Hexahydro-1,3,5-Trinitro-1,3,5-Triazine (Rdx) Degradation in Biologically-Active Iron Columns

Flow-through columns were used to evaluate the efficacy of permeable reactive iron barriers to treat ground water contamination by RDX. Three columns were packed with iron filings (Fe⁰) between soil and sand layers, and were fed continuously with unlabeled plus ¹⁴C-labeled RDX to characterize its removal efficiency under different microbial conditions. One column was poison-sterilized to isolate chemical degradation processes, another was not poisoned to allow colonization of the Fe⁰ layer by indigenous microorganisms, and a third column was amended with anaerobic sludge to evaluate the benefits of enhancing biodegradation through bioaugmentation. Extensive RDX removal (>99%) occurred through the Fe⁰layer of all columns for more than one year, although ¹⁴C-label analysis indicated the presence of soluble byproducts such as methylenedinitramine. RDX byproducts accumulated to a lesser extent in biologically active columns, possibly due to enhanced mineralization by the cumulative action of microbial and chemical degradation processes. Denaturing gradient gel electrophoresis (DGGE) profiles and nucleotide sequencing revealed a predominance of Acetobacterium sp. in the iron layer of all columns after 95 days. Such homoacetogenic bacteria probably feed on hydrogen produced during Fe⁰ corrosion and participate on the RDX degradation process. This notion was supported by batch experiments with a mixed homoacetogenic culture isolated from the bioaugmented column, which degraded RDX and produced acetate when H₂ was present. Overall, this work suggests that Fe⁰barriers can effectively intercept RDX plumes, and that treatment efficiency can be enhanced by biogeochemical interactions though bioaugmentation.     

Changes in properties of soil-derived dissolved organic matter induced by biodegradation

Properties of dissolved organic matter (DOM) determine its biodegradation. In turn, biodegradation changes the properties of the remaining DOM, which may be decisive for the formation of stable organic carbon in soil. To gain information on both mechanisms and controlling factors of DOM biodegradation and the properties of biodegraded DOM, we investigated changes in the composition of 13 different DOM samples extracted from maize straw, forest floors, peats, and agricultural soils during a 90-day incubation using UV absorbance, fluorescence emission spectroscopy, FTIR-spectroscopy, ¹H-NMR spectroscopy, pyrolysis-field ionization mass spectroscopy (Py-FIMS), and ¹³C natural abundance before and after incubation. Changes in the DOM properties were related to the extent of biodegradation determined by the release of CO₂. Increasing UV absorption and humification indices deduced from fluorescence emission spectra, and increasing portions of aromatic H indicated relative enrichment of aromatic compounds during biodegradation. This enrichment significantly correlated with the amount of DOC mineralized suggesting that aromatic compounds were relatively stable and slowly mineralized. ¹³C depletion during the incubation of highly degradable DOM solutions indicated an enrichment of lignin-derived aromatic compounds. Py-FI mass spectra indicated increasing contents of phenols and lignin monomers at the expense of lignin dimers and alkylaromatics during incubation. This partial degradation of higher-molecular, lignin-derived DOM compounds was accompanied by relative increases in the proportions of lower-molecular degradation products and microbial metabolites. Carbohydrates, especially when abundant at high initial contents, seem to be the preferred substrate for microorganisms. However, four independent methods suggested also some microbial production of carbohydrates and peptides during DOM degradation. After incubation, the composition of highly degradable DOM samples became similar to relatively stable DOM samples with respect to aromaticity, carbohydrate content, and thermal stability. We conclude that DOM biodegradation seems to result in organic matter properties being a precondition for the formation of stable carbon. These structural changes induced by DOM biodegradation should also result in stronger DOM sorption to the soil matrix additionally affecting DOM stabilization.     

Time‐resolved in‐situ pH measurement in differently treated,saturated and unsaturated soils

The pH‐value is of utmost relevance for soil properties and functioning. Hence, a time‐resolved in‐situ measurement is mandatory but lacking. As an alternative, a two‐probe pH electrode with gel‐covered reference electrode was newly constructed and tested for a continuous, in‐situ pH recording in saturated and unsaturated soil. This was done using samples from a set of 14 soils with different composition and pH$ _{\rm CaCl_2} $ ranging from 3.5 to 7.5 in batch and repacked soil column experiments. In the latter, changes in pH and redox potential were monitored upon transport of citrate‐phosphate buffer and pig slurry through the soil columns. The pH measurements were largely stable even upon substantial shifts in soil moisture content down to air‐dry conditions. The results of the pH measurements agreed with standard methods using settled soil suspensions in electrolyte solutions and the conventional combination (single‐probe) pH electrode. Testing the suspension effect, it was found that measuring pH directly in the soil is recommended. The pH measured in‐situ was closest to pH values determined in 0.01 M CaCl₂ suspensions according to DIN ISO 10390 (DIN, 2005 ). The transport of citrate buffer and pig slurry as pH active substances through soil induced strong effects on the pH and in part on the redox potential; the reversible effects lasted over days, which may affect the mobility and speciation of nutrients and pollutants as well as microbial processes.     

Temperature and pH effects on biodegradation of hexachlorocyclohexane isomers in water and a soil slurry

This study was conducted to monitor the biodegradation of alpha-, beta-, gamma-, and delta-hexachlorocyclohexane (HCH) isomers in liquid culture by a Pandoraea species and determine the influence of pH and temperature on the biodegradation of alpha- and gamma-HCH in liquid as well as in soil slurry cultures. The Pandoraea species degraded 79.4% delta-HCH and 34.3% gamma-HCH in liquid culture at 4 weeks of incubation. alpha- and beta-HCH exhibited almost identical rates (41.6 and 42.4%, respectively) of degradation. The highest degradation of alpha- and gamma-HCH (67.1 and 60.2%, respectively) was observed at an initial pH of 8.0 in liquid; 58.4 and 51.7% rates of degradation of alpha- and gamma-HCH, respectively, at an initial pH of 9.0 were found in soil slurry cultures. An incubation temperature of 30 degrees C was optimum for effective degradation of alpha- and gamma-HCH isomers (62.5 and 57.7%, respectively) in liquid culture, and 54.3 and 51.9% rates of degradation of alpha- and gamma-HCH isomers, respectively, were found in a soil slurry. Increasing the soil/water ratio decreased the extent of degradation of both HCH isomers. Degradation of HCH isomers occurred concomitant with bacterial growth. Byproducts of growth from Pandoraea species significantly decreased the pH of the liquid and the soil slurry during the growth on HCH isomers. The results of this study suggest that this bacterial strain may effectively be used for remediating polluted sites and water contaminated with different HCH isomers over a range of environmental conditions.     

Dynamics and origin of the non-hydrolysable organic fraction in a forest and a cultivated temperate soil, as determined by isotopic and microscopic studies

We isolated the non‐hydrolysable macromolecular organic fraction (insoluble fraction resistant to drastic laboratory hydrolyses) from a temperate, loamy, forest soil (Lacadée, France) and from the soil of an adjacent plot cleared 35 years ago and continuously cropped with maize. The quantitative, morphological (light, scanning and transmission electron microscopy) and isotopic (bulk δ¹³C, individual compound δ¹³C and radiocarbon dating) features of these two non‐hydrolysable fractions were determined and compared. It appeared that: (i) extensive degradation of the non‐hydrolysable material inherited from the forest soil occurred upon cropping, revealing that its resistance to drastic laboratory hydrolyses is not paralleled by a great resistance to natural biodegradation triggered by change in land use; (ii) only small inputs of maize‐derived compounds occurred in the non‐hydrolysable fraction of the cultivated soil so that, in spite of extensive degradation, the forest‐inherited carbon still predominates; (iii) the non‐hydrolysable fractions of both soils comprise the same components (wood debris, spores, pollen, and, predominantly, granular organic aggregates), which correlate with the previously identified chemical components (charcoal, aliphatic lipid components and melanoidin‐like components); (iv) the non‐hydrolysable fraction of the cropped soil shows a greater contribution of aliphatic moieties, reflecting differential degradation of the components of the non‐hydrolysable material inherited from the forest soil; (v) this degradation resulted in enrichment in the oldest components; and (vi) no relationship is observed, in the two Lacadée soils, between resistance to drastic laboratory hydrolyses, on the one hand, and stability towards biodegradation in situ, on the other. These observations, added to recent ones on other types of soils, suggest that such a conspicuous uncoupling between non‐hydrolysable and stable carbon is probably a general feature of organic matter in soil as opposed to sedimentary organic matter.     

Enrichment of functional microbes and genes during pyrene degradation in two different soils

Purpose

Stimulating microbial degradation is a promising strategy for the remediation of soils contaminated with polycyclic aromatic hydrocarbons (PAHs). To better understand the functional microbial populations and processes involved in pyrene biodegradation in situ, the dynamics of pyrene degradation and functional microbial abundance were monitored during pyrene incubation in soils. We hope our findings will provide new insights into in situ pyrene biodegradation in soils and help to identify functional microbes from soils.

Materials and methods

Pyrene (60 mg kg^?1) was incubated with two different soils, one is lower PAH-containing agricultural soil (LS), and the other is higher PAH-containing industrial soil (HS). During incubation, triplicate samples were collected on days 0, 3, 7, 14, and 35. Pyrene in soil samples was analyzed using an Agilent gas chromatograph (7890A) equipped with a mass-selective detector (model 5897). DNA in soils was extracted with a FastDNA Spin kit for soil (Bio101, USA). The abundance of functional microbes and genes was monitored by a Taqman or SYBR Green based real-time PCR quantification using an iCycler iQ5 themocycler (Bio-Rad, USA). The diversity of PAH-RHDα GP genes was evaluated by constructing clone libraries and sequencing.

Results and discussion

In both soils, more than 80 % of the added pyrene was degraded within 35 days. After 35-day incubation, there was a significant enrichment of Gram-positive bacteria harboring PAH-ring hydroxylation dioxygenase (PAH-RHD_α GP) genes, and the abundance of Mycobacterium increased significantly. In PAH-RHD_α GP clone libraries from two soils, Mycobacterium was detected, while most sequences were closely related to uncultured Gram-positive bacteria. In addition, two pyrene catabolic pathways might be involved in pyrene degradation, as pyrene dioxygenase genes, nidA and nidA3, were dramatically enriched during incubation. Moreover, the abundance and diversity of potential degraders in two soils showed significantly difference in responding to pyrene stress. This result indicates that soil condition can significantly affect functional microbial populations and biological process for pyrene biodegradation.

Conclusions

These results revealed that Mycobacterium as well as uncultured Gram-positive PAH-RHD_α genotypes may be the important group of pyrene degraders in soils, and two pyrene catabolic pathways, targeted by nidA and nidA3, might potentially contribute to in situ biodegradation of pyrene. This study characterized the response pattern of potential pyrene degraders to pyrene stress in two different soils, which would increase our understanding of the indigenous processes of pyrene biodegradation in soil environment.

     

In Situ partitioning and biomagnification of mercury in industrially polluted Husainsagar Lake,Hyderabad, India

The distribution of Hg in water, sediment, plankton and fish along with the in situ biomagnification of the metal in Husainsagar Lake, India were measured. The concentration of Hg in the water was 10 μg L^?1 and exceeded the water quality criteria standards. Sediments of the lake at different sites had Hg concentrations several thousand fold more than that of the overlying water column and showed relationship with the water content of the wet sediment. Sediments in the lake acted as a `sink' for the metal. The concentrations of Hg increased from nanoplankton → phytoplankton -> zooplankton. Fish, an end organism in the food chain, had Hg concentrations higher than that of nanoplankton and lower than those of phytoplankton and zooplankton. This trend did not illustrate the expected pattern of food chain enrichment in the classical sense as noted for chlorinated hydrocarbons (DDT).     

Application of an Enhanced Electrokinetic Ion Injection System to Bioremediation

Recently, electrokinetic injection has been applied in bioremediationto provide nutrients and TEAs in low permeable soil. However, an effective pH control system and a uniform injection system have yet to be developed. This study investigated an enhanced EK injection system, which is combined with electrolyte circulation and electrode polarity reversal on kaolinite. Soil pH was maintained continuously only by circulation of electrolytes in each chamber without any buffering solutions. Existing ions were distributed more uniformly in soil by electrodepolarity reversal. In view of microbiological degradation, the polarity reversal system resulted in better ion injection. Therefore, the novel electrokinetic nutrient and TEAs injectionsystem can be applied to in situ biodegradation more effectively than the conventional technique.     

Non-additive effects of mixing different sources of dissolved organic matter on its biodegradation

To examine the potential impact of plant species richness on ecosystems, we studied non-additive effects of different plant litters on the biodegradation rate of dissolved organic matter (DOM) when mixing plant leaf-derived DOM derived from different plant species. A full factorial biodegradation experiment (31 possible singular and multiple combinations of five litter type-derived DOM sources) was conducted using plant litters from the five most abundant plant species in a subtropical watershed ecosystem, from which dissolved organic carbon (DOC) disappearance was measured. Loss of DOC over time was considered biodegradable DOC. We tested whether DOM diversity, measured as source species richness and composition, would affect biodegradation rates. Overall, we found significant non-additive (synergistic) effects of DOM diversity on biodegradation rates of DOM, which were explained both by plant species richness and composition. Across all treatments, a significantly higher biodegradation rate was correlated with the presence of DOM from higher nitrogen (N) containing plant litters; conversely, the presence of lower N decreased these rates. The N content and chemical characteristic of DOM might influence the magnitude of the synergistic effect. Our results suggest that loss of plant litter species diversity would not affect DOC biodegradation rate, provided that at least two species are conserved. However, the variability in DOC biodegradation rate across the treatments decreased with increased DOM diversity at three incubation time points. Our results also indicate that in an ecosystem with low plant biodiversity, loss of key species such as Lophostemon confertus could reduce the synergistic effects on DOC biodegradation rate.     

Soils with complex organic profiles on the Vasyugan Plain

Soils with intricate patterns of their humus profiles developing in the neutral-calcium landscapes of the southern taiga of Western Siberia under highly dynamic paleogeographic, climatic, and weather conditions are characterized. The specific features of these soils comprise the diverse modern humus horizons along with the relic ones of different preservation rates, shallow leaching of carbonates, and a weak development of the middle-profile soil horizons. Specifying these organo-accumulative soils is substantiated by their high humus content against the geochemical background of the clayey calcareous parent rocks. The conjugated series of soils reflect different stages of the soil evolution (the humus profile degradation, the development of eluvial process, and the increase of contrasts in the acid-base conditions) and the hydromorphic transformation accompanied by the formation of organic horizons making the humus profile more complicated. In accordance with the diagnostic horizons, the position of the soils studied was determined in the Classification and Diagnostics of Soils of Russia. The relic enrichment of the humus horizon is proposed to be used as a specific feature of these soils.     

运用双液相系统进行三氯苯的生物降解

Due to easy volatilization of volatile organic compounds from water,it is difficult to monitor their aerobic biodegradation in the traditional single water system.Whether a two-liquid-phase system(TLPS) could overcome this obstacle and enhance the degradation of volatile contaminants? In this study,a TLPS composed of silicone oil and water was employed to investigate the biodegradation of volatile compounds,trichlorobenzenes(TCBs),by the adapted microorganisms in an activated soil.The degradation and volatilization of TCBs in TLPS and in a single water system were compared.The results showed that due to volatilization losses of TCBs,the mass balance of TCBs in a single water system was very low.In contrast,using TLPS could effectively inhibit the volatilization losses of TCBs and achieved a very good mass balance during the biodegradation process.Meanwhile,the TLPS could increase microbial activity and microbial growth during the degradation process.With TLPS,the TCB degradation was in descending order of 1,2,4-TCB> 1,2,3-TCB>> 1,3,5-TCB,which was related to the exposed concentration of the contaminants in soil.This study showed that TLPS could be employed as an effective tool to evaluate the biodegradation of volatile hydrophobic organic compounds,which could not be achieved with the traditional single water system.     

Biodegradation of chlorinated phenols in subsurface soils

Microcosm studies were employed to determine the subsurface biodegradation rates of phenol, 2-chlorophenol (2-CP), 2,4-dichlorophenol (2,4-DCP), 2,4,6-trichlorophenol (2,4,6-TCP), and pentachlorophenol (PCP). Soil samples were taken from sites in Pennsylvania and Virginia from depths up to 31 m, and all samples contained significant microbial populations. Soil from both sites readily biodegraded all five compounds. Biodegradation rates increased as initial concentrations increased, and all biodegradation rates appeared to follow first-order kinetics with regard to the initial compound concentrations. Biodegradation rates for the five compounds followed the order: phenol = 2-CP > 2,4,6-TCP > 2,4-DCP. PCP was degraded more slowly than phenol or 2-CP, but similarly to 2,4,6-TCP and 2,4-DCP. Different soils exhibited different degradation rates, and the soil characteristics that may influence the rates are discussed. The data suggest that biological degradation is a significant attenuation mechanism for phenol and its chlorinated derivatives in subsurfaces saturated and unsaturated zones.     

Desorption and Transformation of Nitroaromatic (TNT) and Nitramine (RDX and HMX) Explosive Residues on Detonated Pure Mineral Phases

Explosive compounds, including known toxicants 2,4,6-trinitrotoluene (TNT) and hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), are loaded to soils during military training. Their fate in soils is ultimately controlled by soil mineralogical and biogeochemical processes. We detonated pure mineral phases with Composition B, a mixture of TNT and RDX, and investigated the fate of detonation residues in aqueous slurries constructed from the detonated minerals. The pure minerals included Ottawa sand (quartz and calcite), microcline feldspar, phlogopite mica, muscovite mica, vermiculite clay, beidellite (a representative of the smectite clay group), and nontronite clay. Energy-dispersive X-ray spectrometry, X-ray diffraction, and gas adsorption surface area measurements were made of the pristine and detonated minerals. Batch slurries of detonated minerals and deionized water were sampled for 141?days and TNT, RDX, and TNT transformation products were measured from the aqueous samples and from the mineral substrates at day?141. Detonated samples generally exhibited lower gas adsorption surface areas than pristine ones, likely from residue coating, shock-induced compaction, sintering, and/or partial fusion. TNT and RDX exhibited analyte loss in almost all batch solutions over time but loss was greater in vermiculite, beidellite, and phlogopite than in muscovite and quartz. This suggests common phyllosilicate mineral substrates could be used on military training ranges to minimize off-site migration of explosive residues. We present a conceptual model to represent the physical and chemical processes that occurred in our aqueous batches over time.     

含根量与土壤抗剪强度增加值关系的试验研究

通过对裸地、车桑子灌木林地不同深度土壤在天然含水量情况下的直剪试验，及对车桑子根系沿垂直方向的分布情况的调查，研究了含根量对非饱和土抗剪强度的影响。结果表明，植物根系具有提高非饱和土抗剪强度的作用，含根量沿垂直方向呈指数函数规律分布，黏聚力和内摩擦角沿垂直方向的增加值均与含根量成正相关关系。     

Sequential Microaerophilic-Oxic Phase Mineralization of Azo Dyes by a Monoculture of Pseudomonas Aeruginosa Strain AWF Isolated from Textile Wastewater

A novel dye degrading bacterium capable of decolorizing and mineralizing four different dyes (Methyl red, Orange II, Direct red 80, and Direct blue 71) was isolated from textile industrial wastewater using the selective enrichment technique. The bacterium was identified as Pseudomonas aeruginosa. More than 80 % decolorization of Direct red 80 was obtained under microaerophilic conditions in 48 h, whereas only 10 % color removal was obtained under oxic conditions at the same time. Subsequent aeration of the decolorized medium resulted in the mineralization of the metabolic intermediates generated after azo bond cleavage by P. aeruginosa as confirmed by total organic carbon content and high-performance liquid chromatography analyses. The degradation products were characterized by Fourier transform infrared spectrometer and nuclear magnetic resonance techniques whereas the biotoxicity profile of the samples were evaluated using the brine shrimp lethality test assay. Data from this study provide evidence of dye mineralization and detoxification by a monoculture of P. aeruginosa in successive microaerophilic/oxic stages.     

Adsorption effect on the degradation of carbaryl, mecoprop, and paraquat by anodic fenton treatment in an SWy-2 montmorillonite clay slurry

The Fenton reaction-based anodic Fenton treatment (AFT) was applied to three widely used organic agrochemicals, carbaryl, mecoprop, and paraquat, in a clay slurry. The adsorption and degradation behaviors of these neutral (carbaryl), anionic (mecoprop), and cationic (paraquat) agrochemicals were studied in a slurry of SWy-2 Na(+)-montmorillonite clay, and adsorption isotherms were obtained at given experimental conditions. The d spacing (d 001) of the clay layer before and after adsorption or degradation was measured by X-ray diffraction (XRD). On the basis of the change of d spacing, molecular disposition at the clay interlayer was inferred: both mecoprop and paraquat form a monolayer sitting flat and parallel to the clay siloxane surfaces. Results show that, due to different adsorption mechanisms, the adsorption effect on chemical degradation by AFT varies with pesticide: strong and tight adsorption of paraquat at the clay interlayer protects paraquat from being attacked by hydroxyl radicals; loosely adsorbed carbaryl or mecoprop is readily degraded. XRD analysis clearly indicates that AFT is capable of effectively degrading interlayer noncationic organic chemicals that are not usually available for biodegradation.     

Crude Oil-Contaminated Soil Phytoremediation by Using Cyperus brevifolius (Rottb.) Hassk

The degradation of total oil and grease (TOG) in crude oil-contaminated soil in the presence of Cyperus brevifolius (Rottb.) Hassk was investigated in a net house study. C. brevifolius plants were transplanted in to spiked soil containing 8% (w/w) crude oil. The capability of plant for enhancing the biodegradation process was tested in pots containing fertilized and unfertilized soil over a 360-day period. Analysis of the degradation of hydrocarbon contaminants, plant growth, and biomass was conducted at 60-day interval. In the presence of contaminants, plant biomass and height were significantly reduced. The specific root surface area was reduced under the effects of crude oil. Concerning TOG content in soil, C. brevifolius could decrease up to 86.2% in TA (crude oil-contaminated soil with fertilizer) and 61.2% in TC (crude oil-contaminated soil without fertilizer). In the unvegetated pots, the reduction of TOG was 13.7% in TB (crude oil-contaminated soil with fertilizer) and 12.5% in TD (crude oil-contaminated soil without fertilizer). However, biodegradation was significantly more in vegetated pots than in unvegetated pots (p?=?0.05). The addition of fertilizer had positive effect on TOG degradation in the presence of C. brevifolius compared to the unfertilized treatments. Thus, there was evidence of C. brevifolius enhancing the biodegradation of crude oil in soil under the conditions of this experiment.     

Effects of nutrient concentration on the biodegradation of crude oil and associated microbial populations in the soil

The influence of nutrient amendments on the biodegradation of a crude oil and on associated microbial populations in an agricultural soil was studied in microcosms during a 150-day experiment. Concentration and chemical composition of residual hydrocarbons (HC) were periodically monitored in series of polluted soils unfertilized and fertilized with increasing concentrations of nutrients. The decrease in HC concentration was effective in all soils over time. The maximal biodegradation extent was 62%, meanwhile the natural attenuation contributed to 47% of degradation. A permanent inhibition of hydrocarbons assimilation was recorded with a high input of nutrients. The biodegradation of saturates, aromatics and polars was respectively, permanently, temporally and not reduced by excessive fertilization in soil. Accumulation of polar metabolic by-products was demonstrated. Enumerations of total heterotrophic bacteria and hydrocarbon-adapted bacteria showed a strong stimulation in both populations. Maximum stimulation was observed with the highest input of nutrients. However, the extents of biodegradation were not concurrently improved, thus indicating that the microbial degraders were selected depending on the nutrient supply. The permanent and/or temporally inhibition of the saturated and unsaturated HC assimilation revealed that different nutrient supplies were optimum for the degradation of aliphatic and aromatic HC.     

Variability of communities and physiological characteristics between free-living bacteria and attached bacteria during the PAH biodegradation in a soil/water system

The biodegradation of polycyclic aromatic hydrocarbons (PAHs) via free-living and attached micro-organisms in soil/water systems was observed in order to examine the variability in the community dynamics and physiological profiles of the micro-organisms. As determined by fluorescence in situ hybridization (FISH), the Domain Bacteria, consisting of three phyla α-, β- and γ-Proteobacteria, reached 41.27–56.05% of all organisms in the soil/water system for PAH biodegradation. Among the free-living species, Proteobacteria, including Brevundimonas (Pseudomonas) diminuta, Caulobacter spp., Mycoplana bullata, Acidovorax spp. and Pseudomonas aeruginosa were found to be dominant—making up 93.51–99.80% of the population—and therefore seem to be associated with PAH biodegradation. Total plate count numbers and the count of Pseudomonas sp. present in the free-living population increased to between 10³ and 10⁶ CFU ml⁻¹ when clay with very low organic matter content was used as the matrix for PAH degradation. However, total plate count microbial numbers increased to only 10¹–10² CFU ml⁻¹ using natural soil from Taichung containing 1.883% organic matter. The soil organic content (SOM) seemed to affect the mass transfer of PAH in soil, leading to the difference in PAH biodegradation. Two different approaches, which included community-level physiological profiling (CLPP) and ectoenzymatic activities, were used to explain the functional diversity between free-living and attached bacteria. The free-living and attached bacterial communities from the clay system showed proportionately greater differences using CLPP. Relatively high levels of esterases, aminopeptidases and some specific glycolysis-gluconeogenesis enzymes gave an identifiable correlation with PAH biodegradation. The differences in bacterial composition, numbers and physiological characteristics show that free-living and attached micro-organisms may play different biochemical roles in PAH degradation in soil.     

Part 1: Vadose-Zone Column Studies of Toluene (Enhanced Bioremediation) in a Shallow Unconfined Aquifer

The objectives of the laboratory study described in this paper were (1) to determine the effectiveness of four nutrient solutions and a control in stimulating the microbial degradation of toluene in the unsaturated zone as an alternative to bioremediation methodologies such as air sparging, in situ vitrification, or others (Part I), and (2) to compare the effectiveness of the addition of the most effective nutrient solution from Part I (modified Hoagland type, nitrate-rich) and hydrogen peroxide (H₂O₂) on microbial degradation of toluene for repeated, simulated spills in the unsaturated zone (Part II). For Part 1, fifteen columns (30-cm diameter by 150-cm height), packed with air-dried, 0.25-mm, medium-fine sand, were prepared to simulate shallow unconfined aquifer conditions. Toluene (10 mL) was added to the surface of each column, and soil solution and soil gas samples were collected from the columns every third day for 21 days. On day 21, a second application of toluene (10 mL) was made, and the experiment was run for another 21 days. Solution 4 was the most effective for microbial degradation in Part I. For Part II, three columns were designated nutrient-rich 3-day toluene columns and received toluene injections every 3 days; three columns were designated as nutrient-rich 7-day columns and received toluene injections every 7 days; and two columns were used as controls to which no nutrient was added. As measured by CO₂ respiration, the initial benefits for aerobic organisms from the O₂ enhancement were sustained by the bacteria for only a short period of time (about 8 days). Degradation benefits from the nutrient solution were sustained throughout the experiment. The O₂ and nutrient-enhanced columns degraded significantly more toluene than the control columns when simulating repeated spills onto the unsaturated zone, and demonstrated a potentially effective in situ bioremediation technology when used immediately or within days after a spill. The combined usage of H₂O₂ and nitrate-rich nutrients served to effectively maximize natural aerobic and anaerobic metabolic processes that biodegrade hydrocarbons in petroleum-contaminated media. Applications of this technology in the field may offer economical advantages to other, more intrusive abatement technologies.