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Paper

for National Conference

on Household

Wake Treatment Technology,

at Hindustan College ofSc. And Tech. Farah, Mathura 00 24, 2S July 2008

Moringa oleifera Seed as Antibacterial Agent in Water Treatment

Jadhav Swapnali Mohan', Bipinraj N K2, Milind R Gidde3*

Abstract
Water extract of Moringa o/eifera seed, a natural anti-coagulant, was checked for its ability to reduce the total bacterial count in ground water. Using MPN method different concentration of the seed extract was tested for antibacterial disinfectant. potency. The results were compared with chlorine, which is the commonly used

MPN levels were decreased to greater extent with the treatment seed extract. Maximum

reduction of 77.27% in MPN was found when the ground water was treated for 30 min. with 50mg/ml of seed extract. An attempt was made to find out the MIC seed extract against E. coli by TVC method and

found out that 12.5mg/ml concentration of seed extract could reduce >999% E.coli count. The ability of the extract reduced when heated at 80c for 30 min. but normal conditions (30c) it act as an antibacterial agent.

This study highlights commonly

an environmental

friendly method of ground water disinfection.

Moringa

is a

used vegetable and widely available in all part of India. Moreover the seed extract is also

reported as an anti coagulant and used for water treatment in several parts of the world. These reports strongly suggest that Moringa seed extract can replace Chlorine in water treatment after standardization the method. of

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Key words - Water, Moringa seed extract ground water.

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BVIEER, Bharati Vidyapeeth University, Pune Dept. of /cell and Molecular Biology, Rajiv Gandhi Institute of IT and Biotechnology, Vidyapeeth University, Pune Bharati

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Professor,

Dept. of Civil Engineering,

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Bharati Vidyapeeth University College of Engineering,

Dhanakawadi,

Pune - 411043 (Maharashtra) author E-mail: milindgidde@yahoo.co.in

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Corresponding

1. Introduction
Water is the unique molecule that preserves the life on the Earth. All life forms on the Earth depend on

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water. About 70% of Earth is covered with water in that 97% is part of Oceans. Only a small percentage of the total water is fresh water, which is used by humans for drinking, farming and washing. Safe drinking water is a major requirement of any society. Ensuring people's access to safe drinking-water and adequate sanitation will improve the quality of life of millions of individuals. According to WHO report "Safe water for better health" One in every 10 diseases and 6% of all deaths globally are caused by unsafe water and improper hygiene. In India alone, 1.03 crore people die annually of which, nearly 7.5% deaths are related to

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water, sanitation and hygiene. Diarrhoeal diseases make up a major part of this number causing 4.02 lakh deaths. According to the report, 88% of cases of diarrhea worldwide are attributable to unsafe water. Diarrhoeal diseases like cholera, typhoid and dysentery caused mainly by the ingestion of pathogens result in 1.5 million deaths each year. This large number of death due to diarrhea is mainly due to drinking contaminated water. Lack of adequate drainage system and distribution of clean drinking water are the main reasons of high mortality due to waterborne diseases in developing countries. Natural sources of drinking water are ponds, lakes, streams, rivers and rain. Although the pond water can be directly consumed it can transmit harmful pathogen. And it increases the risk for diseases in human as well as animal body. Ground water is another important source of drinking water. Collected through well or tubes wells they are rich source of minerals, but due to industrial pollution ground water also getting
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contaminated with several heavy metals and non-metals. An average person may consume not more than 2 to 3 lit water. However, the per capita consumption of water drawn from public supply is quite large. Untreated or partially treated domestic and industrial wastes released into the water sources affect the health of consumers and causes death by different pathogenic microorganisms.

Chlorination is the most widely used method for water treatment. However, chlorination has numerous disadvantages such as production of trihalomethanes and chlorinated hydrocarbons which are considered health hazards. It combines with inorganic material in water to form chloride salts, and with organic _material in water to form chlorinated organic chemicals. These chlorinated compounds are less likely to degrade and can cause the same hazards as that of chlorine. Chlorination also produce large amount of
,

sludge which is again an environmental pollutant. Due to these demerits of chlorine and high expense of other physical method there is a requirement of safe and cheap water treatment method.

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Naturally occurring coagulants and disinfectants can be an alternative to chemical and physical water treatment method. There are several biological agents, mainly plants, reported for its coagulating activity especially removal of microorganisms. Coagulation effect ofMO seed on turbid water has been reported by a number of researchers (John, 1988; Sutherland et aI., 1990; Nkhata, 2001; Muyibi and Evison, 1995). Seeds of the Moringa o/eifera found to be highly effective in removing suspended particle from water with medium to high levels of turbidity (ECHO, 17391 Durance Rd., North Ft. Myers FL 33917, USA). These studies propose that Moringa o/eifera seed powder can be an alternative to chemical and physical method.

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However, effectiveness of waste water treatment also depends on the removal of pathogenic microorganisms that cause water born diseases. The present study shows activity of Moringa o/eifera seed powder as an antimicrobial agent.

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2. Materials

and Methods of Moringa olelfera seed extract

2.1 Preparation

The Moringa oleifera seed of dry pod were used for the extraction. The seed were removed from the pod, and stored at room temperature.
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The winged seed cover was shelled just before the extraction. The kernel

was ground to a fine powder by using a mixer and 5gm of the seed powder was mixed with 100 ml of distilled water. The suspension was stirred using a Cyclo Mixer for 10 min. The solution was then filtered through a muslin cloth. The filtered solution was used for disinfection experiment immediately.

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2.2 MOST PROBABLE NUMBER (MPN) METHOD method to find out the water quality. This broth. Presence of coli forms occurs

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Most probable number (MPN) is a routine microbiological

method checks the presence of coli forms in the water by using MacConky's

is an indirect proof of the possible occurrence of dangerous pathogen, since coli form contamination

through fecal matters. In this experiment MPN of the ground water treated with Moringa seed extract was checked and compared with non-treated water. MPN of the treated and non-treated water has performed in
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fOUT replicates. In each experiment 5 ml of seed extract (50 mg/ ml) has mixed with the ground water and checked for MPN at 0 hr. and after 30 min. of incubation. Ground water sample without any treatment and sterilized distilled water with 5Omg/ml seed powder were used as controls.

2.3 Effect of seed extract on Bacterial

growth

The effect of seed extract on bacterial growth was studied using E. coli as test organism. In this experiment

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E. coli (1 % 1 aD) was inoculated in nutrient broth containing different concentration (2.5, 5, 10, 50 mg) of seed extract. The growth of the bacteria was checked by measuring optical density and compared with the control flasks inoculated with E. coli.

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2.4 MIC (Minimum Inhibitory Concentration) (MlC) is the lowest concentration of an antimicrobial that will inhibit

Minimum inhibitory concentration the growth of a microorganism. of an antimicrobial

Minimum inhibitory concentrations are important to find out the efficiency

agent. MIC of the seed extract was found out using double dilution method. Initial

concentration of 50 mg/ml was diluted to 25, 12.5, 6.25, 3.12, 1.56 and 0.78 mg/ml in nutrient broth. All these tubes were then inoculated with 0.02 ml of 1 Ol) E. coli suspension and incubated for 18 hr.

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2.5 Comparison

between Chlorine and M.oleifera

Chlorine is the most widely used chemical for water treatment. It can also inhibit microbial growth in the

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water. The ability of Moringa seed extract to inhibit the growth of bacteria was compared with that of chlorine. For this experiment ability of 5% seed extract to inhibit the bacterial growth was compared with chlorine demand of the ground water (5mgIL). The comparison was performed by MPN method.

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3. Result and Discussions 3.1MPN MPN results showed that seed extract was highly effective in reducing the MPN. In the case of ground water control the MPN was 110 and sterilized distilled water with seed powder showed and MPN of 23. Ground water treated with seed extract showed an MPN of 94 at 0 hr incubation while after 30 min. the MPN is found to be 23. This value was equal to that obtained from sterile distilled water (Table 1). Table I: MPN of treated and untreated samples. SET Nos. Ground water Test (Raw water with seed extract) at 0 hr. Test (Raw water with seed extract) after 30 miD. I Sterile distilled water with seed extract 23 23 23 23

m
AVERAGE

no no no no
growth

94 70 94 86

23 23 23 23

3.2 Effect of seed extract on Bacterial

Moringa seed powder extract clearly inhibited the growth of E. coli (Figure 1). After 8 hours of
incubation the growth of control flask increased to 1 OD while the OD of the bacteria in flask containing seed extract remained same as that Ohr. same result was observed with all

concentrations r

of seed extract tested. These results indicate that Moringa seed extract can be used

as an excellent natural antibacterial agent in treating the ground water. These results were similar to the results Canarelli,Florian observed in other earlier reports(Mougli Suarez,Marisa Haenni,Ste phane

Fisch,Pierre Chodanowski,published.Sept.2005p.3847-3857)

1.2 1

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0.8 0.6

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5mg(ml seed extract with e.coIi

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0.4

0.2

o~~~==~===*==~==~~==~==~
1 2 3 4 nilE (IIin..) 5 6 7 8

Figure 1: growth ofbacteria in presence of 5 mg/ml seed extrac

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3.3MIC Tubes with 12.5 and 25 mg/ml seed extracts showed minimum optical density (0.01 aD) after 18hrs incubation. Only 300 viable celli m1were found in the plate when checked the TVC, whereas in the case control the TVC was 106 ceillmi (Table 2.). 12.5-mglml seed extraction reduced the growth by more 99%. (Lausanne, feverier 2004.) Table 2: Total Viable Count Tune for incubation 19hr 19hr
rConcentration of seed powder

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Total viable oount/ ml

12.5mg Control as KcoIi

300
10"

3.4 Comparison with chlorine

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Chlorine at 4.5 mg/l concentration reduced the MPN to zero, whereas Moringa seed extract could reduce the MPN to 23. Chlorine is widely used for water treatment because it can treat any kind of water and highly reactive. However due to its high reactivity it can cause severe environmental and health related problems. The rural people are not aware of the hazardous of chemical disinfectant and use them indiscriminately to treat drinking water. Chlorine forms the HIM (tri-halo methane), which is known carcinogen (D.W.Page, J.A. van Leeuwen, K.M.Spark, M.Drikas, Withers, D.E.Mulcahy; WMer Research

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36(2002) 4884-4892).

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Table 2. Comparison of seed extract and chlorine treatment Untreated ground water NO. Of tubes giving positive reaction 50flOml 5 50flml 3 50fO.lml 1 MPN
110

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Ground water treated with seed extract (5%) Ground water treated with
Chlorine

NO. Of tubes giving positive reaction 50flOml 5 Sof Iml

0

5ofO.lml
0

MPN 23

NO. Of tubes giving positive reaction 50flOml

0

50flml
0

50fO.lml
0

MPN 0

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4. Conclusion This project has indicated that groundwater can be treated considerably with Moringa oleifera seed extract. In this study it was found that the MPN of groundwater was reduced from 110 to 23 (79010 reduction) by

the treatment with seed extract. It can also inhibit the growth of E.coli and the MIC was found between 12.5 to 25 mg/ml of seed powder. Although this figure is higher than the earlier report, by standardization of different parameters affecting the disinfection it can be made more effective as that of chlorine.

Moreover Moringa is widely grown in India for use as a vegetable, so the seeds will be easily available for treatment of water. This will help in the development of rural water treatment using Moringa seed extract. In conclusion Moringa seed extract is proposed to be an effective environmental chlorine in ground water treatment and can be used in rural ground water treatment. 5. Acknowledgement The authors are thankful to the heads of the institutions of Bharati Vidyapeeth University in which they are friendly alternative for

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