of endoplasmic reticulum (ER) stress PERK kinase. Endpoint plasma specimens were collected to quantify the circulating levels of trimethylamine N-oxide (TMAO). Results: Post-DVT probiotic therapy with live AKK effectively mitigated venous wall fibrosis in comparison to BHI culture media gavage alone (fibrotic area: 61.12±6.64 vs 53.17±5.99; p<0.05). TMAO, the only microbial metabolite associated with thromboembolic risk, was significantly reduced in plasma following AKK probiotic therapy. Furthermore, phosphorylation of ER stress PERK, which is directly activated by TMAO and critically contributes to myofibroblast activation and organ fibrosis, was inhibited in the AKK -treated IVC (435.09±26.49 vs 326.86±17.61; p<0.01). Conclusion: Our current study supports the potential use of Post-DVT AKK probiotic therapy for the treatment of PTS, possibly via modulating the TMAO-PERK axis.

Anaerobic cryoEM protocols for air-sensitive nitrogenase proteins.

Single-particle cryo-electron microscopy (cryoEM) provides an attractive avenue for advancing our atomic resolution understanding of materials, molecules and living systems. However, the vast majority of published cryoEM methodologies focus on the characterization of aerobically purified samples. Air-sensitive enzymes and microorganisms represent important yet understudied systems in structural biology. We have recently demonstrated the success of an anaerobic single-particle cryoEM workflow applied to the air-sensitive nitrogenase enzymes. In this protocol, we detail the use of Schlenk lines and anaerobic chambers to prepare samples, including a protein tag for monitoring sample exposure to oxygen in air. We describe how to use a plunge freezing apparatus inside of a soft-sided vinyl chamber of the type we routinely use for anaerobic biochemistry and crystallography of oxygen-sensitive proteins. Manual control of the airlock allows for introduction of liquid cryogens into the tent. A custom vacuum port provides slow, continuous evacuation of the tent atmosphere to avoid accumulation of flammable vapors within the enclosed chamber. These methods allowed us to obtain high-resolution structures of both nitrogenase proteins using single-particle cryoEM. The procedures involved can be generally subdivided into a 4 d anaerobic sample generation procedure, and a 1 d anaerobic cryoEM sample preparation step, followed by conventional cryoEM imaging and processing steps. As nitrogen is a substrate for nitrogenase, the Schlenk lines and anaerobic chambers described in this procedure are operated under an argon atmosphere; however, the system and these procedures are compatible with other controlled gas environments.

Rumen microbes: Exploring its potential for productivity and commercial use

Ruminant animals are known for their dairy and meat products worldwide. They are the best converters of poor quality fibrous feed ingredients, and presence of rumen, the anaerobic chamber that harbours vast category of microbes, is attributable to this phenomenon. The microbes include bacteria, fungi, protozoa, archaea and bacteriophages that work on synergistically for optimal performance of ruminant animals. These microbes help not only in digestion of fibrous materials, but also involved in various biological functions, such as probiotic activity, antimicrobial metabolite production, synthesis of health promoting bioactive fatty acid molecules, biomass conversion, etc. Earlier, the probiotic organisms used in food animals (calves, sheep, goat, swine and poultry) were mainly originated from dairy products but today organisms of autochthonous origin are being used, as they show better adaptability. Since, rumen do possess organisms with probiotic and fibre utilising activity, these organisms are now explored for their suitability as a probiotic and fibrolytic agent in monogastric food animals. Diversity of rumen microbes was not properly understood through the conventional culture methods, however with advancement in ‘Omic’ technologies, researchers could identify new class of organisms from the rumen and their potential use for the commercial and industrial purposes.

P135 Mixed probiotics containing Bifidobacterium longum and its metabolites inhibit the formation of NLRP3 inflammasomes by suppressing GBP5 expression in macrophages in ulcerative colitis

Abstract Background Ulcerative Colitis (UC) is a non-specific chronic inflammation of the intestine. As an intestinal disease with a gradually increasing incidence in recent years and increasingly valued by medical staff, UC has a close relationship with intestinal flora. At present, domestic and foreign guidelines point out that probiotics can be used to induce remission in ulcerative colitis, but there are no clear instructions on the selection of probiotics, the use of dosage forms, and the dosage of probiotics, and there is a lack of relevant clinical and basic research. This study aims to explore the distinct therapeutic effects of mixed probiotics and individual strains of probiotics (Bifidobacterium longum), as well as elucidate the underlying mechanisms responsible for these variations. Methods A total of 90 patients with ulcerative colitis (UC) were enrolled in our center, who were subsequently divided into three groups: control group, single strain probiotic group (Bifidobacterium longum), and mixed probiotic group. After three months of adjuvant probiotic therapy, serum and fecal samples were collected for comparative analysis of therapeutic effects using ELISA and fecal 16s rDNA and metabolome sequence analysis. Mixed probiotic and Bifidobacterium longum were cultured in BHIs in an anaerobic chamber. The supernatant obtained after bacterial cultivation was subjected to metabolome sequence analysis. Human colonic organoids and Caco-2 cells were co-cultured with mixed probiotic and Bifidobacterium longum subsequent to inflammation induction, and cytotoxicity, permeability, and inflammatory cytokine release were assessed. THP1 cells were also to co-cultured with mixed probiotic and Bifidobacterium longum, and expression of virulence genes was analyzed using RT-qPCR, transcriptome sequencing, and western blot. GBP5 gene manipulated cell and mouse model were used to evaluate inflammation and apoptosis. Results The utilization of mixed probiotic therapy in patients with UC demonstrated significantly superior therapeutic efficacy compared to those receiving a single strain probiotic (Bifidobacterium longum). Differential metabolite 8-Deoxylactucinwas identified among various groups through the metabolome sequence analysis of clinical patients’ feces, bacterial culture supernatant, and mouse feces. Through in vitro and in vivo experiments, it has been demonstrated that 8-Deoxylactucin exhibits inhibitory effects on the expression of GBP5 in THP-1 cells and ameliorates inflammation. Conclusion Collectively, these findings indicate that mixed probiotics and its metabolites suppress GBP5 expression in macrophages in ulcerative colitis.

P150 Ulcerative Colitis Patients’ Luminal Zinc Induces Clostridioides difficile Virulence and Exacerbates Inflammatory Responses

Abstract Background The refractory acute severe UC patients are often complicated by C. difficile infection (CDI), resulting in higher surgery and mortality rates. However, few studies have observed C. difficile’s pathogenicity in real UC patients’ gastrointestinal microenvironments, and the actual role of C. difficile in UC development remains unknown. We found zinc concentrations among UC patients’ feces were significantly higher than those among Crohn’s disease patients and healthy subjects. Additionally, we previously reported that excessive zinc would promote the growth and virulence of C. difficile. Therefore, we hypothesized that the increased UC patients’ luminal zinc may up-regulate C. difficile virulence and promote intestinal inflammatory responses, leading to an exacerbated UC progression. Methods UC patients’ stool samples (n=40) were collected and analyzed by qPCR and ICP-MS to determine the presence of C. difficile and the fecal zinc concentrations. C. difficile was cultured in BHIs with or without zinc (ZnSO4) in an anaerobic chamber. Expression of virulence genes was analyzed using RT-qPCR, transcriptome sequencing, and western blot. Human colonic organoids and Caco-2 cells were co-cultured with C. difficile or zinc-treated C. difficile, and cytotoxicity, permeability, and inflammatory cytokine release were assessed. TLR5 knock-out Caco-2 cells were constructed using sgRNA-guided Cas9 nuclease and co-cultured with C. difficile or zinc-treated C. difficile to verify whether zinc-induced C. difficile’s pathogenicity was mainly through flagellar formation. Results C. difficile was detected in ~35% of UC patients’ fecal samples. Luminal zinc concentrations (~915μM, p<0.01) were significantly higher among UC patients than CD patients or healthy subjects. The UC patients’ luminal concentration of zinc promoted C. difficile growth, suppressed its exotoxins release, and induced higher expression of its flagella-related genes, resulting in higher cytotoxicity and inflammatory responses in human colonic organoids and Caco-2 cells. However, zinc-treated C. difficile did not induce higher inflammation and pathogenicity in TLR5 knock-out Caco-2 cells. Conclusion The association between C. difficile and UC aggravation may partially stem from UC patients’ excess luminal zinc promoting C. difficile flagellin expression, inducing inflammatory responses, and worsening UC progression.

A flexible high-throughput cultivation protocol to assess the response of individuals' gut microbiota to diet-, drug-, and host-related factors.

The anaerobic cultivation of fecal microbiota is a promising approach to investigating how gut microbial communities respond to specific intestinal conditions and perturbations. Here, we describe a flexible protocol using 96-deepwell plates to cultivate stool-derived gut microbiota. Our protocol aims to address gaps in high-throughput culturing in an anaerobic chamber. We characterized the influence of the gas phase on the medium chemistry and microbial physiology and introduced a modular medium preparation process to enable the testing of several conditions simultaneously. Furthermore, we identified a medium formulation that maximized the compositional similarity of ex vivo cultures and donor microbiota while limiting the bloom of Enterobacteriaceae. Lastly, we validated the protocol by demonstrating that cultivated fecal microbiota responded similarly to dietary fibers (resistant dextrin, soluble starch) and drugs (ciprofloxacin, 5-fluorouracil) as reported in vivo. This high-throughput cultivation protocol has the potential to facilitate culture-dependent studies, accelerate the discovery of gut microbiota-diet-drug-host interactions, and pave the way to personalized microbiota-centered interventions.

Investigating Iron-Sulfur Proteins in Infectious Diseases: A Review of Characterization Techniques

Iron-sulfur [Fe-S] clusters, comprising coordinated iron and sulfur atoms arranged in diverse configurations, play a pivotal role in redox reactions and various biological processes. Diverse structural variants of [Fe-S] clusters exist, each possessing distinct attributes and functions. Recent discovery of [Fe-S] clusters in infectious pathogens, such as Mycobacterium tuberculosis, and in viruses, such as rotavirus, polyomavirus, hepatitis virus, mimivirus, and coronavirus, have sparked interest in them being a potential therapeutics target. Recent findings have associated these [Fe-S] cluster proteins playing a critical role in structural and host protein activity. However, for a very long time, metalloenzymes containing iron-sulfur clusters have been prone to destabilization in the presence of oxygen, which led to a delayed understanding of [Fe-S] proteins compared to other non-heme iron-containing proteins. Consequently, working with [Fe-S] proteins require specialized equipment, such as anaerobic chambers to maintain cofactor integrity, and tools like ultraviolet visible (UV-Vis) spectroscopy, mass spectrometry, X-ray crystallography, nuclear magnetic resonance (NMR), electron paramagnetic resonance (EPR), Mössbauer spectroscopy and electrochemical characterization. Many of these [Fe-S] cluster proteins have been misannotated as Zinc-binding proteins when purified aerobically. Moreover, the assembly of these iron-sulfur cluster cofactors have not been fully understood since it is a multi-step assembly process. Additionally, disruptions in this assembly process have been linked to human diseases. With rapid advancements in anaerobic gloveboxes and spectroscopic techniques, characterization of these [Fe-S] cluster-containing proteins that are essential for the pathogens can open up new avenues for diagnostics and therapeutics.

Changes in Microbiota Composition during the Anaerobic Digestion of Macroalgae in a Three-Stage Bioreactor.

The use of microalgae as a raw material for biogas production is promising. Macroalgae were mixed with cattle manure, wheat straw, and an inoculant from sewage sludge. Mixing macroalgae with co-substrates increased biogas and methane yield. The research was carried out using a three-stage bioreactor. During biogas production, the dynamics of the composition of the microbiota in the anaerobic chamber of the bioreactor was evaluated. The microbiota composition at different organic load rates (OLRs) of the bioreactor was evaluated. This study also demonstrated that in a three-stage bioreactor, a higher yield of methane in biogas was obtained compared to a single-stage bioreactor. It was found that the most active functional pathway of methane biosynthesis is PWY-6969, which proceeds via the TCA cycle V (2-oxoglutarate synthase). Microbiota composition and methane yield depended on added volatile solids (VSadded). During the research, it was found that after reducing the ORL from 2.44 to 1.09 kg VS/d, the methane yield increased from 175.2 L CH4/kg VSadded to 323.5 L CH4/kg VSadded.

Performance evaluation of gravity-driven bioreactor (GDB) for simultaneous treatment of black liquor and domestic wastewater.

A lab-scale gravity-driven bioreactor (GDB) was designed and constructed to evaluate the simultaneous treatment of black liquor and domestic wastewater. The GDB was operated with a mixture of black liquor and domestic wastewater at a ratio of 1:1 and maintained at an average organic loading rate of 1235mg-COD/L-Day. The wastewater was fed to the primary sedimentation tank at a flow rate of approximately 12mL/min and subsequently passed through serially connected anaerobic and aerobic chambers with the same flow rate. Each wastewater sample was allowed to undergo a hydraulic retention time of approximately 72h, ensuring effective treatment. The GDB was actively operated for nine samples (W1-W9) at a weekly frequency. The entire process was conducted within the workstation's ambient temperature range of 30-35°C to sustain microbial activity and treatment efficiency in an open environment. The performance of the GDB was evaluated in terms of various pollution indicators, including COD, BOD5, lignin removal, TDS, TSS, EC, PO43-, SO42-, microbial load (CFU/mL and MPN index), total nitrogen, and color reduction. The results showed that the GDB achieved promising treatment efficiencies: 84.5% for COD, 71.80% for BOD5, 82.8% for TDS, 100% for TSS, 74.71% for E.C., 67.25% for PO43-, 81% for SO42-, and 69.36% for TN. Additionally, about 80% reduction in lignin content and 57% color reduction were observed after the treatment. The GDB substantially reduced microbial load in CFU/mL (77.98%) and MPN (90%). This study marks the first to report on wastewater treatment from two different sources (black liquor and domestic wastewater) using a simple GDB design. Furthermore, it highlights the GDB's potential as a cost-effective, environmentally friendly, and efficient solution for wastewater treatment, with no need for supplementary chemical or physical agents and zero operational costs.