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­­­Adenovirus, Cryptosporidium, Coxsackie Virus, You-Name-It Pathogen, Here We Come

3/10/2009 7:52:00 AM

In Newmark’s new Level II Disinfection Research Laboratory, researchers will take on the world’s biggest threats to safe drinking water         

By Joyce Mast

Water is life; without it we die.  If water is contaminated, we may get sick—or die. Population and industrial growth, global warming, and climate change stress the world’s water supply and increasingly necessitate quicker re-use. Re-use requires the careful control of human pathogens such as Adenovirus, Cryptosporidium, Coxsackie virus, and others that may emerge in the future. The new Level II Disinfection Research Laboratory in CEE will allow researchers to safely study pathogenic viruses and their human host cells to establish data describing how viruses are inactivated by disinfectants, thus providing a technological key to world-wide drinking water sanitation.

Professor Benito Mariñas, a champion of safe drinking water, conceived of reclaiming the area formerly occupied by the cooling towers on the roof of Newmark Civil Engineering Laboratory to create a biological-safety Level II laboratory. Level II is a federal designation specifying the type of pathogens allowed (in this case, primarily opportunistic pathogens—or those that can make you sick but are not dangerous unless your immune system is already compromised), as well as space (must be non-circulating—only one door), equipment, and safety training regulations. The new disinfection lab with “cold room” and microscopy suite occupies 1,700 square feet including a mechanical equipment room, and there is approximately 2,800 square feet of unfinished space for future labs and/or student offices. Funded by the Civil and Environmental Engineering (CEE) department, the College of Engineering, the National Science Foundation through the WaterCAMPWS (Center of Advanced Materials for the Purification of Water with Systems), and the Provost’s office, construction began in May. Completion is expected by the end of 2008.

Currently, only one student can work at any given time in the very cramped space of the old two-person Level II lab. In the new lab, with its increased safety features, this stringent certification process will allow up to eight properly trained students to work simultaneously in a more open space. Pairs of students studying the same virus will share one of four bio-safety hoods/workstations equipped with laminar air flow filtration system and  ultraviolet (UV) lamps to prevent contamination of the students and their equipment.  After use, the hoods are closed and UV-irradiated to sterilize the work area. Human cells and highly concentrated virus stocks can be manipulated safely in the bio-safety cabinets. Each workstation will be equipped with its own incubators and pH meters to minimize cross-contamination. The lab also contains a water tank to allow high-flow experiments with bench-scale UV reactors or ozone contactors. A solar simulator system that can mimic sunlight in different regions or at different times of day will also be used in the lab.
How does the new lab fit into the grand scheme of water disinfection? Water scarcity dictates re-use. Direct re-use—toilet-to-tap—is technically possible and potentially safe but not yet socially acceptable. Currently in the US, water is indirectly re-used, for example in Minneapolis and St. Louis, where treated wastewater is discharged into a river and then re-used downstream as part of the water source for a drinking water treatment plant.  In California, where there is more demand than water, new treatment strategies are needed to shorten the gap between wastewater and drinking water.  The most urgent concern is pathogens from human, agricultural and even wild animal wastes. These pathogens must be removed and/or inactivated by the re-use treatment processes.

Throughout the 20th century, the standard water disinfectant has been free chlorine (household bleach). However, free chlorine degrades in sunlight and is powerless against the common waterborne pathogen Cryptosporidium, a protozoan parasite that causes gastrointestinal illness and has the potential to produce large waterborne disease outbreaks such as the one that affected more than 400,000 people in Milwaukee, Wis., in 1993.  Free chlorine also reacts with organic matter to form potentially carcinogenic disinfection byproducts. In 2006 the EPA called for municipalities to control some of these byproducts, resulting in the increased use of alternative disinfection technologies.

 One option, UV light, is effective against Cryptosporidium but doesn’t provide residual protection. Monochloramine, or combined chlorine—chlorine plus ammonia with more ammonia than chlorine—has been around since the 1930s but is becoming more popular because it is more stable than free chlorine and forms fewer regulated byproducts.  However, monochloramine is a less efficient disinfectant, requiring too much contact time to control viruses at most treatment plants.  Applied sequentially, UV light and monochloramine can control Cryptosporidium and regulated disinfection byproducts effectively but will not control waterborne viruses at typical doses.  Mariñas and his students continue to study this sequential treatment in order to develop data demonstrating its ineffectiveness against viruses and discover what new byproducts it might form.

 Researchers in the new lab, besides Mariñas’ group, will include collaborators through the WaterCAMPWS in areas of microbiology, chemistry, materials science, and genetics; and those working on water quality in developing countries, for instance, professors Benito Corona Vasquez and Erick Bandala from the Universidad de las Américas-Puebla in Mexico who collaborated with Mariñas and his CEE 449 Integrated Design Project/Laboratory Experience class on a project in Los Llanos over spring break 2008 and are interested in building a biosafety Level II lab in Puebla.

The first to use the new lab will be three of Mariñas’ group of 14 grads and 5 undergrads: Amanda Poole (BS 08), Theresa Vonderhaar and their laboratory mentor, Martin Page (BS 03, MS 04). They represent a new generation of top-level students who, in addition to working on pressing issues relating to disinfection worldwide, dedicate a portion of their work to improving access to safe water in developing countries.  Poole is a first-year grad student; Vonder Haar, second-year; and Page is planning to complete his Ph.D. in March.

Page, in collaboration with Professor Joanna Shisler in Microbiology, is developing molecular approaches to discover which part of Adenovirus type 2 (a UV-resistant virus that causes respiratory illness) is being damaged by disinfectants such as UV light and chlorine. This information will help to figure out how to best transform the Adenovirus genes or  proteins to an extent that it cannot infect human cells, cells which may even help the virus to fix itself.  He is building on the research of a recent doctoral recipient from the Mariñas group, Kwanrawee (Joy) Sirikanchana (MS 03, PhD 07).

Poole is researching how Adenovirus is inactivated with SoChlor (solar/chlorine) using the lab’s solar simulator to mimic conditions in tropical regions. When chlorine is added to water containing ammonia, a common co-contaminant in developing regions, monochloramine is formed, which favors virus survival. If the bottle is exposed to sunlight on a roof, the SoChlor disinfection time, even under cloudy conditions, ranges from one to five hours, whereas disinfection from the sun or chlorine alone requires up to two days.
Last January, Vonderhaar began work on the Coxsackie virus B5, which causes respiratory diseases, miscarriages, heart problems, aseptic meningitis, diabetes and heart problems. Coxsackie is not quite as resistant as Adenovirus to UV light but is more resistant to free chlorine.

The goal of Vonderhaar and all virus researchers is to find an optimal, multifaceted system for disinfecting all types of viruses.  The department’s new Level II lab is a fitting venue for pursuing, and possibly attaining, this pressing objective.    

Photo: Environmental Engineering and Science students (left to right) Amanda Poole, Theresa Vonderhaar and Martin Page pose in front of one of four biosafety hoods that will be used in the new lab.    Photo by Joyce Mast.