Cadmium Selenide Formation Influences the Production and Characteristics of Extracellular Polymeric Substances of Anaerobic Granular Sludge.

Feeding cadmium (II) and selenium (IV) simultaneously to anaerobic granular sludge with the aim of synthesizing cadmium selenide (CdSe) nanoparticles induces compositional changes in the extracellular polymeric substances (EPS) matrix of this sludge. A methanogenic anaerobic granular sludge was repeatedly exposed to Cd(II) (10-50 mg L-1) and selenite (79 mg L-1) for 300 days at pH 7.3 and 30 °C in a fed-batch feeding regime for enrichment of Se-reducing bacteria and synthesis of CdSe nanoparticles. EPS fingerprints of the granular sludge, obtained by size exclusion chromatography coupled to a fluorescence detector, showed a significant increase in the intensity of protein-like substances with > 100 kDa apparent molecular weight (aMW) upon repeated exposure to Cd(II) and Se(VI). This was accompanied by a prominent decrease in protein-like substances of aMW < 10 kDa. The fingerprint of the humic-like substances showed emergence of a new peak with aMW of 13 to 300 kDa in the EPS extracted from the Cd/Se fed granular sludge. Experiments on metal(loid)-EPS interactions showed that the CdSe nanoparticles interact mainly with loosely bound EPS (LB-EPS). This study showed that the formation of Se(0) and CdSe nanoparticles occurs in the LB-EPS fraction of the granular sludge and repeated exposure to Cd and Se induces compositional changes in the EPS matrix.

Making waves: Wastewater surveillance of SARS-CoV-2 for population-based health management.

Worldwide, clinical data remain the gold standard for disease surveillance and tracking. However, such data are limited due to factors such as reporting bias and inability to track asymptomatic disease carriers. Disease agents are excreted in the urine and feces of infected individuals regardless of disease symptom severity. Wastewater surveillance - that is, monitoring disease via human effluent - represents a valuable complement to clinical approaches. Because wastewater is relatively inexpensive and easy to collect and can be monitored at different levels of population aggregation as needed, wastewater surveillance can offer a real-time, cost-effective view of a community's health that is independent of biases associated with case-reporting. For SARS-CoV-2 and other disease-causing agents we envision an aggregate wastewater-monitoring system at the level of a wastewater treatment plant and exploratory or confirmatory monitoring of the sewerage system at the neighborhood scale to identify or confirm clusters of infection or assess impact of control measures where transmission has been established. Implementation will require constructing a framework with collaborating government agencies, public or private utilities, and civil society organizations for appropriate use of data collected from wastewater, identification of an appropriate scale of sample collection and aggregation to balance privacy concerns and risk of stigmatization with public health preservation, and consideration of the social implications of wastewater surveillance.

Microbial transformation of Se oxyanions in cultures of Delftia lacustris grown under aerobic conditions.

Delftia lacustris is reported for the first time as a selenate and selenite reducing bacterium, capable of tolerating and growing in the presence of ≥ 100 mM selenate and 25 mM selenite. The selenate reduction profiles of D. lacustris were investigated by varying selenate concentration, inoculum size, concentration and source of organic electron donor in minimal salt medium. Interestingly, the bacterium was able to reduce both selenate and selenite under aerobic conditions. Although considerable removal of selenate was observed at all concentrations investigated, D. lacustris was able to completely reduce 0.1 mM selenate within 96 h using lactate as the carbon source. Around 62.2% unaccounted selenium (unidentified organo-selenium compounds), 10.9% elemental selenium and 26.9% selenite were determined in the medium after complete reduction of selenate. Studies of the enzymatic activity of the cell fractions show that the selenite/selenate reducing enzymes were intracellular and independent of NADPH availability. D. lacustris shows an unique metabolism of selenium oxyanions to form elemental selenium and possibly also selenium ester compounds, thus a potential candidate for the remediation of selenium-contaminated wastewaters in aerobic environments. This novel finding will advance the field of bioremediation of selenium-contaminated sites and selenium bio-recovery and the production of potentially beneficial organic and inorganic reactive selenium species.

Optimization of Soil Washing to Reduce the Selenium Levels of Seleniferous Soil from Punjab, Northwestern India.

Seleniferous soil collected from the wheat ( L.)-grown agricultural land in Punjab, India, was characterized and the Se concentration in various soil fractions was determined by sequential extraction. The soil had a total Se content of 4.75 (±0.02) mg kg, of which 44% was observed in the oxidizable soil fraction. Soil flushing as an in situ technique was performed to simulate the Se migration pattern in case of rainfall or irrigation. Significant migration of Se from the upper layer to the lower layers was observed during water percolation through the soil column at a flow rate of 1 mL min, which could be attributed to Se reduction in the lower anoxic layers of the soil column. For ex situ treatment, the soil washing technique was optimized by varying different parameters such as treatment time, temperature, pH, liquid to solid (L:S) ratio, and presence of competing ions and oxidizing agents. Selenium extraction from soil was significantly improved by the presence of oxidizing agents in the washing solution: ∼38% Se was removed from the soil in the presence of 0.5% KMnO. In contrast, parameters such as treatment time, temperature, pH, L:S ratio, and competing ions did not significantly enhance the Se extraction efficiency. In this research, laboratory-scale in situ and ex situ treatment techniques for Se removal from soil were studied and optimized. The results provide an insight for large-scale Se removal and recovery from seleniferous soils.

Biological treatment of selenium-laden wastewater containing nitrate and sulfate in an upflow anaerobic sludge bed reactor at pH 5.0.

This study investigated the removal of selenate (SeO42-), sulfate (SO42-) and nitrate (NO3-) at different influent pH values ranging from 7.0 to 5.0 and 20 °C in an upflow anaerobic sludge blanket (UASB) reactor using lactate as an electron donor. At pH 5.0, the UASB reactor showed a 20-30% decrease in reactor performance compared to operation at pH 5.5 to 7.0, reaching removal efficiencies of 79%, 15%, 43% and 61% for NO3-, SO42-, Setotal and Sediss, respectively. However, the reactor stability was an issue upon lowering the pH to 5.0 and further experiments are recommended. The sludge formed during low pH operation had a fluffy, floc-like appearance with filamentous structure, possibly due to the low polysaccharide (PS) to protein (PN) ratio (0.01 PS/PN) in the soluble extracellular polymeric substances (EPS) matrix of the biomass. Scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM-EDX) analysis of the sludge confirmed Se oxyanion reduction and deposition of Se0 particles inside the biomass. Microbial community analysis using Illumina MiSeq sequencing revealed that the families of Campylobacteraceae and Desulfomicrobiaceae were the dominant phylotypes throughout the reactor operation at approximately 23% and 10% relative abundance, respectively. Furthermore, approximately 10% relative abundance of both Geobacteraceae and Spirochaetaceae was observed in the granular sludge during the pH 5.0 operation. Overall, this study demonstrated the feasibility of UASB operation at pH values ranging from 7.0 to 5.0 for removing Se and other oxyanions from wastewaters.

Formation of Se(0), Te(0), and Se(0)-Te(0) nanostructures during simultaneous bioreduction of selenite and tellurite in a UASB reactor.

Simultaneous removal of selenite and tellurite from synthetic wastewater was achieved through microbial reduction in a lab-scale upflow anaerobic sludge blanket reactor operated with 12 h hydraulic retention time at 30 °C and pH 7 for 120 days. Lactate was supplied as electron donor at an organic loading rate of 528 or 880 mg COD L-1 day-1. The reactor was initially fed with a synthetic influent containing 0.05 mM selenite and tellurite each (phase I, day 1-60) and subsequently with 0.1 mM selenite and tellurite each (phase II, day 61-120). At the end of phase I, selenite and tellurite removal efficiencies were 93 and 96%, respectively. The removal percentage dropped to 87 and 81% for selenite and tellurite, respectively, at the beginning of phase II because of the increased influent concentrations. The removal efficiencies of both selenite and tellurite were gradually restored within 20 days and stabilized at ≥ 97% towards the end of the experiment. Powder X-ray diffraction and Raman spectroscopy confirmed the formation of biogenic Se(0), Te(0), and Se(0)-Te(0) nanostructures. Scanning transmission electron microscopy coupled with energy-dispersive X-ray spectroscopy showed aggregates comprising of Se(0), Te(0), and Se-Te nanostructures embedded in a layer of extracellular polymeric substances (EPS). Infrared spectroscopy confirmed the presence of chemical signatures of the EPS which capped the nanoparticle aggregates that had been formed and immobilized in the granular sludge. This study suggests a model for technologies for remediation of effluents containing Se and Te oxyanions coupled with biorecovery of bimetal(loid) nanostructures.

Biotechnology in the management and resource recovery from metal bearing solid wastes: Recent advances.

Solid metalliferous wastes (sludges, dusts, residues, slags, red mud and tailing wastes) originating from ferrous and non-ferrous metallurgical industries are a serious environmental threat, when waste management practices are not properly followed. Metalliferous wastes generated by metallurgical industries are promising resources for biotechnological extraction of metals. These wastes still contain significant amounts of valuable non-ferrous metals, sometimes precious metals and also rare earth elements. Elemental composition and mineralogy of the metallurgical wastes is dependent on the nature of mining site and composition of primary ores mined. Most of the metalliferous wastes are oxidized in nature and contain less/no reduced sulfidic minerals (which can be quite well processed by biohydrometallurgy). However, application of biohydrometallurgy is more challenging while extracting metals from metallurgical wastes that contain oxide minerals. In this review, origin, elemental composition and mineralogy of the metallurgical solid wastes are presented. Various bio-hydrometallurgical processes that can be considered for the extraction of non-ferrous metals from metal bearing solid wastes are reviewed.

Environmental impact and bioremediation of seleniferous soils and sediments.

Selenium concentrations in the soil environment are directly linked to its transfer in the food chain, eventually causing either deficiency or toxicity associated with several physiological dysfunctions in animals and humans. Selenium bioavailability depends on its speciation in the soil environment, which is mainly influenced by the prevailing pH, redox potential, and organic matter content of the soil. The selenium cycle in the environment is primarily mediated through chemical and biological selenium transformations. Interactions of selenium with microorganisms and plants in the soil environment have been studied in order to understand the underlying interplay of selenium conversions and to develop environmental technologies for efficient bioremediation of seleniferous soils. In situ approaches such as phytoremediation, soil amendment with organic matter and biovolatilization are promising for remediation of seleniferous soils. Ex situ remediation of contaminated soils by soil washing with benign leaching agents is widely considered for removing heavy metal pollutants. However, it has not been applied until now for remediation of seleniferous soils. Washing of seleniferous soils with benign leaching agents and further treatment of Se-bearing leachates in bioreactors through microbial reduction will be advantageous as it is aimed at removal as well as recovery of selenium for potential re-use for agricultural and industrial applications. This review summarizes the impact of selenium deficiency and toxicity on ecosystems in selenium deficient and seleniferous regions across the globe, and recent research in the field of bioremediation of seleniferous soils.

Chlorination induced damage and recovery in marine diatoms: Assay by SYTOX® Green staining.

Phytoplankton entrained into cooling water systems of coastal power stations are subjected to acute chemical stress due to biocides (chlorine) used for biofouling control. They are subsequently released into the environment, where they may survive/recover or succumb. Experiments were conducted to evaluate the susceptibility of a centric (Chaetoceros lorenzianus) and pennate (Navicula sp.) diatom to in-plant administered concentrations of chlorine (0.2-0.5mg/L, TRO). Viability of cells exposed to chlorine was assessed by SYTOX® Green fluorimetry and was compared with other conventional end points like total cell counts, chlorophyll a content and cellular autofluorescence. Results showed a concentration-dependant reduction in viability, chlorophyll a and autofluorescence. C. lorenzianus cells were more susceptible to chlorine compared to Navicula sp. SYTOX® Green staining appears to be a sensitive method to assess chlorine-induced damages. The data show that in-use levels of chlorination can potentially impact entrained organisms; however, they can recover when returned to coastal waters.

Continuous removal and recovery of tellurium in an upflow anaerobic granular sludge bed reactor.

Continuous removal of tellurite (TeO32-) from synthetic wastewater and subsequent recovery in the form of elemental tellurium was studied in an upflow anaerobic granular sludge bed (UASB) reactor operated at 30°C. The UASB reactor was inoculated with anaerobic granular sludge and fed with lactate as carbon source and electron donor at an organic loading rate of 0.6g CODL-1d-1. After establishing efficient and stable COD removal, the reactor was fed with 10mg TeO32-L-1 for 42 d before increasing the influent concentration to 20mg TeO32-L-1. Tellurite removal (98 and 92%, respectively, from 10 and 20mg TeL-1) was primarily mediated through bioreduction and most of the removed Te was retained in the bioreactor. Characterization using XRD, Raman spectroscopy, SEM-EDX and TEM confirmed association of tellurium with the granular sludge, typically in the form of elemental Te(0) deposits. Furthermore, application of an extracellular polymeric substances (EPS) extraction method to the tellurite reducing sludge recovered up to 78% of the tellurium retained in the granular sludge. This study demonstrates for the first time the application of a UASB reactor for continuous tellurite removal from tellurite-containing wastewater coupled to elemental Te(0) recovery.

Selenium: environmental significance, pollution, and biological treatment technologies.

Selenium is an essential trace element needed for all living organisms. Despite its essentiality, selenium is a potential toxic element to natural ecosystems due to its bioaccumulation potential. Though selenium is found naturally in the earth's crust, especially in carbonate rocks and volcanic and sedimentary soils, about 40% of the selenium emissions to atmospheric and aquatic environments are caused by various industrial activities such as mining-related operations. In recent years, advances in water quality and pollution monitoring have shown that selenium is a contaminant of potential environmental concern. This has practical implications on industry to achieve the stringent selenium regulatory discharge limit of 5µgSeL(-1) for selenium containing wastewaters set by the United States Environmental Protection Agency. Over the last few decades, various technologies have been developed for the treatment of selenium-containing wastewaters. Biological selenium reduction has emerged as the leading technology for removing selenium from wastewaters since it offers a cheaper alternative compared to physico-chemical treatments and is suitable for treating dilute and variable selenium-laden wastewaters. Moreover, biological treatment has the advantage of forming elemental selenium nanospheres which exhibit unique optical and spectral properties for various industrial applications, i.e. medical, electrical, and manufacturing processes. However, despite the advances in biotechnology employing selenium reduction, there are still several challenges, particularly in achieving stringent discharge limits, the long-term stability of biogenic selenium and predicting the fate of bioreduced selenium in the environment. This review highlights the significance of selenium in the environment, health, and industry and biotechnological advances made in the treatment of selenium contaminated wastewaters. The challenges and future perspectives are overviewed considering recent biotechnological advances in the management of these selenium-laden wastewaters.

Selenium biomineralization for biotechnological applications.

Selenium (Se) is not only a strategic element in high-tech electronics and an essential trace element in living organisms, but also a potential toxin with low threshold concentrations. Environmental biotechnological applications using bacterial biomineralization have the potential not only to remove selenium from contaminated waters, but also to sequester it in a reusable form. Selenium biomineralization has been observed in phylogenetically diverse microorganisms isolated from pristine and contaminated environments, yet it is one of the most poorly understood biogeochemical processes. Microbial respiration of selenium is unique because the microbial cells are presented with both soluble (SeO(4)(2-) and SeO(3)(2-)) and insoluble (Se(0)) forms of selenium as terminal electron acceptor. Here, we highlight selenium biomineralization and the potential biotechnological uses for it in bioremediation and wastewater treatment.

Disruption of microbial biofilms by an extracellular protein isolated from epibiotic tropical marine strain of Bacillus licheniformis.

BACKGROUND: Marine epibiotic bacteria produce bioactive compounds effective against microbial biofilms. The study examines antibiofilm ability of a protein obtained from a tropical marine strain of Bacillus licheniformis D1. METHODOLOGY/PRINCIPAL FINDINGS: B. licheniformis strain D1 isolated from the surface of green mussel, Perna viridis showed antimicrobial activity against pathogenic Candida albicans BH, Pseudomonas aeruginosa PAO1 and biofouling Bacillus pumilus TiO1 cultures. The antimicrobial activity was lost after treatment with trypsin and proteinase K. The protein was purified by ultrafiltration and size-exclusion chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) analysis revealed the antimicrobial agent to be a 14 kDa protein designated as BL-DZ1. The protein was stable at 75°C for 30 min and over a pH range of 3.0 to 11.0. The sequence alignment of the MALDI-fingerprint showed homology with the NCBI entry for a hypothetical protein (BL00275) derived from B. licheniformis ATCC 14580 with the accession number gi52082584. The protein showed minimum inhibitory concentration (MIC) value of 1.6 µg/ml against C. albicans. Against both P. aeruginosa and B. pumilus the MIC was 3.12 µg/ml. The protein inhibited microbial growth, decreased biofilm formation and dispersed pre-formed biofilms of the representative cultures in polystyrene microtiter plates and on glass surfaces. CONCLUSION/SIGNIFICANCE: We isolated a protein from a tropical marine strain of B. licheniformis, assigned a function to the hypothetical protein entry in the NCBI database and described its application as a potential antibiofilm agent.

Disruption of Yarrowia lipolytica biofilms by rhamnolipid biosurfactant.

BACKGROUND: Yarrowia lipolytica is an ascomycetous dimorphic fungus that exhibits biofilm mode of growth. Earlier work has shown that biosurfactants such as rhamnolipids are efficient dispersants of bacterial biofilms. However, their effectiveness against fungal biofilms (particularly Y. lipolytica) has not been investigated. The aim of this study was to determine the effect of rhamnolipid on a biofilm forming strain of Y. lipolytica. Two chemical surfactants, cetyl-trimethyl ammonium bromide (CTAB) and sodium dodecyl sulphate (SDS) were used as controls for comparison. RESULTS: The methylene blue dye exclusion assay indicated an increase in fungal cell permeability after rhamnolipid treatment. Microtiter plate assay showed that the surfactant coating decreased Y. lipolytica biofilm formation by 50%. Rhamnolipid treatment disrupted pre-formed biofilms in a more effective manner than the other two surfactants. Confocal laser scanning microscopic studies showed that biofilm formation onto glass surfaces was decreased by 67% after sub-minimum inhibitory concentration (sub-MIC) treatment with rhamnolipids. The disruption of biofilms after rhamnolipid treatment was significant (P<0.05) when compared to SDS and CTAB. CONCLUSION: The results indicate a potential application of the biological surfactant to disrupt Y. lipolytica biofilms.

Formation of aerobic granules in the presence of a synthetic chelating agent.

This paper examines the development of aerobic granular sludge in the presence of a synthetic chelating agent, nitrilotriacetic acid (NTA), in sequencing batch reactors (SBR). The growth of seed sludge at 0.26 mM, 0.52 mM and 1.05 mM of NTA was found to be significantly lower as compared to that in the absence of NTA. Aerobic granulation was significantly enhanced in the three SBRs (R2, R3 and R4), which were fed with 0.26 mM, 0.52 mM and 1.05 mM of NTA as a co-substrate, in comparison to the acetate-alone fed SBR (R1). After 2 months of operation, the mean diameter of the biomass stabilized at 0.35 mm in R1 (acetate alone), as compared to 2.18 mm in R4 (1.05 mM NTA+acetate). NTA degradation was established in SBRs, with almost complete removal during the SBR cycle. Batch experiments also showed efficient degradation of NTA by the aerobic granules.

Bioaugmentation of aerobic microbial granules with Pseudomonas putida carrying TOL plasmid.

This paper describes results of a successful bioaugmentation experiment on aerobic granular sludge using Pseudomonas putida KT2442 cells bearing the TOL (pWWO) plasmid. The methodology was designed to monitor incorporation of the added donor cells into pre-existent microbial granules and the subsequent plasmid transfer to the autochthonous microbial community using shake flask microcosms. Expression of reporter proteins (GFP and DsRed) allowed in situ monitoring of donor cell attachment and plasmid transfer to the recipient cells using confocal laser scanning microscopy. Concomitant with donor integration and transconjugant proliferation in the granules, a significant increase in degradation of benzyl alcohol (used as sole substrate) was observed in the augmented microcosms. In contrast, control microcosms (with non-augmented granules) did not show any noticeable increase in the degradation of the substrate. This study shows that bioaugmentation of aerobic granular sludge via donor colonization and plasmid transfer is feasible for enhanced biodegradation.

Single cell level microalgal ecotoxicity assessment by confocal microscopy and digital image analysis.

In ecotoxicological studies involving environmental contaminants, rapid and multi-parametric optical detection based methods have definite advantages over traditional growth inhibition assays. In this context, a confocal laser scanning microscopy (CLSM) based method to assess ecotoxicity arising out of biocide insult to marine microalgae is reported. Using this technique, the effect of in-use concentrations of chlorine (an oxidizing biocide) on a marine diatom (Cocconeis scutellum Ehrenb) was determined based on inhibition of chlorophyll autofluorescence and esterase activity (probed by fluorescein diacetate (FDA) staining). Determination of mean fluorescence intensity (MFI) per cell by collecting auto-fluorescence from single cells in x, y and z dimensions permitted reproducible toxicity evaluation at single-cell level. Chlorine-induced inhibition of autofluorescence in laboratory cultures was dose-dependent. Additional data on metabolic activity of the diatom cells following chlorine exposure was collected by FDA staining. Our results demonstrate that chlorine, an antifouling biocide commonly used in cooling water systems, causes significant reduction in chlorophyll autofluorescence and esterase activity in diatoms in short-term exposure experiments. Tests employing multiple organisms and multiple toxicity endpoints are superior to standard algal growth inhibition assays for they provide a better understanding of algal-algal interactions and real impact in the environment. The combined autofluorescence-FDAtechnique described here is rapid and has clear advantages in terms of using environmentally relevant toxicant and cell concentrations. Additional microalgal species and toxicity end points can be employed in order to develop multi-species and multiparameter bioassay using confocal microscopy.