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Antimicrobial Mitigation via Saponin Intervention on Escherichia coli and Growth and Development of Hydroponic Lettuce


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Various saponins have demonstrated allelochemical effects such as bactericidal impacts as well as antimycotic activity against some plant pathogenic fungi, thereby acting to benefit plant growth and development. A commercial saponin solution was evaluated for bactericidal effects against Escherichia coli and growth of lettuce ( Lactuca sativa ) in a hydroponic system. E. coli (P4, P13, and P68) inoculum at final concentration of 10 ⁸ colony-forming units (cfu)/mL was added to 130 L of a fertilized solution recirculating in a nutrient film technique (NFT) system used to grow ‘Rex’ lettuce. After 5 weeks in the NFT system, E. coli populations were lowest in the inoculated treatment that did not contain any saponin addition (0.89 log cfu/mL) when compared with all other inoculated treatments ( P < 0.001). The treatment containing 100 µg·mL ⁻¹ saponin extract had an E. coli population of 4.61 log cfu/mL after 5 weeks that was higher than treatments containing 25 µg·mL ⁻¹ or less ( P < 0.0001). Thus, higher E. coli populations were observed at higher saponin concentrations. Plant growth was also inhibited by increasing saponin concentrations. Fresh and dry shoot weight were both higher in the inoculated and uninoculated treatments without the saponin addition after 5 weeks in the NFT system ( P < 0.0001). Lettuce head diameter was smaller when exposed to saponin treatments with concentrations of 50 and 100 µg·mL ⁻¹ ( P < 0.0001). Lettuce leaves were also tested for the potential of E. coli to travel systemically to the edible portions of the plant. No E. coli was found to travel in this manner. It was concluded that steroidal saponins extracted from mojave yucca ( Yucca schidigera ) are not an acceptable compound for use in mitigation of E. coli in hydroponic fertilizer solution due to its ineffectiveness as a bactericide and its negative impact on lettuce growth.
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Antimicrobial Mitigation via Saponin
Intervention on Escherichia coli and Growth
and Development of Hydroponic Lettuce
Nathan J. Eylands
, Michael R. Evans
, and Angela M. Shaw
ADDITIONAL INDEX WORDS. bactericide, foodborne diarrheal disease
SUMMARY. Various saponins have demonstrated allelochemical effects such as bac-
tericidal impacts as well as antimycotic activity against some plant pathogenic fungi,
thereby acting to benefit plant growth and development. A commercial saponin
solution was evaluated for bactericidal effects against Escherichia coli and growth of
lettuce (Lactuca sativa) in a hydroponic system. E. coli (P4, P13, and P68) inoculum
at final concentration of 10
colony-forming units (cfu)/mL was added to 130 L of
a fertilized solution recirculating in a nutrient film technique (NFT) system used to
grow ‘Rex’ lettuce. After 5 weeks in the NFT system, E. coli populations were lowest
in the inoculated treatment that did not contain any saponin addition (0.89 log
cfu/mL) when compared with all other inoculated treatments (P<0.001). The
treatment containing 100 mg
saponin extract had an E. coli population of
4.61 log cfu/mL after 5 weeks that was higher than treatments containing 25
or less (P<0.0001). Thus, higher E. coli populations were observed at
higher saponin concentrations. Plant growth was also inhibited by increasing sa-
ponin concentrations. Fresh and dry shoot weight were both higher in the in-
oculated and uninoculated treatments without the saponin addition after 5 weeks in
the NFT system (P<0.0001). Lettuce head diameter was smaller when exposed to
saponin treatments with concentrations of 50 and 100 mg
Lettuce leaves were also tested for the potential of E. coli to travel systemically to the
edible portions of the plant. No E. coli was found to travel in this manner. It was
concluded that steroidal saponins extracted from mojave yucca (Yucca schidigera)
are not an acceptable compound for use in mitigation of E. coli in hydroponic fer-
tilizer solution due to its ineffectiveness as a bactericide and its negative impact on
lettuce growth.
Every year, 48 million Ameri-
cans become infected from
a foodborne disease; 128,000
of whom require hospitalization
resulting in 3000 deaths [Centers
for Disease Control and Prevention
(CDC), 2019b]. Although healthy
foods are an important part of
a well-rounded diet, there are con-
cerns, as fruits, vegetables, and nuts
accounted for 23% of reported human
foodborne illness outbreaks between
2009 and 2015 (CDC, 2017a,
2017b). Escherichia coli is one of the
most prominent causes of foodborne
diarrheal disease in humans. It is also
a leading contributor to bacterial in-
fections and extraintestinal infections
in humans and animals alike (Njage
and Buys, 2014). A primary source of
E. coli infection in the United States is
through contaminated agricultural
products. In 2019, the CDC reported
two multistate outbreaks related to E.
coli O157:H7 in leafy greens (CDC,
Controlled environment agricul-
ture (CEA) production facilities, such
as greenhouses and plant factories,
present advantages over traditional
field production, such as crop space
efficiency and year-round growth,
and they also provide a reduced risk
for food safety issues (Holvoet et al.,
2015). Although contamination risks
are reduced in CEA, they are not
eliminated (Orozco et al., 2008) and
therefore water quality used for irri-
gating crops is a concern, as contam-
inated source water (municipal,
holding pond, or well) or fertilizer
solution can splash onto crops during
production and harvesting, leading to
E. coli infection in humans (Solomon
et al., 2003). Additional risks within
CEA pre- and postharvest production
include pest and wild animal manage-
ment, planting substrates, unsanitary
equipment and buildings, and human
handling (Holvoet et al., 2015).
Within CEA, an operational
choice of production system estab-
lishes the likelihood of microbial
spread in the event of contamination.
Hydroponic production is a popular
practice among CEA producers, add-
ing greater efficiency and control to
their cultivation processes over tradi-
tional farming practices. E. coli ex-
hibits the ability to thrive in fertilizer
solutions in a hydroponic system
(Shaw et al., 2016). This presents
a unique problem for growers who
use a recirculating hydroponic sys-
tem. Because a fertilizer solution is
collected and recycled through irriga-
tion lines, a microbial contaminant
has the potential to infect not some,
but the entire crop, continually recy-
cling a solution of pathogens (Premuzic
et al., 2007).
Although contamination poten-
tial remains low in CEA, preventive
measures to disinfest source water
must be a focal point for food safety
and public health. Cultural practices,
such as personal hygiene and sick
employee protocols, will always re-
main an important area in produce
production, but further disinfestation
To convert U.S. to SI,
multiply by U.S. unit SI unit
To convert SI to U.S.,
multiply by
29,574 fl oz mL 3.3814 ·10
29.5735 fl oz mL 0.0338
3.7854 gal L 0.2642
2.54 inch(es) cm 0.3937
1 mmho/cm dSm
1 ppm mgL
1 ppm mgmL
(F – 32) O1.8 FC(C·1.8) + 32
Received for publication 7 Oct. 2020. Accepted for
publication 28 Dec. 2020.
Published online 28 January 2021.
Horticulture Section, College of Agriculture and Life
Sciences, Cornell University, 135 Plant Science Build-
ing, Ithaca, NY 14853
School of Plant and Environmental Sciences, Virginia
Polytechnic Institute and State University, 328 Smyth
Hall, Blacksburg, VA 24061
Department of Food Science and Human Nutrition,
Iowa State University, 2577 Food Sciences Building,
Ames, IA 50011
N.J.E. is the corresponding author. E-mail: nje9@
This is an open access article distributed under the CC
BY-NC-ND license (
licenses/by-nc-nd/4.0/). 1of7
measures will help safeguard a crop to
be free of microbial pathogens like E.
coli. Current techniques used to mit-
igate microbial pathogens can be ef-
fective but have cost and complexity
limitations that prohibit their use to
many farmers. Ultraviolet radiation is
effective and widely used; however,
small pathogens may pass by the light
waves in the shadow of debris and
remain active, therefore filtration of
the water and cleaning of the ultravi-
olet lamp (Garibaldi et al., 2004) are
paramount to this method’s useful-
ness. Biofiltration may reduce patho-
gens, but does not eliminate them
(Belbahri et al., 2007; Wohanka,
1995). Chlorination may cause phyto-
toxic symptoms to plants (Premuzic
et al., 2007) and produce the by-
product trihalomethane, which is clas-
sified by the U.S. Environmental Pro-
tection Agency as a potential human
carcinogen (Symons et al., 1981).
Natural antimicrobials are be-
coming more prevalent among mi-
crobial disinfestation methods in the
food industry (Zhu et al., 2015).
Plant-based isolated compounds con-
tain secondary metabolites that are
known to retard or inhibit the growth
of bacteria, yeasts, and molds (Tiwari
et al., 2009). Saponins are secondary
metabolites widely distributed
throughout the plant kingdom and
have been documented to exhibit
natural antibacterial properties (Lokesh
et al., 2016; Wallace, 2004). Saponins
are nonionic detergents that have an
assortment of biological properties.
Their structure is composed of a steroi-
dal or triterpenoid aglycone skeleton
attached to one or more sugar chains
(Arabski et al., 2011). This diversity in
structure is what leads to the great
diversity in biological properties. Be-
yond their bactericidal functions, sapo-
nins also display antifungal, hemolytic,
membrane-depolarizing, ammonia-
binding, antiyeast, antimold (Arabski
et al., 2011; Oleszek, 1996), and many
other natural biological properties.
Their effects are generally credited to
their ability to permeate cellular mem-
branes (Francis et al., 2002).
The following study was con-
ducted to investigate the antimicro-
bial properties of steroidal saponins
extracted from mojave yucca (Yucca
schidigera) on gram-negative E. coli in
a hydroponic fertilizer solution over
time. In addition, the growth and
development of lettuce (Lactuca sativa)
grown in an NFT hydroponic system
was evaluated for yield parameters.
Materials and methods
fore each replication, all materials
(lettuce seedlings, municipal tap wa-
ter, and hydroponic equipment) used
in this study were analyzed (using the
same enumeration protocol in the
section ‘‘Data Collection and Bacte-
rial Enumeration’’) and found to be
negative for the presence of E. coli cfu
(data not shown; detection limit was
100 cfu/mL).
isolates of nonpathogenic E. coli (P4,
P13, and P68) were obtained from
the culture collection of the Microbial
Food Safety Laboratory, Iowa State
University, Ames, IA. Isolate selec-
tions were based on behavioral simi-
larities to E. coli O157:H7 (Marshall
et al., 2005). All strains were adapted
to grow in the presence of 80 mgmL
rifampicin (Thermo Fisher Scientific,
Waltham, MA), through stepwise ex-
posure (Parnell et al., 2005). Parnell
procedure: Briefly, 100 mL of an over-
night culture was spread onto plate
count agar containing antibiotic. Af-
ter incubation for 24 h at 37 C,
isolated colonies were selected from
the plate containing the highest level
of antibiotic and cultured overnight
in nutrient broth. This procedure was
repeated until a variant resistant to 80
rifampicin was obtained.
Growth curves of the parent and variant
strains were similar in tryptic soy broth
(Difco Laboratories, Detroit, MI) (data
not shown). Bacterial strains were sub-
sequently combined into a cocktail sus-
pended in a cryoprotective glycerol
solution and stored at –21 C. Frozen
cultures were thawed in cold water,
(BPW), and incubated at 37 C for 24
mL. The resulting solution was used to
inoculate irrigation water in the follow-
ing experiment.
SOLUTION.Under ambient green-
house light, foam hydroponic seed
germination media (276-cell count
Horticubes; Smithers Oasis, Kent,
OH) was placed in sub-irrigated hy-
droponic propagation trays (Ameri-
can Hydroponics, Arcata, CA), where
they were leached, and seeded with
‘Rex’ lettuce (Johnny’s Selected
Seeds, Winslow, ME). Greenhouse
temperature setpoints were set to
cool at 21 C and heat at 18 C.
Average recorded daily light integral
across all experimental replications
was 18 molm
. Before seedling
transplantation, six separate NFT sys-
tems were filled with 130 L tap water
and allowed to recirculate for 24 h. At
this point, selected systems were in-
oculated with 20 mL of E. coli cocktail
to obtain a population of 10
mL. Dissolved fertilizer salts and 1 M
sulfuric acid (H
) were added to
create 130 L of fertilizer solution with
an electrical conductivity of 1.4
and a pH of 5.9, which was
maintained daily in all systems
throughout the study. Subsequently,
a saponin intervention was added.
The saponin product used in this
study was supplied in a premixed so-
lution (Micro-Aid Liquid 50; DPI
Global, Porterville, CA), and con-
tained sapogenin with a steroidal
aglycone structure extracted from
mojave yucca. The solution was cer-
tified by the Organic Materials Re-
view Institute and contained 14%
saponins. It was supplied to each 130-
L irrigation reservoir at treatment rates
of 0, 12.5, 25, 50, or 100 mgmL
Saponin treatments were randomly
assigned to each NFT system. The
methods reported here were repeated
in three experimental replications. Sys-
tem water loss due to transpiration and
evaporation was replenished weekly
with a commensurate amount of sapo-
nin solution. Seedlings were trans-
planted into the NFT systems at the
four-true-leaf stage.
solution samples of 25 mL were taken
at 1 h after inoculation and then 1,
168, 336, 504, 672, and 840 h after
saponin intervention. To ensure a ho-
mogeneous sample, 5-mL aliquots
were taken from five separate loca-
tions within each system: top half of
the nutrient reservoir, lower half of
the nutrient reservoir, drain collector,
NFT channel, and dripper emitter.
These samples were used to evaluate
viable E. coli populations in each NFT
Enumeration of E. coli popula-
tions was determined by serial dilu-
tions using BPW as the dilution
solution. Dilutions were plated on
MacConkey agar (0.1% rifampicin)
containing a tryptic soy agar (TSA)
overlay using a spread plate technique.
Plates were incubated at 37 C for 24 h
before manual counts. Presence of
rifampicin was to ensure bacteria were
accurately detected in the presence of
high natural flora.
Five lettuce plants were selected
using a random number generator
and collected for analysis from each
treatment after 5 weeks (840 h) post-
transplant into the NFT systems. The
first three plants selected were evalu-
ated for growth characteristics and
the subsequent two plants were tested
for the presence of E. coli.
Plants that had been designated
for measuring growth characteristics
were weighed immediately to deter-
mine fresh shoot weight on a digital
balance (AP250D; Ohaus, Parsippany,
NJ). Lettuce plant diameter was mea-
sured at the widest point before plants
were placed inside a paper bag and into
an oven. Plants were heated at 70 C
for 2 d to fully desiccate before obtain-
ing dry weights.
Plants that had been designated
to be tested for E. coli presence were
harvested as described earlier and
then immediately transferred into
14 ·19-inch sterile sample bags
(Nasco, Fort Atkinson, WI). Plant
weight was determined using a digital
balance (AP250D) to create a 1:10
(w/v) dilution with deionized water.
The bag contents were then manually
stomached to suspend internal micro-
organisms. The resulting solution was
used to determine presence or ab-
sence of E. coli on or within the edible
portions of the lettuce leaves by the
enumeration techniques described
cation of E. coli concentration sam-
ples were log (log
) transformed
before analysis. Each sample was uni-
laterally increased by one to prevent
syntax error to any zero counts. The
noninoculated treatment without sa-
ponin addition was removed from the
analysis due to lack of variability and
influence on the remaining dataset.
The factorial analysis was performed
as a repeated measure using a Stu-
dent’s ttest least significant difference
to examine mean separation. Plant
growth and development data were
normalized by examining each mea-
surement as a percentage of the
noninoculated treatment without
saponin addition mean for that block.
A one-way analysis of variance was
performed at each time point to eval-
uate mean differences. Mean separa-
tion was determined using a Tukey’s
honestly significant difference. All
analyses were performed using JMP
Pro (version 14.0.0; SAS Institute,
Cary, NC).
GROWTH OF E. COLI.Lettuce plants
that were evaluated for the presence
of E. coli every week were not found
to have any recoverable populations
compartmentalized within the edible
portions of the plant (data not
shown). The timing of treatment
and treatment itself were significant
effects in the analysis (P<0.0001, P<
0.0001). Table 1 displays the effects
of saponin on E. coli at the various
time periods. Through the experi-
ment, the treatment with no inocu-
lum and no saponins remained with
no recoverable E. coli at all time
periods. After 1 h from inoculation
of the recirculating tap water in the
NFT systems, all inoculated treat-
ments had no saponin addition and
contained E. coli at 0.6 to 0.83 log
cfu/mL (P= 0.74).
After 2 h from inoculation (1 h
from the saponin addition), all in-
oculated treatments containing a sa-
ponin addition had similar amounts
of E. coli between the treatment levels
[<0.001–1.11 log cfu/mL (P=
After 168 h (1 week) from the
saponin addition, all treatments in-
creased E. coli cfus by at least 2 logs
(2.49–5.28 log cfu/mL) and were
different (P= 0.001). The inoculated
treatment without saponin (2.49 log
cfu/mL) was similar to the treat-
ments with saponin concentrations
of 12.5 and 25 mgmL
, which had
3.57 and 3.7 log cfu/mL, respec-
tively. Similarly, the treatments with
the highest concentrations of saponin
(50 and 100 mgmL
) yielded the
highest amount of E. coli ranging
from 5.08 to 5.28 log cfu/mL, which
were similar results.
Treatment differences persisted
after 336 h (2 weeks) from the sapo-
nin addition (P= 0.0001). The in-
oculated treatment without saponin
had 2.17 log cfu/mL, which was
similar to the other treatments with
saponin concentrations of 12.5 and
25 mgmL
(3.3 and 3.45 log cfu/
mL). The inoculated treatments con-
taining saponin concentrations of 50
and 100 mgmL
had the highest
yields of E. coli populations with
5.02 and 5.61 log cfu/mL, respec-
tively, and were different from all
other treatments.
After 504 h (3 weeks) from
the saponin addition, differences
remained (P= 0.0001). The inocu-
lated treatment without saponin ex-
perienced a reduction in E. coli
population from the previous week
to 1.53 log cfu/mL, which was lower
than all other inoculated treatments.
The inoculated treatment containing
a saponin concentration of 12.5
had 3.48 log cfu/mL and
was similar to the inoculated treat-
ments with saponin additions of 25
and 50 mgmL
, but not to the
treatment with a saponin concentra-
tion of 100 mgmL
. The inoculated
treatment with a saponin concentra-
tion of 100 mgmL
had a slight re-
duction in E. coli population from the
previous week, yet still carried the
highest amount at the 3-week time
period with 5.23 log cfu/mL.
After 672 h (4 weeks) from the
saponin addition, treatment differ-
ences persisted (P= 0.0001). The
inoculated treatment without sapo-
nin reduced in population to 0.89 log
cfu/mL, which was lower and differ-
ent from all other treatments. The
inoculated treatments with saponin
additions of 12.5 and 25 mgmL
contained E. coli populations of 2.84
and 2.91 log cfu/mL, respectively.
These treatments experienced a re-
duction for the first time and were
also similar to each other. The in-
oculated treatments with saponin ad-
ditions of 50 and 100 mgmL
populations of E. coli at 4.39 and 4.77
log cfu/mL, which were similar to
one another and different from treat-
ments containing saponins at concen-
trations of 25 mgmL
or lower.
At the final time point, 840 h (5
weeks) from the saponin addition,
treatment differences remained (P=
0.0001). The inoculated treatment
without saponin maintained a low
population (0.89 log cfu/mL),
whereas inoculated treatments con-
taining lower saponin concentrations
of 12.5 and 25 mgmL
reduced in
recoverable E. coli populations and
were similar to the inoculated 3of7
treatment without saponin and con-
tained populations that ranged from
2.15 to 2.32 log cfu/mL. The in-
oculated treatments with saponin ad-
ditions of 50 and 100 mgmL
contained lower amounts of E. coli,
yet were higher than other treatments
with 3.21 and 4.61 log cfu/mL and
were similar to one another.
840 h (5 weeks) in the NFT system,
the noninoculated treatment that did
not contain saponins and the inocu-
lated treatment that did not contain
saponins were the highest in terms of
fresh shoot weight and dry shoot
weight (Table 2) and were only sim-
ilar to the inoculated treatment with
a saponin addition of 25 mgmL
0.0001 and P<0.0001, respectively).
The inoculated treatment containing
a saponin concentration of 12.5
was similar in fresh and dry
shoot weight to the treatment with
25 mgmL
of saponins. The inocu-
lated treatment with a saponin addition
of 50 mgmL
had lower fresh and dry
shoot weight than the lower concen-
trations of saponins; however, it had
a higher fresh and dry shoot weight
than the inoculated treatment with
a saponin addition of 100 mgmL
Lettuce head diameter after 840
noninoculated treatment without sa-
ponins and the inoculated treatment
without saponins (Table 2). How-
ever, they were similar (P<0.0001)
to the two inoculated treatments
with the lower levels of saponins
(12.5 and 25 mgmL
). The inocu-
lated treatment with a saponin addi-
tion of 50 mgmL
had a smaller
head diameter than the treatments
with no saponins and lower-level
saponins. The inoculated treatment
with a saponin addition of 100 mgmL
had a head diameter that was smaller
than all other treatments evaluated.
Saponin concentration had the
most influential effect on growth of
E. coli. Over time, all treatments
exhibited growth and decline (Fig.
1). The rate of growth and decline
were affected by the presence and
level of the saponin treatment. The
experimental hypothesis was that
saponins would have an antibacterial
effect on E. coli. This would suggest
that more saponins would equate to
less E. coli. The resulting outcome of
the experiment was the opposite.
The greatest population of E. coli
was consistently found in the inoc-
ulated treatment containing the
highest concentration of saponins.
At its highest population (336 h),
this treatment produced 3 log in-
creases over treatment 2, which con-
tained no saponin addition. This
result was reliably seen at every time
point beyond the initial first hours
of the experiment.
These results were consistent
with those found in the work of
Arabski et al. (2011) on triterpenoid
saponins who also observed an en-
hancement of E. coli growth when
exposed to saponins. The current
experiment was conducted using
steroidal saponins extracted from
mojave yucca found in the southwest
United States and northwest Mexico.
As discussed previously in this article,
the aglycone structure of each sapo-
nin compound determines its biolog-
ical properties. Using the results from
this study and those found by Arabski
et al. (2011), both steroid and triter-
penoid saponins react similarly to
stimulate the growth of E. coli. The
leading postulate to the reasoning of
increased bacterial growth is that sa-
ponins increase cell permeability and
the influx of nutrients (Arabski et al.,
2011). Instead of opening inter-
cellular space to potentially harmful
Table 2. Mean fresh and dry shoot weight and plant diameter of lettuce reported
as a percentage of the control grown in a hydroponic fertilizer solution with 0,
12.5, 25, 50, and 100 mg
(ppm) saponin treatment at time of harvest (5
Plant characteristic
Treatment Fresh wt Dry wt Diam
E. coli
Saponin (mg
) (% control)
0 100 a
100 a 100 a
+ 0 101 a 95 a 98 a
+ 12.5 75 b 70 b 89 a
+ 25 80ab 87ab 88a
+ 50 44c 46c 74b
+ 100 14 d 16 d 48 c
Positive (+) indicates presence of Escherichia coli inoculated at an initial population of 10
colony-forming units
(cfu)/mL, negative (–) indicates E. coli not present; 1 cfu/mL = 29.5735 cfu/fl oz.
Mean responses displayed as percent of noninoculated treatment without saponin (without E. coli, without
saponin addition). All data were pooled from three replications with three subsamples each (n = 9).
Means with different letter(s) are significantly different using a Tukey’s honestly significance difference test at P£0.05.
Table 1. Comparison of Escherichia coli colony-forming unit (cfu) grown in hydroponic fertilizer solution with 0, 12.5, 25,
50, and 100 mg
(ppm) saponin treatment over time.
Time (h)
Treatment 1
168 336 504 672 840
E. coli
Saponin (mg
) Mean (log cfu/mL D1)
0 0 000000
+0<0.001 a
<0.001 a 2.49 a 2.17 a 1.53 a 0.89 a 0.89 a
+ 12.5 0.16 a 0.42 a 3.57 a 3.30 a 3.48 b 2.84 b 2.32 ab
+ 25 0.32 a 0.79 a 3.70 ab 3.45 a 4.01 bc 2.91 b 2.15 ab
+ 50 0.83 a 0.88 a 5.08 bc 5.02 b 4.70 bc 4.39 c 3.21 bc
+ 100 0.72 a 1.11 a 5.28 c 5.61 b 5.23 c 4.77 c 4.61 c
Saponin treatment intervention not yet applied.
Enumeration occurred 1 h post saponin treatment intervention.
Positive (+) indicates presence of E. coli inoculated at an initial population of 10
cfu/mL, negative (–) indicates E. coli not present.
1 cfu/mL = 29.5735 cfu/fl oz.
Means with different letter(s) are significantly different using a Student’s ttest at P£0.05. All data were pooled from three replications with two subsamples each (n = 6).
extracellular conditions, newly formed
pores in bacterial membranes allow the
passage of nutrients to flow into the
cell, allowing E. coli to prosper.
Another possible explanation to
the higher populations of E. coli in
higher concentrations of saponins re-
volves around bacterial structure. E.
coli are gram-negative bacteria, and in
research are harder to kill than gram-
positive bacteria with a peptidoglycan
layer. Previous experimenters have
elucidated the antibacterial effects of
saponins against other gram-negative
bacterium (Khan et al., 2018; Mandal
et al., 2005). A large difference be-
tween those experiments and this
experiment is the addition of plants
into the system ecology. A plant’s
rhizosphere can contain up to 100
times the amount of microorganisms
found in soil without plants (Haas
et al., 2002). This rich biodiversity of
microbes is home to a group known
as rhizobacteria, which produce ben-
eficial secondary metabolites that en-
hance plant growth through a variety
of mechanisms (Sturz and Christie,
2003). A few notable rhizobacteria
are found within the genera Pseudo-
monas,Streptomyces, and Bacillus
(Emmert and Handelsman, 1999;
Haas et al., 2002). Brown et al.
(1976) were able to isolate naturally
occurring sulfur-containing carbox-
ylic acids from strains of Streptomyces,
which are very potent inhibitors of E.
coli. Soetan et al. (2006) reported that
saponins only produced inhibitory
effects on gram-positive bacteria,
contrary to Khan et al. (2018) and
Mandal et al. (2005). As previously
noted, E. coli are gram-negative;
however, Streptomyces is a gram-pos-
itive bacterium, leading the investiga-
tor in the current study to postulate
that higher saponin concentrations
inhibited beneficial rhizobacteria
like Streptomyces, which allowed E.
coli to survive in a less-competitive
It is important to note that early
time points in this experiment had
very low populations of E. coli to
report. A study by Cooper et al.
(2001) involving E. coli thermal de-
pendence also indicated that most
bacterial loss was seen in early stages
of the experiment, when adaptation
is the most rapid. Bacterial injury
was observed on a great deal of the
TSA plates. Typical colony morphol-
ogy appeared circular, convex, and
smooth. E. coli that were recovered
were irregular in shape and size.
Initially this experiment used Mac-
Conkey agar without the TSA over-
lay. Recovery became increasingly
lower as water temperatures dropped
in the nutrient reservoirs due
to changing seasons. E. coli will
grow across the temperature range
of 10 to 49 C, but it will grow at
a progressively slower rate when tem-
perature is raised above 40 Cor
below 20 C (Cooper et al., 2001;
Jones et al., 1987). Water temperature
readings were below 20 C for the
early stages of the first two replica-
tions. E. coli was present (indicated by
subsequent aliquots), but in low num-
bers and in some cases undetectable. A
pre-enrichment step was deemed nec-
essary to facilitate bacterial recovery
(McKillip, 2001). In this case, it was
the addition of TSA to the MacCon-
key plates in the form of an overlay.
This gave injured bacteria an opportu-
nity to repair themselves in the nutri-
ent-rich environment and increased
laboratory success in proper enumera-
tion of E. coli (Smith et al., 2013).
Results for E. coli presence within
the edible portions of lettuce were
omitted from the statistical analysis
due to the simplicity of the findings.
An E. coli presence or absence screen-
ing was conducted on a total of 172
lettuce plants throughout the duration
of the experiment. No contaminated
plants were found, indicating that E.
coli cannot be internalized from the
rhizosphere into the root system grow-
ing in a hydroponic system. This evi-
dence is contrary to that found by
Solomon et al. (2002). The discrepancy
of the before-mentioned study and this
study could be the result of differing
identification techniques. Solomon
et al. (2002) used sophisticated micros-
copy for detection of bacterial internal-
ization. They also grew plants in soil
rather than a hydroponic system. How-
ever, this study is supported by Hora
et al. (2005) who did not find internal-
ization in aerial plant portions of spin-
ach (Spinacia oleracea) when roots had
been inoculated in soil containers.
Although mean separations were
found at individual time points for
lettuce growth and development pa-
rameters, the most important time
point to address is 840 h (week 5). This
time point reflects the most accurate
time of maturation for ‘Rex’ lettuce and
therefore conveys the most fundamen-
tal information to a grower considering
the use of saponins in a recirculating
hydroponic NFT system. Under every
growth measurement, the noninocu-
lated treatment without saponin addi-
tion and the inoculated treatment
without saponin addition produced
the highest yields on average. The fact
that these treatments were the only
Fig. 1. Growth curves of Escherichia coli populations grown in hydroponic
fertilizer solution with 0, 12.5, 25, 50, and 100 mg
saponin treatment over
time. All data were pooled from three replications with two subsamples each (n =
6); 1 mg
=1 ppm, 1 cfu/mL =29.5735 cfu/fl oz. 5of7
treatments tested that did not include
the saponin intervention indicates the
economic impracticality of this treat-
ment as a mitigation tool for E. coli or
tuce in an NFT system.
Reduced growth of lettuce was
clearly related to an increase in sapo-
nin solution. It is difficult to say
whether this reduced growth pattern
was due to the active ingredient (ste-
roid saponins) or other ingredients
within the solution or a combination
of these factors. The provided sapo-
nin solution used in this experiment is
not currently on the market; however,
there are similar products available to
consumers from the manufacturer.
These similar products are used as
supplements for livestock feed to
control ammonia and other noxious
gasses in the immediate environment,
conveying air-quality improvements.
The formulation of the tested saponin
extract product is not necessarily
engineered for plant growth in a hy-
droponic NFT system.
The most likely cause of limited
plant growth at higher concentrations
of saponins is an increase in damaged
plant cell membranes. Saponins are
nonionic surfactants, which have phy-
totoxic effects on plant membranes
by increasing permeability (Riechers
et al., 1994). The damage caused to
the root zone may have inhibited
nutrient uptake and retarded the
growth cycle.
Another postulate worth consid-
ering encompasses dissolved oxygen
(DO) in the nutrient water. Unfortu-
nately, due to equipment failures, DO
was not measured across all replica-
tions of the experiment and therefore
not included in the statistical analysis.
Lettuce grows sufficiently at DO
levels of at least 4 ppm (4 mgL
(Brechner et al., 2013). Using the
limited measurements recorded and
averaged over time in this study, DO
levels are lower as higher saponin
concentrations are added to the
NFT system (Fig. 2). Levels did not
fall below 4 mgL
until saponins
were added at a concentration of 50
and above. Saponins are
well-known for their ability to foam
in aqueous solutions (Francis et al.,
2002) as detergent-like compounds.
Increased amounts of foam were ob-
served at increasing saponin levels in
this experiment. The amounts of
foam were large enough to obstruct
gas exchange between the nutrient
reservoir of the NFT system and the
atmosphere. A correlation cannot be
stated, but appears to be consistent
with DO levels, saponin treatment,
and plant growth.
The primary objectives of this
research study were to identify
whether steroidal saponins from
mojave yucca could be used as a natu-
ral bactericide for E. coli and what, if
any, effects that would have on plant
growth and development of ‘Rex’
lettuce grown in a hydroponic NFT
system. The fact of the matter is that
increasing saponin levels not only
failed to elicit a bactericidal effect
but promoted the growth of E. coli.
All the while, plant health and vigor
suffered in the presence of increasing
amounts of saponin levels. Based on
these results, this chemical interven-
tion technique would not be recom-
mended for the intended use of
bacterial mitigation in hydroponic
irrigation water.
Although data were not taken,
another observation was that the sa-
ponin solution was rather unpleasant
to work with because of a foul odor
and equipment-clogging issues.
Pumps and irrigation lines required
extensive cleaning between experi-
mental replications to prevent occlu-
sions from manifesting.
Another important takeaway was
that E. coli does not appear to travel
from the rhizosphere into the edible
portions of lettuce plants by systemic
means. E. coli also did not affect
lettuce growth. In this study, the
noninoculated treatment without sa-
ponin was juxtaposed to the inocu-
lated treatment without saponin and
found no differences in fresh shoot
weight (P= 0.74). This indicates that
E. coli living in the fertilizer solution
and interacting with the vast commu-
nity of microorganisms surrounding
the root zone do not negatively impact
the growth and development of ‘Rex’
lettuce in a hydroponic NFT system.
E. coli recovery was inadequate
when using MacConkey agar growth
media. Due to sublethal bacterial in-
jury, a pre-enrichment step should be
implemented in future research to
ensure proper recovery and enumer-
ation of bacteria. A TSA overlay on
MacConkey agar was used in this
experiment and is recommended for
future study.
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The n-butanol purified saponin extract of sorghum bicolor were screened for anti-bacterial activity against three pathogenic microbes; Escherichia coli, Staphylococcus aureus and Candida albicans. The extract inhibited the growth of the S. aureus. It was concluded that the saponins have inhibitory effect on gram-positive organism but not on gram negative organism and the fungi.