Chih-Ching Chien

Application of real-time PCR, DGGE fingerprinting, and culture-based method to evaluate the effectiveness of intrinsic bioremediation on the control of petroleum-hydrocarbon plume.

Real-time polymerase chain reaction (PCR), denaturing gradient gel electrophoresis (DGGE), and the culture-based method were applied in the intrinsic bioremediation study at a petroleum-hydrocarbon contaminated site. The genes of phenol hydroxylase (PHE), ring-hydroxylating toluene monooxygenase (RMO), naphthalene dioxygenase (NAH), toluene monooxygenase (TOL), toluene dioxygenase (TOD), and biphenyl dioxygenase (BPH4) were quantified by real-time PCR. Results show that PHE gene was detected in groundwater contaminated with benzene, toluene, ethylbenzene, xylene isomers (BTEX) and methyl tert-butyl ether (MTBE), and this indicates that intrinsic bioremediation occurred at this contaminated site. Results from DGGE analyses reveal that the petroleum-hydrocarbon plume caused the variation in microbial communities. In this study, MTBE degraders including Pseudomonas sp. NKNU01, Bacillus sp. NKNU01, Klebsiella sp. NKNU01, Enterobacter sp. NKNU01, and Enterobacter sp. NKNU02 were isolated from the contaminated groundwater using the cultured-based method. Results from MTBE biodegradation experiment show that the isolated bacteria were affected by propane. This indicates that propane may influence the metabolic pathway of MTBE by these bacteria. Knowledge and comprehension obtained from this study will be helpful in evaluating the occurrence and effectiveness of intrinsic bioremediation on the remediation of petroleum-hydrocarbon contaminated groundwater.

Assessing of natural attenuation and intrinsic bioremediation rates at a petroleum-hydrocarbon spill site: laboratory and field studies.

Natural attenuation is a passive remedial approach that depends upon natural processes to degrade and dissipate contaminants in soil and groundwater. Intrinsic bioremediation is believed to be the major process among the natural attenuation mechanisms that account for the reduction of contaminant concentrations. In this study, a mass flux approach was used to calculate the contaminant mass reduction at a petroleum-hydrocarbon spill site. The mass flux technique is a simplified mass balance procedure, which is accomplished using the differences in total contaminant mass flux across two cross sections of the contaminant plume. The mass flux calculation results show that up to 86% of the dissolved total benzene, toluene, ethylbenzene, and xylene (BTEX) isomers removal was observed via natural attenuation at this site. Evidence for the occurrence of natural attenuation was the decreased contaminant mass flux through the plume cross sections along the transport path and limited spreading of the BTEX plume. Evi...

Inactivation of dhaD and dhaK abolishes by-product accumulation during 1,3-propanediol production in Klebsiella pneumoniae

Abstract1,3-Propanediol (1,3-PD) can be used for the industrial synthesis of a variety of compounds, including polyesters, polyethers, and polyurethanes. 1,3-PD is generated from petrochemical and microbial sources. 1,3-Propanediol is a typical product of glycerol fermentation, while acetate, lactate, 2,3-butanediol, and ethanol also accumulate during the process. Substrate and product inhibition limit the final concentration of 1,3-propanediol in the fermentation broth. It is impossible to increase the yield of 1,3-propanediol by using the traditional whole-cell fermentation process. In this study, dhaD and dhaK, the genes for glycerol dehydrogenase and dihydroxyacetone kinase, respectively, were inactivated by homologous recombination in Klebsiella pneumoniae. The dhaD/dhaK double mutant (designated TC100), selected from 5,000 single or double cross homologous recombination mutants, was confirmed as a double cross by using polymerase chain reaction. Analysis of the cell-free supernatant with high-performance liquid chromatography revealed elimination of lactate and 2,3-butanediol, as well as ethanol accumulation in TC100, compared with the wild-type strain. Furthermore, 1,3-propanediol productivity was increased in the TC100 strain expressing glycerol dehydratase and 1,3-PDO dehydrogenase regulated by the arabinose PBAD promoter. The genetic engineering and medium formulation approaches used here should aid in the separation of 1,3-propanediol from lactate, 2,3-butanediol, and ethanol and lead to increased production of 1,3-propanediol in Klebsiella pneumoniae.

Isolation of polyhydroxyalkanoates‐producing bacteria using a combination of phenotypic and genotypic approach

Aims:  To develop an efficient approach using a combination of phenotypic and genotypic methods for isolation of environmental bacteria that produce mid‐chain‐length polyhydroxyalkanoates (mcl‐PHAs).

Removal of cadmium ions during stationary growth phase by an extremely cadmium‐resistant strain of Stenotrophomonas sp.

Stenotrophomonas sp. CD02 was isolated from a site that previously had been contaminated with high concentrations of the heavy metals cadmium (3 mg kg(-1)) and chromium (115 mg kg(-1)). This strain was able to grow on complex (Luria Bertani) medium containing high concentrations of cadmium ion (up to 4 mM). Additionally, it could remove up to 80% of the dissolved ions but only after reaching stationary growth phase. Strain CD02 also tolerated high concentrations of other heavy metals such as chromium, zinc, copper, nickel, and lead at levels more than 2 mM. Although strain CD02 can tolerate much higher cadmium concentrations than the three Stenotrophomonas maltophilia strains tested, they all possess resistance to the same range of antibiotics. This suggests that strain CD02 possesses a mechanism that allows it to tolerate and remove cadmium differently from those conferring resistance to antibiotics. Strain CD02 can be a suitable candidate for heavy metal bioremediation in contaminated environment because it is able to tolerate high concentration of heavy metals and remove cadmium aerobically.

Enhanced polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli

Aim:  To develop an approach to enhance polyhydroxybutyrate (PHB) production via the coexpressed phaCAB and vgb genes controlled by arabinose PBAD promoter in Escherichia coli.

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.

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.

Biodegradation of methyl tert-butyl ether (MTBE) by Enterobacter sp. NKNU02

We previously isolated and identified Enterobacter sp. NKNU02 as a methyl tert-butyl ether (MTBE)-degrading bacterial strain from gasoline-contaminated water. In this study, tert-butyl alcohol, acetic acid, 2-propanol, and propenoic acid were detected using gas chromatography/mass spectrometry when MTBE was degraded by rest cells of Enterobacter sp. NKNU02 cells. We also found that biodegradation of MTBE was decreased, but not totally inhibited in mixtures of benzene, toluene, ethylbenzene and xylene. The effects of MTBE on the biology of Enterobacter sp. NKNU02 were elucidated using 2D proteomic analysis. The cytoplasmic proteins isolated from these MTBE-treated and -untreated cells were carried out for proteomic analysis. Results showed that there were 6 differential protein spots and 8 differential protein spots, respectively, as compared to their corresponding control (without MTBE addition), at the indicated incubation times when 40% and 60% of 100 mg/L of MTBE had been removed, Among these proteins, nine were successfully identified with matrix-assisted laser desorption ionization-time of flight-mass spectrometry. Proteins identified included extracellular solute-binding protein, periplasmic-binding protein ytfQ, cationic amino acid ABC transporter, isocitrate dehydrogenase, cysteine synthase A, alkyl hydroperoxide reductase (AhpC), transaldolase, and alcohol dehydrogenase. Based on these differential proteins, we discuss the bacterial responses to MTBE at the molecular level.

Isolation and characterization of an environmental cadmium‐ and tellurite‐resistant Pseudomonas strain

A Pseudomonas strain (TeU), resistant to tellurite (TeO(2)(3)(-) and cadmium (Cd(2+)) ions, was isolated from heavy-metal-contaminated sediments by enrichment. Black precipitates, presumably the product of the reduction of tellurite, such as tellurium, occurred in cultures of the isolate after growth in medium containing tellurite. Quantitative determination of the TeO(2)(3)(-) concentration in the liquid culture demonstrated a decreased concentration of tellurite (to less than 100 µM) from initial concentrations of approximately 1,000 µM within 24 h of growth. Strain TeU was resistant to TeO(2)(3)(-) and Cd(2+) concentrations as high as 2,000 µM and 500 µM, respectively. Transposon mutagenesis of strain TeU resulted in mutants exhibiting Cd(2+) sensitivity (Strain BU21) and one with decreased ability to reduce tellurite (strain AU08). Strain BU21 was less tolerant to Cd(2+) (100 µM) compared with the wild-type strain TeU (500 µM) but was still able to reduce tellurite to 80% of that of strain TeU. Although strain AU08 possesses the ability for Cd(2+) resistance, it reduced less than 20% of the initial concentrations of tellurite compared with strain TeU. Genes encoding an HflKC complex and a putative metallopeptidase were associated with the bacteriums capacity for tellurite reduction and Cd resistance, respectively. The ability to reduce tellurite therefore may not be necessary for this bacteriums heavy metal and metalloid tellurite resisting ability.