Technologies developed by NASA's Office of Biological and Physical Research to keep air, water, and food safe for astronauts in space can also help protect people on Earth from bioterrorism.

Moreover, NASA now shares a responsibility (along with other federal agencies) to contribute to this nation's homeland security as part of its greater mission to understand and protect the planet. In a troubled world that now includes threats of biohazards and bioterror, NASA's long experience in monitoring air, water, and food also may play a significant role in helping to protect the home planet, whether from natural diseases or the deliberate acts of individuals or groups.

Above: This miniature closed-system greenhouse used for growing green plants in microgravity, designed by Principal Investigator Weijia Zhou and his colleagues at the Wisconsin Center for Space Automation and Robotics, is equipped with a "scrubber" to remove ethylene (a natural gas byproduct emitted by plants) from the chamber's atmosphere. (Seedlings inside the chamber are bathed in the pink glow of grow lights; the two large white circles are openings to gloves that allow astronauts access to the plants.) Credit: Wisconsin Center for Space Automation and Robotics, University of Wisconsin.

In September and October 2001, several U.S. post offices and two U.S. Senate buildings were shut down when anonymous envelopes containing mysterious powders were found to contain spores of the bacterium Bacillus anthracis, which cause the disease anthrax. Since then, public officials and others have openly discussed the potential threat of future acts of bioterrorism: the unleashing of "weaponized" biological agents such as Bacillus anthracis, the smallpox virus, or Clostridium botulinum (which produces the botulinum toxin that causes botulism poisoning).

In August 2002, NASA Administrator Sean O'Keefe appointed a senior adviser for homeland security, Amy Donahue, as a liaison to the Office — now the Department — of Homeland Security (see sidebar on page 24, NASA's Liaisons for Homeland Security). Donahue capitalized upon efforts at NASA Headquarters and NASA centers to identify technologies valuable not only to NASA's space mission but also possibly to homeland security. By early 2003, NASA's list of potential dual-use technologies had topped 275 research projects and was still growing.

Many of NASA's dual-use projects fall under the purview of the Office of Biological and Physical Research (OBPR), which has long funded research into ensuring the safety of air, water, and food for astronauts, especially for long-duration missions.

Two Purposes, One Challenge

The technical challenges of devising early-warning systems for compromised air, water, and food in space and on Earth are surprisingly similar, according to Jitendra Joshi, deputy program manager for advanced human support technology at NASA Headquarters. And those challenges are tough ones.

For long-duration missions during which astronauts would need to recycle air and water and perhaps grow their own food, a principal concern is a slow buildup of trace toxic elements or microorganisms. Monitoring a slow buildup requires extraordinarily sensitive instruments operating continually, explains Darrell Jan, manager of NASA's Advanced Environmental Monitoring and Control Program Element. "On the other hand, any leak of hazardous gases can spread very rapidly, so we need quick detection. And the instruments must give reliable results."

As with any other payload on spacecraft, Joshi adds, "instruments also need to be compact (low-mass and low-volume), autonomous (low-maintenance), and low-power. They must also consume few expendable supplies and generate little waste. Lastly, they must require little expertise to operate, so astronauts can devote most of their time to tasks other than tending life-support systems."

Back on Earth, what instruments are needed for regular testing of air coming into buildings, drinking water flowing into treatment plants from reservoirs, and food prepared in a processing plant or restaurant? They, too, need to operate continually and be sensitive, fast, and autonomous. Moreover, police, fire, and health-care workers responding to an emergency would also need instruments that are compact, low-power, reliable, durable, and straightforward to operate, which also use few consumables and produce little waste.

Protecting Air

Modern energy-conserving office buildings are essentially closed systems. Windows are hermetically sealed and more than 80 percent of the air inside the buildings is recycled; thus, some large buildings draw in scarcely a fifth of their volume of fresh air from the outside daily. Although recycling air minimizes heating and cooling costs, it also increases the potential that contaminants (such as outgassing from insulation or car exhaust from an underground garage) might accumulate in the indoor air.

But what if there is a conscious, calculated plan to inflict suffering and fear by intentionally introducing an airborne biological agent such as anthrax spores or a chemical toxin such as nerve gas? An energy-efficient building's air recirculation system will tend to transport the agent — which may well be colorless and odorless — throughout the entire building, actually impeding its removal and posing a serious threat to everyone within. What technology could destroy such airborne agents?

One possibility is a compact machine based on an ethylene scrubber originally devised by Weijia Zhou, director of the Wisconsin Center for Space Automation and Robotics (WCSAR) at the University of Wisconsin, Madison. Zhou and his colleagues developed a machine to eliminate contaminant hydrocarbons — primarily ethylene — normally produced by growing plants, which can pose problems in an enclosed environment. Growing green plants in microgravity as possible vegetable crops for astronauts on long-duration missions is significant to NASA.

Although the Russians in Space Station Mir allowed their experimental green plants to share the cabin air with cosmonauts, Zhou explains, since the 1980s NASA has emphasized that plant research and experiments conducted aboard the space shuttle or the International Space Station (ISS) should be enclosed in chambers. Air must not be exchanged between plant chambers and the orbiter cabin without sufficient filtration. As a result, each growth chamber has its own subsystems for controlling nutrient delivery, lighting, humidity, temperature, and atmospheric composition, with stringent limits on consumable supplies and the disposal of waste heat, moisture, and gases — a tall order for months-long missions on the ISS.

Now, plant physiologists have long known that growing plants produce ethylene, an odorless gas that, to plants, acts as a hormone, causing them to mature early and go to seed (ethylene gas is what ripens bananas in a few hours if they are kept enclosed in a paper bag). Indeed, plants are so sensitive to ethylene that concentrations as low as 50 parts per billion (ppb) may affect plant reproduction (seed development), Zhou says, and concentrations of 200 ppb may interfere with the pollen process and even abort the formation of seeds. On Earth, the ethylene diffuses into the atmosphere and is dispersed. But in an enclosed growth chamber, its concentration could build up to lethal levels for the plants within days.

To safeguard the plants on space shuttle flights of only a week or two, filter paper impregnated with chemicals that absorb ethylene does the trick. "But chemical absorbents are consumables that must be replenished. So [for ISS missions] our folks had to develop non-consumable degradation technology," recalls Zhou. The heart of their ethylene scrubber is the white pigment titanium dioxide (TiO2), which is not consumed because it is a photocatalyst. "Under ultraviolet irradiation, the scrubber fully oxidizes ethylene and other hydrocarbons into carbon dioxide (CO2) and water vapor (H2O). Thus, not only did we get rid of the ethylene, we also broke it down into byproducts not harmful to plant growth — which is why this technique is pretty good!" Zhou exclaims. Over the past decade, WCSAR plant growth chambers equipped with photocatalytic ethylene scrubbers have flown on nine space shuttle flights, three ISS missions, and even once on Mir.

Right: The ethylene-scrubber technology led to the development of an air filtration device, the AiroCide TiO2, which can kill dangerous microbes, including spores of the bacterium that causes anthrax. A fan (black circle at the far end) draws in the room air and forces it through a maze of tubes, where chemically reactive hydroxyl radicals and high-energy ultraviolet light attack and kill pathogens. Credit: KES Science and Technology Inc..

In 1998, Zhou's ethylene scrubber caught the attention of John Hayman Jr., chairman of KES Science and Technology Inc. in Kennesaw, Georgia. Two decades ago, Hayman invented the produce-misting systems widely used in grocery stores. Now Hayman has licensed WCSAR's technology to develop a commercial ethylene scrubber for florists and grocers seeking to lengthen the shelf life of flowers, fruits, and vegetables kept in cold storage (any refrigerator is also an enclosed system). He added some fans to circulate air through the unit and some germicidal lights inside the unit to kill airborne bacteria and molds, called his development the Bio-KES, and began marketing it to the perishable foods and floral industries.

But shortly after the first anthrax attacks in late 2001, Hayman recalls, "We were having a managers' meeting, and one manager asked, 'Gee, boss, do you think the Bio-KES could kill anthrax spores?'" When a first test of an unmodified Bio-KES killed 83 percent of Bacillus thuringiensis (a harmless bacillus bacterium with spores that are similar to Bacillus anthracis and widely accepted in the scientific world as a sufficient substitute), ramping up further development "was a no-brainer," Hayman says. After replacing the Bio-KES's germicidal bulbs with 52 ultraviolet lamps so that spores and other airborne pathogens would be bombarded by high-energy UV photons as air is drawn through the unit, the kill rate was raised to 99.99998 percent of spores. "Killing spores, with their basketball-like hard shells, is 50 times harder than killing any vegetative [live] bacteria, such as those that cause tuberculosis or staph infections, or even viruses — so the process also should be effective against smallpox," Hayman points out.

By March 2002, NASA and Hayman jointly announced the modified Bio-KES under the name AiroCide TiO2 as a solution for antiterrorism and emergency preparedness. In January 2003 it gained clearance from the Food and Drug Administration as a class II medical device (a device allowed to be used in hospitals), and it is now undergoing pilot tests. Hayman envisions that the AiroCide should find uses in hospitals, schools, and daycare centers as well as in police headquarters, mobile military vehicles, and emergency response team headquarters.

Monitoring Water

For the purposes of bioterrorism, a major city's water supply could be a prime target. Contaminating a few central reservoirs could have a dramatic effect in terrifying the local population, even though most microbes and toxins would be neutralized at the water treatment plant before being pumped to homes and offices. But contaminating the supply after it leaves the water treatment plant for individual neighborhoods could have grave consequences.

The technology currently used aboard the ISS for detecting the presence of pathogens in the astronauts' recycled drinking water is "frankly, pretty archaic," remarks James Lambert, supervisor of the Intelligent Instruments group at the Jet Propulsion Laboratory (JPL) in Pasadena, California. NASA specifies that on the space shuttle, astronauts' drinking water must contain no more than 100 colony-forming units (CFUs) — that is, no more than 100 live bacteria of any kind capable of forming a colony — per 100 milliliters (about a third of a cup). Although this is actually less rigorous than the Environmental Protection Agency's standard for drinking water on Earth, NASA finds it acceptable, as long as the water is tested regularly — maybe once a day. Astronauts test their drinking water by drawing 100-milliliter samples through a filter impregnated with a growth medium to trap any bacteria, and culturing the bacteria (allowing them to multiply so they are detectable) for five days. The colonies are grown with an indicator that causes them to show up on the filter as purple spots, whose number is estimated by comparison to a chart.

With a five-day culture necessary to detect the bacteria, however, "it may be a horserace between whether the astronauts turn green or the filter turns purple first," Lambert observes wryly. "What you really want is a test that detects what is there in five minutes."

Lambert's team has recently developed a prototype of a one-step test strip that he has designed to be as simple to use as an over-the-counter pregnancy test kit. "With a pregnancy test, you dip a chemically-treated stick into a urine sample and wait for five minutes to see if it turns blue," Lambert explains. "It is a simple yes-no test: if it turns color, you are pregnant; if it does not, you are not."

Technically known as a quantitative lateral flow assay (QLFA), Lambert's test strip consists of an absorbent membrane on a stiff backing. When a water sample is applied to one end of the strip, the water diffuses along the length of the strip, passing several stripes (narrow regions) impregnated with high concentrations of specific antibodies. Each of the stripes is chemically treated with different compounds so that they will turn color in the presence of specific antigens. When bacteria with the target antigen flow into the stripe, they are bound by the antibodies, causing the stripe to change color, revealing their presence. Depending on the QLFA's design and the specific antibodies used, in minutes the test strip yields not only a rough count of the total CFUs in the water sample, but also a preliminary classification as to the types of organisms present — for example, viruses versus different major classes of bacteria (there are no universal antibodies that can detect every type of organism). "Similar strip tests can be developed for testing water in public water supplies for specific strains of bacteria or toxins," Lambert declares.

Above: The Quantitative Lateral Flow Assay (QLFA) is a "test strip" for identifying biological organisms in an analyte (liquid sample), which flows from the sample pad to the wicking pad. On the conjugate pad, specific antibodies (Y shapes) tagged with chemical markers (yellow ovals) bind to the target antigen (red sunbursts) in the sample and flow toward the wicking pad. At the test line, other immobilized antibodies bind the antigens to produce a positive test result, revealed as fluorescing color. A control line antibody confirms the test ran successfully; that is, the sample flowed the length of the test strip. Credit: NASA.

Above: As easy to read as a home pregnancy test, three QLFA strips used to test water for E. coli show different results. The brightly glowing control line on the far right of each strip indicates that all three tests ran successfully. But the glowing test line on the left in the middle and bottom strips reveal their samples were contaminated with E. coli bacteria at two different concentrations (the color intensity of the lines correlates with concentration). Credit: NASA.

Meanwhile, keeping the bacteria count low in drinking water is a concern for Marc Porter, Jim Fritz, and their research team (Bob Lipert, Dan Gazda, and Lisa Ponton) at Iowa State Univeristy's Microanalytical Instrumentation Center (MIC), in Ames, Iowa. In the space shuttle or the ISS, that task is surprisingly tricky. "It is not possible to sterilize water enough to store it for extended periods without having bacteria grow," Porter explains. "You really need to add a biocide" — that is, a chemical to kill bacteria. Although chlorine is commonly used on Earth, NASA uses molecular iodine while the Russians use silver ions.

The concentration of the biocide itself needs to be closely monitored, however, not only to keep it above a minimum effective level for killing bacteria, but also to keep it below a level harmful to the astronauts. For example, Porter says, too much iodine has been linked to thyroid problems; too much silver, on the other hand, "irreversibly turns your skin blue," an unusual condition called argyria.

To monitor biocide levels, the MIC team has developed and flight-tested a chemical sensor system called a colorimetric solid phase extractor (CSPE). "You flow 10 milliliters of water through a 1-centimeter-diameter disk of organic polymeric material, which extracts the biocide and collects it," Porter says. "The disk is preloaded with a dye that selectively binds with silver or iodine and turns color. When the disk is put into a diffuse reflectance spectrometer, it measures the color's intensity, which can be correlated with the biocide's concentration."

Porter and his co-workers are now working on a CSPE using several disks, arranged in series or parallel, preloaded with dyes sensitive to other undesirable water contaminants that tend to build up in the frequently recycled drinking water aboard spacecraft. Example contaminants include heavy metals (lead, iron) or organic materials such as propylene glycol (antifreeze) or polyvinyl chloride (a carcinogenic byproduct of plastics). With such a multitasking CSPE, one could test water for "multiple compounds with both high sensitivity and high selectivity, down to the parts-per-billion level," Porter says, with results available in about 60 seconds. In a battlefield, customized CSPEs could aid troops screening local water for the decomposition products of toxins used in chemical or biological warfare. It could also help personnel decontaminate buildings after an attack by allowing them to test the wash water residue frequently and thereby detect when the area is indeed clean again.

Right: The heart of a colorimetric solid-phase extractor (CSPE) test kit quickly measures the concentration of the biocides silver or iodine in astronauts' drinking water to determine whether concentrations are safe. When 10 ml of water is drawn through the disk, the disk will turn color (yellow in the photo) indicating the presence of the biocide. The device could some day be used to test water safety at reservoirs and water treatment plants on Earth. Credit: Microanalytical Instrumentation Center, Iowa State Universit.

Ultimately, Porter and his MIC colleagues want to make a CSPE able to detect pathogenic microbes as well as biocides — something useful in homeland defense for monitoring the potability of groundwater or the safety of food (in which case "you rinse the food and analyze the rinse," Porter explains). The MIC team also envisions CSPEs' doing routine screening at reservoirs and pipes exiting water treatment plants.

Food

From a bioterrorist's viewpoint, the food supply would be the most difficult to attack because of the previous administration's implementation of the strict Hazard Analysis and Critical Control Point (HACCP) system, which specifies that every handler in the food-processing chain, from farm to table, has legal liability for any compromise to food safety. The HAACP system means a farmer is legally liable, say, for growing crops at a contaminated site, as is a middleman for transporting raw product without proper refrigeration to a processor, all the way to a retail grocer selling goods past their shelf-life date. In the United States, the HACCP system also pertains to any foods imported from other nations.

Because of HACCP, cleanliness procedures in the food-processing industry are strict and penalties for noncompliance severe. But if it takes a day or two to culture and identify harmful microbes, how could one guard against possible bioterrorism as an "inside job" by a food-processing plant employee? Ideally, one should be able to test the cleanliness of conveyor belts and other facilities instantaneously several times a day.

Technology developed by Kasthuri Venkateswaran (who prefers to be called Venkat), a microbiologist in JPL's Biotechnology and Planetary Protection Group, could help food-processing plants ensure they are absolutely clean with no contamination. Venkat's device, an adaptation of an assay invented by Kikkoman Corporation in Japan (maker of soy sauce and other Japanese foods), relies on the fact that all earthly living organisms, single-cell and higher, rely on the molecule adenosine triphosphate (ATP) as a source of stored energy.

Traditionally, people detect microbes by swabbing an area and placing the sample in a dish of nutrient to see what grows. The problem is that "different 'bugs' grow in different nutrients, so it is virtually impossible to grow all 'bugs' at once" in one dish because "there is no universal nutrient," Venkat says. Worse, the growth may take several days — too slow for daily testing of food-processing facilities. Kikkoman's original ATP test, however, can detect ATP from all dead or living microbes in half a minute; because of its speed, it was originally applied by NASA to ascertain the cleanliness of the floors, walls, and air of JPL's clean rooms, where spacecraft intended to explore Mars are assembled. Venkat's modification of the assay, however, can differentiate what proportion of those microbes are alive. (Because larger organisms contain more ATP, guessing the number of microbes from the amount of ATP in either assay is problematic.)

Both versions of the assay are roughly the size of a kitchen wall telephone. In Kikkoman's original assay, a user takes a sterile cotton swab, rubs it over 25 square centimeters (4 square inches) of the target area, and inserts the swab into 2 milliliters of sterile water. Any microbial ATP on the swab is released and immediately combines with luciferase — the same enzyme that lights up fireflies — and begins to glow. The number of photons detected is proportional to the amount of ATP in the sample, regardless of whether the ATP is from dead or living microbes. In Venkat's modification, another enzyme is first introduced to degrade the ATP from dead microbes into other chemicals (a process taking about half an hour), so that the only ATP eventually detected is that from living organisms.

Venkat is now adapting his ATP assay for detecting microbes in the drinking water used aboard the ISS to reconstitute the astronauts' food (done essentially by replacing the sterile water in the ATP assay with a sample of the drinking water). If an automated water- or air-collection apparatus is installed at the front end, he also envisions that his ATP assay could be stationed at the water intakes of reservoirs or the air intakes of buildings, and could alert early responders to unusual spikes in the total amount of ATP (and thus the total "bio burden" of microbes detected), possibly signaling a bioterrorism attack. The ATP assay could also be used by grocery stores and consumers to assure themselves of the safety of heat-and-eat foods that do not require cooking.

Because of HACCP's stringent procedures and penalties, Venkat and other sources expressed less concern about the possibility of bioterrorism in food-processing plants than about what George May, director of ProVision Technologies (a NASA research partnership center at Stennis Space Center in Mississippi), calls "agriterrorism." Agriterrorism is "anything people could do to disrupt the food or feed supply at its source" with the goal of throwing the industry into disarray rather than trying to kill consumers eating a final product, May explains.

Of some concern is the possibility of intentional contamination of crops with molds and fungi. Although such organisms occur naturally in food and feed, they can spoil a field or shipment. The Department of Agriculture specifies tolerances for certain molds and fungi in crops such as corn, soybeans, and peanuts. Currently, to ensure transported crops are within the tolerances, samples of grain from different parts of a shipment are taken to a laboratory for chemical analysis, a process requiring up to 24 hours. The analysis and related delay in shipment cost money, and if crops were tampered with could contribute to a serious disruption in supply.

Right: Some molds on corn, soybean, or other food or feed products produce toxins poisonous to humans, so any crops or shipments containing more than a few infected kernels must be destroyed. A hyperspectral sensor system based on NASA remote-sensing technology, however, allows harmful molds to be differentiated quickly from harmless ones using differences in their spectral signatures (patterns of color). Here the physical appearances of three molds in petri dishes (top) are shown with their corresponding hyperspectral images (bottom). Credit: ProVision Technologies.

Enter hyperspectral imaging (HSI), a technique JPL invented two decades ago for remote sensing satellites. HSI captures hundreds of images of a target at very close wavelengths (colors), picking up subtle changes and variations in the sample imperceptible to other techniques. May and his colleagues at ProVision Technologies have been developing compact HSI scanners that operate all the way from the ultraviolet through visible wavelengths and into the shortwave infrared for use on Earth. Specifically, they are pilot-testing a commercial device for the poultry industry that quickly recognizes the subtle difference in the spectral signature (wavelength composition) of light reflected off a raw chicken carcass that is clean versus one contaminated with fecal matter. The key word is "quickly," May says, because an assembly line in a commercial poultry factory passes by the HSI scanner "at the rate of 70 birds a minute, giving less than a second for each test."

Responding to fears about agriterrorism, May is now adapting HSI to identify various molds that attack grains and other crops. May's goal is to develop a dipstick-like probe that could be pushed down into different places in a shipment of grain and give instantaneous readings as to the presence or absence of harmful molds.

Easy Use = Tall Challenges

For equipment both to advance the frontiers of science in space and to be useful in checking bioterrorism on Earth, the laundry list of technical requirements (compact, low-power, etc.) is monumental. "The detector and sensor are the easiest parts," declares John Hines, technology development manager of the Fundamental Biology Research Program at NASA Ames Research Center in Moffett Field, California. "The hardest parts are preparing the sample without human intervention, and miniaturizing and automating the complex analytical process."

For example, once a molecule has been tagged for fluorescence (as in Venkat's ATP assay using the firefly enzyme), Hines explains, "all I need is a detector to see the light — a mature technology. But how do I get a sample to emit light specific only to the one analyte I am looking for? How do I filter, heat, add reagents [substances used to detect or measure an analyte], separate, and tag the sample according to well-accepted protocols before it can even be presented to the sensor? How do I design the reservoirs of reagents, valves, and pumps for each stage in the process? And how can all those intricate functions — usually done by trained personnel in a major laboratory — be done automatically in the volume of a shoebox?"

In the context of bioterrorism, data acquisition and dissemination are also essential. Autonomous sensors that continuously monitor air and water from office building air intakes or reservoir water intakes must be equipped with wireless devices for communicating their readings in real time to a local data-collection point. Moreover, data acquisition and dissemination are vital to first responders in an actual terrorist attack because "their protective gear has to be completely self-contained," Hines points out. Sensors embedded in protective suits could ascertain whether or not they have been exposed — even helping responders decide whether they have to wear bulky protective gear at all. If they have been exposed, sensors with real-time communications to databases could even determine "to what they have been exposed, and to what degree," Hines continues.

The technical challenges of preventing, detecting, and combating bioterrorism are great. But the technical challenges of keeping astronauts healthy in space for months at a time are also great — and the similarity of the challenges means NASA has much to contribute to protecting our home planet.

"Although NASA is not in the business of fighting terrorism on Earth, NASA is in the business of keeping astronauts safe," Joshi states. "But whatever works for astronauts can improve life on Earth. And if we are successful, that can translate very well to meeting today's challenges."