Effect of the Opuntia (ficus-indica) cactus mucilage extract in reducing sediment and
bacteria contamination from water.
Kathy Tavakoli
Department of Biological Sciences
Saddleback College
Mission Viejo, California 92692
Although nearly all newly derived water purification methods have improved the water
quality in developing countries, few have been accepted and maintained for long-term use.
Field studies indicate that the most beneficial methods use indigenous resources, as they are
both accessible and accepted by communities they help. In an effort to implement a
material that will meet community needs, three fractions of mucilage gum were extracted
from the Opuntia ficus-indica cactus and tested as flocculation agents against sediment and
bacterial contamination. Column tests containing suspensions of the sediment kaolin
exhibited particle flocculation and settling rates up to 15.0 cm/min with mucilage versus
control settling rates of 1.0 cm/min (p=6.94 x 10-5 ANOVA). Escherichia coli tests displayed
flocculation and improved settling times with mucilage concentrations lower than 5 l and
removal rates between 96 and 91% were observed for high bacteria concentration tests
(>108 cells/ml) (p = 9.5 x 10-4 ANOVA). These results indicate the cactus mucilage extract
can reduce sediment and bacteria from water. This natural material not only displays
water purification abilities, but it is also affordable, renewable and readily available.
Introduction
The United Nations has estimated
that 1.1 billion people lack access to potable
water (Bugbee and Reigel 2008). With so
many people living on the brink of illness, a
great deal of attention has been drawn to
designing and implementing new and
innovative methods of water purification,
particularly in developing countries.
Gradually, the goal of bringing safe water to
the world has developed into a series of
goals from educating to finding a method of
purification that will be culturally accepted
and sustainable (Bugbee and Reigel 2008).
In an attempt to circumvent problems
associated with implementing purification
methods based solely on technology, the
Opuntia ficus-indica cactus (also known as
the Nopal or Prickly Pear) indigenous to
Mexico is being tested as a flocculating
Buttice (2009) observed, kaolin settling rates
observed in columns treated with mucilage
agent for waterborne contaminant removal.
Through simple extraction processes, two
fractions of mucilage gum can be obtained
from fresh cut Opuntia ficus- indica pads
including a Gelling Extract (GE) and NonGelling Extract (NE) (Cook, 2000).
Mucilage consists of up to 55 sugars, mainly
arabinose, galactose, rhamnose, xylose,
glucose, and uronic acids, the percentage of
which varies with mucilage type (Edward
2010). Literature has indicated that these
extracts, particularly the GE, undergo
property alterations including viscosity
changes in the presence of diatomic ions
such as Ca2+ (Edward 2010). University of
Florida scientific team demonstrated that
cactus mucilage is an effective tool for
separating sediments (clay and mud
particles) represented by kaolin, from
deionized
(DI)
water
suspensions.
and aluminum sulfate (Al2(SO4)3), a
common commercially used flocculent,
were compared and mucilage was concluded
to induce a greater increase in settling rate
(Young 2011). Related tests also indicate
that mucilage extracted from the Opuntia
spp. acts as an efficient coagulant in
surrogate turbid water (Young 2011). In this
work, the efficiency of mucilage to remove
kaolin suspended in DI-Water was
evaluated. Aside from sediment intrusion,
another common problem associated with
drinking water has been bacterial
contamination. Even in more developed
countries where purification and distribution
systems are technologically advanced and
water is closely monitored, cases of
waterborne illnesses caused by bacteria are
occasionally observed (Cook, 2000). In
developing countries, issues with bacterial
contamination are more severe. Escherichia
coli, a Gram-positive, spore- forming, nonpathogenic, soil-dwelling rod (∼1 by 3 μm),
was chosen to study bacterial aggregation
with mucilage for its ease of use and
potential as a possible surrogate for
waterborne bacteria with similar structural
characteristics. Data on its effects could
impact the status of this widely used product
and may have influence in water purification
industries.
Material and Method
Pads were obtained from Opuntia
ficus-indica cactus, originally purchased
from Crown Valley Market, located in
Crown valley Parkway, Mission Viejo. This
method and procedure is replicate of the
project conducted by University of Southern
Florida, Buttice (2009) study. The 3 cactus
pads (average weight 252 g) were diced,
heated with Nuova stir-hot-plate for 20
minutes at 85 oC, (water bath) and then the
solution was naturalized to pH 7-7.5 with 4
mL of 1 M NaOH. The pads were liquidized
by Braun blender with high speed (range 4)
for 5 minutes. The pH adjusted to 7 using
approximately 4 mL of 1 M NaOH solution
and the solid separated using Clay Adams
Compact II Centrifuge and 12 - test tube
placed (6 test tubes at a time) in there with
3200-RPM speed for 10 minutes. The
precipitate was removed and used in the
extraction of GE and the supernatant was
reserved for the acquisition of NE. In
precipitate, a 50 mM NaOH with 0.75%
(w/w) was added until the precipitate was
covered and then stirred with Nuova stirhot-plate for 30 min. The pH was adjusted to
2 using 12 mL of 0.1 M HCl solution, the
mixture centrifuged, and the supernatant
discarded. The precipitate was re-suspended
in DI water, and the pH was increased to 8
using 5 mL of 1 M NaOH. This suspension
was filtered via vacuum filtration using #41
Whatman filter. The portion of the original
suspension that was reserved for extraction
of NE was mixed with 200 mL of 1 M NaCl
solution and filtered using #41 Whatman
filter and a vacuum filtration system. The
filtrates of both the NE and GE were
separately mixed with acetone (1:1 v/v),
spread upon watch-glass dishes and left
overnight for allowing water to evaporate.
The recovered mucilage was removed and
washed in isopropanol (1:1 v/v), and some
NE (v/v) were mixed with GE mucilage to
make CG (combined- gelling). The mucilage
that spread upon watch-glass dishes, dried,
ground using mortar and pestle and some of
them stored for further experiments. Unused
mucilage suspension was stored in the
refrigerator
for
future
use.
The two distinct fractions of mucilage gum
were
studied
for
their
different
characteristics and removal abilities. Simple
test tubes were used to evaluate the
flocculation effect of the mucilage. The test
tube contents (mucilage and DI- water) were
Result
Kaolin Removal with Cactus
Mucilage. Kaolin suspension in DI water
was treated with 0, 2 l, 5 l and 10 l
concentrations for each GE (gelling
mucilage), NE (non gelling mucilage) and
CG (combined gelling mucilage), which is
demonstrated two characteristic of the
mucilage induced settling (n=10). Frist it
was observed that as mucilage concentration
increased, for all NE, GE, and CG, so did
the removing rate of the kaolin (Figure 2).
Initially (without any mucilage), the
relationship between concentration and
settling rate appeared to be linear (Figure 1).
concentrations. The final and initial
concentration
read
by
Marienfeld
Hemocytometer plate and Nikon microscope
(For accurate result, count ten (10) of the
1/16 sq. mm squares. Calculate the average
value and multiply by 1.6 X 105 to
determine the number of cells per milliliter).
E.coli did not from a visible interface while
settling. The time when flocs began to
appear as small white flecks in the otherwise
turbid water time that the flocs ceased to fall
was recorded. Box plot were used to
represent settling times, where the bottom of
the box indicates the time when flocs were
no longer falling in the column. The dotted
lines represent the start (lower line) and
completion time (upper line) of the control
columns containing only E.coli. Once the
flocs had completed their descent, a 1 mL
sample was taken from the top of the
column and final cell counts were evaluated
using hemocytometer plate and microscope
microscope.
The
resulting
bacteria
concentration was compared to the initial
and final concentration and a removal rate
was
obtained
base
on
clarity.
Kaolin Settling Rate (cm/min)
mixed to 10 mL in 15 mL centrifuge tubes,
vortexed and poured in to column arrays,
which were observed over a period of time.
Kaolin (hydrated aluminum silicate)
was ordered online from “Allied Products
Wholesale” via “eBay”. The suspension
with final concentration 25g/500 L were set
up in the Di-water at least 24 h prior to the
run of the experiment to allow kaolin
particles thorough hydration time. In test
tubes tests of specific concentrations, kaolin
has been observed to from a clear interface
which was read every minute fro up 60 min,
and the column marker at the interface was
recorded for ate plotting. These plots were
truncated where compression in the column
began and settling rate in cm/min was
obtained.
The Escherichia coli were cultured
overnight, with final column cell count 108
cell/mL. The final concentration was
determined with direct counting chamber
and Hemocytometer plate. 8 dilution tubes
were taken, each containing 9.0 mL of
sterile saline. Aseptically diluted 1.0 mL of
a sample of E. coli. Dilution was repeated
for total 10 final tubes each containing 108
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
Concentration (ppm)
Figure 1. The effect of the control solution (nonmucilage) on the settling rate of kaolin (50 g/L)
suspended in DI water.
15
highest removable solution, and after CG for
86% (15 cm initial to 2.1 cm final), and NE
(15 cm initial to 3.5 cm final) with 79%,
each remove kaolin from water (Figure 3).
Kaolin Settling Rate
(cm/min)
9.4
9.2
9.0
8.8
8.6
8.4
16.0
8.2
NE (2 uL)
NE (5 uL)
NE (10 uL)
Kaolin Settling Rate
(cm/min)
8.0
7.5
7.0
6.5
14.0
12.0
Average CG(cm/min)
10.0
Average GE (cm/min)
Contorl cm
Average NE (cm/min)
8.0
6.0
4.0
2.0
0.0
6.0
GE (2uL)
Kaolin Settling Rate
(cm/min)
Kaolin Settling Rate (cm/min)
8.0
GE (5uL)
GE (10 uL)
9.0
8.8
8.6
8.4
8.2
8.0
CG (2 uL)
CG (5 uL)
CG (10 uL)
Figure 2. The effect of the mucilage concentration,
for all NE, GE, and CG, on the settling rate of kaolin
(50 g/L) suspended in DI water. As the concentration
increase, settling rate of kaolin decrease (reducing
rate increase).
Second, it was observed that with no
mucilage treatment, all kaolin suspension
settled at rate close to 15 cm/min, indicating
that any differences observed among the
treated suspensions were primarily due to
ion interaction with the mucilage. Base on
the result, it can be indicate the mucilage
extract did reduce the kaolin from water.
There was significant statistically difference
between GE, NE, and CE mucilage in
removing kaolin (p = 6.94 x 10-5 ANOVA).
GE mucilage observed 92% removable
kaolin sediment from water in 60 min (15
cm initial to 1.2 cm final), which became a
0
20
40
60
Time ( min )
Figure 3. Comparing the average Kaolin settling rate
(cm/min) with removing sediments time in each 1
minute, for 60 minutes, with respect to the GE, NE,
CG and control (non-gelling) solutions (n=10). GE
mucilage observed most removable kaolin sediment
from water in 60 minutes (15 cm initial to 1.2 cm
final). There was significant statistically difference
between GE, NE, and CE mucilage in removing
kaolin (p = 6.94 x 10-5 ANOVA).
Escherichia coli Removal with Cactus
Mucilage.
Arrange
of
mucilage
concentration were tested to evaluate the
effect of mucilage on the settling time. The
settling rate of E. coli with 5 l NE, GE, and
CG were obtained from columns contacting
steamed water and 108 (cell/min) diluted E.
coli (n =10). From comparing the initial
counting E. coli cells and final counting,
indicated that mucilage extract did remove
E. coli from water. The difference in initial
and final concentration shows that there is
statistically difference between NE, GE, and
CG solutions in reducing E. coli from water
(p = 9.5 x 10-4 ANOVA). Difference
concentration in GE was more 0.29
(cell/min), which indicated the most
removable E. coli from water. At GE
concentration of 5 l, signs of flocculation
(14:95 min:sec) occurred faster than NE
(17:34 min:sec) and CG (23:35 min:sec)
mucilage.
Bacteria
removable
rate
associated with 5 l GE, NE, and CG were
determined to be 96.43% , 94.35%. and
91.24% (Figure 4).
Difference in bacteria concentartion
0.35
0.30
0.25
0.20
0.15
0.10
0.05
0.00
NE (5 uL)
GE (5 uL)
CG (5 uL)
Figure 4. The effects of the mucilage extracts (NE,
GE, CG) to the difference concentration of E. coli
(cell/min) (initial - Final) (n=10). Difference
concentration in GE was more 0.29 (cell/min), which
indicated the most removable E. coli from water. The
difference in initial and final concentration shows
that there is statistically difference between NE, GE,
and CG solutions in reducing E. coli from water (p =
9.5 x 10-4 ANOVA). Error bars indicate mean ±
SEM.
Discussion
An important difference between the
mucilage ‘s ability to aggregate kaolin
particle compared to bacteria was that there
existed a concentration where the bacteria
was that reacted to the mucilage in positive
manner. In columns contacting kaolin,
increases in mucilage concentration resulted
in higher settling rate, but no concentrations
were observed to restrict settling as seen in
suspension of E. coli. Also, in columns
containing kaolin, the GE appeared to cause
larger increase in settling rate while the NE
seems to work slightly better as a treatment
for E. coli suspension cause the flocs that
formed appeared as organized, stable, and
larger as those formed in GE, and CG. The
indicated results are equivalent to the
University of Florida studies (2009) studies,
observed that small amount of mucilage that
is required for contaminant removal from
surrogate waters. Although in this research,
as diatomic ions are known to affect both
mucilage and promote cell aggregation,
CaCl2 was studied in conjunction and
compared with mucilage as a bacteria
removal method in three different kind of
water (Hard/Soft/DI), which each contain
different ions and Bacillus cereus was tested
to displayed flocculation and improved
settling times. The experiment that
performed in Saddleback College was base
on the specific sediment (kaolin) and
bacteria (Escherichia coli), with different
concentration in mucilage for kaolin and
same concentration but repeated three times
for bacteria (more accurate result). In
addition in Young (2011) demonstrated the
use of Non-Gelling (NE) and Gelling (GE)
Extract; mucilage fractions for removal of
sediment in DI water. He concluded that
both mucilage fractions act faster in
sediment removal than the controls
containing no flocculating agent, and
solutions treated with the commonly used
Alum. He suggested the combined gelling
solution for future experimentations. In
Saddleback College experiment, the NE, GE
were testing in addition of CG as new
experiment and the results are new, since it
could find in another research experiment
for result comparison. The result that
observed here indicated there is significant
difference in effect of the mucilage extract
(NE, GE, and CG ) in reducing bacteria and
kaolin from water. The result observed the
GE solution had removed more kaolin and
bacteria in less time from water; compare to
NE removed less kaolin and take much long
to flocs. In CG result, it appear to have
removing settling rate and flocculation time
in between GE and NG, from this results, It
appears the CG solution have characteristics
from both gelling and non-gelling, which is
part that we observed in non-experimental
cactus pad. Although these removal rates are
high, the level of bacteria remaining in the
columns renders the water still unsafe to
drink. This is due to the initial cell
concentration (108 cell/mL), which would
not be typically observed in the real world,
but was used to obtain a visual indicator of
flocculation
and
removal.
Future
experimentation will involve optimizing
parameters for bacteria removal at lower
level of contamination. The results discussed
in this work demonstrate the potential of
mucilage extracted from the Opuntia ficusindica as flocculation agent for sediment and
bacteria contamination in ion rich water.
The cactus’ prevalence, affordability, and
cultural acceptance make it an attractive
natural material fro water purification
technologies that could be beneficial in
Mexico and around world. The difference
mucilage fractions have been observed to
provide diversity both in their structural
features as well as in their reactions to the
natural ion concentration in water they are
treating.
Literature Cited
Opuntia Polyacantha (Plains Prickly-Pear)
in Great Plains Grasslands. Great Plains
Research: A Journal of Natural and Social
Sciences. pp 996.
- Bugbee, R.E. and A. Reigel. 2008. The
cactus moth, Melitara dentata (Grote), and
its effect on Opuntia macrorhiza in western
Kansas. pp 1-94.
- Buttice, Audrey Lynn. 2009. Reducing
sediment and bacterial contamination in
water using mucilage extracted from the
Opuntia ficus- indica cactus. Graduate
School Theses and Dissertations.pp 1-9.
- Larsen, N.; Nissen, P.; Willatts. 2007.
The effect of calcium ions on adhesion and
competitive exclusion of Lactobacillus.ssp
and E. coli O138. Int. J. Food Microbiol. ,
114, 113–119.
- Lauenroth, William K.; Dougherty, R. L.
and Singh, J. S. 2009. Precipitation Event
Size Controls on Long-Term Abundance of
Acknowledgements
I would like to acknowledge the generous
support received from Professor Teh who
supplied the necessary equipment and
guidance to make the research happen. In
addition, we thank Saddleback College
Biological Sciences Department for
allowing us to use their facilities in the
study.
- Edward, Lin. 2010. Removal of Sediment
and Bacteria from Water Using Green
Chemistry, Environ. Sci. Technol. 44 (9). pp
3514-3519.
- Grant, V. and K.A. Grant. 2010.
Systematics of the Opuntia phaeacantha
group in Texas. Bot. Gaz. Opuntia
lindheimeri group. Bot. Gaz. pp 1-18.
- Natural Standard Monograph. 2013.
Nopal(Opuntia) The Authority on
Integrative Medicine,accessed 12 FEB 2011.
- Young, N. 2011. The medicago genome
provides insight into the evolution of
rhizobial symbioses. Nature Magazine . 480.
pp 520–524.