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Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water
Physics and Chemistry of the Earth xxx (2017) 1e5
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Physics and Chemistry of the Earth journal homepage: www.elsevier.com/locate/pce
Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water M.I. Ikhile a, *, T.G. Barnard b, J.C. Ngila a, ** a b
Department of Applied Chemistry, University of Johannesburg, Doornfontein Campus, P.O. Box 17011, Doornfontein 2028, Johannesburg, South Africa Water and Health Research Centre, University of Johannesburg, Doornfontein Campus, P.O Box 17011, Doornfontein 2028, Johannesburg, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2016 Received in revised form 12 January 2017 Accepted 31 January 2017 Available online xxx
This work reports a study towards a search for environmentally friendly water disinfectant. The most common method for water treatment is based on chlorine which had a wide application over the years as a water disinfectant, but suffer the disadvantage of reacting with natural organic matter to form disinfection by products. In this study, the potential application of novel ferrocenylimines compounds, namely 4-ferrocenylaniline (1), N-(3-bromo-2-hydroxylbenzylidene)-4-ferrocenylimine (2) and N-(3-bromo-5chlorosalicyl)-4-ferrocenylimine (3) for the elimination of bacteria in water was investigated by evaluating their antibacterial properties against twelve different bacterial strains using microdilution method in sterile 96 well micro titer plates. The in vitro antibacterial activity revealed that the ferrocenylimines compound exhibit higher antibacterial activity than ferrocene, which is one of the starting materials towards the synthesis of this novel ferrocenylimines compounds. The most active ferrocenylimines compound was compound 3 with a minimal inhibitory concentration (MIC) value of 0.30 mg/ml against S. sonnei. In addition, all the ferrocenylimines compounds possessed excellent antibacterial activity against B. cereus with the same MIC value of 0.31 mg/ml. The results obtained so far show great potential in the three tested ferrocenylimines compounds for use in water treatment in killing bacteria in water. © 2017 Published by Elsevier Ltd.
Keywords: Bacteria Ferrocenylimines Non-toxic Pollution Water
1. Introduction Developing a new method for the elimination of bacteria in water is very important because water is essential to life. Safe water that is free from bacteria is very important to the development of man and his environment (West, 2006). Water has a wide range of applications including agriculture, domestic and industry. As the human population continues to increase, the use of water also increases. Due to improper drainage system, the pollution of water (surface and underground) has increased as a result of improper practices such as direct deposit of faeces on surface water and rainfall runoff from agriculture to surface and underground water (Medema et al., 2003). The consumption of contaminated water with bacteria, is the leading worldwide cause of deaths and diseases, accounting for the deaths of more than 14,000 people daily (West, 2006; Westblade et al., 2015). Microbial waterborne diseases affect both the developing and
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M.I. Ikhile), [email protected] (J.C. Ngila).
developed countries (Medema et al., 2003). The bacteria are usually present in human and animal faeces and transmitted through water contaminated with faeces (George et al., 2001). The most important bacterial diseases transmitted through water are acute diarrheas, gastroenteritis, typhoid fever, cholera and bacillary dysentery (Grabow, 1996). These are some of the bacterial agent responsible for these waterborne diseases: Vibrio cholera, Salmonella enterica, Shigella boydii, Shigella sonnei, Enterobacter cloacae and Escherichia coli (Cabral, 2010). Infact acute microbial diarrheal diseases caused by E. coli are among the major public health problem in developing countries (Cabral, 2010). An estimated deaths of 1.5 million children have been reported, each year from diarrheal diseases (Fenwick, 2006). Children under five years are usually the most affected by waterborne diseases in Asian and African countries (Seas et al., 2000). Hence, there is need for an effective and environmentally friendly water disinfectant, in order to prevent waterborne diseases. Chlorination which is the process of adding chlorine to water, is one of the methods used to disinfect water and to prevent waterborne disease caused by pathogens in water. Pathogens are harmful microorganisms that may cause diseases in humans. The use of free
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Please cite this article in press as: Ikhile, M.I., et al., Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water, Physics and Chemistry of the Earth (2017), http://dx.doi.org/10.1016/j.pce.2017.01.022
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M.I. Ikhile et al. / Physics and Chemistry of the Earth xxx (2017) 1e5
chlorine had found a wide application over the years, as a disinfectant for water treatment, but suffers a drawback because it reacts with natural organic matter (NOM) to form disinfection by-products (DBPs) which are mostly chlorinated organic compounds such as trichloroethylene (TCE), carbon tetrachloride (CT), chlorophenols, polychlorinated biphenyls and other halogenated organics (Westrick et al., 1984). The DBPs are said to be endocrine disruptors as they are highly toxic and persist in the environment (Vogel et al., 1987). Their direct exposure to humans can lead to cancer, miscarriages and nervous system complications (Vogel et al., 1987). Therefore it is very crucial to develop new methods that are able to remove bacteria in water without causing toxin effects. Over the year the synthesis of compounds based on ferrocene compounds such as ferrocenylimines, have gained wide interest because of the unique properties and applications associated with it (Ikhile et al., 2013). The chemical stability, biological activities and non-toxicity properties of ferrocene has made it to be molecule of interest to incorporate into an organic compound (Ornelas, 2011). Recently, Qin et al. (2013) reviewed the application of imines also known as Schiff bases, in organic synthesis especially their biological activities. Studies by Zaheer et al. (2011) on some ferrocenyl Schiff bases showed that they exhibit antioxidant, antibacterial, antifungal, DNA protection and low cytotoxicity activities. Therefore reacting ferrocene with imines might enhance their biological activities. As part of an ongoing search to develop an environmentally friendly water disinfectants, novel ferrocenylimines compounds (Fig. 1) were easily synthesized (Ikhile and Ngila, 2015), according to a modified procedure (Zaheer et al., 2011; Ping et al., 2001). Their potential application for the elimination of bacteria in water was
investigated by evaluating their antibacterial properties against twelve test bacterial strains. The antibacterial activity of ferrocenylimines compounds 1e3 were also compared with the antimicrobial activity of ferrocene (Fig. 1), which is one of the starting materials towards the synthesis of ferrocenylimines compounds. However, to our knowledge, their use in biological water treatment has not been explored. 2. Materials and methods 2.1. Bacterial strains The microorganisms selected in this study have been implicated in transmission of waterborne diseases, which is a global major concern (Craun, 1986; Gauthier and Archibald, 2001). The twelve bacterial strains used for the experiments were obtained from the American Type Culture Collection (ATCC) as shown in Table 1 below. 2.2. Maintenance and growth of bacterial strains All the bacterial strains were plated and maintained on MullerHinton agar (Oxoid) during the experiments. The plates were incubated at 37 C overnight and stored at 4 C when not in use. Bacterial strains were grown in liquid culture by inoculating Mueller-Hinton broth (HI Media) with a colony from a freshly grown plate. All strains were grown at 37 C with mild agitation (100 RPM) until an optical density of 600 nm (OD600) was reached. These cell suspensions, in media, were used for the anti-bacterial testing.
Fig. 1. Chemical structure of ferrocene and ferrocenylimines compounds 1e3.
Please cite this article in press as: Ikhile, M.I., et al., Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water, Physics and Chemistry of the Earth (2017), http://dx.doi.org/10.1016/j.pce.2017.01.022
M.I. Ikhile et al. / Physics and Chemistry of the Earth xxx (2017) 1e5 Table 1 Summary of strains used for the testing of the nano-composites. Organism
ATCC Nr
Gram stain
Motility
Escherichia coli Escherichia coli Klebsiella pneumoniae Klebsiella pneumoniae Klebsiella oxytoca Pseudomonas aeruginosa Proteus mirabilis Shigella sonnei Shigella boydii Enterococcus faecalis Bacillus cereus Enterobacter cloacae
25,922 11,775 13,882 31,488 8724 27,853 12,453 25,931 9207 7080 10,876 13,047
Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram
Motile Motile Non-motile Non-motile Non-motile Motile Motile Non-motile Non-motile Motile Motile Motile
negative negative negative negative negative negative negative negative negative positive positive negative
2.3. Anti-bacterial testing procedures All tests were performed in sterile 96 well micro titer plates with lids and one plate was used to test a compound against selected bacterial strains. Sterile distilled water (50 ml) was added to each of the wells after which, 50 ml of the test solutions (which comprised of ferrocene and compounds 1e3 (Fig. 1.) in DMSO at a concentration of 5 mg/ml) was added to the first well, mixed and 50 ml transferred to the next well. The serial dilutions were continued four more times until the middle of the plate was reached after which the process was started again. To this 50 ml of the bacterial cultures were added prepared as described above to obtain a typical experimental set-up as shown in Fig. 2. As a control the impact of dilutions of the solvents as well as a known antibiotic (Cefepime hydrochloride monohydrate Powder; Alfa Aesar) on the bacterial strains was tested to ensure the inhibitory effect of solvents and to show that the experiment is working (antibiotics). The covered microplates were incubated overnight at 37 C. To indicate bacterial growth, 50 ml of p-iodonitrotetrazohium violet
3
(INT; Fluka) was dissolved in water and added to the microplate wells and incubated at 37 C for 30e60 min (Eloff, 1998). Active bacterial cells reduces the INT to purple colour indicating bacterial survival and thus no inhibition. It must be noted that Fig. 2 shows the dilutions as two times up to sixty-four times and correlate to a percentage of the original compound added. The Minimum inhibitory concentration (MIC) is used for the results. The MIC is the concentration of the highest dilution well in which there is no bacteria growth. The comparisons and the correlation between dilution factor and MIC values is given in Table 2. 3. Results The results is presented in two manner. Firstly we show the results as read directly from the plates (Table 3) that includes the results for the solvents used and antibiotics. The results were then simplified to show only the results obtained after the influence of the solvents was taken into consideration (Table 4). This means that tests that might have been seen as positive might show no reaction since it could simply have been due to the influence of the solvent. Most of the articles we sourced reported that the solvent has no significant influence on the bacterial strains, something that was not confirmed with these experiments. As seen in Table 3, the tested compounds and the solvent (DMSO) showed some activity against the tested bacteria. In addition the tested compounds and DMSO showed similar activity against K. pneumoniae and one of the E. coli strains. Therefore removing the effect of the solvent as showed in Table 4, no MIC values was reported for K. pneumoniae and one of the E. coli strains. In addition the tested compounds showed similar activity against four bacterial strains E. coli (MIC ¼ 1.25 mg/ml), K. oxytoca (MIC ¼ 1.25 mg/ml), B. cereus (MIC ¼ 0.31 mg/ml), S. boydii (MIC ¼ 1.25 mg/ml). Compound 2 alone showed activity against P. aeruginsoa (MIC ¼ 1.25 mg/ml) and E. cloacae (MIC ¼ 2.50 mg/ml).
Fig. 2. Example of the typical 96 well micro titre plate layout for the testing of ferrocenylimines compounds. The clear wells shows inhibition of bacterial growth and the purple indicates actives cells reducing the INT. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Comparison of dilution factor and the percentage of ferrocenylimines compounds/MIC values in each well. Ferroenylimines compounds Dilution factor Percentage original compound Initial concentration 5 mg/ml (MIC)
2 50% 2.5 mg/ml
4 25% 1.25 mg/ml
8 12.5% 0.625 mg/ml
16 6.25% 0.3125 mg/ml
32 3.125% 0.1563 mg/ml
64 1.5625% 0.0763 mg/ml
MIC e Minimum inhibiting concentration.
Please cite this article in press as: Ikhile, M.I., et al., Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water, Physics and Chemistry of the Earth (2017), http://dx.doi.org/10.1016/j.pce.2017.01.022
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Table 3 Summary of the highest dilution of the compounds that inhibited the bacterial growth as determined by the INT assay. The table includes the data observed for the antibiotic control as well as solvent control. The results were not adjusted based on the solvent results. Test compound
E. faecalis
K. pneumoniae
K. pneumoniae
p. aeruginosa
E. coli
S. sonnei
E. coli
P. mirabilis
K. oxytoca
B. cereus
S. boydii
E. cloacae
Antibiotica DMSOb Ferrocene 1 2 3
64 16 32 16 16 16
2 8 16 8 8 16
4 8 8 8 8 8
2 4 4 8 2 4
4 8 8 8 8 8
4 8 8 8 8 16
4 4 4 8 8 8
2 4 4 4 4 4
8 4 4 8 8 8
2 4 8 64 64 64
2 8 8 16 64 64
2 8 4 16 8 8
a b
Antibiotic control. Dimethyl sulfoxide e solvent control.
Table 4 The minimum inhibitory concentration (MIC) in mg/ml of the compounds that inhibited the bacterial growth as determined by the INT assay. The results have been adjusted with the influence of the solvent control. Test compound
E. faecalis
K. pneumoniae
K. pneumoniae
p. aeruginosa
E. coli
S. sonnei
E. coli
P. mirabilis
K. oxytoca
B. cereus
S. boyddi
E. cloacae
Ferrocene 1 2 3
0.31 e e e
0.63 e e 0.63
e e e e
e e 1.25 e
e e e e
e e e 0.30
e 1.25 1.25 1.25
e e e e
e 1.25 1.25 1.25
e 0.31 0.31 0.31
e 1.25 1.25 1.25
e e 2.50 e
Also only compound 3 showed activity against S. sonnei (MIC ¼ 0.31 mg/ml). 4. Discussions The results presented in Table 3 showed the bacteria inhibition as read directly from the plate. Interestingly, the solvent (DMSO) also showed some antibacterial activity against the bacterial strains. This confirmed the effect of the solvent on the antibacterial testing. As a result, when the influence of the solvent was removed from the data in Table 3 and presented in Table 4 with the MIC values of the compounds tested that inhibited bacterial growth. Compounds 1e3 showed no significant activity against K. Pneumoniae, E. coli and P. mirabilis. Also, Zaheer et al. reported no significant antibacterial activity of some ferrocenyl Schiff bases against six bacterial strains which was attributed to the lipophilic characteristics of their Schiff bases. The results from the experimental work clearly indicated that the solvents used can and do have an effect on the bacterial strains. It was also noted that, depending on the order the reagents were added, there was precipitation of the media in the wells. This could be due to the solvent or reagents and was difficult to deduce since the solvents was always present in some concentration. As shown in Table 4, compound 2 exhibited anti-bacterial activity against the bacterial strains tested, similar to what was seen with compound 3. Ferrocene alone showed very low antibacterial activity towards some of the test strains as compared to compounds 1e3. The compounds 1e3 were able to inhibit the growth of one or more of the tested microorganisms. Compound 3 showed the highest activity with MIC value of 0.30 mg/ml against S. sonnei. This could be attributed to the presence of the imine group (C]N) in compound 3, although it is also present in compound 2. However, the presence of electron withdrawing chlorine atom in compound 3 might have contributed to the high antibacterial activity. In comparison to our study, Mathiyalagan et al. (2012) had shown that the presence of two electron withdrawing atoms in their molecules, was attributed to the high antimicrobial activities. The strongest activity of the ferrocenylimines compounds 1e3 was seen with Gram positive B. cereus. Low activities were seen with negative P. aeruginosa and no MIC values was obtained for P. mirabilis with all the compounds tested. In general, there seems
to be low-level anti-microbial activity against some of the strains. It should be noted that the anti-bacterial activity might have been underestimated due to the effects of the solvents and alternative methods will have to be investigated to get more concrete answers. It is worth noting that the ferrocenylimines compounds 2 exhibited greater antibacterial activity against E. cloacae with MIC values of 2.50 mg/ml in comparison with some chlorine-releasingagents (CRA) against E. cloacae with MIC values of 420 mg/ml (Mazzola et al., 2009). The ferrocenylimines compounds are very stable in solution and easy to handle without decomposition when expose to air (Ikhile and Ngila, 2015). The non-toxicity of the ferrocenylimines compounds have also been reported (Gupta et al., 2014; Qin et al., 2013). In contrast to all form of chlorine used in water disinfection such as chlorine gas, calcium hypochlorite, bromium chloride and hypochloride solution (Casey et al., 1998). These are highly corrosive and toxic, which pose safety risk when handled (Darby et al., 1995). The activity of the ferrocenylimines compounds to both Grampositive and Gram-negative may be attributed to the chemical structure of the compound. Thus the ferrocenyl derived compounds have been reported to have high antibacterial activity (Biot et al., 2000; Zhang, 2008). Imines (also known as Schiff bases) too are known to exhibit high antibacterial activities (Qin et al., 2013). Karthikeyan et al. (2006) reported the inhibition of the growth of S. aureus, E. coli, P. aeruginosa and K. pneumonia with 2,4-dichloro5-flurophenyl Schiff base moiety. Thus, the combination of biological active ferrocenyl and imines in compounds 2 and 3 contributed to their activity towards the tested bacterial strains. The Bacterial cell wall are surrounded by lipid membrane which only favor the passage of lipid soluble materials (Abu-Dief and Mohamed, 2015). Thus antibacterial activity of ferrocenylimines compounds increases as the lipophilicity nature increases, which affords the permeation of the ferrocenylimines compounds into the bacterial cell wall and inhibit the bacterial growth (Raman et al., 2009). The toxicology studies of these ferrocenylimines compounds are currently being investigated. It will be interesting to observe their effects on bacteria when applied in water treatment as potential disinfectants. We however caution that, from the literature reports, it has been observed that ferrocenyl derived compounds do possess low toxicity (Popova et al., 1993; Snegur et al., 2004; Simenel et al., 2008).
Please cite this article in press as: Ikhile, M.I., et al., Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water, Physics and Chemistry of the Earth (2017), http://dx.doi.org/10.1016/j.pce.2017.01.022
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5. Conclusion The ferrocenylimine compounds 1e3 were subjected to tests for antimicrobial activity against twelve different bacterial strains: E. coli, E. coli, K. pneumoniae, K. pneumoniae, K. oxytoca, P. aeruginosa, P. mirabilis, S. sonnei, S. boydii, E. faecalis, B. cereus, E. cloacae and the MIC values have been reported. The tested compounds shown similar activity against four bacterial strains E. coli (MIC ¼ 1.25 mg/ ml), K. oxytoca (MIC ¼ 1.25 mg/ml), B. cereus (MIC ¼ 0.31 mg/ml), S. boydii (MIC ¼ 1.25 mg/ml). Compound 2 alone shown activity against P. aeruginsoa (MIC ¼ 1.25 mg/ml) and E. cloacae (MIC ¼ 2.50 mg/ml). Also compound 3 exhibit excellent activity against S. sonnei (MIC ¼ 0.31 mg/ml). These results revealed the ferrocenylimine compounds to exhibit antibacterial activity. Therefore, the ferrocenylimines compounds could serve as a good potential for water disinfectants. Further studies are currently ongoing to investigate the toxicology of ferrocenylimine compounds 1e3. Acknowledgements The Faculty of Science, University of Johannesburg is highly appreciated for financial support and Department of Applied Chemistry, Water and Health Research centre for availing its facilities. The Centre for Nanomaterials Science Research (CNSR) is acknowledged for providing running cost of this project. References Abu-Dief, A.M., Mohamed, I.M.A., 2015. A review on versatile applications of transition metal complexes incorporating Schiff bases. BJBAS 4, 119e133. Biot, C., Franccois, N., Maciejewski, L., Brocard, J., Poulain, D., 2000. Synthesis and antifungal activity of a ferrocene-fluconazole analogue. Bioorg. Med. Chem. Lett. 10, 839e841. Cabral, J.P.S., 2010. Water microbiology. Bacterial pathogens and water. Int. J. Environ. Res. Public Health 7, 3657e3703. Casey, P., Mackne, C., Lake, A., 1998. Chlorine Disinfection. http://www.nesc.wvu. edu/pdf/ww/publications/eti/chl_dis_gen.pdf. Accessed 04 July 2016. Craun, G.F., 1986. Waterborne Disease in the United States. CRC Press, Inc, Boca Raton, FL. Darby, J., Heath, M., Jacangelo, J., Loge, F., Swaim, P., Tchobanoglous, G., 1995. Comparison of UV Irradiation to Chlorination: Guidance for Achieving Optimal UV Performance. Water Environment Research Foundation, Alexandria, Virginia. Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med. 64, 711e713. Fenwick, A., 2006. Waterborne Diseases- could they be consigned to History? Science 313, 1077e1081. Gauthier, F., Archibald, F., 2001. The ecology of “Faecal indicator” Bacteria commonly found in pulp and paper mill water systems. Water Res. 35, 2207e2218. George, I., Crop, P., Servais, P., 2001. Use of b-D-Galactosidase and b-D-Glucuronidase activities for quantitative detection of total and faecal coliforms in wastewater. Can. J. Microbiol. 47, 670e675. Grabow, W.O.K., 1996. Waterborne diseases: update on water quality assessment
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Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water M.I. Ikhile a, *, T.G. Barnard b, J.C. Ngila a, ** a b
Department of Applied Chemistry, University of Johannesburg, Doornfontein Campus, P.O. Box 17011, Doornfontein 2028, Johannesburg, South Africa Water and Health Research Centre, University of Johannesburg, Doornfontein Campus, P.O Box 17011, Doornfontein 2028, Johannesburg, South Africa
a r t i c l e i n f o
a b s t r a c t
Article history: Received 28 April 2016 Received in revised form 12 January 2017 Accepted 31 January 2017 Available online xxx
This work reports a study towards a search for environmentally friendly water disinfectant. The most common method for water treatment is based on chlorine which had a wide application over the years as a water disinfectant, but suffer the disadvantage of reacting with natural organic matter to form disinfection by products. In this study, the potential application of novel ferrocenylimines compounds, namely 4-ferrocenylaniline (1), N-(3-bromo-2-hydroxylbenzylidene)-4-ferrocenylimine (2) and N-(3-bromo-5chlorosalicyl)-4-ferrocenylimine (3) for the elimination of bacteria in water was investigated by evaluating their antibacterial properties against twelve different bacterial strains using microdilution method in sterile 96 well micro titer plates. The in vitro antibacterial activity revealed that the ferrocenylimines compound exhibit higher antibacterial activity than ferrocene, which is one of the starting materials towards the synthesis of this novel ferrocenylimines compounds. The most active ferrocenylimines compound was compound 3 with a minimal inhibitory concentration (MIC) value of 0.30 mg/ml against S. sonnei. In addition, all the ferrocenylimines compounds possessed excellent antibacterial activity against B. cereus with the same MIC value of 0.31 mg/ml. The results obtained so far show great potential in the three tested ferrocenylimines compounds for use in water treatment in killing bacteria in water. © 2017 Published by Elsevier Ltd.
Keywords: Bacteria Ferrocenylimines Non-toxic Pollution Water
1. Introduction Developing a new method for the elimination of bacteria in water is very important because water is essential to life. Safe water that is free from bacteria is very important to the development of man and his environment (West, 2006). Water has a wide range of applications including agriculture, domestic and industry. As the human population continues to increase, the use of water also increases. Due to improper drainage system, the pollution of water (surface and underground) has increased as a result of improper practices such as direct deposit of faeces on surface water and rainfall runoff from agriculture to surface and underground water (Medema et al., 2003). The consumption of contaminated water with bacteria, is the leading worldwide cause of deaths and diseases, accounting for the deaths of more than 14,000 people daily (West, 2006; Westblade et al., 2015). Microbial waterborne diseases affect both the developing and
* Corresponding author. ** Corresponding author. E-mail addresses: [email protected] (M.I. Ikhile), [email protected] (J.C. Ngila).
developed countries (Medema et al., 2003). The bacteria are usually present in human and animal faeces and transmitted through water contaminated with faeces (George et al., 2001). The most important bacterial diseases transmitted through water are acute diarrheas, gastroenteritis, typhoid fever, cholera and bacillary dysentery (Grabow, 1996). These are some of the bacterial agent responsible for these waterborne diseases: Vibrio cholera, Salmonella enterica, Shigella boydii, Shigella sonnei, Enterobacter cloacae and Escherichia coli (Cabral, 2010). Infact acute microbial diarrheal diseases caused by E. coli are among the major public health problem in developing countries (Cabral, 2010). An estimated deaths of 1.5 million children have been reported, each year from diarrheal diseases (Fenwick, 2006). Children under five years are usually the most affected by waterborne diseases in Asian and African countries (Seas et al., 2000). Hence, there is need for an effective and environmentally friendly water disinfectant, in order to prevent waterborne diseases. Chlorination which is the process of adding chlorine to water, is one of the methods used to disinfect water and to prevent waterborne disease caused by pathogens in water. Pathogens are harmful microorganisms that may cause diseases in humans. The use of free
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chlorine had found a wide application over the years, as a disinfectant for water treatment, but suffers a drawback because it reacts with natural organic matter (NOM) to form disinfection by-products (DBPs) which are mostly chlorinated organic compounds such as trichloroethylene (TCE), carbon tetrachloride (CT), chlorophenols, polychlorinated biphenyls and other halogenated organics (Westrick et al., 1984). The DBPs are said to be endocrine disruptors as they are highly toxic and persist in the environment (Vogel et al., 1987). Their direct exposure to humans can lead to cancer, miscarriages and nervous system complications (Vogel et al., 1987). Therefore it is very crucial to develop new methods that are able to remove bacteria in water without causing toxin effects. Over the year the synthesis of compounds based on ferrocene compounds such as ferrocenylimines, have gained wide interest because of the unique properties and applications associated with it (Ikhile et al., 2013). The chemical stability, biological activities and non-toxicity properties of ferrocene has made it to be molecule of interest to incorporate into an organic compound (Ornelas, 2011). Recently, Qin et al. (2013) reviewed the application of imines also known as Schiff bases, in organic synthesis especially their biological activities. Studies by Zaheer et al. (2011) on some ferrocenyl Schiff bases showed that they exhibit antioxidant, antibacterial, antifungal, DNA protection and low cytotoxicity activities. Therefore reacting ferrocene with imines might enhance their biological activities. As part of an ongoing search to develop an environmentally friendly water disinfectants, novel ferrocenylimines compounds (Fig. 1) were easily synthesized (Ikhile and Ngila, 2015), according to a modified procedure (Zaheer et al., 2011; Ping et al., 2001). Their potential application for the elimination of bacteria in water was
investigated by evaluating their antibacterial properties against twelve test bacterial strains. The antibacterial activity of ferrocenylimines compounds 1e3 were also compared with the antimicrobial activity of ferrocene (Fig. 1), which is one of the starting materials towards the synthesis of ferrocenylimines compounds. However, to our knowledge, their use in biological water treatment has not been explored. 2. Materials and methods 2.1. Bacterial strains The microorganisms selected in this study have been implicated in transmission of waterborne diseases, which is a global major concern (Craun, 1986; Gauthier and Archibald, 2001). The twelve bacterial strains used for the experiments were obtained from the American Type Culture Collection (ATCC) as shown in Table 1 below. 2.2. Maintenance and growth of bacterial strains All the bacterial strains were plated and maintained on MullerHinton agar (Oxoid) during the experiments. The plates were incubated at 37 C overnight and stored at 4 C when not in use. Bacterial strains were grown in liquid culture by inoculating Mueller-Hinton broth (HI Media) with a colony from a freshly grown plate. All strains were grown at 37 C with mild agitation (100 RPM) until an optical density of 600 nm (OD600) was reached. These cell suspensions, in media, were used for the anti-bacterial testing.
Fig. 1. Chemical structure of ferrocene and ferrocenylimines compounds 1e3.
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M.I. Ikhile et al. / Physics and Chemistry of the Earth xxx (2017) 1e5 Table 1 Summary of strains used for the testing of the nano-composites. Organism
ATCC Nr
Gram stain
Motility
Escherichia coli Escherichia coli Klebsiella pneumoniae Klebsiella pneumoniae Klebsiella oxytoca Pseudomonas aeruginosa Proteus mirabilis Shigella sonnei Shigella boydii Enterococcus faecalis Bacillus cereus Enterobacter cloacae
25,922 11,775 13,882 31,488 8724 27,853 12,453 25,931 9207 7080 10,876 13,047
Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram Gram
Motile Motile Non-motile Non-motile Non-motile Motile Motile Non-motile Non-motile Motile Motile Motile
negative negative negative negative negative negative negative negative negative positive positive negative
2.3. Anti-bacterial testing procedures All tests were performed in sterile 96 well micro titer plates with lids and one plate was used to test a compound against selected bacterial strains. Sterile distilled water (50 ml) was added to each of the wells after which, 50 ml of the test solutions (which comprised of ferrocene and compounds 1e3 (Fig. 1.) in DMSO at a concentration of 5 mg/ml) was added to the first well, mixed and 50 ml transferred to the next well. The serial dilutions were continued four more times until the middle of the plate was reached after which the process was started again. To this 50 ml of the bacterial cultures were added prepared as described above to obtain a typical experimental set-up as shown in Fig. 2. As a control the impact of dilutions of the solvents as well as a known antibiotic (Cefepime hydrochloride monohydrate Powder; Alfa Aesar) on the bacterial strains was tested to ensure the inhibitory effect of solvents and to show that the experiment is working (antibiotics). The covered microplates were incubated overnight at 37 C. To indicate bacterial growth, 50 ml of p-iodonitrotetrazohium violet
3
(INT; Fluka) was dissolved in water and added to the microplate wells and incubated at 37 C for 30e60 min (Eloff, 1998). Active bacterial cells reduces the INT to purple colour indicating bacterial survival and thus no inhibition. It must be noted that Fig. 2 shows the dilutions as two times up to sixty-four times and correlate to a percentage of the original compound added. The Minimum inhibitory concentration (MIC) is used for the results. The MIC is the concentration of the highest dilution well in which there is no bacteria growth. The comparisons and the correlation between dilution factor and MIC values is given in Table 2. 3. Results The results is presented in two manner. Firstly we show the results as read directly from the plates (Table 3) that includes the results for the solvents used and antibiotics. The results were then simplified to show only the results obtained after the influence of the solvents was taken into consideration (Table 4). This means that tests that might have been seen as positive might show no reaction since it could simply have been due to the influence of the solvent. Most of the articles we sourced reported that the solvent has no significant influence on the bacterial strains, something that was not confirmed with these experiments. As seen in Table 3, the tested compounds and the solvent (DMSO) showed some activity against the tested bacteria. In addition the tested compounds and DMSO showed similar activity against K. pneumoniae and one of the E. coli strains. Therefore removing the effect of the solvent as showed in Table 4, no MIC values was reported for K. pneumoniae and one of the E. coli strains. In addition the tested compounds showed similar activity against four bacterial strains E. coli (MIC ¼ 1.25 mg/ml), K. oxytoca (MIC ¼ 1.25 mg/ml), B. cereus (MIC ¼ 0.31 mg/ml), S. boydii (MIC ¼ 1.25 mg/ml). Compound 2 alone showed activity against P. aeruginsoa (MIC ¼ 1.25 mg/ml) and E. cloacae (MIC ¼ 2.50 mg/ml).
Fig. 2. Example of the typical 96 well micro titre plate layout for the testing of ferrocenylimines compounds. The clear wells shows inhibition of bacterial growth and the purple indicates actives cells reducing the INT. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Table 2 Comparison of dilution factor and the percentage of ferrocenylimines compounds/MIC values in each well. Ferroenylimines compounds Dilution factor Percentage original compound Initial concentration 5 mg/ml (MIC)
2 50% 2.5 mg/ml
4 25% 1.25 mg/ml
8 12.5% 0.625 mg/ml
16 6.25% 0.3125 mg/ml
32 3.125% 0.1563 mg/ml
64 1.5625% 0.0763 mg/ml
MIC e Minimum inhibiting concentration.
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Table 3 Summary of the highest dilution of the compounds that inhibited the bacterial growth as determined by the INT assay. The table includes the data observed for the antibiotic control as well as solvent control. The results were not adjusted based on the solvent results. Test compound
E. faecalis
K. pneumoniae
K. pneumoniae
p. aeruginosa
E. coli
S. sonnei
E. coli
P. mirabilis
K. oxytoca
B. cereus
S. boydii
E. cloacae
Antibiotica DMSOb Ferrocene 1 2 3
64 16 32 16 16 16
2 8 16 8 8 16
4 8 8 8 8 8
2 4 4 8 2 4
4 8 8 8 8 8
4 8 8 8 8 16
4 4 4 8 8 8
2 4 4 4 4 4
8 4 4 8 8 8
2 4 8 64 64 64
2 8 8 16 64 64
2 8 4 16 8 8
a b
Antibiotic control. Dimethyl sulfoxide e solvent control.
Table 4 The minimum inhibitory concentration (MIC) in mg/ml of the compounds that inhibited the bacterial growth as determined by the INT assay. The results have been adjusted with the influence of the solvent control. Test compound
E. faecalis
K. pneumoniae
K. pneumoniae
p. aeruginosa
E. coli
S. sonnei
E. coli
P. mirabilis
K. oxytoca
B. cereus
S. boyddi
E. cloacae
Ferrocene 1 2 3
0.31 e e e
0.63 e e 0.63
e e e e
e e 1.25 e
e e e e
e e e 0.30
e 1.25 1.25 1.25
e e e e
e 1.25 1.25 1.25
e 0.31 0.31 0.31
e 1.25 1.25 1.25
e e 2.50 e
Also only compound 3 showed activity against S. sonnei (MIC ¼ 0.31 mg/ml). 4. Discussions The results presented in Table 3 showed the bacteria inhibition as read directly from the plate. Interestingly, the solvent (DMSO) also showed some antibacterial activity against the bacterial strains. This confirmed the effect of the solvent on the antibacterial testing. As a result, when the influence of the solvent was removed from the data in Table 3 and presented in Table 4 with the MIC values of the compounds tested that inhibited bacterial growth. Compounds 1e3 showed no significant activity against K. Pneumoniae, E. coli and P. mirabilis. Also, Zaheer et al. reported no significant antibacterial activity of some ferrocenyl Schiff bases against six bacterial strains which was attributed to the lipophilic characteristics of their Schiff bases. The results from the experimental work clearly indicated that the solvents used can and do have an effect on the bacterial strains. It was also noted that, depending on the order the reagents were added, there was precipitation of the media in the wells. This could be due to the solvent or reagents and was difficult to deduce since the solvents was always present in some concentration. As shown in Table 4, compound 2 exhibited anti-bacterial activity against the bacterial strains tested, similar to what was seen with compound 3. Ferrocene alone showed very low antibacterial activity towards some of the test strains as compared to compounds 1e3. The compounds 1e3 were able to inhibit the growth of one or more of the tested microorganisms. Compound 3 showed the highest activity with MIC value of 0.30 mg/ml against S. sonnei. This could be attributed to the presence of the imine group (C]N) in compound 3, although it is also present in compound 2. However, the presence of electron withdrawing chlorine atom in compound 3 might have contributed to the high antibacterial activity. In comparison to our study, Mathiyalagan et al. (2012) had shown that the presence of two electron withdrawing atoms in their molecules, was attributed to the high antimicrobial activities. The strongest activity of the ferrocenylimines compounds 1e3 was seen with Gram positive B. cereus. Low activities were seen with negative P. aeruginosa and no MIC values was obtained for P. mirabilis with all the compounds tested. In general, there seems
to be low-level anti-microbial activity against some of the strains. It should be noted that the anti-bacterial activity might have been underestimated due to the effects of the solvents and alternative methods will have to be investigated to get more concrete answers. It is worth noting that the ferrocenylimines compounds 2 exhibited greater antibacterial activity against E. cloacae with MIC values of 2.50 mg/ml in comparison with some chlorine-releasingagents (CRA) against E. cloacae with MIC values of 420 mg/ml (Mazzola et al., 2009). The ferrocenylimines compounds are very stable in solution and easy to handle without decomposition when expose to air (Ikhile and Ngila, 2015). The non-toxicity of the ferrocenylimines compounds have also been reported (Gupta et al., 2014; Qin et al., 2013). In contrast to all form of chlorine used in water disinfection such as chlorine gas, calcium hypochlorite, bromium chloride and hypochloride solution (Casey et al., 1998). These are highly corrosive and toxic, which pose safety risk when handled (Darby et al., 1995). The activity of the ferrocenylimines compounds to both Grampositive and Gram-negative may be attributed to the chemical structure of the compound. Thus the ferrocenyl derived compounds have been reported to have high antibacterial activity (Biot et al., 2000; Zhang, 2008). Imines (also known as Schiff bases) too are known to exhibit high antibacterial activities (Qin et al., 2013). Karthikeyan et al. (2006) reported the inhibition of the growth of S. aureus, E. coli, P. aeruginosa and K. pneumonia with 2,4-dichloro5-flurophenyl Schiff base moiety. Thus, the combination of biological active ferrocenyl and imines in compounds 2 and 3 contributed to their activity towards the tested bacterial strains. The Bacterial cell wall are surrounded by lipid membrane which only favor the passage of lipid soluble materials (Abu-Dief and Mohamed, 2015). Thus antibacterial activity of ferrocenylimines compounds increases as the lipophilicity nature increases, which affords the permeation of the ferrocenylimines compounds into the bacterial cell wall and inhibit the bacterial growth (Raman et al., 2009). The toxicology studies of these ferrocenylimines compounds are currently being investigated. It will be interesting to observe their effects on bacteria when applied in water treatment as potential disinfectants. We however caution that, from the literature reports, it has been observed that ferrocenyl derived compounds do possess low toxicity (Popova et al., 1993; Snegur et al., 2004; Simenel et al., 2008).
Please cite this article in press as: Ikhile, M.I., et al., Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water, Physics and Chemistry of the Earth (2017), http://dx.doi.org/10.1016/j.pce.2017.01.022
M.I. Ikhile et al. / Physics and Chemistry of the Earth xxx (2017) 1e5
5. Conclusion The ferrocenylimine compounds 1e3 were subjected to tests for antimicrobial activity against twelve different bacterial strains: E. coli, E. coli, K. pneumoniae, K. pneumoniae, K. oxytoca, P. aeruginosa, P. mirabilis, S. sonnei, S. boydii, E. faecalis, B. cereus, E. cloacae and the MIC values have been reported. The tested compounds shown similar activity against four bacterial strains E. coli (MIC ¼ 1.25 mg/ ml), K. oxytoca (MIC ¼ 1.25 mg/ml), B. cereus (MIC ¼ 0.31 mg/ml), S. boydii (MIC ¼ 1.25 mg/ml). Compound 2 alone shown activity against P. aeruginsoa (MIC ¼ 1.25 mg/ml) and E. cloacae (MIC ¼ 2.50 mg/ml). Also compound 3 exhibit excellent activity against S. sonnei (MIC ¼ 0.31 mg/ml). These results revealed the ferrocenylimine compounds to exhibit antibacterial activity. Therefore, the ferrocenylimines compounds could serve as a good potential for water disinfectants. Further studies are currently ongoing to investigate the toxicology of ferrocenylimine compounds 1e3. Acknowledgements The Faculty of Science, University of Johannesburg is highly appreciated for financial support and Department of Applied Chemistry, Water and Health Research centre for availing its facilities. The Centre for Nanomaterials Science Research (CNSR) is acknowledged for providing running cost of this project. References Abu-Dief, A.M., Mohamed, I.M.A., 2015. A review on versatile applications of transition metal complexes incorporating Schiff bases. BJBAS 4, 119e133. Biot, C., Franccois, N., Maciejewski, L., Brocard, J., Poulain, D., 2000. Synthesis and antifungal activity of a ferrocene-fluconazole analogue. Bioorg. Med. Chem. Lett. 10, 839e841. Cabral, J.P.S., 2010. Water microbiology. Bacterial pathogens and water. Int. J. Environ. Res. Public Health 7, 3657e3703. Casey, P., Mackne, C., Lake, A., 1998. Chlorine Disinfection. http://www.nesc.wvu. edu/pdf/ww/publications/eti/chl_dis_gen.pdf. Accessed 04 July 2016. Craun, G.F., 1986. Waterborne Disease in the United States. CRC Press, Inc, Boca Raton, FL. Darby, J., Heath, M., Jacangelo, J., Loge, F., Swaim, P., Tchobanoglous, G., 1995. Comparison of UV Irradiation to Chlorination: Guidance for Achieving Optimal UV Performance. Water Environment Research Foundation, Alexandria, Virginia. Eloff, J.N., 1998. A sensitive and quick microplate method to determine the minimal inhibitory concentration of plant extracts for bacteria. Planta Med. 64, 711e713. Fenwick, A., 2006. Waterborne Diseases- could they be consigned to History? Science 313, 1077e1081. Gauthier, F., Archibald, F., 2001. The ecology of “Faecal indicator” Bacteria commonly found in pulp and paper mill water systems. Water Res. 35, 2207e2218. George, I., Crop, P., Servais, P., 2001. Use of b-D-Galactosidase and b-D-Glucuronidase activities for quantitative detection of total and faecal coliforms in wastewater. Can. J. Microbiol. 47, 670e675. Grabow, W.O.K., 1996. Waterborne diseases: update on water quality assessment
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Please cite this article in press as: Ikhile, M.I., et al., Potential application of synthesized ferrocenylimines compounds for the elimination of bacteria in water, Physics and Chemistry of the Earth (2017), http://dx.doi.org/10.1016/j.pce.2017.01.022