2013 Sustainable Nanotechnology Organization Conference Effects of nanomaterial disposal on wastewater treatment microbial communities and toxicity implications Yanjun Ma Jacob Metch, Eric Vejerano, Amy Pruden, Linsey Marr Department of Civil & Environmental Engineering Virginia Tech 2013-11-5
Background Potential for Engineered Nanomaterials to enter wastewater treatment plants Engineered nanomaterials are becoming more common in consumer products
Background Engineered Nanomaterials measured in wastewater influents Brar et al., Waste Management, 2010
Background What are potential concerns about nanomaterials in wastewater?
Background Typical wastewater treatment process Biological treatment Fixed or suspended film growth systems Remove soluble organic matter Stabilize insoluble organic matter Convert soluble inorganic matter (e.g. nitrification)
Background Typical wastewater treatment process - Nitrification Ammonification: heterotrophic bacteria Nitrification: Ammonia oxidizing bacteria (AOB) Nitrosomonas, Nitrosospira Nitrite oxidizing bacteria (NOB) Nitrobacter, Nitrospira
Background What are potential concerns about nanomaterials in wastewater? Potential toxicity to microbes during biological treatment Nanomaterial toxicity in biological treatment Inhibition to nitrifying bacteria Impact on microbial community level Antimicrobial effects of nanomaterials in many previous studies Toxicity of nano-ag on Nitrifying bacteria cultures (Arnaout and Gunsh,2012) (Choi and Hu, 2009)
Background What are potential concerns about nanomaterials in wastewater? Potential toxicity to microbes during biological treatment Persistence: Toxicity of original particles Aggregation: Reduce the toxicity Transformation: Change the toxicity
Goal of this study Investigate effects of engineered nanomaterials on nitrifying microbial communities during biological wastewater treatment
Experimental Design 1. Lab-scale sequencing batch reactors (SBRs) to simulate typical nitrifying activated sludge treatment Fed with synthetic wastewater: Influent COD=450mg/L Influent Total Nitrogen=55mg/L Solids retention time (SRT): 14 days Hydraulic retention time (HRT): 2 days Seeded with return activated sludge from local wastewater treatment plants Reactors were re-started after each testing of one type of nanoparticle
Experimental Design 2. Dosing of representative nanomaterials to the SBRs Nanoparticles Nano-Ag Nano zero-valent iron (NZVI) Nano-TiO 2 Nano-CeO 2 Source Synthesized by citrate reduction method Commercial aqueous dispersion with a combination of a biodegradable organic and inorganic stabilizer Commercial anatase powder, dispersed in nanopure water by ultrasound Commercial powder, dispersed in nanopure water by ultrasound Size characterization by TEM 52±12 nm 46±10 nm 21±12 nm 33±12 nm
Experimental Design 2. Dosing of representative nanomaterials to the SBRs SBR set 1 SBR set 2 SBR set 3 No-dosing control No-dosing control Ag + as AgNO 3 Fe 2+ as FeSO 4 Nano-Ag NZVI No-dosing control No-dosing control BulkTiO 2 Nano-TiO 2 BulkCeO 2 Nano-CeO 2
Experimental Design 2. Dosing of representative nanomaterials to the SBRs Dosing timeline Experiment 1: Sequential Load 14 days 14 days 14 days 14 days 0.1mg/L 1mg/L 10mg/L 20mg/L Experiment 2: High Load (Currently completed testing of nanoag and nanoceo 2 ) 42 days 20mg/L
Monitoring and Analysis Monitor reactor performances: ph, COD, MLSS, MLVSS Monitor nitrification performance: concentration of nitrate, nitrite and ammonia Quantify partitioning of nanoparticles into water and sludge: Ag, Fe, Ti and Ce analysis by ICP-AES Nanoparticle characterization: TEM-EDS mapping DNA were extracted from sludge samples Microbial community profiling: pyrosequencing Abundance of nitrifying bacteria: qpcr
Results 1. SBR Nitrification performance Influent Total Nitrogen The reactors failed Nitrification was inhibited by Ag + in the high load experiment No accumulation of ammonia and nitrite was observed in other SBRs
Results 2. Abundance of Nitrifying bacteria Genes specific to nitrifying bacteria were quantified by Quantitative Polymerase Chain Reaction (qpcr) - amoa gene encoding the active-site polypeptide of ammonia monooxygenase Ammonia oxidizing bacteria - Specific 16S rrna gene of Nitrobacter - Specific 16S rrna gene of Nitrospira Nitrite oxidizing bacteria
Results High Load amoa genes (high load of nano-ag) Significant decrease of amoa genes in SBRs dosed with Ag +
Results Sequential Load amoa genes (sequential load of nano-ag) Specific genes of Nitrobacter (sequential load of nano-ag) 0.1mg/L 1mg/L 10mg/L 20mg/L 0.1mg/L 1mg/L 10mg/L 20mg/L Significant decrease of amoa genes in SBRs dosed with nano-ag and Ag + Significant decrease of Nitrobacter specific genes in SBRs dosed with nano-ag
Results Sequential Load amoa genes (sequential load of nano-ceo 2 ) amoa genes (sequential load of NZVI) 0.1mg/L 1mg/L 10mg/L 20mg/L 0.1mg/L 1mg/L 10mg/L 20mg/L Significant decrease of amoa genes in SBRs dosed with nano-ceo 2, bulk-ceo 2, Fe 2+ and NZVI at the end of 20 mg/l dosing Ammonia oxidizing bacteria appeared to be more sensitive to the dosing of nanomaterials and bulk/ionic materials than nitrite oxidizing bacteria
Results 3. Response of microbial community - Pyrosequencing Sampling Experiment 1: Sequential Load 14 days 14 days 14 days 14 days 0.1mg/L 1mg/L 10mg/L 20mg/L Experiment 2: High Load 42 days 20mg/L Target universal bacterial 16S rrna genes with primer set: 341f/907r An average of 3000 16S rrna sequences were obtained per sample Sequences were processed and classified to operational taxonomy unit (OTU) Multidimensional Scaling (MDS) analysis was used to compare the relative similarities of the microbial community compositions
Results Nano-Ag experiments: SBR dosed with Ag + were distinct SBRs dosed with Ag + SBRs dosed with Ag + Phylogenetic similarity revealed by Multidemensional Scaling (MDS) analysis
Results Nano-CeO 2 experiments: No significant difference between nano- CeO 2 and controls End of dosing End of dosing Before dosing Before dosing Phylogenetic similarity revealed by Multidemensional Scaling (MDS) analysis
Results End of dosing NZVI experiment: No significant difference between NZVI and controls Before dosing Before dosing End of dosing Nano-TiO 2 experiment: No significant difference between nano-tio 2 and controls
Results 4. Partitioning and Characterization of nanomaterials in water effluent and sludge > 99% of nanomaterials and bulk/ionic chemicals dosed to the SBRs partitioned into the activated sludge Characterization of nanoparticles in the sludge Sludge at the end of dosing Freeze-dried Prepare TEM grids TEM with EDS to detect nanoparticles and analyze the chemical composition TEM = Transmission electron microscope EDS = Energy Dispersive Spectroscopy
Results Nano-Ag: remained mostly dispersed TEM Imaging EDS mapping Ag S
Results NZVI NZVI, nano-tio 2 and nano-ceo 2 : mostly aggregated TEM Imaging EDS mapping Fe Nano-TiO 2 Ti
Results Nano-CeO 2 NZVI, nano-tio 2 and nano-ceo 2 : mostly aggregated EDS mapping TEM Imaging Ce Aggregation state did not appear to be associated with toxicity
Results Summary Nitrification inhibited by high load of Ag + Ammonia oxidizing bacteria was more sensitive than nitrite oxidizing bacteria Microbial community composition impacted by Ag + in both sequential and high load experiments
Conclusions and future work Limited extent of toxicity of nano-ag, NZVI, nano-tio 2 and nano-ceo 2 was shown during nitrifying activated sludge treatment as revealed by the nitrification function, abundance of nitrifying bacteria and response of microbial communities. -> Cautious optimism for disposal of nano-products into WWTPs However, this study is just a start: Short-term Four type of nanomaterials One type of wastewater treatment process Simplified wastewater matrix
Conclusions and future work Future Interests Long-term study Other nanomaterials Varied nanomaterial properties (size, shape, coatings, etc.) Other wastewater treatment process Disposal of sludge containing concentrated nanomaterials
Acknowledgements This research is funded by EPA Star Grant 834856 Special thanks to: My advisor: Dr. Amy Pruden My committee: Dr. Peter Vikesland, Dr. Linsey Marr, Dr. John Novak Elizabeth Smiley, Julie Petruska, Jeffery Parks Dr. Pruden s group VTSuN group
Results All samples: Variation associated with dosing of nanomaterials was no greater than the differences observed among initial communities nzvi sequential Load nanoceo 2 sequential Load nanotio 2 sequential Load nanoceo 2 High Load nanoag sequential Load nanoag High Load