Allotropy of selenium nanoparticles: Colourful transition, synthesis, and biotechnological applications.

Elemental selenium (Se0 ) nanomaterials undergo allotropic transition from thermodynamically-unstable to more stable phases. This process is significantly different when Se0 nanoparticles (NPs) are produced via physico-chemical and biological pathways. While the allotropic transition of physico-chemically synthesized Se0 is fast (minutes to hours), the biogenic Se0 takes months to complete. The biopolymer layer covering biogenic Se0 NPs might be the main factor controlling this retardation, but this still remains an open question. Phylogenetically-diverse bacteria reduce selenium oxyanions to red amorphous Se0 allotrope, which has low market value. Then, red Se0 undergoes allotropic transition to trigonal (metallic grey) allotrope, the end product having important industrial applications (e.g. semiconductors, alloys). Is it not yet clear whether biogenic Se0 presents any biological function, or it is mainly a detoxification and respiratory by-product. The better understanding of this transition would benefit the recovery of Se0 NPs from secondary resources and its targeted utilization with respect to each allotropic stage. This review article presents and critically discusses the main physico-chemical methods and biosynthetic pathways of Se0 (bio)mineralization. In addition, the article proposes a conceptual model for the resource recovery potential of trigonal selenium nanomaterials in the context of circular economy.

Genomic insight into iron acquisition by sulfate-reducing bacteria in microaerophilic environments.

Historically, sulfate-reducing bacteria (SRB) have been considered to be strict anaerobes, but reports in the past couple of decades indicate that SRB tolerate exposure to O2 and can even grow in aerophilic environments. With the transition from anaerobic to microaerophilic conditions, the uptake of Fe(III) from the environment by SRB would become important. In evaluating the metabolic capability for the uptake of iron, the genomes of 26 SRB, representing eight families, were examined. All SRB reviewed carry genes (feoA and feoB) for the ferrous uptake system to transport Fe(II) across the plasma membrane into the cytoplasm. In addition, all of the SRB genomes examined have putative genes for a canonical ABC transporter that may transport ferric siderophore or ferric chelated species from the environment. Gram-negative SRB have additional machinery to import ferric siderophores and ferric chelated species since they have the TonB system that can work alongside any of the outer membrane porins annotated in the genome. Included in this review is the discussion that SRB may use the putative siderophore uptake system to import metals other than iron.

Interplay between arsenic and selenium biomineralization in Shewanella sp. O23S.

Bacteria play crucial roles in the biogeochemical cycle of arsenic (As) and selenium (Se) as these elements are metabolized via detoxification, energy generation (anaerobic respiration) and biosynthesis (e.g. selenocysteine) strategies. To date, arsenic and selenium biomineralization in bacteria were studied separately. In this study, the anaerobic metabolism of As and Se in Shewanella sp. O23S was investigated separately and mixed, with an emphasis put on the biomineralization products of this process. Multiple analytical techniques including ICP-MS, TEM-EDS, XRD, Micro-Raman, spectrophotometry and surface charge (zeta potential) were employed. Shewanella sp. O23S is capable of reducing selenate (SeO42-) and selenite (SeO32-) to red Se(-S)0, and arsenate (AsO43-) to arsenite (AsO33-). The release of H2S from cysteine led to the precipitation of AsS minerals: nanorod AsS and granular As2S3. When As and Se oxyanions were mixed, both As-S and Se(-S)0 biominerals were synthesized. All biominerals were extracellular, amorphous and presented a negative surface charge (-24 to -38 mV). Kinetic analysis indicated the following reduction yields: SeO32- (90%), AsO43- (60%), and SeO42- (<10%). The mix of SeO32- with AsO43- led to a decrease in As removal to 30%, while Se reduction yield was unaffected (88%). Interestingly, SeO42- incubated with AsO43- boosted the Se removal (71%). The exclusive extracellular formation of As and Se biominerals might indicate an extracellular respiratory process characteristic of various Shewanella species and strains. This is the first study documenting a complex interplay between As and Se oxyanions: selenite decreased arsenate reduction, whereas arsenate stimulated selenate reduction. Further investigation needs to clarify whether Shewanella sp. O23S employs multi-substrate respiratory enzymes or separate, high affinity enzymes for As and Se oxyanion respiration.

Selenium respiration in anaerobic bacteria: Does energy generation pay off?

Selenium (Se) respiration in bacteria was revealed for the first time at the end of 1980s. Although thermodynamically-favorable, energy-dense and documented in phylogenetically-diverse bacteria, this metabolic process appears to be accompanied by a number of challenges and numerous unanswered questions. Selenium oxyanions, SeO42- and SeO32-, are reduced to elemental Se (Se0) through anaerobic respiration, the end product being solid and displaying a considerable size (up to 500 nm) at the bacterial scale. Compared to other electron acceptors used in anaerobic respiration (e.g. N, S, Fe, Mn, and As), Se is one of the few elements whose end product is solid. Furthermore, unlike other known bacterial intracellular accumulations such as volutin (inorganic polyphosphate), S0, glycogen or magnetite, Se0 has not been shown to play a nutritional or ecological role for its host. In the context of anaerobic respiration of Se oxyanions, biogenic Se0 appears to be a by-product, a waste that needs proper handling, and this raises the question of the evolutionary implications of this process. Why would bacteria use a respiratory substrate that is useful, in the first place, and then highly detrimental? Interestingly, in certain artificial ecosystems (e.g. upflow bioreactors) Se0 might help bacterial cells to increase their density and buoyancy and thus avoid biomass wash-out, ensuring survival. This review article provides an in-depth analysis of selenium respiration (model selenium respiring bacteria, thermodynamics, respiratory enzymes, and genetic determinants), complemented by an extensive discussion about the evolutionary implications and the properties of biogenic Se0 using published and original/unpublished results.

PbS biomineralization using cysteine: Bacillus cereus and the sulfur rush.

Bacillus sp. Abq, belonging to Bacillus cereus sensu lato, was isolated from an aquifer in New Mexico, USA and phylogenetically classified. The isolate possesses the unusual property of precipitating Pb(II) by using cysteine, which is degraded intracellularly to hydrogen sulfide (H2S). H2S is then exported to the extracellular environment to react with Pb(II), yielding PbS (galena). Biochemical and growth tests showed that other sulfur sources tested (sulfate, thiosulfate, and methionine) were not reduced to hydrogen sulfide. Using equimolar concentration of cysteine, 1 mM of soluble Pb(II) was removed from Lysogeny Broth (LB) medium within 120 h of aerobic incubation forming black, solid PbS, with a removal rate of 2.03 µg L-1 h-1 (∼8.7 µM L-1 h-1). The mineralogy of biogenic PbS was characterized and confirmed by XRD, HRTEM and EDX. Electron microscopy and electron diffraction identified crystalline PbS nanoparticles with a diameter <10 nm,  localized in the extracellular matrix and on the surface of the cells. This is the first study demonstrating the use of cysteine in Pb(II) precipitation as insoluble PbS and it may pave the way to PbS recovery from secondary resources, such as Pb-laden industrial effluents.

Desulfurization: Critical step towards enhanced selenium removal from industrial effluents.

Selenium (Se) removal from synthetic solutions and from real Flue Gas Desulfurization (FGD) wastewater generated by a coal-fired power plant was studied for the first time using a commercial iron oxide impregnated strong base anion exchange resin, Purolite® FerrIX A33E. In synthetic solutions, the resin showed high affinity for selenate and selenite, while sulfate exhibited a strong competition for both oxyanions. The FGD wastewater investigated is a complex system that contains Se (∼1200 µg L-1), SO42- (∼1.1 g L-1), Cl- (∼9.5 g L-1), and Ca2+ (∼5 g L-1), alongside a broad spectrum of toxic trace metals including Cd, Cr, Hg, Ni, and Zn. The resin performed poorly against Se in the raw FGD wastewater and showed moderate to good removal of several trace elements such as Cd, Cr, Hg, and Zn. In FGD effluent, sulfate was identified as a powerful competing anion for Se, having high affinity for the exchange active sites of the resin. The desulfurization of the FGD effluent using BaCl2 led to the increase in Se removal from 3% (non-desulfurized effluent) to 80% (desulfurized effluent) by combined precipitation and ion exchange treatment. However, complete desulfurization using equimolar BaCl2 could not be achieved due to the presence of bicarbonate that acts as a sulfate competitor for barium. In addition to selenium and sulfate removal, several toxic metals were efficiently removed (Cd: 91%; Cr: 100%; Zn: 99%) by the combined (desulfurization and ion exchange) treatment.

Progress toward clonable inorganic nanoparticles.

Pseudomonas moraviensis stanleyae was recently isolated from the roots of the selenium (Se) hyperaccumulator plant Stanleya pinnata. This bacterium tolerates normally lethal concentrations of SeO3(2-) in liquid culture, where it also produces Se nanoparticles. Structure and cellular ultrastructure of the Se nanoparticles as determined by cellular electron tomography shows the nanoparticles as intracellular, of narrow dispersity, symmetrically irregular and without any observable membrane or structured protein shell. Protein mass spectrometry of a fractionated soluble cytosolic material with selenite reducing capability identified nitrite reductase and glutathione reductase homologues as NADPH dependent candidate enzymes for the reduction of selenite to zerovalent Se nanoparticles. In vitro experiments with commercially sourced glutathione reductase revealed that the enzyme can reduce SeO3(2-) (selenite) to Se nanoparticles in an NADPH-dependent process. The disappearance of the enzyme as determined by protein assay during nanoparticle formation suggests that glutathione reductase is associated with or possibly entombed in the nanoparticles whose formation it catalyzes. Chemically dissolving the nanoparticles releases the enzyme. The size of the nanoparticles varies with SeO3(2-) concentration, varying in size form 5 nm diameter when formed at 1.0 µM [SeO3(2-)] to 50 nm maximum diameter when formed at 100 µM [SeO3(2-)]. In aggregate, we suggest that glutathione reductase possesses the key attributes of a clonable nanoparticle system: ion reduction, nanoparticle retention and size control of the nanoparticle at the enzyme site.

Selenium hyperaccumulators harbor a diverse endophytic bacterial community characterized by high selenium resistance and plant growth promoting properties.

Selenium (Se)-rich plants may be used to provide dietary Se to humans and livestock, and also to clean up Se-polluted soils or waters. This study focused on endophytic bacteria of plants that hyperaccumulate selenium (Se) to 0.5-1% of dry weight. Terminal restriction fragment length polymorphism (T-RFLP) analysis was used to compare the diversity of endophytic bacteria of hyperaccumulators Stanleya pinnata (Brassicaceae) and Astragalus bisulcatus (Fabaceae) with those from related non-accumulators Physaria bellii (Brassicaceae) and Medicago sativa (Fabaceae) collected on the same, seleniferous site. Hyperaccumulators and non-accumulators showed equal T-RF diversity. Parsimony analysis showed that T-RFs from individuals of the same species were more similar to each other than to those from other species, regardless of plant Se content or spatial proximity. Cultivable endophytes from hyperaccumulators S. pinnata and A. bisulcatus were further identified and characterized. The 66 bacterial morphotypes were shown by MS MALDI-TOF Biotyper analysis and 16S rRNA gene sequencing to include strains of Bacillus, Pseudomonas, Pantoea, Staphylococcus, Paenibacillus, Advenella, Arthrobacter, and Variovorax. Most isolates were highly resistant to selenate and selenite (up to 200 mM) and all could reduce selenite to red elemental Se, reduce nitrite and produce siderophores. Seven isolates were selected for plant inoculation and found to have plant growth promoting properties, both in pure culture and when co-cultivated with crop species Brassica juncea (Brassicaceae) or M. sativa. There were no effects on plant Se accumulation. We conclude that Se hyperaccumulators harbor an endophytic bacterial community in their natural seleniferous habitat that is equally diverse to that of comparable non-accumulators. The hyperaccumulator endophytes are characterized by high Se resistance, capacity to produce elemental Se and plant growth promoting properties.

Removal of colloidal biogenic selenium from wastewater.

Biogenic selenium, Se(0), has colloidal properties and thus poses solid-liquid separation problems, such as poor settling and membrane fouling. The separation of Se(0) from the bulk liquid was assessed by centrifugation, filtration, and coagulation-flocculation. Se(0) particles produced by an anaerobic granular sludge are normally distributed, ranging from 50 nm to 250 nm, with an average size of 166±29 nm and a polydispersity index of 0.18. Due to its nanosize range and protein coating-associated negative zeta potential (-15 mV to -23 mV) between pH 2 and 12, biogenic Se(0) exhibits colloidal properties, hampering its removal from suspension. Centrifugation at different centrifugal speeds achieved 22±3% (1500 rpm), 73±2% (3000 rpm) and 91±2% (4500 rpm) removal. Separation by filtration through 0.45 µm filters resulted in 87±1% Se(0) removal. Ferric chloride and aluminum sulfate were used as coagulants in coagulation-flocculation experiments. Aluminum sulfate achieved the highest turbidity removal (92±2%) at a dose of 10(-3) M, whereas ferric chloride achieved a maximum turbidity removal efficiency of only 43±4% at 2.7×10(-4) M. Charge repression plays a minor role in particle neutralization. The sediment volume resulting from Al2(SO3)4 treatment is three times larger than that produced by FeCl3.

Electrocoagulation of colloidal biogenic selenium.

Colloidal elemental selenium (Se(0)) adversely affects membrane separation processes and aquatic ecosystems. As a solution to this problem, we investigated for the first time the removal potential of Se(0) by electrocoagulation process. Colloidal Se(0) was produced by a strain of Pseudomonas fluorescens and showed limited gravitational settling. Therefore, iron (Fe) and aluminum (Al) sacrificial electrodes were used in a batch reactor under galvanostatic conditions. The best Se(0) turbidity removal (97 %) was achieved using iron electrodes at 200 mA. Aluminum electrodes removed 96 % of colloidal Se(0) only at a higher current intensity (300 mA). At the best Se(0) removal efficiency, electrocoagulation using Fe electrode removed 93 % of the Se concentration, whereas with Al electrodes the Se removal efficiency reached only 54 %. Due to the less compact nature of the Al flocs, the Se-Al sediment was three times more voluminous than the Se-Fe sediment. The toxicity characteristic leaching procedure (TCLP) test showed that the Fe-Se sediment released Se below the regulatory level (1 mg L(-1)), whereas the Se concentration leached from the Al-Se sediment exceeded the limit by about 20 times. This might be related to the mineralogical nature of the sediments. Electron scanning micrographs showed Fe-Se sediments with a reticular structure, whereas the Al-Se sediments lacked an organized structure. Overall, the results obtained showed that the use of Fe electrodes as soluble anode in electrocoagulation constitutes a better option than Al electrodes for the electrochemical sedimentation of colloidal Se(0).

Sensitive, selective analysis of selenium oxoanions using microchip electrophoresis with contact conductivity detection.

The common selenium oxoanions selenite (SeO3(2-)) and selenate (SeO4(2-)) are toxic at intake levels slightly below 1 mg day(-1). These anions are currently monitored by a variety of traditional analytical techniques that are time-consuming, expensive, require large sample volumes, and/or lack portability. To address the need for a fast and inexpensive analysis of selenium oxoanions, we present the first microchip capillary zone electrophoresis (MCE) separation targeting these species in the presence of chloride, sulfate, nitrate, nitrite, chlorate, sulfamate, methanesulfonate, and fluoride, which can be simultaneously monitored. The chemistry was designed to give high selectivity in nonideal matrices. Interference from common weak acids is avoided by operating near pH 4. Separation resolution from chloride was enhanced to improve tolerance of high-salinity matrices. As a result, selenate can be quantified in the presence of up to 1.5 mM NaCl, and selenite analysis is even more robust against chloride. Using contact conductivity detection, detection limits for samples with conductivity equal to the background electrolyte are 53 nM (4.2 ppb Se) and 380 nM (30 ppb) for selenate and selenite, respectively. Analysis time, including injection, is ∼2 min. The MCE method was validated against ion chromatography (IC) using spiked samples of dilute BBL broth and slightly outperformed the IC in accuracy while requiring <10% of the analysis time. The applicability of the technique to real samples was shown by monitoring the consumption of selenite by bacteria incubated in LB broth.