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Growth media have been developed to facilitate the enrichment and isolation of acidophilic and acid-tolerant sulfate-reducing bacteria (aSRB) from environmental and industrial samples, and to allow their cultivation in vitro. The main features of the ‘standard’ solid and liquid devised media are as follows: (i) use of glycerol rather than an aliphatic acid as electron donor; (ii) inclusion of stoichiometric concentrations of zinc ions to both buffer pH and to convert potentially harmful hydrogen sulphide produced by the aSRB to insoluble zinc sulphide; (iii) inclusion of Acidocella aromatica (an heterotrophic acidophile that does not metabolize glycerol or yeast extract) in the gel underlayer of double layered (overlay) solid media, to remove acetic acid produced by aSRB that incompletely oxidize glycerol and also aliphatic acids (mostly pyruvic) released by acid hydrolysis of the gelling agent used (agarose). Colonies of aSRB are readily distinguished from those of other anaerobes due to their deposition and accumulation of metal sulphide precipitates. Data presented illustrate the effectiveness of the overlay solid media described for isolating aSRB from acidic anaerobic sediments and low pH sulfidogenic bioreactors. The paper describes how bacteria that live in acidic environments, and that form hydrogen sulphide from sulfate, may be isolated and grown in the laboratory. Graphical Abstract Figure. The paper describes how bacteria that live in acidic environments, and that form hydrogen sulphide from sulfate, may be isolated and grown in the laboratory.

Summary Two continuous‐flow bench‐scale bioreactor systems populated by mixed communities of acidophilic sulfate‐reducing bacteria were constructed and tested for their abilities to promote the selective precipitation of transition metals (as sulfides) present in synthetic mine waters, using glycerol as electron donor. The objective with the first system (selective precipitation of copper from acidic mine water containing a variety of soluble metals) was achieved by maintaining a bioreactor pH of ∼2.2–2.5. The second system was fed with acidic (pH 2.5) synthetic mine water containing 3 mM of both zinc and ferrous iron, and varying concentrations (0.5–30 mM) of aluminium. Selective precipitation of zinc sulfide was possible by operating the bioreactor at pH 4.0 and supplementing the synthetic mine water with 4 mM glycerol. Analysis of the microbial populations in the bioreactors showed that they changed with varying operational parameters, and novel acidophilic bacteria (including one sulfidogen) were isolated from the bioreactors. The acidophilic sulfidogenic bioreactors provided ‘proof of principle’ that segregation of metals present in mine waters is possible using simple online systems within which controlled pH conditions are maintained. The modular units are versatile and robust, and involve minimum engineering complexity.

Three strains of sulfate-reducing bacteria (M1 T , D, and E) were isolated from acidic sediments (White river and Tinto river) and characterized phylogenetically and physiologically. All three strains were obligately anaerobic, mesophilic, spore-forming straight rods, stained Gram-negative and displayed variable motility during active growth. The pH range for growth was 3.8–7.0, with an optimum at pH 5.5. The temperature range for growth was 15–40 °C, with an optimum at 30 °C. Strains M1 T , D, and E used a wide range of electron donors and acceptors, with certain variability within the different strains. The nominated type strain (M1 T ) used ferric iron, nitrate, sulfate, elemental sulfur, and thiosulfate (but not arsenate, sulfite, or fumarate) as electron acceptors, and organic acids (formate, lactate, butyrate, fumarate, malate, and pyruvate), alcohols (glycerol, methanol, and ethanol), yeast extract, and sugars (xylose, glucose, and fructose) as electron donors. It also fermented some substrates such as pyruvate and formate. Strain M1 T tolerated up to 50 mM ferrous iron and 10 mM aluminum, but was inhibited by 1 mM copper. On the basis of phenotypic, phylogenetic, and genetic characteristics, strains M1 T , D, and E represent a novel species within the genus Desulfosporosinus , for which the name Desulfosporosinus acididurans sp. nov. is proposed. The type strain is M1 T (=DSM 27692 T  = JCM 19471 T ). Strain M1 T was the first acidophilic SRB isolated, and it is the third described species of acidophilic SRB besides Desulfosporosinus acidiphilus and Thermodesulfobium narugense .

DNA vaccination has emerged as a promising tool against infectious diseases of farmed fish. Oral delivery allows stress-free administration that is ideal for mass immunization and of paramount importance for infectious pancreatic necrosis (IPN) and other viral disease that affect young salmonids and cause economic losses in aquaculture worldwide. We describe the development and in vivo assessment of an “in-feed” formulation strategy for oral immunization with liposomal DNA vaccines, by delivering a vaccine construct coding for an immunogenic region of the VP2 capsid protein. A challenge against IPNV was carried out to determine the vaccine efficacy, by comparing the mortality of pre-smolt Atlantic salmons immunized and non-immunized with the oral vaccine. The antibody response (ELISA) and hematological parameters after immunization were examined, as well as the vaccine effect on the growth and internal structures of fry salmons (histological analysis). The vaccine distribution in the experimental tank after oral administration was investigated by HPLC and PCR amplification. The oral vaccine induced detectable levels of VP2-specific antibodies and conferred significant protection following IPNV challenge, with relative percent survivals (RPS) of 58.2%, for single dose (1mgpDNA/kgfish⋅d), and 66% for double dose (2mgpDNA/kgfish⋅d). We further provide evidence in favour of the vaccine safety to fish and demonstrated absence of pDNA in the tank water, but presence of vaccine residues in faeces and unconsumed feed sediments (solid wastes). The delivery platform for liposomal DNA vaccination via feed was successfully proved against IPNV in Atlantic salmon, showing the oral vaccine to be immunogenic and safe for fish, and providing significant protection after oral administration. The “in-feed” technology for oral DNA vaccination holds potential to be applied against IPNV and other pathogens that currently threaten the aquaculture worldwide.

An oxidized lateritic ore which contained 0.8 % (by weight) copper was bioleached in pH- and temperature-controlled stirred reactors under acidic reducing conditions using pure and mixed cultures of the acidophilic chemolithotrophic bacterium Acidithiobacillus ferrooxidans. Sulfur was provided as the electron donor for the bacteria, and ferric iron present in goethite (the major ferric iron mineral present in the ore) acted as electron acceptor. Significantly more copper was leached by bacterially catalysed reductive dissolution of the laterite than in aerobic cultures or in sterile anoxic reactors, with up to 78 % of the copper present in the ore being extracted. This included copper that was leached from acid-labile minerals (chiefly copper silicates) and that which was associated with ferric iron minerals in the lateritic ore. In the anaerobic bioreactors, soluble iron in the leach liquors was present as iron (II) and copper as copper (I), but both metals were rapidly oxidized (to iron (III) and copper (II)) when the reactors were aerated. The number of bacteria added to the reactors had a critical role in dictating the rate and yield of copper solubilised from the ore. This work has provided further evidence that reductive bioprocessing, a recently described approach for extracting base metals from oxidized deposits, has the potential to greatly extend the range of metal ores that can be biomined.

It is anticipated that copper mining output will significantly increase over the next 20 years because of the more intensive use of copper in electricity-related technologies such as for transport and clean power generation, leading to a significant increase in the impacts on water resources if stricter regulations and as a result cleaner mining and processing technologies are not implemented. A key concern of discarded copper production process water is sulfate. In this study we aim to transform sulfate into sulfur in real mining process water. For that, we operate a sequential 2-step membrane biofilm reactor (MBfR) system. We coupled a hydrogenotrophic MBfR (H 2 -MBfR) for sulfate reduction to an oxidizing MBfR (O 2 -MBfR) for oxidation of sulfide to elemental sulfur. A key process improvement of the H 2 -MBfR was online pH control, which led to stable high-rate sulfate removal not limited by biomass accumulation and with H 2 supply that was on demand. The H 2 -MBfR easily adapted to increasing sulfate loads, but the O 2 -MBfR was difficult to adjust to the varying H 2 -MBfR outputs, requiring better coupling control. The H 2 -MBfR achieved high average volumetric sulfate reduction performances of 1.7–3.74 g S/m 3 -d at 92–97% efficiencies, comparable to current high-rate technologies, but without requiring gas recycling and recompression and by minimizing the H 2 off-gassing risk. On the other hand, the O 2 -MBfR reached average volumetric sulfur production rates of 0.7–2.66 g S/m 3 -d at efficiencies of 48–78%. The O 2 -MBfR needs further optimization by automatizing the gas feed, evaluating the controlled removal of excess biomass and S 0 particles accumulating in the biofilm, and achieving better coupling control between both reactors. Finally, an economic/sustainability evaluation shows that MBfR technology can benefit from the green production of H 2 and O 2 at operating costs which compare favorably with membrane filtration, without generating residual streams, and with the recovery of valuable elemental sulfur.

Nitrate, a major groundwater pollutant from anthropogenic activities, poses serious health risks when present in drinking water. Denitrification using bio-electrochemical reactors (BER) offers an innovative technology, eco-friendly solution for nitrate removal from groundwater. BER use electroactive bacteria to reduce inorganic compounds like nitrate and bicarbonate by transferring electrons directly from the cathode. In our work, two batch BER were implemented at 1V and 2V, using anaerobic digestate from a full-scale wastewater treatment plant as inoculum. Nitrate, nitrite, sulfate, total ammoniacal nitrogen, and 16S rRNA analysis of bacterial community, were monitored during BER operation. The results showed effective nitrate removal in all BERs, with denitrification rate at 1V and 2V higher than the Control system, where endogenous respiration drove the process. At 1V, complete nitrate conversion to N 2 occurred in 4 days, while at 2V, it took 14 days. The slower rate at 2V was likely due to O 2 production from water electrolysis, which competed with nitrate as final electron acceptor. Bacterial community analysis confirmed the electroactive bacteria selection like the genus Desulfosporosinus and Leptolinea , confirming electrons transfer without an electroactive biofilm. Besides, Hydrogenophaga was enhanced at 2V likely due to electrolytically produced H 2 . Sulfate was not reduced, and total ammoniacal nitrogen remained constant indicating no dissimilatory nitrite reduction of ammonia. These results provide a significant contribution to the scaling up of electro-assisted autotrophic denitrification and its application in groundwater remediation, utilizing a simple reactor configuration-a single-chamber, membrane-free design- and a conventional power source instead of a potentiostat.

Reactive pyritic mine tailings can be populated by chemolithotrophic prokaryotes that enhance the solubilities of many metals, though iron-reducing heterotrophic microorganisms can inhibit the environmental risk posed by tailings by promoting processes that are the reverse of those carried out by pyrite-oxidising autotrophic bacteria. A strain (IT2) of Curtobacterium ammoniigenes, a bacterium not previously identified as being associated with acidic mine wastes, was isolated from pyritic mine tailings and partially characterized. Strain IT2 was able to reduce ferric iron under anaerobic conditions, but was not found to catalyse the oxidation of ferrous iron or elemental (zero-valent) sulfur, and was an obligate heterotrophic. It metabolized monosaccharides and required small amounts of yeast extract for growth. Isolate IT2 is a mesophilic bacterium, with a temperature growth optimum of 30 °C and is moderately acidophilic, growing optimally at pH 4.0 and between pH 2.7 and 5.0. The isolate tolerated elevated concentrations of many transition metals, and was able to grow in the cell-free spent medium of the acidophilic autotroph Acidithiobacillus ferrooxidans, supporting the hypothesis that it can proliferate in acidic mine tailings. Its potential role in mitigating the production of acidic, metal-rich drainage waters from mine wastes is discussed.