Microbially Mediated Rubber Recycling to Facilitate the Valorization of Scrap Tires.

The recycling of scrap tire rubber requires high levels of energy, which poses challenges to its proper valorization. The application of rubber in construction requires significant mechanical and/or chemical treatment of scrap rubber to compatiblize it with the surrounding matrix. These methods are energy-consuming and costly and may lead to environmental concerns associated with chemical leachates. Furthermore, recent methods usually call for single-size rubber particles or a narrow rubber particle size distribution; this, in turn, adds to the pre-processing cost. Here, we used microbial etching (e.g., microbial metabolism) to modify the surface of rubber particles of varying sizes. Specifically, we subjected rubber particles with diameters of 1.18 mm and 0.6 mm to incubation in flask bioreactors containing a mineral medium with thiosulfate and acetate and inoculated them with a microbial culture from waste-activated sludge. The near-stoichiometric oxidation of thiosulfate to sulfate was observed in the bioreactors. Most notably, two of the most potent rubber-degrading bacteria (Gordonia and Nocardia) were found to be significantly enriched in the medium. In the absence of added thiosulfate in the medium, sulfate production, likely from the desulfurization of the rubber, was also observed. Microbial etching increased the surface polarity of rubber particles, enhancing their interactions with bitumen. This was evidenced by an 82% reduction in rubber-bitumen separation when 1.18 mm microbially etched rubber was used. The study outcomes provide supporting evidence for a rubber recycling method that is environmentally friendly and has a low cost, promoting pavement sustainability and resource conservation.

Implementation of in situ aerobic cometabolism for groundwater treatment: State of the knowledge and important factors for field operation.

In situ aerobic cometabolism of groundwater contaminants has been demonstrated to be a valuable bioremediation technology to treat many legacy and emerging contaminants in dilute plumes. Several well-designed and documented field studies have shown that this technology can concurrently treat multiple contaminants and reach very low cleanup goals. Fundamentally different from metabolism-based biodegradation of contaminants, microorganisms that cometabolically degrade contaminants do not obtain sufficient carbon and energy from the degradation process to support their growth and require an exogenous growth supporting primary substrate. Successful applications of aerobic cometabolic treatment therefore require special considerations beyond conventional in situ bioremediation, such as competitive inhibition between growth-supporting primary substrate(s) and contaminant non-growth substrates, toxic effects resulting from contaminant degradation, and differences in microbial population dynamics exhibited by biostimulated indigenous consortia versus bioaugmentation cultures. This article first provides a general review of microbiological factors that are likely to affect the rate of aerobic cometabolic biodegradation. We subsequently review fourteen well documented field-scale aerobic cometabolic bioremediation studies and summarize the underlying microbiological factors that may affect the performance observed in these field studies. The combination of microbiological and engineering principles gained from field testing leads to insights and recommendations on planning, design, and operation of an in situ aerobic cometabolic treatment system. With a vision of more aerobic cometabolic treatments being considered to tackle large, dilute plumes, we present several novel topics and future research directions that can potentially enhance technology development and foster success in implementing this technology for environmental restoration.

Acetylene Tunes Microbial Growth During Aerobic Cometabolism of Trichloroethene.

Microbial aerobic cometabolism is a possible treatment approach for large, dilute trichloroethene (TCE) plumes at groundwater contaminated sites. Rapid microbial growth and bioclogging pose a persistent problem in bioremediation schemes. Bioclogging reduces soil porosity and permeability, which negatively affects substrate distribution and contaminant treatment efficacy while also increasing the operation and maintenance costs of bioremediation. In this study, we evaluated the ability of acetylene, an oxygenase enzyme-specific inhibitor, to decrease biomass production while maintaining aerobic TCE cometabolism capacity upon removal of acetylene. We first exposed propane-metabolizing cultures (pure and mixed) to 5% acetylene (v v-1) for 1, 2, 4, and 8 d and we then verified TCE aerobic cometabolic activity. Exposure to acetylene overall decreased biomass production and TCE degradation rates while retaining the TCE degradation capacity. In the mixed culture, exposure to acetylene for 1-8 d showed minimal effects on the composition and relative abundance of TCE cometabolizing bacterial taxa. TCE aerobic cometabolism and incubation conditions exerted more notable effects on microbial ecology than did acetylene. Acetylene appears to be a viable approach to control biomass production that may lessen the likelihood of bioclogging during TCE cometabolism. The findings from this study may lead to advancements in aerobic cometabolism remediation technologies for dilute plumes.

High Efficacy Two-Stage Metal Treatment Incorporating Basic Oxygen Furnace Slag and Microbiological Sulfate Reduction.

Lignocellulosic sulfate-reducing biochemical reactors (SRBRs) can be implemented as passive treatment for mining-influenced water (MIW) mitigating the potentially deleterious effects of MIW acidic pH, and high concentrations of metal(loid)s and SO42-. In this study, a novel two-stage treatment for MIW was designed, where basic oxygen furnace slag (slag stage) and microbial SO42- reduction (SRBR stage) were incorporated in series. The SRBRs contained spent brewing grains or sugarcane bagasse as sources of lignocellulose. The slag reactor removed >99% of the metal(loid) concentration present in the MIW (130 ± 40 mg L-1) and increased MIW pH from 2.6 ± 0.2 to 12 ± 0.3. The alkaline effluent pH of the slag reactor was mitigated by remixing slag effluent with acidic MIW before SRBR treatment. The SRBR stage removed the bulk of SO42- from MIW, additional metal(loid)s, and yielded a circumneutral effluent pH. Cadmium, copper, and zinc showed high removal rates in SRBRs (≥96%) and likely precipitated as sulfide minerals. The microbial communities developed in SRBRs were enriched in hydrolytic, fermentative, and sulfate-reducing taxa. However, the SRBRs developed distinct community compositions due to the different lignocellulose sources employed. Overall, this study underscores the potential of a two-stage treatment employing steel slag and SRBRs for full-scale implementation at mining sites.

Butanol as a major product during ethanol and acetate chain elongation.

Chain elongation is a relevant bioprocess in support of a circular economy as it can use a variety of organic feedstocks for production of valuable short and medium chain carboxylates, such as butyrate (C4), caproate (C6), and caprylate (C8). Alcohols, including the biofuel, butanol (C4), can also be generated in chain elongation but the bioreactor conditions that favor butanol production are mainly unknown. In this study we investigated production of butanol (and its precursor butyrate) during ethanol and acetate chain elongation. We used semi-batch bioreactors (0.16 L serum bottles) fed with a range of ethanol concentrations (100-800 mM C), a constant concentration of acetate (50 mM C), and an initial total gas pressure of ∼112 kPa. We showed that the butanol concentration was positively correlated with the ethanol concentration provided (up to 400 mM C ethanol) and to chain elongation activity, which produced H2 and further increased the total gas pressure. In bioreactors fed with 400 mM C ethanol and 50 mM C acetate, a concentration of 114.96 ± 9.26 mM C butanol (∼2.13 g L-1) was achieved after five semi-batch cycles at a total pressure of ∼170 kPa and H2 partial pressure of ∼67 kPa. Bioreactors with 400 mM C ethanol and 50 mM C acetate also yielded a butanol to butyrate molar ratio of 1:1. At the beginning of cycle 8, the total gas pressure was intentionally decreased to ∼112 kPa to test the dependency of butanol production on total pressure and H2 partial pressure. The reduction in total pressure decreased the molar ratio of butanol to butyrate to 1:2 and jolted H2 production out of an apparent stall. Clostridium kluyveri (previously shown to produce butyrate and butanol) and Alistipes (previously linked with butyrate production) were abundant amplicon sequence variants in the bioreactors during the experimental phases, suggesting the microbiome was resilient against changes in bioreactor conditions. The results from this study clearly demonstrate the potential of ethanol and acetate-based chain elongation to yield butanol as a major product. This study also supports the dependency of butanol production on limiting acetate and on high total gas and H2 partial pressures.

Decoupling Fe0 Application and Bioaugmentation in Space and Time Enables Microbial Reductive Dechlorination of Trichloroethene to Ethene: Evidence from Soil Columns.

Fe0 is a powerful chemical reductant with applications for remediation of chlorinated solvents, including tetrachloroethene and trichloroethene. Its utilization efficiency at contaminated sites is limited because most of the electrons from Fe0 are channeled to the reduction of water to H2 rather than to the reduction of the contaminants. Coupling Fe0 with H2-utilizing organohalide-respiring bacteria (i.e., Dehalococcoides mccartyi) could enhance trichloroethene conversion to ethene while maximizing Fe0 utilization efficiency. Columns packed with aquifer materials have been used to assess the efficacy of a treatment combining in space and time Fe0 and aD. mccartyi-containing culture (bioaugmentation). To date, most column studies documented only partial conversion of the solvents to chlorinated byproducts, calling into question the feasibility of Fe0 to promote complete microbial reductive dechlorination. In this study, we decoupled the application of Fe0 in space and time from the addition of organic substrates andD. mccartyi-containing cultures. We used a column containing soil and Fe0 (at 15 g L-1 in porewater) and fed it with groundwater as a proxy for an upstream Fe0 injection zone dominated by abiotic reactions and biostimulated/bioaugmented soil columns (Bio-columns) as proxies for downstream microbiological zones. Results showed that Bio-columns receiving reduced groundwater from the Fe0-column supported microbial reductive dechlorination, yielding up to 98% trichloroethene conversion to ethene. The microbial community in the Bio-columns established with Fe0-reduced groundwater also sustained trichloroethene reduction to ethene (up to 100%) when challenged with aerobic groundwater. This study supports a conceptual model where decoupling the application of Fe0 and biostimulation/bioaugmentation in space and/or time could augment microbial trichloroethene reductive dechlorination, particularly under oxic conditions.

Using radish (Raphanus lativus L.) germination to establish a benchmark dose for the toxicity of ozonated-petroleum byproducts in soil.

The concentration-response relationship between the germination outcome of radish (Raphanus lativus L.) and ozonated petroleum residuals was determined experimentally. The outcomes were used to produce an ecological risk assessment model to predict the extra risk of adverse outcomes based on the concentration of ozonated residuals. A test soil with low organic matter (0.5% w/w) was mixed with raw crude oil, artificially weathered, and treated at three doses of ozone (O3) gas (5 g, 10 g, and 40 g O3 per 600 g of soil). Total petroleum hydrocarbons (TPH) and produced dissolved organic carbon (DOC) were measured. TREATMENT categories (control, petroleum, petroleum + 5 g O3, petroleum + 10 g O3, and petroleum + 40 g O3) were then used to create a dilution series using different proportions of the test soil and a commercially available potting mix (∼75% w/w organic matter) to evaluate the effects of background organic matter (b-ORGANIC) in conjunction with TPH and DOC. Multivariable logistic regression was performed on the adverse germination outcome as a function of TPH, DOC, TREATMENT, and b-ORGANIC. The parameters controlling germination were the continuous variable DOC and the categorical variables TREATMENT and b-ORGANIC. Radish germination was strongly harmed by DOC from ozonation, but DOC's ecotoxicity decreased with increasing O3 dose and the presence of b-ORGANIC beyond 10% (w/w). We used the germination outcome of radish to produce a logistic regression model that computes margins of DOC (± std. error) that create 10%, 25%, and 50% extra risk of adverse germination effects.

Organic carbon metabolism is a main determinant of hydrogen demand and dynamics in anaerobic soils.

Hydrogen (H2) is a crucial electron donor for many processes in the environment including nitrate-, sulfate- and, iron-reduction, homoacetogenesis, and methanogenesis, and is a major determinant of microbial competition and metabolic pathways in groundwater, sediments, and soils. Despite the importance of H2 for many microbial processes in the environment, the total H2 consuming capacity (or H2 demand) of soils is generally unknown. Using soil microcosms with added H2, the aims of this study were 1) to measure the H2 demand of geochemically diverse soils and 2) to define the processes leading to this demand. Study results documented a large range of H2 demand in soil (0.034-1.2 millielectron equivalents H2 g-1 soil). The measured H2 demand greatly exceeded the theoretical demand predicted based on measured concentrations of common electron acceptors initially present in a library of 15 soils. While methanogenesis accounted for the largest fraction of H2 demand, humic acid reduction and acetogenesis were also significant contributing H2-consuming processes. Much of the H2 demand could be attributed to CO2 produced during incubation from fermentation and/or acetoclastic methanogenesis. The soil initial total organic carbon showed the strongest correlation to H2 demand. Besides external additions, H2 was likely generated or cycled in the microcosms. Apart from fermentative H2 production, carboxylate elongation to produce C4-C7 fatty acids may have accounted for additional H2 production in these soils. Many of these processes, especially the organic carbon contribution is underestimated in microbial models for H2 consumption in natural soil ecosystems or during bioremediation of contaminants in soils.

Biodegradation of petroleum hydrocarbons in a weathered, unsaturated soil is inhibited by peroxide oxidants.

Field-weathered crude oil-containing soils have a residual concentration of hydrocarbons with complex chemical structure, low solubility, and high viscosity, often poorly amenable to microbial degradation. Hydrogen peroxide (H2O2)-based oxidation can generate oxygenated compounds that are smaller and/or more soluble and thus increase petroleum hydrocarbon biodegradability. In this study, we assessed the efficacy of H2O2-based oxidation under unsaturated soil conditions to promote biodegradation in a field-contaminated and weathered soil containing high concentrations of total petroleum hydrocarbons (25200 mg TPH kg-1) and total organic carbon (80900 mg TOC kg-1). Microcosms amended with three doses of 48 g H2O2 kg-1 soil (unactivated or Fe2+-activated) or 24 g sodium percarbonate kg-1 soil and nutrients did not show substantial TPH changes during the experiment. However, 7.6-41.8% of the TOC concentration was removed. Furthermore, production of DOC was enhanced and highest in the microcosms with oxidants, with approximately 20-40-fold DOC increase by the end of incubation. In the absence of oxidants, biostimulation led to > 50% TPH removal in 42 days. Oxidants limited TPH biodegradation by diminishing the viable concentration of microorganisms, altering the composition of the soil microbial communities, and/or creating inhibitory conditions in soil. Study's findings underscore the importance of soil characteristics and petroleum hydrocarbon properties and inform on potential limitations of combined H2O2 oxidation and biodegradation in weathered soils.

Continuous-mode acclimation and operation of lignocellulosic sulfate-reducing bioreactors for enhanced metal immobilization from acidic mining-influenced water.

Lignocellulosic sulfate-reducing bioreactors are an inexpensive passive approach for treatment of mining-influenced water (MIW). Typically, microbial community acclimation to MIW involves bioreactor batch-mode operation to initiate lignocellulose hydrolysis and fermentation and provide electron donors for sulfate-reducing bacteria. However, batch-mode operation could significantly prolong bioreactor start-up times (up to several months) and select for slow-growing microorganisms. In this study we assessed the feasibility of bioreactor continuous-mode acclimation to MIW (pH 2.5, 6.5 mM SO42-, 18 metal(loid)s) as an alternate start-up method. Results showed that bioreactors with spent brewing grains and sugarcane bagasse achieved acclimation in continuous mode at hydraulic retention times (HRTs) of 7-12-d within 16-22 days. During continuous-mode acclimation, extensive SO42- reduction (80 ± 20% -91 ± 3%) and > 98% metal(loid) removal was observed. Operation at a 3-d HRT further yielded a metal(loid) removal of 97.5 ± 1.3 -98.8 ± 0.9% until the end of operation. Sulfate-reducing microorganisms were detected closer to the influent in the spent brewing grains bioreactors, and closer to the effluent in the sugarcane bagasse bioreactors, giving insight as to where SO42- reduction was occurring. Results strongly support that a careful selection of lignocellulose and bioreactor operating parameters can bypass typical batch-mode acclimation, shortening bioreactor start-up times and promoting effective MIW metal(loid) immobilization and treatment.

An Ion Chromatography Method for Simultaneous Quantification of Chromate, Arsenate, Selenate, Perchlorate, and Other Inorganic Anions in Environmental Media.

Chromium (Cr) (VI) is a toxic, mutagenic, and carcinogenic water pollutant. The standard ion chromatography (IC) method for quantification of Cr (VI) in water samples is Environmental Protection Agency Method 218.7, which requires postcolumn derivatization with 1,5-diphenylcarbazide and UV-Vis spectroscopy detection. Method 218.7 is Cr (VI) specific; thus, it does not allow detection of co-occurring natural and anthropogenic anions in environmental media. In this study, we developed an isocratic IC method with suppressed conductivity detection, a Metrohm Metrosep A Supp 7 column, and sodium carbonate/acetonitrile as mobile phase for simultaneous quantification of Cr (VI), C l O 4 - , As (V) as arsenate, Se (VI) as selenate, and the common anions F-, Cl-, N O 2 - , N O 3 - , and S O 4 2 - . The determination coefficient for every analyte was >0.99 and the method showed good accuracy in quantification. Cr (VI), As (V), Se (VI), and C l O 4 - limit of detection and limit of quantification were 0.1-0.6 µg/L and 0.5-2.1 µg/L, respectively. Recovery of Cr (VI) in various aqueous samples (tap water, surface water, groundwater, and wastewater) was between 97.2% and 102.8%. Overall, most analytes showed acceptable recovery (80-120%) in the environmental samples tested. The IC method was applied to track Cr (VI) and other anion concentrations in laboratory batch microcosms experiments with soil, surface water, and anaerobic medium. The IC method developed in this study should prove useful to environmental practitioners, academic and research organizations, and industries for monitoring low concentrations of multiple anions in environmental media, helping to decrease the sample requirement, time, and cost of analysis.

Microbial Chain Elongation and Subsequent Fermentation of Elongated Carboxylates as H2-Producing Processes for Sustained Reductive Dechlorination of Chlorinated Ethenes.

In situ anaerobic groundwater bioremediation of trichloroethene (TCE) to nontoxic ethene is contingent on organohalide-respiring Dehalococcoidia, the most common strictly hydrogenotrophic Dehalococcoides mccartyi (D. mccartyi). The H2 requirement for D. mccartyi is fulfilled by adding various organic substrates (e.g., lactate, emulsified vegetable oil, and glucose/molasses), which require fermenting microorganisms to convert them to H2. The net flux of H2 is a crucial controlling parameter in the efficacy of bioremediation. H2 consumption by competing microorganisms (e.g., methanogens and homoacetogens) can diminish the rates of reductive dechlorination or stall the process altogether. Furthermore, some fermentation pathways do not produce H2 or having H2 as a product is not always thermodynamically favorable under environmental conditions. Here, we report on a novel application of microbial chain elongation as a H2-producing process for reductive dechlorination. In soil microcosms bioaugmented with dechlorinating and chain-elongating enrichment cultures, near stoichiometric conversion of TCE (0.07 ± 0.01, 0.60 ± 0.03, and 1.50 ± 0.20 mmol L-1 added sequentially) to ethene was achieved when initially stimulated by chain elongation of acetate and ethanol. Chain elongation initiated reductive dechlorination by liberating H2 in the conversion of acetate and ethanol to butyrate and caproate. Syntrophic fermentation of butyrate, a chain-elongation product, to H2 and acetate further sustained the reductive dechlorination activity. Methanogenesis was limited during TCE dechlorination in soil microcosms and absent in transfer cultures fed with chain-elongation substrates. This study provides critical fundamental knowledge toward the feasibility of chlorinated solvent bioremediation based on microbial chain elongation.

The occurrence and ecology of microbial chain elongation of carboxylates in soils.

Chain elongation is a growth-dependent anaerobic metabolism that combines acetate and ethanol into butyrate, hexanoate, and octanoate. While the model microorganism for chain elongation, Clostridium kluyveri, was isolated from a saturated soil sample in the 1940s, chain elongation has remained unexplored in soil environments. During soil fermentative events, simple carboxylates and alcohols can transiently accumulate up to low mM concentrations, suggesting in situ possibility of microbial chain elongation. Here, we examined the occurrence and microbial ecology of chain elongation in four soil types in microcosms and enrichments amended with chain elongation substrates. All soils showed evidence of chain elongation activity with several days of incubation at high (100 mM) and environmentally relevant (2.5 mM) concentrations of acetate and ethanol. Three soils showed substantial activity in soil microcosms with high substrate concentrations, converting 58% or more of the added carbon as acetate and ethanol to butyrate, butanol, and hexanoate. Semi-batch enrichment yielded hexanoate and octanoate as the most elongated products and microbial communities predominated by C. kluyveri and other Firmicutes genera not known to undergo chain elongation. Collectively, these results strongly suggest a niche for chain elongation in anaerobic soils that should not be overlooked in soil microbial ecology studies.

Synergistic Zerovalent Iron (Fe0) and Microbiological Trichloroethene and Perchlorate Reductions Are Determined by the Concentration and Speciation of Fe.

Trichloroethene (TCE) and perchlorate (ClO4-) are cocontaminants at multiple Superfund sites. Fe0 is often used during TCE bioremediation with Dehalococcoides mccartyi to establish anoxic conditions in the aquifer. However, the synergy between Fe0 abiotic reactions and microbiological TCE and ClO4- reductions is poorly understood and seldom addressed in the literature. Here, we investigated the effects of Fe0 and its oxidation product, Fe2+, at field-relevant concentrations in promoting microbial TCE and ClO4- reductions. Using semibatch microcosms with a Superfund site soil and groundwater, we showed that the high Fe0 concentration (16.5 g L-1) expected during Fe0in situ injection mostly yielded TCE abiotic reduction to ethene/ethane. However, such concentrations obscured dechlorination by D. mccartyi, impeded ClO4- reduction, and enhanced SO42- reduction and methanogenesis. Fe2+ at 0.25 g L-1 substantially delayed conversion of TCE to ethene when compared to no-Fe controls. A low concentration of aged-Fe0 synergistically promoted microbiological TCE dechlorination to ethene while achieving complete ClO4- reduction. Collectively, these results illustrate scenarios relevant at or downstream of Fe0 injection zones when Fe0 is used to facilitate microbial dechlorination. Results also underscore the potential detrimental effects of Fe0 and bioaugmentation cultures coinjection for in situ treatment of chlorinated ethenes and ClO4-.

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.

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.

The effects of CO2 and H2 on CO metabolism by pure and mixed microbial cultures.

BACKGROUND: Syngas fermentation, the bioconversion of CO, CO2, and H2 to biofuels and chemicals, has undergone considerable optimization for industrial applications. Even more, full-scale plants for ethanol production from syngas fermentation by pure cultures are being built worldwide. The composition of syngas depends on the feedstock gasified and the gasification conditions. However, it remains unclear how different syngas mixtures affect the metabolism of carboxidotrophs, including the ethanol/acetate ratios. In addition, the potential application of mixed cultures in syngas fermentation and their advantages over pure cultures have not been deeply explored. In this work, the effects of CO2 and H2 on the CO metabolism by pure and mixed cultures were studied and compared. For this, a CO-enriched mixed culture and two isolated carboxidotrophs were grown with different combinations of syngas components (CO, CO:H2, CO:CO2, or CO:CO2:H2). RESULTS: The CO metabolism of the mixed culture was somehow affected by the addition of CO2 and/or H2, but the pure cultures were more sensitive to changes in gas composition than the mixed culture. CO2 inhibited CO oxidation by the Pleomorphomonas-like isolate and decreased the ethanol/acetate ratio by the Acetobacterium-like isolate. H2 did not inhibit ethanol or H2 production by the Acetobacterium and Pleomorphomonas isolates, respectively, but decreased their CO consumption rates. As part of the mixed culture, these isolates, together with other microorganisms, consumed H2 and CO2 (along with CO) for all conditions tested and at similar CO consumption rates (2.6 ± 0.6 mmol CO L-1 day-1), while maintaining overall function (acetate production). Providing a continuous supply of CO by membrane diffusion caused the mixed culture to switch from acetate to ethanol production, presumably due to the increased supply of electron donor. In parallel with this change in metabolic function, the structure of the microbial community became dominated by Geosporobacter phylotypes, instead of Acetobacterium and Pleomorphomonas phylotypes. CONCLUSIONS: These results provide evidence for the potential of mixed-culture syngas fermentation, since the CO-enriched mixed culture showed high functional redundancy, was resilient to changes in syngas composition, and was capable of producing acetate or ethanol as main products of CO metabolism.

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.

Evolution of microbial communities growing with carbon monoxide, hydrogen, and carbon dioxide.

Microbial anaerobic conversion of carbon monoxide (CO) and syngas (mainly composed of CO, CO2 and H2) leads to the production of important industrial products, such as acetate and ethanol. The composition of CO- and syngas-converting microbial communities and the microbial interactions involved are still largely unknown. The main objectives of this study were (i) to understand the effects of CO, CO2, and H2 on the structure and function of a CO-consuming microbial community, and (ii) to identify key carboxidotrophs in the mixed culture. For this, sludge was anaerobically enriched with CO as the sole carbon/energy source at incrementally increasing CO partial pressures (PCO). Phylotypes of Methanobacteriaceae and methane production were detected at PCO ≤ 44.1 kPa. At higher PCO, enriched phylotypes were Acetobacterium, Oscillospira and Pleomorphomonas, and acetate was the main end product. The addition of CO2/HCO3- or H2 to CO fermentation increased the acetate/ethanol ratio and species diversity, compared to growth with CO as sole substrate. Phylotypes associated with Pleomorphomonas and Acetobacterium increased in relative abundance during exponential CO utilization. The Pleomorphomonas-like isolate produced H2:CO2, whereas the Acetobacterium-like isolate produced ethanol, when CO was the only electron/carbon source. These findings shed light on the interplay between syngas components and microbial communities.

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.