Bioaugmenting the poplar rhizosphere to enhance treatment of 1,4-dioxane.

1,4-Dioxane is a highly mobile and persistent groundwater pollutant that often forms large dilute plumes. Because of this, utilizing aggressive pump-and-treat and ex-situ technologies such as advanced oxidation can be prohibitively expensive. In this study, we bioaugmented the poplar rhizosphere with dioxane-degrading bacteria Mycobacterium dioxanotrophicus PH-06 or Pseudonocardia dioxanivorans CB1190 to enhance treatment of 1,4-dioxane in bench-scale experiments. All treatments tested removed 10 mg/L dioxane to near health advisory levels (<4 µg/L). However, PH-06-bioaugmented poplar significantly outperformed all other treatments, reaching <4 µg/L in only 13 days. Growth curve experiments confirmed that PH-06 could not utilize root extract as an auxiliary carbon source for growth. Despite this limitation, our findings suggest that PH-06 is a strong bioaugmentation candidate to enhance the treatment of dioxane by phytoremediation. In addition, we confirmed that CB1190 could utilize both 1,4-dioxane and root extract as substrates. Finally, we demonstrated the large-scale production of these two strains for use in the field. Overall, this study shows that combining phytoremediation and bioaugmentation is an attractive strategy to treat dioxane-contaminated groundwater to low risk-based concentrations (~1 µg/L).

Spirulina platensis in Japanese quail feeding alters fatty acid profiles and improves egg quality: Benefits to consumers.

The aim of this study was to investigate whether microalgae in Japanese quail feed alters performance, fatty acid profiles in the eggs and egg quality. One hundred quails were distributed in four groups and five replicates of five birds per experimental group. The treatments consisted of four levels of Spirulina platensis microalgae (0%, 5%, 10%, and 15%) in the diets. We evaluated the productive performance and chemical-physical characteristics of eggs, the oxidant/antioxidant status in egg yolks, and the fatty acid profile in the diet and egg yolks. Microalgae in the diet did not influence egg production; however, it increased the yolk index as well as the color intensity of the yolk. Saturated and polyunsaturated fatty acid levels decreased in egg yolks, and monounsaturated fatty acid levels increased in the yolks. Lipid peroxidation levels were lower and total antioxidant capacity was higher in egg yolks of quails receiving microalgae in the diet. PRACTICAL APPLICATIONS: Microalgae in quail diets improves egg quality and provides benefits to consumer health, acting as an antioxidant and immune-stimulant. Microalgae in quail diets had positive effects on egg quality. This is because it reduced levels of saturated fatty acids that are undesirable, and increased monounsaturated fatty acid levels that are beneficial to the health of consumers. Finally, antioxidants increased in egg yolks, consequently reducing lipid peroxidation.

Going Viral: Emerging Opportunities for Phage-Based Bacterial Control in Water Treatment and Reuse.

Water security to protect human lives and support sustainable development is one of the greatest global challenges of this century. While a myriad of water pollutants can impact public health, the greatest threat arises from pathogenic bacteria that can be harbored in different components of water treatment, distribution, and reuse systems. Bacterial biofilms can also promote water infrastructure corrosion and biofouling, which substantially increase the cost and complexity of many critical operations. Conventional disinfection and microbial control approaches are often insufficient to keep up with the increasing complexity and renewed relevance of this pressing challenge. For example, common disinfectants cannot easily penetrate and eradicate biofilms, and are also relatively ineffective against resistant microorganisms. The use of chemical disinfectants is also curtailed by regulations aimed at minimizing the formation of harmful disinfection byproducts. Furthermore, disinfectants cannot be used to kill problematic bacteria in biological treatment processes without upsetting system performance. This underscores the need for novel, more precise, and more sustainable microbial control technologies. Bacteriophages (phages), which are viruses that exclusively infect bacteria, are the most abundant (and perhaps the most underutilized) biological resource on Earth, and hold great promise for targeting problematic bacteria. Although phages should not replace broad-spectrum disinfectants in drinking water treatment, they offer great potential for applications where selective targeting of problematic bacteria is warranted and antimicrobial chemicals are either relatively ineffective or their use would result in unintended detrimental consequences. Promising applications for phage-based biocontrol include selectively suppressing bulking and foaming bacteria that hinder activated sludge clarification, mitigating proliferation of antibiotic resistant strains in biological wastewater treatment systems where broad-spectrum antimicrobials would impair pollutant biodegradation, and complementing biofilm eradication efforts to delay corrosion and biofouling. Phages could also mitigate harmful cyanobacteria blooms that produce toxins in source waters, and could also serve as substitutes for the prophylactic use of antibiotics and biocides in animal agriculture to reduce their discharge to source waters and the associated selective pressure for resistant bacteria. Here, we consider the phage life cycle and its implications for bacterial control, and elaborate on the biochemical basis of such potential application niches in the water supply and reuse cycle. We also discuss potential technological barriers for phage-based bacterial control and suggest strategies and research needs to overcome them.

1,4-Dioxane-degrading consortia can be enriched from uncontaminated soils: prevalence of Mycobacterium and soluble di-iron monooxygenase genes.

Two bacterial consortia were enriched from uncontaminated soil by virtue of their ability to grow on 1,4-dioxane (dioxane) as a sole carbon and energy source. Their specific dioxane degradation rates at 30°C, pH = 7 (i.e. 5.7 to 7.1 g-dioxane per g-protein per day) were comparable to those of two dioxane-metabolizing archetypes: Pseudonocardia dioxanivoransCB1190 and Mycobacterium dioxanotrophicusPH-06. Based on 16S rRNA sequencing, Mycobacterium was the dominant genus. Acetylene inhibition tests suggest that dioxane degradation was mediated by monooxygenases. However, qPCR analyses targeting the tetrahydrofuran/dioxane monooxygenase gene (thmA/dxmA) (which is, to date, the only sequenced dioxane monooxygenase gene) were negative, indicating that other (as yet unknown) catabolic gene(s) were responsible. DNA sequence analyses also showed threefold to sevenfold enrichment of group 5 and group 6 soluble di-iron monooxygenase (SDIMO) genes relative to the original soil samples. Whereas biodegradation of trace levels of dioxane is a common challenge at contaminated sites, both consortia degraded dioxane at low initial concentrations (300 µg l-1 ) below detectable levels (5 µg l-1 ) in bioaugmented microcosms prepared with impacted groundwater. Overall, this work shows that dioxane-degrading bacteria (and the associated natural attenuation potential) exist even in some uncontaminated soils, and may be enriched to broaden bioaugmentation options for sites experiencing insufficient dioxane catabolic capacity.

Assessment of N2O emission from a photobioreactor treating ammonia-rich swine wastewater digestate.

This study investigated the interactions between naturally occurring bacteria and the microalgae Chlorella vulgaris within a lab scale photobioreactor treating ammonia-rich swine wastewater digestate effluent. Nitrification and denitrification were assessed by targeting ammonia monoxygenases (amoA), nitrate (narG), nitrite (nirS), nitric oxide (norB) and nitrous oxide (nosZ) reductases genes. Oxygen produced from microalgae photosynthesis stimulated nitrification. Under limiting carbon availability (i.e., <1.44 for mg TOC/mg NO2-N and 1.72 for mg TOC/mg NO3-N), incomplete denitrification led to accumulation of NO2 and NO3. Significant N2O emission (up to 118 µg N2O-N) was linked to NO2 metabolism in Chlorella. The addition of acetate as external carbon source recovered heterotrophic denitrification activity suppressing N2O emission. Effluent methane concentrations trapped within photobioreactor was removed concomitantly with ammonia. Overall, closed photobioreactors can be built to effectively remove nitrogen and mitigate simultaneously greenhouse gases emissions that would occur otherwise in open microalgae-based wastewater treatment systems.

Analytical model for BTEX natural attenuation in the presence of fuel ethanol and its anaerobic metabolite acetate.

Flow-through column studies were conducted to mimic the natural attenuation of ethanol and BTEX mixtures, and to consider potential inhibitory effects of ethanol and its anaerobic metabolite acetate on BTEX biodegradation. Results were analyzed using a one-dimensional analytical model that was developed using consecutive reaction differential equations based on first-order kinetics. Decrease in pH due to acetogenesis was also modeled, using charge balance equations under CaCO(3) dissolution conditions. Delay in BTEX removal was observed and simulated in the presence of ethanol and acetate. Acetate was the major volatile fatty acid intermediate produced during anaerobic ethanol biodegradation (accounting for about 58% of the volatile fatty acid mass) as suggested by the model data fit. Acetate accumulation (up to 1.1 g/L) near the source zone contributed to a pH decrease by almost one unit. The anaerobic degradation of ethanol (2 g/L influent concentration) at the source zone produced methane at concentrations exceeding its solubility (~/=26mg/L). Overall, this simple analytical model adequately described ethanol degradation, acetate accumulation and methane production patterns, suggesting that it could be used as a screening tool to simulate lag times in BTEX biodegradation, changes in groundwater pH and methane generation following ethanol-blended fuel releases.

Field metabolomics and laboratory assessments of anaerobic intrinsic bioremediation of hydrocarbons at a petroleum-contaminated site.

Field metabolomics and laboratory assays were used to assess the in situ anaerobic attenuation of hydrocarbons in a contaminated aquifer underlying a former refinery. Benzene, ethylbenzene, 2-methylnaphthalene, 1,2,4- and 1,3,5-trimethylbenzene were targeted as contaminants of greatest regulatory concern (COC) whose intrinsic remediation has been previously reported. Metabolite profiles associated with anaerobic hydrocarbon decay revealed the microbial utilization of alkylbenzenes, including the trimethylbenzene COC, PAHs and several n-alkanes in the contaminated portions of the aquifer. Anaerobic biodegradation experiments designed to mimic in situ conditions showed no loss of exogenously amended COC; however, a substantive rate of endogenous electron acceptor reduction was measured (55 ± 8 µM SO(4) day(-1)). An assessment of hydrocarbon loss in laboratory experiments relative to a conserved internal marker revealed that non-COC hydrocarbons were being metabolized. Purge and trap analysis of laboratory assays showed a substantial loss of toluene, m- and o-xylene, as well as several alkanes (C(6)-C(12)). Multiple lines of evidence suggest that benzene is persistent under the prevailing site anaerobic conditions. We could find no in situ benzene intermediates (phenol or benzoate), the parent molecule proved recalcitrant in laboratory assays and low copy numbers of Desulfobacterium were found, a genus previously implicated in anaerobic benzene biodegradation. This study also showed that there was a reasonable correlation between field and laboratory findings, although with notable exception. Thus, while the intrinsic anaerobic bioremediation was clearly evident at the site, non-COC hydrocarbons were preferentially metabolized, even though there was ample literature precedence for the biodegradation of the target molecules.

Microbial community response to a release of neat ethanol onto residual hydrocarbons in a pilot-scale aquifer tank.

The microbial community response to a neat ethanol release (E100, 76 l) onto residual hydrocarbons in sandy soil was evaluated in a continuous-flow 8 m(3) pilot-scale aquifer tank, simulating a release at a bulk fuel terminal. Microbial genotypic shifts were assessed using quantitative real-time PCR analysis. High ethanol concentrations in the capillary fringe at potentially toxic levels, exceeding 100,000 mg l(-1), were tolerated by the microbial community. The high biochemical oxygen demand exerted by ethanol rapidly induced anaerobic conditions, and both methane production (up to 1.2 mg l(-1)) and growth of putative methanogenic Archaea (up to 10(6) gene copies per g of soil) were observed in shallow groundwater and soil samples 75 cm down gradient from the source. Aerobic conditions returned after ethanol was flushed out of the system, approximately 45 days after the spill (less than 7.5 pore volumes flushed). Total Bacteria growth coincided with ethanol migration and availability, which was restricted to a relatively thin layer at the capillary fringe and water table interface. The concentrations of bacteria harbouring the aerobic catabolic genes dmpN (coding for phenol hydroxylase) and to dC1 (coding for toluene dioxygenase) increased (up to 100x) down gradient from the source, likely as a result of both fortuitous growth on ethanol and on aromatic hydrocarbons mobilized by ethanol. Growth of hydrocarbon degraders was corroborated by denaturing gradient gel electrophoresis analysis showing proliferation of Azospirillum and Brevundimonas spp., which are bacteria commonly associated with microaerophilic hydrocarbon degradation. Nevertheless, the relative abundance of hydrocarbon-specific degraders (as a fraction of total Bacteria) decreased as other bacteria grew to a higher extent. Overall, the observed growth of hydrocarbon degraders suggests a potential enhancement in aerobic natural attenuation in shallow aquifers after ethanol and its degradation by-products are degraded or flushed from sites impacted by ethanol-blended fuels.

Aerobic bioremediation of chlorobenzene source-zone soil in flow-through columns: performance assessment using quantitative PCR.

Flow-through aquifer columns were operated for 12 weeks to evaluate the benefits of aerobic biostimulation for the bioremediation of source-zone soil contaminated with chlorobenzenes (CBs). Quantitative Polymerase Chain Reaction (qPCR) was used to measure the concentration of total bacteria (16S rRNA gene) and oxygenase genes involved in the biodegradation of aromatic compounds (i.e., toluene dioxygenase, ring hydroxylating monooxygenase, naphthalene dioxygenase, phenol hydroxylase, and biphenyl dioxygenase). Monochlorobenzene, which is much more soluble than dichlorobenzenes, was primarily removed by flushing, and biostimulation showed little benefit. In contrast, dichlorobenzene removal was primarily due to biodegradation, and the removal efficiency was much higher in oxygen-amended columns compared to a control column. To our knowledge, this is the first report that oxygen addition can enhance CB source-zone soil bioremediation. Analysis by qPCR showed that whereas the biphenyl and toluene dioxygenase biomarkers were most abundant, increases in the concentration of the phenol hydroxylase gene reflected best the higher dichlorobenzene removal due to aerobic biostimulation. This suggests that quantitative molecular microbial ecology techniques could be useful to assess CB source-zone bioremediation performance.

Assessment of anaerobic benzene degradation potential using 16S rRNA gene-targeted real-time PCR.

Benzene is a common groundwater pollutant that is often recalcitrant under the anaerobic conditions that prevail at hydrocarbon-contaminated aquifers. Thus, determining the potential for anaerobic benzene degradation is important to assess the feasibility of intrinsic bioremediation. In this work we developed a 16S rRNA biomarker to estimate the concentration of putative benzene degraders in a methanogenic consortium that has been enriched on benzene for several years. Primers were designed based on phylogenetic information from this consortium. The primers and probe were obtained by sequencing the dominant denaturing gradient gel electrophoresis band of this consortium, which corresponded to Desulfobacterium sp. clone OR-M2. No hybridization was observed with DNA samples from negative controls (i.e. toluene-degrading and dehalorespiring methanogenic consortia that do not degrade benzene). Samples from an anaerobic aquifer column that was bioaugmented with this benzene-degrading consortium showed a strong correlation between benzene degradation activity and the concentration of the target organism. Although our data do not prove that Desulfobacterium sp. is a benzene degrader, its enrichment as a result of benzene consumption and its correlation to anaerobic benzene degradation activity suggest that it either initiates benzene degradation or is a critical (commensal) partner. Therefore, the utility of this primers and probe set to assess anaerobic benzene degradation potential was demonstrated. This is the first report of the use of real-time quantitative PCR for forensic analysis of anaerobic benzene degradation. Whether this biomarker will be adequately selective and broadly applicable to assess benzene degradation potential under strongly anaerobic (sulfate reducing and methanogenic) conditions will require further research.

Effect of simulated rhizodeposition on the relative abundance of polynuclear aromatic hydrocarbon catabolic genes in a contaminated soil.

Microcosms were used to investigate whether soil exposure to mulberry root extracts (rich in phenolic compounds) select for bacteria that degrade polynuclear aromatic hydrocarbons (PAHs). Unlike previous studies with freshly spiked soil, the present experiments were conducted with soils aged for 518 d with [14C]phenanthrene to decrease bioavailability and avoid exaggerating the selective pressure exerted by PAHs relative to the rhizosphere effect. Microcosms simulating contaminated planted soil were exposed to carbon at 20 mg/L/week of mulberry root extract for 211 d to simulate rhizodeposition. Contaminated bulk soils microcosms were amended with a C-free mineral medium to discern the effect of rhizodeposition. Uncontaminated soil controls also were exposed to similar dose regimes. Real-time quantitative polymerase chain reaction was used to enumerate total bacteria and PAH degraders harboring the genes nahAc (coding for naphthalene dioxygenase), todC1 (coding for toluene/benzene/chlorobenzene dioxygenase), bmoA (coding for hydroxylating monooxygenases), and dmpN (coding for phenol hydroxylase). Exposure to root extracts enhanced the growth of total bacteria and PAH degraders in both contaminated and uncontaminated rhizosphere microcosms. The relative abundance of PAH-degrader gene copies (as a fraction of the total bacteria) was similar for different treatments, suggesting that the root extracts did not select for PAH degraders. Overall, these results suggest that rhizodeposition from phenolic releasers contributes to the fortuitous (but not selective) proliferation of PAH degraders, which may enhance phytoremediation.

Enhanced anaerobic biodegradation of BTEX-ethanol mixtures in aquifer columns amended with sulfate, chelated ferric iron or nitrate.

Flow-through aquifer columns were used to investigate the feasibility of adding sulfate, EDTA-Fe(III) or nitrate to enhance the biodegradation of BTEX and ethanol mixtures. The rapid biodegradation of ethanol near the inlet depleted the influent dissolved oxygen (8 mg l(-1)), stimulated methanogenesis, and decreased BTEX biodegradation efficiencies from > 99% in the absence of ethanol to an average of 32% for benzene, 49% for toluene, 77% for ethylbenzene, and about 30% for xylenes. The addition of sulfate, EDTA-Fe(III) or nitrate suppressed methanogenesis and significantly increased BTEX biodegradation efficiencies. Nevertheless, occasional clogging was experienced by the column augmented with EDTA-Fe(III) due to iron precipitation. Enhanced benzene biodegradation (> 70% in all biostimulated columns) is noteworthy because benzene is often recalcitrant under anaerobic conditions. Influent dissolved oxygen apparently played a critical role because no significant benzene biotransformation was observed after oxygen was purged out of the influent media. The addition of anaerobic electron acceptors could enhance BTEX biodegradation not only by facilitating their anaerobic biodegradation but also by accelerating the mineralization of ethanol or other substrates that are labile under anaerobic conditions. This would alleviate the biochemical oxygen demand (BOD) and increase the likelihood that entraining oxygen would be used for the biotransformation of residual BTEX.

Enhanced anaerobic biodegradation of benzene-toluene-ethylbenzene-xylene-ethanol mixtures in bioaugmented aquifer columns.

Methanogenic flowthrough aquifer columns were used to investigate the potential of bioaugmentation to enhance anaerobic benzene-toluene-ethylbenzene-xylene (BTEX) degradation in groundwater contaminated with ethanol-blended gasoline. Two different methanogenic consortia (enriched with benzene or toluene and o-xylene) were used as inocula. Toluene was the only hydrocarbon degraded within 3 years in columns that were not bioaugmented, although anaerobic toluene degradation was observed after only 2 years of acclimation. Significant benzene biodegradation (up to 88%) was observed only in a column bioaugmented with the benzene-enriched methanogenic consortium, and this removal efficiency was sustained for 1 year with no significant decrease in permeability due to bioaugmentation. Benzene removal was hindered by the presence of toluene, which is a more labile substrate under anaerobic conditions. Real-time quantitative PCR analysis showed that the highest numbers of bssA gene copies (coding for benzylsuccinate synthase) occurred in aquifer samples exhibiting the highest rate of toluene degradation, which suggests that this gene could be a useful biomarker for environmental forensic analysis of anaerobic toluene bioremediation potential. bssA continued to be detected in the columns 1 year after column feeding ceased, indicating the robustness of the added catabolic potential. Overall, these results suggest that anaerobic bioaugmentation might enhance the natural attenuation of BTEX in groundwater contaminated with ethanol-blended gasoline, although field trials would be needed to demonstrate its feasibility. This approach may be especially attractive for removing benzene, which is the most toxic and commonly the most persistent BTEX compound under anaerobic conditions.