Anaerobic Chamber Research Articles

Effects of Novel Dental Composites on Streptococcus mutans Biofilms.

With the phase-out of amalgam and the increase in minimally invasive dentistry, there is a growing need for high-strength composite materials that can kill residual bacteria and promote tooth remineralization. This study quantifies how antibacterial polylysine (PLS) and re-mineralizing monocalcium phosphate monohydrate (MCPM) affect Streptococcus mutans biofilms and the strength of dental composites. For antibacterial studies, the MCPM-PLS filler percentages were 0-0, 8-4, 12-6, and 16-8 wt% of the composite filler phase. Composite discs were immersed in 0.1% sucrose-supplemented broth containing Streptococcus mutans (UA159) and incubated in an anaerobic chamber for 48 h. Surface biomass was determined by crystal violet (CV) staining. Growth medium pH was measured at 24 and 48 h. Biofilm bacterial viability (CFU), exo-polysaccharide (water-soluble glucan (WSG) and water-insoluble glucan (WIG)), and extracellular DNA (eDNA) were quantified. This was by serial dilution plate counting, phenol-sulfuric acid microassay, and fluorometry, respectively. The biaxial flexural strengths were determined after water immersion for 1 week, 1 month, and 1 year. The MCPM-PLS wt% were 8-4, 8-8, 16-4 and 16-8. The normalized biomass, WSG, and WIG showed a linear decline of 66%, 64%, and 55%, respectively, as the PLS level increased up to 8%. The surrounding media pH (4.6) was all similar. A decrease in bacterial numbers with the 12-6 formula and a significant reduction with 16-8 compared to the 0-0 formulation was observed. The eDNA concentrations in biofilms formed on 12-6 and 16-8 formulations were significantly less than the 0-0 control and 8-4 formulations. Doubling MCPM and PLS caused a 14 and 19% reduction in strength in 1 week, respectively. Average results were lower at 1 month and 1 year but affected less upon doubling MCPM and PLS levels. Moreover, a 4% PLS may help to reduce total biomass and glucan levels in biofilms on the above composites. Higher levels are required to reduce eDNA and provide bactericidal action, but these can decrease early strength.

The Influences of Traces of Oxygen and Sulfide on the Corrosion of Copper Under Canadian Nuclear Waste Disposal Conditions

The Canadian repository concept for the permanent disposal of used nuclear fuel is to seal it in Cu-coated steel containers and dispose of it in a deep geological repository (DGR) surrounded by compacted bentonite clay. The Cu coating will be directly bonded on the steel vessel and lid using electro-deposition (ED-Cu), with the final container closure weld sealed with a cold-sprayed Cu deposit (CS-Cu). To assess the durability of such a container, long-term corrosion experiments have been performed on CS-Cu and ED-Cu specimens, and compared with those performed on wrought Cu specimens provided by the Swedish Nuclear Fuel and Waste Management Company (SKB-Cu).Experiments were conducted in 3 M NaCl solutions containing traces of sulfide (< 10- 6 M) for exposure periods of ≥150 days. These conditions reflect a generic groundwater that may influence container corrosion in a Canadian DGR Since conditions in a DGR will evolve from initially oxic to eventually anoxic, experiments were conducted under three sets of redox conditions: (i) aerated (oxic) conditions; (ii) Ar-purged (oxic to anoxic transition conditions (with a dissolved [O2] ~ 10- 6 M)); and (iii) anaerobic chamber (anoxic) conditions ([O2] < 1.3 × 10- 9 M). The exposure process was followed using open circuit (i.e., corrosion) potential and polarization resistance (RP) measurements. Specimens were analyzed before and after corrosion using optical microscopy, scanning electron microscopy on surfaces and focused ion beam cut cross sections, Raman spectroscopy, and X-ray photoelectron/Auger spectroscopy.In all environments, all three types of specimens (ED-Cu, CS-Cu, SKB-Cu) showed similar behaviour. Under anoxic conditions, minor corrosion, indicated by large RP values, leading to the deposition of traces of chalcocite (Cu2S) (identified by Raman spectroscopy) and observed by SEM, indicated that corrosion could be attributed to reaction with the trace amounts of sulfide present in NaCl. In Ar-purged solutions, the RP values were approximately one order of magnitude lower (i.e., the corrosion rate was one order of magnitude higher) than those measured under anoxic conditions, and significant corrosion damage was observed in SEM micrographs. Both Cu2O and Cu2S were detected by Raman spectroscopy. The detection of oxidized sulfur species (SO3 2 - and SO4 2 -) on the corroded surface by XPS/Auger confirmed that even trace amounts of dissolved O2 led to sulfide oxidation and the possible formation of potentially more corrosive species such as thiosulfate (S2O3 2 -). Under aerated conditions, extensive corrosion occurred leading to the formation of green CuII products (atacamite (Cu2(OH)3Cl)) with no detectable Cu2S formation.Within the DGR, corrosion of Cu under anoxic conditions will be limited by the available supply of sulfide, which may be produced remotely at the host rock/clay interface and/or in the porewater within the rock fractures. In the presence of even trace of dissolved O2, the corrosion process is significantly accelerated with the formation of potentially aggressive oxidized sulfur species. Under aerated conditions, extensive corrosion due to reaction with O2 leads to the deposition of CuII deposits. On the time scale of these experiments with only trace of sulfide present, no detectable conversion of copper oxide to Cu2S was observed. Keywords: Copper; Sulfide; Corrosion; Nuclear waste disposal

High-throughput anaerobic screening for identifying compounds acting against gut bacteria in monocultures or communities.

The human gut microbiome is a key contributor to health, and its perturbations are linked to many diseases. Small-molecule xenobiotics such as drugs, chemical pollutants and food additives can alter the microbiota composition and are now recognized as one of the main factors underlying microbiome diversity. Mapping the effects of such compounds on the gut microbiome is challenging because of the complexity of the community, anaerobic growth requirements of individual species and the large number of interactions that need to be quantitatively assessed. High-throughput screening setups offer a promising solution for probing the direct inhibitory effects of hundreds of xenobiotics on tens of anaerobic gut bacteria. When automated, such assays enable the cost-effective investigation of a wide range of compound-microbe combinations. We have developed an experimental setup and protocol that enables testing of up to 5,000 compounds on a target gut species under strict anaerobic conditions within 5 d. In addition, with minor modifications to the protocol, drug effects can be tested on microbial communities either assembled from isolates or obtained from stool samples. Experience in working in an anaerobic chamber, especially in performing delicate work with thick chamber gloves, is required for implementing this protocol. We anticipate that this protocol will accelerate the study of interactions between small molecules and the gut microbiome and provide a deeper understanding of this microbial ecosystem, which is intimately intertwined with human health.

Integrated CO2 capture and conversion via H2-driven CO2 biomethanation: Cyclic performance and microbial community response

This study investigated the use of H2-driven CO2 biomethanation for integrated CO2 capture and conversion (iCCC). Anaerobic chambers containing Na2CO3-amended microbial growth medium provided with H2 were inoculated with anaerobic granular sludge. Microorganisms were enriched that could regenerate carbonate by using the bicarbonate formed from CO2 absorption to generate methane. Multiple absorption–regeneration cycles were performed and effective restoration of CO2 absorption capacity and stable carbonate recycling via CO2 biomethanation were observed for CO2 absorbents adjusted to three different pH values (9.0, 9.5, and 10.0). The pH = 10.0 group had the highest CO2 absorption capacity; 65.3 mmol/L in the 5th cycle. A slight alkaline inhibition of acetoclastic methanogenesis occurred near the end of regeneration, but had limited impact on the cyclic performance of the iCCC process. Microbial communities were dominated by H2-utilizing and alkali-tolerant species that could participate in CO2 biomethanation and survive under alternating neutral and alkaline conditions.

Novel role of folate (vitamin B9) released by fermenting bacteria under Human Intestine like environment

The anaerobic region of the gastrointestinal (GI) tract has been replicated in the anaerobic chamber of a microbial fuel cell (MFC). Electroactive biomolecules released by the facultative anaerobes (Providencia rettgeri) under anoxic conditions have been studied for their potential role for redox balance. MALDI study reveals the presence of vitamin B9 (folate), 6-methylpterin, para-aminobenzoic acid (PABA) and pteroic acid called pterin pool. ATR-FTIR studies further confirm the presence of the aromatic ring and side chains of folate, 6-methylpterin and PABA groups. The photoluminescence spectra of the pool exhibit the maximum emission at 420, 425, 440, and 445 nm when excited by 310, 325, 350, and 365 nm wavelengths (day 20 sample) highlighting the presence of tunable bands. The cyclic voltammetric studies indicate the active participation of pterin pool molecules in the transfer of electrons with redox potentials at − 0.2 V and − 0.4 V for p-aminobenzoate and pterin groups, respectively. In addition, it is observed that under prolonged conditions of continuous oxidative stress (> 20 days), quinonoid tetrahydrofolate is formed, leading to temporary storage of charge. The results of the present study may potentially be useful in designing effective therapeutic strategies for the management of various GI diseases by promoting or blocking folate receptors.

Identification of acacia gum fermenting bacteria from pooled human feces using anaerobic enrichment culture.

Commercial acacia gum (AG) used in this study is a premium-grade free-flowing powder. It is a gummy exudate composed of arabinogalactan branched polysaccharide, a biopolymer of arabinose and galactose. Also known as food additive, acacia gum (E414), which is presently marketed as a functional dietary fiber to improve overall human gut health. The health effects may be related to the luminal pH regulation from the short-chain fatty acids (SCFA) production. Studies suggested that amylolytic and butyrogenic pathways are the major factors determining the SCFA outcome of AG in the lower gut. However, the primary bacteria involved in the fermentation have not been studied. This study aimed to investigate the putative primary degraders of acacia gum in the gut ecosystem. Isolation and identification of gum-fermenting bacteria were performed through enrichment culture fermentation. The experiment was conducted in an anaerobic chamber for 144 h in three stages. The study was conducted in triplicate using an anaerobic chamber system. This culture system allows specific responses to support only bacteria that are responsible for gum fermentation among the gut microbiota. Five bacterial strains were isolated and found to be gum-fermenting bacteria. Based on the 16s RNA sequence, the isolates matched to butyrate-producing Escherichia fergusonii, ATCC 35469.

Enzyme-functionalized alginate microparticles enable anaerobic culture under ambient oxygen.

Methods for culturing oxygen-sensitive cells and organisms under anaerobic conditions are vital to biotechnology research. Here, we report a biomaterial-based platform for anaerobic culture that consists of glucose oxidase (GOX) functionalized alginate microparticles (ALG-GOX), which are designed to deplete dissolved [O2 ] through enzymatic activity. ALG-GOX microparticles were synthesized via a water-in-oil emulsion and had a size of 132.0 ± 51.4 µm. Despite having a low storage modulus, the microparticles remained stable under aqueous conditions due to covalent crosslinking through amide bonds. Enzyme activity was tunable based on the loaded GOX concentration, with a maximum activity of 3.6 ± 0.3 units/mg of microparticles being achieved at an initial loading concentration of 5 mg/mL of GOX in alginate precursor solution. High enzyme activity in ALG-GOX microparticles resulted in rapid oxygen depletion, producing a suitable environment for anaerobic culture. Microparticles loaded with both GOX and catalase (ALG-GOX-CAT) to reduce H2 O2 buildup exhibited sustained activity for potential long-term anaerobic culture. ALG-GOX-CAT microparticles were highly effective for the anaerobic culture of Bacteroides thetaiotaomicron, with 10 mg/mL of ALG-GOX-CAT microparticles supporting the same level of growth in an aerobic environment compared to an anaerobic chamber after 16 h (8.70 ± 0.96 and 10.03 ± 1.03 million CFU, respectively; N.S. p = 0.07). These microparticles could be a valuable tool for research and development in biotechnology.

In Vitro Biofilm Formation on Zirconia Implant Surfaces Treated with Femtosecond and Nanosecond Lasers.

(1) Background: The purpose of this study was to evaluate how a zirconia implant surface treated with laser technology affects the degree of biofilm formation. (2) Methods: Experimental titanium (Ti) disks were produced that were sandblasted with large grit and acid-etched (T), and they were compared with zirconia (ZrO2) discs with a machined (M) surface topography; a hydrophilic surface topography with a femtosecond laser (HF); and a hydrophobic surface topography with a nanosecond laser (HN) (N = 12 per surface group). An in vitro three-species biofilm sample (Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Prevotella intermedia (Pi)) was applied to each disc type, and bacterial adhesion was assessed after 48 and 72 h of incubation using an anaerobic flow chamber model. Statistical significance was determined using the Kruskal-Wallis H test, with Bonferroni correction used for the post-hoc test (α = 0.05). (3) Results: Compared to the T group, the M group exhibited more than twice as many viable bacterial counts in the three-species biofilm samples (p < 0.05). In comparison to the T group, the HF group had significantly higher viable bacterial counts in certain biofilm samples at 48 h (Aa and Pi) and 72 h (Pi) (p < 0.05). The HN group had higher viable bacterial counts in Pi at 48 h (5400 CFU/mL, p < 0.05) than the T group (4500 CFU/mL), while showing significantly lower viable bacterial counts in Pg at both 48 (3010 CFU/mL) and 72 h (3190 CFU/mL) (p < 0.05). (4) Conclusions: The surface treatment method for zirconia discs greatly influences biofilm formation. Notably, hydrophobic surface treatment using a nanosecond laser was particularly effective at inhibiting Pg growth.

Diversity and functional annotation of microorganisms in anaerobic chamber treating nitrate-rich wastewater.

The vertical flow constructed wetlands (VFCW) for the treatment of domestic wastewater has become a conventional and cost effective treatment system with one of the major disadvantage of elevated nitrate concentrations. The present study makes an effort in providing a new design of anaerobic denitrification unit termed as anaerobic chamber (AC) whichwas introduced after two-stage VFCW to remove nitrates from the treated wastewater (WW). The AC provided all the essential conditions of effective denitrification such as anaerobic environment with enough carbon and nitrogen source. To understand the pollutant removal mechanism in AC, microbial diversity and functional annotation was studied by metagenomic analysis of sequences obtained from biofilm formed in AC. The efficiency of AC was measured with respect to physicochemical wastewater quality parameters. The removal efficiencies were 88, 65, 43 and 27% for total nitrogen, nitrate (NO3), ammoniacal-nitrogen (NH4) andortho-phosphate respectively. The microbial flora was much more diverse and unique pertaining to anaerobic microbes in AC compared to WW with total of 954 and 1191 genuses respectively with minimum abundance of 10 hits. The metagenomes exhibited 188% more Archaea in the AC than WW where Crenarchaeota, Euryarchaeota, Korarchaeota, Nanoarchaeota and Thaumarchaeota were major phyla with 60 genuses. The nitrogen metabolism was reported in terms of assimilatory nitrate reductase. As the class, Proteobacteria, Actinobacteria were prominent in WW, whereas Proteobacteria, Chloroflexi in AC were abundant. From functional annotation of sequences, the microbial flora in AC has the potential of removal of pollutants present in the form of carbon, nitrogen, and phosphorus.

Isoxanthohumol improves obesity and glucose metabolism via inhibiting intestinal lipid absorption with a bloom of Akkermansia muciniphila in mice

ObjectivePolyphenols have health-promoting effects, such as improving insulin resistance. Isoxanthohumol (IX), a prenylated flavonoid found in beer hops, has been suggested to reduce obesity and insulin resistance; however, the mechanism remains unknown. MethodsHigh-fat diet-fed mice were administered IX. We analyzed glucose metabolism, gene expression profiles and histology of liver, epididymal adipose tissue and colon. Lipase activity, fecal lipid profiles and plasma metabolomic analysis were assessed. Fecal 16s rRNA sequencing was obtained and selected bacterial species were used for in vitro studies. Fecal microbiota transplantation and monocolonization were conducted to antibiotic-treated or germ-free (GF) mice. ResultsThe administration of IX lowered weight gain, decreased steatohepatitis and improved glucose metabolism. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption by decreasing the expression of the fatty acid transporter CD36 in the small intestine, which was confirmed by increased lipid excretion in feces. IX administration increased markers of intestinal barrier function, including thickening the mucin layer and increasing caludin-1, a tight-junction related protein in the colon. In contrast, the effects of IX were nullified by antibiotics. As revealed using 16S rRNA sequencing, the microbial community structure changed with a significant increase in the abundance of Akkermansia muciniphila in the IX-treated group. An anaerobic chamber study showed that IX selectively promoted the growth of A. muciniphila while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct effect of A. muciniphila on lipid and glucose metabolism, we monocolonized either A. muciniphila or Bacteroides thetaiotaomicron to GF mice. A. muciniphila monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism, with decreased levels of multiple classes of fatty acids determined using plasma metabolomic analysis. ConclusionsOur study demonstrated that IX prevents obesity and enhances glucose metabolism by inhibiting dietary fat absorption. This mechanism is linked to suppressing pancreatic lipase activity and shifts in microbial composition, notably an increase in A. muciniphila. These highlight new treatment strategies for preventing metabolic syndrome by boosting the gut microbiota with food components.

Next-generation Sequencing Results Require Higher Inoculum for Cutibacterium acnes Detection Than Conventional Anaerobic Culture.

Cutibacterium acnes has been described as the most common causative microorganism in prosthetic shoulder infections. Conventional anaerobic culture or molecular-based technologies are usually used for this purpose, but little to no concordance between these methodologies (k = 0.333 or less) has been observed. (1) Is the minimum C. acnes load for detection higher for next-generation sequencing (NGS) than for anaerobic conventional culture? (2) What duration of incubation is necessary for anaerobic culture to detect all C. acnes loads? Five C. acnes strains were tested for this study: Four strains were causing infection and were isolated from surgical samples. Meanwhile, the other was a reference strain commonly used as a positive and quality control in microbiology and bioinformatics. To create inoculums with varying degrees of bacterial load, we began with a standard bacterial suspension at 1.5 x 10 8 colony-forming units (CFU)/mL and created six more diluted suspensions (from 1.5 x 10 6 CFU/mL to 1.5 x 10 1 CFU/mL). Briefly, to do so, we transferred 200 µL from the tube with the highest inoculum (for example, 1.5 x 10 6 CFU/mL) to the following dilution tube (1.5 x 10 5 CFU/mL; 1800 µL of diluent + 200 µL of 1.5 x 10 6 CFU/mL). We serially continued the transfers to create all diluted suspensions. Six tubes were prepared per strain. Thirty bacterial suspensions were tested per assay. Then, 100 µL of each diluted suspension was inoculated into brain heart infusion agar with horse blood and taurocholate agar plates. Two plates were used per bacterial suspension in each assay. All plates were incubated at 37°C in an anaerobic chamber and assessed for growth after 3 days of incubation and daily thereafter until positive or Day 14. The remaining volume of each bacterial suspension was sent for NGS analysis to identify bacterial DNA copies. We performed the experimental assays in duplicate. We calculated mean DNA copies and CFUs for each strain, bacterial load, and incubation timepoint assessed. We reported detection by NGS and culture as a qualitative variable based on the identification or absence of DNA copies and CFUs, respectively. In this way, we identified the minimum bacterial load detected by NGS and culture, regardless of incubation time. We performed a qualitative comparison of detection rates between methodologies. Simultaneously, we tracked C. acnes growth on agar plates and determined the minimum incubation time in days required for CFU detection in all strains and loads examined in this study. Growth detection and bacterial CFU counting were performed by three laboratory personnel, with a high intraobserver and interobserver agreement (κ > 0.80). A two-tailed p value below 0.05 was considered statistically significant. Conventional cultures can detect C. acnes at a load of 1.5 x 10 1 CFU/mL, whereas NGS can detect bacteria when the concentration was higher, at 1.5 x 10 2 CFU/mL. This is represented by a lower positive detection proportion (73% [22 of 30]) for NGS than for cultures (100% [30 of 30]); p = 0.004). By 7 days, anaerobic cultures were able to detect all C. acnes loads, even at the lowest concentrations. When NGS is negative and culture is positive for C. acnes , there is likely a low bacterial load. Holding cultures beyond 7 days is likely unnecessary. This is important for treating physicians to decide whether low bacterial loads necessitate aggressive antibiotic treatment or whether they are more likely contaminants. Cultures that are positive beyond 7 days likely represent contamination or bacterial loads even below the dilution used in this study. Physicians may benefit from studies designed to clarify the clinical importance of the low bacteria loads used in this study at which both methodologies' detection differed. Moreover, researchers might explore whether even lower C. acnes loads have a role in true periprosthetic joint infection.

1672-P: Isoxanthohumol Improves Obesity and Glucose Metabolism via Regulation of Gut Barrier and Intestinal Lipid Absorption with a Bloom of Akkermansia muciniphila

Dysbiosis is an important factor that leads to metabolic disorders via disruption of the gut barrier function. A decrease in Akkermansia muciniphila (AM) is a phenotype of obesity-induced disruption of the gut barrier. Although interventions to increase AM have been reported to improve glucose metabolism, the underlying mechanism has not been fully understood. The administration of IX suppressed weight gain and steatohepatitis and improved glucose metabolism in diet-induced obese mice. Mechanistically, IX inhibited pancreatic lipase activity and lipid absorption via decreased expression of the lipid absorption transporter, CD36, in the jejunum. Moreover, IX administration improved the gut barrier function and reduced metabolic endotoxemia. In contrast, the observed effects of IX were cancelled by antibiotics and reproduced by fecal microbiota transplantation to GF mice. 16S rRNA sequencing revealed that the microbial community structure changed with a significant increase in AM in the IX-administration group. An anaerobic chamber study showed that IX promoted the growth of AM, while exhibiting antimicrobial activity against some Bacteroides and Clostridium species. To further explore the direct impact of AM on lipid and glucose metabolism in monocolonized mice, we transplanted either AM or Bacteroides thetaiotaomicron to GF mice. AM monocolonization decreased CD36 expression in the jejunum and improved glucose metabolism with decreased multiple classes of fatty acids in plasma metabolomics analysis. In summary, IX contributes to metabolic improvement by decreasing CD36 and lipid absorption in the small intestine which is associated with a bloom of AM, suggesting an important interaction between food ingredients and AM abundance in controlling metabolism. Disclosure Y.Watanabe: None. S.Fujisaka: None. S.Watanabe: None. A.Nishimura: None. T.Kado: None. M.Aslam: None. M.Bilal: None. Y.Morinaga: None. K.Tobe: Other Relationship; MSD K.K., Novo Nordisk Pharma Ltd., Takeda Pharmaceutical Company Limited, Daiichi Sankyo, Mitsubishi Tanabe Pharma Corporation, Suntory Global Corporation, Ltd.,, Taiho Pharmaceutical Co. Ltd., Japan Diabetes Foundation, Japan Association for Diabetes Education and Care. Funding Japan Society for the Promotion of Science (21K20896, 22K16424); Lotte Foundation; Yakult Bio-Science Foundation

In-situ sludge degradation and kinetics of a full scale modified activated sludge system achieving near zero sludge production

This work reports on the performance of a full scale modified AS system that consists of anaerobic/anoxic/oxic and a settler with internal waste activated sludge (WAS) recycle that achieves sludge reduction through in situ cell lysis. This modification is termed as sludge process reduction activated sludge (SPRAS) process. The SPRAS plant achieved significant sludge reduction with sludge yield of 0.0823 kg TSS/kg COD which is much lower than the sludge yield of a conventional activated sludge process of 0.3–1.2 kg TSS/kg COD. The TSS concentration in the SPRAS tank was nearly constant at 4500 mg/L showing that there was no sludge build up, a sludge volume index of 80–120 mg/L implied adequate sludge settling ability in the settling chamber which facilitated sludge separation for subsequent degradation through anaerobic digestion. In addition, the process achieved ammonia and COD reductions above 85 %. Comparative FTIR analysis of the sludge in the anaerobic and oxic chambers indicated degradation of the proteinaceous biomass cell wall in the anaerobic chamber leading to release of carbohydrates that could easily be degraded in the oxic chamber. The maximum sludge yield and sludge decay coefficient were calculated as 0.103 kg TSS/kg COD and 0.0015 thus the technology is suitable for handling municipal wastewater at minimal sludge handling cost.

Passive removal of sulphate and heavy metals from acid mine drainage using sewage sludge and fly ash

This study aims to calculate the removal efficiency (%RE) of metals and sulphate and the constant rate of sulphate reduction from acid mine drainage (AMD) based on passive treatment principles named permeable reactive barrier (PRB) in batch test prior column test. Three media mixtures containing a variety of domestic sewage sludge (SE), mess hall compost (CO), cocopeat (CP), and fly ash (FA) were simulated. All reactive materials are solid waste from other operating units. M1 and M2 were contains organic and inorganic waste, M3 was only contain organic waste. The AMD was collected from a copper mining waste rock dump of which contained high sulphate, metals, and low pH. Batch tests were conducted in a series of glass bottles in an anaerobic chamber, and sub-samplings were taken on days 0, 7, 14, and 28. At the end of treatment, it indicated that M1 mixture resulting in the highest sulphate (SO4 2-) removal (44%), highest alkalinity generation of 1431 mg/L (as CaCO3) and %RE of Al, Cd, Co, Cu, Fe, and Zn were expected to be 100%, Ni 82%, Se 57%, and Mn 98%. Additional of FA for treatment such in M1 and M2 releasing more As in the final result compare to M3 that only contain organic substrates. The primary mechanism controlling the reaction from the M1 was a combination of sulphide precipitation enhanced by Sulphate Reducing Bacteria (SRB) activities supported by pH buffering and hydroxide precipitation. The sulphate reduction mechanism assumed to be the first-order reaction with highest rate constants found as 0.0208 d−1 from M1 reactors, 0.0144 d−1, 0.0161 d−1 for M2 and M3 respectively.

Removal of chloramphenicol and resistance gene changes in electric-integrated vertical flow constructed wetlands

The performance of an electric-integrated vertical flow constructed wetland (E-VFCW) for chloramphenicol (CAP) removal, changes in microbial community structure, and the fate of antibiotic resistance genes (ARGs) were evaluated. CAP removal in the E-VFCW system was 92.73% ± 0.78% (planted) and 90.80% ± 0.61% (unplanted), both were higher than the control system which was 68.17% ± 1.27%. The contribution of anaerobic cathodic chambers in CAP removal was higher than the aerobic anodic chambers. Plant physiochemical indicators in the reactor revealed electrical stimulation increased oxidase activity. Electrical stimulation enhanced the enrichment of ARGs in the electrode layer of the E-VFCW system (except floR). Plant ARGs and intI1 levels were higher in the E-VFCW than in the control system, suggesting electrical stimulation induces plants to absorb ARGs, reducing ARGs in the wetland. The distribution of intI1 and sul1 genes in plants suggests that horizontal transfer may be the main mechanism dispersing ARGs in plants. High throughput sequencing analysis revealed electrical stimulation selectively enriched CAP degrading functional bacteria (Geobacter and Trichlorobacter). Quantitative correlation analysis between bacterial communities and ARGs confirmed the abundance of ARGs relates to the distribution of potential hosts and mobile genetic elements (intI1). E-VFCW is effective in treating antibiotic wastewater, however ARGs potentially accumulate.

Wastewater Treatment and Electricity Generation with Different Cathode Solutions in MFC

A microbial fuel cell (MFC) with cathode and anode chambers was utilized to generate power while simultaneously removing COD from wastewater. By utilizing various oxidant solutions, it is possible to increase the generated voltage. The anode chamber was used for anaerobic treatment of synthetic wastewater (approximately 1000 mg/L), whereas the cathode chamber contained various oxidant solutions such as dilute hydrogen peroxide (300 mg/L), KMnO4 (300 mg/L), K2Cr2O7 (300 mg/L) and Fenton reagent (H2O2/Fe(II), 300/20 mg/L). Aerobic wastewater treatment and intermittent ozonation were also tested in the cathode chamber. With intermittent ozonation of the cathode chamber, the highest power output (382 mW/m2) was obtained. At the conclusion of the operation period, the COD concentration in the anode chamber decreased from 1170 mg/L to 650 mg/L, resulting in nearly 45% COD removal. In the cathode chamber, the use of diluted KMnO4 and H2O2 solutions produced high power densities of 35 and 23 W/m2, respectively, while the other oxidants produced low power densities. At the end of 72 hours, the COD content of the anaerobic chamber decreased from 800 mg/L to nearly 333 mg/L, resulting in nearly 59% COD removal for the KMnO4 solution. Considering the high cost of ozonation, it is recommended that either aerobic wastewater treatment or dilute KMnO4/H2O2 solutions should be used in the cathode chamber for high power generation.

Improved Biological Phosphorus Removal under Low Solid Retention Time Regime in Full-Scale Sequencing Batch Reactor

Enhanced biological phosphorus removal (EBPR) is an obscure but economical and helpful technology for removing phosphorus biologically from wastewater. A 3-MLD capacity pre-anoxic selector-attached sequencing batch reactor (SBR) treated municipal wastewater from the residents of IIT Roorkee. The treatment in the plant satisfied the effluent discharge standards in all respects except phosphorus, observed during an intensive two-year study. An elaborated 80-day study was performed to enhance and improve the plant’s performance in terms of phosphorus removal specifically, with run 1: solid retention times (SRT) reduced from 56 to 20 days (t = 35 d), run 2: lowering the diffuser’s running time from 15 min to 10 min in anoxic cum anaerobic selector chambers (dissolved oxygen (DO) concentration reduced to &lt;0.15 mg/L) along with reducing SRT to 15 days (t = 25 d), and run 3:intensive reduction in SRT to ≤10 days (t = 20 d). During run 3, the increment in the enhanced biological phosphorus removal (EBPR) efficiency was three times that of the initial run (ηmax~65%) with a readily biodegradable chemical oxygen demand to total phosphorus ratio (rbCOD/TP) of 7.8. The 16SrRNA sequencing revealed the microbial community structure before and after the changes in SRT and EBPR efficiencies, to correlate the biochemical processes and functional organisms.

Effects of pre‐treated sludge on sludge stabilization and electricity generation in microbial fuel cell

AbstractIn this study, based on the advantage of having high organic content of sludge both sludge treatment and electricity generation in the microbial fuel cell were investigated. The hydrolysis step of anaerobic digestion which is known as rate‐limiting step was tried to accelerate with the pre‐treatment methods such as Fenton and thermal/alkaline for sludge stabilization in the microbial fuel cell. The chemical oxygen demand and organic matter removal were 53% and 64% in the anaerobic chamber of the microbial fuel cell fed with raw sludge, respectively. In the microbial fuel cell fed with pre‐treated sludge using Fenton Process, these removal efficiencies were found to be 59% and 60%, respectively, while in thermal/alkaline microbial fuel cells were 83.3% and 85%, respectively. The current densities in microbial fuel cells fed with raw sludge, Fenton process sludge and thermal/alkaline sludge were 0.002, 0.221 and 38 mA/cm2, respectively. As a result, it can be concluded that, the microbial fuel cell fed with pre‐treated sludge can be used for both the sludge treatment and the electricity generation by improving the decomposition of organic matter and accelerating the rate‐limiting step in the anaerobic chamber.

Assessment of Surfactant Removal Capacity and Microbial Community Diversity in a Greywater-Treating Constructed Wetland

Surfactants are among the main chemical contaminants in greywater (GW) and can cause severe health issues in humans and aquatic organisms. We assessed the performance of a multistage constructed wetland system (EvaTAC) for GW treatment and capacity of the microbial community in linear alkyl benzene sulfonate (LAS) biodegradation. Physicochemical analyses were performed over 497 d, and biomass samples were collected for high-throughput DNA sequencing. The system was predominated by anaerobic conditions and received an average chemical oxygen demand (COD) and LAS of 374 and 32 mg·L−1, with removal rates of 66% and 43%, respectively. A positive correlation between COD and LAS suggested COD as a design parameter for LAS removal. We identified microbial genera participating in hydrolysis, fermentation, syntrophy, acetogenesis, methanogenesis, surfactant degradation, and sulphate reduction. Among the 15 surfactant-degrading genera, Pseudomonas was predominant. Community richness and diversity indices were comparable between subsystems, with a slight decrease in diversity observed towards the outlet. Among the LAS degraders, Rhodopseudomonas palustris had the highest relative abundance of operational taxonomic unit (OTU)s in all samples and the highest richness in the anaerobic chamber. The patterns in microbial community composition and environmental conditions suggest that LAS biodegradation occurred throughout the EvaTAC system.

Mercury methylation potential and bioavailability in the sediments of two distinct aquatic systems

This study explored mercury (Hg) methylation potential in two distinct aquatic systems. Fourmile Creek (FMC) was historically polluted with Hg effluents from groundwater as it is a typical gaining stream, where organic matter and microorganisms in streambed are continuously winnowed. The H02 constructed wetland only receives atmospheric Hg and is rich in organic matter and microorganisms. Both systems receive Hg from atmospheric deposition now. Surface sediments were collected from FMC and H02, spiked with inorganic Hg, and cultivated in an anaerobic chamber to stimulate microbial Hg methylation reactions. Total mercury (THg) and methylmercury (MeHg) concentrations were measured at each spiking stage. Mercury methylation potential (MMP, %MeHg in THg) and Hg bioavailability were assessed with the deployment of diffusive gradients in thin films (DGTs). During the methylation process and at the same incubation stage, FMC sediment showed faster increasing rates of %MeHg and higher MeHg concentrations than H02, demonstrating a stronger MMP in the FMC sediment. Similarly, higher Hg bioavailability was observed in FMC sediment compared to the H02 as indicated by DGT-Hg concentrations. In conclusion, the H02 wetland with high levels of organic matter and microorganisms presented low MMP. But the Fourmile Creek as a gaining stream and a historical site of Hg pollution showed strong MMP and high Hg bioavailability. A related study on microbial community activities characterized the microorganisms between FMC and H02, which is attributed to be the main reason for their different methylation capabilities. Our study further brought up the considerations on remediated sites from Hg contamination: Hg bioaccumulation and biomagnification can still be elevated and higher than the surrounding environment due to lagged changes in microbial community structures. This study supported the sustainable ecological modifications of legacy Hg contamination and raised the necessity of long-term monitoring actions even after executing a remediation plan.