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Applied Microbiology and Biotechnology (v.63, #3)
Bioleaching review part A: by T. Rohwerder; T. Gehrke; K. Kinzler; W. Sand (pp. 239-248).
Bioleaching of metal sulfides is caused by astonishingly diverse groups of bacteria. Today, at least 11 putative prokaryotic divisions can be related to this phenomenon. In contrast, the dissolution (bio)chemistry of metal sulfides follows only two pathways, which are determined by the acid-solubility of the sulfides: the thiosulfate and the polysulfide pathway. The bacterial cell can effect this sulfide dissolution by “contact” and “non-contact” mechanisms. The non-contact mechanism assumes that the bacteria oxidize only dissolved iron(II) ions to iron(III) ions. The latter can then attack metal sulfides and be reduced to iron(II) ions. The contact mechanism requires attachment of bacteria to the sulfide surface. The primary mechanism for attachment to pyrite is electrostatic in nature. In the case of Acidithiobacillus ferrooxidans, bacterial exopolymers contain iron(III) ions, each complexed by two uronic acid residues. The resulting positive charge allows attachment to the negatively charged pyrite. Thus, the first function of complexed iron(III) ions in the contact mechanism is mediation of cell attachment, while their second function is oxidative dissolution of the metal sulfide, similar to the role of free iron(III) ions in the non-contact mechanism. In both cases, the electrons extracted from the metal sulfide reduce molecular oxygen via a complex redox chain located below the outer membrane, the periplasmic space, and the cytoplasmic membrane of leaching bacteria. The dominance of either At. ferrooxidans or Leptospirillum ferrooxidans in mesophilic leaching habitats is highly likely to result from differences in their biochemical iron(II) oxidation pathways, especially the involvement of rusticyanin.
Bioleaching review part B: by G. J. Olson; J. A. Brierley; C. L. Brierley (pp. 249-257).
This review describes the historical development and current state of metals leaching and sulfide mineral biooxidation by the minerals industries. During the past 20 years commercial processes employing microorganisms for mineral recovery have progressed from rather uncontrolled copper dump leaching to mineral oxidation and leaching in designed bioheaps for oxidation of refractory gold ores and for copper recovery. Also during this period of time, stirred tank bioleaching has been commercialized for cobalt recovery and for biooxidation of refractory gold ores. Chalcopyrite bioleaching in stirred tanks is on the verge of commercialization. Commercial applications of biohydrometallurgy have advanced due to favorable process economics and, in some cases, reduced environmental problems compared to conventional metal recovery processes such as smelting. Process development has included recognition of the importance of aeration of bioheaps, and improvements in stirred tank reactor design and operation. Concurrently, knowledge of the key microorganisms involved in these processes has advanced, aided by advances in molecular biology to characterize microbial populations.
Bacteria engineered for fuel ethanol production: current status by B. S. Dien; M. A. Cotta; T. W. Jeffries (pp. 258-266).
The lack of industrially suitable microorganisms for converting biomass into fuel ethanol has traditionally been cited as a major technical roadblock to developing a bioethanol industry. In the last two decades, numerous microorganisms have been engineered to selectively produce ethanol. Lignocellulosic biomass contains complex carbohydrates that necessitate utilizing microorganisms capable of fermenting sugars not fermentable by brewers' yeast. The most significant of these is xylose. The greatest successes have been in the engineering of Gram-negative bacteria: Escherichia coli, Klebsiella oxytoca, and Zymomonas mobilis. E. coli and K. oxytoca are naturally able to use a wide spectrum of sugars, and work has concentrated on engineering these strains to selectively produce ethanol. Z. mobilis produces ethanol at high yields, but ferments only glucose and fructose. Work on this organism has concentrated on introducing pathways for the fermentation of arabinose and xylose. The history of constructing these strains and current progress in refining them are detailed in this review.
Optimization of recombinant aminolevulinate synthase production in Escherichia coli using factorial design by L. Xie; D. Hall; M. A. Eiteman; E. Altman (pp. 267-273).
The production of recombinant Rhodobacter sphaeroides aminolevulinate (ALA) synthase was optimized in two strains of Escherichia coli: the wild-type strain MG1655, and a ptsG mutant AFP111. The effects of initial succinate, glucose and isopropyl-β-d-thiogalactopyranoside (IPTG) concentrations and the time of induction on enzyme activity were studied. One-way analysis was used to approximate the optimal ranges for these factors, followed by a full factorial design to quantify the effects of each factor and the interactions between the factors. Initial succinate, glucose, and IPTG concentration were observed to be the key factors affecting ALA synthase activity with the optimal levels determined to be above 6 g/l succinate, 0 g/l glucose, and 0.10 mM IPTG. ALA synthase activity was generally lower with AFP111 than with MG1655, and the effect of these three key factors was also lower with AFP111 than with MG1655. Based on the full factorial design results, a fermentation was completed that yielded 296 mU/mg protein with a final ALA concentration of 5.2 g/l (39 mM).
Conversion of aliphatic 2-acetoxynitriles by nitrile-hydrolysing bacteria by U. Heinemann; C. Kiziak; S. Zibek; N. Layh; M. Schmidt; H. Griengl; A. Stolz (pp. 274-281).
The enzymatic hydrolysis of the nitrile group of different 2-acetoxynitriles was investigated in order to obtain catalysts that chemoselectively hydrolyse nitriles in the presence of ester groups. The biotransformation of four 2-acetoxynitriles [2-acetoxybutenenitrile (ABN), 2-acetoxyheptanenitrile (AHN), 2-acetoxy-2-(2-furyl)acetonitrile (AFN), and 2-acetoxy-2,3,3-trimethylbutanenitrile (ATMB)] by different bacterial strains that synthesise nitrilases or nitrile hydratases was studied. ABN, AHN and AFN were converted by various microorganisms belonging to different bacterial genera (e.g. Pseudomonas or Rhodococcus) expressing either nitrilase or nitrile hydratase activities. In contrast, no metabolism of the sterically hindered substrate ATMB was observed. All wild-type strains investigated formed considerable amounts of cyanide and aldehydes from the 2-acetoxynitriles. This indicated the presence of esterases converting the 2-acetoxynitriles to 2-hydroxynitriles, which then spontaneously decomposed to the corresponding aldehydes and cyanide. In order to suppress unwanted side-reactions, biotransformations were performed with recombinant Escherichia coli strains that heterologously expressed nitrilase activities originating from Pseudomonas, Rhodococcus, or Synechocystis strains. The attempted conversion of the 2-acetoxynitriles to almost stoichiometric amounts of the corresponding 2-acetoxycarboxylic acids was finally achieved by using either a recombinant E. coli strain that highly overexpressed the nitrilase gene from the pseudomonad or the purified enzyme derived from this strain.
Improved GA1 production by Fusarium fujikuroi by J. L. Oller-López; J. Avalos; A. F. Barrero; J. E. Oltra (pp. 282-285).
Recent studies into gibberellin A1 (GA1) showed it to be physiologically more active than GA3 in plants of great agricultural interest, such as tomatoes, rice, peas, and sweet cherries. We describe here a simple procedure for obtaining large quantities of GA1 (1,500 mg/l) by incubating the FKMC1995 strain of Fusarium fujikuroi in a standard complex medium (SCM). We also compare the GA production of this strain with that of two other wild-type strains of F. fujikuroi (IMI58289, m567) in SCM and low-nitrogen medium and discuss the possible biogenetic mechanisms involved in the over-accumulation of GA1 by FKMC1995.
Synthesis of α-galactooligosaccharides with α-galactosidase from Lactobacillus reuteri of canine origin by G. Tzortzis; A. J. Jay; M. L. A. Baillon; G. R. Gibson; R. A. Rastall (pp. 286-292).
Crude cell-free extracts from Lactobacillus reuteri grown on cellobiose, maltose, lactose and raffinose were assayed for glycosidic activities. When raffinose was used as the carbon source, α-galactosidase was produced, showing the highest yield at the beginning of the stationary growth phase. A 64 kDa enzyme was purified by ultra- and gel filtration, and characterized for its hydrolytic and synthetic activity. Highest hydrolytic activity was found at pH 5.0 at 50 °C (K M 0.55 mM, V max 0.80 μmol min−1 mg−1 of protein). The crude cell-free extract was further used in glycosyl transfer reactions to synthesize oligosaccharides from melibiose and raffinose. At a substrate concentration of 23% (w/v) oligosaccharide mixtures were formed with main products being a trisaccharide at 26% (w/w) yield from melibiose after 8 h and a tetrasaccharide at 18% (w/w) yield from raffinose after 7 h. Methylation analysis revealed the trisaccharide to be 6′ α-galactosyl melibiose and the tetrasaccharide to be stachyose. In both cases synthesis ceased when hydrolysis of the substrate reached 50%.
Overexpression of the ATP-dependent helicase RecG improves resistance to weak organic acids in Escherichia coli by P. Steiner; U. Sauer (pp. 293-299).
Increased resistance to several weak organic acids was conferred on Escherichia coli by overexpression of the ATP-dependent helicase RecG and, to a lesser extent, by overexpressing the helicase RuvAB. This property of helicases was identified by reproducible selection of recG-bearing clones from genomic libraries of the acetate-resistant species Acetobacter aceti and Staphylococcus capitis. We show that overexpression of RecG from both species, but also from E. coli, increased the maximum biomass concentration attained by E. coli cultures that were grown in the presence of various weak organic acids and uncouplers. Furthermore, overexpression of RecG from A. aceti significantly improved the maximum growth rates of E. coli under weak organic acid challenge. Based on the known role of RecG in DNA replication/repair, our data provide a first indication that weak organic acids negatively affect DNA replication and/or repair, and that these negative effects may be counteracted by helicase activity.
A δ-endotoxin encoded in Pseudomonas fluorescens displays a high degree of insecticidal activity by R. Peng; A. Xiong; X. Li; H. Fuan; Q. Yao (pp. 300-306).
The short field-life of Bacillus thuringiensis (Bt) insecticidal crystal protein has limited its use. When the Bt toxin is produced in Pseudomonas fluorescens it can be encapsulated and retain its effectiveness for two to three times longer than other Bt formulations. In order to improve Bt expression, we have synthesized cryIA(c) Bt δ-endotoxin encoding region (GenBank AF537267) according to the usage codon of P. fluorescens and transformed the Bt toxin expression cassette into P. fluorescens strains. T7 RNA polymerase and the T7 promoter system were used to control expression of Bt toxin. SDS-PAGE and Western blotting assay revealed that the δ-endotoxin was expressed as 8% of the total protein in P. fluorescens. In in vitro tests, release of toxin from dead bacteria was demonstrated. Supplementation of diets with Bt toxin-containing Pseudomonas bacterium resulted in high mortality of cabbage butterfly (Pieris brassicae) larvae.
Pathways of methanol conversion in a thermophilic anaerobic (55 °C) sludge consortium by P. L. Paulo; A. J. M. Stams; J. A. Field; C. Dijkema; J. B. van Lier; G. Lettinga (pp. 307-314).
The pathway of methanol conversion by a thermophilic anaerobic consortium was elucidated by recording the fate of carbon in the presence and absence of bicarbonate and specific inhibitors. Results indicated that about 50% of methanol was directly converted to methane by the methylotrophic methanogens and 50% via the intermediates H2/CO2 and acetate. The deprivation of inorganic carbon species [Σ(HCO3 −+CO2)] in a phosphate-buffered system reduced the rate of methanol conversion. This suggests that bicarbonate is required as an electron (H2) sink and as a co-substrate for the efficient and complete removal of the chemical oxygen demand. Nuclear magnetic resonance spectroscopy was used to investigate the route of methanol conversion to acetate in bicarbonate-sufficient and bicarbonate-depleted environments. The proportions of [1,2-13C]acetate, [1-13C]acetate and [2-13C]acetate were determined. Methanol was preferentially incorporated into the methyl group of acetate, whereas HCO3 − was the preferred source of the carboxyl group. A small amount of the added H13CO3 − was reduced to form the methyl group of acetate and a small amount of the added 13CH3OH was oxidised and found in the carboxyl group of acetate when 13CH3OH was converted. The recovery of [13C]carboxyl groups in acetate from 13CH3OH was enhanced in bicarbonate-deprived medium. The small amount of label incorporated in the carboxyl group of acetate when 13CH3OH was converted in the presence of bromoethanesulfonic acid indicates that methanol can be oxidised to CO2 prior to acetate formation. These results indicate that methanol is converted through a common pathway (acetyl-CoA), being on the one hand reduced to the methyl group of acetate and on the other hand oxidised to CO2, with CO2 being incorporated into the carboxyl group of acetate.
Hydrogenases in sulfate-reducing bacteria function as chromium reductase by B. Chardin; M.-T. Giudici-Orticoni; G. De Luca; B. Guigliarelli; M. Bruschi (pp. 315-321).
The ability of sulfate-reducing bacteria (SRB) to reduce chromate VI has been studied for possible application to the decontamination of polluted environments. Metal reduction can be achieved both chemically, by H2S produced by the bacteria, and enzymatically, by polyhemic cytochromes c 3. We demonstrate that, in addition to low potential polyheme c-type cytochromes, the ability to reduce chromate is widespread among [Fe], [NiFe], and [NiFeSe] hydrogenases isolated from SRB of the genera Desulfovibrio and Desulfomicrobium. Among them, the [Fe] hydrogenase from Desulfovibrio vulgaris strain Hildenborough reduces Cr(VI) with the highest rate. Both [Fe] and [NiFeSe] enzymes exhibit the same K m towards Cr(VI), suggesting that Cr(VI) reduction rates are directly correlated with hydrogen consumption rates. Electron paramagnetic resonance spectroscopy enabled us to probe the oxidation by Cr(VI) of the various metal centers in both [NiFe] and [Fe] hydrogenases. These experiments showed that Cr(VI) is reduced to paramagnetic Cr(III), and revealed inhibition of the enzyme at high Cr(VI) concentrations. The significant decrease of both hydrogenase and Cr(VI)-reductase activities in a mutant lacking [Fe] hydrogenase demonstrated the involvement of this enzyme in Cr(VI) reduction in vivo. Experiments with [3Fe-4S] ferredoxin from Desulfovibrio gigas demonstrated that the low redox [Fe-S] (non-heme iron) clusters are involved in the mechanism of metal reduction by hydrogenases.
Sulfate-reducing bacterial community structure and their contribution to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions by S. Okabe; T. Ito; H. Satoh (pp. 322-334).
The community structure of sulfate-reducing bacteria (SRB) and the contribution of SRB to carbon mineralization in a wastewater biofilm growing under microaerophilic conditions were investigated by combining molecular techniques, molybdate inhibition batch experiments, and microelectrode measurements. A 16S rDNA clone library of bacteria populations was constructed from the biofilm sample. The 102 clones analyzed were grouped into 53 operational taxonomic units (OTUs), where the clone distribution was as follows: Cytophaga-Flexibacter-Bacteroides (41%), Proteobacteria (41%), low-G+C Gram-positive bacteria (18%), and other phyla (3%). Three additional bacterial clone libraries were also constructed from SRB enrichment cultures with propionate, acetate, and H2 as electron donors to further investigate the differences in SRB community structure due to amendments of different carbon sources. These libraries revealed that SRB clones were phylogenetically diverse and affiliated with six major SRB genera in the delta-subclass of the Proteobacteria. Fluorescent in situ hybridization (FISH) analysis revealed that Desulfobulbus and Desulfonema were the most abundant SRB species in this biofilm, and this higher abundance (ca. 2–4×109 cells cm–3 and 5×107 filaments cm–3, respectively) was detected in the surface of the biofilm. Microelectrode measurements showed that a high sulfate-reducing activity was localized in a narrow zone located just below the oxic/anoxic interface when the biofilm was cultured in a synthetic medium with acetate as the sole carbon source. In contrast, a broad sulfate-reducing zone was found in the entire anoxic strata when the biofilm was cultured in the supernatant of the primary settling tank effluent. This is probably because organic carbon sources diffused into the biofilm from the bulk water and an unknown amount of volatile fatty acids was produced in the biofilm. A combined approach of molecular techniques and batch experiments with a specific inhibitor (molybdate) clearly demonstrated that Desulfobulbus is a numerically important member of SRB populations and the main contributor to the oxidation of propionate to acetate in this biofilm. However, acetate was preferentially utilized by nitrate-reducing bacteria but not by acetate-utilizing SRB.