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Large volumes of pharmaceuticals and their metabolites, mostly from human waste flushed down the toilet, are exiting municipal wastewater treatment plants intact and, by some estimates, contaminating almost 25% of the world’s rivers and lakes. About 10,000 drugs are on the market in Western countries. Many have been detected in the environment in concentrations ranging from nanograms to micrograms per liter. All are biologically active by design; many are bioaccumulative and persistent.
When the oral contraceptive 17α-ethinylestradiol is discharged from waste treatment plants into rivers, lakes, or bays, it can cause endocrine disruption in aquatic animals. It is a prime suspect in feminization of male fish even at concentrations in the low parts per trillion, according to scientists from Carnegie Mellon University and Brunel University London.
Antibiotics in wastewater, meanwhile, could be contributing to antibiotic resistance, some researchers say. The antibiotic tetracycline has been found in wastewater in the U.S. and Canada in concentrations between 0.02 and 1.0 µg/L, the highest level anywhere in the world.
Techniques typically used to clean up wastewater—reverse osmosis and nanofiltration—aren’t able to catch parts-per-million levels of pharmaceuticals. And not all compounds succumb to biological degradation. The risk these compounds pose to human health and the environment remains largely undocumented. But regulators in Europe want to remove them to head off any potential long-term adverse effects.
So in 2012, the European Union helped fund a $3.5 million program by six European technology firms and academic institutions to develop a method for degrading antibiotics and other pharmaceuticals in wastewater using oxidative enzymes. Those involved in the project say it is the first time anyone has attempted to develop such a system that could be used at large scale.
Although the project partners established proof of concept for the enzyme technology, not everything went according to plan. The main goal of the project, named Enzymatic Decontamination Technology (ENDETech), was to develop a commercially viable process. But after the program ended in January of this year, the scientists concluded that doing so will require more selective and powerful enzymes.
Biotech firms participating in the project were Paris-based Da Volterra, the German firm c-LEcta, and ChiralVision of the Netherlands. The project’s research groups were the European Membrane Institute (EMI) of Montpellier, France; the Catalan Institute for Water Research, in Spain; and Goethe University Frankfurt, in Germany.
c-LEcta, a custom enzymes developer, had not expected the project to be so tough. “We thought we would find a lot of suitable enzymes in our libraries that would inactivate the antibiotics,” says Rico Czaja, head of biodiversity for c-LEcta.
The partners targeted four commonly used but recalcitrant antibiotics—tetracycline, erythromycin, sulfamethoxazole, and ciprofloxacin—that are not readily degraded in conventional wastewater treatment plants. c-LEcta searched several hundred thousand oxidative enzyme clones from environmental libraries including soils, sheep stomachs, and tree canopies, looking for effectiveness at inactivating multiple antibiotics.
The firm used high-throughput, highly automated cluster screening to identify 70 enzyme candidates. But none of them proved effective even against individual antibiotics.
c-LEcta scientists subsequently attempted to engineer copper-containing laccase enzymes, which had previously been shown to break down antibiotics. But they were unable to identify any effective at pH 7, the typical pH of wastewater. “We hit walls at every turn,” Czaja says. In the end, the team came away empty-handed.
The ENDETech researchers concluded that a broad set of enzymes will be needed to inactivate the plethora of antibiotics and other drugs present in wastewater, according to Martin Wagner, an environmental toxicologist at Goethe University’s department of aquatic ecotoxicology.
The researchers did make progress in developing the supports needed to ensure that enzymes aren’t washed away by wastewater. To make a model system, they used an off-the-shelf but costly industrial laccase enzyme that degrades tetracycline—but none of the other target pharmaceuticals.
Researchers at EMI immobilized this laccase on the surface of a tubular ceramic membrane by applying a thin layer of gelatin and glutaraldehyde solution.
The membrane, which had a dual role as a filtration medium and biocatalyst support, was tested on a wastewater solution containing 20 ppm of tetracycline. Less than 7% of the antibiotic was degraded, indicating the need to position membrane reactors in series, states José Sanchez-Marcano, deputy director of EMI, and colleagues in a paper published in the journal Water Research (2015, DOI: 10.1016/j.watres.2015.01.012).
To apply the system commercially, a much more reactive enzyme will be required so that the size of the relatively expensive membrane system can be reduced, Sanchez-Marcano tells C&EN. “We haven’t succeeded yet,” he says.
ChiralVision, another ENDETech partner, covalently attached the laccase on acrylic beads using epoxides and an additional cross-linking substance based on glutaraldehyde. The system achieved a similar level of performance to EMI’s ceramic membranes.
“We did recycle experiments, and calculations show that the enzyme is stable enough to last at least three to six months, which brings the operating cost down to just a few cents per cubic meter of wastewater,” says ChiralVision’s managing director, Rob Schoevaart. Projected costs for the ceramic membrane approach are similar, he adds.
The ENDETech team says the most cost-effective use of the enzyme technology would be to treat sewage emerging from hospitals, retirement homes, and industrial facilities where pharmaceuticals are at their highest concentrations. Rather than treating raw wastewater, water treatment facilities should introduce the technology after standard treatment, the researchers recommend. The approach could also be used to treat concentrates of drugs and drug metabolites collected using nanofiltration systems, Sanchez-Marcano says.
A chemical solution may also be in the cards. A study published in Scientific Reports by researchers at Carnegie Mellon and Brunel shows that a group of iron(III) tetraamido macrocyclic ligand complexes, when combined with hydrogen peroxide, is effective at breaking down pharmaceuticals in wastewater (2015, DOI: 10.1038/srep10511). The researchers showed that these small-molecule homogeneous oxidation catalysts were able to degrade 17α-ethinylestradiol, its intermediate compounds, and other drugs and pesticides.
“Our results provide a starting point for a future process in which tens of thousands of tons of wastewater could be treated per kilogram of catalyst,” the researchers wrote.
ENDETech no longer has any funding, and none of the participating companies are actively hunting the oxidative enzymes required. But those who participated in the project remain confident that their approach can—with some development—be commercially viable. “Initially I thought it was crazy,” Wagner says. “But then after participating firsthand, I am quite convinced by the approach.”
Sanchez-Marcano is also convinced the ENDETech approach is worth pursuing, even if a cocktail of enzymes is required. Researchers at Mexico’s Monterrey Institute of Technology, with whom Sanchez-Marcano’s lab is collaborating, may even have discovered the enzymes the ENDETech partners were seeking. “They have very, very active enzymes at lab scale,” an optimistic Sanchez-Marcano says.
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