Microbial & Biochemical Technology
Monica et al. J Microbial Biochem Technol 2011, 3:3
Research Article Open Access
Research Article Open Access
Formulation of Effective Microbial Consortia and its Application for
S Monica, L Karthik, S Mythili and A Sathiavelu*
School of Biosciences and Technology, VIT University, Vellore, India
The present study was conducted for sewage treatment using effective microbial consortium. The Effective
Microorganisms (EM) like Lactobacillus, Pseudomonas, Aspergillus, Saccharomyces and Streptomyces were
isolated from respective sources. The microbial consortium was formulated using molasses as medium at pH 3.8
and incubated at 37°C for 3 days. The sewage treatment was carried out with the addition of 3 ml/l EM solution under
aerobic condition. The BOD, COD, TDS and TSS were reduced by 85%, 82%, 55%, and 91% respectively after 3
days of treatment. The results showed that the formulated EM was efficient for sewage treatment and thereby it
reduced the environmental impact.
Keywords: Effective Microorganisms; Molasses; Optimization;
Sewage; BOD; COD; TDS; TSS
Abbreviation: EM: Effective Microorganisms
Sewage treatment is one of the major problems faced by
municipalities. Sewage is the wastewater comprising 99.9% water and
0.1% solid particles. The domestic sewage has high amount of organic
and inorganic pollutants . The untreated sewage causes foul smell
. The improper disposal of sewage causes pollution and destroys the
aquatic organisms due to high organic content and biological oxygen
demand (BOD) concentration . So, the sewage has to be treated to
reduce the environmental impact. The chemically treated water causes
harmful effects due to toxic chemicals than the organisms which
are originally present in the sewage . The organisms present in
wastewater degrade organic matter  and helps for further treatment.
In conventional treatment method, bacteria remove the organic content
of wastewater but the solid particle remains as sludge. The sludge can
be used as fertilizer or incinerated, disposed into ocean or landfill. The
conventional sewage treatment processes are expensive to operate and
maintain  and causes pollution.
Effective Microorganism (EM) is the consortia of beneficial
and naturally occurring microorganisms which are not chemically
synthesized or genetically modified. The EM technology was developed
by Professor Dr. Teruo Higa at University of Ryukus, Okinawa, Japan
in 1970s. The EM solution is the blending of effective microorganisms
in molasses at low pH. Initially EM was developed to increase the crop
yield by enhancing the soil activity . But later, it has its application
in wastewater treatment . The EM has its wide application in the
field of agriculture, natural farming, livestock, gardening, composting
, bioremediation , algal control and prawn culture. The EM
suppresses soil borne pathogen and pest, promotes plant growth,
improves soil fertility and yield of crops and used as feed additive for
livestock. The EM treated sludge is used as fertilizer and the EM treated
waste water is used in crop production as it is enriched with beneficial
The EM secretes organic acids and enzymes which acts on sewage
and degrades complex organic matter into simpler ones . The
antioxidant substances produced by EM enhances the breakdown of
solids and reduces the sludge volume . Missouri river in Jefferson
City, North America was polluted by run off from industries and
cities and generates foul odour. The application of EM for one month reduced the foul odour . In Thailand, EM was sprayed 3 to 4 times
on 3000-4000 metric tons of garbage which were dumped daily at a site
just outside Bangkok in Ladkra Bhan. The EM reduced the foul odour
and flies .
The EM used in this study comprises Lactobacillus, Pseudomonas,
Aspergillus, Saccharomyces and Streptomyces. The lactic acid bacteria
enhance the breakdown of organic matter such as lignin and cellulose.
Yeast produces antimicrobial substances and their metabolites are used
as substrate for lactic acid bacteria and actinomycete. The bioactive
substance produced by yeast promotes plant growth. Pseudomonas
releases bioactive compounds which act on the sewage and precipitates
or detoxifies the metal. Aspergillus decomposes organic matter
rapidly and produces alcohol, esters and antimicrobial substances.
Actinomycete produces antimicrobial substances from amino acids
derived from organic matter for suppressing harmful fungi and
The main objective of this study was to develop low cost and eco-
friendly sewage treatment process using effective microbial consortia.
Materials and Methods
Collection of samples
The respective samples were collected for isolation of various
microorganisms. The curd sample was used for isolation of Lactobacillus.
The oil spilled soil and moist soil at the depth of 10 cm was aseptically
collected in a sterile polythene bag from VIT University, Vellore, Tamil
Nadu for isolation of Pseudomonas and Streptomyces, respectively. The
dry yeast granules were used for isolation of Saccharomyces. The boiled
rice sample was maintained in closed container for 3 days under sterile
condition until the fungal mat was observed and used as inoculum for
isolation of Aspergillus. The samples were refrigerated at 4°C for further use.
Isolation of effective microorganisms
The curd sample and oil spilled soil sample were serially diluted, 10-4,
10-5 and 10-6 dilutions of sample were inoculated on de Man Rogosa
Sharpe Agar and King’s B Agar and incubated at 37°C for 24 hours
to isolate Lactobacillus and Pseudomonas, respectively. The moist soil
sample was serially diluted, 10-3, 10-4 and 10-5 dilutions were inoculated
on Kenknight’s Agar and incubated at 37°C for 3 days to isolate
Streptomyces. The obtained inoculum from rice was inoculated on
Czapek’s Dox Agar by hyphal tip technique and incubated at 28°C for
3 days to isolate Aspergillus. The loop full of inoculum was inoculated
on Potato Dextrose Agar and incubated at 37°C for overnight period to
isolate Saccharomyces. The obtained colonies were subcultured to get
pure culture as described by Cappuccino and Sherman .
Characterization of effective microorganisms
The isolates were identified by morphological and biochemical
studies. Biochemical tests like catalase test, oxidase test, IMViC
test, sugar fermentation tests, Triple Sugar Iron test, urease test and
hydrolysis tests were performed as described by Cappuccino and
Formulation of EM
The isolated microorganisms were cultured together in a medium
(molasses) at various pH, temperature and concentration of molasses.
The optimal physical conditions for formulating EM was analysed by
culturing microbial consortia at pH of 6.5-8, temperature of 28°C and
37°C and molasses concentration of 1-10%.
Sewage treatment using EM
The raw sewage sample was collected from VIT University,
Vellore, Tamil Nadu. The floating particles were removed from sample
and collected in a clean container. The container was washed using
sodium hypochlorite and water followed by rinsing of sample before
collection. 20 litres of sewage water was collected, divided into six
equal parts and maintained one as control and rest five for inoculating
different concentrations of EM. The pH, total dissolved solids (TDS),
total suspended solids (TSS), biological oxygen demand (BOD) and chemical oxygen demand (COD) of sample were analysed according
to the standard protocol of APHA  within 2 hours of collection.
Then the formulated EM solution was added to sewage at various
concentration ranged from 1-10 ml/l. The EM inoculated water was
analysed daily to determine the effect of EM in treating sewage.
All the experiments were done in triplicates. The data was analysed
statistically using Microsoft Excel 2007 and reported as mean ±
standard deviation (SD).
Results and Discussion
Characterization of EM
The isolated microorganisms were characterized according to
Bergey’s manual (Table 1). Erdogrul and Erbilir  stated that
Lactobacillus is gram positive rods, catalase and oxidase negative.
Pseudomonas was identified as gram negative motile rods and showed
positive for catalase, oxidase and citrate tests . Praveen and Jain
 reported that Streptomyces is gram positive rods and can hydrolyse
casein. The species of Streptomyces exhibited variation in colour of
substrate mycelium depending on the media composition .
Table 1: Characteristics of Effective Microorganisms.
Formulation of EM
Effect of pH and temperature: The growth of EM was observed at
pH of 6.5 to 8 and temperature of 28°C and 37°C. The Pseudomonas
may grow in a wide pH range of 4-10 at 27°C and 37°C but the optimal
condition is pH 8 and 37°C . The fungal species isolated from
Antarctic soil was observed to grow at temperature between 4°C and
35°C and exhibited variation in growth pattern . Praveen and Jain
 stated that Streptomyces sampsonii shows its growth at pH of 5-10
and temperature of 15-42°C.
Effect of molasses concentration: The growth of microbial
consortia was observed at various molasses concentration of 1-10%.
The lowest concentration of molasses facilitated the growth of EM and
the increased concentration inhibited the growth and survival of EM.
It is observed from Table 2 that 1% to 3% of molasses is favourable for EM. The growth of Lactobacillus and Saccharomyces was observed even at highest molasses concentration of 10%. The growth inhibition may be due to osmotic pressure created by molasses. 8.2
Effect of incubation period: The incubation period has greatest effect on microbial consortia formulation. At longer incubation period, the growth of microorganisms was inhibited due to depletion
of nutrients, accumulation of toxic end products and change in pH. The optimal incubation period was 72 hours as growth of all the five 7.4
organisms was observed (Table 3).
The pH is an important parameter for preparation of EM solution.
Figure 1 depicts the variation in pH of EM solution during incubation.
The pH was decreased from 7 to 2.9 in 5 days of incubation by fermenting
the molasses. After 5 days of incubation, the pH was constant as the
organisms utilised the entire energy source and there was no further
growth of organisms. The organisms was not able survive at high acidic
pH; hence EM solution was used after 3 days of incubation (pH 3.8).
Analysis of EM treated sewage
Biological oxygen demand: The EM reduced the BOD of sewage
from 374.5 to 55.9 mg/l with mean reduction of 85%. The EM
showed the effective result when compare to control while treated
at a concentration of 3 ml/l for 3 days. The control showed the
decrease in BOD from 374.5 to 248.6 mg/l in 5 days (Figure 3). The
acetogenic bacteria strain BP103 reduced the BOD by 58.5–82.2% . Mongkolthanaruk and Dharmsthiti  formulated bacterial
consortium including Pseudomonas, Bacillus and Acinetobacter using
molasses for treating lipid rich wastewater and the consortium reduced
BOD from 448 to 72 mg/l. Kumar  used the bacterial consortium of
Pseudomonas aeruginosa, Bacillus megaterium and Stenotrophomonas
maltophilia for treating paper and pulp mill effluent and observed BOD
reduction from 87 to 89%.
Figure 2: Effect of EM treatment on pH of sewage.
Figure 3: Effect of EM treatment on BOD reduction of sewage.
Chemical oxygen demand: The EM reduced the COD of sewage
from 570.4 to 99.8 mg/l with mean reduction of 82%. The EM reduced
the COD effectively while treated at concentration of 3 ml/l for 3 days.
The control showed the decrease in COD from 570.4 to 409.3 mg/l
in 5 days (Figure 4). The EM reduced the COD of wastewater from
Nestle and Trebor companies to 76% in 11 days at a concentration
1 ml/l . The acetogenic bacteria strain BP103 reduced the COD
by 35.5–71.2% . Stanley  reported that whey disposed from
cheese manufacturing industry was treated using Kluyveromyces
fragilis which reduced the COD by 29% and 37% in 16 and 20 hours,
respectively after the growth of culture. Kumar  used the bacterial
consortium of Pseudomonas aeruginosa, Bacillus megaterium and
Stenotrophomonas maltophilia for treating paper and pulp mill effluent
and observed COD reduction from 67% to 71%. The consortium of
five white-rot fungi, Phanerochaete chrysosporium, Pleurotus ostreatus,
Lentinus edodes, Trametes versicolor and S22 removed 71% of lignin
content and 48% of COD from wastewater .
Figure 4: Effect of EM treatment on COD reduction of sewage.
Total dissolved solids: The EM reduced the TDS of sewage from 2460 to 1084 mg/l with mean reduction of 55%. The EM showed the
effective reduction of TDS while treated at concentration of 3 ml/l for 3
days. The control showed the decrease in TDS from 2460 to 2309 mg/l
in 5 days (Figure 5).
Total suspended solids: The EM reduced the TSS of sewage from
486.6 to 43.3 mg/l with mean reduction of 91%. The EM showed the
effective reduction of TSS while treated at concentration of 3 ml/l for 3
days. The control showed the decrease in TSS from 486.6 to 433 mg/l
in 5 days (Figure 6). The acetogenic bacteria strain BP103 reduced
the total solid content by 59.1% . Okuda and Higa  used EM to
reduce the total solid content of wastewater by 94%.
pH: The EM did not show any considerable change in pH of
sewage. The fluctuation in pH was due to the natural environmental
factors (Figure 2).
At higher concentration of EM, the BOD and COD was increased
due to high microbial population. Hence 3 ml/l is the efficient
concentration of EM for the effective treatment of sewage. After 3 days
of treatment, the dissolved oxygen was decreased due to depletion of
nutrients. So the treated water has to be left for chlorination.
The white rot fungi and brown rot fungi in presence of glucose
reduced the BOD and COD of wastewater. If Streptomyces is cultured
along with these fungi there was increase in the decolourisation to 85%
. The microorganisms exhibit efficient treatment in consortium
than the sole organism.
The COD, BOD, TDS and TSS reduction of domestic wastewater
by sedimentation, aeration, activated sludge and sand filter was 92.17%,
97.66%, 32.38% and 97.58%, respectively . The sludge released by
these process causes environmental impact and also it is expensive. But
there is no release of sludge in EM treatment and the sewage can be
The Effective Microbial consortium was formulated and its
efficiency for sewage treatment was studied. The results showed that
the EM treatment of sewage reduced BOD, COD, TDS and TSS by 85%,
82%, 55% and 91% respectively. The malodour and turbidity of sewage
was reduced. The treatment process is highly viable and economical.
The EM treated water is non-toxic and safe to dispose as it contains
beneficial microorganisms. The EM reduces the environmental impact
of conventional methods.
Authors wish to thank management of VIT University, Vellore, TN, India, for
providing necessary facilities and support for the completion of this work.
1. Elliot HA (1986) Land application of municipal sewage sludge. Journal of Soil
and Water Conservation 41: 5-10.
2. Kulkarni GJ (1997) Water supply and sanitary engineering. 10th ed. Farooq
Kitab Ghar Karachi pp497.
3. Ronald MA, Richard B (1981) Microbial Ecology Fundamental and application
Addison-Wesley Publishing Company Sydney pp560.
4. Kurihara H (1990) Water quality of reusing waste water. Journal of Japan
Sewage Works Association 27: 38-41
5. Taylor C, Yahner J, Jones D, Dunn A (1997) Wastewater Pipeline. 8.
6. Mazumder D, Roy B (2000) Low cost options for treatment and reuse of
municipal wastewater. Indian J Environ Prot 20: 529-532.
7. Higa T, Parr JF (1994) Beneficial and Effective Microorganisms for a Sustainable
Agriculture and Environment. International Nature Farming Research Centre,
Atami, Japan, pp16.
8. Okuda A, Higa T (1995) Purification of Waste Water with Effective
Microorganisms and its Utilization in Agriculture, University of the Ryukyus,
9. Daly MJ, Arnst B (2005) The use of an innovative microbial technology (EM) for
enhancing vineyard production and recycling waste from the winery back to the
land, The 15th IFOAM Organic World Congress Adelaide.
10. Miyajima M, Nagano N, Higa T (1995) Suppression of dioxin generation in the
garbage incinerator, using EM (Effective Microorganisms), EM-Z, and EM-Z
ceramics Powder, College of Agriculture, University of Ryukyus.
11. Higa T (1996) Effective Microorganisms -Their role in Kyusei Nature Farming
and sustainable agriculture. In Proceedings of the Third International
Conference on Kyusei Nature Farming. Washington, USA pp20-24.
12. Freitag DG (2000) The use of Effective Microorganisms (EM) in Organic Waste
13.Higa T, Chinen N (1998) EM Treatments of Odor, Waste Water and
Environmental Problems College of Agriculture University of Ryukyus Okinawa
da Silva AB, Sanches AB, Kinjo S (1997) Use of Effective Microorganisms for
treatment of domestic sewage by the activated sludge process. Mokichi Ohada
Foundation lpeuna SP Brazil.
Cappuccino JG, Sherman N (1996) Microbiology - a Laboratory Manual 159-
Anon (1992) Standard methods of water and wastewater examination 18th Ed,
American Public Health Association NW Washington, DC 2-127.
Erdogrul O, Erbilir F (2006) Isolation and characterization of Lactobacillus
bulgaricus and Lactobacillus casei from Various Foods. Turk J Biol 30: 39-44.
18.Hussein H, Moawad H, Farag S (2004) Isolation and characterization of
Pseudomonas resistant to heavy metals contaminants. Arab J Biotech 7: 13-
19. Jain PK, Jain PC (2007) Isolation characterization and antifungal activity of
Streptomyces sampsonii GS 1322 Indian. J Exp Biology 45: 203-206.
20. Hassan AM, YI El-Shahed K, El-Monaem A, El-Nakkadi M (2009) Isolation,
screening and identification of newly isolated soil Streptomyces (Streptomyces
sp. NRC-35) for β-Lactamase inhibitor production. World Applied Science
Journal 5: 637-646.
21. Murad S, Hasan F, Ali Shah A, Hameed A, Ahmed S (2007) Isolation of phthalic
acid degrading Pseudomonas sp. p1 from soil. Pak J Bot 39: 1833-1841
22. Kostadinova N, Krumova E, Tosi S, Pashova, Angelova M (2009) Isolation and
identification of filamentous fungi from island Livingston Antarctica Biotechnol
11th anniversary scientific conference. pp267-270.
23.Sirianuntapiboon S, Phothilangka P, Ohmomo S (2004) Decolourization of
molasses wastewater by a strain No. BP 103 of acetogenic bacteria. Bioresour
Technol 92: pp31-39.
24.Mongkolthanaruk W, Dharmsthiti S (2002) Biodegradation of lipid-rich
wastewater by a mixed bacterial consortium. Int Biodeterior Biodegradation
25.Kumar A, Kumar R, Tiku DK, Sharma P, Chaturvedi R (2007) Biological
process for reducing chemical and biochemical oxygen demand of pulp and
paper industrial effluent. United States Patent 7267772-B2.
26. Gede Ngurah Wididana (1994) Preliminary experiment of EM technology on
wastewater treatment Indonesian Kyusei Nature Farming Society Indonesia.
27. Gilliland SE (1979) Measuring Chemical Oxygen Demand of Cottage Cheese
Whey cultured with Kluyveromyces fragilis. J Dairy Sci 62: 882-887.
28. Juan Wu, Xiao YZ, Yu HQ (2005) Degradation of lignin in pulp mill wastewaters
by white-rot fungi on biofilm. Bioresour Technol 96: 1357-1363.
29. Bowling M, Adams P (2003) Method of wastewater treatment utilizing white rot
and brown rot fungi, United States patent.
30. Al-Jlil S (2009) COD and BOD reduction of waste water using activated sludge
sand filters and activated carbon in Saudi Arabia. Biotechnol 8: 473-477.
Copyright: © 2011 Monica S, et al. This is an open-access article distributed under
the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and
source are credited
Full Text available: http://www.omicsonline.org/1948-5948/JMBT-03-051.pdf