Effects of<Emphasis Type="Italic"> Vitreoscilla</Emphasis> hemoglobin on the 2,4-dinitrotoluene (2,4-DNT) dioxygenase activity of<Emphasis Type="Italic"> Burkholderia</Emphasis> and on 2,4-DNT degradation in two-phase bioreactors

Expression of vgb, encoding Vitreoscilla hemoglobin (VHb), in Burkholderia strain YV1 was previously shown to improve cell growth and enhance 2,4-dinitrotoluene (2,4-DNT) degradation compared with control strain DNT, especially under hypoxic conditions. In the work reported here, the ratio of 2,4-DNT degraded to oxygen uptake was approximately 5-fold larger for strain YV1 than for strain DNT. The addition of purified VHb to cytosolic fractions of strain DNT increased 2,4-DNT degradation 1.5-fold, compared with 1.1-fold for control bovine Hb, but increased the 2,4-DNT degradation 2.7-fold when added to partially purified 2,4-DNT dioxygenase, compared with 1.3-fold for bovine Hb. This suggests a direct transfer of oxygen from VHb to the oxygenase. In a bioreactor at high 2,4-DNT concentration (using 100 ml oleyl alcohol containing 2 g 2,4-DNT as the second phase) with 1.5 l culture, both strains could remove 0.8 g 2,4-DNT by 120 h; and, under the same conditions in a fed-batch reactor, the degradation increased to 1 g for strain YV1 but not for strain DNT.     

Cloning and expression of Vitreoscilla hemoglobin gene in Burkholderia sp. strain DNT for enhancement of 2,4-dinitrotoluene degradation

The gene (vgb) encoding the hemoglobin (VHb) of Vitreoscilla sp. was cloned into a broad host range vector and stably transformed into Burkholderia (formerly Pseudomonas) sp. strain DNT, which is able to degrade and metabolize 2,4-dinitrotoluene (DNT). Vgb was stably maintained and expressed in functional form in this recombinant strain (YV1). When growth of YV1, in both tryptic soy broth and minimal salts broth containing DNT and yeast extract, was compared with that of the untransformed strain, YV1 grew significantly better on a cell mass basis (A(600)) and reached slightly higher maximum viable cell numbers. YV1 also had roughly twice the respiration as strain DNT on a cell mass basis, and in DNT-containing medium, YV1 degraded DNT faster than the untransformed strain. YV1 cells pregrown in medium containing DNT plus succinate showed the fastest degradation: 100% of the initial 200 ppm DNT was removed from the medium within 3 days.     

Enhancement of 2,4-dinitrotoluene biodegradation by Burkholderia sp. in sand bioreactors using bacterial hemoglobin technology

Continuous flow sand column bioreactor experiments were conducted to investigate the effect of 2,4-dinitrotoluene (DNT) concentration (i.e. DNT loading rate) and influent dissolved oxygen (DO) concentration on aerobic biodegradation of DNT by wild type (DNT) and recombinant (YV1) Burkholderia sp., the latter containing plasmid pSC160 which carries the gene (vgb) encoding the hemoglobin (VHb) from the bacterium Vitreoscilla. The experiments were conducted in two continuous flow packed bed sand column bioreactors, one growing the wild type strain and the other growing YV1. Under oxygen-rich feed conditions (6.8 mg DO/L in the feed) with an influent DNT concentration of 99.6 mg/L (DNT loading rate approximately = 9.2 mg/m2/day), the effluent DNT concentration from the wild type bioreactor reached 0.7 mg DNT/L in 40 days whereas it was less than 0.2 mg DNT/L for the YV1 bioreactor in about 25 days. When influent DNT concentration was increased to 214 mg/L (DNT loading rate approximately = 20.3 mg/m2/day) while maintaining the same influent DO level of 6.8 mg/L, the effluent DNT concentration increased to about 5 mg/L for the wild type bioreactor whereas it was maintained at less than 0.2 mg/L for the YV1 bioreactor. Additionally, when influent DO was reduced from 6.8 mg/L to 3.1 mg/L while the influent DNT concentration remained at 214 mg/L, the effluent DNT concentration increased to more than 20 mg/L for the wild type bioreactor but up to only 1.7 mg/L for the YV1 bioreactor. A subsequent increase of influent DO back to 6.6 mg/L reduced the effluent DNT concentration to about 5 mg/L for the wild type bioreactor and to 0.10-0.19 mg/L for the YV1 bioreactor. These results confirm the utility of vgb technology to enhance biodegradation of aromatic compounds under hypoxic conditions and also that this enhancement can be maintained over extended periods of time as evidenced by plasmid stability in Burkholderia YV1.     

Saturation mutagenesis of 2,4-DNT dioxygenase of Burkholderia sp. strain DNT for enhanced dinitrotoluene degradation

2,4-Dinitrotoluene (2,4-DNT) and 2,6-DNT are priority pollutants, and 2,4-DNT dioxygenase of Burkholderia sp. strain DNT (DDO) catalyzes the initial oxidation of 2,4-DNT to form 4-methyl-5-nitrocatechol and nitrite but has significantly less activity on other dinitrotoluenes and nitrotoluenes (NT). Hence, oxidation of 2,3-DNT, 2,4-DNT, 2,5-DNT, 2,6-DNT, 2NT, and 4NT were enhanced here by performing saturation mutagenesis on codon I204 of the alpha subunit (DntAc) of DDO and by using a membrane agar plate assay to detect catechol formation. Rates of degradation were quantified both by the formation of nitrite and by the formation of the intermediates with high performance liquid chromatography. The degradation of both 2,3-DNT and 2,5-DNT were achieved for the first time (no detectable activity with the wild-type enzyme) using whole Escherichia coli TG1 cells expressing DDO variants DntAc I204L and I204Y (0.70 +/- 0.03 and 0.22 +/- 0.02 nmol/min/mg protein for 2,5-DNT transformation, respectively). DDO DntAc variant I204L also transformed both 2,6-DNT and 2,4-DNT 2-fold faster than wild-type DDO (0.8 +/- 0.6 nmol/min/mg protein and 4.7 +/- 0.5 nmol/min/mg protein, respectively). Moreover, the activities of DDO for 2NT and 4NT were also enhanced 3.5-fold and 8-fold, respectively. Further, DntAc variant I204Y was also discovered with comparable rate enhancements for the substrates 2,4-DNT, 2,6-DNT, and 2NT but not 4NT. Sequencing information obtained during this study indicated that the 2,4-DNT dioxygenases of Burkholderia sp. strain DNT and B. cepacia R34 are more closely related than originally reported. This is the first report of engineering an enzyme for enhanced degradation of nitroaromatic compounds and the first report of degrading 2,5-DNT.     

TNT and nitroaromatic compounds are chemoattractants for Burkholderia cepacia R34 and Burkholderia sp. strain DNT

Nitroaromatic compounds are toxic and potential carcinogens. In this study, a drop assay was used to detect chemotaxis toward nitroaromatic compounds for wild-type Burkholderia cepacia R34, wild-type Burkholderia sp. strain DNT, and a 2,4-dinitrotoluene (2,4-DNT) dioxygenase mutant strain (S5). The three strains are chemotactic toward 2,4,6-trinitrotoluene (TNT), 2,3-DNT, 2,4-DNT, 2,5-DNT, 2-nitrotoluene (NT), 4NT, and 4-methyl-5-nitrocatechol (4M5NC), but not toward 2,6-DNT. Of these, only 2,4-DNT is a carbon and energy source for B. cepacia R34 and Burkholderia sp. strain DNT, and 4M5NC is an intermediate in the 2,4-DNT degradation pathway. It was determined that the 2,4-DNT dioxygenase genes are not required for the chemotaxis for these nitroaromatic compounds because the DNT DDO mutant S5 has a chemotactic response toward 2,4-DNT although 2,4-DNT is not metabolized by S5; hence, 2,4-DNT itself is the chemoattractant. This is the first report of chemotaxis toward TNT, 2,3-DNT, 2,4-DNT, 2,5-DNT, 2NT, 4NT, and 4M5NC.     

Engineering Pseudomonas fluorescens for biodegradation of 2,4-dinitrotoluene

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10 degrees C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.     

Aerobic degradation of dinitrotoluenes and pathway for bacterial degradation of 2,6-dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2, 4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2, 6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2, 4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Aerobic Degradation of Dinitrotoluenes and Pathway for Bacterial Degradation of 2,6-Dinitrotoluene

An oxidative pathway for the mineralization of 2,4-dinitrotoluene (2,4-DNT) by Burkholderia sp. strain DNT has been reported previously. We report here the isolation of additional strains with the ability to mineralize 2,4-DNT by the same pathway and the isolation and characterization of bacterial strains that mineralize 2,6-dinitrotoluene (2,6-DNT) by a different pathway. Burkholderia cepacia strain JS850 and Hydrogenophaga palleronii strain JS863 grew on 2,6-DNT as the sole source of carbon and nitrogen. The initial steps in the pathway for degradation of 2,6-DNT were determined by simultaneous induction, enzyme assays, and identification of metabolites through mass spectroscopy and nuclear magnetic resonance. 2,6-DNT was converted to 3-methyl-4-nitrocatechol by a dioxygenation reaction accompanied by the release of nitrite. 3-Methyl-4-nitrocatechol was the substrate for extradiol ring cleavage yielding 2-hydroxy-5-nitro-6-oxohepta-2,4-dienoic acid, which was converted to 2-hydroxy-5-nitropenta-2,4-dienoic acid. 2,4-DNT-degrading strains also converted 2,6-DNT to 3-methyl-4-nitrocatechol but did not metabolize the 3-methyl-4-nitrocatechol. Although 2,6-DNT prevented the degradation of 2,4-DNT by 2,4-DNT-degrading strains, the effect was not the result of inhibition of 2,4-DNT dioxygenase by 2,6-DNT or of 4-methyl-5-nitrocatechol monooxygenase by 3-methyl-4-nitrocatechol.     

Kinetic analysis of simultaneous 2,4-dinitrotoluene (DNT) and 2, 6-DNT biodegradation in an aerobic fluidized-bed biofilm reactor.

We previously reported on the mineralization of 2,4-dinitrotoluene (2,4-DNT) and 2,6-dinitrotoluene (2,6-DNT) in an aerobic fluidized-bed bioreactor (FBBR) (Lendenmann et al. 1998 Environ Sci Technol 32:82-87). The current study examines the kinetics of 2, 4-DNT and 2,6-DNT mineralization at increasing loading rates in the FBBR with the goal of obtaining system-independent kinetic parameters. At each steady state, the FBBR was subjected to a set of transient load experiments in which substrate flux in the biofilm and bulk substrate concentrations were measured. The pseudo-steady-state data were used to estimate the biokinetic parameters for 2,4-DNT and 2,6-DNT removal using a mechanistic mathematical biofilm model and a routine that minimized the sum of the squared residuals (RSS). Estimated kinetic parameters varied slightly for each steady-state; retrieved parameters for qm were 0. 83 to 0.98 g DNT/g XCOD d for 2,4-DNT removal and 0.14 to 0.33 g DNT/g XCOD d for 2,6-DNT removal. Ks values for 2,4-DNT removal (0. 029 to 0.36 g DNT/m3) were consistently lower than Ks values for 2, 6-DNT removal (0.21 to 0.84 g DNT/m3). A new approach was introduced to estimate the fundamental biofilm kinetic parameter S*b,min from steady-state performance information. Values of S*b,min indicated that the FBBR performance was limited by growth potential. Adequate performance of the examined FBBR technology at higher loading rates will depend on an improvement in the growth potential. The obtained kinetic parameters, qm, Ks, and S*b,min, can be used to aid in the design of aerobic FBBRs treating waters containing DNT mixtures.     

Engineering Pseudomonas fluorescens for Biodegradation of 2,4-Dinitrotoluene

Using the genes encoding the 2,4-dinitrotoluene degradation pathway enzymes, the nonpathogenic psychrotolerant rhizobacterium Pseudomonas fluorescens ATCC 17400 was genetically modified for degradation of this priority pollutant. First, a recombinant strain designated MP was constructed by conjugative transfer from Burkholderia sp. strain DNT of the pJS1 megaplasmid, which contains the dnt genes for 2,4-dinitrotoluene degradation. This strain was able to grow on 2,4-dinitrotoluene as the sole source of carbon, nitrogen, and energy at levels equivalent to those of Burkholderia sp. strain DNT. Nevertheless, loss of the 2,4-dinitrotoluene degradative phenotype was observed for strains carrying pJS1. The introduction of dnt genes into the P.fluorescens ATCC 17400 chromosome, using a suicide chromosomal integration Tn5-based delivery plasmid system, generated a degrading strain that was stable for a long time, which was designated RE. This strain was able to use 2,4-dinitrotoluene as a sole nitrogen source and to completely degrade this compound as a cosubstrate. Furthermore, P. fluorescens RE, but not Burkholderia sp. strain DNT, was capable of degrading 2,4-dinitrotoluene at temperatures as low as 10°C. Finally, the presence of P. fluorescens RE in soils containing levels of 2,4-dinitrotoluene lethal to plants significantly decreased the toxic effects of this nitro compound on Arabidopsis thaliana growth. Using synthetic medium culture, P. fluorescens RE was found to be nontoxic for A.thaliana and Nicotiana tabacum, whereas under these conditions Burkholderia sp. strain DNT inhibited A.thaliana seed germination and was lethal to plants. These features reinforce the advantageous environmental robustness of P. fluorescens RE compared with Burkholderia sp. strain DNT.     

Assessment of the in vivo genotoxicity of isomers of dinitrotoluene using the alkaline Comet and peripheral blood micronucleus assays

Dinitrotoluene (DNT) is a nitroaromatic explosive that exists as six isomers; two major isomers (2,4- and 2,6-DNT) and four minor isomers (2,3-, 2,5-, 3,4-, and 3,5-DNT). DNT has been found in soil, surface water, and groundwater near ammunition production plants. The major isomers of DNT are classified as "likely to cause cancer in humans."In vitro studies have provided conflicting data regarding the genotoxicity of the minor isomers. Studies indicate that metabolism in the gut and liver are necessary to convert DNT to genotoxic compounds. As such, in the present study the genotoxicity of isomers of DNT was assessed using two in vivo genotoxicity assays. The Comet assay was used to detect DNA damage in liver cells from male Sprague-Dawley rats following oral exposure (14-day) to individual isomers of DNT. The micronucleus assay was conducted using flow cytometric analysis to detect chromosomal damage in peripheral blood. Treatment with 2,3-, 3,4-, 2,4-, 2,5- and 3,5-DNT did not induce DNA damage in liver cells or increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood at the doses tested. Treatment with 2,6-DNT induced DNA damage in liver tissue at all doses tested, but did not increase the frequency of micronucleated reticulocytes (MN-RET) in peripheral blood. Thus, 2,4-DNT and the minor isomers were not genotoxic under these test conditions, while 2,6-DNT was genotoxic in the target tissue, the liver. These results support previous research which indicated that the hepatocarcinogenicity of technical grade DNT (TG-DNT) could be attributed to the 2,6-DNT isomer.     

Phytotransformation of 2,4-dinitrotoluene in arabidopsis thaliana: toxicity, fate, and gene expression studies in vitro

Basic knowledge of the plant transformation pathways and toxicity of 2,4-dinitrotoluene (2,4-DNT) will assist in the design and assessment of a phytoremediation strategy. This study presents the toxicity and fate of 2,4-DNT and gene expression in response to 2,4-DNT exposure using the model plant Arabidopsis thaliana, an increasingly popular system for genetic and biochemical studies of phytotransformation of explosives. From the results of biomass and root growth assays for toxicity, 2,4-DNT was toxic to the plants at concentrations as low as 1 mg/L. In the uptake study, 95% of the initial 2,4-DNT was removed by 15-day-old seedlings from liquid media regardless of the initial 2,4-DNT concentrations while 30% accounted for the adsorption to the autoclaved plant materials. The mass balance was over 86% using [U-14C]2,4-DNT, and the mineralization by the plants was less than 1% under sterile conditions during 14 days of exposure. The percentage of the bound radioactivity increased from 49% to 72% of the radioactivity in the plants, suggesting transformed products of 2,4-DNT may be incorporated into plant tissues such as lignin and cellulose. Monoaminonitrotoluene isomers and unknown metabolites with short retention times were detected as transformed products of 2,4-DNT by the plants. Most (68%) of the radioactivity taken up by the plants was in the root tissues in nonsterile hydroponic cultures. Glutathione and expression of related genes (GSH1 and GSH2) in plants exposed to 2,4-DNT were 1.7-fold increased compared to untreated plants. Genes of a glutathione S-transferase and a cytochrome P450, which were induced by 2,4,6-trinitrotoluene exposure in previous studies, were upregulated by 10- and 8-fold, respectively. The application of phytoremediation and the development of transgenic plants for 2,4-DNT may be based on TNT phytotransformation pathway characteristics because of the similar fate and gene expression in plants.     

Microbial consortia that degrade 2,4-DNT by interspecies metabolism: isolation and characterisation

Two consortia, isolated by selective enrichment from a soil sample of anitroaromatic-contaminated site, degraded 2,4-DNT as their sole nitrogensource without accumulating one or more detectable intermediates. Thoughoriginating from the same sample, the optimised consortia had no commonmembers, indicating that selective enrichment resulted in different end points.Consortium 1 and consortium 2 contained four and six bacterial speciesrespectively, but both had two members that were able to collectivelydegrade 2,4-DNT. Variovorax paradoxus VM685 (consortium 1)and Pseudomonas sp. VM908 (consortium 2) initiate the catabolismof 2,4-DNT by an oxidation step, thereby releasing nitrite and forming4-methyl-5-nitrocatechol (4M5NC). Both strains contained a gene similarto the dntAa gene encoding 2,4-DNT dioxygenase. They subsequentlymetabolised 4M5NC to 2-hydroxy-5-methylquinone (2H5MQ) and nitrite,indicative of DntB or 4M5NC monooxygenase activity. A second consortiummember, Pseudomonas marginalis VM683 (consortium 1) and P.aeruginosa VM903, Sphingomonas sp. VM904, Stenotrophomonasmaltophilia VM905 or P. viridiflava VM907 (consortium 2), was foundto be indispensable for efficient growth of the consortia on 2,4-DNT and forefficient metabolisation of the intermediates 4M5NC and 2H5MQ. Knowledgeabout the interactions in this step of the degradation pathway is rather limited.In addition, both consortia can use 2,4-DNT as sole nitrogen and carbon source.A gene similar to the dntD gene of Burkholderia sp. strain DNT that catalyses ring fission was demonstrated by DNA hybridisation in the secondmember strains. To our knowledge, this is the first time that consortia are shownto be necessary for 2,4-DNT degradation.     

Chromosomal integration of the Vitreoscilla hemoglobin gene in Burkholderia and Pseudomonas for the purpose of producing stable engineered strains with enhanced bioremediating ability

Using the pUT-miniTn5 vector system developed by the laboratory of K.N. Timmis, the Vitreoscilla hemoglobin gene (vgb) was integrated into the chromosomes of Pseudomonas aeruginosa and Burkholderia cepacia; Vitreoscilla hemoglobin (VHb) was expressed at 8.8 and 0.8 nmol/g wet weight of cells in the respective engineered strains. The vgb-bearing P. aeruginosa outgrew wild-type P. aeruginosa and degraded benzoic acid faster than the latter strain at both normal and low aeration. In contrast, the vgb-bearing B. cepacia strain had a growth advantage over the wild-type strain at ca. 90 ppm, but not at ca. 120 ppm 2,4-dinitrotoluene (DNT); no difference in DNT degradation was seen between the two strains at either normal or low aeration. The results demonstrate the practicality of enhancing bioremediation with vgb stably integrated into the chromosome, but also suggest that a minimal level of VHb expression is required for its beneficial effects to be seen. Journal of Industrial Microbiology & Biotechnology (2001) 27, 27–33. Received 20 October 2000/ Accepted in revised form 04 May 2001     

Metabolism of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes sp. JS867 under oxygen limited conditions

Transformation of 2,4-dinitrotoluene (2,4-DNT) by Alcaligenes JS867 undervarying degrees of oxygen limitation was examined. Complete 2,4-DNT removalwas observed under oxygen excess with near stoichiometric release (83%) of nitrite.Average kinetic parameters were estimated based on a dual-Monod biokinetic modelwith 2,4-DNT and O₂ as growth limiting substrates. The negative impact of nitrite accumulation on the reaction rate was adequately described by inclusion of a noncompetitive inhibition term for NO₂ ^-. Under aerobic conditions, _max, K_sDNT, andK_iNO were 0.058(0.004) hr^-1, 3.3(±1.3) mg 2,4-DNT/L, and 1.2(±pm0.2) hr^-1, respectively. At increasing oxygen limitation, rates of 2,4-DNT disappearance and nitrite production decreased and incomplete removal of 2,4-DNT commenced. JS867 was able to use NO₂ ^- as a terminal electron acceptor whengrown on glucose or succinate under anaerobic conditions. However, during growthon 2,4-DNT and under O₂-limited conditions, JS867 did not use released nitrite as electron acceptor. The nearly constant molar ratios of DNT removed over NO₂ ^- released under various degrees of oxygen limitation suggested that oxygenolytic denitration pathways continued. No evidence of nitroreduction was obtained under the examined oligotrophic conditions. JS867 displayed a high affinity for oxygen consumption with K_SO2 value of 0.285(±0.198) mg O₂/L. Our results indicate thatunder oligotrophic conditions with 2,4-DNT as dominant carbon source, oxygen availability and nitrite accumulation may limit 2,4-DNT biomineralization, but the accumulation of reduced 2,4-DNT transformation products will be small.     

Effect of glucose on the amount of bacteria mineralizing 2,4-dichlorophenoxyacetic acid in soil

Plate numbers of bacteria and relative incidence of strains capable of mineralization of 2,4-dichlorophenoxyacetic acid (2,4-D) in chernozem samples incubated for 14 d with the herbicide (50 ppm) in the presence or absence of glucose (1000 ppm) were compared. Whereas the total number of bacteria increased 1.2-fold in the variant with 2,4-D and 2.4-fold in the variant with glucose and the herbicide, the number of 2,4-D-mineralizing bacteria increased 12.1-fold and 34.2-fold, respectively. In a collection of 96 isolates of soil bacteria substantially more strains capable of degradation of 2,4-D in the presence of glucose were detected as compared with the variant without it, indicating that processes of cometabolic type are involved during the degradation of this herbicide in the soil.     

The influence of cosubstrate and aeration on xylitol formation by recombinantSaccharomyces cerevisiae expressing theXYL1 gene

Xylitol formation by a recombinantSaccharomyces cerevisiae strain containing theXYL1 gene fromPichia stipitis CBS 6054 was investigated under three sets of conditions: (a) with glucose, ethanol, acetate, or glycerol as cosubstrates, (b) with different oxygenation levels, and (c) with different ratios of xylose to cosubstrate. With both glucose and ethanol the conversion yields were close to 1 g xylitol/g consumed xylose. Decreased aeration increased the xylitol yield on the basis of consumed cosubstrate, while the rate of xylitol formation decreased. The xylitol yield based on consumed cosubstrate also increased with increased-xylose:cosubstrate ratios. The transformant utilized the cosubstrate more efficiently than did a reference strain in terms of utilization rate and growth rate, implying that the regeneration of NAD(P)⁺ during xylitol formation by the transformant balanced the intracellular redox potential.     

Degradation of Chlorophenols by Alcaligenes eutrophus JMP134(pJP4) in Bleached Kraft Mill Effluent

The ability of Alcaligenes eutrophus JMP134(pJP4) to degrade 2,4-dichlorophenoxyacetic acid, 2,4,6-trichlorophenol, and other chlorophenols in a bleached kraft mill effluent was studied. The efficiency of degradation and the survival of strain JMP134 and indigenous microorganisms in short-term batch or long-term semicontinuous incubations performed in microcosms were assessed. After 6 days of incubation, 2,4-dichlorophenoxyacetate (400 ppm) or 2,4,6-trichlorophenol (40 to 100 ppm) were extensively degraded (70 to 100%). In short-term batch incubations, indigenous microorganisms were unable to degrade such of compounds. Degradation of 2,4,6-trichlorophenol by strain JMP134 was significantly lower at 200 to 400 ppm of compound. This strain was also able to degrade 2,4-dichlorophenoxyacetate, 2,4,6-trichlorophenol, 4-chlorophenol, and 2,4,5-trichlorophenol when bleached Kraft mill effluent was amended with mixtures of these compounds. On the other hand, the chlorophenol concentration and the indigenous microorganisms inhibited the growth and survival of the strain in short-term incubations. In long-term (>1-month) incubations, strain JMP134 was unable to maintain a large, stable population, although extensive 2,4,6-trichlorophenol degradation was still observed. The latter is probably due to acclimation of the indigenous microorganisms to degrade 2,4,6-trichlorophenol. Acclimation was observed only in long-term, semicontinuous microcosms.     

厌氧条件下Shewanella oneidensis MR-1对2,4-二硝基甲苯的还原转化

【目的】研究Shewanella oneidensis MR-1厌氧生物转化2,4-二硝基甲苯(2,4-DNT)的能力、转化过程和影响因素。【方法】以乳酸钠为电子供体, 2,4-DNT为电子受体, S. oneidensis MR-1为降解菌, 黄素为胞外电子载体, 设立四个不同的对照体系并监测各体系在转化过程中2,4-DNT及其产物的动态变化。同时研究不同2,4-DNT浓度下细胞的生长情况, 以及不同黄素浓度下2,4-DNT的降解情况。【结果】S. oneidensis MR-1菌能够高效还原转化2,4-DNT为4-氨基-2-硝基甲苯(4A2NT)和2-氨基-4-硝基甲苯(2A4NT), 并将其进一步还原为2,4-二氨基甲苯(2,4-DAT), 黄素能加速转化过程。【结论】S. oneidensis MR-1菌具备高效还原转化2,4-DNT的能力, 为实际环境中硝基苯污染的原位修复提供科学依据。     

Improvement of bioremediation by Pseudomonas and Burkholderia by mutants of the Vitreoscilla hemoglobin gene (vgb) integrated into their chromosomes

Using genetic engineering, the Vitreoscilla (bacterial) hemoglobin gene (vgb) was integrated stably into the chromosomes of Pseudomonas aeruginosa and Burkholderia sp. strain DNT. This was done for both wild type vgb and two site-directed mutants of vgb that produce Vitreoscilla hemoglobin (VHb) with lowered oxygen affinities; in all cases functional VHb was expressed. Similar to previous results, the wild type VHb improved growth for both species and degradation of 2,4-dinitrotoluene (Burkholderia sp.) or benzoic acid (P. aeruginosa) under both normal and low aeration conditions. Both mutant vgbs enhanced these parameters compared to wild type vgb, and the improvement was seen in both species. The enhancements were generally greater at low aeration than at normal aeration. The results demonstrate the possibility that the positive effects provided by VHb may be augmented by protein engineering.