Intensification of Nickel Bioleaching with Neutrophilic Bacteria Guyparkeria halophila as an Approach to Limitation of Sulfuric Acid Pollution

Hydrometallurgical production of valuable and non-ferrous metals is traditionally accompanied with acid waste effluents/acid mine drainage leading to acidification of the mining areas. The traditional cause of this pollution is the well-known technology based on the recovery of metals with acid solutions and the application of strong acidophilic leaching bacteria for the oxidation of sulfide ores. In our experiments, we used neutrophilic autotrophic bacteria (NAB) stimulated with formic acid or coupled with acidophilic bacteria. The first approach was based on using formic acid as an energetic substrate by autotrophic bacteria. In the second case, the NAB provided initial biogenic acidification for the following growth of the inoculated acidophilic bacteria. Our experiments resulted in increased nickel recovery from the low-grade sulfide ores, which was provided by the NAB in a medium supplemented with formic acid. Bioleaching resulted in 1116 mg Ni/L (69.75%) in the medium with formate and only 35.4 mg Ni/L without formate in 43 days. As a whole, our bench scale experiments showed that the stimulated NAB can be effective at pH 7–5. Partially replacing sulfuric acid with formic acid could also give benefits via the following natural degradation of acid wastes. As a whole, this approach is more environmentally friendly than conventional bioleaching techniques.

Oligo-heterotrophic activity of Marinobacter subterrani creates an indirect Fe(II)-oxidation phenotype in gradient tubes

Autotrophic bacteria utilizing Fe(II) as their energy and electron sources for growth affect multiple biogeochemical cycles. Some chemoheterotrophic bacteria have also been considered to exhibit an Fe(II) oxidation phenotype. For example, several Marinobacter strains have been reported to oxidize Fe(II) based on formation of oxidized iron bands in semi-solid gradient tubes that produce opposing concentration gradients of Fe(II) and oxygen. While gradient tubes are a simple and visually compelling method to test for Fe(II) oxidation, this method alone cannot confirm if, and to what extent, Fe(II) oxidation is linked to metabolism in chemoheterotrophic bacteria. Here we probe the possibility of protein-mediated and metabolic byproduct-mediated Fe(II) oxidation in Marinobacter subterrani JG233, a chemoheterotroph previously proposed to oxidize Fe(II). Results from conditional and mutant studies, along with measurements of Fe(II) oxidation rates suggest M. subterrani is unlikely to facilitate Fe(II) oxidation under microaerobic conditions. We conclude that the Fe(II) oxidation phenotype observed in gradient tubes inoculated with M. subterrani JG233 is a result of oligo-heterotrophic activity, shifting the location where oxygen dependent chemical Fe(II) oxidation occurs, rather than a biologically-mediated process. Importance Gradient tubes are the most commonly used method to isolate and identify neutrophilic Fe(II)-oxidizing bacteria. The formation of oxidized iron bands in gradient tubes provides a compelling assay to ascribe the ability to oxidize Fe(II) to autotrophic bacteria whose growth is dependent on Fe(II) oxidation. However, the physiological significance of Fe(II) oxidation in chemoheterotrophic bacteria is less well understood. Our work suggests that oligo-heterotrophic activity of certain bacteria may create a false-positive phenotype in gradient tubes by altering the location of the abiotic, oxygen-mediated oxidized iron band. Based on the results and analysis presented here, we caution against utilizing gradient tubes as the sole evidence for the capability of a strain to oxidize Fe(II) and that additional experiments are necessary to ascribe this phenotype to new isolates.

Dissolved inorganic carbon accumulating complexes from autotrophic bacteria from extreme environments

In nature, concentrations of dissolved inorganic carbon (DIC; = CO 2 + HCO 3 - + CO 3 2- ) can be low, and autotrophic organisms adapt with a variety of mechanisms to elevate intracellular DIC concentrations to enhance CO 2 fixation. Such mechanisms have been well-studied in Cyanobacteria , but much remains to be learned about their activity in other phyla. Novel multi-subunit membrane-spanning complexes capable of elevating intracellular DIC were recently described in three species of bacteria. Homologs of these complexes are distributed among 17 phyla in Bacteria and Archaea, and are predicted to consist of one, two, or three subunits. To determine whether DIC accumulation is a shared feature of these diverse complexes, seven of them, representative of organisms from four phyla, from a variety of habitats, and with three different subunit configurations were chosen for study. A high-CO 2 requiring, carbonic anhydrase-deficient ( yadF - cynT - ) strain of E. coli Lemo21(DE3), which could be rescued via elevated intracellular DIC concentrations, was created for heterologous expression and characterization of the complexes. Expression of all seven complexes rescued the ability of E. coli Lemo21(DE3) yadF - cynT - to grow under low CO 2 conditions, and six of the seven generated measurably elevated intracellular DIC concentrations when their expression was induced. For complexes consisting of two or three subunits, all subunits were necessary for DIC accumulation. Isotopic disequilibrium experiments clarified that CO 2 was the substrate for these complexes. In addition, the presence of an ionophore prevented the accumulation of intracellular DIC, suggesting that these complexes may couple proton potential to DIC accumulation. IMPORTANCE To facilitate the synthesis of biomass from CO 2 , autotrophic organisms use a variety of mechanisms to increase intracellular DIC concentrations. A novel type of multi-subunit complex has recently been described, which has been shown to generate measurably elevated intracellular DIC concentrations in three species of bacteria, begging the question of whether these complexes share this capability across the 17 phyla of Bacteria and Archaea where they are found. This study shows that DIC accumulation is a trait shared by complexes with varied subunit structures, from organisms with diverse physiologies and taxonomies, suggesting that this trait is universal among them. Successful expression in E. coli suggests the possibility of their expression in engineered organisms synthesizing compounds of industrial importance from CO 2 .

The Impact of Fertilizer Amendments on Soil Autotrophic Bacteria and Carbon Emissions in Maize Field on the Semiarid Loess Plateau

Soil autotrophic bacteria play a crucial role in regulating CO2 fixation and crop productivity. However, the information is limited to how fertilization amendments alter soil autotrophic bacterial community, crop yield, and carbon emission efficiency (CEE). Here, we estimated the impact of the structure and co-occurrence network of soil autotrophic bacterial community on maize yield and CEE. A long-term field experiment was conducted with five fertilization treatments in semiarid Loess Plateau, including no amendment (NA), chemical fertilizer (CF), chemical fertilizer plus commercial organic fertilizer (SC), commercial organic fertilizer (SM), and maize straw (MS). The results showed that fertilization amendments impacted the structure and network of soil Calvin–Benson–Bassham (CBB) (cbbL) gene-carrying bacterial community via changing soil pH and NO3–N. Compared with no amendment, the cbbL-carrying bacterial diversity was increased under the SC, SM, and MS treatments but decreased under the CF treatment. Soil autotrophic bacterial network contained distinct microbial modules that consisted of closely associated microbial species. We detected the higher abundances of soil cbbL-carrying bacterial genus Xanthobacter, Bradyrhizobium, and Nitrosospira. Structural equation modeling further suggested that the diversity, composition, and network of autotrophic bacterial community had strongly positive relationships with CEE and maize yield. Taken together, our results suggest that soil autotrophic bacterial community may drive crop productivity and CEE, and mitigate the atmospheric greenhouse effect.

Production of biopolymer precursors beta-alanine and L-lactic acid from CO2 with metabolically versatile Rhodococcus opacus DSM 43205

Hydrogen oxidizing autotrophic bacteria are promising hosts for CO2 conversion into chemicals. In this work, we engineered the metabolically versatile lithoautotrophic bacterium Rhodococcus opacus strain DSM 43205 for synthesis of polymer precursors. Aspartate decarboxylase (panD) or lactate dehydrogenase (ldh) were expressed for beta-alanine or L-lactic acid production, respectively. The heterotrophic cultivations on glucose produced 25 mg L-1 beta-alanine and 742 mg L-1 L-lactic acid, while autotrophic cultivations with CO2, H2 and O2 resulted in the production of 1.8 mg L-1 beta-alanine and 146 mg L-1 L-lactic acid. Beta-alanine was also produced at 345 µg L-1 from CO2 in electrobioreactors, where H2 and O2 were provided by water electrolysis. This work demonstrates that R. opacus DSM 43205 can be readily engineered to produce chemicals from CO2 and provides base for its further metabolic engineering.

A novel oxidase from Alcaligenes sp. HO-1 oxidizes hydroxylamine to N2

Hydroxylamine is a key intermediate of microbial ammonia oxidation and plays an important role in the biogeochemical cycling of N-compounds. Hydroxylamine is oxidized to NO or N2O by hydroxylamine oxidases or cytochrome P460 from heterotrophic or autotrophic bacteria, but its enzymatic oxidation to N2 has not yet been observed. Here, we report on the discovery of a novel oxidase that converts hydroxylamine to N2 from the newly isolated heterotrophic nitrifier Alcaligenes strain HO-1. Strain HO-1 accumulated hydroxylamine and produced N2 from ammonia oxidation. Using transcriptome analysis and heterologous expression via fosmid library screening, we identified three genes (dnfABC) of strain HO-1 that enabled E. coli cells not only to produce hydroxylamine from 15N-labelled ammonium but also to further convert it to 15N2. The three genes were individually cloned and expressed, and their translational products DnfA, DnfB, and DnfC were purified. In vitro DnfA bound to hydroxylamine and catalyzed the conversion of hydroxylamine to N2 in the presence of FAD, NADH and O2. Thus, DnfA was identified as a novel hydroxylamine oxidase and catalyzed a previously unknown N-N bond forming reaction with a yet-to-be discovered mechanism. DnfA homologs were detected in different bacterial groups, suggesting that hydroxylamine oxidation to nitrogen might occur in additional microbial taxa.

Surveying Denitrification Efficacy in Up-Flow Packed Bed Bioreactor Operated under Heterotrophic Condition Using Autotrophic Bacteria

Introduction: The biological denitrification process is an interesting cost-effective technique to remove nitrate from water supplies. Acetic acid can be used as a carbon source in this process, but its consumption rate is a critical issue and, in some cases, it is quite different from stoichiometric constants. The current study aimed to investigate the nitrate removal in an up-flow packed bed bioreactor. Furthermore, various parameters affecting this process were investigated and optimized. In this study, the autotrophic bacteria were used for the heterotrophic process. Materials and Methods: Initially, the autotrophic bacteria were cultured and used for the following heterotrophic conditions in distinct reactors. A pilot-scale anoxic up flow bioreactor packed was constructed using the polyethylene media and applied to remove nitrate from the aqueous environment. Consequently, the effects of hydraulic retention times (HRT) and different acetic acid concentrations as carbon source were evaluated. During the study, the amounts of alkalinity, pH, temperature, and nitrate were checked. Results: The designed bioreactor removed an average of over 88% of nitrate, while the acetic acid consumption was 2 mg/mg NO3-N, which was lower than the stoichiometric constant for heterotrophic process. Moreover, in the three studied HRTs (1.5, 3, and 5 h), the Alkalinity increased from 14.2 to 19.8 %. Conclusion: The results of this study showed high efficiency in nitrate removal via heterotrophic denitrification using acetic acid as carbon source for autotrophic bacteria.

Pyrite oxidization accelerates bacterial carbon sequestration in copper mine tailings

Polymetallic mine tailings have great potential as carbon sequestration tools to stabilize atmospheric CO2 concentrations. However, previous studies focused on carbonate mineral precipitation, whereas the role of autotrophic bacteria in mine tailing carbon sequestration has been neglected. In this study, carbon sequestration in two samples of mine tailings treated with FeS2 was evaluated using 13C isotope, pyrosequencing and DNA-based stable isotope probing (SIP) analyses to identify carbon fixers. Mine tailings treated with FeS2 exhibited a higher percentage of 13C atoms (1.76±0.06 % for Yangshanchong and 1.36±0.01 % for Shuimuchong) than did controls over a 14-day incubation, which emphasized the role of autotrophs in carbon sequestration with pyrite addition. Pyrite treatment also led to changes in the composition of bacterial communities, and several autotrophic bacteria increased, including Acidithiobacillus and Sulfobacillus. Furthermore, pyrite addition increased the relative abundance of the dominant genus Sulfobacillus by 8.86 % and 5.99 % in Yangshanchong and Shuimuchong samples, respectively. Furthermore, DNA SIP results indicated a 8.20–16.50 times greater gene copy number for cbbL than cbbM in 13C-labeled heavy fractions, and a Sulfobacillus-like cbbL gene sequence (cbbL-OTU1) accounted for 30.11 %–34.74 % of all cbbL gene sequences in 13C-labeled heavy fractions of mine tailings treated with FeS2. These findings highlight the importance of the cbbL gene in bacterial carbon sequestration and demonstrate the ability of chemoautotrophs to sequester carbon during sulfide mineral oxidation in mine tailings. This study is the first to investigate carbon sequestration by autotrophic bacteria in mine tailings through the use of isotope tracers and DNA SIP.