Fast Kinetics of Fe2+ Oxidation in Packed-Bed Reactors

Thiobacillus ferrooxidans was used in fixed-film bioreactors to oxidize ferrous sulfate to ferric sulfate. Glass beads, ion-exchange resin, and activated-carbon particles were tested as support matrix materials. Activated carbon was tested in both a packed-bed bioreactor and a fluidized-bed bioreactor; the other matrix materials were used in packed-bed reactors. Activated carbon displayed the most suitable characteristics for use as a support matrix of T. ferrooxidans fixed-film formation. The reactors were operated within a pH range of 1.35 to 1.5, which effectively reduced the amount of ferric iron precipitation and eliminated diffusion control of mass transfer due to precipitation. The activated-carbon packed-bed reactor displayed the most favorable biomass holdup and kinetic performance related to ferrous sulfate oxidation. The fastest kinetic performance achieved with the activated-carbon packed-bed bioreactor was 78 g of Fe²⁺ oxidized per liter per h (1,400 mmol of Fe²⁺ oxidized per liter per h) at a true dilution rate of 40/h, which represents a hydraulic retention time of 1.5 min.     

Sensitivity of Iron-Oxidizing Bacteria, Thiobacillus ferrooxidans and Leptospirillum ferrooxidans, to Bisulfite Ion

When grown on iron-salt medium supplemented with the bisulfite ion, Leptospirillum ferrooxidans was much more sensitive to the ion than was Thiobacillus ferrooxidans. The causes of the sensitivity of L. ferrooxidans to the bisulfite ion were studied. The bisulfite ion completely inhibited the iron-oxidizing activities of L. ferrooxidans and T. ferrooxidans at 0.02 and 0.2 mM, respectively. A trapping reagent for the bisulfite ion, formaldehyde, completely reversed the inhibition. The treatment of intact cells with 1.0 mM bisulfite ion for 1 h and washing the bisulfite ion from the cells had no harmful effects on the iron-oxidizing activity of T. ferrooxidans. However, the treatment of L. ferrooxidans with 0.1 mM bisulfite ion for 1 h completely destroyed the iron-oxidizing activity. T. ferrooxidans had sulfite:ferric ion oxidoreductase activity. In contrast, a quite low level of sulfite:ferric ion oxidoreductase activity was found in L. ferrooxidans, suggesting that it is much more difficult for L. ferrooxidans to oxidize the bisulfite ion to the less harmful sulfate than it is for T. ferrooxidans. These results suggest that the sensitivity of L. ferrooxidans to the bisulfite ion is due to a lack of an active sulfite:ferric ion oxidoreductase and the sensitivity of its iron oxidase to bisulfite ion.     

Characterization of Jarosite Formed upon Bacterial Oxidation of Ferrous Sulfate in a Packed-Bed Reactor

A packed-bed bioreactor with activated-carbon particles as a carrier matrix material inoculated with Thiobacillus ferrooxidans was operated at a pH of 1.35 to 1.5 to convert ferrous sulfate to ferric sulfate. Despite the low operating pH, trace amounts of precipitates were produced in both the reactor and the oxidized effluent. X-ray diffraction and chemical analyses indicated that the precipitates were well-ordered potassium jarosite. The chemical analyses also revealed a relative deficiency of Fe and an excess of S in the reactor sample compared with the theoretical composition of potassium jarosite.     

Biological ferrous sulfate oxidation by A. ferrooxidans immobilized on chitosan beads

The immobilization of Acidithiobacillus ferrooxidans cells on chitosan and cross-linked chitosan beads and the biooxidation of ferrous iron to ferric iron in a packed-bed bioreactor were studied. The biofilm formation was carried out by using a glass column reactor loaded with chitosan or cross-linked chitosan beads and 9 K medium previously inoculated with A. ferrooxidans cells. The immobilization cycles on the carrier matrix with the bioreactor operating in batch mode were compared. Then, the reactor was operated using a continuous flow of 9 K medium at different dilution rates. The results indicate that the packed-bed reactor allowed increasing the flow rate of medium approximately two fold (chitosan) and eight fold (chitosan cross-linked) without cells washout, compared to a free cell suspension reactor used as control, and to reach ferric iron productivities as high as 1100 and 1500 mg l(-1) h(-1) respectively. Scanning electron microscopy micrographs of the beads, infrared spectroscopy and the X-ray diffraction patterns of precipitates on the chitosan beads were also investigated.     

Existence of a Hydrogen Sulfide:Ferric Ion Oxidoreductase in Iron-Oxidizing Bacteria

The existence of a hydrogen sulfide:ferric ion oxidoreductase, which catalyzes the oxidation of elemental sulfur with ferric ions as an electron acceptor to produce ferrous and sulfite ions, was assayed with washed intact cells and cell extracts of various kinds of iron-oxidizing bacteria, such as Thiobacillus ferrooxidans 13598, 13661, 14119, 19859, 21834, 23270, and 33020 from the American Type Culture Collection, Leptospirillum ferrooxidans 2705 and 2391 from the Deutsche Sammlung von Mikroorganismen, L. ferrooxidans BKM-6-1339 and P3A, and moderately thermophilic iron-oxidizing bacterial strains BC1, TH3, and Alv. It was found that hydrogen sulfide:ferric ion oxidoreductase activity comparable to that of T. ferrooxidans AP19-3 was present in all iron-oxidizing bacteria tested, suggesting a wide distribution of this enzyme in iron-oxidizing bacteria.     

Ferric iron reduction by Thiobacillus ferrooxidans at extremely low pH-values

When ferrous iron and sulfur were supplied, cells of T. ferrooxidans in a well-aerated medium started growth by oxidizing ferrous iron. After ferrous iron depletion a lagphase followed before sulfur oxidation started. During sulfur oxidation at pH-values below 1.3 (±0,2) the ferrous iron concentration increased again, although the oxygen saturation of the medium amounted to more than 95%. The number of viable cells did not increase. Thus resting cells of T. ferrooxidans, which are oxidizing sulfur to maintain their proton balance, reduce ferric to ferrous iron. The ferrous iron-oxidizing system seemed to be inhibited at pH-values below 1.3. At a pH-value of 1.8 the ferrous iron was reoxidized at once. A scheme for the linkage of iron- and sulfur metabolism is discussed.     

生物造粒流化床微生物落结构及其动态变化

为了研究生物造粒流化床污水处理反应器颗粒污泥中微生物群落结构及其动态变化,分别从生物造粒流化床10、60、110cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA。以细菌和古细菌16S rRNA基因通用引物530F/1490R,对活性污泥中提取的细菌基因组DNA进行PCR扩增,长约1kb的PCR扩增产物纯化后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱。结果显示,生物造粒流化床反应器颗粒污泥中的微生物群落非常丰富,在10cm处微生物的种属达到23种,60cm处为21种,110cm处为20种;生物造粒流化床不同高度都有一些各自的特有种属和共有种属,反应器不同高度的微生物群落演替不明显,微生物群落相似性为83.1%,群落结构较为稳定。     

Heterotrophic cultivation of Paracoccus denitrificans in a horizontal rotating tubular bioreactor

In this work, the heterotrophic cultivation of bacterium Paracoccus denitrificans has been studied in a horizontal rotating tubular bioreactor (HRTB). After development of a microbial biofilm on the inner surface of the HRTB, conditions for one-step removal of acetate and ammonium ion were created. The effect of bioreactor process parameters [medium inflow rate (F) and bioreactor rotation speed (n)] on the bioprocess dynamics in the HRTB was studied. Nitrite and nitrogen oxides (NO and N₂O) were detected as intermediates of ammonium ion degradation. The biofilm thickness and the nitrite concentration were gradually reduced with increase of bioreactor rotation speed when the medium inflow rate was in the range of 0.5–1.5 l h⁻¹. Further increase of inflow rate (2.0–2.5 l h⁻¹) did not have a significant effect on the biofilm thickness and nitrite concentration along the HRTB. Complete acetate consumption was observed when the inflow rate was in the range of 0.5–1.5 l h⁻¹ at all bioreactor rotation speeds. Significant pH gradient (cca 1 pH unit) along the HRTB was only observed at the highest inflow rate (2.5 l h⁻¹). The results have clearly shown that acetate and ammonium ion removal by P. denitificans can be successfully conducted in a HRTB as a one-step process.     

Selective Adhesion of Thiobacillus ferrooxidans to Pyrite

Bacterial adhesion to mineral surfaces plays an important role not only in bacterial survival in natural ecosystems, but also in mining industry applications. Selective adhesion was investigated with Thiobacillus ferrooxidans by using four minerals, pyrite, quartz, chalcopyrite, and galena. Escherichia coli was used as a control bacterium. Contact angles were used as indicators of hydrophobicity, which was an important factor in the interaction between minerals and bacteria. The contact angle of E. coli in a 0.5% sodium chloride solution was 31°, and the contact angle of T. ferrooxidans in a pH 2.0 sulfuric acid solution was 23°. E. coli tended to adhere to more hydrophobic minerals by hydrophobic interaction, while T. ferrooxidans selectively adhered to iron-containing minerals, such as pyrite and chalcopyrite. Ferrous ion inhibited the selective adhesion of T. ferrooxidans to pyrite competitively, while ferric ion scarcely inhibited such adhesion. When selective adhesion was quenched by ferrous ion completely, adhesion of T. ferrooxidans was controlled by hydrophilic interactions. Adhesion of E. coli to pyrite exhibited a liner relationship on langmuir isotherm plots, but adhesion of T. ferrooxidans did not. T. ferrooxidans recognized the reduced iron in minerals and selectively adhered to pyrite and chalcopyrite by a strong interaction other than the physical interaction.     

Identification of the dominant bacterium of two-stage biooxidation of gold-arsenic concentrate

In the process of biooxidation at 39°C in a continuous mode of the gold-arsenic concentrate from the Olympiadinskoe deposit, which was pretreated by chemical leaching with ferric ions, by a microbial association from the BIO department reactors of the Polyus gold mining company, a bacterial culture designated as strain HT-4 was isolated. The bacterium was a spore-forming rod 0.5–0.6 × 1.4–2.0 μm with a flagellum. The optimal temperature for growth and Fe²⁺ oxidation was 55°C. The strain grew in the pH range from 1.21 to 2.10 with the optimum at pH 1.6. The organism was incapable of lithotrophic and organotrophic growth. It grew mixotrophically by Fe²⁺ oxidation in the presence of 0.02% yeast extract. The DNA G+C base content was 48.6 mol %. Based on comparative phylogenetic analysis of 1472-bp nucleotide sequences of 16S rRNA genes, strain HT-4 was classified as Sulfobacillus thermosulfidooxidans. Analysis by pulse-field gel electrophoresis revealed a unique profile of the NotI fragments of the chromosomal DNA. These results demonstrate the strain and species diversity of sulfobacilli in microbial associations involved in biooxidation of concentrates in different technological conditions. The strain “S. olympiadicus S-5” dominated in the process of biooxidation of original concentrate not treated with ferric iron, while S. thermosulfidooxidans HT-4 was predominant in biooxidation of the chemically leached concentrate.     

Role of a Ferric Ion-Reducing System in Sulfur Oxidation of Thiobacillus ferrooxidans

The properties of a ferric ion-reducing system which catalyzes the reduction of ferric ion with elemental sulfur was investigated with a pure strain of Thiobacillus ferrooxidans. In anaerobic conditions, washed intact cells of the strain reduced 6 mol of Fe³⁺ with 1 mol of elemental sulfur to give 6 mol of Fe²⁺, 1 mol of sulfate, and a small amount of sulfite. In aerobic conditions, the 6 mol of Fe²⁺ produced was immediately reoxidized by the iron oxidase of the cell, with a consumption of 1.5 mol of oxygen. As a result, Fe²⁺ production was never observed under aerobic conditions. However, in the presence of 5 mM cyanide, which completely inhibits the iron oxidase of the cell, an amount of Fe²⁺ production comparable to that formed under anaerobic conditions was observed under aerobic conditions. The ferric ion-reducing system had a pH optimum between 2.0 and 3.8, and the activity was completely destroyed by 10 min of incubation at 60°C. A short treatment of the strain with 0.5% phenol completely destroyed the ferric ion-reducing system of the cell. However, this treatment did not affect the iron oxidase of the cell. Since a concomitant complete loss of the activity of sulfur oxidation by molecular oxygen was observed in 0.5% phenol-treated cells, it was concluded that the ferric ion-reducing system plays an important role in the sulfur oxidation activity of this strain, and a new sulfur-oxidizing route is proposed for T. ferrooxidans.     

Energy Transduction by Anaerobic Ferric Iron Respiration in Thiobacillus ferrooxidans

Formate-grown cells of the obligately chemolithoautotrophic acidophile Thiobacillus ferrooxidans were capable of formate- and elemental sulfur-dependent reduction of ferric iron under anaerobic conditions. Under aerobic conditions, both oxygen and ferric iron could be simultaneously used as electron acceptors. To investigate whether anaerobic ferric iron respiration by T. ferrooxidans is an energy-transducing process, uptake of amino acids was studied. Glycine uptake by starved cells did not occur in the absence of an electron donor, neither under aerobic conditions nor under anaerobic conditions. Uptake of glycine could be driven by formate- and ferrous iron-dependent oxygen uptake. Under anaerobic conditions, ferric iron respiration with the electron donors formate and elemental sulfur could energize glycine uptake. Glycine uptake was inhibited by the uncoupler 2,4-dinitrophenol. The results indicate that anaerobic ferric iron respiration can contribute to the energy budget of T. ferrooxidans.     

Molecular Relationship between Two Groups of the Genus Leptospirillum and the Finding that Leptospirillum ferriphilum sp. nov. Dominates South African Commercial Biooxidation Tanks That Operate at 40°C

Iron-oxidizing bacteria belonging to the genus Leptospirillum are of great importance in continuous-flow commercial biooxidation reactors, used for extracting metals from minerals, that operate at 40°C or less. They also form part of the microbial community responsible for the generation of acid mine drainage. More than 16 isolates of leptospirilla were included in this study, and they were clearly divisible into two major groups. Group I leptospirilla had G+C moles percent ratios within the range 49 to 52% and had three copies of rrn genes, and based on 16S rRNA sequence data, these isolates clustered together with the Leptospirillum ferrooxidans type strain (DSM2705 or L15). Group II leptospirilla had G+C moles percent ratios of 55 to 58% and had two copies of rrn genes, and based on 16S rRNA sequence data, they form a separate cluster. Genome DNA-DNA hybridization experiments indicated that three similarity subgroups were present among the leptospirilla tested, with two DNA-DNA hybridization similarity subgroups found within group I. The two groups could also be distinguished based on the sizes of their 16S-23S rRNA gene spacer regions. We propose that the group II leptospirilla should be recognized as a separate species with the name Leptospirillum ferriphilum sp. nov. Members of the two species can be rapidly distinguished from each other by amplification of their 16S rRNA genes and by carrying out restriction enzyme digests of the products. Several, but not all, isolates of the group II leptospirilla, but none from group I (L. ferrooxidans), were capable of growth at 45°C. All the leptospirilla isolated from commercial biooxidation tanks in South Africa were from group II.     

Fluorescent protein biosensors applied to microphysiological systems

This mini-review discusses the evolution of fluorescence as a tool to study living cells and tissues in vitro and the present role of fluorescent protein biosensors (FPBs) in microphysiological systems (MPSs). FPBs allow the measurement of temporal and spatial dynamics of targeted cellular events involved in normal and perturbed cellular assay systems and MPSs in real time. FPBs evolved from fluorescent analog cytochemistry (FAC) that permitted the measurement of the dynamics of purified proteins covalently labeled with environmentally insensitive fluorescent dyes and then incorporated into living cells, as well as a large list of diffusible fluorescent probes engineered to measure environmental changes in living cells. In parallel, a wide range of fluorescence microscopy methods were developed to measure the chemical and molecular activities of the labeled cells, including ratio imaging, fluorescence lifetime, total internal reflection, 3D imaging, including super-resolution, as well as high-content screening. FPBs evolved from FAC by combining environmentally sensitive fluorescent dyes with proteins in order to monitor specific physiological events such as post-translational modifications, production of metabolites, changes in various ion concentrations, and the dynamic interaction of proteins with defined macromolecules in time and space within cells. Original FPBs involved the engineering of fluorescent dyes to sense specific activities when covalently attached to particular domains of the targeted protein. The subsequent development of fluorescent proteins (FPs), such as the green fluorescent protein, dramatically accelerated the adoption of studying living cells, since the genetic “labeling” of proteins became a relatively simple method that permitted the analysis of temporal–spatial dynamics of a wide range of proteins. Investigators subsequently engineered the fluorescence properties of the FPs for environmental sensitivity that, when combined with targeted proteins/peptides, created a new generation of FPBs. Examples of FPBs that are useful in MPS are presented, including the design, testing, and application in a liver MPS.     

Polymorphism of <Emphasis Type="Italic">Sulfobacillus thermosulfidooxidans</Emphasis> strains dominating in processes of high-temperature oxidation of gold-arsenic concentrate

The composition was studied of the microbial association involved in tank biooxidation of the concentrate of a refractory pyrrhotite-containing pyrite-arsenopyrite gold-arsenic ore from the Olympiadinskoe deposit at 50°C. The two Sulfobacillus thermosulfidooxidans strains predominant in the association were phylogenetically different from the strains used as inocula. The isolates were found to differ significantly both from each other and from the strains that dominated in the processes of biooxidation of a similar concentrate by traditional tank technology at 39°C or at 39°C with treatment of the concentrate with ferric iron prior to biooxidation. These results indicate the strain and species diversity of sulfobacilli in microbial associations involved in biooxidation of the concentrates under different technological modes.     

Biofilm-Specific Cross-Species Induction of Antimicrobial Compounds in Bacilli

An air-membrane surface (AMS) bioreactor was designed to allow bacteria to grow attached to a surface as a biofilm in contact with air. When Bacillus licheniformis strain EI-34-6, isolated from the surface of a marine alga, was grown in this reactor, cells produced antimicrobial compounds which they did not produce when they were grown in shake flask cultures. An unidentified red pigment was also produced by surface-grown cells but not by planktonically grown cells. Glycerol and ferric iron were important for the production of antimicrobial compounds and the red pigment. Release of these secondary metabolites was not due to the onset of sporulation. Cell-free spent medium recovered from beneath the reactor membrane could induce production of antimicrobial compounds and red pigment in shake flask cultures. Neither glycerol nor ferric iron was required for production of these inducer compounds. Spent medium from beneath the membrane of an AMS bioreactor culture of Bacillus subtilis strain DSM10^T and Bacillus pumilus strain EI-25-8 could also induce production of antimicrobial compounds and a red pigment in B. licheniformis isolate EI-34-6 grown in shake flask cultures; however, the corresponding spent medium from shake flask cultures of DSM10^T and EI-25-8 could not. These results suggest that there is a biofilm-specific cross-species signaling system which can induce planktonically grown cells to behave as if they were in a biofilm by regulating the expression of pigments and antimicrobial compounds.     

生物造粒流化床污水处理反应器的微生物多样性

为了研究生物造粒流化床污水处理反应器颗粒污泥的微生物种群多样性,分别从生物造粒流化床10、60和110cm处取颗粒污泥,通过细胞裂解直接提取颗粒污泥细菌基因组DNA,PCR扩增后经变性梯度凝胶电泳(DGGE)分离,获得微生物群落的DNA特征指纹图谱,对特征条带进行序列测定及序列同源性分析。16S rRNA序列分析表明,获得的18个OTUs均属于细菌域,其中61%属于变形菌,17%属于放线菌,11%属于低G C革兰氏阳性菌,11%属于其它未知细菌。     

Copper Ions Stimulate Polyphosphate Degradation and Phosphate Efflux in Acidithiobacillus ferrooxidans

For some bacteria and algae, it has been proposed that inorganic polyphosphates and transport of metal-phosphate complexes could participate in heavy metal tolerance. To test for this possibility in Acidithiobacillus ferrooxidans, a microorganism with a high level of resistance to heavy metals, the polyphosphate levels were determined when the bacterium was grown in or shifted to the presence of a high copper concentration (100 mM). Under these conditions, cells showed a rapid decrease in polyphosphate levels with a concomitant increase in exopolyphosphatase activity and a stimulation of phosphate efflux. Copper in the range of 1 to 2 μM greatly stimulated exopolyphosphatase activity in cell extracts from A. ferrooxidans. The same was seen to a lesser extent with cadmium and zinc. Bioinformatic analysis of the available A. ferrooxidans ATCC 23270 genomic sequence did not show a putative pit gene for phosphate efflux but rather an open reading frame similar in primary and secondary structure to that of the Saccharomyces cerevisiae phosphate transporter that is functional at acidic pH (Pho84). Our results support a model for metal detoxification in which heavy metals stimulate polyphosphate hydrolysis and the metal-phosphate complexes formed are transported out of the cell as part of a possibly functional heavy metal tolerance mechanism in A. ferrooxidans.     

Biooxidation of Gold-, Silver,and Antimony-Bearing Highly Refractory Polymetallic Sulfide Concentrates,and its Comparison with Abiotic Pretreatment Techniques

Effectiveness of different pure and mixed cultures of three moderately thermophilic, extremely acidophilic bacterial strains (Acidimicrobium ferrooxidans ICP, Sulfobacillus sibiricus N1, Acidithiobacillus caldus KU) were investigated for biooxidation of highly refractory polymetallic gold ore concentrates. Despite of its complex mineralogy and the presence of a mixture of potentially inhibitory metals and metalloids, the concentrate was readily dissolved in defined mixed cultures including both iron and sulfur oxidizers, releasing as much as 80% of soluble Fe and 61% of soluble As. Factors to affect microbial mineral dissolution efficiencies (i.e. microbial As(III) oxidation ability, formation of secondary mineral precipitation (e.g. jarosite, elemental sulfur, scorodite, anglesite), and microbial population dynamics during biooxidation) were studied, based on which roles of individual microbes and their synergistic interactions during biooxidation were discussed. Applying the biooxidation pretreatment using the most efficient mixed cultures containing all three strains significantly improved the recovery of both Au (from 1.1% to 86%) and Ag (from 3.2% to 87%). Finally, this study provides one of the very few available comparisons of the effectiveness of different pretreatment techniques for refractory gold ore concentrates: Compared with other abiotic pretreatment approaches (roasting, pressure oxidation, and alkali dissolution), biooxidation was shown to be one of the most effective options in terms of the recovery of Au and Ag.