Doyun Shin

Distribution of the Microbial Community Structure in Sulfur-Based Autotrophic Denitrification Columns

Substrate-dependent evolution of a bacterial community capable of transforming nitrate was examined in sulfur-based autotrophic denitrification columns. The 16S rRNA genes and denaturing gradient gel electrophoresis (DGGE) analysis revealed that the initial bacterial consortium was well adapted to column operation time and distribution of nitrate concentration. In the lower part of a 200-day operated column where nitrate was introduced, a bacterial strain designated OTU DE-1 was abundant, occupying 92% of the community. The species was identified as Thiobacillus denitrificans with a similarity of 97% by BLAST search. A heterotroph designated as OTU DE-2 showing a similarity of 94% to Cenibacterium arsenoxidans was then enriched in the middle part of the column occupying 82% of the community, indicating the presence of organic electron donors. Interestingly, OTU DE-5 with a similarity of 98% to Chlorobium limicola, which is commonly present in hydrogen sulfide-rich environments, was found in the upper part...

Use of reporter-gene based bacteria to quantify phenanthrene biodegradation and toxicity in soil

A phenanthrene-degrading bacterium, Sphingomonas paucimobilis EPA505 was used to construct two fluorescence-based reporter strains. Strain D harboring gfp gene was constructed to generate green fluorescence when the strain started to biodegrade phenanthrene. Strain S possessing gef gene was designed to die once phenanthrene biodegradation was initiated and thus to lose green fluorescence when visualized by a live/dead cell staining. Confocal laser scanning microscopic observation followed by image analysis demonstrates that the fluorescence intensity generated by strain D increased and the intensity by strain S decreased linearly at the phenanthrene concentration of up to 200 mg/L. Such quantitative increase and decrease of fluorescence intensity in strain D (i.e., from 1 to 11.90 ± 0.72) and strain S (from 1 to 0.40 ± 0.07) were also evident in the presence of Ottawa sand spiked with the phenanthrene up to 1000 mg/kg. The potential use of the reporter strains in quantitatively determining biodegradable or toxic phenanthrene was discussed.

Potential use of a self-dying reporter bacterium to determine the bioavailability of aged phenanthrene in soil: Comparison with physicochemical measures

The potential bioavailability of phenanthrene aged in soil was determined by using a self-dying reporter bacterium, and the results were compared to two physicochemical measures, Tenax TA(®) bead-assisted desorption, and hydroxypropyl-β-cyclodextrin (HPCD) extraction. The reporter bacterium, capable of degrading phenanthrene as a sole carbon and energy source, was genetically reconstructed to die when it degrades phenanthrene. Therefore, population change of the reporter cells can be viewed as the quantification of bioavailable phenanthrene. When Ottawa sand was used as an aging matrix, the amounts of bioavailable phenanthrene (i.e. little gradual decrease) were similar, regardless of aging time, and consistent between the reporter bacterium and the two physicochemical measures. However, decrease in bioavailable phenanthrene with aging was readily evident in sandy loam with organic matter of 11.5%, with all three measures. More importantly, when the reporter bacterium was used, a rapid and significant decrease in the bioavailable fraction from 1.00 to 0.0431 was observed. The extent of decrease in bioavailable fraction was less than 40% in the two physicochemical measures, but was nearly 100% in the reporter bacterium, during the first 3 months of aging. Our results suggest that the phenanthrene fraction available to bacterial degradation, and probably the fraction that really manifests toxicity, may be much smaller than the fractions predicted with the physicochemical measures.

Microbial succession in response to 1,4-dioxane exposure in activated sludge reactors: Effect of inoculum source and extra carbon addition

Bacterial community succession related to 1,4-dioxane exposure was investigated in two different activated sludge-inoculated reactors (municipal wastewater and dye industrial wastewater sludge), with or without additional carbon source, for 7 weeks. The denaturing gradient gel electrophoresis (DGGE) analysis revealed that microbial succession varied according to the inoculum sludge sources and the presence or absence of the extra carbon source. In the reactor inoculated with the municipal sludge, bacterial species belonging to α - and γ -Proteobacteria and Nitrospira class were dominant over time. On the other hand, bacterial species showing significant homology to β -Proteobacteria (e.g., Methylibium petroleiphilum PM1) and Actinobacteria class, who have been reported to have 1,4-dioxane degradation potential, were found in the industrial sludge-inoculated reactors. The appearance of these bacteria demonstrates that the microbial community structure of the inoculum and the presence of an extra carbon source affect the microbial succession in the system exposed to 1,4-dioxane.

Effect of the redox dynamics on microbial-mediated As transformation coupled with Fe and S in flow-through sediment columns

Arsenic (As) biogeochemistry coupled with iron (Fe) and sulfur (S) was studied using columns packed with As(V)-contaminated sediments under two phases: a reduction phase followed by an oxidation phase. During the reduction phase, four identical columns inoculated with G. sulfurreducens were stimulated with 3mM acetate for 60days. The As(III) in the effluent rapidly increased then gradually decreased. The Fe(II) and sulfate concentration indicated ferrous sulfide precipitation inside the column after day 14 and X-ray absorption near edge structure spectra showed that As(III) was enriched at the column outlet. The genera Desulfosporosinus and Anaeromyxobacter as well as the Geobacter inoculum played a primary role in As reduction. During the oxidation phase, dissolved oxygen was consumed by heterotrophic aerobes belonging to the phylum Cloroflexi in the column with acetate, resulting in more As in the effluent. When only nitrate was injected, sulfur-oxidizing bacteria such as Thiobacillus thioparus instantly oxidized the sulfide formed during the first phase, resulting in less As(V) in the aqueous phase compared to the column with dissolved oxygen alone. This study showed that redox gradients and dynamics linked to Fe and S biogeochemistry have an important role in controlling As mobility in subsurface environments.

Field Applicability Study of Landfarming for Petroleum Hydrocarbons Contaminated Soils

ABSTRACT The landfarming treatment for the remediation of the petroleum contaminated soil at the returned U.S. Military bases wasinvestigated in this study. Specifically, the bioaugmentation performance using various commercially available petroleum-degrading bacteria was evaluated and the directions for enhancing the performance of the landfarming treatment weresuggested. The environmental factors of the soils at the returned U.S. Military bases chosen for remediation indicate thatthe landfarming treatment can be used as the remediation technique; however, the addition of nitrogen or phosphorus isrequired. The lab-scale landfarming treatment tests using the model soil and the site soil showed that the degradationefficiency was greater with the model soil than the site soil and that the treatment performance was not affected by thenumber of bacteria present in the soil in the range of 10 6 -10 12 CFU/g. These results suggest that the successful landfarmingtreatment depends on the petroleum degradability of bacteria used and the environmental conditions during the treatmentrather than the number of petroleum-degrading bacteria used. Key words: Landfarming, Petroleum-contaminated soil, Soil remediation, Petroleum-degrading bacteria, Contaminantaging

Determination of phenanthrene bioavailability by using a self-dying reporter bacterium: Test with model solids and soil

The present study was conducted to investigate the performance and feasibility of a self-dying reporter bacterium to visualize and quantify phenanthrene bioavailability in soil. The self-dying reporter bacterium was designed to die on the initiation of phenanthrene biodegradation. The viability of the reporter bacterium was determined by a fluorescence live/dead cell staining method and visualized by confocal laser scanning microscopic observation. Phenanthrene was spiked into four types of model solids and a sandy loam. The bioavailability of phenanthrene to the reporter bacterium was remarkably declined with the hydrophobicity of the model solids: essentially no phenanthrene was biodegraded in the presence of 9-nm pores and about 35.8% of initial phenanthrene was biodegraded without pores. Decrease in bioavailability was not evident in the nonporous hydrophilic bead, but a small decrease was observed in the porous hydrophilic bead at 1000 mg/kg of phenanthrene. The fluorescence intensity was commensurate with the extent of phenanthrene biodegradation by the reporter bacterium at the concentration range from 50 to 500 mg/kg. Such a quantitative relationship was also confirmed with a sandy loam spiked up to 1000 mg/kg of phenanthrene. This reporter bacterium may be a useful means to determine phenanthrene bioavailability in soil.

Enhancement of Denitrification Capacity of Pseudomonas sp. KY1 through the Optimization of C/N ratio of Liquid Molasses and Nitrate

This study was conducted to identify an optimal ratio of carbon to nitrogen (C/N ratio) for denitrification of nitrate using molasses as an external carbon source. A series of batch and column tests was conducted using an indigenous bacterium Pseudomonas sp. KY1 isolated from a nitrate-contaminated soil. For the initial nitrate-nitrogen concentration of 100 mg-N/L, batch test results indicated that C/N ratio of 3/1 was the optimal ratio with a relatively high pseudo-first-order reaction constant of 0.0263 hr. At C/N ratio of 3/1, more than 80% of nitrate-nitrogen concentration of 100 mg-N/L was removed in 100 hrs. Results of column tests with a flow velocity of 0.3 mL/min also indicated that the C/N ratio of 3/1 was optimal for denitrification with minimizing remaining molasses concentrations. After 172 hrs of column operation (35 pore volumes) with an influent nitrate-nitrogen concentration of 100 mg-N/L, the effluent met the drinking water standard (i.e., 10 mg NO3-N/L).

Potential use of a novel reporter bacterium to determine phenanthrene biodegradation and toxicity in a model solid

Laser photostimulation of inoculum of selected fungi could be also supporting factor of stimulation of mycorhisis moulds and adaptation of the infected roots of seedlings to contaminated soil. Proper photostimulation of inoculum of selected moulds and bacteria could also biodegradation of some organic pollutants of soil and water. Laser biotechnology seems to be a new tool of sustainable development of different kind of the regions (industrial as we as rural), including contribution to prevention against food in the rivers regions as well as for more effective protection of aquatic ecosystems against euthrophisation.