2017 Excellence in Environmental Engineering and Science® Awards Competition Winner

Grand Prize - University Research

Novel and Advanced Hybrid Oxidation and Enzymatic Technologies for Emerging Trace Environmental Contaminants

Entrant: Institut National de la Recherche Scientifique
Engineer in Charge: Dr. Satinder Kaur Brar and Dr. Rao Y. Surampalli, P.E., BCEE
Location: Quebec, Canada
Media Contact: Dr. Satinder Kaur Brar and Dr. Rao Y. Surampalli, P.E., BCEE

Entrant Profile

Dr. S. K. Brar is Professor at Institut National de la Recherche Scientifique at Québec city in Canada. Her broader research expertise lies in biovalorization of wastes and fate of trace environmental contaminants. Her research has led to the successful supervision of 20 Ph.Ds, 6 M.Scs, 6 Post-docs and 4 Research Assistants. Dr. Brar is a recipient of the ASCE State-of-the-Art Civil Engineering award (2007), Rudolf Hering Medal (2008) and YWCA "women in science excellence" award (2015). In 2014, she was elected member of the College of New Scholars, Artists and Scientists of the Royal Society of Canada. She recently initiated Nanotechnology for Environmental Engineering as Editor-in-Chief with Springer Publishers. She has more than 230 research publications comprising 7 books, 40 book chapters, 125 research papers, 90 conferences and 2 patents to her credit. She also researches the fate of endocrine disrupters, pharmaceuticals, nanoparticles and other emerging contaminants in wastewater and wastewater sludge and finding suitable biological decontamination technologies. She has developed the original scientific concept, drafted the research programme on emerging contaminants and their holistic removal methods and obtained funding in collaboration with Dr. Rao Y. Surampalli.

Dr. Rao Y. Surampalli who is a retired engineer director from US EPA and currently is President and CEO of the Global Institute for Energy, Environment and Sustainability.

The research was executed in the laboratory by a team of researchers:

Ph.Ds:
- D Mohapatra
- F Gassara
- GS Dhillon
- R Pulicharla
- L Lonappan
- M Taheran
- RK Das
- M Nagdhi
- V Pachapur
Post-Doc:
- T Rouissi
- SJ Sarma
- M. Cledon who also compiled the research results into publications.

Project Description

Progressive lifestyle of the world population has led to the release of myriad of emerging trace contaminants (ETCs) in the environment, such as plasticizers, pharmaceutically active compounds (PhACs), among others. Most importantly, the transformation products of these ETCs can be found in air, water and soil and they can have potentially adverse impacts on living organisms. In fact, their wide use for humans and animals can lead to their passage into waterways and soil through wastewater treatment plants (WWTPs) and application of animal manure and WWTP biosolids to agricultural lands. The released PhACs are present at very low concentration and may cause antibiotic resistance, fish feminization or other disorders in different organisms. Very poor degradation happens in WWTPs, so that developing new treatment methods for degradation of the released compounds in air, aqueous and soil media is crucial.

Different removal processes, such as physical, chemical, biological, and their combination systems for the ETCs exist. However, chemical processes, such as ozonation and electro-oxidation are generally expensive and produce more toxic by-products while microbial biological processes suffer from slow reaction and stability problems. Physical separation systems, such as adsorbent materials and membranes showed high removal efficiency though they do not degrade the compounds and need regeneration or complementary systems. Therefore, combining these systems can innovate integrated technologies for ETCs removal in wastewater and drinking water.

In this team project, various rapid analytical methods and mass flow rates (Fig. 1) for ETCs in complex environmental matrices, such as wastewaters and wastewater sludge was developed. Further, enzymatic degradation using highly oxidative ligninolytic enzyme (laccase) was employed in different configurations for degradation of representative ETCs in wastewaters. The configurations include coupling enzymatic treatment with ultrasonication, encapsulation of enzyme in nano-sized/micro-sized adsorbent particles i.e. biochar and integrating enzyme and adsorbent particles into electrospun membrane. The first configuration is designed to extract the compounds from solid media and increase their mobility with ultrasonic waves and production of radicals to accelerate the reaction. In the second configuration, the adsorbent materials act as a support for immobilization of enzyme and adsorption of ETCs to increase contact time with the enzyme. In the third configuration, the membrane act as a support for enzyme immobilization, increases contact time and removes pathogens. Besides transformation of ETCs to innocuous products, green chemistry principles, such as less harmful process, renewable feedstock and energy efficiency are followed in these methods

Figure 1. Estimation of mass flow rate of some ETCs.

Figure 2. Analytical procedure for ETCs in wastewater and wastewater sludge.

In the performed experiments, a wide concentration range (10 ppt to 100 ppm) of ETCs were tested and three compounds represented PhACs i.e. Chlortetracycline (antibiotics), Carbamazepine (antiepileptic) and Diclofenac (anti-inflammatory) and Bisphenol-A as a common plasticizer. Many treatment technologies, such as hydrolysis, Fenton oxidation, ultrasonication and ferro-sonication degraded BPA with 98% efficiency, when combined with enzymes. In fact, biochar can efficiently adsorb the three PhACs (>90% removal) and the enzymatic treatment can degrade them with moderate efficiency (45%) and high efficiency (95%) in absence and presence of enzyme mediator, respectively. In addition, the immobilized enzyme onto biochar and electrospun membrane retained at least 80 % and 40 % of its original activity after one month and ten cycles of standard substrate oxidation, respectively. Degradation of PhACs using immobilized enzyme onto biochar and electrospun membrane showed that they can remove the compounds by up to 95 % efficiency and they preserve at least 35 % of their effectiveness after 5 cycles. Yeast estrogen screen (YES) assay was performed for evaluating the toxicity of the ETCs by-products obtained in different systems. The results showed that the tested compounds and the by-products obtained in ultrasonication process are toxic but the by-products obtained in enzymatic treatment in both free and immobilized forms are not toxic. It confirmed the advantage of hybrid enzymatic treatment over the advanced oxidation processes.

Combination of enzymatic treatment with advanced oxidation processes, membrane separation and nanosized adsorbent materials for removal of PhACs are being tested for the first time. These new systems can retrofit the tertiary treatment in WWTPs, drinking water treatment plants and pharmaceutical industries. Also, they can be applied for in-situ and ex-situ treatment of contaminated soils with ETCs and it has been tested in soils with amended biosolids.

Likewise, as ETCs are present in nanogram or lower concentrations, their detection using the chromatography-mass spectroscopy methods is challenging in complex media. Extraction and cleanup with appropriate method e.g. solid phase extraction, liquid-liquid extraction, etc. is crucial prior to analysis. Also, selection of appropriate derivatization of solid ETCs to use gas chromatography or finding appropriate eluent mixture for using liquid chromatography is necessary for analysis. The complexation of compounds with metals or transforming to their isomers that depend on pH and temperature complicate the detection and treatment methods. In this project, a new technique called laser diode thermal desorption (LDTD) was successfully tested to substitute the chromatography methods that eliminate derivatization and eluent selection (Fig.2). Likewise, hybrid advanced oxidation-enzymatic methods tackled metal complexes which have been proven to cause toxicity and persistence in the environment. Production of laccase at reasonable cost was another challenge as the synthetic media costs can be 40-60% of the overall costs. Hence, cost-effective apple pomace was used for copious laccase production.

We can develop compact hybrid technology modules for the removal of ETCs for the urban municipalities and remote communities. Likewise, this research project provided federal, state and local agencies, industry and others a better understanding to address the issues regarding ETCs in drinking water and wastewater and frame regulations for their release and control. Limited space and significant change of infrastructure which act as main cost restraint for the establishment of drinking water/wastewater treatment plants to treat ETCs can be overcome by these hybrid technologies. Further, they will offer potential growth opportunities to the environmental sector which is a $ 14 billion market with 6% annual growth rate. This will reinforce the next generation wastewater/drinking water treatment infrastructure and management strategy in the coming decade. Finally, the project will help maintain safe and secure wastewater/drinking water treatment systems adapting to growth, climate change and ensuring health safety for the world.

Click images to enlarge in separate window.


Layout of the treatment steps of wastewater treatment plant located in Quebec (Quebec), Canada with different sampling points (1: Influent; 2: Grit influent; 3: Primary sludge;4: Secondary sludge; 5: Effluent; 6: Mixed thickened sludge;7: Dewatered sludge). The samples were taken at these points to analyze different emerging contaminants in the two phases: solid and liquid obtained by centrifugation of the samples to determine their concentration in each of the phases	Effect of pre-treatments on rheology and bisphenol-A degradation: RS: raw sludge; US: ultrasonication; FO: Fenton oxidation; FS: Fenton sonication; TO: thermal oxidation; TAO: Thermal alkaline oxidation; AH: alkaline hydrolysis; TAH: thermal alkaline hydrolysis; TH: thermal hydrolysis. Among the five pre-treatments, lower zeta potential in TO and TAO pre-treatment was due to addition of acid (lower pH) which resulted in neutralization of charge within the medium. Meanwhile, higher zeta potential in AH and TAH was due to addition of alkali (higher pH) that results in net increase in negative charge. As the size of the flocs decreased with increasing zeta potential, overall availability of sorbed BPA molecules to react withOH–ion increased resulting in higher degradation of BPA during TAH pre-treatment.

Effect of pre-treatment of municipal wastewater sludge on growth of Rhizobium meliloti used as a biofertilizer. As we move from RS (raw sludge) to Fenton oxidation (FO) and Ferro-sonication (FS2), there is an increase in the number of microorganisms which is an indicator of enhanced solubilization of organic matter and hence easy assimilation by the microorganisms. Furthermore, due to the increase in microbial growth, there was in-situ production of oxidative enzymes, such as monooxygenases and esterases, among others which increased the degradation of bisphenol-A which was found to be 90-95%. This proved the fact that moderate oxidation processes can aid in solubilization of organic matter, hence value- addition of wastewater sludge to a bioproduct, such as biofertilizer with simultaneous degradation of bisphenol-A.	Scanning electron micrographs of: (a) nano-TiO2 without lactoserum, (b) nanoTiO2 with lactoserum; (c) nano-ZnO without lactoserum, (d) nano-ZnO without lactoserum. The lactoserum stabilized ZnO nanoparticles degraded 100% of bisphenol A from medium after 1 h of reaction time as compared to 45% in the absence of lactoserum. The lactoserum enhanced stability of the nanoparticles and further enhanced rate of photocatalysis by the surface area effect.

Yeast Esterogenicity Assay (YES) of E2 (17-β estradiol) (row A) and ultrasonication (US), Fenton Oxidation (FO) and Fenton Sonication (FS) pre-treated wastewater spiked (row B-D) and unspiked (row E-G) with carbamazepine and blank (row H). The estrogenic activity of US, FO and FS pre-treated sample with CBZ and its by-products was carried out by Yeast Estrogen Screen (YES) assay method. Based upon the YES test results, none of the pre-treated samples showed estrogenic activity. YES is used for in vitro detection of estrogenic activities of natural and synthetic compounds, mixtures and environmental samples. Estrogenic activity can lead to disruption of the endocrine system which regulates the growth, reproduction and metabolism of the human system.	Effect of chlortetracycline (CTC) metal complexation on viability of Gram positive bacterium, Bacillus thuringiensis. A) Control without CTC; B) CTC=0.5 mg/L; C) Cu-CTC metal complex; D) Mg-CTC metal complex. Mg ion which is biologically important in bacteria showed higher toxicity after complexing with CTC when compared to Cu-CTC metal complex and free CTC. Hence, CTC-metal complexes are more toxic than free CTC, if the metal has more importance in biological activities of bacteria.

Effect of chlortetracycline on nitrification in aerobic reactor. High concentrations of CTC (50 and 100 mg CTC/L) are salted out from aerobic reactor during nitrification experiments. Even though CTC is highly soluble in water (8.6 mg/L), higher metal complexing and high adsorption property to sludge solids results in lower solubility which lead to salting out of CTC.	CTC estimation in ultra-pressure liquid chromatography-mass spectrophotometry after complexing with Fe metal. Basic mobile phase is unable to break the CTC-Fe metal complex which led to 10% recovery of CTC. This helped establish a unique method for analysis of antibiotic-metal complexes which is a common phenomenon in wastewater treatment plants where the emerging contaminants and metals co- exist.

Chromatograms; representative peaks of diclofenac in: (a) wastewater with Liquid Chromatography-Electrospray Ionization-Mass Spectrophotometry-Mass Spectrophotometry (LC-ESI-MS/MS); (b) wastewater with Laser Diode Thermal Desorption/Atmospheric Pressure Chemical Ionization Tandem Mass Spectrometry Analysis (LDTD-APCI-MS/MS),; (c) wastewater sludge with LC-ESI-MS/MS; (b) wastewater sludge with LDTD-APCI-MS/MS. Chromatograms proves that matrix interferences were minimal with LDTD-APCI-MS/MS over LC-ESI-MS/MS. The analysis time has been reduced to few seconds by LDTD-APCI-MS/MS over LC-ESI-MS/MS having 12 minutes' analysis time per sample.	Adsorption characteristics of diclofenac with different biochars. Method of preparation as well as the feedstock influences the properties, such as functional groups and surface area of biochar. Biochars originated from pig manure and pinewood was tested for adsorption potential for diclofenac before it subjected to immobilization. Pine wood biochar showed endothermic adsorption and pH had significant effect on adsorption process. Moreover, adsorption was monolayer. However, pinewood biochar exhibited endothermic adsorption with minimal pH influences and the process was multilayer adsorption.

Scaling up process of laccase production using Terrafors-IS solid state bioreactor. Figure (a) represents solid state bioreactor and (b) represents growth of laccase producing fungi Trametes Versicolor over agro-industrial residue apple pomace which was used as the substrate for laccase production.	Scanning electron micrographs (SEM) micrographs of nanofibrous membranes (NFMs), a) smooth and randomly-oriented fibers in NFM- 0%, b & c) entrapment of biochar among fibers in NFM-0.5% and NFM-1%, d & e) NFM-1.5% at different magnifications and F) formation of beads in NFM-2%. At 1.5% of biochar loading, the surface area reached the maximum value of 12.4 m2/g and beyond this loading value, agglomeration of particles inhibited fine interaction with nanofibrous matrix. Upto 95% removal was obtained for chlortetracycline with an initial concentration of 200ppb by NFM-1.5% (fig d & e)