Microbial community degradation of widely used quaternary ammonium disinfectants.

Benzalkonium chlorides (BACs) are disinfectants widely used in a variety of clinical and environmental settings to prevent microbial infections, and they are frequently detected in nontarget environments, such as aquatic and engineered biological systems, even at toxic levels. Therefore, microbial degradation of BACs has important ramifications for alleviating disinfectant toxicity in nontarget environments as well as compromising disinfectant efficacy in target environments. However, how natural microbial communities respond to BAC exposure and what genes underlie BAC biodegradation remain elusive. Our previous metagenomic analysis of a river sediment microbial community revealed that BAC exposure selected for a low-diversity community, dominated by several members of the Pseudomonas genus that quickly degraded BACs. To elucidate the genetic determinants of BAC degradation, we conducted time-series metatranscriptomic analysis of this microbial community during a complete feeding cycle with BACs as the sole carbon and energy source under aerobic conditions. Metatranscriptomic profiles revealed a candidate gene for BAC dealkylation, the first step in BAC biodegradation that results in a product 500 times less toxic. Subsequent biochemical assays and isolate characterization verified that the putative amine oxidase gene product was functionally capable of initiating BAC degradation. Our analysis also revealed cooperative interactions among community members to alleviate BAC toxicity, such as the further degradation of BAC dealkylation by-products by organisms not encoding amine oxidase. Collectively, our results advance the understanding of BAC aerobic biodegradation and provide genetic biomarkers to assess the critical first step of this process in nontarget environments.

Microbial community adaptation to quaternary ammonium biocides as revealed by metagenomics.

Quaternary ammonium compounds (QACs) represent widely used cationic biocides that persist in natural environments. Although microbial degradation, sensitivity and resistance to QACs have been extensively documented, a quantitative understanding of how whole communities adapt to QAC exposure remain elusive. To gain insights into these issues, we exposed a microbial community from a contaminated river sediment to varied levels of benzalkonium chlorides (BACs, a family of QACs) for 3 years. Comparative metagenomic analysis showed that the BAC-fed communities were dramatically decreased in phylogenetic diversity compared with the control (no BAC exposure), resulting presumably from BAC toxicity, and dominated by Pseudomonas species (> 50% of the total). Time-course metagenomics revealed that community adaptation occurred primarily via selective enrichment of BAC-degrading Pseudomonas populations, particularly P. nitroreducens, and secondarily via amino acid substitutions and horizontal transfer of a few selected genes in the Pseudomonas populations, including a gene encoding a PAS/PAC sensor protein and ring-hydroxylating dioxygenase genes. P. nitroreducens isolates were reproducibly recoverable from communities after prolonged periods of no-BAC exposure, suggesting that they are robust BAC-degraders. Our study provides new insights into the mechanisms and tempo of microbial community adaptation to QAC exposure and has implications for treating QACs in biological engineered systems.

Long-term exposure to benzalkonium chloride disinfectants results in change of microbial community structure and increased antimicrobial resistance.

The effect of benzalkonium chlorides (BACs), a widely used class of quaternary ammonium disinfectants, on microbial community structure and antimicrobial resistance was investigated using three aerobic microbial communities: BACs-unexposed (DP, fed a mixture of dextrin/peptone), BACs-exposed (DPB, fed a mixture of dextrin/peptone and BACs), and BACs-enriched (B, fed only BACs). Long-term exposure to BACs reduced community diversity and resulted in the enrichment of BAC-resistant species, predominantly Pseudomonas species. Exposure of the two microbial communities to BACs significantly decreased their susceptibility to BACs as well as three clinically relevant antibiotics (penicillin G, tetracycline, ciprofloxacin). Increased resistance to BACs and penicillin G of the two BACs-exposed communities is predominantly attributed to degradation or transformation of these compounds, whereas resistance to tetracycline and ciprofloxacin is largely due to the activity of efflux pumps. Quantification of several key multidrug resistance genes showed a much higher number of copies of these genes in the DPB and B microbial communities compared to the DP community. Collectively, our findings indicate that exposure of a microbial community to BACs results in increased antibiotic resistance, which has important implications for both human and environmental health.

Aerobic biotransformation of n-tetradecylbenzyldimethylammonium chloride by an enriched Pseudomonas spp. community.

The biotransformation of n-tetradecylbenzyldimethylammonium chloride (C(14)BDMA-Cl), a quaternary ammonium compound (QAC), under aerobic conditions by an enriched microbial community growing on benzalkonium chlorides (BACs) was investigated. Biotransformation of C(14)BDMA-Cl commenced with cleavage of the C(alkyl)-N bond and formation of benzyldimethylamine (BDMA). BDMA was further degraded, but in contrast to a previously reported BAC biotransformation pathway, neither benzylmethylamine (BMA) nor benzylamine (BA) was detected as a BDMA biotransformation product. Kinetic assays further confirmed that BMA and BA were not intermediates of C(14)BDMA-Cl transformation by the enriched community. Thus, BDMA is thought to be transformed to dimethylamine and benzoic acid via debenzylation. The biomass-normalized rate of C(14)BDMA-Cl biotransformation was 0.09 µmol/[mg of volatile suspended solids (VSS)·h]. The Microtox acute toxicity EC(50) value of BDMA was 500 times higher than that of C(14)BDMA-Cl. Thus, the aerobic biotransformation of C(14)BDMA-Cl to BDMA results in substantial toxicity reduction. Phylogenetic analysis of Bacteria diversity indicated that the majority of the sequenced clones (98% of the clone library) belonged to the genus Pseudomonas.

Anaerobic co-digestion of municipal sludge with fat-oil-grease (FOG) enhances the destruction of sludge solids.

The objective of this study was to investigate the benefits of co-digestion of a sludge-mix of primary sludge (PS)/thickened waste activated sludge (TWAS) with concentrated fat-oil-grease (FOG) over a wide range of FOG/sludge-mix volumetric feed ratios. The biodegradability (i.e., COD to methane conversion) of PS, TWAS, sludge-mix, and FOG was 43.0, 38.6, 41.8, and 97.7%, respectively, with a pseudo first-order rate of 0.13, 0.12, 0.13, and 0.18 d-1, respectively. Batch co-digestion of sludge-mix and FOG at COD ratios ranging from 93.2:6.8 to 27.3:72.7% resulted in methane production linearly correlated to both the total waste blend and FOG COD feed concentration. An enhanced extent of degradation of the sludge-mix COD to as much as 10.9% (increased from 42.2 to 53.1%) and an increased degradation rate by 17% (increased from 0.12 to 0.14 d-1) was observed when the feed FOG COD was 18.5% of the total waste COD feed. Overall, co-digestion of mixed municipal sludge with FOG is feasible and recommended to meet target biogas/methane levels at municipal wastewater treatment facilities taking into account the trade-off between energy production and solids destruction to fit their particular needs.

Porous iron material for TcO4- and ReO4- sequestration from groundwater under ambient oxic conditions.

Technetium-99 (99Tc) is a major contaminant at nuclear power plants and several US Department of Energy sites. Its most common aqueous species, pertechnetate (TcO4-), is very mobile in the environment, and currently there are no effective technologies for its sequestration. In this work, a porous iron (pFe) material was investigated for TcO4- and perrhenate (ReO4-) sequestration from artificial groundwater. The pFe was significantly more effective than granular iron for both TcO4- and ReO4- sequestration under oxic conditions. The Tc removal capacity was 27.5 mg Tc/g pFe at pH ˜6.8, while the Re removal capacity was 23.9 mg Re/g pFe at pH ˜10.6. Tc K-edge XANES and EXAFS analyses indicated that the removed Tc species was 70-80% Tc(IV) that was likely incorporated into Fe corrosion products (i.e., Fe(OOH), Fe3O4) and 20-30% unreduced TcO4-. In contrast, the removed Re species was ReO4- only, without detectable Re(IV). In addition, the sequestered ReO4- was not extracted (<3%) by 0.1 M Na2SO4 and 1 M KI solution, which indicated that ReO4- and by chemical analogy, unreduced TcO4-, was likely incorporated into Fe corrosion products. This inexpensive pFe material may be applied to the sequestration and stabilization of 99TcO4- from contaminated environments and nuclear waste streams.

Performance comparison of a pilot-scale UASB and DHS system and activated sludge process for the treatment of municipal wastewater.

This study compares the performance of a pilot-scale combination of UASB and DHS system to that of activated sludge process (ASP) for the treatment of municipal sewage. Both systems were operated in parallel with the same sewage as influent. The study was conducted for more than 300 days, which revealed that organic removal efficiency of UASB+DHS system was comparable to that of ASP. Unfiltered BOD removal by both systems was more than 90%. However, UASB+DHS system outperformed ASP for pathogen removal. In addition, volume of excess sludge production from UASB+DHS was 15 times smaller than that from ASP. Moreover, unlike ASP, there is no requirement of aeration for the operation of UASB+DHS system, which makes it an economical treatment system. Considering the above observations, it was concluded that UASB+DHS system can be a cost-effective and viable option for the treatment of municipal sewage over ASP, especially for low-income countries.

Effective partial nitrification to nitrite by down-flow hanging sponge reactor under limited oxygen condition.

Combining the processes of partial nitrification and anaerobic ammonium oxidation (ANAMMOX) is an attractive wastewater-treatment technology for nitrogen removal. In this study we investigated partial nitrification by implementing a closed down-flow hanging sponge (DHS) reactor operated at controlled oxygen concentrations. Basic concept of DHS process is similar to that of trickling filter, in which oxygen concentration can be easily manipulated by controlling airflow to the reactor. The closed reactor was fed with artificial wastewater containing NH(4)Cl and operated with an HRT of 1.5h at 30 degrees C. Oxygen inside the reactor was maintained below 3% (1.2mgDO x L(-1)) (DO-dissolved oxygen) except during the startup periods. Five months of continuous operation showed that there was a strong relationship between oxygen concentration and nitrite production. The ratio of nitrite produced relative to ammonium oxidized increased by decreasing oxygen concentration. Partial nitrification was satisfactorily accomplished under oxygen limitation at around 0.5% in the gas phase (0.2mgDOL(-1)). The system showed a high ammonium-removal rate, at a maximum of 1.46kg NH(4)(+)-Nm(-3)day(-1), even at limited oxygen concentration. We also found that oxygen concentration played an important role in the production of nitrous oxide, which increased with decreasing oxygen concentration.

Microbial community that catalyzes partial nitrification at low oxygen atmosphere as revealed by 16S rRNA and amoA genes.

We previously reported that partial nitrification in the down-flow hanging sponge (DHS) system was satisfactorily accomplished under oxygen-limited conditions [Chuang et al., Water Res., 41, 295-302 (2007)]. In this study, we investigated the microbes that are responsible for the partial nitrification in this unique system by 16S rRNA- and amoA-based cloning analyses and fluorescence in situ hybridization. Microbes related to Nitrosomonas species were found to be chiefly responsible for catalyzing the partial nitrification. Microbes affiliated with the uncultivated phyla OP10 and Bacteroidetes were also numerous in the DHS, but their ecological niches are still unknown.

Co-digestion of municipal sludge and external organic wastes for enhanced biogas production under realistic plant constraints.

A bench-scale investigation was conducted to select external organic wastes and mixing ratios for co-digestion with municipal sludge at the F. Wayne Hill Water Resources Center (FWHWRC), Gwinnett County, GA, USA to support a combined heat and power (CHP) project. External wastes were chosen and used subject to two constraints: a) digester retention time no lower than 15 d; and b) total biogas (methane) production not to exceed a specific target level based on air permit constraints on CO2 emissions. Primary sludge (PS), thickened waste activated sludge (TWAS) and digested sludge collected at the FWHWRC, industrial liquid waste obtained from a chewing gum manufacturing plant (GW) and dewatered fat-oil-grease (FOG) were used. All sludge and waste samples were characterized and their ultimate digestibility was assessed at 35 °C. The ultimate COD to methane conversion of PS, TWAS, municipal sludge (PS + TWAS; 40:60 w/w TS basis), GW and FOG was 49.2, 35.2, 40.3, 72.7, and 81.1%, respectively. Co-digestion of municipal sludge with GW, FOG or both, was evaluated using four bench-scale, mesophilic (35 °C) digesters. Biogas production increased significantly and additional degradation of the municipal sludge between 1.1 and 30.7% was observed. Biogas and methane production was very close to the target levels necessary to close the energy deficit at the FWHWRC. Co-digestion resulted in an effluent quality similar to that of the control digester fed only with the municipal sludge, indicating that co-digestion had no adverse effects. Study results prove that high methane production is achievable with the addition of concentrated external organic wastes to municipal digesters, at acceptable higher digester organic loadings and lower retention times, allowing the effective implementation of CHP programs at municipal wastewater treatment plants, with significant cost savings.

Transition of municipal sludge anaerobic digestion from mesophilic to thermophilic and long-term performance evaluation.

Strategies for the transition of municipal sludge anaerobic digestion from mesophilic to thermophilic were assessed and the long-term stability and performance of thermophilic digesters operated at a solids retention time of 30days were evaluated. Transition from 36°C to 53.3°C at a rate of 3°C/day resulted in fluctuation of the daily gas and volatile fatty acids (VFAs) production. Steady-state was reached within 35days from the onset of temperature increase. Transitions from either 36 or 53.3°C to 60°C resulted in relatively stable daily gas production, but VFAs remained at very high levels (in excess of 5000mg COD/L) and methane production was lower than that of the mesophilic reactor. It was concluded that in order to achieve high VS and COD destruction and methane production, the temperature of continuous-flow, suspended growth digesters fed with mixed municipal sludge should be kept below 60°C.

Development of a sixth-generation down-flow hanging sponge (DHS) reactor using rigid sponge media for post-treatment of UASB treating municipal sewage.

A sixth-generation down-flow hanging sponge reactor (DHS-G6), using rigid sponge media, was developed as a novel aerobic post-treatment unit for upflow anaerobic sludge blanket (UASB) treating municipal sewage. The rigid sponge media were manufactured by copolymerizing polyurethane with epoxy resin. The UASB and DHS system had a hydraulic retention time (HRT) of 10.6 h (8.6 h for UASB and 2 h for DHS) when operated at 10-28 °C. The system gave reasonable organic and nitrogen removal efficiencies. The final effluent had a total biochemical oxygen demand of only 12 mg/L and a total Kjeldahl nitrogen content of 6 mg/L. The DHS reactor gave particularly good nitrification performance, which was attributed to the new rigid sponge media. The sponge media helped to provide a sufficient HRT, and retained a high biomass concentration, extending the solids retention time. The DHS reactor maintained a high dissolved oxygen concentration under natural ventilation.

Aerobic biotransformation potential of a commercial mixture of naphthenic acids.

The biotransformation potential of a commercial naphthenic acid (NA) mixture (NA sodium salt; TCI Chemicals) under aerobic conditions was investigated using mixed aerobic cultures developed under various levels and duration of NA exposure. A culture enriched using the commercial NA mixture as the sole carbon source degraded NAs in a range of NA concentrations, regardless of culture age and the presence of a co-substrate; however, only 28.5% of the NA-carbon was detected as CO2 while 44% was utilized for biomass growth. A fraction of the NA mixture (15-26%) was persistent under all conditions studied. In contrast, a culture fed with a degradable synthetic wastewater only (NA un-amended culture) and another culture fed with the same wastewater and 50 mg NA/L (NA-amended culture), over time lost their ability to degrade NAs. Analysis of the 16S rRNA gene based clone library revealed that 80% of the NA-enriched culture belonged to the γ-Proteobacteria class and was largely dominated by phylotypes most closely related to known NA and hydrocarbon degraders such as Pseudomonas and Microbulbifer. The results of this study indicate that although significant NA degradation is possible, only a small fraction of the NA mixture is completely mineralized to CO2. Further investigation into the biotransformation products and conditions affecting NA biodegradation under realistic refinery and environmental conditions will help to design effective treatment and bioremediation processes.

Inhibition and biotransformation potential of naphthenic acids under different electron accepting conditions.

Naphthenic acids (NAs) are a complex group of alkyl-substituted acyclic, monocyclic and polycyclic carboxylic acids present in crude oil, oil sands process water and tailings ponds, as well as in refinery wastewater. Bioassays were performed to investigate the biotransformation potential and inhibitory effect of a commercial NA mixture to nitrification, denitrification and fermentation/methanogenesis using mixed cultures not previously exposed to NAs. NAs inhibited nitrification in a mixed aerobic heterotrophic/nitrifying culture at concentrations as low as 80 mg NA/L, whereas, an enriched nitrifying culture was only affected at 400 mg NA/L. The lower nitrification inhibition in the latter assay is attributed to the higher population size of nitrosofying and nitrifying bacteria compared to the mixed heterotrophic/nitrifying culture. The NA mixture was not inhibitory to denitrifiers up to 400 mg/L. At higher NA concentrations, cell lysis was pronounced and lysis products were the main source of degradable carbon driving denitrification in culture series prepared without an external carbon source. In the presence of a degradable external carbon source, no difference was observed in nitrate reduction rates or nitrogen gas production at all NA concentrations tested. Methanogenesis was completely inhibited at NA concentrations equal to or higher than 200 mg/L. Methanogenic culture series amended with 80 mg NA/L were transiently inhibited and methane production in culture series prepared with NAs and an external carbon source or NAs only recovered in 136 and 41 days, respectively. Accumulation of volatile fatty acids was observed at inhibitory NA concentrations; however, carbon dioxide production was not affected by NAs, indicating that fermentation and acidogenesis were not affected by NAs. NAs were not degraded under nitrate-reducing or fermentative/methanogenic conditions used in the present study, regardless of the presence or not of another, degradable carbon/energy source.

Simultaneous carbon removal, denitrification and power generation in a membrane-less microbial fuel cell.

A membrane-less microbial fuel cell (ML-MFC) was developed to investigate the simultaneous carbon removal and denitrification. The removal rates of 0.64 kg COD m(-3) of liquid cathode volume (LCV) d(-1) and 0.186 g NO3(-)-N m(-3) of LCV d(-1) were achieved, which resulted in the maximal COD and nitrate removal rates of 100% and 36.7%, respectively. The ML-MFC also achieved a maximal power output of 0.0712 W m(-3) of LCV and 0.844 A m(-3) of LCV in approximately 24h. The maximal coulombic efficiency of anode (CEAn) and cathode (CECa) was 5.1% and 475%, respectively. The anodic gas phase was consisted of 77.2±4.0% CH4, 3.9±0.5% CO2, and 3.9±1.5% N2, which indicated that the low anode coulombic efficiency was due to anodic methane production. The results of this study demonstrated the potential application of ML-MFC in simultaneous carbon and nitrogen removal and energy (electricity) production.

Biological chromium(VI) reduction in the cathode of a microbial fuel cell.

The biocathode of a microbial fuel cell (MFC) offers a promising potential for the reductive treatment of oxidized pollutants. In this study, we demonstrated biological Cr(VI) reduction in the cathode of a MFC and identified putative Cr(VI) reducing microorganisms. The MFC was continuously monitored for Cr(VI) reduction and power generation. Acetate was provided to the anode compartment as substrate and bicarbonate was added to the cathode compartment as the sole external carbon source. The contribution of biomass decay and abiotic processes on Cr(VI) reduction was minimal, confirming that most of the Cr(VI) reduction was assisted by microbial activity in the cathode, which utilizes electrons and protons generated from the oxidation of acetate in the anode compartment. Relatively fast Cr(VI) reduction was observed at initial Cr(VI) concentrations below 80 mg/L. However, at 80 mg Cr(VI)/L, Cr(VI) reduction was extremely slow. A maximum Cr(VI) reduction rate of 0.46 mg Cr(VI)/g VSS.h was achieved, which resulted in a current and power density of 123.4 mA/m(2) and 55.5 mW/m(2), respectively. The reduced chromium was nondetectable in the supernatant of the catholyte which indicated complete removal of chromium as Cr(OH)(3) precipitate. Analysis of the 16S rRNA gene based clone library revealed that the cathode biomass was largely dominated by phylotypes closely related to Trichococcus pasteurii and Pseudomonas aeruginosa, the putative Cr(VI) reducers.