Marcio Luis Busi da Silva

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

ABSTRACT 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.

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.

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.

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 SO4 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 (C6–C12). 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.

Enhancement of nutrient removal from swine wastewater digestate coupled to biogas purification by microalgae Scenedesmus spp.

This work investigated the effects of swine wastewater-derived biogas on microalgae biomass production and nutrient removal rates from piggery wastewater concomitantly with biogas filtration. Photobioreactors with dominant Scenedesmus spp. were prepared using non-sterile digestate and exposed to different photoperiods. In the presence of biogas and autotrophic conditions microalgae yield of 1.1±0.2 g L(-1) (growth rate of 141.8±3.5 mg L(-1) d(-1)) was obtained leading to faster N-NH3 and P-PO4(3-) assimilation rate of 21.2±1.2 and 3.5±2.5 mg L(-1) d(-1), respectively. H2S up to 3000 ppmv was not inhibitory and completely removed. Maximum CO2 assimilation of 219±4.8 mg L(-1) d(-1) was achieved. Biological consumption of CH4 up to 18% v/v was verified. O2 up to 22% v/v was controlled by adding acetate to exacerbate oxygen demand by microorganisms. Microalgae-based wastewater treatment coupled to biogas purification accelerates nutrient removal concomitantly producing valuable biomass and biomethane.

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.

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.

Enrichment and acclimation of an anaerobic mesophilic microorganism's inoculum for standardization of BMP assays.

Appropriate enrichment of anaerobic microorganisms consortium is crucial for accurate biochemical methane potential (BMP) assays. An alternative method to produce and maintain a mesophilic methanogenic inoculum was demonstrated. Three sources of inoculum were mixed and acclimated for 857days in order to reach steady conditions (pH=7.90±0.46; VS/TS>50%; VFA/alkalinity=0.16±0.04gAcetic Acid/ [Formula: see text] ). Biogas yield >80% was obtained after 70days of inoculum acclimation in comparison to standard cellulose (>600mLN/gVS). Methanogen community analysis based on 16S rDNA of the inoculum revealed Archaea concentration of 3×10(12) gene copies/g (Methanobacteriales 8×10(10); Methanomicrobiales 8×10(10); and Methanosarcinales 4×10(11) gene copies/g). The proposed method for development and maintenance of microorganism enrichment inoculum demonstrates consistent BMP data which is a requirement for dependable prediction of biogas production at field scale operations.

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.

Protocols for In Vitro Culture and Phytochemical Analysis of Phyllanthus Species (Euphorbiaceae)

We developed reproducible protocols for micropropagation, callus culture, and root culture of the medicinal plant Phyllanthus urinaria, P. niruri, P. tenellus, P. corcovadensis, P. caroliniensis, P. stipulatus, and P. fraternus by using single node explants. Genotype-linked differences are visible among the Phyllanthus species concerning shoot culture, callus culture, and root culture growth. The protocols developed for phytochemical screening of callus and root extracts of P. urinaria, P. caroliniensis, P. stipulatus, and P. fraternus have shown the production of sterols and triterpenes. Both compounds are known to account for the antinociceptive activity of the methanolic extracts as glochidone and stigmasterol have strong activity against neurogenic and inflammatory pain. Similarly, methanolic callus extracts of P. tenellus, P. niruri and P. corcovadensis have potent analgesic properties, however phenolics are major compounds isolated from these species. The optimized micropropagation, callus culture, and root culture protocols offer the possibility to use cell/root culture techniques for vegetative propagation and secondary metabolite studies.