Development of innovative and green adsorbents for in situ cleanup of fluoride-polluted groundwater: Mechanisms and field-scale studies.

In this study, the magnesium oxide (MgO)-based adsorbents [granulated MgO aggregates (GA-MgO) and surface-modified MgO powder (SM-MgO)] were developed to remediate a fluoride-contaminated groundwater site. Both GA-MgO and SM-MgO had porous, spherical, and crystalline structures. Diameters for GA-MgO and SM-MgO were 1-1.7 mm and 1-10 µm, respectively. The pseudo second-order dynamic adsorption and the Freundlich isotherm could be applied to express the chemical adsorption phenomena. The monolayer adsorption was the dominant mechanism at the initial adsorption period. During the latter part of fluoride adsorption, the multilayer adsorption became the dominant mechanism for fluoride removal from the water phase, which also resulted in the increased adsorption capacity. Higher hydroxide, phosphate, and carbonate concentrations caused a decreased fluoride removal efficiency due to the competition of sorption sites between fluoride and other anions with similar electronic properties. Fluoride removal mechanism using GA-MgO and SM-MgO as the adsorbents was mainly carried out by the chemical adsorption. Reaction paths contained two main processes: (1) formation of magnesium hydroxide after the reaction of MgO with water, and (2) the hydroxyl group of the magnesium hydroxide was replaced by fluoride ions to form magnesium fluoride precipitation. Results from column tests show that up to 61 and 73% of fluoride removal (initial fluoride concentration = 9.3 mg/L) could be obtained after 50 pore volumes of groundwater pumping with GA-MgO and SM-MgO injection, respectively. The GA-MgO system could be applied to contain and remediate fluoride-contaminated groundwater, and SM-MgO could be applied as an immediate fluoride removal alternative to achieve a rapid pollutant removal for emergency responses. Up to 71% of fluoride removal (fluoride concentration = 10.8 mg/L) could be obtained with GA-MgO injection after 30 days of operation. The developed GA-MgO system is a potential and green remediation alternative to contain the fluoride plume significantly.

Cleanup of Cr(VI)-polluted groundwater using immobilized bacterial consortia via bioreduction mechanisms.

Cr(VI) bioreduction has become a remedial alternative for Cr(VI)-polluted site cleanup. However, lack of appropriate Cr(VI)-bioreducing bacteria limit the field application of the in situ bioremediation process. In this study, two different immobilized Cr(VI)-bioreducing bacterial consortia using novel immobilization agents have been developed for Cr(VI)-polluted groundwater remediation: (1) granular activated carbon (GAC) + silica gel + Cr(VI)-bioreducing bacterial consortia (GSIB), and (2) GAC + sodium alginate (SA) + polyvinyl alcohol (PVA) + Cr(VI)-bioreducing bacterial consortia (GSPB). Moreover, two unique substrates [carbon-based agent (CBA) and emulsified polycolloid substrate (EPS)] were developed and used as the carbon sources for Cr(VI) bioreduction enhancement. The microbial diversity, dominant Cr-bioreducing bacteria, and changes of Cr(VI)-reducing genes (nsfA, yieF, and chrR) were analyzed to assess the effectiveness of Cr(VI) bioreduction. Approximately 99% of Cr(VI) could be bioreduced in microcosms with GSIB and CBA addition after 70 days of operation, which caused increased populations of total bacteria, nsfA, yieF, and chrR from 2.9 × 108 to 2.1 × 1012, 4.2 × 104 to 6.3 × 1011, 4.8 × 104 to 2 × 1011, and 6.9 × 104 to 3.7 × 107 gene copies/L. In microcosms with CBA and suspended bacteria addition (without bacterial immobilization), the Cr(VI) reduction efficiency dropped to 60.3%, indicating that immobilized Cr-bioreducing bacteria supplement could enhance Cr(VI) bioreduction. Supplement of GSPB led to a declined bacterial growth due to the cracking of the materials. The addition of GSIB and CBA could establish a reduced condition, which favored the growth of Cr(VI)-reducing bacteria. The Cr(VI) bioreduction efficiency could be significantly improved through adsorption and bioreduction mechanisms, and production of Cr(OH)3 precipitates confirmed the occurrence of Cr(VI) reduction. The main Cr-bioreducing bacteria included Trichococcus, Escherichia-Shigella, and Lactobacillus. Results suggest that the developed GSIB bioremedial system could be applied to cleanup Cr(VI)-polluted groundwater effectively.

Isolation of an ectoine-producing Sinobaca sp. and identification of genes that are involved in ectoine biosynthesis.

A moderate halophilic bacterium that could accumulate ectoine and hydroxyectoine was isolated from soil near a salt mine and was identified as a Sinobaca sp. (designed strain H24) according to 16S rRNA gene sequence analysis. The bacterium grew well in the presence of 1-2 M NaCl, while growth in a medium that contained 2 M NaCl led to higher accumulation of ectoines. The yields of ectoine and hydroxyectoine by Sinobaca sp. H24 reached 11.27 mg/l and 1.34 mg/l, respectively, when cultured in the following medium: NaCl (2 M), peptone (5 g/l), yeast extract (1 g/l), NH4Cl (0.02 M), KH2PO4 (1 M), K2HPO4 (0.1 M), and glycerol (1% w/v). Genes that are involved in ectoine biosynthesis of Sinobaca sp. H24 were also identified, and their sequences were determined by a metagenomics approach. The results demonstrated that Sinobaca sp. H24 possesses ectoine metabolism genes for both ectoine biosynthesis (ectA, ectB, ectC, and ectD) and ectoine degradation (doeA). Genes that are related to ectoine biosynthesis, such as lysC and asd, were also characterized. The identification and characterization results for ectoine/hydroxyectoine biosynthesis genes are in agreement with the physiology of Sinobaca sp. H24 as a potential candidate for ectoine production for industrial applications. This report established for the first time the accumulation of ectoine/hydroxyectoine in Sinobaca sp. and characterized the genes that are involved in ectoine/hydroxyectoine biosynthesis in Sinobaca sp. H24.

Cleanup chlorinated ethene-polluted groundwater using an innovative immobilized Clostridium butyricum column scheme: A pilot-scale study.

In this study, the developed innovative immobilized Clostridium butyricum (ICB) (hydrogen-producing bacteria) column scheme was applied to cleanup chlorinated-ethene [mainly cis-1,2-dichloroethene (cis-DCE)] polluted groundwater in situ via the anaerobic reductive dechlorinating processes. The objectives were to assess the effectiveness of the field application of ICB scheme on the cleanup of cis-DCE polluted groundwater, and characterize changes of microbial communities after ICB application. Three remediation wells and two monitor wells were installed within the cis-DCE plume. In the remediation well, a 1.2-m PVC column (radius = 2.5 cm) (filled with ICB beads) and 20 L of slow polycolloid-releasing substrate (SPRS) were supplied for hydrogen production enhancement and primary carbon supply, respectively. Groundwater samples from remediation and monitor wells were analyzed periodically for cis-DCE and its degradation byproducts, microbial diversity, reductive dehalogenase, and geochemical indicators. Results reveal that cis-DCE was significantly decreased within the ICB and SPRS influence zone. In a remediation well with ICB injection, approximately 98.4% of cis-DCE removal (initial concentration = 1.46 mg/L) was observed with the production of ethene (end-product of cis-DCE dechlorination) after 56 days of system operation. Up to 0.72 mg/L of hydrogen was observed in remediation wells after 14 days of ICB and SPRS introduction, which corresponded with the increased population of Dehalococcoides spp. (Dhc) (increased from 3.76 × 103 to 5.08 × 105 gene copies/L). Results of metagenomics analyses show that the SPRS and ICB introduction caused significant impacts on the bacterial communities, and increased Bacteroides, Citrobacter, and Desulfovibrio populations were observed, which had significant contributions to the reductive dechlorination of cis-DCE. Application of ICB could effectively result in increased populations of Dhc and RDase genes, which corresponded with improved dechlorination of cis-DCE and vinyl chloride. Introduction of ICB and SPRS could be applied as a potential in situ remedial option to enhance anaerobic dechlorination efficiencies of chlorinated ethenes.

A protein containing the DUF1471 domain regulates biofilm formation and capsule production in Klebsiella pneumoniae.

BACKGROUND/PURPOSE: Biofilms formed by Klebsiella pneumoniae on medical devices increase infection risk. Fimbriae and capsule polysaccharides (CPSs) are important factors involved in biofilm formation. KP1_4563 in K. pneumoniae NTUH-K2044, a small protein containing the DUF1471 domain, was reported to inhibit type 3 fimbriae function. In this study, we aimed to determine whether the KP1_4563 homolog is conserved in each K. pneumoniae isolate and what role it has in Klebsiella biofilms. METHODS: The genomes of K. pneumoniae NTUH-K2044, CG43, MGH78578, KPPR1 and STU1 were compared. The KP1_4563 homolog in K. pneumoniae STU1 was named orfX. Biofilms of wild-type and orfX mutant strains from K. pneumoniae STU1 and one clinical isolate, 83535, were quantified. Transcription levels of the type 3 fimbrial genes, mrkA and mrkH, were investigated by RT-qPCR. MrkA of the wild-type and orfX mutant were observed by Western blotting. The morphology of bacterial cells was observed by transmission electron microscopy (TEM). Bacterial CPSs were quantified. RESULTS: The gene and upstream region of orfX were conserved among the five K. pneumoniae isolates. Deletion of orfX enhanced Klebsiella biofilm formation. However, the amount of mRNA from mrkA and mrkH and the level of MrkA protein were not different between the wild type and orfX mutant. In contrast, the amount of CPS in orfX mutants was increased, compared to their parental strains, STU1 and 83535. CONCLUSION: The role of orfX is speculated to be conserved in most K. pneumoniae isolates. OrfX negatively controlled biofilm formation by reducing CPS, not type 3 fimbriae, production.

Propolis alleviates 4-aminobiphenyl-induced oxidative DNA damage by inhibition of CYP2E1 expression in human liver cells.

4-Aminobiphenyl (4-ABP) may cause DNA damage in human liver cells (HepG2 and L-02). Propolis exhibits antioxidant properties through reactive oxygen species (ROS) scavenging. We determined the effects of propolis in alleviating 4-ABP -induced DNA damage using the comet assay. Results revealed that propolis could significantly alleviated oxidative damaged DNA by 4-ABP. Furthermore, we proved that inhibition of cytochrome P450 2E1 (CYP2E1) expression by propolis could contribute to the decreased oxidative DNA damage in the treated cells, as the conversion of 4-ABP into its metabolite, N-hydroxy-ABP (HOABP), was blocked; after all, HOABP showed more genotoxic than its parent chemical, 4-ABP. With the homologous recombination assay, propolis failed to induce DNA repair enzymes. Furthermore, the expression of RAD51, Ku70/Ku80, and OGG1 in treated cells were determined with the western blot, revealing that the expression of these protein were unchanged in comparison with those in nontreated cells. However, propolis could protect the treated cells from DNA damage. In conclusion, propolis could antagonize 4-ABP-induced oxidative DNA damage though the removal of ROS and inhibition of CYP2E1 expression in the treated cells.

The PTS Components in Klebsiella pneumoniae Affect Bacterial Capsular Polysaccharide Production and Macrophage Phagocytosis Resistance.

Capsular polysaccharide (CPS) is a crucial virulence factor for Klebsiella pneumoniae infection. We demonstrated an association of CPS production with two phosphoenolpyruvate:carbohydrate phosphotransferase systems (PTSs). Deficiency of crr, encoding enzyme IIA of PTS, in K. pneumoniae enhanced the transcriptional activities of galF, wzi and gnd, which are in the cps gene cluster, leading to high CPS production. A crr mutant exhibited a higher survival rate in 1% hydrogen peroxide than the wild-type. The crr mutant showed less sensitivity to engulfment by macrophage (RAW 264.7) than the wild-type by observing the intracellular bacteria using confocal laser scanning microscopy (CLSM) and by calculating the colony-forming units (CFU) of intracellular bacteria. After long-term incubation, the survival rate of the intracellular crr mutant was higher than that of the wild-type. Deficiency of crr enhanced the transcriptional activities of etcABC which encodes another putative enzyme II complex of a PTS. Deletion of etcABC in the crr mutant reduced CPS production and the transcriptional activities of galF compared to those of the crr mutant. These results indicated that one PTS component, Crr, represses CPS production by repressing another PTS component, EtcABC, in K. pneumoniae. In addition, PTS plays a role in bacterial resistance to macrophage phagocytosis.

Polyethylene Glycol6000/carbon Nanodots as Fluorescent Bioimaging Agents.

Photoluminescent nanomaterials have immense potential for use in biological systems due to their excellent fluorescent properties and small size. Traditional semiconductor quantum dots are heavy-metal-based and can be highly toxic to living organisms, besides their poor photostability and low biocompatibility. Nano-sized carbon quantum dots and their surface-modified counterparts have shown improved characteristics for imaging purposes. We used 1,3, 6-trinitropyrene (TNP) and polyethylene glycol6000 (PEG6000) in a hydrothermal method to prepare functional polyethylene glycol6000/carbon nanodots (PEG6000/CDs) and analyzed their potential in fluorescent staining of different types of bacteria. Our results demonstrated that PEG6000/CDs stained the cell pole and septa of gram-positive bacteria B. Subtilis and B. thuringiensis but not those of gram-negative bacteria. The optimal concentration of these composite nanodots was approximately 100 ppm and exposure times varied across different bacteria. The PEG6000/CD composite had better photostability and higher resistance to photobleaching than the commercially available FM4-64. They could emit two wavelengths (red and green) when exposed to two different wavelengths. Therefore, they may be applicable as bioimaging molecules. They can also be used for differentiating different types of bacteria owing to their ability to differentially stain gram-positive and gram-negative bacteria.

Application of enhanced bioreduction for hexavalent chromium-polluted groundwater cleanup: Microcosm and microbial diversity studies.

Hexavalent chromium (Cr6+) is a commonly found heavy metal at polluted groundwater sites. In this study, the effectiveness of Cr6+ bioreduction by the chromium-reducing bacteria was evaluated to remediate Cr6+-contaminated groundwater. Microcosms were constructed using indigenous microbial consortia from a Cr6+-contaminated aquifer as the inocula, and slow-releasing emulsified polycolloid-substrate (ES), cane molasses (CM), and nutrient broth (NB) as the primary substrates. The genes responsible for the bioreduction of Cr6+ and variations in bacterial diversity were evaluated using metagenomics assay. Complete Cr6+ reduction via the biological mechanism was observed within 80 days using CM as the carbon source under anaerobic processes with the increased trivalent chromium (Cr3+) concentrations. Cr6+ removal efficiencies were 83% and 59% in microcosms using ES and NB as the substrates, respectively. Increased bacterial communities associated with Cr6+ bioreduction was observed in microcosms treated with CM and ES. Decreased bacterial communities were observed in NB microcosms. Compared to ES, CM was more applicable by indigenous Cr6+ reduction bacteria and resulted in effective Cr6+ bioreduction, which was possibly due to the growth of Cr6+-reduction related bacteria including Sporolactobacillus, Clostridium, and Ensifer. While NB was applied for specific bacterial selection, it might not be appropriate for electron donor application. These results revealed that substrate addition had significant impact on microbial diversities, which affected Cr6+ bioreduction processes. Results are useful for designing a green and sustainable bioreduction system for Cr6+-polluted groundwater remediation.

Enhanced reductive dechlorination of trichloroethene with immobilized Clostridium butyricum in silica gel.

Deteriorated environmental conditions during the bioremediation of trichloroethene (TCE)-polluted groundwater cause decreased treatment efficiencies. This study assessed the effect of applying immobilized Clostridium butyricum (a hydrogen-producing bacterium) in silica gel on enhancing the reductive dechlorination efficiency of TCE with the slow polycolloid-releasing substrate (SPRS) supplement in groundwater. The responses of microbial communities with the immobilized system (immobilized Clostridium butyricum and SPRS amendments) were also characterized by the metagenomics assay. A complete TCE removal in microcosms was obtained within 30 days with the application of this immobilized system via reductive dechlorination processes. An increase in the population of Dehalococcoides spp. was observed using the quantitative polymerase chain reaction (qPCR) analysis. Results of metagenomics assay reveal that the microbial communities in the immobilized system were distinct from those in systems with SPRS only. Bacterial communities associated with TCE biodegradation also increased in microcosms treated with the immobilized system. The immobilized system shows a great potential to promote the TCE dechlorination efficiency, and the metagenomics-based approach provides detailed insights into dechlorinating microbial community dynamics. The results would be helpful in designing an in situ immobilized system to enhance the bioremediation efficiency of TCE-contaminated groundwater.

Ectoine production with indigenous Marinococcus sp. MAR2 isolated from the marine environment.

Ectoine has fostered the development of products for skin care and cosmetics. In this study, we employed the marine bacterial strain Marinococcus sp. MAR2 to increase ectoine production by optimizing medium constituents using Response Surface Methodology (RSM) and a fed-batch strategy. The results from the steepest ascent and central composite design indicated that 54 g/L of yeast extract, 14.0 g/L of ammonium acetate, 74.4 g/L of sodium glutamate, and 6.2 g/L of sodium citrate constituted the optimal medium with maximum ectoine production (3.5 g/L). In addition, we performed fed-batch culture in the bioreactor, combining pH and dissolved oxygen to produce ectoine by Marinococcus sp. MAR2. The ectoine production, content, and productivity of 5.6 g/L, 10%, and 3.9 g/L/day were further reached by a fed-batch culture. Thus, the ectoine production by Marinococcus sp. MAR2 using RSM and fed-batch strategy shows its potential for industrial production.

Proteomic analysis of ametryn toxicity in zebrafish embryos.

Ametrym (AMT) is the most widely used herbicide and frequently detected in the aquatic environment. AMT also represent a potential health risk to aquatic organisms and animals, including humans. However, little data are available on their toxicity to zebrafish (Danio rerio). The aim of the present study was to evaluate the toxicological effects of AMT exposure on zebrafish embryos. In the acute toxicity test, 6 hpf embryos were exposed to various concentrations of AMT for 24 or 48 h. The results indicated that AMT induced malformation in larvae. To investigate the toxicological mechanism on the protein expression level. A proteomic approach was employed to investigate the proteome alterations of zebra fish embryos exposed to 20 mg/L AMT for 48 h. Among 2925 unique proteins identified, 298 differential proteins (> or <1.3-fold, P < 0.05) were detected in the treated embryos as compared to the corresponding proteins in the untreated embryos. Gene ontology analysis showed that these up-regulated proteins were most involved in glycolysis, lipid transport, protein polymerization, and nucleotide binding, and the down-regulated proteins were related to microtubule-based process, protein polymerization, oxygen transport. Moreover, KEGG pathway analysis indicated that tight junction, ribosome, and oxidative phosphorylation were inhibited in the treated embryos. These findings provide new insight into the mechanisms of toxicity induced by AMT.

The integrated analysis of transcriptome and proteome for exploring the biodegradation mechanism of 2, 4, 6-trinitrotoluene by Citrobacter sp.

Citrobacter sp. has been shown to degrade 2,4,6-trinitrotoluene (TNT). However, the mechanism of its TNT biodegradation is poorly understood. An integrated proteome and transcriptome analysis was performed for investigating the differential genes and differential proteins in bacterial growth at the onset of experiments and after 12 h treatment with TNT. With the RNA sequencing, we found a total of 3792 transcripts and 569 differentially expressed genes (≥2 fold, P < 0.05) by. Genes for amino acid transport, cellular metabolism and stress-shock proteins were up-regulated, while carbohydrate transport and metabolism were down-regulated. A total of 42 protein spots (≥1.5 fold, P < 0.05) showed differential expression on two-dimensional gel electrophoresis and these proteins were identified by mass spectrometry. The most prominent proteins up-regulated were involved in energy production and conversion, amino acid transport and metabolism, posttranslational modification, protein turnover and chaperones. Proteins involved in carbohydrate transport and metabolism were down-regulated. Most notably, we observed that nemA encoding N-ethylmaleimide reductase was the most up-regulated gene involved in TNT degradation, and further proved that it can transform TNT to 4-amino-2,6-dinitrotoluene (4-ADNT) and 2-amino-4,6-dinitrotoluene (2-ADNT). This study highlights the molecular mechanisms of Citrobacter sp. for TNT removal.

Biotransformation of trinitrotoluene by Citrobacter sp. YC4 and evaluation of its cyto-toxicological effects.

Trinitrotoluene (TNT) is an explosive chemical generally used for military, civil and industrial purposes. Therefore, TNT residue can be found in soil and ground water as an environmental pollutant. The environmental control of TNT pollution has become a critical issue because of its potential toxicity and carcinogenicity. The aim of this study is to evaluate the cyto-toxicological effects of TNT after bioremediation. Citrobacter sp. YC4 is able to utilize TNT as a sole nitrogen source. Citrobacter sp. YC4 cells grown in medium with TNT as the sole nitrogen source (TNT-N) were able to rapidly degrade TNT, in contrast to cells grown in Luria Bertani medium as determined by resting cell suspension. The concentration of TNT decreased from 100 to 0 ppm within 10 h in the solution containing TNT mixed with TNT-N-grown YC4. The cytotoxicity of TNT and its degradation products generated by TNT-N-grown YC4 were assessed by WST-1-based cell cytotoxicity assays. Our results showed that the cytotoxic potential of solutions containing TNT decreased almost to the level of the control after a 1-h incubation with TNT-N-grown YC4 cells. The rapid conversion of TNT into possibly less toxic products by Citrobacter sp. YC4 proposes a bioremediation prospection.

The Negative Effects of KPN00353 on Glycerol Kinase and Microaerobic 1,3-Propanediol Production in Klebsiella pneumoniae.

1,3-Propanediol (1,3-PD) is a valuable chemical intermediate in the synthesis of polyesters, polyethers, and polyurethanes, which have applications in various products such as cloth, bottles, films, tarpaulins, canoes, foam seals, high-resilience foam seating, and surface coatings. Klebsiella pneumoniae can produce 1,3-PD from glycerol. In this study, KPN00353, an EIIA homologue in the phosphoenolpyruvate (PEP):carbohydrate phosphotransferase system (PTS), was found to play a negative regulatory role in 1,3-PD production under microaerobic conditions via binding to glycerol kinase (GlpK). The primary sequence of KPN00353 is similar to those of the fructose-mannitol EIIA (EIIFru and EIIAMtl) family. The interaction between KPN00353 and GlpK resulted in inhibition of the synthesis of glycerol-3-phosphate (G3P) and correlated with reductions in glycerol uptake and the production of 1,3-PD. Based on structure modeling, we conclude that residue H65 of KPN00353 plays an important role in the interaction with GlpK. We mutated this histidine residue to aspartate, glutamate, arginine and glutamine to assess the effects of each KPN00353 variant on the interaction with GlpK, on the synthesis of G3P and on the production of 1,3-PD. Our results illuminate the role of KPN00353 in 1,3-PD production by K. pneumoniae under microaerobic conditions.

Enhancement of microbial 2,4,6-trinitrotoluene transformation with increased toxicity by exogenous nutrient amendment.

In this study, the bacterial strain Citrobacter youngae strain E4 was isolated from 2,4,6-trinitrotoluene (TNT)-contaminated soil and used to assess the capacity of TNT transformation with/without exogenous nutrient amendments. C. youngae E4 poorly degraded TNT without an exogenous amino nitrogen source, whereas the addition of an amino nitrogen source considerably increased the efficacy of TNT transformation in a dose-dependent manner. The enhanced TNT transformation of C. youngae E4 was mediated by increased cell growth and up-regulation of TNT nitroreductases, including NemA, NfsA and NfsB. This result indicates that the increase in TNT transformation by C. youngae E4 via nitrogen nutrient stimulation is a cometabolism process. Consistently, TNT transformation was effectively enhanced when C. youngae E4 was subjected to a TNT-contaminated soil slurry in the presence of an exogenous amino nitrogen amendment. Thus, effective enhancement of TNT transformation via the coordinated inoculation of the nutrient-responsive C. youngae E4 and an exogenous nitrogen amendment might be applicable for the remediation of TNT-contaminated soil. Although the TNT transformation was significantly enhanced by C. youngae E4 in concert with biostimulation, the 96-h LC50 value of the TNT transformation product mixture on the aquatic invertebrate Tigriopus japonicas was higher than the LC50 value of TNT alone. Our results suggest that exogenous nutrient amendment can enhance microbial TNT transformation; however, additional detoxification processes may be needed due to the increased toxicity after reduced TNT transformation.

The change of microbial community from chlorinated solvent-contaminated groundwater after biostimulation using the metagenome analysis.

The compositions of bacterial community in one site contaminated with PCE/TCE after the slow polycolloid-releasing substrate (SPRS) (contained vegetable oil, cane molasses, and surfactants) addition were analyzed. Results show that SPRS caused a rapid enhancement of reductive dechlorination of TCE. The transformation of PCE/TCE into ethene was observed after 20 days of operation. To compare the change of bacterial communities before and after SPRS addition, 16S rRNA amplicon sequencing using the metagenome analysis was performed. Results demonstrated the detection of the increased amounts of Dehalogenimonas by 2.2-fold, Pseudomonas by 3.4-fold and Sulfuricurvum by 4-fold with the analysis of the ribosomal database project (RDP). Metagenomic DNA was extracted from PCE/TCE-contaminated groundwater after SPRS addition, and subjected to sequencing. Results obtained from metagenomic sequencing indicate that genes from Dehalococcoides mccartyi was ranked as the second abundant bacteria among all of the detected bacteria via the analysis of the lowest common ancestor (LCA). Abundance of these bacterial groups, as shown above suggests their role in TCE biodegradation. Functional analysis of the metagenome, with the specific reference to chloroalkane and chloroalkene degradation, revealed the presence of some genes responsible for TCE biodegradation. Overall, results of this study provided new insights for a better understanding of the potential of biostimulation on TCE-contaminated sites.

Molecular analysis of codon 548 in the rpoB gene involved in Mycobacterium tuberculosis resistance to rifampin.

Most Mycobacterium tuberculosis rifampin-resistant strains have been associated with mutations in an 81-bp rifampin resistance-determining region (RRDR) in the gene rpoB. However, if this region alone were targeted, rifampin-resistant strains with mutations outside the RRDR would not be detected. In this study, among 51 rifampin-resistant clinical isolates analyzed by sequencing 1,681-bp-long DNA fragments containing the RRDR, 47 isolates contained mutations within the RRDR, three isolates contained mutations both within and outside the RRDR, and only one isolate had a single missense mutation (Arg548His) located outside the RRDR. A drug susceptibility test of recombinant Mycobacterium smegmatis and M. tuberculosis isolates carrying mutated rpoB (Arg548His) showed an increased MIC for rifampin compared to that of the control strains. Modeling of the Arg548His mutant RpoB-DNA complex revealed that the His548 side chain formed a more stable hydrogen bond structure than did Arg548, reducing the flexibility of the rifampin-resistant cluster II region of RpoB, suggesting that the RpoB Arg548His mutant does not effectively interact with rifampin and results in bacterial resistance to the drug. This is the first report on the relationship between the mutation in codon 548 of RpoB and rifampin resistance in tuberculosis. The novel mutational profile of the rpoB gene described here will contribute to the comprehensive understanding of rifampin resistance patterns and to the development of a useful tool for simple and rapid drug susceptibility tests.

Activation of MAPK pathways and downstream transcription factors in 2-aminobiphenyl-induced apoptosis.

2-Aminobiphenyls (2-ABP) induces oxidative DNA damage and leads to apoptosis. The precise signaling pathways of inducing apoptosis in vitro are still unknown. This study provides insight into the relationship between 2-ABP-induced apoptosis and the activation of MAPK and downstream transcription factors using pharmacological inhibitors of ERK, p38, and JNK pathways. Results showed that 2-ABP induced the activation of ERK and JNK but not p38. The ERK/JNK pathways downstream transcription factors, c-Jun and ATF-2, were also activated by 2-ABP. The inhibitory effects of ERK inhibitor, U0126, on 2-ABP-induced caspase-3 activity were not detected. However, JNK inhibitor, SP600125, significantly attenuated the caspase-3 activity induced by 2-ABP. The expression of the transcription factors c-Jun and ATF-2 were decreased in 2-ABP treated cells in the presence of ERK/JNK inhibitors, suggesting that the expression of ERK/JNK pathways leads to the downstream activation of c-Jun and ATF-2. N-acetylcysteine, an ROS scavenger, inhibited 2-ABP-induced activation of ERK and JNK, the cell death and caspase-3 activity, which suggested that oxidative stress plays a crucial role in apoptosis through activation of caspase-3 in a ROS/JNK-dependent signaling cascade.

Biotransformation of trinitrotoluene (TNT) by Pseudomonas spp. isolated from a TNT-contaminated environment.

The compound 2,4,6-trinitrotoluene (TNT) is a secondary explosive widely used worldwide for both military and civil purposes. As a result, residual TNT has been detected as an environmental pollutant in both soil and groundwater. The authors have isolated several microbial strains from soil contaminated with TNT by enrichment culture techniques using TNT as a carbon, nitrogen, and energy source. The contaminated soil contained approximately 1860 ppm TNT measured by high-performance liquid chromatography (HPLC). The initial identification of these isolates was determined by 16S rRNA gene comparison. The isolates mainly included species belonging to the genus Pseudomonas. Two strains (Pseudomonas putida strain TP1 and Pseudomonas aeruginosa strain TP6) were selected for further examination. Both strains demonstrated the ability to grow on the medium containing TNT as a carbon, energy, and nitrogen source and also clearly demonstrated the ability to degrade TNT. More than 90% of the TNT in the growth medium was degraded by both strains after 22 d incubation, as determined by HPLC. Additionally, the resting cells of P. putida TP1 and P. aeruginosa TP6 both significantly displayed the ability to transform (metabolize) TNT.