Effect of nickel (II) on the performance of anodic electroactive biofilms in bioelectrochemical systems.

The impact of nickel (Ni2+) on the performance of anodic electroactive biofilms (EABs) in the bioelectrochemical system (BES) was investigated in this study. Although it has been reported that Ni2+ influences microorganisms in a number of ways, it is unknown how its presence in the anode of a BES affects extracellular electron transfer (EET) of EABs, microbial viability, and the bacterial community. Results revealed that the addition of Ni2+ decreased power output from 673.24 ± 12.40 mW/m2 at 0 mg/L to 179.26 ± 9.05 mW/m2 at 80 mg/L. The metal and chemical oxygen demand removal efficiencies of the microbial fuel cells (MFCs) declined as Ni2+ concentration increased, which could be attributed to decreased microbial viability as revealed by SEM and CLSM. FTIR analysis revealed the involvement of various microbial biofilm functional groups, including hydroxyl, amides, methyl, amine, and carboxyl, in the uptake of Ni2+. The presence of Ni2+ on the anodic biofilms was confirmed by SEM-EDS and XPS analyses. CV demonstrated that the electron transfer performance of the anodic biofilms was negatively correlated with the various Ni2+ concentrations. EIS showed that the internal resistance of the MFCs increased with increasing Ni2+ concentration, resulting in a decrease in power output. High-throughput sequencing results revealed a decrease in Geobacter and an increase in Desulfovibrio in response to Ni2+ concentrations of 10, 20, 40, and 80 mg/L. Furthermore, the various Ni2+ concentrations decreased the expression of EET-related genes. The Ni2+-fed MFCs had a higher abundance of the nikR gene than the control group, which was important for Ni2+ resistance. This work advances our understanding of Ni2+ inhibition on EABs, as well as the concurrent removal of organic matter and Ni2+ from wastewater.

Recovery of heavy metals from industrial wastewater using bioelectrochemical system inoculated with novel Castellaniella species.

Water pollution is a global issue that has drastically increased in recent years due to rapid industrial development. Different technologies have been designed for the removal of pollutants from wastewater. However, most of these techniques are expensive, generate new waste, and focus solely on metal removal instead of metal recovery. In this study, novel facultative exoelectrogenic strains designated Castellaniella sp. A5, Castellaniella sp. B3, and Castellaniella sp. A3 were isolated from a microbial fuel cell (MFC). These isolates were utilized as pure and mixed culture inoculums in a bioelectrochemical system (BES) to produce bioelectricity and treat simulated industrial wastewater. A single-chamber MFC inoculated with the mixed culture attained the highest electricity generation (i.e., 320 mW/m2 power density and 3.19 A/m2 current density), chemical oxygen demand removal efficiency (91.15 ± 0.05%), and coulombic efficiency (54.81 ± 4.18%). In addition, the BES containing biofilms of the mixed culture achieved the highest Cu, Cr, and Cd removal efficiencies of 99.89 ± 0.07%, 99.59 ± 0.53%, and 99.91 ± 0.04%, respectively. The Cr6+ and Cu2+ in the simulated industrial wastewater were recovered via microbial electrochemical reduction as Cr3+ and Cu0, respectively. However, Cd2+ precipitated as Cd (OH)2 or CdCO3 on the surface of the cathodes. These results suggest that a mixed culture inoculum of Castellaniella sp. A5, Castellaniella sp. B3, and Castellaniella sp. A3 has great potential as a biocatalyst in BES for heavy metals recovery from industrial wastewater.

Sequentially recover heavy metals from smelting wastewater using bioelectrochemical system coupled with thermoelectric generators.

Smelting wastewater is characterized with high concentration of toxic heavy metals and high acidity, which must be properly treated before discharge. Here, bioelectrochemical system (BES) coupled with thermoelectric generator (TEG) was first demonstrated to simultaneously treat organic wastewater and smelting wastewater by utilizing the simulated waste heat that was abundant in smelting factories. By modulating the input voltage generated from simulated waste heat via TEG to 0, 1.0 and 2.0 V, almost all the Cu2+, Cd2+ and Co2+ in smelting wastewater were sequentially recovered with a respective rate of 121.17, 158.20 and 193.87 mg L-1 d-1. Cu2+ was bioelectrochemically recovered as Cu0. While, Cd2+ and Co2+ were recovered by electrodeposition as Cd(OH)2, CdCO3 or Co(OH)2 on cathodic surface. High throughput sequencing analysis showed that the microbial community of anodic biofilm was greatly shifted after successive treatment by batch-mode. Desulfovibrio (17.00%), Megasphaera (11.81%), Geobacter (10.36%) and Propionibacterium (8.64%) were predominant genera in anodic biofilm enriched from activated sludge in BES before treatment. After successive treatment by batch-mode, Geobacter (34.76%), Microbacter (8.60%) and Desulfovibrio (5.33%) were shifted as the major genera. Economic analysis revealed that it was feasible to use TEG to substitute electrical grid energy to integrate with BES for wastewater treatment. In addition, literature review indicated that it was not uncommon for the coexistence of waste heat with typical pollutants (e.g. heavy metal ions and various biodegradation-resistant organic wastes) that could be treated by BES in different kinds of factories or geothermal sites. This study provides novel insights to expand the application potentials of BES by integrating with TEG to utilize widespread waste heat.

Recovery of Metals from Acid Mine Drainage by Bioelectrochemical System Inoculated with a Novel Exoelectrogen, Pseudomonas sp. E8.

Acid mine drainage (AMD) is a typical source of environmental pollution ascribing to its characteristics of high acidity and heavy metal content. Currently, most strategies for AMD treatment merely focus on metal removal rather than metal recovery. However, bioelectrochemical system (BES) is a promising technology to simultaneously remove and recover metal ions from AMD. In this study, both cupric ion and cadmium ion in simulated AMD were effectively recovered by BES inoculated with a novel exoelectrogen, Pseudomonas sp. E8, that was first isolated from the anodic electroactive biofilm of a microbial fuel cell (MFC) in this study. Pseudomonas sp. E8 is a facultative anaerobic bacterium with a rod shape, 0.43-0.47 µm wide, and 1.10-1.30 µm long. Pseudomonas sp. E8 can agglomerate on the anode surface to form a biofilm in the single-chamber MFC using diluted Luria-Bertani (LB) medium as an energy substrate. A single-chamber MFC containing the electroactive Pseudomonas sp. E8 biofilms has a maximum output voltage of 191 mV and a maximum power density of 70.40 mW/m2, which is much higher than those obtained by most other exoelectrogenic strains in the genus of Pseudomonas. Almost all the Cu2+ (99.95% ± 0.09%) and Cd2+ (99.86% ± 0.04%) in simulated AMD were selectively recovered by a microbial fuel cell (MFC) and a microbial electrolysis cell (MEC). After the treatment with BES, the high concentrations of Cu2+(184.78 mg/L), Cd2+(132.25 mg/L), and total iron (49.87 mg/L) in simulated AMD were decreased to 0.02, 0.19, and 0 mg/L, respectively. Scanning electron micrograph (SEM), energy dispersive X-ray spectrometry (EDXS) and X-ray diffraction (XRD) analysis indicate that the Cu2+ and Cd2+ in simulated AMD were selectively recovered by microbial electrochemical reduction as Cu0 (together with trace amounts of Cu2O) or Cd0 on the cathode surface. Collectively, data suggest that Pseudomonas sp. E8 has great potential for AMD treatment and metal recovery.

Metagenomic Insights into the Effects of Seasonal Temperature Variation on the Activities of Activated Sludge.

It is well acknowledged that the activities of activated sludge (AS) are influenced by seasonal temperature variation. However, the underlying mechanisms remain largely unknown. Here, the activities of activated sludge under three simulated temperature variation trends were compared in lab-scale. The TN, HN3-H, and COD removal activities of activated sludge were improved as temperature elevated from 20 °C to 35 °C. While, the TN, HN3-H, COD and total phosphorus removal activities of activated sludge were inhibited as temperature declined from 20 °C to 5 °C. Both the extracellular polymer substances (EPS) composition (e.g., total amount, PS, PN and DNA) and sludge index of activated sludge were altered by simulated seasonal temperature variation. The variation of microbial community structures and the functional potentials of activated sludge were further explored by metagenomics. Proteobacteria, Actinobacteria, Acidobacteria and Bacteroidetes were the dominant phyla for each activated sludge sample under different temperatures. However, the predominant genera of activated sludge were significantly modulated by simulated temperature variation. The functional genes encoding enzymes for nitrogen metabolism in microorganisms were analyzed. The enzyme genes related to ammonification had the highest abundance despite the changing temperature, especially for gene encoding glutamine synthetase. With the temperature raising from 20 °C to 35 °C. The abundance of amoCAB genes encoding ammonia monooxygenase (EC:1.14.99.39) increased by 305.8%. Meanwhile, all the enzyme genes associate with denitrification were reduced. As the temperature declined from 20 °C to 5 °C, the abundance of enzyme genes related to nitrogen metabolism were raised except for carbamate kinase (EC:2.7.2.2), glutamate dehydrogenase (EC:1.4.1.3), glutamine synthetase (EC:6.3.1.2). Metagenomic data indicate that succession of the dominant genera in microbial community structure is, to some extent, beneficial to maintain the functional stability of activated sludge under the temperature variation within a certain temperature range. This study provides novel insights into the effects of seasonal temperature variation on the activities of activated sludge.

Increased chalcopyrite bioleaching capabilities of extremely thermoacidophilic Metallosphaera sedula inocula by mixotrophic propagation.

Extremely thermoacidophilic Crenarchaeota belonging to the order Sulfolobales, such as Metallosphaera sedula, are metabolically versatile and of great relevance in bioleaching. However, the impacts of extreme thermoacidophiles propagated with different energy substrates on subsequent bioleaching of refractory chalcopyrite remain unknown. Transcriptional responses underlying their different bioleaching potentials are still elusive. Here, it was first showed that M. sedula inocula propagated with typical energy substrates have different chalcopyrite bioleaching capabilities. Inoculum propagated heterotrophically with yeast extract was deficient in bioleaching; however, inoculum propagated mixotrophically with chalcopyrite, pyrite or sulfur recovered 79%, 78% and 62% copper, respectively, in 12 days. Compared with heterotrophically propagated inoculum, 937, 859 and 683 differentially expressed genes (DEGs) were identified in inoculum cultured with chalcopyrite, pyrite or sulfur, respectively, including upregulation of genes involved in bioleaching-associated metabolism, e.g., Fe2+ and sulfur oxidation, CO2 fixation. Inoculum propagated with pyrite or sulfur, respectively, shared 480 and 411 DEGs with chalcopyrite-cultured inoculum. Discrepancies on repertories of DEGs that involved in Fe2+ and sulfur oxidation in inocula greatly affected subsequent chalcopyrite bioleaching rates. Novel genes (e.g., Msed_1156, Msed_0549) probably involved in sulfur oxidation were first identified. This study highlights that mixotrophically propagated extreme thermoacidophiles especially with chalcopyrite should be inoculated into chalcopyrite heaps at industrial scale.

Enhancement of Metallosphaera sedula Bioleaching by Targeted Recombination and Adaptive Laboratory Evolution.

Thermophilic and lithoautotrophic archaea such as Metallosphaera sedula occupy acidic, metal-rich environments and are used in biomining processes. Biotechnological approaches could accelerate these processes and improve metal recovery by biomining organisms, but systems for genetic manipulation in these organisms are currently lacking. To gain a better understanding of the interplay between metal resistance, autotrophy, and lithotrophic metabolism, a genetic system was developed for M. sedula and used to evaluate parameters governing the efficiency of copper bioleaching. Additionally, adaptive laboratory evolution was used to select for naturally evolved M. sedula cell lines with desirable phenotypes for biomining, and these adapted cell lines were shown to have increased bioleaching capacity and efficiency. Genomic methods were used to analyze mutations that led to resistance in the experimentally evolved cell lines, while transcriptomics was used to examine changes in stress-inducible gene expression specific to the environmental conditions.

Identification and Analysis of a Novel Gene Cluster Involves in Fe2+ Oxidation in Acidithiobacillus ferrooxidans ATCC 23270, a Typical Biomining Acidophile.

Iron-oxidizing Acidithiobacillus spp. are applied worldwide in biomining industry to extract metals from sulfide minerals. They derive energy for survival through Fe2+ oxidation and generate Fe3+ for the dissolution of sulfide minerals. However, molecular mechanisms of their iron oxidation still remain elusive. A novel two-cytochrome-encoding gene cluster (named tce gene cluster) encoding a high-molecular-weight cytochrome c (AFE_1428) and a c4-type cytochrome c552 (AFE_1429) in A. ferrooxidans ATCC 23270 was first identified in this study. Bioinformatic analysis together with transcriptional study showed that AFE_1428 and AFE_1429 were the corresponding paralog of Cyc2 (AFE_3153) and Cyc1 (AFE_3152) which were encoded by the extensively studied rus operon and had been proven involving in ferrous iron oxidation. Both AFE_1428 and AFE_1429 contained signal peptide and the classic heme-binding motif(s) as their corresponding paralog. The modeled structure of AFE_1429 showed high resemblance to Cyc1. AFE_1428 and AFE_1429 were preferentially transcribed as their corresponding paralogs in the presence of ferrous iron as sole energy source as compared with sulfur. The tce gene cluster is highly conserved in the genomes of four phylogenetic-related A. ferrooxidans strains that were originally isolated from different sites separated with huge geographical distance, which further implies the importance of this gene cluster. Collectively, AFE_1428 and AFE_1429 involve in Fe2+ oxidation like their corresponding paralog by integrating with the metalloproteins encoded by rus operon. This study provides novel insights into the Fe2+ oxidation mechanism in Fe2+-oxidizing A. ferrooxidans ssp.

Evolution of copper arsenate resistance for enhanced enargite bioleaching using the extreme thermoacidophile Metallosphaera sedula.

Adaptive laboratory evolution (ALE) was employed to isolate arsenate and copper cross-resistant strains, from the copper-resistant M. sedula CuR1. The evolved strains, M. sedula ARS50-1 and M. sedula ARS50-2, contained 12 and 13 additional mutations, respectively, relative to M. sedula CuR1. Bioleaching capacity of a defined consortium (consisting of a naturally occurring strain and a genetically engineered copper sensitive strain) was increased by introduction of M. sedula ARS50-2, with 5.31 and 26.29% more copper recovered from enargite at a pulp density (PD) of 1 and 3% (w/v), respectively. M. sedula ARS50-2 arose as the predominant species and modulated the proportions of the other two strains after it had been introduced. Collectively, the higher Cu2+ resistance trait of M. sedula ARS50-2 resulted in a modulated microbial community structure, and consolidating enargite bioleaching especially at elevated PD.

Increased acid resistance of the archaeon, Metallosphaera sedula by adaptive laboratory evolution.

Extremely thermoacidophilic members of the Archaea such as the lithoautotroph, Metallosphaera sedula, are among the most acid resistant forms of life and are of great relevance in bioleaching. Here, adaptive laboratory evolution was used to enhance the acid resistance of this organism while genomics and transcriptomics were used in an effort to understand the molecular basis for this trait. Unlike the parental strain, the evolved derivative, M. sedula SARC-M1, grew well at pH of 0.90. Enargite (Cu3AsS4) bioleaching conducted at pH 1.20 demonstrated SARC-M1 leached 23.78 % more copper relative to the parental strain. Genome re-sequencing identified two mutations in SARC-M1 including a nonsynonymous mutation in Msed_0408 (an amino acid permease) and a deletion in pseudogene Msed_1517. Transcriptomic studies by RNA-seq of wild type and evolved strains at various low pH values demonstrated there was enhanced expression of genes in M. sedula SARC-M1 encoding membrane complexes and enzymes that extrude protons or that catalyze proton-consuming reactions. In addition, M. sedula SARC-M1 exhibited reduced expression of genes encoding enzymes that catalyze proton-generating reactions. These unique genomic and transcriptomic features support a model for increased acid resistance arising from enhanced control over cytoplasmic pH.

Complete Genome Sequences of Evolved Arsenate-Resistant Metallosphaera sedula Strains.

Metallosphaera sedula is a thermoacidophilic crenarchaeote with a 2.19-Mb genome. Here, we report the genome sequences of several evolved derivatives of M. sedula generated through adaptive laboratory evolution for enhanced arsenate resistance.

Role of an archaeal PitA transporter in the copper and arsenic resistance of Metallosphaera sedula, an extreme thermoacidophile.

Thermoacidophilic archaea, such as Metallosphaera sedula, are lithoautotrophs that occupy metal-rich environments. In previous studies, an M. sedula mutant lacking the primary copper efflux transporter, CopA, became copper sensitive. In contrast, the basis for supranormal copper resistance remained unclear in the spontaneous M. sedula mutant, CuR1. Here, transcriptomic analysis of copper-shocked cultures indicated that CuR1 had a unique regulatory response to metal challenge corresponding to the upregulation of 55 genes. Genome resequencing identified 17 confirmed mutations unique to CuR1 that were likely to change gene function. Of these, 12 mapped to genes with annotated function associated with transcription, metabolism, or transport. These mutations included 7 nonsynonymous substitutions, 4 insertions, and 1 deletion. One of the insertion mutations mapped to pseudogene Msed_1517 and extended its reading frame an additional 209 amino acids. The extended mutant allele was identified as a homolog of Pho4, a family of phosphate symporters that includes the bacterial PitA proteins. Orthologs of this allele were apparent in related extremely thermoacidophilic species, suggesting M. sedula naturally lacked this gene. Phosphate transport studies combined with physiologic analysis demonstrated M. sedula PitA was a low-affinity, high-velocity secondary transporter implicated in copper resistance and arsenate sensitivity. Genetic analysis demonstrated that spontaneous arsenate-resistant mutants derived from CuR1 all underwent mutation in pitA and nonselectively became copper sensitive. Taken together, these results point to archaeal PitA as a key requirement for the increased metal resistance of strain CuR1 and its accelerated capacity for copper bioleaching.

Expression, purification, and characterization of an iron chaperon protein CyaY from Acidithiobacillus ferrooxidans.

CyaY is the bacterial homolog of frataxin, proposed to be involved in the assembly of iron-sulfur clusters. While, the physiological iron donor for the iron-sulfur clusters assembly remains controversial. In this study, the gene of CyaY from Acidithiobacillus ferrooxidans was cloned and expressed in Escherichia coli, the protein was purified by one-step affinity chromatography to homogeneity. The CyaY protein can bind ferric iron and serve as an iron donor for the biogenesis of iron-sulfur clusters on the scaffold protein IscU in the presence of IscS and L-cysteine in vitro.

Expression, purification and molecular structure modeling of thioredoxin (Trx) and thioredoxin reductase (TrxR) from Acidithiobacillus ferrooxidans.

The thioredoxin system consists of thioredoxin (Trx), thioredoxin reductase (TrxR) and NADPH, which plays several key roles in maintaining the redox environment of the cell. In Acidithiobacillus ferrooxidans, thioredoxin system may play important functions in the activity regulation of periplasmic proteins and energy metabolism. Here, we cloned thioredoxin (trx) and thioredoxin reductase (trxR) genes from Acidithiobacillus ferrooxidans, and expressed the genes in Escherichia coli. His-Trx and His-TrxR were purified to homogeneity with one-step Ni-NTA affinity column chromatography. Site-directed mutagenesis results confirmed that Cys33, Cys36 of thioredoxin, and Cys142, Cys145 of thioredoxin reductase were active-site residues.

Glu43 is an essential residue for coordinating the [Fe2S2] cluster of IscR from Acidithiobacillus ferrooxidans.

The iron-sulfur cluster regulator (IscR) was reported to be a repressor of the iscRSUA operon. In vitro transcription reactions revealed that the IscR had a repression effect on the iscR promoter. The IscR contains a [Fe2S2] cluster per each monomer, and three highly conserved cysteines were identified to ligate the [Fe2S2] cluster. It was proposed that a non-cysteine residue might be the fourth ligand for the [Fe2S2] cluster. In this study, using site-directed mutagenesis, Glu43 was found to be the fourth residue that coordinates the [Fe2S2] cluster of IscR.