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Applied Microbiology and Biotechnology (v.54, #5)
Microbial degradation of explosives: biotransformation versus mineralization by J. Hawari; S. Beaudet; A. Halasz; S. Thiboutot; G. Ampleman (pp. 605-618).
The nitroaromatic explosive 2,4,6-trinitrotoluene (TNT) is a reactive molecule that biotransforms readily under both aerobic and anaerobic conditions to give aminodinitrotoluenes. The resulting amines biotransform to give several other products, including azo, azoxy, acetyl and phenolic derivatives, leaving the aromatic ring intact. Although some Meisenheimer complexes, initiated by hydride ion attack on the ring, can be formed during TNT biodegradation, little or no mineralization is encountered during bacterial treatment. Also, although the ligninolytic physiological phase and manganese peroxidase system of fungi can cause some TNT mineralization in liquid cultures, little to no mineralization is observed in soil. Therefore, despite more than two decades of intensive research to biodegrade TNT, no biomineralization-based technologies have been successful to date. The non-aromatic cyclic nitramine explosives hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) and octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) lack the electronic stability enjoyed by TNT or its transformed products. Predictably, a successful enzymatic change on one of the N–NO2 or C–H bonds of the cyclic nitramine would lead to a ring cleavage because the inner C–N bonds in RDX become very weak (<2 kcal/mol). Recently this hypothesis was tested and proved feasible, when RDX produced high amounts of carbon dioxide and nitrous oxide following its treatment with either municipal anaerobic sludge or the fungus Phanaerocheate chrysosporium. Research aimed at the discovery of new microorganisms and enzymes capable of mineralizing energetic chemicals and/or enhancing irreversible binding (immobilization) of their products to soil is presently receiving considerable attention from the scientific community.
Genetic transformation and biotechnological application of the yeast Arxula adeninivorans by T. Wartmann; G. Kunze (pp. 619-624).
The relatively unknown, non-pathogenic, dimorphic, haploid, ascomycetous yeast Arxula adeninivorans exhibits some unusual properties which are of biotechnological interest. The yeast is able to assimilate and ferment many compounds as sole source of carbon and/or nitrogen, it utilises n-alkanes and degrades starch efficiently. A. adeninivorans features such as thermo- and haloresistance as well as the yeast's uncommon growth and secretion behaviour should be especially emphasised. In media containing up to 20% NaCl, A. adeninivorans is able to grow at cultivation temperatures up to 48 °C. Additionally, the dimorphism of the yeast is unusual. Arxula grows at up temperatures of up to 42 °C as budding cells, which turn into mycelia at higher temperatures. This environmentally conditioned dimorphism is reversible and budding is reestablished when the cultivation temperature is decreased below 42 °C. Alteration of morphology correlates with changes in secretion behaviour. Mycelium cultures accumulate two-fold higher protein concentrations and contain two- to five-fold higher glucoamylase and invertase activities in the medium than budding cells. Based on these unusual properties, Arxula adeninivorans is used for heterologous gene expression and as a gene donor to construct more suitable yeasts for biotechnology. For example the Arxula glucoamylase gene was successfully expressed in Saccharomyces cerevisiae and Kluyveromyces lactis. Both transformed yeasts are able to assimilate and ferment starch as carbon source. A transformation system is used for heterologous gene expression which is based on integration of linearised DNA fragments in two to ten copies, e.g. into the 25S rDNA of A. adeninivorans by homologous recombination. The obtained transformants are mitotically stable. The expression of the lacZ gene from E. coli as well as the XylE gene from Pseudomonas putida indicates the suitability of A. adeninivorans as host for heterologous gene expression.
Pseudomonas aeruginosa rhamnolipids: biosynthesis and potential applications by R. M. Maier; G. Soberón-Chávez (pp. 625-633).
Pseudomonas aeruginosa produces and secretes rhamnose-containing glycolipid biosurfactants called rhamnolipids. This review describes rhamnolipid biosynthesis and potential industrial and environmental applications of rhamnolipids. Rhamnolipid production is dependent on central metabolic pathways, such as fatty acid synthesis and dTDP-activated sugars, as well as on enzymes participating in the production of the exopolysaccharide alginate. Synthesis of these surfactants is regulated by a very complex genetic regulatory system that also controls different P. aeruginosa virulence-associated traits. Rhamnolipids have several potential industrial and environmental applications including the production of fine chemicals, the characterization of surfaces and surface coatings, as additives for environmental remediation, and as a biological control agent. Realization of this wide variety of applications requires economical commercial-scale production of rhamnolipids.
Continuous marennin production by agar-entrapped Haslea ostrearia using a tubular photobioreactor with internal illumination by T. Lebeau; P. Gaudin; G.-A. Junter; L. Mignot; J.-M. Robert (pp. 634-640).
The marine diatom Haslea ostrearia was immobilized in a tubular agar gel layer introduced into a photobioreactor of original design with internal illumination for the continuous synthesis of marennin, a blue-green pigment of biotechnological interest. Marennin was produced for a long-term period (27–43 days) and the volumetric productivity was maximum (18.7 mg day−1 l−1 gel) at the highest dilution rate (0.25 day−1) and lowest agar layer thickness (3 mm). Heterogeneous cell distribution in the agar layer revealed diffusional limitation of light and nutrients. However, the 3 mm gel thickness led to a more homogeneous cell distribution during incubation and to an increase of the whole biomass in the agar gel layer.
Continuous production of enantiopure 1,2-epoxyhexane by yeast epoxide hydrolase in a two-phase membrane bioreactor by W. J. Choi; C. Y. Choi; J. A. M. De Bont; C. A. G. M. Weijers (pp. 641-646).
A two-phase membrane bioreactor was developed to continuously produce enantiopure epoxides using the epoxide hydrolase activity of Rhodotorula glutinis. An aqueous/organic cascade, hydrophilic, hollow-fiber membrane bioreactor was used: (1) to carry out large-scale resolution of epoxides, (2) to continuously extract residual enantiopure epoxides from the aqueous phase, and (3) to separate inhibitory formed diol from the yeast cells contained in the aqueous phase. Dodecane was employed to dissolve-feed epoxide as well as to extract residual epoxide. 1,2-Epoxyhexane was used as a model substrate. By use of this membrane bioreactor, enantiopure (S)-1,2-epoxyhexane (>98% enantiomeric excess) was obtained with a volumetric productivity of 3.8 g l−1 h−1. The continuous-production system was operated for 12 days and resulted in 38 g enantiopure (S)-1,2-epoxyhexane.
Characterization of salt-tolerant mutant for enhancement of l-threonine production in Escherichia coli by K.-H. Song; H.-H. Lee; H.-H. Hyun (pp. 647-651).
Escherichia coli strain HS3, metabolically engineered to have Met–, AHVr, IleL and AECr characteristics, produced 58.0 g/l of l-threonine, but it was neither salt-tolerant nor osmotolerant; and the growth and threonine production of the strain were severely inhibited both by the addition of NaCl with a concentration higher than 2% and by the presence of glucose with a concentration higher than 10%. Therefore, salt-tolerant mutants were isolated. The salt-tolerant mutants, HS454 and HS528 which were derived from strain HS3, were both tolerant to salt (2%) and hyperproductive. The growth and l-threonine production by the mutant strain HS454 were almost unaffected by a glucose concentration lower than 10%, but gradually reduced with increasing glucose concentration, up to 15%. However, the mutant strain HS528 showed slightly enhanced growth and l-threonine production with increasing glucose concentration, up to 10–12.5%. Strains HS454 and HS528 produced 69.8 g/l and 74.0 g/l of l-threonine, respectively in a 5-l jar fermentor.
Microbial and cytoplasmic membrane-based potentiometric biosensors for direct determination of organophosphorus insecticides by S. Gäberlein; F. Spener; C. Zaborosch (pp. 652-658).
Potentiometric biosensors for the determination of organophosphorus (OP) insecticides were developed by applying either immobilized whole cells or cytoplasmic membrane fractions of wild-type Flavobacterium sp. on the surface of a glass pH electrode. The ability of Flavobacterium sp. to degrade OP compounds as sole carbon source was demonstrated for parathion with a degradation rate of almost 100% after 30 min and for chlorpyrifos of 33% after 48 h incubation. The products of hydrolysis of these compounds, p-nitrophenol and 3,5,6-trichloro-2-pyridinol, were accumulated in the medium and not used as substrates for growth by Flavobacterium sp. In the course of hydrolysis, which is catalyzed by organophosphorus hydrolase, two protons are released for each substrate molecule hydrolyzed. This stoichiometry forms the electrochemical basis of the potentiometric biosensors. Direct determination without previous extraction of OP was carried out in a stirred measuring cell with a pH electrode as transducer. Poly(carbamoyl sulfonate) (PCS) prepolymer, a hydrogel with good adhesive properties, was used for immobilization of whole cells and membrane-associated organophosphorus hydrolase. The sensor with cytoplasmic membrane fractions was superior to the one with whole cells and showed a linear range for paraoxon from 0.01 to 0.47 mM and 3 weeks' working stability.
Glutathione-mediated mineralization of 14C-labeled 2-amino-4,6-dinitrotoluene by manganese-dependent peroxidase H5 from the white-rot fungus Phanerochaete chrysosporium by B. Van Aken; M. D. Cameron; J. D. Stahl; A. Plumat; H. Naveau; S. D. Aust; S. N. Agathos (pp. 659-664).
Manganese-dependent peroxidase (MnP) H5 from the white-rot fungus Phanerochaete chrysosporium, in the presence of either Mn(II) (10 mM) or GSH (10 mM), was able to mineralize 14C-U-ring-labeled 2-amino-4,6-dinitrotoluene (2-A-4,6-DNT) up to 29% in 12 days. When both Mn(II) and GSH were present, the mineralization extent reached 82%. On the other hand, no significant mineralization was observed in the absence of both Mn(II) and GSH, suggesting the requirement of a mediator [either Mn(II) or GSH] for the degradation of 2-A-4,6-DNT by MnP. Using electron spin resonance (ESR) techniques, it was found that the glutathionyl free radical (GS•) was produced through the oxidation of GSH by MnP in the presence as well as in the absence of Mn(II). GS• was also generated through the direct oxidation of GSH by Mn(III). Our results strongly suggest the involvement of GS• in the GSH-mediated mineralization of 2-A-4,6-DNT by MnP.
Homologous functional expression of cryptic phaG from Pseudomonas oleovorans establishes the transacylase-mediated polyhydroxyalkanoate biosynthetic pathway by N. Hoffmann; A. Steinbüchel; B. H. A. Rehm (pp. 665-670).
Various pseudomonads are capable of the synthesis of polyhydroxyalkanoate (PHA), composed of medium chain length (MCL) 3-hydroxy fatty acids (C6—C14), when grown on simple carbon sources such as, for example, gluconate or acetate. In Pseudomonas putida, the fatty acid de novo synthesis and PHA synthesis are linked by the transacylase PhaG. Southern hybridization experiments with digoxigenin-labeled phaG Pp from P. putida and genomic DNA from various pseudomonads indicate that phaG homologues are present in various other pseudomonads. Although P. oleovorans does not accumulate PHAMCL from non-related carbon sources, its genomic DNA reveals a strong hybridization signal. We employed PCR to amplify this phaG homologue. The respective PCR product comprising the coding region of phaG Po was cloned into pBBR1MCS-2, resulting in plasmid pBHR84. DNA sequencing revealed that putative PhaGPo from P. oleovorans exhibited about 95% amino acid sequence identity to PhaGPp from P. putida. Reverse transcriptase-PCR analysis demonstrated that phaG Po was not transcribed even under inducing conditions, i.e. in the presence of gluconate as carbon source, whereas induction of phaG Pp transcription was obtained in P. putida. When octanoate was used as sole carbon source, only low levels of phaG mRNA were detected in P. putida. Plasmid pBHR84 complemented the phaG-negative mutant PhaGN-21 from P. putida. Interestingly, reintroduction of phaG Po under lac promoter control into the natural host P. oleovorans established PHAMCL synthesis from non-related carbon sources in this bacterium. These data indicated that phaG Po in P. oleovorans is not functionally expressed and does not exert its original function.
Recombinant expression analysis of natural and synthetic bovine alpha-casein in Escherichia coli by S. K. Goda; A. F. Sharman; M. Yates; N. Mann; N. Carr; N. P. Minton; J. K. Brehm (pp. 671-676).
As a prelude to developing a yeast-based fermentation process for the production of phenylalanine-free alpha-casein as a foodstuff for patients suffering from phenylketonuria, we cloned the gene encoding bovine alpha-casein. We synthesised a modified gene sequence encoding the same, but devoid of phenylalanine codons and with a codon bias similar to that of naturally occurring highly expressed genes in Saccharomyces cerevisiae. The results show that both gene sequences are readily expressed in Escherichia coli when cloned in an E. coli bacteriophage T7 promoter-driven plasmid vector. In this host, the natural and synthetic casein proteins were produced at levels equating to 18.0% and 7.6% of the cell's soluble protein, respectively.
Targeted, PCR-based gene disruption in cyanobacteria: inactivation of the polyhydroxyalkanoic acid synthase genes in Synechocystis sp. PCC6803 by G. Taroncher-Oldenburg; G. Stephanopoulos (pp. 677-680).
A PCR-based method is described for the efficient construction of targeted gene disruptions and gene fusions in the cyanobacterium Synechocystis sp. PCC6803. In a simple two-step PCR approach, a gene conversion cassette was synthesized targeting the polyhydroxyalkanoic acid (PHA) synthase genes. Upon transformation, PHA production in Synechocystis under normal as well as high production culture conditions was undetectable. The application of this method to the genetic inactivation of the phaE-C Syn gene cluster demonstrates its potential for genetic engineering of cyanobacteria and the study of functional genomics in Synechocystis.
New ways of determining structural groups in brown coals and their bioconversion products by FT IR spectroscopy by A. Weber; S. Tesch; B. Thomas; H. Schmiers (pp. 681-685).
New methods of determining the structural groups —COOH and —CH2— have been developed. The investigation of carboxyl groups is possible both after derivatization with p-fluorophenacylbromide and by quantitative interpretation of the Fourier transform infrared (FT IR) spectra. There exists a linear relationship between the results of these two methods that is generally valid for the analysis of all brown coal components. The maximum extinction coefficient of the symmetric stretching vibration band of the CH2 groups has been determined using model substances. This allows quantification of this structural group directly from the FT IR spectrum. The results agree with the contents of methylene groups as determined by 13C-cross polarization–magic angle spinning–nuclear magnetic resonance (13C CPMAS NMR) spectroscopy. Using these methods, the COOH and CH2 groups contained in brown coals of the North Rhine region and in their bioconversion products have been quantified.
Differential expression of manganese peroxidase and laccase in white-rot fungi in the presence of manganese or aromatic compounds by T. Scheel; M. Höfer; S. Ludwig; U. Hölker (pp. 686-691).
White-rot fungi (basidiomycetes) play an important role in the degradation of lignin which is, beside cellulose, the major compound of wood. This process is catalyzed by ligninolytic enzymes, which are able to cleave oxidatively aromatic rings in lignin structure. Manganese peroxidase and laccase of white-rot-fungi are the most important of these among the ligninolytic enzymes. In addition, they are able to degrade xenobiotic aromatic polymers, persisting as environmental pollutants. Manganese and aromatic compounds have often been discussed as being inducers, enhancers or mediators of these ligninolytic enzymes. It is known that supplementing the growth medium with either Mn2+, veratryl alcohol or coal-derived humic acids leads to significantly enhanced extracellular ligninolytic activities. Measuring the amount of expressed mRNA of the two enzymes by quantitative RT-PCR provided evidence that the expression of manganese peroxidase was induced in the three tested white-rot fungi, Clitocybula dusenii b11, Nematoloma frowardii b19, and a straw-degrading strain designated i63–2. Laccase, on the other hand, was expressed in all three fungi with a significant basic activity even without inducer added. However, since the level of laccase mRNA was higher in cultures supplemented with any one of the tested inducers, we conclude that both manganese and the aromatic substances also increase the expression of laccase.
Anaerobic biodegradability of alkylphenols and fuel oxygenates in the presence of alternative electron acceptors by L. Puig-Grajales; N. G. Tan; F. van der Zee; E. Razo-Flores; J. A. Field (pp. 692-697).
Alkylphenols and fuel oxygenates are important environmental pollutants produced by the petrochemical industry. A batch biodegradability test was conducted with selected ortho-substituted alkylphenols (2-cresol, 2,6-dimethylphenol and 2-ethylphenol), fuel oxygenates (methyl tert-butyl ether, ethyl tert-butyl ether and tert-amylmethyl ether) and tert-butyl alcohol (TBA) as model compounds. The ortho-substituted alkylphenols were not biodegraded after 100 days of incubation under methanogenic, sulfate-, or nitrate-reducing conditions. However, biodegradation of 2-cresol and 2-ethylphenol (150 mg l−1) was observed in the presence of Mn (IV) as electron acceptor. The biodegradation of these two compounds took place in less than 15 days and more than 90% removal was observed for both compounds. Mineralization was indicated since no UV-absorbing metabolites accumulated after 23 days of incubation. These alkylphenols were also slowly chemically oxidized by Mn (IV). No biodegradation of fuel oxygenates or TBA (1 g l−1) was observed after 80 or more days of incubation under methanogenic, Fe (III)-, or Mn (IV)-reducing conditions, suggesting that these compounds are recalcitrant under anaerobic conditions. The fuel oxygenates caused no toxicity towards acetoclastic methanogens activity in anaerobic granular sludge.
The production of hemicellulases by Thermomyces lanuginosus strain SSBP: influence of agitation and dissolved oxygen tension by S. Singh; J. C. du Preez; B. Pillay; B. A. Prior (pp. 698-704).
Shake-flask cultivation of T. lanuginosus strain SSBP on coarse corn cobs yielded β-xylanase levels of 56,500 nkat/ml at 50 °C, whereas other hemicellulases (β-xylosidase, β-glucosidase, and α-l-arabinofuranosidase) were produced at levels less than 7 nkat/ml. Cultivation on d-xylose yielded much lower levels of xylanase (350 nkat/ml), although other hemicellulase levels were similar to those produced on corn cobs. The influence of agitation rate and dissolved oxygen tension (DOT) on hemicellulase production was studied further in a bioreactor. On xylose, xylanase activities of 4,330 nkat/ml and 4,900 nkat/ml were obtained at stirrer speeds up to 1,400 rpm to control DOT. At a constant stirrer speed of 400 rpm, xylanase activities of 10,930 nkat/ml and 15,630 nkat/ml were obtained when cultivated on xylose and beechwood xylan respectively, despite DOT levels below 5% for the duration of fermentation. The results indicate that there is an interaction between agitation rate and DOT, impacting on xylanase and accessory enzyme production. Higher agitation rates favoured the production of xylosidase, arabinofuranosidase and glucosidase by T. lanuginosus strain SSBP, whereas the lower agitation rates favoured xylanase production. Rheological difficulties precluded cultivation on corn cobs in the bioreactor. Volumetric xylanase productivities of 1,060,000 nkat/l · h and 589,000 nkat/l · h obtained on beechwood xylan and xylose indicate that T. lanuginosus strain SSBP is a hyper-xylanase producer with considerable industrial potential.
Effect of glycine betaine on osmoadaptation of Propionibacterium acidipropionici cultivated in elevated osmolarities by A. K. Kylmä; J. Jokela; M. Leisola (pp. 705-710).
The sensitivity of industrial strains Acetobacter aceti, Gluconobacter frateurii, and Propionibacterium acidipropionici to osmotic stress was studied. Growth of A. aceti and G. frateurii was totally inhibited at 0.4 M NaCl concentration, but P. acidipropionici was able to grow on a medium containing 1.2 M NaCl. Addition of glycine betaine to the medium had no detectable osmoprotective effect on A. aceti and G. frateurii cultivations in elevated NaCl concentrations, but it enabled cells of P. acidipropionici to achieve faster the maximum specific growth rate after the prolonged lag phase and therefore to gain faster the final biomass and product concentrations. The final concentrations of biomass and product of P. acidipropionici were the same as for the cultivations of the bacterium without NaCl and glycine betaine present in the medium. Intracellular accumulation of glycine betaine was detected in P. acidipropionici cells cultivated in the medium containing glycine betaine. The amount accumulated increased with NaCl concentration, suggesting that glycine betaine plays an important role in the osmoadaptation.
The solvent efflux system of Pseudomonas putida S12 is not involved in antibiotic resistance by S. Isken; J. A. M. De Bont (pp. 711-714).
The active efflux system contributing to the solvent tolerance of Pseudomonas putida S12 was characterized physiologically. The mutant P. putida JK1, which lacks the active efflux system, was compared with the wild-type organism. None of 20 known substrates of common multi-drug-resistant pumps had a stronger growth-inhibiting effect on the mutant than on the wild type. The amount of [14C]toluene accumulating in P. putida S12 increased in the presence of the solvent xylene and in the presence of uncouplers. The effect of uncouplers confirms the proton dependency of the efflux system in P. putida S12. Other compounds, potential substrates for the solvent pump, did not affect the accumulation of [14C]toluene. These results show that the efflux system in P. putida S12 is specific for organic solvents and does not export antibiotics or other known substrates of multi-drug-resistant pumps.
Identification of interacting mixed cultures of lactic acid bacteria by their exclusion from a model predicting the acidifying activity of non-interacting mixed cultures by I. Sodini; E. Latrille; G. Corrieu (pp. 715-718).
A model predicting the acidifying activity of mixed cultures of lactic acid bacteria and based on the lack of interaction between the strains has been investigated to identify interacting cultures. Three mixed cultures with Streptococcus thermophilus TH3 and ST7 and Lactobacillus delbrueckii ssp. bulgaricus LB10 were grown on milk. The acidifying activities of the two mixed cultures TH3/LB10 and TH3/ST7 were predicted accurately by the model, with mean prediction errors of 7.7% and 14.1%, respectively. However, the model underestimated the acidifying activity of the mixed culture ST7/LB10, with a mean prediction error of 43.5%, which provides evidence of positive interaction between the strains ST7 and LB10 during acidification.
Reclamation of an activated-sludge microbial consortium by selective biostimulation by K. Watanabe; M. Miyashita; S. Harayama (pp. 719-723).
Our previous study showed that an activated-sludge process broke down at the phenol-loading rate of 1.5 g l−1 day−1, when non-flocculating bacteria (called R6T and R10) overgrew the sludge, resulting in a sludge washout. In this study, we attempted to circumvent this breakdown problem by reclaiming the consortium structure. Activated sludge was fed phenol, and the phenol-loading rate was increased stepwise from 0.5 g l−1 day−1 to 1.0 g l−1 day−1 and then to 1.5 g l−1 day−1. Either galactose or glucose (at 0.5 g l−1 day−1) was also supplied to the activated sludge from the phenol-loading rate of 1.0 g l−1 day−1. Pure culture experiments have suggested galactose to be a preferential substrate for a floc-forming bacterium (R6F) that predominantly degrades phenol under low phenol-loading conditions. Supplying galactose allowed sustainment of the R6F population and suppression of the overgrowth of R6T and R10 at the phenol-loading rate of 1.5 g l−1 day−1. This measure allowed the activated-sludge process to treat phenol at a phenol-loading rate up to 1.5 g l−1 day−1, although it broke down at 2.0 g l−1 day−1. In contrast, supplying glucose reduced the R6F population and allowed the activated-sludge process to break down at the phenol-loading rate of 1.0 g l−1 day−1. This study demonstrated that reclamation of the activated-sludge consortium by selective biostimulation of the floc-forming population improved the phenol-treating ability of the process.