Evaluating a new injection method of liquid/gas mixture spray injection via performing long-term in situ bioremediation tests.

During in situ bioremediation, continuous injection of growth substrates such as carbon sources, electron donors, or electron acceptors inevitably results in microbial growth, resulting in biological clogging in an aquifer. Therefore, for successful bioremediation, development of a new injection method is needed to reduce or alleviate this clogging problem. In this study, we carried out field tracer tests using single-well push-pull tests (SWPPTs), single-well natural gradient drift tests (SWNGDTs), and long-term in situ well-to-well tests to develop and evaluate a new method of liquid/gas mixture spray injection. The effectiveness of the new method was evaluated by estimating the factors as follow: longitudinal dispersivity (αL), radius of influence (RI), shear stress on the surface of aquifer particles (σ), biofilm-shear-loss rate (Rs), and the ratio of volume occupied by cells grown to the original pore volume. At the tested site, the liquid/gas mixture spray injection method turned out to have several advantages compared to the traditional solution injection method: 1) transport of solute to a larger proportion of an aquifer by a factor of 1.3-1.7, 2) application of higher shear stress onto the surface of soil particles by a factor of 4.2-5.0, 3) faster biofilm sloughing rates by a factor of 2.3-2.6, 4) reduction in the ratio of the volume occupied by microorganisms to total pore volume (Volmicrobes/Volpore), and 5) efficient trichloroethylene (TCE) dechlorination for a period of 550 days without any injection problems. This new injection method showed positive effects on the hydrogeological and physical characteristics of the system, thus alleviating the biological clogging problem.

Efficacy of in situ well-based denitrification bio-barrier (WDB) remediating high nitrate flux in groundwater near a stock-raising complex.

This study assessed the feasibility of an in situ well-based denitrification bio-barrier (WDB) for managing groundwater contaminated with high-strength nitrate. To evaluate the efficacy of WDB using fumarate as a carbon source and/or electron donor, three sequential single-well push-pull tests (SWPPTs) were conducted at six test sites. The values of the isotope enrichment factor (ɛ) ranging from -6.5‰ to -22.6‰ and the detection and degradation of nitrite and nitrous oxide confirmed complete in situ denitrification of nitrate to nitrogen gas. The ratio of the first-order rate coefficient of fumarate to nitrate (k1,fum/k1,NO3) was obtained to estimate the amount and frequency of fumarate injection for the effective design of WDB. At three sites, the ratios ranged from 0.67 to 0.80, while the other two sites showed higher ratios of 2.97 and 2.20 than the theoretical values and significant amounts of sulfate reduction, theoretically equivalent to 6.5% of total fumarate consumption. Considering the theoretical mole ratio of fumarate to nitrate of 0.98, the amount and frequency of fumarate injection is site specific. During the operating WDB, the average annual nitrate mass degraded (95% CI) was 2.2 ± 1.0 kg N/yr/well. The amount of N reduced by one well of WDB is equivalent to treating 110 m3 of groundwater at 30 mg N/L to the level of 10 mg N/L for one year. WDB would be an effective remediation option for managing high nitrate flux in groundwater.

Estimating bioaugmentation efficacy of TCE dechlorination using long-term field well-to-well tests in a highly recharged and TCE-contaminated aquifer.

This study demonstrates a combined field method accurately assessing the extent of trichloroethylene (TCE) reductive dechlorination activity and the mass fraction of its by-products. A combined method of injecting a known concentration of 1,1,2-trichloro-2-fluoroethene (TCFE) as a TCE bio-surrogate and a data processing technique of forced mass balance (FMB), considering the sorption effect on the mass fraction of chloroethene was evaluated by performing soil column and field bioaugmentation tests. In the soil column test, the FMB resulted in the mass fraction of 6% TCE, 48.3% cis-1,2-dichloroethene, 18.5% vinyl chloride and 27.2% ethylene. In the field bioaugmentation test, TCFE showed equivalent dechlorination pathways of TCE. The mass fractions estimated by FMB were very similar to those observed in the soil column bioaugmentation tests: 4.5% TCFE, 57.1% 1,2-dichloro-1-fluoroethene, 12% 1-chloro-1-fluoroethene and 26.4% fluoroethene (FE). The FMB method gave ∼50% higher mass fraction for more chlorinated ethenes (i.e., TCFE) and ∼10% lower mass fraction of less chlorinated ethenes (i.e., FE) than those considering only the aqueous concentrations of chlorofluoroethenes. A combined method of TCFE and FMB that could accurately estimate both the extent of dechlorination activities and mass distribution of TCE reductive dechlorination would be highly useful.

Assessing the activity and diversity of fumarate-fed denitrifying bacteria by performing field single-well push-pull tests.

In situ biological denitrification has been proposed as an important metabolic activity in the remediation of nitrate-contaminated groundwater. In this study, the effects of fumarate, an electron donor for biological denitrification, on the in situ denitrifying activity were determined by using three types of single-well push-pull tests; transport, biostimulation and activity tests. During the tests, changes in microbial community composition were also investigated using denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes. Transport test demonstrated that non-reactive tracer and biologically reactive solutes behaved similarly. A biostimulation test was conducted to stimulate the denitrifying activities of native microorganisms, which were monitored by detecting the simultaneous production of CO(2) and drastic degradations of both nitrate and fumarate after the injection of fumarate as an electron donor and/or carbon source, with nitrate as an electron acceptor. A phylogenetic analysis suggested that the taxonomic affiliation of the dominant species before biostimulation was γ-Proteobacteria, including Acinetobacter species and Pseudomonas fluorescens, while the dominant species after biostimulation were affiliated with ß-Proteobacteria, cytophaga-Flavobacterium-Bacteroides and high G+C gram-positive bacteria. These results suggest that the analyses of groundwater samples using a combination of single well push pull tests with DGGE can be applied to investigate the activity, diversity and composition shift of denitrifying bacteria in a nitrate-contaminated aquifer.

In situ field method for evaluating biodegradation potential of BTEX by indigenous heterotrophic denitrifying microorganisms in a BTEX-contaminated fractured-rock aquifer.

Generally different anaerobic degradation potentials for benzene, toluene, ethylbenzene and xylene isomers (BTEX) has been reported due to site specific conditions, such as the indigenous microbial population, electron acceptors (EA) available and concentrations of each BTEX compound. It was of interest to estimate relative biodegradation potential of each BTEX compound during enhanced anaerobic bioremediation of a BTEX-contaminated aquifer. In this study, an in situ method for assessing the degradation potentials of each BTEX compound present as a mixture under NO3-injecting conditions by performing a series of single-well push-pull tests and well-to-well tests (WWTs) was developed. During the 1st and 2nd WWTs, biological heterotrophic dissimilative NO3- denitrification was confirmed by simultaneous detection of both NO2- and N2O and significant production of CO2 during the NO3- degradation. The biodegradation fractions of NO3- injected during the 1st and 2nd WWTs were 1.7% and 5.0%, respectively, with 7.18 and 8.85 mmol N/L/day of in situ zero-order denitrification rate coefficients. The concentrations of benzene, ethylbenzene, and xylenes measured were similar to values calculated when considering only dilution, but the measured concentrations of toluene were significantly lower than the values calculated were. These results indicate that in situ method presented in the study successfully evaluate anaerobic biodegradation potential of individual BTEX compounds by indigenous heterotrophic denitrifying microorganisms.

Assessing the feasibility of sequential aerobic respiration and heterotrophic denitrification of a high-strength mixture of phenol and its derivatives in the field single-well-drift test.

Biological degradation of high strength phenol and its derivatives in groundwater is problematic because these compounds are toxic to human and microbes. To evaluate the feasibility of in situ bioremediation using sequential aerobic respiration and heterotrophic denitrification, a field single-well-drift test (SWDT) was conducted in groundwater contaminated with coal tar distillates. To stimulate indigenous phenol degrading microorganisms, a 1400 L of oxygen-saturated test solution containing bromide (3.96 ±â€¯0.179 mmol-Br/L) and nitrate (5.34 ±â€¯0.187 mmol NO3--N/L) was injected into an aquifer. After injection of the test solution, significant consumption of dissolved oxygen (DO) was immediately observed; then, degradation of the methyl derivatives o-cresol and m,p-cresol was observed with average zero-order rate coefficients of 0.047 mmol/L/d and 0.23 mmol/L/d, respectively. After 73% of the injected DO was consumed, significant NO3- consumption was observed along with degradation of phenol and the dimethyl derivatives 2,4-xylenol and 3,5-xylenol, which had average zero-order rate coefficients of 0.17 mmol/L/d, 0.060 mmol/L/d, and 0.018 mmol/L/d, respectively. The production of CO2, NO2-, and N2O along with significant consumption of DO and NO3- suggest that phenolic compounds were biologically degraded by sequential aerobic respiration and heterotrophic denitrification. The results of 16s RNA analysis revealed that, after injection of the test solution, a bacterium that shared a 99% 16s rRNA sequence similarity with an uncultured bacterium revealed to be Pseudomonas stutzeri, a facultative heterotrophic denitrifier, was found in the aquifer. Thus, these results suggest that simultaneous injection of DO and NO3- is an appropriate in situ bioremediation strategy for degrading mixtures of high-strength phenolic compounds in an aquifer.

Production, characterization, and evaluation of two types of slow-releasing carbon source tablets for in-situ heterotrophic nitrate denitrification in aquifers.

Slow-releasing carbon source tablets were manufactured for an in-situ biological denitrification system. The average zero-order nitrate degradation rates seen, from highest to lowest, were in microcosms to which lactate, fumarate, propionate, and formate had been added. Fumarate was approximately 80% cheaper than lactate, and consequently was determined to be the most optimal slow-releasing carbon source in tablet form. The slow-releasing precipitating tablet (SRPT) and slow-releasing floating tablet (SRFT) were manufactured with hydroxypropyl methylcellulose (HPMC) as the agent of release control, microcrystalline cellulose pH 101 (MCC 101) as the binder, #8 sand as the precipitation agent, and calcium carbonate and citric acid as floating agents. Fourier transform infrared spectroscopy and powder X-ray diffraction indicated that the crystal arrangement in the SRPTs and SRFTs was maintained and ordered in a manner similar to raw excipients. SRFTs floated in water within 30 min and remained so for 5 d due to the buoyancy of carbon dioxide. The carbon source release rate was proportional to the quantity of HPMC added. The longevities of SRPT with 300 mg of HPMC and SRFT with 400 mg of HPMC were 25.4 d and 37.3 d, respectively. This study observed that SRPT and SRFT were manufactured effectively and are suitable for in-situ slow-releasing biological systems.