Syngas as Electron Donor for Sulfate and Thiosulfate Reducing Haloalkaliphilic Microorganisms in a Gas-Lift Bioreactor.

Biodesulfurization processes remove toxic and corrosive hydrogen sulfide from gas streams (e.g., natural gas, biogas, or syngas). To improve the efficiency of these processes under haloalkaline conditions, a sulfate and thiosulfate reduction step can be included. The use of H2/CO mixtures (as in syngas) instead of pure H2 was tested to investigate the potential cost reduction of the electron donor required. Syngas is produced in the gas-reforming process and consists mainly of H2, carbon monoxide (CO), and carbon dioxide (CO2). Purification of syngas to obtain pure H2 implies higher costs because of additional post-treatment. Therefore, the use of syngas has merit in the biodesulfurization process. Initially, CO inhibited hydrogen-dependent sulfate reduction. However, after 30 days the biomass was adapted and both H2 and CO were used as electron donors. First, formate was produced, followed by sulfate and thiosulfate reduction, and later in the reactor run acetate and methane were detected. Sulfide production rates with sulfate and thiosulfate after adaptation were comparable with previously described rates with only hydrogen. The addition of CO marginally affected the microbial community in which Tindallia sp. was dominant. Over time, acetate production increased and acetogenesis became the dominant process in the bioreactor. Around 50% of H2/CO was converted to acetate. Acetate supported biomass growth and higher biomass concentrations were reached compared to bioreactors without CO feed. Finally, CO addition resulted in the formation of small, compact microbial aggregates. This suggests that CO or syngas can be used to stimulate aggregation in haloalkaline biodesulfurization systems.

Immobilization of sulfate and thiosulfate-reducing biomass on sand under haloalkaline conditions.

Biological sulfate and thiosulfate reduction under haloalkaline conditions can be applied to treat waste streams from biodesulfurization systems. However, the lack of microbial aggregation under haloalkaline conditions limits the volumetric rates of sulfate and thiosulfate reducing bioreactors. As biomass retention in haloalkaline bioreactors has not been studied before, sand was chosen as a biomass carrier material to increase cell retention and consequently raise the volumetric rates. The results showed that ~10 fold higher biomass concentrations could be achieved with sand, compared to previous studies without carrier addition. The volumetric rates of sulfate/thiosulfate reduction increased approximately 4.5 times. Biomass attachment to the sand was restricted to cavities within the sand particles. Acetate produced by acetogenic bacteria from H2 and CO2 was used as carbon source for biomass growth, while formate that was also produced from H2 and CO2 enhanced sulfate reduction. The microbial community composition was analyzed by 16S rRNA gene amplicon sequencing, and Tindallia related bacteria were probably responsible for formate formation from hydrogen. The community attached to the sand particles was similar to the suspended fraction, but the relative abundance of sequences most closely related to Desulfohalobiaceae was much higher in the attached fraction compared to the suspended fraction (30% and 13%, respectively). The results indicated that even though the biomass attachment to sand was poor, it still increased the biomass concentration and consequently the sulfate and thiosulfate reduction volumetric rates.

Systems biology of acidophile biofilms for efficient metal extraction.

Society's demand for metals is ever increasing while stocks of high-grade minerals are being depleted. Biomining, for example of chalcopyrite for copper recovery, is a more sustainable biotechnological process that exploits the capacity of acidophilic microbes to catalyze solid metal sulfide dissolution to soluble metal sulfates. A key early stage in biomining is cell attachment and biofilm formation on the mineral surface that results in elevated mineral oxidation rates. Industrial biomining of chalcopyrite is typically carried out in large scale heaps that suffer from the downsides of slow and poor metal recoveries. In an effort to mitigate these drawbacks, this study investigated planktonic and biofilm cells of acidophilic (optimal growth pH < 3) biomining bacteria. RNA and proteins were extracted, and high throughput "omics" performed from a total of 80 biomining experiments. In addition, micrographs of biofilm formation on the chalcopyrite mineral surface over time were generated from eight separate experiments. The dataset generated in this project will be of great use to microbiologists, biotechnologists, and industrial researchers.

Reverse engineering directed gene regulatory networks from transcriptomics and proteomics data of biomining bacterial communities with approximate Bayesian computation and steady-state signalling simulations.

BACKGROUND: Network inference is an important aim of systems biology. It enables the transformation of OMICs datasets into biological knowledge. It consists of reverse engineering gene regulatory networks from OMICs data, such as RNAseq or mass spectrometry-based proteomics data, through computational methods. This approach allows to identify signalling pathways involved in specific biological functions. The ability to infer causality in gene regulatory networks, in addition to correlation, is crucial for several modelling approaches and allows targeted control in biotechnology applications. METHODS: We performed simulations according to the approximate Bayesian computation method, where the core model consisted of a steady-state simulation algorithm used to study gene regulatory networks in systems for which a limited level of details is available. The simulations outcome was compared to experimentally measured transcriptomics and proteomics data through approximate Bayesian computation. RESULTS: The structure of small gene regulatory networks responsible for the regulation of biological functions involved in biomining were inferred from multi OMICs data of mixed bacterial cultures. Several causal inter- and intraspecies interactions were inferred between genes coding for proteins involved in the biomining process, such as heavy metal transport, DNA damage, replication and repair, and membrane biogenesis. The method also provided indications for the role of several uncharacterized proteins by the inferred connection in their network context. CONCLUSIONS: The combination of fast algorithms with high-performance computing allowed the simulation of a multitude of gene regulatory networks and their comparison to experimentally measured OMICs data through approximate Bayesian computation, enabling the probabilistic inference of causality in gene regulatory networks of a multispecies bacterial system involved in biomining without need of single-cell or multiple perturbation experiments. This information can be used to influence biological functions and control specific processes in biotechnology applications.

Metagenomes and metatranscriptomes from boreal potential and actual acid sulfate soil materials.

Natural sulfide rich deposits are common in coastal areas worldwide, including along the Baltic Sea coast. When artificial drainage exposes these deposits to atmospheric oxygen, iron sulfide minerals in the soils are rapidly oxidized. This process turns the potential acid sulfate soils into actual acid sulfate soils and mobilizes large quantities of acidity and leachable toxic metals that cause severe environmental problems. It is known that acidophilic microorganisms living in acid sulfate soils catalyze iron sulfide mineral oxidation. However, only a few studies regarding these communities have been published. In this study, we sampled the oxidized actual acid sulfate soil, the transition zone where oxidation is actively taking place, and the deepest un-oxidized potential acid sulfate soil. Nucleic acids were extracted and 16S rRNA gene amplicons, metagenomes, and metatranscriptomes generated to gain a detailed insight into the communities and their activities. The project will be of great use to microbiologists, environmental biologists, geochemists, and geologists as there is hydrological and geochemical monitoring from the site stretching back for many years.

Comparison of boreal acid sulfate soil microbial communities in oxidative and reductive environments.

Due to land uplift after the last ice age, previously stable Baltic Sea sulfidic sediments are becoming dry land. When these sediments are drained, the sulfide minerals are exposed to air and can release large amounts of metals and acid into the environment. This can cause severe ecological damage such as fish kills in rivers feeding the northern Baltic Sea. In this study, five sites were investigated for the occurrence of acid sulfate soils and their geochemistry and microbiology was identified. The pH and soil chemistry identified three of the areas as having classical acid sulfate soil characteristics and culture independent identification of 16S rRNA genes identified populations related to acidophilic bacteria capable of catalyzing sulfidic mineral dissolution, including species likely adapted to low temperature. These results were compared to an acid sulfate soil area that had been flooded for ten years and showed that the previously oxidized sulfidic materials had an increased pH compared to the unremediated oxidized layers. In addition, the microbiology of the flooded soil had changed such that alkalinity producing ferric and sulfate reducing reactions had likely occurred. This suggested that flooding of acid sulfate soils mitigates their environmental impact.

Deep neural networks outperform human expert's capacity in characterizing bioleaching bacterial biofilm composition.

BACKGROUND: Deep neural networks have been successfully applied to diverse fields of computer vision. However, they only outperform human capacities in a few cases. METHODS: The ability of deep neural networks versus human experts to classify microscopy images was tested on biofilm colonization patterns formed on sulfide minerals composed of up to three different bioleaching bacterial species attached to chalcopyrite sample particles. RESULTS: A low number of microscopy images per category (<600) was sufficient for highly efficient computational analysis of the biofilm's bacterial composition. The use of deep neural networks reached an accuracy of classification of ∼90% compared to ∼50% for human experts. CONCLUSIONS: Deep neural networks outperform human experts' capacity in characterizing bacterial biofilm composition involved in the degradation of chalcopyrite. This approach provides an alternative to standard, time-consuming biochemical methods.

How Rainforest Conversion to Agricultural Systems in Sumatra (Indonesia) Affects Active Soil Bacterial Communities.

Palm oil production in Indonesia increased constantly over the last decades, which led to massive deforestation, especially on Sumatra island. The ongoing conversion of rainforest to agricultural systems results in high biodiversity loss. Here, we present the first RNA-based study on the effects of rainforest transformation to rubber and oil palm plantations in Indonesia for the active soil bacterial communities. For this purpose, bacterial communities of three different converted systems (jungle rubber, rubber plantation, and oil palm plantation) were studied in two landscapes with rainforest as reference by RT-PCR amplicon-based analysis of 16S rRNA gene transcripts. Active soil bacterial communities were dominated by Frankiales (Actinobacteria), subgroup 2 of the Acidobacteria and Alphaproteobacteria (mainly Rhizobiales and Rhodospirillales). Community composition differed significantly between the converted land use systems and rainforest reference sites. Alphaproteobacteria decreased significantly in oil palm samples compared to rainforest samples. In contrast, relative abundances of taxa within the Acidobacteria increased. Most important abiotic drivers for shaping soil bacterial communities were pH, calcium concentration, base saturation and C:N ratio. Indicator species analysis showed distinct association patterns for the analyzed land use systems. Nitrogen-fixing taxa including members of Rhizobiales and Rhodospirillales were associated with rainforest soils while nitrifiers and heat-resistant taxa including members of Actinobacteria were associated with oil palm soils. Predicted metabolic profiles revealed that the relative abundances of genes associated with fixation of nitrogen significantly decreased in plantation soils. Furthermore, predicted gene abundances regarding motility, competition or gene transfer ability indicated rainforest conversion-induced changes as well.

Automated Microscopic Analysis of Metal Sulfide Colonization by Acidophilic Microorganisms.

Industrial biomining processes are currently focused on metal sulfides and their dissolution, which is catalyzed by acidophilic iron(II)- and/or sulfur-oxidizing microorganisms. Cell attachment on metal sulfides is important for this process. Biofilm formation is necessary for seeding and persistence of the active microbial community in industrial biomining heaps and tank reactors, and it enhances metal release. In this study, we used a method for direct quantification of the mineral-attached cell population on pyrite or chalcopyrite particles in bioleaching experiments by coupling high-throughput, automated epifluorescence microscopy imaging of mineral particles with algorithms for image analysis and cell quantification, thus avoiding human bias in cell counting. The method was validated by quantifying cell attachment on pyrite and chalcopyrite surfaces with axenic cultures of Acidithiobacillus caldus, Leptospirillum ferriphilum, and Sulfobacillus thermosulfidooxidans. The method confirmed the high affinity of L. ferriphilum cells to colonize pyrite and chalcopyrite surfaces and indicated that biofilm dispersal occurs in mature pyrite batch cultures of this species. Deep neural networks were also applied to analyze biofilms of different microbial consortia. Recent analysis of the L. ferriphilum genome revealed the presence of a diffusible soluble factor (DSF) family quorum sensing system. The respective signal compounds are known as biofilm dispersal agents. Biofilm dispersal was confirmed to occur in batch cultures of L. ferriphilum and S. thermosulfidooxidans upon the addition of DSF family signal compounds.IMPORTANCE The presented method for the assessment of mineral colonization allows accurate relative comparisons of the microbial colonization of metal sulfide concentrate particles in a time-resolved manner. Quantitative assessment of the mineral colonization development is important for the compilation of improved mathematical models for metal sulfide dissolution. In addition, deep-learning algorithms proved that axenic or mixed cultures of the three species exhibited characteristic biofilm patterns and predicted the biofilm species composition. The method may be extended to the assessment of microbial colonization on other solid particles and may serve in the optimization of bioleaching processes in laboratory scale experiments with industrially relevant metal sulfide concentrates. Furthermore, the method was used to demonstrate that DSF quorum sensing signals directly influence colonization and dissolution of metal sulfides by mineral-oxidizing bacteria, such as L. ferriphilum and S. thermosulfidooxidans.

Indirect DNA extraction method suitable for acidic soil with high clay content.

DNA extraction is an essential procedure when investigating microbial communities in environmental samples by sequencing technologies. High clay soils can be problematic as DNA adsorbs to the clay particles and can thereby be preserved from lysed, non-viable cells for a substantial period of time. In order to accurately estimate the intact and living microbial community in the soil, extracellular DNA from dead, remnant bacterial cells needs to be removed prior to DNA extraction. One possibility is to use a sodium phosphate buffer to release both extracellular DNA and bacterial cells from the clay particles. After removing the extracellular DNA by centrifugation, the remaining viable cells can be harvested and DNA extracted. The described method is a modification of a procedure for separating extracellular DNA and bacterial cells from acidic clay soils. •The modified method increases bacterial cell yields from acidic clay soils, such as acid sulfate soil.•The modified method eliminates some steps from the original method, as only DNA from intact bacterial cells is required.•The indirect DNA extraction method increases the workload compared to standard direct extraction methods, but subsequent downstream analyses will give a more representative picture of the viable microbial community composition in the soil.

Chemical and microbiological evaluation of novel chemical treatment methods for acid sulfate soils.

Naturally occurring sulfide rich deposits are common along the northern Baltic Sea coast that when exposed to air, release large amounts of acid and metals into receiving water bodies. This causes severe environmental implications for agriculture, forestry, and building of infrastructure. In this study, we investigated the efficiency of ultrafine-grained calcium carbonate and peat (both separately and in combination) to mitigate acid and metal release. The experiments were carried out aerobically that mimicked summer conditions when the groundwater level is low and acid sulfate soils are exposed to oxygen, and anaerobically that is similar to autumn to spring conditions. The ultrafine-grained calcium carbonate dissipated well in the soil and its effect alone and when mixed with peat raised the pH and reduced pyrite dissolution while peat alone was similar to the controls and did not halt metal and acid release. High throughput 16S rRNA gene sequencing identified populations most similar to characterized acidophiles in the control and peat treated incubations while the acidophilic like populations were altered in the calcium carbonate alone and calcium carbonate plus peat treated acid sulfate soils. Coupled with the geochemistry data, it was suggested that the acidophiles were inactivated by the high pH in the presence of calcium carbonate but catalyzed pyrite dissolution in the controls and peat incubations. In conclusion, the anaerobic conditions during winter would likely be sufficient to mitigate acid production and metal release from acid sulfate soils and in the summer, treatment with calcium carbonate was the best mitigation method.

Multi-omics Reveals the Lifestyle of the Acidophilic, Mineral-Oxidizing Model Species Leptospirillum ferriphilumT.

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.IMPORTANCELeptospirillum 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.

Weak Iron Oxidation by Sulfobacillus thermosulfidooxidans Maintains a Favorable Redox Potential for Chalcopyrite Bioleaching.

Bioleaching is an emerging technology, describing the microbially assisted dissolution of sulfidic ores that provides a more environmentally friendly alternative to many traditional metal extraction methods, such as roasting or smelting. Industrial interest is steadily increasing and today, circa 15-20% of the world's copper production can be traced back to this method. However, bioleaching of the world's most abundant copper mineral chalcopyrite suffers from low dissolution rates, often attributed to passivating layers, which need to be overcome to use this technology to its full potential. To prevent these passivating layers from forming, leaching needs to occur at a low oxidation/reduction potential (ORP), but chemical redox control in bioleaching heaps is difficult and costly. As an alternative, selected weak iron-oxidizers could be employed that are incapable of scavenging exceedingly low concentrations of iron and therefore, raise the ORP just above the onset of bioleaching, but not high enough to allow for the occurrence of passivation. In this study, we report that microbial iron oxidation by Sulfobacillus thermosulfidooxidans meets these specifications. Chalcopyrite concentrate bioleaching experiments with S. thermosulfidooxidans as the sole iron oxidizer exhibited significantly lower redox potentials and higher release of copper compared to communities containing the strong iron oxidizer Leptospirillum ferriphilum. Transcriptomic response to single and co-culture of these two iron oxidizers was studied and revealed a greatly decreased number of mRNA transcripts ascribed to iron oxidation in S. thermosulfidooxidans when cultured in the presence of L. ferriphilum. This allowed for the identification of genes potentially responsible for S. thermosulfidooxidans' weaker iron oxidation to be studied in the future, as well as underlined the need for new mechanisms to control the microbial population in bioleaching heaps.

Low temperature, autotrophic microbial denitrification using thiosulfate or thiocyanate as electron donor.

Wastewaters generated during mining and processing of metal sulfide ores are often acidic (pH < 3) and can contain significant concentrations of nitrate, nitrite, and ammonium from nitrogen based explosives. In addition, wastewaters from sulfide ore treatment plants and tailings ponds typically contain large amounts of inorganic sulfur compounds, such as thiosulfate and tetrathionate. Release of these wastewaters can lead to environmental acidification as well as an increase in nutrients (eutrophication) and compounds that are potentially toxic to humans and animals. Waters from cyanidation plants for gold extraction will often conjointly include toxic, sulfur containing thiocyanate. More stringent regulatory limits on the release of mining wastes containing compounds such as inorganic sulfur compounds, nitrate, and thiocyanate, along the need to increase production from sulfide mineral mining calls for low cost techniques to remove these pollutants under ambient temperatures (approximately 8 °C). In this study, we used both aerobic and anaerobic continuous cultures to successfully couple inorganic sulfur compound (i.e. thiosulfate and thiocyanate) oxidation for the removal of nitrogenous compounds under neutral to acidic pH at the low temperatures typical for boreal climates. Furthermore, the development of the respective microbial communities was identified over time by DNA sequencing, and found to contain a consortium including populations aligning within Flavobacterium, Thiobacillus, and Comamonadaceae lineages. This is the first study to remediate mining waste waters by coupling autotrophic thiocyanate oxidation to nitrate reduction at low temperatures and acidic pH by means of an identified microbial community.

Acidithiobacillus ferrivorans SS3 presents little RNA transcript response related to cold stress during growth at 8 °C suggesting it is a eurypsychrophile.

Acidithiobacillus ferrivorans is an acidophilic bacterium that represents a substantial proportion of the microbial community in a low temperature mining waste stream. Due to its ability to grow at temperatures below 15 °C, it has previously been classified as 'psychrotolerant'. Low temperature-adapted microorganisms have strategies to grow at cold temperatures such as the production of cold acclimation proteins, DEAD/DEAH box helicases, and compatible solutes plus increasing their cellular membrane fluidity. However, little is known about At. ferrivorans adaptation strategies employed during culture at its temperature extremes. In this study, we report the transcriptomic response of At. ferrivorans SS3 to culture at 8 °C compared to 20 °C. Analysis revealed 373 differentially expressed genes of which, the majority were of unknown function. Only few changes in transcript counts of genes previously described to be cold adaptation genes were detected. Instead, cells cultured at cold (8 °C) altered the expression of a wide range of genes ascribed to functions in transcription, translation, and energy production. It is, therefore, suggested that a temperature of 8 °C imposed little cold stress on At. ferrivorans, underlining its adaptation to growth in the cold as well as suggesting it should be classified as a 'eurypsychrophile'.

Electricity generation from an inorganic sulfur compound containing mining wastewater by acidophilic microorganisms.

Sulfide mineral processing often produces large quantities of wastewaters containing acid-generating inorganic sulfur compounds. If released untreated, these wastewaters can cause catastrophic environmental damage. In this study, microbial fuel cells were inoculated with acidophilic microorganisms to investigate whether inorganic sulfur compound oxidation can generate an electrical current. Cyclic voltammetry suggested that acidophilic microorganisms mediated electron transfer to the anode, and that electricity generation was catalyzed by microorganisms. A cation exchange membrane microbial fuel cell, fed with artificial wastewater containing tetrathionate as electron donor, reached a maximum whole cell voltage of 72 ± 9 mV. Stepwise replacement of the artificial anolyte with real mining process wastewater had no adverse effect on bioelectrochemical performance and generated a maximum voltage of 105 ± 42 mV. 16S rRNA gene sequencing of the microbial consortia resulted in sequences that aligned within the genera Thermoplasma, Ferroplasma, Leptospirillum, Sulfobacillus and Acidithiobacillus. This study opens up possibilities to bioremediate mining wastewater using microbial fuel cell technology.

RNA transcript sequencing reveals inorganic sulfur compound oxidation pathways in the acidophile Acidithiobacillus ferrivorans.

Acidithiobacillus ferrivorans is an acidophile implicated in low-temperature biomining for the recovery of metals from sulfide minerals. Acidithiobacillus ferrivorans obtains its energy from the oxidation of inorganic sulfur compounds, and genes encoding several alternative pathways have been identified. Next-generation sequencing of At. ferrivorans RNA transcripts identified the genes coding for metabolic and electron transport proteins for energy conservation from tetrathionate as electron donor. RNA transcripts suggested that tetrathionate was hydrolyzed by the tetH1 gene product to form thiosulfate, elemental sulfur and sulfate. Despite two of the genes being truncated, RNA transcripts for the SoxXYZAB complex had higher levels than for thiosulfate quinone oxidoreductase (doxDAgenes). However, a lack of heme-binding sites in soxX suggested that DoxDA was responsible for thiosulfate metabolism. Higher RNA transcript counts also suggested that elemental sulfur was metabolized by heterodisulfide reductase (hdrgenes) rather than sulfur oxygenase reductase (sor). The sulfite produced as a product of heterodisulfide reductase was suggested to be oxidized by a pathway involving the sat gene product or abiotically react with elemental sulfur to form thiosulfate. Finally, several electron transport complexes were involved in energy conservation. This study has elucidated the previously unknown At. ferrivorans tetrathionate metabolic pathway that is important in biomining.