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Applied Microbiology and Biotechnology (v.48, #5)
Biohydrometallurgical processing of solids: a patent review by Christoph Brombacher; Reinhard Bachofen; Helmut Brandl (pp. 577-587).
Autotrophic as well as heterotrophic bacteria and fungi play an important role for the industrial recovery of metals from low-grade ore or, in general, from low-grade mineral resources. The same inorganic bacterial pathways that are responsible for huge and expensive corrosion problems can be used for economical biohydrometallurgical applications. Metals and metalloids can be microbially transformed by oxidation, reduction, alkylation, dealkylation, solubilization, and␣precipitation mechanisms. Biohydrometallurgy, a branch of classical metallurgy, is not as widely publicized as other areas of metallurgy, e.g. pyrometallurgical or hydrometallurgical processes. Since 1990, approximately 15 international patent applications concerning biohydrometallurgical techniques have been claimed under the Patent Cooperation Treaty in contrast to a large number of patents concerning pyro- and hydrometallurgical techniques. Nevertheless, it is a very important field of investigation, especially for the future when aspects of a sustainable development have to be considered. New processes to support this development are applicable, at least on a laboratory scale. Bacterial leaching processes for the recovery of metals from solid residues are applied for low-grade ore, and, more recently, for fly ash, galvanic sludges, or, in general, for industrial wastes. It is possible to recover leached metals and to recycle them in metal-manufacturing industries. In addition, by removing the metals from residues, the environmental quality is improved, and the material can be re-used for construction purposes.
Efficient production of medium-chain-length poly(3-hydroxyalkanoates) from octane by Pseudomonas oleovorans: economic considerations by W. Hazenberg; B. Witholt (pp. 588-596).
Poly(3-hydroxyalkanoates) (PHA) have the potential to become a biodegradable alternative for conventional plastics. In order to produce PHA at competitive costs in comparison with commonly used plastics, efficient PHA production systems will have to be developed. Poly(3-hydroxybutyrate) fermentations are well developed and in actual use on an industrial scale; medium-chain-length PHA (mcl-PHA) production is less well described, although the vast majority of all PHA known today are mcl-PHA. This paper compares and describes mcl-PHA production systems with respect to the volumetric productivity, the cellular PHA content and the polymer yield on carbon substrates. Nitrogen was shown to be the most effective limitation to trigger PHA formation in P. oleovorans after different nutrient limitations had been compared. By using an economic model for the calculation of PHA production costs, we show that it should be possible to produce octane-based mcl-PHA on a large scale (more than 1000 tonnes/year) at costs below U.S. $ 10 kg−1.
Influence of acetic acid on the growth of Escherichia coli K12 during high-cell-density cultivation in a dialysis reactor by K. Nakano; M. Rischke; S. Sato; H. Märkl (pp. 597-601).
High-cell-density cultivations of Escherichia coli K12 in a dialysis reactor with controlled levels of dissolved oxygen were carried out with different carbon sources: glucose and glycerol. Extremely high cell concentrations of 190 g/l and 180 g/l dry cell weight were obtained in glucose medium and in glycerol medium respectively. Different behaviour was observed in the formation of acetic acid in these cultivations. In glucose medium, acetic acid was formed during the earlier phase of cultivation. However, in glycerol medium, acetic acid formation started later and was particularly rapid at the end of the cultivation. In order to estimate the influence of acetic acid during these high-cell-density cultivations, the inhibitory effect of acetic acid on cell growth was investigated under different culture conditions. It was found that the inhibition of cell growth by acetic acid in the fermentor was much less than that in a shaker culture. On the basis of the results obtained in these investigations of the inhibitory effect of acetic acid, and the mathematical predictions of cell growth in a dialysis reactor, the influence of acetic acid on high-cell-density cultivation is discussed.
Keywords: Key wordsEscherichia coli; High-cell-density cultivation; Acetic acid; Inhibition; Dialysis reactor
Purification of the main manganese peroxidase isoenzyme MnP2 from the white-rot fungus Nematoloma frowardii b19 by I. Schneegaß; M. Hofrichter; K. Scheibner; W. Fritsche (pp. 602-605).
The main manganese peroxidase isoenzyme MnP2 of the South American white-rot fungus Nematoloma frowardii b19 was purified to homogeneity using anion-exchange chromatography (Mono Q) and preparative isoelectric focusing. The purified enzyme has a molecular mass of 44 kDa and a pI of 3.2.
Expression of the cefG gene is limiting for cephalosporin biosynthesis in Acremonium chrysogenum by S. Gutiérrez; J. Velasco; A. T. Marcos; F. J. Fernández; F. Fierro; J. L. Barredo; B. Díez; J. F. Martín (pp. 606-614).
The conversion of deacetylcephalosporin C to cephalosporin C is inefficient in most Acremonium chrysogenum strains. The cefG gene, which encodes deacetylcephalosporin C acetyltransferase, is expressed very poorly in A. chrysogenum as compared to other genes of the cephalosporin pathway. Introduction of additional copies of the cefG gene with its native promoter (in two different constructions with upstream regions of 1056 bp and 538 bp respectively) did not produce a significant increase of the steady-state level of the cefG transcript. Expression of the cefG gene from the promoters of (i) the glyceraldehyde-3-phosphate dehydrogenase (gpd ) gene of Aspergillus nidulans, (ii) the glucoamylase (gla) gene of Aspergillus niger, (iii) the glutamate dehydrogenase (gdh) and (iv) the isopenicillin N synthase ( pcbC ) genes of Penicillium chrysogenum, led to very high steady-state levels of cefG transcript and to increased deacetylcephalosporin-C acetyltransferase protein concentration (as shown by immunoblotting) and enzyme activity in the transformants. Southern analysis showed that integration of the new constructions occurred at sites different from that of the endogenous cefG gene. Cephalosporin production was increased two- to threefold in A. chrysogenum C10 transformed with constructions in which the cefG gene was expressed from the gdh or gpd promoters as a result of a more efficient acetylation of deacetylcephalosporin C.
Anthracyclines: isolation of overproducing strains by the selection and genetic recombination of putative regulatory mutants of Streptomycespeucetius var. caesius by D. Segura; C. Santana; R. Gosh; L. Escalante; S. Sanchez (pp. 615-620).
In Streptomyces peucetius var. caesius, the production of anthracyclines was suppressed either by 330 mM d-glucose or 25 mM phosphate. In addition, the anthracycline doxorubicin and the glucose analogue 2-deoxyglucose inhibited the growth of this microorganism at concentrations of 0.025 mM and 10 mM respectively. Spontaneous and induced mutants, resistant to the action of these compounds, were isolated, tested and chosen by their ability to overproduce anthracyclines. Genetic recombination between representative mutants was carried out by the protoplast fusion technique. Some recombinants carrying resistance to doxorubicin, phosphate and 2-deoxyglucose produced more than 40-fold greater levels of anthracyclines than those obtained with the parental strain. This improvement resulted in total antibiotic titres of more than 2 g/l culture medium at 6 days of fermentation.
Transformation system for a wastewater treatment yeast, Hansenula fabianii J640: isolation of the orotidine-5′-phosphate decarboxylase gene (URA3 ) and uracil auxotrophic mutants by M. Kato; H. Iefuji; K. Miyake; Y. Iimura (pp. 621-625).
A transformation system for Hansenula fabianii J640, a commonly used wastewater treatment yeast, was constructed. As a host cell, a uracil auxotrophic mutant designated as H. fabianii J640 u-1, which was confirmed to have a mutation at the locus of the gene for orotidine-5′-phosphate (OMP) decarboxylase (URA3), was obtained by positive selection using 5-fluoroorotic acid. A plasmid named pHFura3, which includes a 795-bp open-reading frame of the OMP decarboxylase H. fabianii, was obtained by complementation of the Escherichia colipyrF mutant. pHFura3 could transform H. fabianii J640 u-1 by a non-homologous and frequently multicopy integration into the host genomic DNA.
Variations in xanthan production by antibiotic-resistant mutants of Xanthomonas campestris by H. Rodríguez; L. Aguilar; M. LaO (pp. 626-629).
Mutants resistant to different antibiotics (streptomycin, tetracycline, ampicillin and penicillin) were obtained from several strains of Xanthomonas campestris and evaluated for xanthan production. Most of the mutants showed alterations in their polysaccharide production, either increasing, decreasing or totally losing their polymer-production capacity. The existence of two types of antibiotic-resistance mechanisms for the assayed drugs is suggested: one that affects xanthan production and another that does not. Differences in outer-membrane protein patterns of mutants that were simultaneously altered in antibiotic resistance and xanthan production were found, in comparison with their parental strains. These findings suggest the existence of a genetic relationship between antibiotic-resistance mechanisms and xanthan production. Some of the mutants obtained showed significant increases in broth viscosity and xanthan concentration. These results suggest that resistance to streptomycin and ampicillin can be used to obtain improved strains in plate screening assays.
The influence of a specific microelemental environment in alginate gel beads on the course of propionic acid fermentation by K. Czaczyk; K. Trojanowska; W. Grajek (pp. 630-635).
In this work the exchange of calcium, cobalt, iron, magnesium, zinc and manganese ions between alginate gel beads and casein medium was investigated. The high release of calcium ions from alginate to the medium and the biosorption of some metal ions were observed. The pure alginate gel adsorbed all the metal ions examined, from a fermentative medium. Gel with immobilized cells of two strains of Propionibacterium freudenreichii subsp. shermanii showed an active ability to adsorb only cobalt, iron and zinc ions. In this way, a special microelemental environment was created in the alginate gel. This resulted in an increase of propionic acid production and a decrease of vitamin B12 biosynthesis.
Effects of Ni(II) on respirometric oxygen uptake by M. (Lüle) Albek; Ü. Yetiş; C. F. Gökçay (pp. 636-641).
The effects of Ni(II), substrate and initial biomass concentrations on biochemical oxygen demand (BOD) were studied by using an electrolytic respirometer. The effects of Ni(II) (2.5, 5.0, 10.0, 25.0 mg/l) and substrate (325, 650, 1300 mg/l as chemical oxygen demand) in a synthetic wastewater with differing initial biomass concentrations (1, 10, 100 mg/l) were investigated. The biomass-to-metal ratio was found to be the most important parameter affecting the measured BOD values. The maximum specific growth rates were calculated and the results of batch respirometric experiments were analysed both by graphical and statistical methods. In statistical analyses, a factorial experimental design approach was followed and results were treated by multiple regression techniques. A mathematical model was developed to express the maximum oxygen uptake in terms of nickel, substrate and initial biomass concentrations and their magnitudes of their effects were compared. The biomass-to-metal ratio was found to be very significant so that another model that expresses oxygen uptake in relation to the biomass-to-metal ratio and also to substrate concentration was developed. Finally, the effect of Ni(II) was demonstrated to depend on both substrate and initial biomass concentrations. This effect was stimulatory at low concentrations of Ni(II), and complete inhibition was never observed even at the highest concentration of Ni(II) studied, which was 25.0 mg/l.
Effect of solvent adaptation on the antibiotic resistance in Pseudomonas putida S12 by S. Isken; P. M. A. C. Santos; J. A. M. de Bont (pp. 642-647).
The effect of the adaptation to toluene on the␣resistance to different antibiotics was investigated in the␣solvent-resistant strain Pseudomonas putida S12. We␣followed the process of the solvent adaptation of P.␣putida S12 by cultivating the strain in the presence␣of␣increasing concentrations of toluene and studied␣the correlation of this gradual adaptation to the resistance towards antibiotics. It was shown that the tolerance to various chemically and structurally unrelated antibiotics, with different targets in the cell, increased during this gradual adaptation. The survival of P. putida S12 in the presence of antibiotics like tetracycline, nigericin, polymyxin B, piperacillin or chloramphenicol increased 30- to and 1000-fold after adaptation to 600 mg/l toluene. However, cells grown in the absence of any solvents lost their adaptation to toluene even when grown in the presence of antibiotics. Results are discussed in terms of the physico-chemical properties of membranes as affected by the observed cis/trans isomerization of unsaturated fatty acids, as well as in terms of the active efflux of molecules from the cytoplasmic membrane.
Substrate inhibition under stationary growth conditions – nutristat experiments with Ralstonia eutropha JMP 134 during growth on phenol and 2,4-dichlorophenoxyacetate by R. H. Müller; D. Simon; H. J. Große; W. Babel (pp. 648-655).
Ralstonia eutropha (formerly Alcaligenes eutrophus) JMP 134 was continuously grown on phenol and 2,4-dichlorophenoxyacetate at elevated levels of stationary substrate concentration by using the nutristat principle in order to study the physiological impact exerted by these toxic substrates. Growth at stationary concentrations of both the substrates resulted in the reduction of growth efficiency and growth rate. The growth yield data revealed a pronounced dependence on the substrate concentration, and the growth yield increasingly diminished with rising substrate concentration. Inhibition was more pronounced with 2,4-dichlorophenoxyacetate, which reduced the growth yield coefficient by 50% at a substrate concentration of 0.1–0.25 mM. The same effect was obtained with phenol at about 5 mM. The growth rate profile had two distinct phases: after an initially strong reduction, the rate levelled-off at higher substrate concentrations. Standardizing the inhibition profiles, by taking into account the maximum effect after extrapolating the data to zero growth yield, revealed an almost identical pattern with both substrates, indicating some common mechanism. The growth yield data show that an increased amount of energy is required for both growth and maintenance. Homeostatic work was increased by a factor of 8 at 75% inhibition; growth collapsed once this amount of energy was no longer available. The effects are discussed with respect to the properties of these substrates functioning as potential uncouplers of energy conservation.
Growth yield coefficients of Sphingomonas sp. strain P5 on various chlorophenols in chemostat culture by M. Rutgers; A. M. Breure; J. G. van Andel; W. A. Duetz (pp. 656-661).
A polychlorophenol-degrading bacterium, Sphingomonas sp. strain P5, was grown in 2,6-dichlo-rophenol(26-DCP)-limited, 2,3,6-trichlorophenol(236-TCP)-limited, 2,4,6-trichlorophenol(246-TCP)-limited, 2,3,4,6-tetrachlorophenol(2346-TeCP)-limited, and pentachlorophenol(PCP)-limited chemostat cultures at a dilution rate of 0.02 ± 0.002 h−1. The cultures were analyzed for the yield coefficient for growth on chlorophenol during steady-state conditions. The average growth yields coefficients (as carbon conversion efficiencies) were 0.252, 0.230, 0.219, 0.157, and 0.121 mol C mol C−1 for 26-DCP, 236-TCP, 246-TCP, 2346-TeCP, and PCP respectively. The differences in growth yield can be interpreted in terms of the energetics of chlorinated carbon metabolism; i.e. substitution of the phenol moiety reduces the available metabolic energy by one electron per chlorine. The growth yield coefficients on chlorinated phenols were lower than the yield coefficients of heterotrophic growth reported in the literature on non-chlorinated and aliphatic compounds. Metabolic origins for low growth yield coefficients on (chlorinated) aromatic compounds are postulated.
Effect of aeration and carbon/nitrogen ratio on the molecular mass of the biodegradable polymer poly-β-hydroxybutyrate obtained from Azotobacter chroococcum 6B by J. C. Quagliano; Silvia S. Miyazaki (pp. 662-664).
The bacterial polyester poly-β-hydroxybutyrate (PHB) was quantified and characterized on an isolate␣of the nitrogen-fixing bacteria Azotobacter chroococcum 6B on the basis of its average molecular mass, determined from the relative viscosity at different aeration rates and carbon/nitrogen ratios during culture in fermentors. A higher value for the molecular mass (1100 kDa) was obtained with the lower aeration rates investigated, which diminished, significantly at the highest aeration rate of 2.5 vvm (a 100-fold decrease). The yield of PHB relative to the amount of glucose consumed increased with the C/N ratio (a maximum of 0.16 g PHB/g glucose consumed with a carbon/nitrogen ratio of 137.7), but the molecular mass was lowered from 800 kDa to nearly 100 kDa. The maximum PHB content was 63.5% (on a cellular dry-weight basis) after 47 h in fed-batch culture with an initial C/N ratio of 68.9 and aeration at a rate of 0.5 vvm. Calorimetric measurements on the isolated PHB showed a melting point near 175 °C.
Enhancing the mineralization of [U-14C]dibenzo-p-dioxin in three different soils by addition of organic substrate or inoculation with white-rot fungi by P. Rosenbrock; R. Martens; F. Buscot; F. Zadrazil; J. C. Munch (pp. 665-670).
The potential for aerobic mineralization of [U-14C]dibenzo-p-dioxin (DD) was investigated in samples of three different agricultural soils already contaminated with polychlorinated dibenzo-p-dioxins and dibenzofurans (PCDD/F) by industrial activities. The influence of amendments, i.e. wheat straw and compost, and of soil treatment by inoculation with lignolytic fungi, grown on wheat straw substrate, was tested. All the soils tested contained an indigenous DD-mineralizing microflora. The soil characterized by the highest organic matter content and the highest content of soil microbial biomass displayed the best DD mineralization of 36.6% within 70 days, compared with the two organic-matter-poor soils with an endogenous DD mineralization of 19.5% and 23.3% respectively. Amendments with compost increased DD mineralization up to 28% in both soils with low organic matter and microbial biomass content, but did not affect mineralization in the organic-matter-rich soil. Addition of wheat straw had no constant influence on DD mineralization in the soils tested. The best DD mineralization resulted from inoculation with lignolytic white-rot fungi (Phanerochaete chrysosporium, Pleurotus sp. Florida, Dichomitus squalens) and with an unidentified lignolytic fungus, which was isolated originally from a long-term PCDD/F-contaminated soil. A mineralization of up to 50% within 70 days was reached by this treatment. The influence of inoculated fungi on mineralization differed between the soils investigated.
Continuous vapor-phase trichloroethylene biofiltration using hydrocarbon-enriched compost as filtration matrix by S. Sukesan; M. E. Watwood (pp. 671-676).
Two sources of finished compost material were examined for the capacity to support trichloroethylene(TCE)-degrading microbial populations in a gas-phase bioreactor. Gaseous hydrocarbon was passed through the bioreactor to stimulate cometabolic oxidation of TCE. Significant differences in TCE removal efficiencies were observed between the two compost types, and between hydrocarbon-stimulated and non-stimulated compost. At an average column retention time of 5.6 min, deciduous leaf debris compost removed more than 95% of a 5–50 ppm (by vol.) TCE gas stream, whereas less than 15% removal was observed under similar conditions with a woodchip and bark compost. Trichloroethylene removal efficiency varied with the hydrocarbon-stimulation regime employed, although propane and methane stimulated TCE degradation equally well. Amendment of compost with granular activated carbon substantially increased biological TCE removal. Differences in TCE removal efficiencies observed between the two compost types and between hydrocarbon-stimulated and non-stimulated composts were investigated in terms of changes in the overall heterotrophic microbial populations by using community-level physiological profile analysis.