Feasibility of Ultraviolet Technology to Disinfect ...

49th International Conference on Environmental Systems ICES-2019-118 7-11 July 2019, Boston, Massachusetts

Copyright © 2018 KBRwyle Inc.

Feasibility of Ultraviolet Technology to Disinfect Spacecraft

Water Systems

Audry Almengor 1, Susan N. Gilbert2, Kristina Todd 3

KBRwyle, Houston, TX, 77058, U.S.A

and

Niklas Adam4, Michael Callahan5, C. Mark Ott6

NASA Johnson Space Center, Houston, TX, 77058, U.S.A

and

Anthony Hanford7

HX5, LLC, Houston, TX, 77058, U.S.A

As the National Aeronautics and Space Administration (NASA) expands its scope and

begins to venture into long-duration, manned space flights, the function and maintenance of

spacecraft water systems becomes increasingly critical and difficult to achieve. New mission

requirements will limit opportunities for resupply and demand extended periods of uncrewed

operations. Based on lessons learned from the International Space Station (ISS), one

particular challenge of future spacecraft water systems will be maintaining adequate

microbial control, especially in water subsystems and component-level elements where

effective long-duration biocontrol strategies do not currently exist. To ensure the reliability

and redundancy in these systems, new technologies will be needed in order to ensure mission

success. This paper summarizes a feasibility study conducted to look into commercial off-the-

shelf (COTS) ultraviolet (UV) reactor systems intended to aid in slowing the progress of

microbial and biofilm growth via the implementation of a single pass, point of use and/or

recirculation UV device. Using this technology may reduce the need for consumable resupply,

such as filters or biocides, as well as minimize crew time needed to make the repairs on

exhausted and/or compromised systems. The ultimate rationale behind developing a UV

disinfection system is to increase the stability of water systems as requirements for sterility

and microbial control become more stringent for deep space missions. The resulting data from

this study will be used to narrow down possible technology demonstrations for selected ISS

locations in order to assess the use of UV technology on future exploration-class spacecraft

systems.

Nomenclature

β = Constant = c•I•V [mW•cm•s/min] or [mJ•cm/min]

c = Conversion Constant = 60 s/min[s/min]

°C = Degrees Celsius

1 Microbiologist, JES Tech – Biomedical and Environment Research Department, KBRwyle (2101 NASA Pkwy) 2 Project Engineer, KBRwyle – Human Systems Engineering Department, KBRwyle (2400 NASA Pkwy) 3 Project Engineer, KBRwyle – Human Systems Engineering Department, KBRwyle (2400 NASA Pkwy) 4 UV Disinfection Lead – NASA JSC, Crew and Thermal Systems Division, Mail Stop: EC3 (2101 NASA Pkwy) 5 Water Technology Lead – NASA JSC, Crew and Thermal Systems Division, Mail Stop: EC3 (2101 NASA Pkwy) 6 Microbiology Laboratory Lead – NASA JSC, Biomedical Research and Environmental Sciences Directorate, Mail

Stop: SK4 (2101 NASA Pkwy) 7 Analyst – JETS Contract, Crew and Thermal Systems Division, Mail Stop: JE-5EA (2224 Bay Area Blvd)

International Conference on Environmental Systems

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CFU = Colony Forming Units

COTS = Commercial Off-the-Shelf

DI = Deionized Water

EMU = Extravehicular Mobility Unit

Hg = Mercury

HV = Hand Valve

I = Ultraviolet (UV) Intensity [mW/cm²]

ISS = International Space Station

k = Inactivation Rate Constant [cm²/mW•s] or [cm²/mJ]

LED = Light Emitting Diode

LEO = Low Earth Orbit

MCV = Microbial Check Valve

mL = Milliliter

N = Final Bacterial Concentration [CFU/mL]

NASA = National Aeronautics and Space Administration

nm = Nanometer

No = Initial Bacterial Concentration [CFU/mL]

Np = Bacterial Concentration Masked by Particles [CFU/mL]

OGA = Oxygen Generation Assembly

OGS = Oxygen Generation System

PWD = Potable Water Dispenser

Q = Volumetric Flowrate [mL/min] or [cm³/min]

QD = Quick Disconnect

SETi = Sensor Electronics Technology, Incorporated

t = UV Exposure Time or Residence Time [s]

UV = Ultraviolet

V = Reactor Volume [cm³ or mL]

W = Watts

WAL = Water Analysis Laboratory

WPA = Water Processor Assembly

I. Introduction and Background

s the National Aeronautics and Space Association (NASA) expands its scope and begins to venture into long-

duration, manned space flights, water reclamation systems become increasingly critical and difficult to maintain.

Visiting vehicles can no longer replenish water once spaceflight moves beyond low earth orbit (LEO) and into deep

space. Deep space transport vehicles will need to contain and reuse all life support essentials (e.g. air and water) for

the duration of the flight, which for Mars could last up to 2-3 years. Looking specifically at water, the reclamation

system must perform flawlessly for the entire mission by reclaiming wastewater and continuously producing safe,

clean drinking water. Several subsystems and components will need breakthrough upgrades in order to achieve this

feat. The first area of concern with the current ISS water system (the baseline for the deep space transport vehicle

water system) is bacterial growth in which multiple challenges must be overcome to achieve a complete and effective,

long-term bio-control strategy.

One major issue regarding microbial control is continued biofilm growth in the Water Processor Assembly (WPA)

wastewater storage tank upstream of the water treatment system. Biofilm formation in the tank can result in

downstream issues such as clogged lines/filters/valves. A second issue specific to long-duration missions is the use of

consumables in the protection of the potable water system. An iodine biocide is added as part of the WPA water

treatment and serves as a secondary barrier to mitigate potential bacterial regrowth in the potable water storage tank

and the downstream potable water distribution system. Water then passes through a third protective barrier, a 0.2-

micron filter, before being dispensed per the crew’s needs. This multi-barrier approach is very effective, but comes

at the cost of consumables.

Finally, there are areas both in the current and future water systems, where immediate solutions to maintain

adequate microbial control are necessary. One such area is the recirculation line in the oxygen generation system

(OGS), where current biocides are not compatible with the system. A second is the microbial check valve (MCV)

used on the reject line of the WPA. The reject line allows processed water not meeting specifications to be redirected

back through the WPA. When the clean and dirty side of the line are hydraulically connected, the MCV prevents

A


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microbial contamination on the “dirty” side of the WPA from passing through to the potable side. The current MCV

system uses iodine biocide released from an ion-exchange material. For future potable water systems, silver is being

considered as the biocide. However, iodine is not compatible with silver, and, currently, no MCV solution exists for

a silver biocide based system. In total, the maintenance required to correct issues such as blockages and replace

consumable components can be frequent, require materials and spares, and take significant crew time. These resources

are expected to be extremely limited on a long-duration flight. As such, alternative disinfection approaches are of

interest for future spaceflight.

UV disinfection has remained a technology of interest, but historically deemed a challenge to implement on

spacecraft. UV disinfection systems in use today are almost exclusively mercury-lamp based. For a number of reasons,

including an effective wavelength output, price, and availability, mercury-based UV designs have been and continue

to be the state-of-the-art for terrestrial applications. For spaceflight, however, conventional UV disinfection

technology has been less attractive due to issues of volume, mass, power, maintenance, lifetime, and, most importantly,

environmental toxicity. This is where UV disinfection based on light emitting diodes (LEDs) has sparked renewed

interest. After only becoming commercially available in the last decade, LED-based UV disinfection technology is

still in its development infancy stage, but continuing to yield promise in almost every element of implementation.

Table 1 summarizes the advantages of UV-LED lamp technology over conventional mercury (Hg) systems. The

advantages include environmental risks, robustness, size, lifetime, startup and cycling, wavelength selectivity, overall

system configurability, and temperature. Leveraging the advantages of UV-LED technology, new systems can now

be considered for spacecraft applications and designed into compact, ruggedized, reliable systems that require low

power, minimal maintenance, and no hazardous chemicals. This paper describes results from an initial feasibility

assessment of UV-LED disinfection technology for spacecraft. The feasibility assessment had three main goals.

These included: (1) to demonstrate the core UV disinfection technology against spacecraft microbes, (2) assess the

technology against specific spacecraft water system applications, and (3) initiate forward plans and recommendations

toward future development and potential technology demonstrations on the ISS. Spaceflight drives specific

requirements when it comes to selecting a commercial off-the-shelf (COTS) device, and, in a perfect world, this would

include a system with the following attributes: LED-based, compact, variable flow capacity (up to 4 L/min), a

disinfection efficiency, 255-285 nanometers (nm) wavelength, non-flow limiting, self-contained unit with no

additional accessory hardware needed, and approved material composition. However, for the testing performed for

this paper, vendors were selected to test the core technology against spacecraft specific bacteria to identify feasibility

and design changes that might be required for spacecraft specific hardware.

Table 1: LED-Based UV vs. Conventional Hg Lamps

Element LED-based UV (primary choice) Conventional Hg-lamp

Environmental Mercury-free ‒ No environmental risk to

water, crew or cabin

Mercury-based, generally contained in

fragile glass elements

Robustness Solid State Fragile glass tube and filaments

susceptible to mechanical shock and

vibration

Footprint Slim Profiles Bulky lamp and containment structure

requiring heavy ballasts

Lifetime 10,000 to 20,000 hrs. ≈6000 hours

Startup/Cycling Instant ON and unlimited ON/OFF

cycling

Up to 30 minutes, limited cycling life

Wavelength Specificity Selectable, can be used to efficiently

target microbes

Emit at 254 nm, not the most efficient

germicidal wavelength

Configurability Customizable arrangements to achieve

high power densities in small footprints

Specific, unalterable geometries, typically

shell in tube arrangement

Surface temp Low, no flowrate dependency Heat buildup on lamp surface is

dependent on fluid flowrate for cooling

II. Materials and Methods

The feasibility study started with a market survey to identify potential vendors of UV-LED technology for water

disinfection. For initial studies, the plan was to procure some commercially available systems and collect both

fundamental and application specific data to inform the use of technology in spacecraft. Note, the COTS devices

(AquiSense and SETi) provide a different dose. To understand how individual typical spacecraft bacteria respond to


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varying UV stimuli, collimated beam experiments were conducted. To implement all possible UV reactor applications

found on station, testing was broken down into three different configurations: Single-pass flow-through, recirculation,

and microbial check valve. A breakdown of the tests and their corresponding parameters is displayed in Table 2. The

results of that testing are reported herein. Also considered in the assessment was the form and fit of current UV-LED

systems integration into the spacecraft environment. Since this part of this assessment involved specifics about the

reactor systems designs and operations, those results are not provided in this paper.

Table 2: Test Parameters

A. Market Survey

A preliminary market survey for companies with

readily available UV-LED water disinfection

systems came back with only two companies. The

first company, Sensor Electronics Technology,

Incorporated (SETi), has developed a UV LED-

based prototype drinking water disinfection device

intended for battlefield and/or other military

applications. A key attraction of the technology was

the prototype design had been ruggedized for use in

harsh environments. The second company,

AquiSense Technologies, has a series of

commercially available water disinfection devices

for use in multiple applications, including medical

devices, life sciences, remote communities, defense,

emergency response, transportation, and commercial

water. The current product line has size ranges,

dependent upon application and specified flow rates.

The specific design of the UV reactors are not

discussed in this paper. Renderings of each system

are shown in Figure 1 for illustrative purposes only. In general, it can be stated that both types of devices can handle

varying flow rates and are relatively low power devices, less than 16 W. Because information on the AquiSense

reactors are publicly available, some features offered in their devices included (1) a modular/removable LED bank for

easy change out and swappable range of UV wavelengths, (2) real-time monitoring of lamp intensity and alarm

conditions, and (3) remote start/stop.

a) b)

Figure 1. Renderings of example UV reactors provided

by (a) SETi and (b) AquiSense. Note: relative images

are not to scale.


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B. Ersatz Development Within the Life Support Systems Branch at NASA, the Water Analysis Laboratory (WAL) was asked to prepare

ersatz solutions simulating the Water Processor Assembly (WPA) influent and effluent water. WAL provided the

ersatz solutions in the form of concentrates, where they were filtered, sterilized, and diluted using aseptic techniques

prior to testing.

C. Bacterial Selection

Bacteria were chosen based off their likelihood of contaminating the “potable water and environmental control life

support systems in space due to some of their unique abilities to resist antimicrobials”.1 This consortium of five

organisms included Ralstonia insidiosa, Burkholderia multivorans, Cupriavidus metallidurans, Methylobacterium

fujisawaense, and Pseudomonas aeruginosa. With the exception of P. aeruginosa, these bacteria are frequently found

in water aboard the International Space Station (ISS). However, all organisms, including P. aeruginosa, are known

formers of biofilms.

D. Microbial Growth and Analysis

Bacteria were grown in R2A agar at 35°C for 18-24 hours and were suspended in the desired test fluid to 3.33

McFarland units using a Densichek Plus instrument (bioMerieux). This equates to an approximate bacterial

concentration of 109 colony forming units (CFU) per milliliter (mL). 106 CFU/mL and 103 CFU/mL solutions were

made by diluting the original suspension 1:1000 in sterile test fluid one or two times, respectively. The consortium

consisted of an equal mixture of bacteria with a total concentration of 103 CFU/mL or 106 CFU/mL.

Planktonic bacteria were enumerated by either filtering the test fluid onto 0.22 µm mixed cellulose ester or 0.2 µm

cellulose nitrate membranes (Milliflex or Nalgene, respectively) and transferring the membranes to R2A agar or spread

plating 100 µL of the test fluid or dilutions directly onto R2A agar. Bacteria were allowed to grow at 35°C for 2-7

days before counting the colonies.

Biofilms were enumerated by immersing coupons in 3 mL sterile Butterfield’s phosphate buffer, sonicating for 10

minutes, and spread plating 100 µL of the suspension or dilutions onto R2A agar. Bacteria were allowed to grow at

35°C for 7 days before counting the colonies.

E. Inactivation Rate Constant

Schmelling et al. notes that except at very low UV intensities, effective UV dose to achieve microbial inactivation

is independent of UV intensity.2 In other words, the same UV dose may be delivered by a low UV intensity over a

longer duration as may be delivered by a high UV intensity over a shorter duration. However, at very low UV

intensities, microbes may repair some damage from UV irradiation before microbial inactivation is complete. This is

not to imply that microbes may not be inactivated using low UV intensities, but rather that a longer duration and higher

overall UV dose is required at low UV intensities than with moderate to high UV intensities.

The inactivation rate constant, k [cm²/mJ], may be extracted as the slope when plotting the UV dose. This value is

a function both of microbial species within the water sample and the primary wavelength of the ultraviolet irradiation

source.

Abu-ghararah describes the UV disinfection process as first-order with respect to both surviving organisms and

ultraviolet light intensity.3 Meulemans agrees with these assessments.4 Given the following definitions:

N Final Bacterial Concentration [CFU/mL]

Np Bacterial Concentration Masked by Particles [CFU/mL]

No Initial Bacterial Concentration [CFU/mL]

k Inactivation Rate Constant [cm²/mW•s] or [cm²/mJ]

I Ultraviolet (UV) Intensity [mW/cm²]

t UV Exposure Time or Residence Time [s]

An appropriate model then is:

𝑁 = 𝑁𝑝 + (𝑁𝑜 − 𝑁𝑝)𝑒−𝑘•𝐼•𝑡 (1)

Rearranging:

(𝑁 − 𝑁𝑝) = (𝑁𝑜 − 𝑁𝑝)𝑒−𝑘•I•t (2)


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(𝑁 − 𝑁𝑝)

(𝑁𝑜 − 𝑁𝑝)= 𝑒−𝑘•I•t

(3)

ln [

𝑁 − 𝑁𝑝

𝑁𝑜 − 𝑁𝑝

] = −𝑘 • I • t

(4)

ln [


𝑁 − 𝑁𝑝

] = 𝑘 • I • t

(5)

So, plotting ℓn [(No ─ Np) / (N ─ Np)] as a function of I•t, the UV dose, should yield a linear plot that passes

through the origin with a slope of k, the inactivation rate constant. When Abu-ghararah applied this form to data

collected at a municipal water treatment plant, he found k ≈ 590 cm²/W•s for temperatures from 20 °C to 40 °C,

k = 350 cm²/W•s at 10 °C, and k = 770 cm²/W•s at 45 °C.3

More generally, the local slope of a plot of ℓn [(No ─ Np) / (N ─ Np)] as a function of I•t, the UV dose, is the local

inactivation rate constant, k. Because No and N are equivalent when the UV dose is zero, (No ─ Np) / (N ─ Np) is

unity and ℓn [(No ─ Np) / (N ─ Np)] is zero, so the plot passes through the origin. While a linear plot for

ℓn [(No ─ Np) / (N ─ Np)] as a function of UV dose is convenient, realistically the inactivation rate constant is sensitive

to many factors, including microbial species. So, while a linear model fits well for a limited range of either

ℓn [(No ─ Np) / (N ─ Np)] or UV dose, a general relationship for a broad range of measurements may be nonlinear.

Guo et al., for example, has similar results.5

Equation 5 works well for microbes subjected to collimated ultraviolet light beam testing where the UV intensity

and UV exposure time are well known. For a flow-through cell, an expression in terms of flowrate is easier to use.

Thus, the residence time is defined as:

𝑡 =𝑐 • V

𝑄 (6)

Where:

c Conversion Constant = 60 s/min [s/min]

V Reactor Volume [cm³ or mL]

Q Volumetric Flowrate [mL/min] or [cm³/min]

Substituting into Equation 5:

ln [


𝑁 − 𝑁𝑝

] =𝑘 • c • I • V

𝑄

(7)

Or:

ln [


𝑁 − 𝑁𝑝

] =𝑘 • β

𝑄

(8)

Where:

β A constant equal to c•I•V [mW•cm•s/min] or [mJ•cm/min]

Plotting ℓn [(No ─ Np) / (N ─ Np)] as a function of (β/Q) should yield a linear plot that again passes through the origin

with a slope of k, the inactivation rate constant. For optically clear solutions, Np ≈ 0, to which Equation 5 and

Equation 8 reduce to:


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ln [

𝑁𝑜

𝑁] = 𝑘 • I • t

(9)

ln [

𝑁𝑜

𝑁] =

𝑘 • β

𝑄

(10)

F. Collimated Beam – Dose Response

Collimated beam testing was developed to assess individual bacterial responses to varying UV stimuli, as well as

perform the control verifications needed to baseline the standard equipment being used. To gain additional confidence

that the flow-through and recirculation applications worked as marketed, collimated beam testing was a beneficial

method in breaking down elements into smaller units to quantitatively answer fundamental questions.

Dose response curves were established using the AquiSense

PearlBeam (Fig. 2). The PearlBeam collimated UV light at a

wavelength of 265 nm or 285 nm onto a petri dish of test water

inoculated with bacteria. Each of the dose response curves included

six UV doses, chosen to yield a 3- to 4-log10 reduction for each

bacterial species, and three technical replicates. The five bacteria

were tested individually at two bacterial concentrations (103 and 106

CFU/mL), three water qualities (deionized water, WPA influent

ersatz, and WPA effluent ersatz), and two biological replicates at 265

nm and room temperature.

A reduced matrix was performed for testing at 285 nm. In this

case, the five bacterial species were tested individually at two

bacterial concentrations (103 and 106 CFU/mL), a single water

quality (WPA influent ersatz), and two biological replicates at room

temperature. Additional tests were completed at 265 nm and 10°C

to evaluate the effect of temperature. Four dose response curves were performed with Burkholderia multivorans at

two concentrations (103 and 106 CFU/mL) with two biological replicates and a single water quality, WPA influent

ersatz. An upper temperature limit (40-45°C) could not be assessed due to the bacteria being sensitive to incubation

in the ersatz at elevated temperatures.

G. Single–Pass Flow Through

The single-pass flow-through test was used to assess the bacterial count reduction across the UV Reactor. The test

fluid, inoculated with bacteria, was pumped through a UV reactor at various flow rates (Fig. 3). Bacterial counts were

obtained from samples taken before (N0) and after (N) the UV reactor to determine the log reduction.

Figure 2. The AquiSense PearlBeam was

used to assess individual bacteria

characteristics.

Figure 3. Flow-through testing configuration.


HV = Hand Valve


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Twenty-four tests were performed for flow-through testing. Variables included two UV reactors providing

different doses, a single bacterial consortium of all five bacteria, two bacterial concentrations (103 and 106 CFU/mL),

two biological replicates, and three water qualities (deionized water, WPA influent ersatz, and WPA effluent ersatz).

Each test consisted of three flow rates (0.25, 1, 4.5 L/min) and three technical replicates.

H. Recirculation Recirculation testing addressed the efficacy of using ultraviolet irradiation to inactivate microbial species in a large

volume, such as a storage tank, by continuously recirculating the tank’s fluid through an ultraviolet light irradiation

reactor. The recirculation test stand was set up to replicate the water flowing through the ISS wastewater tank. The

test fluid (WPA influent ersatz), inoculated with bacteria, flowed through the UV reactor first and then through a

biofilm reactor (CDC Biofilm Reactor, Biosurface Technologies) which acted as a water storage tank (Fig. 4). The

biofilm reactor contained coupon holder rods, allowing coupons to be suspended in the test fluid and exposed to

bacteria on both sides. The coupons could then be aseptically removed and sampled for biofilm growth.

The desired outcome was the UV reactor would kill most, if not all, bacteria and prevent biofilm growth

throughout the system. Similar to the single-pass flow-through test, bacterial counts were determined from samples

taken before (N0) and after (N) the UV reactor to determine the log reduction. The stainless steel coupons placed in

the biofilm reactor were sampled in duplicate three times per week.

There was a test stand for each UV reactor system, as well as a control test stand, without a UV reactor, that

provided an uninterrupted data set. This test not only assessed the efficacy of the disinfection science, but also

delineated how well each of the UV reactors performed in comparison with each other.

Three tests were performed for recirculation testing, one for each reactor system and a control test stand not

containing a UV reactor. These tests incorporated a single bacterial consortium, one starting bacterial concentration

(106 CFU/mL), one biological replicate (with the exception of biofilm coupon duplicates), and one water quality (WPA

influent ersatz). Each test consisted of three technical replicates.

The recycle loop, which passed through the UV reactor and a biofilm reactor, operated at a volumetric flowrate of

50 mL/min. Fresh solution, with bacteria, entered the recycle loop at 0.50 mL/min (500 μL/min). Thus, the ratio of

recycle to influent was 100 to 1. Initially, the recycle loop was microbe free. Microbes were introduced via the influent

line from an inlet reservoir holding microbial solution. The initial concentration of the microbial solution was

106 CFU/mL when freshly prepared, but this concentration varied as the solution aged within the reservoir. The inlet

reservoir was augmented with fresh solution roughly twice a week.

Figure 4. Recirculation testing configuration. Green arrows dictate the flow path during testing.


HV = Hand Valve


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I. Microbial Check Valve The goal of this test configuration was to assess whether each UV device could act as a barrier between dirty water

and clean water such that it would prevent microbial growth to the clean side of the UV device. In this setup, a COTS

UV reactor separated 2 reservoirs of test fluid; one reservoir was sterile and the other contained 106 CFU/mL of

bacteria. The reactor was connected to each reservoir by a short length of Tygon tubing (Fig. 5). The test fluid remained

stagnant throughout the test with the exception of fluid being drawn out of the tubing on either side of the UV reactor

for sampling purposes.

Three tests were performed for microbial check valve/static testing, one for each reactor system and a control test

stand not containing a UV reactor. These tests incorporated a single bacterial consortium, one starting bacterial

concentration (106 CFU/mL), one biological replicate, and one water quality (WPA influent ersatz). Each test

consisted of three technical replicates.

III. Results

A. Collimated Beam At both wavelengths, Cupriavidus metallidurans (Fig. 6a), Pseudomonas aeruginosa (Fig. 6b), and Ralstonia

insidiosa (Fig. 6c) are relatively sensitive to UV light, whereas Burkholderia multivorans (Fig. 7a) is approximately

twice as resistant and Methylobacterium fujisawaense (Fig. 7b) is approximately five times as resistant as the other

species. Minor effects were observed when subjecting the bacteria to different water qualities (data not shown) and

bacterial loads (Fig. 6 and 7).

For similar levels of inactivation of all microbial species, ℓn (No/N), tests using 285 nm required a greater UV

dose than corresponding trials at 265 nm (Fig. 6 and 7). Thus, ultraviolet irradiation at 285 nm requires a greater UV

dose than ultraviolet irradiation at 265 nm to accomplish the same microbial inactivation. This lack of efficiency was

compensated for by an increase in intensity of the 285 nm LEDs used in the collimated beam apparatus.

As noted, both Abu-ghararah and Meulemans report that microbial inactivation rate is unaffected by temperatures

ranging from 20°C to 40°C.3, 4 Abu-ghararahn reported a change in microbial inactivation rate at temperatures below

20°C or above 40°C.4 Within this study, considering ultraviolet irradiation at 265 nm for Burkholderia multivorans in

WPA influent, Figure 8 displays results at 23°C and at 10°C on a single plot. For the test points executed, there appears

to be no variation due to temperature between the data collected at 10°C compared to the data collected at 23°C within

the scatter of the data collected. The effect of a higher temperature (40-45°C) could not be tested, as both B.

multivorans and P. aeruginosa could not tolerate the heat when incubated in the WPA influent solution.

Figure 5. Microbial check valve testing configuration.


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a)

b)

c)

Figure 6. Collimated Beam Test Results at 265 nm

and 285 nm versus Cupriavidus metallidurans (a),

Pseudomonas aeruginosa (b), and Ralstonia insidiosa

(c) in WPA Influent.

a)

b)

Figure 7. Collimated Beam Test Results at 265 nm and

285 nm versus Burkholderia multivorans (a), and

Methylobacterium fujisawaense (b) in WPA Influent.

Figure 8. Collimated Beam Test Results at 265 nm, and

10 °C and 23 °C versus Burkholderia multivorans in

WPA Influent.


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B. Single-Pass Flow Through

When sized appropriately, the UV reactors were highly effective at reducing a population of bacteria in flow-

through testing. For the UV reactor that delivered a higher dose, essentially all the effluent samples, the samples taken

after passing through the UV reactor, had undetectable levels of bacteria. Initially, a few bacteria were detected in

these samples, but it was determined to be due to contamination from the positive control, where bacteria were pumped

through the test stand prior to startup of the UV reactor. When it was observed that the reactor was highly effective,

this practice was terminated in order to keep from contaminating the effluent samples. Due to time constraints, not all

of the initial tests could be repeated without a positive control to get more accurate results. Furthermore, there is a

possibility that there were unaccounted-for Methylobacterium present in filtered effluent samples due to the R2A

cassettes manufactured by Milliflex not supporting its growth. This is especially true in cases where counts for the

effluent samples were low and filtration was necessary to get an accurate count.

Most samples from the higher dosage reactor were taken at 10 mL (in triplicate) containing a final concentration

less than 0.1 CFU/mL. At a 106 CFU/mL starting concentration, this equates to a greater than 7-log10 fold reduction.

Table 3 provides an example of quantitative results for a minimum log10 inactivation achieved for each flow rate

within a test based on the sampled volume and the initial bacteria concentration. The higher the initial concentration

and the sample volume, the larger the minimum log reduction. Had a larger volume been sampled (e.g., 1 L vs 10

mL), a larger log reduction may have been seen. Despite this limitation, all samples showed at least 4-log10

inactivation.

For the lower dosage UV reactor, microbial reduction (or inactivation) tended to be described well by a natural

logarithm function as a function of flowrate (Fig. 9). At higher flow rates, the efficiency of the reactor diminishes.

This is expected since flow rate is inversely proportional to retention time and therefore UV dosage. Interestingly, the

bacteria found in the outlet ports of the test stand were predominately Burkholderia multivorans and Methylobacterium

fujisawaense, the two most UV-resistant organisms used in this test. A subtle trend of reduced inactivation with poorer

water quality was also observed. In this configuration, the reactor would be useful in a number of applications.

Table 3. Example of minimum log10 inactivation of samples from flow-through test stand.

Base Liquid Bacteria Concentration

(CFU/mL)

Flow Rate (L/min) Log10 Inactivation

DI Water

103

0.25 4.41

1 4.18

4.5 4.01

106

0.25 5.54

1 5.65

4.5 5.65

WPA Effluent

103

0.25 4.15

1 4.15

4.5 4.39

106

0.25 7.09

1 7.09

4.5 7.33

WPA Influent

103

0.25 3.97

1 3.97

4.5 4.21

106

0.25 6.73

1 6.73

4.5 6.70


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Noting that the slope of a plot of ℓn (No/N) as a

function of (β/Q) is the inactivation rate constant, k,

generally the slope of the fit logarithm function is:

𝑑

𝑑(βQ

)[ln (

𝑁𝑜

𝑁)] = 𝑠𝑙𝑜𝑝𝑒 • (

𝑄

β)

(11)

For the WPA influent at 106 CFU/mL, in figure 9, the

specific result is:

For example if reactor volume, V, is 143.71 cm³ and the

measured ultraviolet light irradiation, I, is

0.555 mW/cm², the corresponding constant β becomes

4,786 mJ•cm/min. The previous expression then becomes:

𝑑

𝑑(βQ

)[ln (

𝑁𝑜

𝑁)] = (4.066X10−4 min • cm²/mJ • cm³) Q)

(13)

or:

𝑘 = (4.066X10−4 min • cm²/mJ • cm³) Q) (14)

For the flowrates used during testing, k = 0.1016 cm²/mJ for 0.25 L/min, k = 0.4066 cm²/mJ for 1.0 L/min, and

k = 1.8297 cm²/mJ for 4.5 L/min.

C. Recirculation Testing

Use of a UV system in a recirculation system is of interest for spacecraft applications. For such systems,

recirculation past a UV device may be a way of preventing microbial growth and/or biofilm issues, especially in

systems where biocides are not a viable option, e.g., the oxygen generation assembly (OGA) or a wastewater storage

tank. In feasibility testing, active UV irradiation in a recirculation configuration provided significant microbial

reduction versus the control test stand. For the control, the logarithm reduction is roughly zero, which is the expected

result when No equals N near the end of the testing. The control test stand was populated by bacteria almost

immediately following inoculation (Fig. 10a). The UV recirculated test stands showed a delayed response in their

microbial population, but bacteria on the outlet side of the UV reactor was observed to grow exponentially on the

coupons found within the biofilm reactor in both cases. An example of the microbial response is shown in Fig. 10b

and 10c, respectively. Both UV irradiated test stand configurations showed generally lower average biofilm counts

than the corresponding control test stand as a function of test duration. Considering all three measurements, the

microbial concentration increased until test day 11, after which it appears to decrease steadily. Fresh microbial solution

was added to each recirculation test stand inlet reservoir on day 10, noting that the initial microbial concentrations,

No, in all of the test stands increased on day 11. The steady decrease in microbial concentrations for all measurements

related to the control test stand suggests that other impacts besides active ultraviolet irradiation controlled microbial

growth within the recirculation test stands. Microbes may have attached to and propagated in the tubing between the

ultraviolet light irradiation reactor and the biofilm reactor, leading to higher microbial concentrations in the effluent

samples, effectively lowering the measured logarithmic reduction for the ultraviolet light irradiation reactors. The data

for the control test stand appears to support this hypothesis, as the downstream measurements are much higher than

the upstream measurements for samples taken near the end of the test. Another conjecture, based on the test results, is

𝑑

𝑑(βQ

)[ln (

𝑁𝑜

𝑁)] = 1.946 • (

𝑄

β)

(12) Figure 9: Example of Log10 inactivation as a function

of flowrate using WPA influent.


13

that active microbes may have used dead

microbial biomass as a shield to bypass

ultraviolet irradiation and allow

colonization of the biofilm reactor.

Throughout the recirculation testing, it

was noticed that the water in the systems

with the UV Reactors was accumulating a

brownish tint while the control system

water remained clear. Additionally,

biofilm had formed in a low point of the

tubing at the location of the pump head on

all three test stands. Correlating to the

color of the water, the biofilm of the

control test stand was clear/white in color,

while the biofilm in the other two test

stands was dark brown/black.

Consequently, upon completion of

testing, the effluent water from each test

stand was sampled and filtered through a

0.2 µm filter to determine if the water

discoloration was due to the presence of

tinted bacteria. The effluent water from

the UV reactor test stands remained

discolored when compared to the control.

This suggests that the effect is not due to

the presence of whole cells, but it cannot

rule out the presence of tinted cellular

components or fermentation products that

are small enough to pass through the filter.

Further analysis would be necessary to

determine if the water discoloration was

due to the presence of the bacteria or a

component of the ersatz. Additional

testing in recirculation mode is also being

proposed in alternative configurations.

D. Microbial Check Valve Testing

In the control test stand, the system

without a UV reactor, bacteria from the

inoculated reservoir quickly populated the

lines in the test stand as seen by the rapid

rise in bacterial population in both the

inlet (N0) and outlet (N) sample ports in

Figure 11a. The logarithmic reduction

values for the control test stand, hover

around zero as expected without any

ultraviolet irradiation. For the UV reactor

test cases, results were mixed. In one

case, bacterial population on the

inoculated side of one UV reactor test

case (N0) rose quickly in the test stand

(Fig. 11b). However, there was a

significant delay (more than a week) in

the bacteria crossing over into the outlet

side of the UV reactor. Once the bacteria

a)

b)

c) Figure 10. Bacterial concentration (per volume or per coupon) as a

function of time for the Control recirculation test stand (a), and

for the reactor systems tested, (b) and (c).


14

passed the UV reactor, their population

increased exponentially on the outlet

side. Interestingly, while the bacteria

found on the inlet side of the UV reactor

were a mixture of organisms,

Burkholderia multivorans dominated

growth on the outlet side. One theory is

that this may be due to the ability of B.

multivorans to repair itself long after

exposure to UV light. Once a cell is

damaged in the reactor, it can

diffuse/migrate out of the reactor and

repair itself on the other side. Notably, in the test case of the second

reactor, the test stand remained sterile

throughout the entire test at both sample

ports. Twice, the supply reservoir was

sampled to enumerate the bacteria. On

day six, the tank contained 1.06 x 106

CFU/mL, and on the final day of testing,

the tank contained 2.27 x 105 CFU/mL.

Points for this test case could not be

graphed because the No and N input

values are zero and the logarithm of the

ratio of two zero values is undefined. One

theory as to why bacteria were not found

in the inlet sample port is that the UV

light (either directly or indirectly) was

capable of killing bacteria just outside the

reactor. This phenomenon was also

observed in the recirculation test stand,

where it took a week before bacteria

appeared on the inlet side of that system

test, and the first bacteria to appear were

solely Methylobacterium, the most UV-

resistant organism used in this test.

Another theory is that the other factors in

the local environment, e.g., temperatures

and or residual chemicals resulted in

uninhabitable conditions for the bacteria.

Follow on testing is being pursued to explore this phenomenon.

E. Overall COTs Evaluation

UV disinfection technology was shown effective against all challenge microorganisms in the spacecraft matrixes

of interest, e.g., common spacecraft isolates in deionized water, WPA influent ersatz, and WPA effluent ersatz. Data

collected to date best supports use of the technology in flow-through/POU applications. Alternative applications for

MCV and in-loop recirculation applications, e.g., tank, extravehicular mobility unit (EMU), and OGA, still show

potential, but additional testing is required. Alternative test configurations for in-tank biofilm prevention are currently

being pursued. Testing supports the UV disinfection theory and scalability – the key parameter is delivery of a UV

dosage (intensity x time). Design flexibility is anticipated to accommodate any of the spacecraft applications

proposed. The available devices as built are considered viable designs, but system modifications are anticipated, e.g.,

thermal management of the LED systems. Spacecraft versions for specific devices are being pursued. Location

assessments best support MCV and potable water dispenser (PWD) applications for ease of implementation.

Alternative locations will likely require exterior mounting. Software and hardware assessments suggest no hard

impediments for adapting to spacecraft application.

a)

b)

Figure 11. Example of MCV testing - Bacterial concentration as a

function of time in a control test stand (a) and UV reactor test stand

(b).


15

IV. Conclusions

After completing a variety of tests to determine the feasibility of using UV LED devices to irradiate

microorganisms within a water system, it is concluded that the UV technology shows promise. Further testing is being

pursued, and modification of COTS units would be expected for spaceflight applications. Design changes would apply

both to application-specific optimization of the base UV technology as well as changes to meet ISS or other vehicle

flight specifications. Based on this initial feasibility study, the recommendation is to continue pursuing this

technology. At present, on-going work is looking at specific applications in the following areas: (1) in-tank UV

disinfection system to prevent biofilm growth in the WPA wastewater tank, (2) development of an ISS flight

technology demonstration for an exploration class PWD, and (3) follow-on MCV testing. If successful, ultraviolet

disinfection technology has the potential to solve a number of microbial control challenges in current and future

spacecraft water systems.

Acknowledgements

The authors wish to express their sincere appreciation to the numerous people who have contributed to this

feasibility study, including, but not limited to Brandon Dunbar, Michelle Green, Javier Jimenez, Melinda Kimmel,

Kelly Mann, Stacey Moller, William O’Hara, Patrick O’Rear, Stuart Pensinger, Shawn Schumacher, Jessica Wells,

and Gina Young. Furthermore, the authors wish to thank Oliver Lawal, Molly McManus, Jennifer Pagan, and Richard

Simmons from AquiSense as well as Peter Barber and Robert Kennedy from SETi for their insightful consultation on

the core technology.

References

1Birmele, M. N., O’Neal, J. A., and Roberts, M. S., “Disinfection of Spacecraft Potable Water Systems by Photocatalytic

Oxidation Using UV-A Light Emitting Diodes,” NASA Technical Reports Server [online server], URL:

https://ntrs.nasa.gov/search.jsp?R=20110014453 2Schmelling, D., Cotton, C., Owen, D., Mackey, E., Wright, H., Linden, K. G., and Malley, Jr., J. P., “Ultraviolet

Disinfection Guidance Manual for the Final Long Term 2 Enhanced Surface Water Treatment Rule,” EPA 815-R-06-007, United

States Environmental Protection Agency, Office of Water, November, 2006. 3Abu‐ghararah, Z. H. “Effect of temperature on the kinetics of wastewater disinfection using ultraviolet radiation,” Journal of

Environmental Science and Health. Part A: Environmental Science and Engineering and Toxicology, Volume 29, Issue 3, 1994,

pp. 585-603. 4Meulemans, C.C.E. “The Basic Principles of UV–Disinfection of Water,” Ozone: Science and Engineering, Volume 9, Issue

4, 1987, pp. 299-313. 5Guo, M., Huang, J., Hu, H., Liu, W., and Yang, J. “UV inactivation and characteristics after photoreactivation of

Escherichia coli with plasmid: Health safety concern about UV disinfection,” Water Research, Volume 46, 2012 pp. 4031-4036,

17 May 2012.

https://ntrs.nasa.gov/search.jsp?R=20110014453

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