Palladium bionanoparticles production from acidic Pd(II) solutions and spent catalyst leachate using acidophilic Fe(III)-reducing bacteria

The acidophilic, Fe(III)-reducing heterotrophic bacteria Acidocella aromatica PFBC^T and Acidiphilium cryptum SJH were utilized to produce palladium (Pd) bionanoparticles via a simple 1-step microbiological reaction. Monosaccharide (or intracellular NADH)-dependent reactions lead to visualization of intra/extra-cellular enzymatic Pd(0) nucleation. Formic acid-dependent reactions proceeded via the first slow Pd(0) nucleation phase and the following autocatalytic Pd(II) reduction phase regardless of the presence or viability of the cells. However, use of active cells (with full enzymatic and membrane protein activities) at low formic acid concentration (5 mM) was critical to allow sufficient time for Pd(II) biosorption and the following enzymatic Pd(0) nucleation, which consequently enabled production of fine, dense and well-dispersed Pd(0) bionanoparticles. Differences of the resultant Pd(0) nanoparticles in size, density and localization between the two bacteria under each condition tested suggested different activity and location of enzymes and membrane “Pd(II) trafficking” proteins responsible for Pd(0) nucleation. Despite the inhibitory effect of leaching lixiviant and dissolved metal ions, Pd(0) bionanoparticles were effectively formed by active Ac. aromatica cells from both acidic synthetic Pd(II) solutions and from the actual spent catalyst leachates at equivalent 18–19 nm median size with comparable catalytic activity.

     

Identification of new secreted proteins and secretion of heterologous amylase by <Emphasis Type="Italic">C. glutamicum</Emphasis>

In this study, secreted Corynebacterium glutamicum proteins were investigated by two-dimensional gel electrophoresis. Around 100 spots observed in the pH range 4.5–5.5 had molecular masses that varied from 10 to 50 kDa. Upon N-terminal amino acid sequence analysis by Edman degradation, two of them were hits to two hypothetical proteins encoded by cgR_1176 and cgR_2070 on C. glutamicum R genome, respectively. Active-form α-amylase derived from Geobacillus stearothermophilus was successfully secreted by using the predicted cgR_1176 and cgR_2070 signal sequences, indicating that these hypothetical proteins were secreted proteins. Analysis using a disruption mutant of the twin-arginine translocation (Tat) export pathway machinery of C. glutamicum suggested that one is Tat pathway dependent secretion while the other is independent of the pathway. Our results demonstrate that C. glutamicum can secrete exoproteins by using its own signal sequences, indicating its potential as a host for protein productions.     

“Bioshrouding”—a novel approach for securing reactive mineral tailings

A novel technique (“bioshrouding”) for safeguarding highly reactive sulfidic mineral tailings deposits is proposed. In this, freshly milled wastes are colonised with ferric iron-reducing heterotrophic acidophilic bacteria that form biofilms on reactive mineral surfaces, thereby preventing or minimising colonisation by iron sulfide-oxidising chemolithotrophs such as Acidithiobacillus ferrooxidans and Leptospirillum spp. Data from initial experiments showed that dissolution of pyrite could be reduced by between 57 and 75% by “bioshrouding” the mineral with three different species of heterotrophic acidophiles (Acidiphilium, Acidocella and Acidobacterium spp.), under conditions that were conducive to microbial oxidative dissolution of the iron sulfide.     

Enumeration and Characterization of Acidophilic Microorganisms Isolated from a Pilot Plant Stirred-Tank Bioleaching Operation

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45°C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.     

Isolation,evaluation and use of two strong,carbon source‐inducible promoters from Corynebacterium glutamicum

Aims: To obtain strong, carbon source‐inducible promoters useful for industrial applications of Corynebacterium glutamicum. Methods and Results: DNA microarray and qRT‐PCR enabled identification of the promoters of cgR_2367 (malE1) and cgR_2459 (git1) as strong, maltose‐ and gluconate‐inducible promoters, respectively, in C. glutamicum. Promoter probe assays revealed that in the presence of the inducing sugars, PmalE1 and Pgit1, respectively, facilitated 3·4‐ and 4·2‐fold increased β‐galactosidase activities compared to the same activity induced by glucose. In addition, PmalE1 was not functional in Escherichia coli, in which Pgit1 function was repressible, which enabled the cloning of a hitherto ‘difficult‐to‐clone’ heterologous gene of a lignocellulolytic enzyme, whose secretion was consequently induced by the carbon sources. Conclusions: PmalE1 and Pgit1 are strong, carbon source‐inducible promoters of C. glutamicum whose characteristics in E. coli are integral to the secretion ability of C. glutamicum to secrete lignocellulolytic enzyme. Significance and Impact of the Study: Corynebacterium glutamicum, like its counterpart industrial workhorses E. coli and Bacillus subtilis, does exhibit strong, carbon source‐inducible promoters, and the functionality of two of which was demonstrated in this study. While this study may be most relevant in the ongoing efforts to establish technologies of the biorefinery, it should also be of interest to general microbiologists exploring the versatility of industrial micro‐organisms. In so doing, the study should impact future advances in industrial microbiology.     

Efficient markerless gene replacement in Corynebacterium glutamicum using a new temperature-sensitive plasmid

Random chemical mutation of a Corynebacterium glutamicum-Escherichia coli shuttle vector derived from plasmid pCGR2 was done using hydroxylamine. It brought about amino acid substitutions G109D and E180K within the replicase superfamily domain of the plasmid's RepA protein and rendered the plasmid highly unstable, especially at higher incubation temperatures. Colony formation of C. glutamicum was consequently completely inhibited at 37 °C but not at 25 °C. G109 is a semi-conserved residue mutation which resulted in major temperature sensitivity. E180 on the other hand is not conserved even among RepA proteins of closely related C. glutamicum pCG1 family plasmids and its independent mutation caused relatively moderate plasmid instability. Nonetheless, simultaneous mutation of both residues was required to achieve temperature-sensitive colony formation. This new pCGR2-derived temperature-sensitive plasmid enabled highly efficient chromosomal integration in a variety of C. glutamicum wild-type strains, proving its usefulness in gene disruption studies. Based on this, an efficient markerless gene replacement system was demonstrated using a selection system incorporating the temperature-sensitive replicon and Bacillus subtilis sacB selection marker, a system hitherto not used in this bacterium. Single-crossover integrants were accurately selected by temperature-dependent manner and 93% of the colonies obtained by the subsequent sucrose selection were successful double-crossover recombinants.     

Enumeration and characterization of acidophilic microorganisms isolated from a pilot plant stirred-tank bioleaching operation

Microorganisms were enumerated and isolated on selective solid media from a pilot-scale stirred-tank bioleaching operation in which a polymetallic sulfide concentrate was subjected to biologically accelerated oxidation at 45 degrees C. Four distinct prokaryotes were isolated: three bacteria (an Acidithiobacillus caldus-like organism, a thermophilic Leptospirillum sp., and a Sulfobacillus sp.) and one archaeon (a Ferroplasma-like isolate). The relative numbers of these prokaryotes changed in the three reactors sampled, and the Ferroplasma isolate became increasingly dominant as mineral oxidation progressed, eventually accounting for >99% of plate isolates in the third of three in-line reactors. The identities of the isolates were confirmed by analyses of their 16S rRNA genes, and some key physiological traits (e.g., oxidation of iron and/or sulfur and autotrophy or heterotrophy) were examined. More detailed studies were carried out with the Leptospirillum and Ferroplasma isolates. The data presented here represent the first quantitative study of the microorganisms in a metal leaching situation and confirm that mixed cultures of iron- and sulfur-oxidizing prokaryotic acidophiles catalyze the accelerated dissolution of sulfidic minerals in industrial tank bioleaching operations. The results show that indigenous acidophilic microbial populations change as mineral dissolution becomes more extensive.     

Novel thermo-acidophilic bacteria isolated from geothermal sites in Yellowstone National Park: physiological and phylogenetic characteristics

Moderately thermophilic acidophilic bacteria were isolated from geothermal (30-83 degrees C) acidic (pH 2.7-3.7) sites in Yellowstone National Park. The temperature maxima and pH minima of the isolates ranged from 50 to 65 degrees C, and pH 1.0-1.9. Eight of the bacteria were able to catalyze the dissimilatory oxidation of ferrous iron, and eleven could reduce ferric iron to ferrous iron in anaerobic cultures. Several of the isolates could also oxidize tetrathionate. Six of the iron-oxidizing isolates, and one obligate heterotroph, were low G+C gram-positive bacteria ( Firmicutes). The former included three Sulfobacillus-like isolates (two closely related to a previously isolated Yellowstone strain, and the third to a mesophilic bacterium isolated from Montserrat), while the other three appeared to belong to a different genus. The other two iron-oxidizers were an Actinobacterium (related to Acidimicrobium ferrooxidans) and a Methylobacterium-like isolate (a genus within the alpha -Proteobacteria that has not previously been found to contain either iron-oxidizers or acidophiles). The other three (heterotrophic) isolates were also alpha-Proteobacteria and appeared be a novel thermophilic Acidisphaera sp. An ARDREA protocol was developed to discriminate between the iron-oxidizing isolates. Digestion of amplified rRNA genes with two restriction enzymes ( SnaBI and BsaAI) separated these bacteria into five distinct groups; this result was confirmed by analysis of sequenced rRNA genes.