A multidisciplinary assessment of the impact of spilled acids on geoecosystems: an overview.

We developed and applied a multidisciplinary approach to the impact of an accidentally spilled acid on the underlying geomedia and subsurface environment, based on the concept of geoecosystem. We used mineralogical, geochemical, microbiological, and ecotoxicological techniques to identify and assess the multiple aspects involved. First, we constructed a conceptual model for the acid interactions with the underlying subsurface environment by introducing the concept of a geoecosystem-a multicomponent system composed of inorganic, organic, and biological components to describe the subsurface environment. Second, we designed and manufactured a two dimensional cell to visualize acid transport through geomedia. Third, we hypothesized that the acids are neutralized through dissolution of minerals and protonation of functional groups on the surfaces of minerals and organic matter. We tested this hypothesis by conducting batch-type geomedia-acid reaction and surface titration experiments. Fourth, we observed changes in soil microbial communities before and after the acid exposure and neutralization treatment. Fifth, we performed flow-through experiments using columns packed with soil samples pre-contaminated with arsenic to investigate potential longer term, secondary effects of remnant acids on geoecosystems. Finally, we conducted ecotoxicological investigations using various geomedia and observed that suitability of the geoecosystem as a habitat deteriorated to different degrees depending on the respective systems' acid neutralizing power. We conclude that a holistic understanding of the interactions among the multiple components of geoecosystems and subsequent estimation of the influenced area requires a multidisciplinary approach such as those used in this study. Based on the findings of this study, we propose geoecosystems' vulnerability defined as the reciprocal of their acid-neutralizing capacity against the moving acid fronts and present this concept as central to a quantitative assessment of the impact of acid spills on geoecosystems. We also inventoried the essential components, factors, and parameters necessary in developing geoecosystems' acid vulnerability assessment system.

Fate and toxicity of spilled chemicals in groundwater and soil environment I: strong acids.

We reviewed the chemical/physical properties, toxicity, environmental fate, and ecotoxicity of strong acids in soil and groundwater environments. We recommend that sulfuric acid and hydrofluoric acid be classified as chemicals of priority control based on volumes used, toxicity, carcinogenicity, and past significant spill events. Understanding the behavior and transport of spilled strong acids in soil and groundwater environments requires a multi-disciplinary approach, as they can undergo a variety of geochemical and biochemical reactions with complex geomedia. The toxicity of spilled acid is dependent on the characteristics of the geomedia exposed to the acid and the amount of residual protons following acid-substrate interaction. Soil texture, cation exchange capacity, mineral composition, bedrock type, and aluminum content may be important factors affecting the toxicity of spilled acid in soil-groundwater environments. We expect that the results of this study will contribute preliminary data for future research on chemical spills.

Soil microbial community responses to acid exposure and neutralization treatment.

Changes in microbial community induced by acid shock were studied in the context of potential release of acids to the environment due to chemical accidents. The responses of microbial communities in three different soils to the exposure to sulfuric or hydrofluoric acid and to the subsequent neutralization treatment were investigated as functions of acid concentration and exposure time by using 16S-rRNA gene based pyrosequencing and DGGE (Denaturing Gradient Gel Electrophoresis). Measurements of soil pH and dissolved ion concentrations revealed that the added acids were neutralized to different degrees, depending on the mineral composition and soil texture. Hydrofluoric acid was more effectively neutralized by the soils, compared with sulfuric acid at the same normality. Gram-negative ß-Proteobacteria were shown to be the most acid-sensitive bacterial strains, while spore-forming Gram-positive Bacilli were the most acid-tolerant. The results of this study suggest that the Gram-positive to Gram-negative bacterial ratio may serve as an effective bio-indicator in assessing the impact of the acid shock on the microbial community. Neutralization treatments helped recover the ratio closer to their original values. The findings of this study show that microbial community changes as well as geochemical changes such as pH and dissolved ion concentrations need to be considered in estimating the impact of an acid spill, in selecting an optimal remediation strategy, and in deciding when to end remedial actions at the acid spill impacted site.

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.

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-ß-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.

Effect of Fenton reagent shock and recovery periods on anaerobic microbial community structure and degradation of chlorinated aliphatics.

This study investigates the effect of Fenton reagent on the structure and function of a microbial consortium during the anaerobic degradation of hexachloroethane (HCA) and tetrachloroethene (PCE). Anaerobic biodegradation tests of HCA and PCE were performed in batch reactors using an anaerobic microbial consortium that had been exposed to Fenton reagent for durations of 0, 0.04, and 2 days and then allowed to recover for periods of 0, 3, and 7 days. The bacterial community structure was determined using culture-independent methods of 16S rRNA gene sequencing and automated ribosomal intergenic spacer analysis. Larger recovery periods partially restored the microbial community structure; however, the recovery periods did not restore the loss of ability to degrade HCA and PCE in cultures shocked for 0.04 days, and PCE in cultures shocked for 2 days. Overall the exposure to Fenton reagent had an impact on bacterial community structure with downstream effects on HCA and PCE degradation. This study highlights that the impacts of short- and long-term shocks on microbial community structure and function can be correlated using a combination of biodegradation tests and community structure analysis tools.

Survival of introduced phosphate-solubilizing bacteria (PSB) and their impact on microbial community structure during the phytoextraction of Cd-contaminated soil.

This study was conducted to investigate whether or not phosphate-solubilizing bacteria (PSB) as a kind of plant growth promoting rhizobacteria enhance the uptake of Cd by plants. In addition, the effect of PSB augmentation during phytoextraction on the microbial community of indigenous soil bacteria was also studied. In the initial Cd-contaminated soil, the major phyla were Proteobacteria (35%), Actinobacteria (38%) and Firmicutes (8%). While Proteobacteria were dominant at the second and sixth week (41 and 54%, respectively) in inoculated soil, Firmicutes (mainly belonging to the Bacilli class-61%), dramatically increased in the eight-week soil. For the uninoculated soil, the proportion of α-Proteobacteria increased after eight weeks (32%). Interestingly, Actinobacteria class, which was originally present in the soil (37%), seemed to disappear during phytoremediation, irrespective of whether PSB was inoculated or not. Cluster analysis and Principal Component Analysis revealed that the microbial community of eight-week inoculated soil was completely separated from the other soil samples, due to the dramatic increase of Bacillus aryabhattai. These findings revealed that it took at least eight weeks for the inoculated Bacillus sp. to functionally adapt to the introduced soil, against competition with indigenous microorganisms in soil. An ecological understanding of interaction among augmented bacteria, plant and indigenous soil bacteria is needed, for proper management of phytoextraction.

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.

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.

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 alpha- and gamma-Proteobacteria and Nitrospira class were dominant over time. On the other hand, bacterial species showing significant homology to beta-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.

Stenotrophomonas koreensis sp. nov., isolated from compost in South Korea.

A Gram-negative, rod-shaped, non-spore-forming bacterium (TR6-01T) was isolated from compost near Daejeon city in South Korea. On the basis of 16S rRNA gene sequence similarity, strain TR6-01T was shown to belong to the class Gammaproteobacteria, related to Stenotrophomonas acidaminiphila (97.1 %) and Stenotrophomonas maltophilia (96.9 %); the phylogenetic distance from any other established species within the genus Stenotrophomonas was less than 97.0 %. Phenotypic and chemotaxonomic data (major ubiquinone Q-8; fatty acid profile) supported the affiliation of strain TR6-01T to the genus Stenotrophomonas. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain TR6-01T from the five Stenotrophomonas species with validly published names. TR6-01T therefore represents a novel species, for which the name Stenotrophomonas koreensis sp. nov. is proposed, with the type strain TR6-01T (= KCTC 12211T = JCM 13256T).

Paracoccus koreensis sp. nov., isolated from anaerobic granules in an upflow anaerobic sludge blanket (UASB) reactor.

A Gram-negative, short rod- to coccus-shaped, non-spore-forming bacterium (Ch05(T)) was isolated from granules in an upflow anaerobic sludge blanket (UASB) reactor. On the basis of 16S rRNA gene sequence similarity, strain Ch05(T) was shown to belong to the subclass alpha-Proteobacteria, being related to Paracoccus solventivorans (97.5%), Paracoccus alkenifer (96.9%) and Paracoccus kocurii (96.4%). The phylogenetic distance from Paracoccus species with validly published names was always less than 96%. Physiological and chemotaxonomic data (major ubiquinone, Q-10; major fatty acids, C(18:1) and C(18:0)) supported the affiliation of strain Ch05(T) to the genus Paracoccus. The results of DNA-DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain Ch05(T) from the 17 Paracoccus species with validly published names. Ch05(T) therefore represents a novel species, for which the name Paracoccus koreensis sp. nov. is proposed. The type strain is Ch05(T) (=KCTC 12238(T)=IAM 15216(T)).