Wolfgang Sand

Sulfur Oxygenase Reductase (Sor) in the Moderately Thermoacidophilic Leaching Bacteria: Studies in Sulfobacillus thermosulfidooxidans and Acidithiobacillus caldus.

The sulfur oxygenase reductase (Sor) catalyzes the oxygen dependent disproportionation of elemental sulfur, producing sulfite, thiosulfate and sulfide. Being considered an “archaeal like” enzyme, it is also encoded in the genomes of some acidophilic leaching bacteria such as Acidithiobacillus caldus, Acidithiobacillus thiooxidans, Acidithiobacillus ferrivorans and Sulfobacillus thermosulfidooxidans, among others. We measured Sor activity in crude extracts from Sb. thermosulfidooxidans DSM 9293T. The optimum temperature for its oxygenase activity was achieved at 75 °C, confirming the “thermophilic” nature of this enzyme. Additionally, a search for genes probably involved in sulfur metabolism in the genome sequence of Sb. thermosulfidooxidans DSM 9293T was done. Interestingly, no sox genes were found. Two sor genes, a complete heterodisulfidereductase (hdr) gene cluster, three tetrathionate hydrolase (tth) genes, three sulfide quinonereductase (sqr), as well as the doxD component of a thiosulfate quinonereductase (tqo) were found. Seven At. caldus strains were tested for Sor activity, which was not detected in any of them. We provide evidence that an earlier reported Sor activity from At. caldus S1 and S2 strains most likely was due to the presence of a Sulfobacillus contaminant.

Mechanical and chemical studies on EPS from Sulfobacillus thermosulfidooxidans : from planktonic to biofilm cells

Bacteria attach to minerals and form biofilms, which can initiate and enhance bioleaching. Extracellular polymeric substances (EPS) play a crucial role during the whole process. Little is known how the cell surface/EPS mechanically and chemically respond to transformation from planktonic to biofilm cells. In this study the attachment and biofilm formation by Sulfobacillus thermosulfidooxidans were followed during pyrite leaching. Adhesiveness and stiffness of the cell/biofilm and the pyrite surface were checked by atomic force microscopy (AFM) in force mapping mode under real living conditions. The EPS components were analysed by colorimetry, fourier transform infrared spectroscopy and energy dispersive X-ray spectroscopy. The results indicate that slimy and soft EPS heterogeneously accumulated in the biofilms and on the surface of pyrite to induce bacterial adhesion and form robust biofilms. After attaching to the pyrite surface, the cells started to change the components of their EPS. Huge amounts of humic substances were detected in the biofilm EPS.

Bacterial and archaeal community distribution and stabilization of anaerobic sludge in a strengthen circulation anaerobic (SCA) reactor for municipal wastewater treatment

In this study, a SCA reactor was employed for municipal wastewater treatment at a mesophilic temperature (30°C) under different hydraulic retention times (HRT) and upflow velocities (Vup) to investigate granule sludge stability and spatial microbial distribution. The stable COD removal efficiency readied at HRT of 15, 12, 9 and 6h, and Vup ranging from 0.6 to 5.9mh-1. EPS fraction analysis of granule sludge shows that municipal wastewater was mainly attributed to the enrichment influence of polysaccharide and tightly bound-EPS. SEM images exhibited that the stability and floating of anaerobic granular sludge may be promoted in the primary three-phase separator area because the channels of the granules was clogged by EPS. The SMA and high-throughput sequencing analysis indicated acetoclastic methanogens and hydrogenotrophic methanogens played an important role in formation and maintenance of the anaerobic granule sludge in low and high organic load rate operation conditions.

Multi-omics reveal the lifestyle of the acidophilic, mineral-oxidizing model species Leptospirillum ferriphilumT.

ABSTRACT Leptospirillum ferriphilum plays a major role in acidic, metal-rich environments, where it represents one of the most prevalent iron oxidizers. These milieus include acid rock and mine drainage as well as biomining operations. Despite its perceived importance, no complete genome sequence of the type strain of this model species is available, limiting the possibilities to investigate the strategies and adaptations that Leptospirillum ferriphilum DSM 14647T (here referred to as Leptospirillum ferriphilumT) applies to survive and compete in its niche. This study presents a complete, circular genome of Leptospirillum ferriphilumT obtained by PacBio single-molecule real-time (SMRT) long-read sequencing for use as a high-quality reference. Analysis of the functionally annotated genome, mRNA transcripts, and protein concentrations revealed a previously undiscovered nitrogenase cluster for atmospheric nitrogen fixation and elucidated metabolic systems taking part in energy conservation, carbon fixation, pH homeostasis, heavy metal tolerance, the oxidative stress response, chemotaxis and motility, quorum sensing, and biofilm formation. Additionally, mRNA transcript counts and protein concentrations were compared between cells grown in continuous culture using ferrous iron as the substrate and those grown in bioleaching cultures containing chalcopyrite (CuFeS2). Adaptations of Leptospirillum ferriphilumT to growth on chalcopyrite included the possibly enhanced production of reducing power, reduced carbon dioxide fixation, as well as elevated levels of RNA transcripts and proteins involved in heavy metal resistance, with special emphasis on copper efflux systems. Finally, the expression and translation of genes responsible for chemotaxis and motility were enhanced. IMPORTANCE Leptospirillum ferriphilum is one of the most important iron oxidizers in the context of acidic and metal-rich environments during moderately thermophilic biomining. A high-quality circular genome of Leptospirillum ferriphilumT coupled with functional omics data provides new insights into its metabolic properties, such as the novel identification of genes for atmospheric nitrogen fixation, and represents an essential step for further accurate proteomic and transcriptomic investigation of this acidophile model species in the future. Additionally, light is shed on adaptation strategies of Leptospirillum ferriphilumT for growth on the copper mineral chalcopyrite. These data can be applied to deepen our understanding and optimization of bioleaching and biooxidation, techniques that present sustainable and environmentally friendly alternatives to many traditional methods for metal extraction.

Anaerobic biodegradation and decolorization of a refractory acid dye by a forward osmosis membrane bioreactor

In this study, the feasibility of utilizing an anaerobic osmotic membrane bioreactor (OMBR) for the treatment of a refractory acid dye, Lanaset red G.GR, is demonstrated. The experimental results show that an increased sludge concentration and reversed salt accumulation exacerbate membrane fouling, which leads to flux decline. The excellent rejection performance of the forward osmosis (FO) membrane and salt accumulation could lead to a reduction of microbial activity and an increase in soluble microbial product (SMP) and extracellular polymeric substance (EPS) contents. These consequences will affect the OMBR performance. Moreover, the FO membrane demonstrated a limited rejection of aniline-type intermediates. These overall findings suggest that the OMBR process is a good option for the treatment of dyeing wastewater. Further improvements on the membrane materials and membrane surface properties to alleviate fouling, salt reverse osmosis as well as the remaining color issues are still necessary before practical application becomes possible.

Investigation on adhesion of Sulfobacillus thermosulfidooxidans via atomic force microscopy equipped with mineral probes

Bacterial adhesion is a key step to prevent environmental problems called acid mine drainage or to improve leaching efficiency in industry, since it initiates and enhances bioleaching. Thus, to analyze bacterial adhesion and to understand this process is crucial. In this study atomic force microscopy equipped with a pyrite or chalcopyrite tip was applied to study the adhesion of Sulfobacillus thermosulfidooxidans. The results illustrate that planktonic cells of both pyrite- and sulfur-grown cells of S. thermosulfidooxidans show more affinity to pyrite than to chalcopyrite (adhesion forces 2u2009nN versus 0.13u2009nN). However, the interactions between bacteria and chalcopyrite can be strengthened, if the bacteria are brought into contact with the chalcopyrite. The biofilm cells show low affinity to either pyrite or chalcopyrite. A high content of proteins in the extracellular polymeric substances collected from planktonic cells of S. thermosulfidooxidans and a low content of proteins collected from biofilm EPS indicates that proteins play an important role in initial adhesion. Analysis of adhesion force-distance curves reveal that adhesion by pyrite-grown cells is a complex interaction involving several bonding forces.

Insights into the biology of acidophilic members of the Acidiferrobacteraceae family derived from comparative genomic analyses

The family Acidiferrobacteraceae (order Acidiferrobacterales) currently contains Gram negative, neutrophilic sulfur oxidizers such as Sulfuricaulis and Sulfurifustis, as well as acidophilic iron and sulfur oxidizers belonging to the Acidiferrobacter genus. The diversity and taxonomy of the genus Acidiferrobacter has remained poorly explored. Although several metagenome and bioleaching studies have identified its presence worldwide, only two strains, namely Acidiferrobacter thiooxydans DSM 2932T, and Acidiferrobacter spp. SP3/III have been isolated and made publically available. Using 16S rRNA sequence data publically available for the Acidiferrobacteraceae, we herein shed light into the molecular taxonomy of this family. Results obtained support the presence of three clades Acidiferrobacter, Sulfuricaulis and Sulfurifustis. Genomic analyses of the genome sequences of A. thiooxydansT and Acidiferrobacter spp. SP3/III indicate that ANI relatedness between the SPIII/3 strain and A. thiooxydansT is below 95-96%, supporting the classification of strain SP3/III as a new species within this genus. In addition, approximately 70% of Acidiferrobacter sp. SPIII/3 predicted genes have a conserved ortholog in A.xa0thiooxydans strains. A comparative analysis of iron, sulfur oxidation pathways, genome plasticity and cell-cell communication mechanisms of Acidiferrobacter spp. are also discussed.

Ligand-Free Nano-Au Catalysts on Nitrogen-Doped Graphene Filter for Continuous Flow Catalysis

In this study, the authors rationally designed a high-performance catalytic filter for continuous flow catalysis. The catalytic filter consisted of ligand-free nanoscale gold (nano-Au) catalysts and nitrogen-doped graphene (N-rGO). The Au catalyst was fabricated in situ onto a pre-formed N-rGO support by the NaBH4 reduction of the Au precursor, and the size of the nano-Au was fine-tuned. A hydrothermal pretreatment of graphene oxide enriched nitrogen-containing species on the surface of two-dimensional graphene supports and enhanced the affinity of Au precursors onto the support via electrocatalytic attraction. The nano-Au catalysts acted as high-performance catalysts, and the N-rGO acted as ideal filter materials to anchor the catalysts. The catalytic activity of the as-designed catalytic filter was evaluated using 4-nitrophenol (4-NP) hydrogenation as a model catalytic reaction. The catalytic filters demonstrated superior catalytic activity and excellent stability, where a complete 4-nitrophenol conversion was readily achieved via a single pass through the catalytic filter. The as-fabricated catalytic filter outperformed the conventional batch reactors due to evidently improved mass transport. Some key operational parameters impacting the catalytic performance were identified and optimized. A similar catalytic performance was also observed for three 4-nitrophenol spiked real water samples (e.g., surface water, tap water, and industrial dyeing wastewater). The excellent catalytic activity of the nano-Au catalysts combined with the two-dimensional and mechanically stable graphene allowed for the rational design of various continuous flow catalytic membranes for potential industrial applications.

Treatment of industrial dyeing wastewater with a pilot-scale strengthened circulation anaerobic reactor

We developed a pilot-scale strengthened circulation anaerobic (SCA) reactor (with an effective volume of 27u202fm3) and applied to the treatment of industrial textile wastewater. The treatment performance and the working mechanism were studied systematically and the key operational parameters were identified. The results demonstrated that a stable and excellent chemical oxygen demand removal efficiency of 62.7% and a maximum chromaticity removal efficiency of 73.5% were obtained at an optimal reflux ratio of 4. Interestingly, the bio-degradability was evidently improved after the SCA reactor treatment. The high throughput sequencing analysis indicated that the diversity of the bacteria or archaebacteria before the treatment was slightly higher than that after the treatment, which may be attributed to the production of certain toxic intermediates and/or characteristic pollutants during the treatment. Enzyme activity test and COD removal show that numerous microorganisms still maintained active in the anaerobic granular sludge even in a severe environment.

Granulation Process in an Expanded Granular Sludge Blanket (EGSB) Reactor for Domestic Sewage Treatment: Impact of Extracellular Polymeric Substances Compositions and Evolution of Microbial Population

In this study, an expanded granular sludge blanket (EGSB) reactor was used for the treatment of low-strength domestic sewage and the sludge granulation process was systematically investigated. At an optimized hydraulic retention time (HRT) of 5u202fh, up-flow velocity (Vup) of 1.9u202fm/h, and organic loading rate (OLR) of 2.16u202fkg COD/m3/d, the average COD removal efficiency was 71.5u202f±u202f2.3%. Completely granular sludge can be observed after 107 d of continuous operation. Analysis of the distribution and composition of the extracellular polymeric substances (EPS) indicates that the tightly bound EPS (TB-EPS) content shows an increasing trend, while the loosely bound EPS (LB-EPS) content did not significantly alter after the granular sludge was formed. The three-dimensional excitation-emission matrix technique (3D-EEM) confirms that aromatic protein-like substances are of key importance to sludge granulation. High-throughput sequencing analysis indicates that the metabolism shifted from hydrogenotrophic (Methanobaterium) to aceticlastic methanogens (Methanosaeta) during sludge granulation.