Abstract
As the numbers of incidences of waterborne disease have globally increased so
has the interest in employing more advanced techniques to reduce biological
contamination of water. It has been demonstrated that ultrasound has a great
influence on microbial activity and this can be seen in two ways, the inactivation
of bacteria and the de-clumping of flocs. A range of frequencies of ultrasound
was employed (20, 40, 580, 864 and 1146 kHz) at different powers to determine
the effects on the pathogenesis of two Gram negative (Escherichia coli and
Klebsiella pneumoniae) and two Gram positive (Staphylococcus aureus and
Bacillus subtilis) bacteria at a laboratory and at a larger-scale. The results were
analysed using both traditional viable plate counts and flow cytometry to analysis
the effect of different ultrasonic frequencies on the viability of different bacterial
species. Calorimetry was employed to determine the actual acoustic power
entering each ultrasonic system. The 864 kHz bath (power setting 80%) resulted
in the highest power input of all systems at 16.59 Watts as determined by
calorimetric measurements. However, this frequency and power was not used in
experiments for disinfection of all the bacterial species because it was difficult to
maintain steady temperatures in the aqueous media and increases in
temperature occurred.
Ultrasound at the lower frequencies of 20 and 40 kHz yielded effective
inactivation by greatly reducing the viability of Gram negative bacteria
(Escherichia coli and Klebsiella pneumoniae) with 86 and 90% reduction in cell
numbers at 20 kHz and 85 and 88% reduction in cell numbers at 40 kHz after 30
minutes sonication. By comparison only a minor de-clumping effect was
observed for Gram positive bacteria (Staphylococcus aureus and Bacillus
subtilis) with effective increases in cell numbers of 12 and 6% at 20 kHz
respectively.
High frequencies of 580 kHz (40 and 80% power setting), 864 kHz (40% power
setting) and 1164 kHz (80% power setting) were employed to treat Gram
negative bacteria (Escherichia coli and Klebsiella pneumoniae).
Frequencies of 864 kHz (40% power setting) and 1164 kHz (80% power setting)
were only employed to treat Escherichia coli. Results demonstrated a small declumping effect resulting in 9% and 3% increase in the number of live cells for
both Escherichia coli and Klebsiella pneumoniae treated with 580 kHz (40%
power setting) for 30 minutes. Small inactivation effects of 9 and 11% were
observed for same bacterial strains when treated at 580 kHz (80% power
setting). Gram positive bacteria (Staphylococcus aureus and Bacillus subtilis)
treated with 580 kHz (80% power setting) also showed small inactivation effects
(14 and 2%) after 30 minutes treatment. Flow cytometry analysis differs from the
plate count treatment in that it can discriminate between and quantify two subpopulations in bacterial suspensions; live cells and dead cells. Flow cytometry
data supported the plate count results for most tests.
The effect of ultrasound (20 kHz) on a mixed culture of two bacterial species one
Gram negative and one Gram positive (Escherichia coli and Staphylococcus
aureus) generated an interesting result in that the response of the bacterial
species was entirely dependent on their previous growth conditions. When the
single bacteria were cultured separately and then mixed immediately prior to
ultrasound treatment (20 kHz probe for 30 minutes) they each responded in a
very similar way as they did in single cultures. Escherichia coli were sensitive to
ultrasonic treatment, with 82% reduction, while Staphylococcus aureus was
highly resistant with only declumping of 36% observed. On the other hand when
a mixture of the two bacteria was cultured together there was a quite different
response. While Escherichia coli continued to show almost complete inactivation
(86%), Staphylococcus aureus bacteria now showed 93% inactivation of cells
when treated with 20 kHz probe for 30 minutes (compared with only a
declumping effect was observed when the two species
separately).
were cultured
The effect of adding 5 ppm sodium hypochlorite (NaOCl) to the water with and
without
ultrasonic
treatment
on
Escherichia
coli,
Bacillus
subtilis
and
Staphylococcus aureus was investigated. Both Escherichia coli and Bacillus
subtilis were highly susceptible showing 97% and 98% reductions respectively
after 30 minutes treatment time compared to Staphylococcus aureus which
showed greater resistance to treatment with only 2% inactivation under the same
conditions. While the effect of 20 kHz probe alone on the single cultural of
(Escherichia coli, Staphylococcus aureus and Bacillus subtilis) illustrated a (89%,
-12% and -6%) reduction in the cells after 30 minutes ultrasonic treatment.
Combined NaOCl (5 ppm) and ultrasound treatment (20 kHz, intensity 0.015 W
cm3) enhanced the inactivation effect of Staphylococcus aureus with 24%
reduction in the number of the live cells following 30 minutes treatment.
The effect of sodium hypochlorite was also studied in combination with a
hydrodynamic cavitation system (Sonolator). The effect of 5 ppm NaOCl on
Escherichia coli bacteria resulted in 90% reduction in the number of live cells
following 5 minutes treatment. The combination of treatments with 5 ppm NaOCl
and hydrodynamic cavitation (Sonolator) resulted in 100% inactivation following 5
minutes flow treatment which equated to only 15 seconds of residence time
within the hydrodynamic cavitation system (Sonolator).
Large-scale commercial system which can operate at 100L/minute for advance
oxidation processes (ozone and ultrasound system (USO 3), Ultra Sonic Systems)
was used to test the potential for combined ozone and ultrasound treatment of
bacterial suspensions. Three single bacterial strains Escherichia coli, Klebsiella
pneumoniae and Staphylococcus aureus each 75 L were treated separately with
ozone alone, ultrasound (612 kHz) alone and a combination of both under flow
conditions for 16 minutes. Results revealed that three bacterial species
(Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus) treated
with ozone alone showed 100% inactivation within the first 2 minutes
(corresponding to a single pass of the 75 L through the reactor). Ultrasonic
treatment at 612 kHz alone resulted in small inactivation of 24%, 11% and 4% for
Escherichia coli, Klebsiella pneumoniae and Staphylococcus aureus respectively.
The combination of ozone and ultrasound, not surprisingly in view of the result
with ozone, also resulted in complete removal of live cells within the first 2
minutes i.e. a single pass. Flow cytometry data generally supported the viable
plate count results showing a reduction in the number of live cells following a
single pass through the reactor. However there appeared to be some cells still in
the quadrant related to the live cells position following treatment at all tests for all
bacterial strains. This may be due to some bacteria still retaining fragments of
cell walls which may take up fluorescent stains specific for live cells (SYTO9)
thus appearing in the live cell position. Ultrasound clearly enhanced the
inactivation effect of ozone via generation and collapse of cavitation bubbles
which leads to cell death, in addition to deaggregating bacterial clumps to
individual cell which makes the cells easer target for oxidation.
An interesting observation was made when the bacteria treated with the ozone
and ultrasound system were observed using Transmission Electron Microscopy
(TEM). The effect of ozone alone on single cultures of Escherichia coli, which
showed complete inactivation after a single pass through the ozone and
ultrasound system (USO3, Ultra Sonic Systems) as indicated by holes in the
bacterial cell walls using TEM analysis. In contrast to this observation the
combined effect of the ultrasound and ozone treatment on the biological structure
of Escherichia coli showed the complete removal of the entire bacterial cell wall.
In addition, the application of TEM following the treatment with ultrasound (20
kHz, intensity 0.015 W cm3) has demonstrated the ability to remove attachment
factors (pili) from Escherichia coli following 30 minutes treatment, along with cell
destruction.