Wat. Res. Vol. 34, No. 12, pp.

30753086, 2000
7 2000 Elsevier Science Ltd. All rights reserved
Printed in Great Britain
PII: S0043-1354(00)00085-3 0043-1354/00/$ - see front matter

www.elsevier.com/locate/watres

GRAYWATER PROCESSING IN RECIRCULATING

HYDROPONIC SYSTEMS: PHYTOTOXICITY, SURFACTANT
DEGRADATION, AND BACTERIAL DYNAMICS
J. L. GARLAND*, L. H. LEVINE, N. C. YORIO, J. L. ADAMS and K. L. COOK
Dynamac Corporation, Mail Code DYN-3, Kennedy Space Center, FL 32899, USA

(First received 1 July 1999; accepted in revised form 1 November 1999)

AbstractIncorporation of human hygiene water (graywater) into hydroponic plant production

systems, and subsequent recovery of the water transpired by the plants, is one potential means for
water purication and recycling in bioregenerative life support systems under development for long
duration space missions. Surfactant phytotoxicity and the potential for growth of human-associated
microorganisms were assessed in studies of wheat and lettuce in controlled environmental chambers to
provide baseline information for future studies with actual graywater streams. Igepon TC-42 (sodium
N-coconut acid-N-methyl taurate), a surfactant designated for use on the International Space Station
and a common ingredient of soaps and detergents, was added to plant systems in three dierent modes:
(1) pulse addition of 875 mg m2 growing area once a day, (2) continuous addition of 875 mg m2
over the course of a day, and (3) variable addition of 03000 mg m2 d1 based on plant water
demand. The survival of three human-associated bacteria (Escherichia coli, Staphylococcus aureus, and
Pseudomonas aeruginosa ) in the plant nutrient delivery systems were monitored following introduction
6 (wheat) or 3 (lettuce) days after planting (DAP). Igepon rapidly disappeared (i.e., a half-life of less
than 1 h) following an initial adaptation period lasting less than 2 days. Microbial degradation of
Igepon was supported by appearance of the degradation intermediate methyl taurine and an increase in
the numbers of bacteria able to grow on media containing Igepon as the sole carbon source in the
Igepon treated systems relative to the control. Wheat growth was not signicantly aected by any of
the Igepon treatments, but lettuce yield was signicantly reduced in the pulse and continuous
treatments. E. coli and S. aureus decreased below detection limits within 35 days within the systems,
but P. aeruginosa persisted in the rhizosphere, nutrient solution, and nutrient delivery system biolm
for the duration of the wheat (70-day) and lettuce (28-day) experiments. 7 2000 Elsevier Science Ltd.
All rights reserved

Key wordsgraywater, Igepon, hydroponics, survival, lettuce, wheat, Lactuca sativa, Triticum aestivum,
Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa

INTRODUCTION for extended space missions (Barta and Henninger,

Just as in terrestrial homes, graywater (non-toilet
wastewater) is projected to be the largest waste
stream (by mass) generated in long-term crewed
space habitats. Although estimates of graywater
production within space systems are much smaller
than values reported for typical terrestrial resi-
dences (27 vs 96172 l person1 day1), this waste
stream will comprise 80% of the total waste output
(Gerba et al., 1995; Wieland, 1994; Wydeven and
Golub, 1990). Recycling this water is necessary to
limit resupply costs. The National Aeronautics and
Space Administration's (NASA) Advanced Life
Support (ALS) program is examining the feasibility
of developing regenerative life support systems,
including physiochemical and biological processes,
*Author to whom all correspondence should be addressed.
Tel.: +1-321-476-4276; fax: +1-407-853-4165; e-mail:
jay.garland-1@ksc.nasa.gov
3075
1994). Direct addition of graywater into plant pro-
duction units under development for ALS systems
(Wheeler et al., 1996) is a simple potential method
for water processing. This approach would rely on
(1) microbial activity in the rhizosphere to degrade
organic material (primarily surfactants), and (2)
plant transpiration and subsequent condensation to
purify water. Graywater processing through plant
production system could increase system eciency
by utilizing the existing food production system for
water processing.
Graywater processing by plants in greenhouses
(Kane, 1981), the landscape (Kourik, 1990; Ludwig,
1995), or specic treatment systems (Gerba et al.,
1995) has become increasingly popular in response
to water shortages. Little information is available
on the rate of surfactant degradation and the extent
of microbial persistence in these systems, two im-
portant parameters aecting potential plant phyto-
toxicity and human health associated with this
3076 J. L. Garland et al.
mode of graywater processing. The balance between
loading rate and degradation rate will control
chronic exposure of the plant to the surfactant.
Acute bioassays with lettuce found that Igepon, a
linear alkyl methyl taurate designated for use on
the International Space Station and a common
ingredient of soaps and detergents, did not exhibit
any toxic eects below 250 ppm (Bubenheim et al.,
1997; Jacquez and Montoya, 1994). Both these stu-
dies also reported that the surfactant was actively
degraded in hydroponic systems, but no specic
degradation rates were reported. These data suggest
that at the surfactant levels anticipated in space-
based systems (i.e., 200300 ppm), direct addition
of graywater would not reduce plant growth, but
additional information is required on chronic tox-
icity thresholds and rates of degradation within the
plant growth system.
Concerns about health hazards from the reuse of
graywater stem due to the presence of a variety of
human-associated microorganisms and the potential
for regrowth and/or persistence of these organisms
(Rose et al., 1991). Gerba et al. (1995) reported that
while levels of fecal and total coliform bacteria
were signicantly reduced by treatment in various
graywater processing systems, including one based
on water hyacinths, further disinfection probably
would be necessary to reduce the concentration of
pathogenic microorganisms. In the proposed ALS-
based graywater processing system, the plants
would serve as a barrier between the contaminated
water and the atmospheric condensate. However,
the persistence of pathogenic microorganisms within
the plant growth system could present health risks
due to human contact, especially during harvesting.
The experiments outlined in this paper evaluated
the eects of adding Igepon and specic human-as-
sociated microorganisms to recirculating hydropo-
nic systems containing two candidate ALS crops,
wheat and lettuce. Plant growth response, surfac-
tant degradation rates, shifts in the density of total
and surfactant-degrading bacteria, and the persist-
ence of the human-associated bacteria were quanti-
ed. These controlled experiments were performed
to provide baseline information for future exper-
iments with actual graywater streams in which the
microbial and chemical inputs are more variable
and dicult to quantify (Loader et al., 1999)
MATERIALS AND METHODS
Environmental conditions
Two tests with wheat (Triticum aestivum L. cv. USU-
Apogee) and two tests with lettuce (Lactuva sativa L. cv.
Waldmann's Green) were conducted in a reach-in con-
trolled environmental growth chamber (EGC Model M-
12, EGC, Inc., Chagrin Falls, OH). Lighting to the
chamber was provided by four 400 W high-pressure
sodium lamps (Lucolux; General Electric Co., Cleveland,
OH) and was maintained near 700 mmol m2 s1 photo-
synthetic photon ux (PPF) with a 24-h light/0-h dark
photoperiod for wheat and 300 mmol m2 s1 PPF with a
18-h light/6-h dark photoperiod for lettuce. For all tests,
atmospheric [CO2] was maintained near 1200 ppm with an
infrared gas analyzer (LiCor Model 6252, LiCor Corp.,
Lincoln, NE) and dedicated computer monitoring and
control system. Air temperature and relative humidity
(RH) were maintained near 228C (wheat), 238C (lettuce)
and 65% (both crops), respectively with the exception that
RH was maintained at 85% for the rst four days of each
experiment to foster germination. Experimental means of
environmental parameters are summarized in Table 1.
Plant cultural techniques
Wheat seed was soaked for 20 min in 5% bleach in
deionized (DI) water (0.26% sodium hypochlorite), rinsed
three times with DI water, wrapped in nylon wicking ma-
terial (Nitex, Tetko, Inc., New York, NY) and moistened
paper towels, and refrigerated at 48C for 24 h prior to
sowing. The surface sterilization was performed to limit
problems associated with Pythium root infections observed
in previous studies with wheat. The imbibed wheat seeds
were sown directly on 0.08 m2 plastic (white acrylonitrile-
butadiene-styrene) hydroponic tray inserts designed to
support wheat growth (Fig. 1) at a density of 164 seeds
per tray (eective planting density was 1800 seeds m2).
White acrylic ``germination'' covers covered each tray to
maintain high humidity during germination. Dry lettuce
seed was sown directly on similarly sized 0.08 m2 plastic
hydroponic tray inserts designed to support lettuce growth
(Fig. 1) at a density of 18 seeds per tray (eective plant-
ing density was 1100 seeds m2). Surface sterilization was
not performed since no disease problems had been
observed in previous lettuce studies. Translucent culture
vessels (Magenta, Sigma Chemical Co., St Louis, Mo.)
were used as ``germination'' covers and inverted over each
lettuce planting position to maintain high humidity during
germination. For both wheat and lettuce hydroponic tray
inserts, Nitex was used to conduct nutrient solution to the
seeds. Seeds and covers were sprayed daily with DI water
and germination covers from all trays were removed at 4
days after planting (DAP) at which time lettuce seedlings
were thinned to 4 plants per tray (50 plants m2).
For both crops, hydroponic tray inserts were positioned
within rectangular 0.08 m2 vinyl hydroponic trays and
were grown in a recirculating nutrient lm technique
(NFT) hydroponic culture system (Fig. 1). The eective
aerial growing area for each tray was 0.10 m2. Each gray-
water treatment consisted of a pair of hydroponic trays,
arranged randomly within the controlled environment
chamber to minimize chamber position eects. Each tray
pair had common plumbing and shared a single 10 l nutri-
ent solution reservoir. Nutrient solution level was auto-
matically maintained in the nutrient solution tanks with
DI water. Flow rate of the nutrient solution was 10.5 l
min1 tray1. For the rst 7 days (wheat) and 5 days (let-
tuce), plants were maintained on modied Hoagland's sol-
ution (Table 2), pH was maintain automatically at 5.8
with the addition of dilute acid (0.2 M HNO3), and electri-
cal conductivity (EC) was maintained near 1200 mS cm1
with the daily addition of a concentrated nutrient replen-
ishment stock solution (Table 2).
Igepon treatments
Igepon addition was initiated at 8 (wheat) and 6 (let-
tuce) DAP. The pulse and continuous addition treatments
received 200 ml of a 0840 ppm Igepon solution prepared
from whole body shampoo and hand cleaner (Ecolab,
Inc., St Paul, MN) The cleanser is composed of 24%
Igepon TC-42 (sodium N-coconut acid-N-methyl taurate).
Igepon solution was added daily via a single batch manual
addition to the pulse treatment, and slowly over 24 h
(1.39 ml/10 min) via a peristaltic pump to the continuous
 Graywater processing in recirculating hydroponic systems 3077

Table 1. Summary of environmental parameters observed in tests of wheat and lettuce grown on graywater containing nutrient solutions.
Values represent mean (SD) of daily averages

Crop/rep Environmental parameter

Air temperature (8C) Relative humidity (%) [CO2] (mmol mol1) PPF (mmol m2 s1)

Wheat/1 22.0 (0.6) 68.7 (10.2) 1168 (140) 686 (140)

Wheat/2 21.9 (0.6) 67.0 (6.2) 1189 (45) 696 (169)
Lettuce/1 23.0 (0.6) 69.9 (8.5) 1208 (220) 299 (31)
Lettuce/2 23.0 (0.4) 68.5 (7.3) 1201 (32) 294 (36)

Fig. 1. Schematic drawing of recirculating hydroponic systems.

3078 J. L. Garland et al.

Table 2. Elemental concentrations of nutrient solution and stock (replenishment) solution of the modied Hoagland's hydroponic nutrient solution (Wheeler et al., 1999) used to grow wheat and lettuce plants (all

Mo (mM)
treatment. Liquid level in the nutrient solution reservoirs

0.01
0.13
was automatically maintained with a 0240 ppm Igepon
solution in the on-demand treatment. The control treat-
ment did not receive any Igepon. All Igepon solutions
were made by adding Igepon TC-42 to deionized water.

B (mM)

7.13
Surfactant analysis

93
Igepon concentration in the bulk solution was measured
as methylene blue active substances (MBAS) according to
Greenberg et al. (1992) using Igepon instead of linear

Cu (mM)
alkylbenzene sulfonate (LAS) for calibration. There was a

1.04
13.5
linear correlation between absorbance and Igepon concen-
tration in the 0.123 ppm range R 2 > 0:9994: Samples
were diluted prior to analysis to fall within the calibrated
range. Methyl taurine was determined using a liquid chro-
matographic system equipped with a photodiode array
Zn (mM)

0.96
detector (Model HP 1100, Hewlett-Packard Analytical,
12.5
Wilmington, DE). Nutrient solution samples were ltered
through a 0.45 mm membrane lter immediately after
retrieval from the system. A 10 ml ltered aliquot was
transferred to a glass vial and a known amount of taurine
Mn (mM)

was added as an internal standard. The samples were lyo-

7.4

philized and reacted with 1 ml of 1 mg/ml 4-(dimethylami-

96

no)azobenzene-4'-sulfonyl chloride (DABSCl) in acetone

and 0.5 ml of 0.5 M sodium bicarbonate at room tem-
perature for 30 min. Upon the completion of reaction, the
mixture was claried by centrifugation and ltration
Fe (mM)

through a 0.45 mm lter. The dabsyl- compounds were

134
60

separated on a SUPELCOSIL LC-18 column (5 mm, 25 cm

 4.6 mm) at room temperature using a segment gradient
chemicals added to deionized water)

of 25 mM potassium dihydrogen phosphate (pH 6.8) and

solvent B (2-propanol:acetonitrile, 25:75) at the ow rate
S (mM)

1 ml/min and quantied at 460 nm. Elution was com-

1.0

pleted by increasing solvent B from 20 to 70% within

10

26 min.
Samples were taken on a weekly basis from all Igepon
treatments immediately prior to the daily checks (i.e.,
nutrient replenishment and addition of Igepon to the
Mg (mM)

pulse treatment). In order to investigate the kinetics of

1.0
10

Igepon and methyl taurine biodegradation, samples were

taken at various time points within a 24-h period after
pulse addition of 0840 ppm Igepon to all three treatments
on DAP 8, 13, and 57 (or at day 0, 13, and 49 after in-
Ca (mM)

itiation of Igepon treatments)

2.5
12

Microbiological analysis
The density of microorganisms associated with plants
roots (i.e., rhizosphere), wetted surfaces of the nutrient
K (mM)

delivery (i.e., biolm), and the nutrient solution were

3.0

removed 35 times during each plant test. Rhizosphere

56

samples were obtained by excising sections (01 cm  1 cm)

of root mat from each tray. Root samples were hand sha-
ken for 2 min in lter-sterilized deionized water and glass
beads (2 mm). Biolm organisms were sampled by remov-
P (mM)

ing polyvinyl chloride (PVC) ``chips'' (01 cm2) submerged

0.5
10

in the nutrient delivery tanks approximately 510 cm

below the nutrient solution level. Chips removed for
dierent sampling days were located within 5 cm of each
other. Suspensions of biolm organisms were obtained by
N (mM)

shaking the chips as described above. Nutrient solution

7.5
70

samples were removed via pipette through a 5 cm diam-

eter sampling port in the nutrient delivery tank lid. Nutri-
ent solution, rhizosphere suspensions, and biolm
suspensions were analyzed for total cell density using acri-
Replenishment solution

dine orange staining and epiuorescent microscopy (Hob-

bie et al., 1977). Igepon-degrading cell density was
analyzed by serial dilution and spread plating on minimal
Nutrient solution

salts media (0.871 g KH2PO4, 2.262 g K2HPO4, 0.608 g

NH4Cl, 0.097 g MgS04, with trace elements) containing
Igepon (750 mg/l) as the sole carbon source. Colony form-
ing units (CFU) on Igepon plates were enumerated after
incubation for 72 h at 378C.
 Graywater processing in recirculating hydroponic systems 3079

Table 3. Number of heads, top dry mass (DM), root DM, head DM, seed DM, and total DM growth response of wheat through on-
demand, continuous, and pulse additions of Igepon compared to Hoagland's control. Values in parentheses indicate S.D. For all growth
parameters, no signicant dierences between treatments were observed based on ANOVA P < 0:05

Growth parameter Hoagland's On-demand Continuous Pulse

No. plant1 % Control No. plant1 % Control No. plant1 % Control No. plant1 % Control

Number of heads 277 (45) 100 222 (44) 80 272 (39) 98 213 (74) 77

g plant1 % Control g plant1 % Control g plant1 % Control g plant1 % Control

Top DM 93.3 (14.4) 100 68.4 (20.5) 73 80.6 (17.1) 86 73.0 (21.2) 78
Root DM 18.8 (5.1) 100 21.1 (6.0) 112 21.0 (8.6) 112 17.9 (4.4) 95
Head DM 221.6 (63.7) 100 191.6 (69.5) 86 204.0 (50.1) 92 180.4 (59.6) 81
Seed DM 131.7 (40.3) 100 110.3 (29.2) 84 128.1 (30.6) 97 112.4 (37.3) 85
Total DM 333.7 (81.5) 100 265.7 (76.4) 80 305.6 (70.9) 92 271.3 (76.4) 81

The persistence of three human-associated bacteria RESULTS

(Pseudomonas aeruginosa, Staphylococcus aureus, and
Escherichia coli ) was assessed by inoculating plant growth The rate of addition in the pulse and continuous
systems with log-phase cultures of the organisms into the treatments was the same for both the lettuce and
nutrient solution six (lettuce) or eight (wheat) days after wheat experiments, although the cumulative amount
planting. The bacterial strains used in the study were: (1)
P. aeruginosa UG2lr, a spontaneous rifampicin-resistant of Igepon added was much greater in the wheat
strain isolated by Dr. Jack Trevors from the University of study (010 g vs 04 g) due to its longer duration.
Guelph, (2) S. aureus ATCC 25923, and (3) E. coli KSC1, Both the rate and cumulative amount of Igepon ad-
a spontaneous rifampicin and streptomycin resistant strain dition in the on-demand treatment was much
of E. coli ATCC 23590. Separate suspensions of each bac-
greater in the wheat study. This dierence was due
teria were grown in Tryptic soy broth (Difco Laboratories,
Detroit, MI) for 1618 h at room temperature with rotary to higher transpiration rates for wheat compared to
shaking (200 rpm) prior to addition (10 ml) to the nutrient lettuce, as reected in the cumulative Igepon values
solution. The density of each organism in the rhizosphere, (Fig. 2) which were calculated by multiplying water
biolm, and nutrient solution was assessed by plating use data by a constant concentration of Igepon
samples described above on selective media and incubating
plates at 378C for 48 h. P. aeruginosa, S. aureus, E.coli
used in on-demand treatment water replenishment
were enumerated using Cetrimide media (Remel, Lenexa, sources (240 ppm). It is important to note that the
KS) supplemented with rifampicin (50 mg ml1), Mannitol relative loading rate in the on-demand vs pulse/con-
Salts media (Remel, Lenexa, KS), and eosin methylene tinuous treatments diered between crops; wheat
blue (EMB) agar (Difco Laboratories, Detroit, MI) sup-
plants in the on-demand treatment were exposed to
plemented with streptomycin (20 mg ml1) and rifampicin
(50 mg ml1), respectively. The use of anitobiotic-resistant greater levels of Igepon than those in the pulse and
S. aureus strains was not necessary due to less growth of continuous treatment, but the opposite was the case
non-target organisms on Mannitol Salts agar vs Cetrimide with the lettuce.
or EMB. ANOVA indicated a signicant eect of gray-
Plant growth measurements water treatments on the growth of lettuce, but not
wheat. Multiple range testing indicated a signicant
Each week PPF was measured manually with a hand-
held quantum sensor (LiCor Model LI-190SB, LiCor, Lin- reduction in dry mass of plants grown in the pulse
coln, NE). Nutrient solution samples were taken each treatment (72% of control), an intermediate re-
week to determine the concentration of plant nutrients. At
70 DAP (wheat) and 28 DAP (lettuce), plants were har-
vested by tray. Wheat plants were separated into roots,
shoots (stems and leaves), and heads. Lettuce plants were
separated into roots and shoots. Fresh mass determi-
nations were made of each plant tissue as well as number
of heads and diameter of lettuce shoots. Dry mass deter-
minations were made after tissues were dried in a forced-
air oven at 708C for 72 h. After drying, wheat heads were
threshed to determine seed dry mass (DM).

Experimental design
The experiment was repeated with trays randomized
within the chamber n 4: Analysis of variance was con-
ducted to test for signicant nutrient solution treatment
eect on the various plant yield parameters identied in
Tables 3 and 4. Duncan's multiple range test was used to
determine signicant dierences among dierent nutrient
solution treatment levels (i..e, control, pulse, continuous,
and on-demand). All analyses were conducted using SPSS Fig. 2. Cumulative addition of Igepon over the duration
software (Chicago, Ill.). Signicant dierences were of plant growth experiments. Data represent means of two
dened at PR0:05: replicate studies.
3080 J. L. Garland et al.

Table 4. Top dry mass (DM), root DM, and total DM growth response of lettuce through on-demand, continuous, and pulse additions
of Igepon compared to Hoagland's control. Values represent means per hydroponic tray n 4: Values in parentheses indicate S.D.
Within a column, values followed by dierent letters are signicantly dierent as determined by ANOVA and Duncan's Mean Separation
Test P < 0:05

Growth parameter Hoagland's On-demand Continuous Pulse

g plant1 % Control g plant1 % Control g plant1 % Control g plant1 % Control

Top DM 31.1 (5.7) a 100 31.6 (4.5) a 102 24.9 (4.1) ab 80 22.3 (4.7) b 72
Root DM 1.7 (0.4) a 100 2.1 (0.6) a 124 1.9 (0.3) a 112 1.8 (0.4) a 106
Total DM 32.8 (6.1) a 100 33.7 (5.1) a 103 26.8 (4.3) ab 82 24.2 (5.0) b 74

sponse in the continuous treatment (80% of con-
trol), and no eect in the on-demand treatment
(102% of control) (Table 3). Root mass was unaf-
fected by any of the treatments. Although no stat-
istically signicant eect was observed in wheat, the
average yield of seed and total mass was reduced
with graywater addition, particularly in the on-
demand (73% and 80% of control seed and total
yield, respectively) and pulse treatment (78% and
81% of control seed and total yield, respectively)
(Table 4).
Igepon persisted at signicant levels in the system
after the rst day of graywater treatment, but was
near or below the detection limit by the third day
of treatment in both the wheat (Table 5) and lettuce
(Table 6) studies. The increasing rate of Igepon loss
from the system was evident in time course
sampling during the wheat study (Fig. 3); nearly
100% of the Igepon was remaining after 24 h on
the rst day of addition, but was gone within 8 h
and 4 h on the 13th and 49th day of addition, re-
spectively. The rates of loss at the 13th and 49th
day of addition t the exponential decay well
R 2 0:810:94), allowing for estimation of the
Igepon half-life of 2.58 h and 0.76 h, respectively.
The rate of appearance (and disappearance) of
methyl taurine, a potential intermediate in the bio-
degradation of Igepon resulting from initial clea-
vage of the amide bond, increased with increasing
rates of Igepon loss from the system (Fig. 4). All
treatments (pulse, continuous, on-demand) were
pulsed with 0840 ppm Igepon 13 and 49 days after
initiation of graywater treatment, but data from all
treatments were pooled for presentation since no
signicant dierences in either Igepon or methyl
taurine dynamics were apparent among the treat-
ments.
The density of bacteria capable of growth on
media containing Igepon as a sole carbon source
are hereafter referred to as Igepon-degraders. The
reported densities are likely underestimations of the
real number of Igepon-degraders due to the general
inability to culture many types of organisms. The
Igepon concentration used in the media (750 ppm)
was higher than exposure levels, but allowed for
growth of larger more readily distinguishable colo-
nies than lower concentration (25 ppm) media (data
not shown). Incubation at 378C is also not repre-
sentative of conditions within the nutrient delivery
systems (0258C), but decreased the number of colo-
nies with spreading edges and concomitant over-
growth among colonies. The data are used to
evaluate relative dierences among treatments with
these limitations in mind.
The numbers of Igepon-degraders were signi-
cantly higher in hydroponic systems treated with
Igepon in both the wheat (Fig. 5) and lettuce
(Fig. 6) studies. The increased density in Igepon
treatments vs the control was greater, on average,
in the lettuce study compared to the wheat study,
and in biolms compared to the rhizosphere or
nutrient solution. The increase in Igepon-degraders
in the biolms was 1050-fold in the wheat study
and 1001000-fold in the lettuce. The increase in
Igepon-degraders in the rhizosphere was 010-fold
in the wheat study and 50100-fold in the lettuce
study. The increase in cells suspended in the nutri-
ent solution was 050-fold in both the lettuce and
wheat studies. The dierential response between
wheat and lettuce is largely due to lower densities

Table 5. Igepon concentration as MBAS (mg/ml) at 24 h following daily graywater treatments in hydroponic medium supporting wheat
growth

Rep 1 Rep 2

DAP Pulse On-demand Continuous Pulse On-demand Continuous

9 8.54 8.73 3.21 9.52 3.71 9.52

12 < MDLa < MDL 7.85 0.20 < MDL < MDL
14 0.10 0.28 0.35 < MDL < MDL 0.19
21 0.10 < MDL 0.11 < MDL 2.66 0.14
33 0.15 0.46 0.14 < MDL 0.15 0.12
47 < MDL 0.82 0.46 < MDL < MDL 0.14
57 0.11 1.18 1.78 0.30 < MDL 0.15

a
MDL (minimum detection limit)=0.08.
 Graywater processing in recirculating hydroponic systems
Table 6. Igepon concentration as MBAS (mg/ml) at 24 h following daily graywater treatments in hydroponic medium supporting lettuce
Rep 1
DAP Pulse On-demand
7 0.39 0.21
9 < MDLa < MDL
14 < MDL < MDL
21 < MDL < MDL
27 < MDL < MDL
a
MDL (minimum detection limit)=0.08.
in the lettuce control plants; the average density in
each of the habitats among Igepon-treated systems
were similar between the two crop types.
As observed with the plate counts, the increase in
total cell density in the Igepon treatments vs the
control was substantially higher in the lettuce study
compared to the wheat study, and in biolms com-
pared to other habitats (Figs 7 and 8). However,
Igepon treatment did not cause as large an increase
in total cell density as in Igepon-degrader density.
In the wheat study, Igepon treatment increased den-
sity (056-fold) only on biolms. In the lettuce
study, Igepon addition resulted in larger increases
(up to 10-fold) in cell density on biolms, and
increased density in the rhizosphere and nutrient
solution (025-fold).
Of the three human-associated bacteria added to
the system, only P. aeruginosa was consistently
detected several days after introduction in both
plant studies. In the wheat study, E. coli was
detected 4 days after addition in all habitats, and 11
days after addition in the rhizosphere and in bio-
lms (Fig. 9). S. aureus was not detected in any of
the habitats within 3 days of addition. In the lettuce
study, neither E. coli nor S. aureus was detected in
any of the habitats within 3 days of addition
Fig. 3. Concentration of Igepon, quantied as methylene
blue active substance, in the nutrient solution at various
days after initiation of Igepon treatment in systems con-
taining wheat. Data represent values from all three Igepon
treatments (on-demand, continuous, pulse) for both of the
wheat studies. Lines represent linear repression model for
day 1 of surfactant addition and exponential decay re-
gression model for days 13 and 49.
3081
Rep 2
Continuous Pulse On-demand Continuous
0.95 13.82 7.10 13.71
0.11 0.23 0.26 0.33
< MDL 0.23 0.32 0.36
< MDL 0.21 0.72 0.43
< MDL 0.30 0.19 0.32
(Fig. 10). P. aeruginosa, on the other hand, was
detected in all habitats for the duration of the
wheat and lettuce experiments, or up to 27 and 59
days after addition, respectively (Figs 9 and 10). P.
aeruginosa persisted at higher levels in the Igepon-
treated vs untreated systems (Fig. 11).
The greater persistence of P. aeruginosa may be
partially attributed to higher levels of inoculation.
The number of cells added to the nutrient solution
varied among the bacterial types as a result of
dierences in the cell density in the 18-h old culture
asks containing P. aeruginosa (1.06  109 2 1.65 
109 cells ml1), E. coli (6.97  108 21.65  108 cells
ml1) or S. aureus (2.99  108 2 1.65  109 cells
ml1). The 10-fold variation in inoculum level is
relatively small compared to the up to 1000-fold
dierences in persistence described above.
The density of the inoculated organisms in the
nutrient solution 10 min after addition were lower
than that predicted by dilution alone. A 1000-fold
dilution (10 ml of inoculum added to 10 l nutrient
solution tank) would yield higher values of P. aeru-
ginosa (01.0  106 cell ml1), E. coli (5  105 cells
ml1) and S. aureus (03  105 cells ml1) than
observed in both the wheat and lettuce studies (Figs
9 and 10). Adherence of the cells to hardware sur-
faces, roots, and wicking material during the 10 min
mixing period may have led to the poor recovery
Fig. 4. Concentration of methyl taurine in the nutrient sol-
ution at various days after initiation of Igepon treatment
in systems containing wheat. Data represent mean and
standard deviation of values from all three Igepon treat-
ments (on-demand, continuous, pulse) for both of the
wheat studies.
3082 J. L. Garland et al.
(i.e., 0110%) of cells in the nutrient solution. The
mixing period length was selected based on the esti-
mated time necessary to complete one turnover of
the nutrient solution tank volume (see Methods for
details).
DISCUSSION
Results indicate that the surfactant, Igepon, is
rapidly degraded within hydroponic systems. The
temporal increase in degradation rate, coupled with
a relative increase in the number of bacteria able to
utilize Igepon as a sole carbon source, indicate that
the degradation is microbially mediated. Additional
evidence for microbial degradation was the rapid
degradation on the rst day of surfactant addition
in the rst lettuce experiment, the only study in
which the hydroponic systems were not cleaned
with bleach prior to planting (Table 6). The corre-
lation between the rate of Igepon disappearance
and methyl taurine appearance (and subsequent dis-
appearance) indicates that the degradation pathway
involves initial cleavage of the amide bond, which is
consistent with previous reports of amidase-
mediated hydrolysis of sulfonated amide surfactants
(Sawyer et al., 1956; Sheers et al., 1967). To the
Fig. 5. Density of Igepon-degrading bacteria in the various
Igepon treatments and Hoagland's over time in the wheat
studies. Densities reported for organisms associated with
roots (rhizosphere), attached to wetted hardware surfaces
within the nutrient delivery system (biolms), and sus-
pended in solution (nutrient solution). Data represent
means and standard deviation of four samples (two from
each study) from each habitat at each sampling. Igepon-
degraders dened as the number of colony forming units
(CFU) on agar media containing minimal salts and
Igepon as a sole carbon source.
best of our knowledge, this is the rst time that
methyl taurine has been detected and quantied
along with Igepon degradation.
Igepon utilization appears to be a common trait
among microorganisms in hydroponic systems, as
evidenced by the signicant numbers of bacteria
(i.e., 0.11% of the total cells) capable of growth
on the Igepon media even in the control systems.
Igepon-degraders were associated with the plant
roots, biolms on hardware surfaces, and the nutri-
ent solution. The relative importance of the dier-
ent habitats in Igepon degradation was not
specically measured, but it can be estimated by
multiplying the measured density of Igepon-degra-
ders by the total size of each habitat (root mass,
wetted area in nutrient delivery system, and sol-
ution volume). For systems containing wheat, ap-
proximately 95% of the Igepon-degraders (as well
as total cells) were associated with plant roots,
while only 3.3 and 1.7% were on biolms and in
solution, respectively. For lettuce, which has a
much smaller root mass, only 60% of Igepon-degra-
ders and total cells were associated with roots,
while 30 and 15% were on biolms or in nutrient
solution, respectively. These estimates were based
on root mass at harvest, so the numbers of root as-
sociated bacteria would be much lower when plants
Fig. 6. Density of Igepon-degrading bacteria in the various
Igepon treatments and Hoagland's over time in the lettuce
studies. Densities reported for organisms associated with
roots (rhizosphere), attached to wetted hardware surfaces
within the nutrient delivery system (biolms), and sus-
pended in solution (nutrient solution). Data represent
means and standard deviation of four samples (two from
each study) from each habitat at each sampling. Igepon-
degraders dened as the number of colony forming units
(CFU) on agar media containing minimal salts and
Igepon as a sole carbon source.
 Graywater processing in recirculating hydroponic systems
are young. This eect would be greater for lettuce
in which most of the root growth occurs during the
last week, compared to wheat in which root mass
reaches maximal levels by the 3rd to 4th week of
growth. The larger root mass of wheat vs lettuce
(020 g vs 02 g at harvest), and concomitant num-
bers of root-associated microbes, may have more
readily assimilated the additional carbon, resulting
in the reduced stimulation in the number of total
and Igepon-degrading microorganisms observed in
the wheat vs lettuce study.
The increase in total cell density in response to
Igepon addition cannot be attributed to the
observed increase in Igepon-degraders; the density
of Igepon-degraders was no more than 10% of the
total cell counts, yet Igepon addition resulted in up
to 50-fold increases in total cell density in the let-
tuce study. Several reasons for this discrepancy can
be proposed. The method for detecting Igepon-
degraders may be capturing only a small fraction of
the total numbers of Igepon-degraders in the sys-
tems. Alternatively, metabolic by-products from the
Igepon-degraders, including intermediates in the
decompositon, may be supporting the growth of
other bacteria. Both of these hypotheses suggest
that the amount of Igepon added to the system rep-
resents a greater carbon ux than root-generated
carbon ux in the lettuce study, but not the wheat
Fig. 7. Total cell density (as estimated by acridine orange
staining and epiuorescent microscopy) in the various
Igepon treatments and Hoagland's over time in the wheat
studies. Densities reported for organisms associated with
roots (rhizosphere), attached to wetted hardware surfaces
within the nutrient delivery system (biolms), and sus-
pended in solution (nutrient solution). Data represent
means and standard deviation of four samples (two from
each study) from each habitat at each sampling.
3083
study. Preliminary evaluation supports this claim. If
one assumes that approximately 5% of net primary
productivity (NPP) is lost from the plants in the
form of root exudates and lysates (Barber and Mar-
tin, 1976; Martin, 1971; Martin and Kemp, 1986),
then root-derived organic material would have
amounted to 15 and 0.4 g in the wheat and lettuce
studies respectively. The cumulative amount of Ige-
pon added to the system was 1015 g in the wheat
study and 24 g in the lettuce study, indicating that
Igepon would represent a 1-fold and a 510-fold
increase in available organic matter in the wheat
and lettuce studies, respectively.
The rapid degradation rates (i.e., half-life <1 h
after initial acclimation period) indicate that the
hydroponic systems are eective bioreactors. The
surfactant loading rate in the batch and continuous
treatments was 0.17 g d1, or, based on plant
growth area, 0.87 g m2 d1. Approximately 10 m2
of growing area could process the per capita surfac-
tant production rates (810 g d1) estimated for
extraterrestial facilities. Higher loading rates to
wheat-based systems are currently being assessed to
quantify the minimum surface area required. Since
wheat evapotranspires an average of 5 l/m2
(Wheeler et al., 1996), at least 56 m2 would be
necessary to handle the per capita volume of
Fig. 8. Total cell density (as estimated by acridine orange
staining and epiuorescent microscopy) in the various
Igepon treatments and Hoagland's over time in the lettuce
studies. Densities reported for organisms associated with
roots (rhizosphere), attached to wetted hardware surfaces
within the nutrient delivery system (biolms), and sus-
pended in solution (nutrient solution). Data represent
means and standard deviation of four samples (two from
each study) from each habitat at each sampling.
3084 J. L. Garland et al.

graywater. Approximately 40 m2 of growing area

would be needed to support per capita caloric
requirements (Wheeler et al., 1996), suggesting that
ALS systems scaled for food production could
readily assimilate graywater uxes.
The percent reduction in total plant yield was
slightly less in wheat (819%) than lettuce (20
28%) at similar levels of graywater exposure but
still appreciable. However, statistical analyses indi-
cated no signicant eect in wheat. These results
could be interpreted as meaning that graywater had
no eect on wheat yield, or that the statistical sig-
nicance of actual eects were obscured by exper-
imental variation. Recently completed follow-up
studies indicated that surfactant loading rates sev-
eral times higher than those used in this study
caused no reduction in wheat growth (data not
shown), supporting the statistical analysis of the
present study.
The lack of phytotoxic eects in the wheat study
would agree with previous acute response studies
indicating a lack of plant growth inhibition at Ige- Fig. 10. Density of introduced human associated bacteria
pon concentrations below 250 ppm (Bubenheim et in the lettuce study over time. Densities reported for
al., 1997; Jacquez and Montoya, 1994). The concen- organisms associated with roots (rhizosphere), attached to
tration of Igepon, even immediately after pulse ad- wetted hardware surfaces within the nutrient delivery sys-
tem (biolms), and suspended in solution (nutrient sol-
ditions, did not exceed 2030 ppm, and the rapid ution). Data represent two samples from all four
microbial degradation prevented accumulation. treatments at each sampling.
However, signicant plant growth inhibition was

Fig. 11. Density of introduced human associated bacteria

Fig. 9. Density of introduced human associated bacteria in
the wheat study over time. Densities reported for organ-
isms associated with roots (rhizosphere), attached to
wetted hardware surfaces within the nutrient delivery sys-
tem (biolms), and suspended in solution (nutrient sol-
ution). Data represent two samples from all four
treatments at each sampling.
in the wheat study over time. Densities reported for organ-
isms associated with roots (rhizosphere), attached to
wetted hardware surfaces within the nutrient delivery sys-
tem (biolms), and suspended in solution (nutrient sol-
ution). Data represent mean and standard deviation of
samples from the control (Hoagland's solution) or from
all Igepon treatments.
 Graywater processing in recirculating hydroponic systems
apparent with lettuce, suggesting secondary toxic
eects, or a chronic eect threshold much lower
than the acute threshold. Na accumulation as a
result of Igepon addition ranged from 1.83.1 after
38 days in the lettuce studies, which is well below
the thresold levels of Na toxicity (20 mM) reported
for lettuce (Francois and Maas, 1999). Secondary
toxic eects (i.e., reduced oxygen availability, pro-
duction of secondary metabolites) may have been
associated with the increased microbial biomass in
the lettuce study. However, the on-demand treat-
ment, in which plant growth was not reduced, had
similar levels of microbial density as the continuous
and pulse treatments. Measurements of microbial
activity, rather than only numbers, could provide
more insight.
Rapid adsorption of Igepon to roots may lead to
greater phytotoxic eects than predicted from sol-
ution concentration. In fact, a 2535% reduction in
Igepon concentration in the bulk hydroponic med-
ium was observed within the rst 10 min after ad-
dition. The initial Igepon loss appears to be mainly
due to foaming and/or adsoprtion of Igepon to
hardware surfaces since 25% reductions were
observed even when added to systems with very
young lettuce plants in which root mass was negli-
gible. The dierential plant growth response, despite
similar rates of Igepon addition and degradation in
the lettuce and wheat studies, suggests that lettuce
is more sensitive to the surfactant, and may not be
a good candidate species for this mode of graywater
recycling.
The rapid numerical decline in E. coli and S. aur-
eus in the hydroponic systems is similar to 56 log
decreases within 510 days reported for introduc-
tion of these organisms into seawater (Cornax et
al., 1990) and lake water (Scheurman et al., 1988).
The long term persistence of P. aeruginosa is coun-
ter to the 5 log reduction within 14 days observed
in seawater (Cornax et al., 1990), but it is in agree-
ment with reports of its long term persistence in
various habitats, including bottled water (Tamag-
nini and Gonzalez, 1997; Warburton et al., 1994),
vegetables (Correa et al., 1991), tap water (Ferroni
et al., 1998), and water-retaining bath toys (Buttry
et al., 1998). More specically, the results agree
with previous short-term (i.e., 7 day) studies indi-
cating the P. aeruginosa persisted on the hydroponi-
cally grown wheat seedlings to a greater degree
than either E. coli or S. aureus (Morales et al.,
1996). Although the overall density declined during
the study, closer analysis of the data suggests that
P. aeruginosa may have been actively growing in
the system. Similar or even slightly declining density
in the rhizosphere would reect increases in total
cell number since roots were growing. Numerical
increases in the rhizosphere were particularly signi-
cant in the lettuce study since the density of P. aer-
uginosa was relatively constant in 7 and 30 day old
plants, but total root mass increased at least 10-
3085
fold. P. aeruginosa may have been using Igepon as
a growth media based on its increased survival in
the Igepon treated systems and our previous obser-
vations that strains of P. aeruginosa isolated from
human shower water could grow on Igepon sole
carbon source media (data not shown).
These data suggest that human-health risks may
be associated with this graywater recycling
approach. P. aeruginosa, while not a signicant risk
to healthy individuals, causes 1011% of all noso-
comial infections in hospitals (Botzenhart and Dor-
ing, 1993) and has the highest fatality rate of all
hospital-acquired bacteremias (Bryan et al., 1983).
Susceptibility to opportunistic pathogens may be
greater in ALS systems given reports of decreased
immune response under spaceight conditions
(Konstantinova et al., 1993; Taylor, 1993). Methods
for mitigating the health risks depends on expected
exposure routes. The risk of food contamination is
minimal from a crop like wheat in which the edible
portion (seed) is not in contact with the nutrient
solution and is cooked prior to eating. However,
harvesting the systems could lead to exposure of
human pathogens persisting on the plant roots.
These data indicate that this risk can be reduced by
stopping graywater addition for 0a week prior to
harvest, but not eliminated. Further studies with
actual human shower water are needed to quantify
the human health risks. Use of the molecular-based
approaches to assess persistence is also needed to
determine the risk associated with viable but non-
culturable cells (Roszak and Colwell, 1987).
CONCLUSIONS
1. Igepon added to recirculating hydroponic sys-
tems was rapidly removed from solution (i.e.,
half life <1 h) via microbially-mediated degra-
dation involving initial amide cleavage of the sur-
factant molecule.
2. Igepon loading rates of up to 1 g m2 growing
area d1 signicantly reduced the growth of let-
tuce, but not wheat. The reason for the dieren-
tial growth response is unknown, but is unlikey
to be related to direct phytotoxicity of the surfac-
tant since Igepon concentrations were, at worst,
an order of magnitude less than reported acute
toxicity thresholds.
3. Surfactant loading increased total microbial
abundance in the system. Stimulation was greater
in the lettuce vs wheat studies, and on biolms
colonizing hardware surfaces compared to on
root surfaces or in suspension.
4. Two common bacterial contaminants of gray-
water, E. coli and S. aureus decreased below
detection limits within 411 days after introduc-
tion to the system, while a third, P. aeruginosa,
persisted for the duration of the experiments (27
or 57 days). These data suggest that human
3086 J. L. Garland et al.
health risks associated with harvesting the system
can be greatly minimized, but not completely
eliminated, by stopping graywater addition sev-
eral days prior to harvest.
AcknowledgementsThe authors wish to thank Lisa M.
Rue, Nathan A. Cranston, and Hollie R. Kagie for hor-
ticultural assistance with this study, J. Scott Young for en-
gineering support and development of Fig. 1 in the text,
Holly D. Loesel for engineering support of the growth
chamber and graywater control systems, Teresa H.
Englert, Steven P. Black, and John R. Levy for inorganic
chemistry analysis of nutrient solutions, and Maureen
Johnson for assistance in the identication of introduced
bacteria.
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