Formulation of Effective Microbial Consortia and its Application for Sewage Treatment

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


Introduction
Sewage treatment is one of the major problems faced by municipalities. Sewage is the wastewater comprising 99.9% water and aquatic organisms due to high organic content and biological oxygen demand (BOD) concentration [3]. So, the sewage has to be treated to wastewater degrade organic matter [5] and helps for further treatment. In conventional treatment method, bacteria remove the organic content conventional sewage treatment processes are expensive to operate and maintain [6] and causes pollution. and naturally occurring microorganisms which are not chemically by Professor Dr. Teruo Higa at University of Ryukus, Okinawa, Japan in molasses at low pH. Initially EM was developed to increase the crop yield by enhancing the soil activity [7]. But later, it has its application suppresses soil borne pathogen and pest, promotes plant growth, improves soil fertility and yield of crops and used as feed additive for microorganisms [11].
antioxidant substances produced by EM enhances the breakdown of on 3000-4000 metric tons of garbage which were dumped daily at a site Lactobacillus, Pseudomonas, Aspergillus, Saccharomyces and Streptomyces. enhance the breakdown of organic matter such as lignin and cellulose. Yeast produces antimicrobial substances and their metabolites are used substance produced by yeast promotes plant growth. Pseudomonas releases bioactive compounds which act on the sewage and precipitates 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 bacteria.

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 [15].

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 Sherman [15].

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 [16] 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.

Statistical analysis
All the experiments were done in triplicates. The data was analysed statistically using Microsoft Excel 2007 and reported as mean ± standard deviation (SD).

Characterization of EM
The isolated microorganisms were characterized according to Bergey's manual (Table 1). Erdogrul and Erbilir [17] 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 [18]. Praveen and Jain [19] 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 [20].

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 [21]. 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 [22]. Praveen and Jain [19] 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  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.

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 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% [23]. Mongkolthanaruk and Dharmsthiti [24] 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 [25] 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%.

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 [26]. The acetogenic bacteria strain BP103 reduced the COD by 35.5-71.2% [23]. Stanley [27] 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 [25] 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 [28].    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% [23]. Okuda and Higa [8] 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% [29]. 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 [30]. 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 treated economically.

Conclusion
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.

Okuda A, Higa T (1995) Purification of Waste Water with Effective
Microorganisms and its Utilization in Agriculture, University of the Ryukyus, Okinawa, Japan.