Anca G. Delgado

Role of bicarbonate as a pH buffer and electron sink in microbial dechlorination of chloroethenes

BackgroundBuffering to achieve pH control is crucial for successful trichloroethene (TCE) anaerobic bioremediation. Bicarbonate (HCO3−) is the natural buffer in groundwater and the buffer of choice in the laboratory and at contaminated sites undergoing biological treatment with organohalide respiring microorganisms. However, HCO3− also serves as the electron acceptor for hydrogenotrophic methanogens and hydrogenotrophic homoacetogens, two microbial groups competing with organohalide respirers for hydrogen (H2). We studied the effect of HCO3− as a buffering agent and the effect of HCO3−-consuming reactions in a range of concentrations (2.5-30 mM) with an initial pH of 7.5 in H2-fed TCE reductively dechlorinating communities containing Dehalococcoides, hydrogenotrophic methanogens, and hydrogenotrophic homoacetogens.ResultsRate differences in TCE dechlorination were observed as a result of added varying HCO3− concentrations due to H2-fed electrons channeled towards methanogenesis and homoacetogenesis and pH increases (up to 8.7) from biological HCO3− consumption. Significantly faster dechlorination rates were noted at all HCO3− concentrations tested when the pH buffering was improved by providing 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) as an additional buffer. Electron balances and quantitative PCR revealed that methanogenesis was the main electron sink when the initial HCO3− concentrations were 2.5 and 5 mM, while homoacetogenesis was the dominant process and sink when 10 and 30 mM HCO3− were provided initially.ConclusionsOur study reveals that HCO3− is an important variable for bioremediation of chloroethenes as it has a prominent role as an electron acceptor for methanogenesis and homoacetogenesis. It also illustrates the changes in rates and extent of reductive dechlorination resulting from the combined effect of electron donor competition stimulated by HCO3− and the changes in pH exerted by methanogens and homoacetogens.

Selective enrichment yields robust ethene-producing dechlorinating cultures from microcosms stalled at cis-dichloroethene.

Dehalococcoides mccartyi strains are of particular importance for bioremediation due to their unique capability of transforming perchloroethene (PCE) and trichloroethene (TCE) to non-toxic ethene, through the intermediates cis-dichloroethene (cis-DCE) and vinyl chloride (VC). Despite the widespread environmental distribution of Dehalococcoides, biostimulation sometimes fails to promote dechlorination beyond cis-DCE. In our study, microcosms established with garden soil and mangrove sediment also stalled at cis-DCE, albeit Dehalococcoides mccartyi containing the reductive dehalogenase genes tceA, vcrA and bvcA were detected in the soil/sediment inocula. Reductive dechlorination was not promoted beyond cis-DCE, even after multiple biostimulation events with fermentable substrates and a lengthy incubation. However, transfers from microcosms stalled at cis-DCE yielded dechlorination to ethene with subsequent enrichment cultures containing up to 109 Dehalococcoides mccartyi cells mL−1. Proteobacterial classes which dominated the soil/sediment communities became undetectable in the enrichments, and methanogenic activity drastically decreased after the transfers. We hypothesized that biostimulation of Dehalococcoides in the cis-DCE-stalled microcosms was impeded by other microbes present at higher abundances than Dehalococcoides and utilizing terminal electron acceptors from the soil/sediment, hence, outcompeting Dehalococcoides for H2. In support of this hypothesis, we show that garden soil and mangrove sediment microcosms bioaugmented with their respective cultures containing Dehalococcoides in high abundance were able to compete for H2 for reductive dechlorination from one biostimulation event and produced ethene with no obvious stall. Overall, our results provide an alternate explanation to consolidate conflicting observations on the ubiquity of Dehalococcoides mccartyi and occasional stalling of dechlorination at cis-DCE; thus, bringing a new perspective to better assess biological potential of different environments and to understand microbial interactions governing bioremediation.

Archaea and Bacteria Acclimate to High Total Ammonia in a Methanogenic Reactor Treating Swine Waste

Inhibition by ammonium at concentrations above 1000 mgN/L is known to harm the methanogenesis phase of anaerobic digestion. We anaerobically digested swine waste and achieved steady state COD-removal efficiency of around 52% with no fatty-acid or H2 accumulation. As the anaerobic microbial community adapted to the gradual increase of total ammonia-N (NH3-N) from 890 ± 295 to 2040 ± 30 mg/L, the Bacterial and Archaeal communities became less diverse. Phylotypes most closely related to hydrogenotrophic Methanoculleus (36.4%) and Methanobrevibacter (11.6%), along with acetoclastic Methanosaeta (29.3%), became the most abundant Archaeal sequences during acclimation. This was accompanied by a sharp increase in the relative abundances of phylotypes most closely related to acetogens and fatty-acid producers (Clostridium, Coprococcus, and Sphaerochaeta) and syntrophic fatty-acid Bacteria (Syntrophomonas, Clostridium, Clostridiaceae species, and Cloacamonaceae species) that have metabolic capabilities for butyrate and propionate fermentation, as well as for reverse acetogenesis. Our results provide evidence countering a prevailing theory that acetoclastic methanogens are selectively inhibited when the total ammonia-N concentration is greater than ~1000 mgN/L. Instead, acetoclastic and hydrogenotrophic methanogens coexisted in the presence of total ammonia-N of ~2000 mgN/L by establishing syntrophic relationships with fatty-acid fermenters, as well as homoacetogens able to carry out forward and reverse acetogenesis.

Treatment of Heavy, Long-Chain Petroleum-Hydrocarbon Impacted Soils Using Chemical Oxidation

AbstractChemical oxidation is a promising approach for in situ or ex situ treatment of heavy, long-chain (C12−C40) petroleum-hydrocarbon impacted soils. Aqueous chemical oxidation treatments (sodium percarbonate, hydrogen peroxide, sodium persulfate, chlorine dioxide, sodium permanganate, and ozone) using two oxidant concentrations were tested in batch tests on soils containing C12–C40 total petroleum hydrocarbon (TPH) concentrations of 1.6 and 2.0% weight/weight (w/w) resulting in TPH reductions from 20 to 90%. Gas chromatography with flame ionization detector (GC-FID) chromatograms for hydrocarbons were obtained and presented as chain-length fractions. Sodium percarbonate and hydrogen peroxide achieved the highest TPH reduction. There was little difference between 1 and 10% weight/volume (w/v) for all oxidant doses on TPH removal. Soluble organics in the liquid supernatants after oxidation of the TPH-containing soils were characterized by TPH analysis and excitation-emission matrix fluorescence spectros...

Interpreting Interactions between Ozone and Residual Petroleum Hydrocarbons in Soil

We evaluated how gas-phase O3 interacts with residual petroleum hydrocarbons in soil. Total petroleum hydrocarbons (TPH) were 18 ± 0.6 g/kg soil, and TPH carbon constituted ∼40% of the dichloromethane-extractable carbon (DeOC) in the soil. At the benchmark dose of 3.4 kg O3/kg initial TPH, TPH carbon was reduced by nearly 6 gC/kg soil (40%), which was accompanied by an increase of about 4 gC/kg soil in dissolved organic carbon (DOC) and a 4-fold increase in 5-day biochemical oxygen demand (BOD5). Disrupting gas channeling in the soil improved mass transport of O3 to TPH bound to soil and increased TPH removal. Ozonation resulted in two measurable alterations of the composition of the organic carbon. First, part of DeOC was converted to DOC (∼4.1 gC/kg soil), 75% of which was not extractable by dichloromethane. Second, the DeOC containing saturates, aromatics, resins, and asphaltenes (SARA), was partially oxidized, resulting in a decline in saturates and aromatics, but increases in resins and asphaltenes. Ozone attack on resins, asphaltenes, and soil organic matter led to the production of NO3-, SO42-, and PO43-. The results illuminate the mechanisms by which ozone gas interacted with the weathered petroleum residuals in soil to generate soluble and biodegradable products.

Impact of Ammonium on Syntrophic Organohalide-Respiring and Fermenting Microbial Communities

Contamination with ammonium and chlorinated solvents has been reported in numerous subsurface environments, and these chemicals bring significant challenges for in situ bioremediation. Dehalococcoides mccartyi is able to reduce the chlorinated solvent trichloroethene to the nontoxic end product ethene. Fermentative bacteria are of central importance for organohalide respiration and bioremediation to provide D. mccartyi with H2, their electron donor, acetate, their carbon source, and other micronutrients. In this study, we found that high concentrations of ammonium negatively correlated with rates of trichloroethene reductive dehalogenation and fermentation. However, detoxification of trichloroethene to nontoxic ethene occurred even at ammonium concentrations typical of those found in animal waste (up to 2 g liter−1 NH4+-N). To date, hundreds of subsurface environments have been bioremediated through the unique metabolic capability of D. mccartyi. These findings extend our knowledge of D. mccartyi and provide insight for bioremediation of sites contaminated with chlorinated solvents and ammonium. ABSTRACT Syntrophic interactions between organohalide-respiring and fermentative microorganisms are critical for effective bioremediation of halogenated compounds. This work investigated the effect of ammonium concentration (up to 4 g liter−1 NH4+-N) on trichloroethene-reducing Dehalococcoides mccartyi and Geobacteraceae in microbial communities fed lactate and methanol. We found that production of ethene by D. mccartyi occurred in mineral medium containing ≤2 g liter−1 NH4+-N and in landfill leachate. For the partial reduction of trichloroethene (TCE) to cis-dichloroethene (cis-DCE) at ≥1 g liter−1 NH4+-N, organohalide-respiring dynamics shifted from D. mccartyi and Geobacteraceae to mainly D. mccartyi. An increasing concentration of ammonium was coupled to lower metabolic rates, longer lag times, and lower gene abundances for all microbial processes studied. The methanol fermentation pathway to acetate and H2 was conserved, regardless of the ammonium concentration provided. However, lactate fermentation shifted from propionic to acetogenic at concentrations of ≥2 g liter−1 NH4+-N. Our study findings strongly support a tolerance of D. mccartyi to high ammonium concentrations, highlighting the feasibility of organohalide respiration in ammonium-contaminated subsurface environments. IMPORTANCE Contamination with ammonium and chlorinated solvents has been reported in numerous subsurface environments, and these chemicals bring significant challenges for in situ bioremediation. Dehalococcoides mccartyi is able to reduce the chlorinated solvent trichloroethene to the nontoxic end product ethene. Fermentative bacteria are of central importance for organohalide respiration and bioremediation to provide D. mccartyi with H2, their electron donor, acetate, their carbon source, and other micronutrients. In this study, we found that high concentrations of ammonium negatively correlated with rates of trichloroethene reductive dehalogenation and fermentation. However, detoxification of trichloroethene to nontoxic ethene occurred even at ammonium concentrations typical of those found in animal waste (up to 2 g liter−1 NH4+-N). To date, hundreds of subsurface environments have been bioremediated through the unique metabolic capability of D. mccartyi. These findings extend our knowledge of D. mccartyi and provide insight for bioremediation of sites contaminated with chlorinated solvents and ammonium.

Carbonaceous nano-additives augment microwave-enabled thermal remediation of soils containing petroleum hydrocarbons

Remediating soils contaminated with heavy hydrocarbons (C12–C40) from petrochemical exploration activities is a major environmental challenge across the globe. This study evaluated microwave irradiation in the presence of nano- and macro-scale graphitic additives as a rapid remediation technology for removing heavy hydrocarbons from soil. Adding inert materials (i.e., glass wool fibers or washed silica sand) as controls had no effect on total petroleum hydrocarbons (TPH) removal upon microwave irradiation. Carbonaceous nanomaterials (i.e., carbon nanotubes, graphene nanosheets, and carbon nanofibers) because of their favorable dielectric properties showed extraordinary heating performances when mixed with soil and microwave irradiated. As a result, adding these carbonaceous nanomaterials to contaminated soils removed more TPH compared with macro-scale carbonaceous additives. TPH concentrations decreased from 11 000 to between 2000 and 6000 mg TPH kg−1 soil within one minute using carbon nanomaterial additives and a 2.45 GHz, 1000 W conventional microwave oven. In separate experiments, this technology decreased TPH from 2500 to 650 mg TPH kg−1 soil from soils containing recalcitrant, non-biodegradable fractions of TPH. Large scale microwave systems are available and hold promise for remediating soils when used in conjunction with carbon nanomaterials.

Impacts of moisture content during ozonation of soils containing residual petroleum

We tested the effect of soil moisture content on the efficiency of gas-phase ozonation for two types of soils containing residual petroleum. For the first soil (BM2), having a total petroleum hydrocarbons (TPH) concentration of 18,000mg/kg soil, a moisture content of 5% benefited oxidation, giving the highest efficiency of ozonation for TPH removal and for producing soluble and biodegradable products. In contrast, higher moisture content hindered O3 from oxidizing reactive materials in the second soil (BM3), which had a higher TPH concentration, 33,000mg/kg soil. This trend was documented by less TPH removal, less generation of soluble and biodegradable organic products, and a carbon balance that showed retarded carbon oxidation. An unexpected phenomenon was smoldering during ozonation of air-dried (<1% moisture) BM3, which did not occur with the same moisture conditions for BM2. BM3 smoldered was due to its higher TPH content, low heat buffering, and more release of volatiles with low self-ignition points. Smoldering did not occur for ≥ 5% water content, as it suppressed the temperature increase needed to volatilize the organics that initiated smoldering. The findings underscore the importance of controlling water content during ozonation to optimize the effectiveness of ozonation and prevent smoldering.

Coupling Bioflocculation of Dehalococcoides mccartyi to High-Rate Reductive Dehalogenation of Chlorinated Ethenes

Continuous bioreactors operated at low hydraulic retention times have rarely been explored for reductive dehalogenation of chlorinated ethenes. The inability to consistently develop such bioreactors affects the way growth approaches for Dehalococcoides mccartyi bioaugmentation cultures are envisioned. It also affects interpretation of results from in situ continuous treatment processes. We report bioreactor performance and dehalogenation kinetics of a D. mccartyi-containing consortium in an upflow bioreactor. When fed synthetic groundwater at 11-3.6 h HRT, the upflow bioreactor removed >99.7% of the influent trichloroethene (1.5-2.8 mM) and produced ethene as the main product. A trichloroethene removal rate of 98.51 ± 0.05 me- equiv L-1 d-1 was achieved at 3.6 h HRT. D. mccartyi cell densities were 1013 and 1012 16S rRNA gene copies L-1 in the bioflocs and planktonic culture, respectively. When challenged with a feed of natural groundwater containing various competing electron acceptors and 0.3-0.4 mM trichloroethene, trichloroethene removal was sustained at >99.6%. Electron micrographs revealed that D. mccartyi were abundant within the bioflocs, not only in multispecies structures, but also as self-aggregated microcolonies. This study provides fundamental evidence toward the feasibility of upflow bioreactors containing D. mccartyi as high-density culture production tools or as a high-rate, real-time remediation biotechnology.

Optical fiber-mediated photosynthesis for enhanced subsurface oxygen delivery

Remediation of polluted groundwater often requires oxygen delivery into subsurface to sustain aerobic bacteria. Air sparging or injection of oxygen containing solutions (e.g., hydrogen peroxide) into the subsurface are common. In this study visible light was delivered into the subsurface using radially emitting optical fibers. Phototrophic organisms grew near the optical fiber in a saturated sand column. When applying light in on-off cycles, dissolved oxygen (DO) varied from super saturation levels of >15 mg DO/L in presence of light to under-saturation (<5 mg DO/L) in absence of light. Non-photosynthetic bacteria dominated at longer radial distances from the fiber, presumably supported by soluble microbial products produced by the photosynthetic microorganisms. The dissolved oxygen variations alter redox condition changes in response to light demonstrate the potential to biologically deliver oxygen into the subsurface and support a diverse microbial community. The ability to deliver oxygen and modulate redox conditions on diurnal cycles using solar light may provide a sustainable, long term strategy for increasing dissolved oxygen levels in subsurface environments and maintaining diverse biological communities.