1. Introduction
1.1 Research question
What is the difference in antibacterial properties between marine flora – Codium duthieae, Sargassopsis
heteromorphum and Cystoseira trinodis – and terrestrial flora – Melaleuca alternifolia and Eucalyptus
globulus, and how do these natural extracts compare to common, modern treatments – Betadine
(Povidone-iodine) and 70% Ethanol – as measured by the inhibition zone (mm ±0.5mm) of Escherichia
coli (K-12 strain) growth after 16 hours of incubation (30oC) using the Kirby-Bauer method on 20% Luria
Bertani agar?
1.2 Aim
The aim of this study is to firstly, identify the three native Australian seaweeds with the greatest
antibacterial properties. Secondly, to compare the antibacterial properties of these identified marine flora to
those of native Australian terrestrial flora. And finally, to compare the antibacterial properties of these
natural plant extracts to those of modern, human-made treatments.
1.3 Background Information
Plants – both marine and terrestrial – have been used for medicinal purposes for centuries by numerous
indigenous cultures, in particular, Australian Indigenous tribes (Shannon and Abu-Ghannam, 2016). Due to
the increasing exhibition of bacterial resistance, there is a rising demand for novel antibacterial substances
that can be used alone or in conjunction with current treatments (Tajbakhsh et al, 2011). Many believe
plants to be a natural source of antibacterial properties that have untapped potential (Sandsdalen et al,
2003; Kim and Chojnacka, 2015). It is further believed that natural antimicrobial agents contain fewer side
effects, minimal toxicity and greater bioavailability – the rate and degree of absorption by the body –
compared to modern treatments (Tajbakhsh et al, 2011). However, whilst many terrestrial plants are used in
modern medicine, marine algae generally remain uncommon in medicines despite findings that marine
plants generally exhibit greater antibacterial properties than terrestrial plants (Kim and Chojnacka, 2015).
The preliminary experiments of this investigation aimed to identify the three seaweeds with the greatest
antibacterial properties from the selected Australian species – Codium duthieae, Sargassopsis
heteromorphum, Halopteris paniculata and Cystoseira trinodis. Of the selected species, C. duthieae, C. trinodis,
and S. heteromorphum have been identified as the species possessing antibacterial potential. Research
showed that of the algae selected, C. trinodis exhibits the greatest antibacterial properties with a low
minimum inhibitory concentration (MIC) of 0.4125% against E. coli (Tajbakhsh et al, 2011). MIC is a
common method of determining antibacterial properties and is the minimum concentration at which a
substance inhibits bacteria, thus, the lower the MIC the greater the antibacterial properties of a substance
(Tajbakhsh et al, 2011). Likewise, C. duthieae demonstrated antibacterial properties with an inhibition zone
of 6.2mm against E. coli (Vlachos et al., 1997).
This investigation further compared the antibacterial properties of marine and terrestrial flora. The
Australian terrestrial plants used in this investigation were Melaleuca alternifolia (Tea tree) and Eucalyptus
globulus (Eucalyptus), both identified as species exhibiting antibacterial properties (Prabuseenivasan et al.,
2006). M. alternifolia exhibits the greatest antibacterial properties of all selected plant species with a MIC of
0.25%, compared to E. globulus’s MIC of 0.8%, for E. coli specifically amongst other Gram-positive and Gram-
negative bacteria (Harkenthal et al, 1999; Hendry et al, 2009). E. globulus possesses weaker antibacterial
properties, however, Ghalem and Mohamed (2008) found that E. globulus exhibited a mean inhibition zone
of 15mm against E. coli, demonstrating relatively strong antibacterial properties although weaker than those
of M. alternifolia. These results are supported by those of Trivedi and Hotchandani (2004) and Hendry et al
(2009) who both observed E. globulus’ antibacterial properties. Since M. alternifolia and E. globulus were
identified as common Australian terrestrial species possessing antibacterial properties – and are also used
medicinally by Indigenous Australians as the chosen seaweed species were – they were selected for this
1
, investigation (Kamenev, 2011). This investigation used the Kirby-Bauer disk diffusion method, a universally
accepted methodology, to determine inhibition zones as an estimate of antibacterial properties.
Due to the “biological and chemical diversities” in marine habitats, marine algae contain various “biologically
active compounds” not present in terrestrial plants (Sandsdalen et al., 2003; TÜney et al., 2006). The marine
environment contains a greater variety of bacteria against which seaweeds must protect themselves, which
many believe to be a contributing factor to differences in the structures of marine and terrestrial plants
(Wang et al., 2009). Due to these differences, algae generally contain more hydroxyl groups than terrestrial
plants (Shannon and Abu-Ghannam, 2016). Thus, algae generally produce more hydrogen peroxide, which is
toxic to bacteria, than their terrestrial counterparts (Shannon and Abu-Ghannam, 2016). Many believe that
due to such differences in structure and secondary metabolites, algae generally exhibit greater antibacterial
properties than terrestrial flora (Kim and Chojnacka, 2015). Specifically, M. alternifolia inhibits E. coli growth
by preventing glucose-dependent respiration and stimulating K+ ion leakage (Cox et al., 1998). Contrastingly,
marine plants, with antibacterial properties, inhibit bacteria by producing hydrogen peroxide which oxidizes
bacteria’s cell walls – known as lysis – thus disrupting chemical structures (Shannon and Abu-Ghannam,
2016; Miller, 1969). This is considered a more effective method of inhibiting bacteria; thus, algae generally
possess greater antibacterial properties (Shannon and Abu-Ghannam, 2016).
1.4 Hypotheses
Null hypothesis (1H0): There will be lesser inhibition zones of E. coli when marine plant extracts are used
compared to terrestrial plant extracts.
Experimental hypothesis (1H1): There will be greater inhibition zones in E. coli growth when marine algae
extracts are used compared to terrestrial plant extracts.
Second null hypothesis (2H0): There will be lesser inhibition zones of E. coli when natural extracts are used
compared to modern antibacterial treatments.
Second experimental hypothesis (2H1): There will be equal or greater inhibition zones in E. coli growth
when natural treatments are used compared to modern antibacterial treatments.
2. Methodology
2.1 Variables
The independent variable for this investigation is the solution paper disks are dipped in. The four seaweed
extracts used in preliminary experiments are C. duthieae, S. heteromorphum, H. paniculata and C. trinodis.
The seven solutions used in the main body are C. duthieae, C. trinodis, S. heteromorphum, M. alternifolia, E.
globulus, Betadine, and 70% Ethanol. The dependent variable is the inhibition zone (mm ±0.5mm) of E. coli
(K-12) growth, measured after 16 hours of incubation at 30oC using the Kirby-Bauer disk diffusion method. E.
coli was chosen as it is a common bacterium, Gram Negative and relatively strong (CDCP, 2018). Thus, these
results will suggest whether these extracts’ antibacterial properties can inhibit a broader spectrum of
bacteria. The controlled and uncontrolled variables, along with methods of controlling, are outlined below:
1. Bacteria species: E. coli (K-12 strain) was used to in all trials ensure comparability of results as bacteria
exhibit varying resistance (Patra et al., 2009).
2. Extract concentration: Extracts were diluted to 50%, thus ensuring a fair test, as seaweed extracts were
prepared at a 50% dilution, and inhibition zones of appropriate size relative to Petri dish size (90mm).
3. Filter paper disks: Standardized filter paper disks were used to ensure the disk diameter (5.5mm) was
constant and the amount of extract used is standardized. Furthermore, all disks were sterilized using
UV irradiation by the research technician to ensure E. coli was the only present bacterium.
4. Incubation conditions: Plates were incubated at ~30oC for 16 hours in the same Thermoline incubator.
5. Agar type: 2.8% Luria Bertani (LB) agar was used in all trials to ensure no variation in the conditions of
E. coli growth as agars affect rates of bacteria growth and recovery (Patterson, 1988).
6. Seasonality of seaweed samples: As seasonality can affect antibacterial properties, all seaweeds were
personally collected from Redcliffe Bay on the same day at the first low tide (Inácio et al., 2016).
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1.1 Research question
What is the difference in antibacterial properties between marine flora – Codium duthieae, Sargassopsis
heteromorphum and Cystoseira trinodis – and terrestrial flora – Melaleuca alternifolia and Eucalyptus
globulus, and how do these natural extracts compare to common, modern treatments – Betadine
(Povidone-iodine) and 70% Ethanol – as measured by the inhibition zone (mm ±0.5mm) of Escherichia
coli (K-12 strain) growth after 16 hours of incubation (30oC) using the Kirby-Bauer method on 20% Luria
Bertani agar?
1.2 Aim
The aim of this study is to firstly, identify the three native Australian seaweeds with the greatest
antibacterial properties. Secondly, to compare the antibacterial properties of these identified marine flora to
those of native Australian terrestrial flora. And finally, to compare the antibacterial properties of these
natural plant extracts to those of modern, human-made treatments.
1.3 Background Information
Plants – both marine and terrestrial – have been used for medicinal purposes for centuries by numerous
indigenous cultures, in particular, Australian Indigenous tribes (Shannon and Abu-Ghannam, 2016). Due to
the increasing exhibition of bacterial resistance, there is a rising demand for novel antibacterial substances
that can be used alone or in conjunction with current treatments (Tajbakhsh et al, 2011). Many believe
plants to be a natural source of antibacterial properties that have untapped potential (Sandsdalen et al,
2003; Kim and Chojnacka, 2015). It is further believed that natural antimicrobial agents contain fewer side
effects, minimal toxicity and greater bioavailability – the rate and degree of absorption by the body –
compared to modern treatments (Tajbakhsh et al, 2011). However, whilst many terrestrial plants are used in
modern medicine, marine algae generally remain uncommon in medicines despite findings that marine
plants generally exhibit greater antibacterial properties than terrestrial plants (Kim and Chojnacka, 2015).
The preliminary experiments of this investigation aimed to identify the three seaweeds with the greatest
antibacterial properties from the selected Australian species – Codium duthieae, Sargassopsis
heteromorphum, Halopteris paniculata and Cystoseira trinodis. Of the selected species, C. duthieae, C. trinodis,
and S. heteromorphum have been identified as the species possessing antibacterial potential. Research
showed that of the algae selected, C. trinodis exhibits the greatest antibacterial properties with a low
minimum inhibitory concentration (MIC) of 0.4125% against E. coli (Tajbakhsh et al, 2011). MIC is a
common method of determining antibacterial properties and is the minimum concentration at which a
substance inhibits bacteria, thus, the lower the MIC the greater the antibacterial properties of a substance
(Tajbakhsh et al, 2011). Likewise, C. duthieae demonstrated antibacterial properties with an inhibition zone
of 6.2mm against E. coli (Vlachos et al., 1997).
This investigation further compared the antibacterial properties of marine and terrestrial flora. The
Australian terrestrial plants used in this investigation were Melaleuca alternifolia (Tea tree) and Eucalyptus
globulus (Eucalyptus), both identified as species exhibiting antibacterial properties (Prabuseenivasan et al.,
2006). M. alternifolia exhibits the greatest antibacterial properties of all selected plant species with a MIC of
0.25%, compared to E. globulus’s MIC of 0.8%, for E. coli specifically amongst other Gram-positive and Gram-
negative bacteria (Harkenthal et al, 1999; Hendry et al, 2009). E. globulus possesses weaker antibacterial
properties, however, Ghalem and Mohamed (2008) found that E. globulus exhibited a mean inhibition zone
of 15mm against E. coli, demonstrating relatively strong antibacterial properties although weaker than those
of M. alternifolia. These results are supported by those of Trivedi and Hotchandani (2004) and Hendry et al
(2009) who both observed E. globulus’ antibacterial properties. Since M. alternifolia and E. globulus were
identified as common Australian terrestrial species possessing antibacterial properties – and are also used
medicinally by Indigenous Australians as the chosen seaweed species were – they were selected for this
1
, investigation (Kamenev, 2011). This investigation used the Kirby-Bauer disk diffusion method, a universally
accepted methodology, to determine inhibition zones as an estimate of antibacterial properties.
Due to the “biological and chemical diversities” in marine habitats, marine algae contain various “biologically
active compounds” not present in terrestrial plants (Sandsdalen et al., 2003; TÜney et al., 2006). The marine
environment contains a greater variety of bacteria against which seaweeds must protect themselves, which
many believe to be a contributing factor to differences in the structures of marine and terrestrial plants
(Wang et al., 2009). Due to these differences, algae generally contain more hydroxyl groups than terrestrial
plants (Shannon and Abu-Ghannam, 2016). Thus, algae generally produce more hydrogen peroxide, which is
toxic to bacteria, than their terrestrial counterparts (Shannon and Abu-Ghannam, 2016). Many believe that
due to such differences in structure and secondary metabolites, algae generally exhibit greater antibacterial
properties than terrestrial flora (Kim and Chojnacka, 2015). Specifically, M. alternifolia inhibits E. coli growth
by preventing glucose-dependent respiration and stimulating K+ ion leakage (Cox et al., 1998). Contrastingly,
marine plants, with antibacterial properties, inhibit bacteria by producing hydrogen peroxide which oxidizes
bacteria’s cell walls – known as lysis – thus disrupting chemical structures (Shannon and Abu-Ghannam,
2016; Miller, 1969). This is considered a more effective method of inhibiting bacteria; thus, algae generally
possess greater antibacterial properties (Shannon and Abu-Ghannam, 2016).
1.4 Hypotheses
Null hypothesis (1H0): There will be lesser inhibition zones of E. coli when marine plant extracts are used
compared to terrestrial plant extracts.
Experimental hypothesis (1H1): There will be greater inhibition zones in E. coli growth when marine algae
extracts are used compared to terrestrial plant extracts.
Second null hypothesis (2H0): There will be lesser inhibition zones of E. coli when natural extracts are used
compared to modern antibacterial treatments.
Second experimental hypothesis (2H1): There will be equal or greater inhibition zones in E. coli growth
when natural treatments are used compared to modern antibacterial treatments.
2. Methodology
2.1 Variables
The independent variable for this investigation is the solution paper disks are dipped in. The four seaweed
extracts used in preliminary experiments are C. duthieae, S. heteromorphum, H. paniculata and C. trinodis.
The seven solutions used in the main body are C. duthieae, C. trinodis, S. heteromorphum, M. alternifolia, E.
globulus, Betadine, and 70% Ethanol. The dependent variable is the inhibition zone (mm ±0.5mm) of E. coli
(K-12) growth, measured after 16 hours of incubation at 30oC using the Kirby-Bauer disk diffusion method. E.
coli was chosen as it is a common bacterium, Gram Negative and relatively strong (CDCP, 2018). Thus, these
results will suggest whether these extracts’ antibacterial properties can inhibit a broader spectrum of
bacteria. The controlled and uncontrolled variables, along with methods of controlling, are outlined below:
1. Bacteria species: E. coli (K-12 strain) was used to in all trials ensure comparability of results as bacteria
exhibit varying resistance (Patra et al., 2009).
2. Extract concentration: Extracts were diluted to 50%, thus ensuring a fair test, as seaweed extracts were
prepared at a 50% dilution, and inhibition zones of appropriate size relative to Petri dish size (90mm).
3. Filter paper disks: Standardized filter paper disks were used to ensure the disk diameter (5.5mm) was
constant and the amount of extract used is standardized. Furthermore, all disks were sterilized using
UV irradiation by the research technician to ensure E. coli was the only present bacterium.
4. Incubation conditions: Plates were incubated at ~30oC for 16 hours in the same Thermoline incubator.
5. Agar type: 2.8% Luria Bertani (LB) agar was used in all trials to ensure no variation in the conditions of
E. coli growth as agars affect rates of bacteria growth and recovery (Patterson, 1988).
6. Seasonality of seaweed samples: As seasonality can affect antibacterial properties, all seaweeds were
personally collected from Redcliffe Bay on the same day at the first low tide (Inácio et al., 2016).
2
