Isolation of Microorganisms from Dairy Effluent

Aim: Every Dairy industry has problems of effluent treatment. This can be revealed by effective treatment of the effluent. The effective treatment can be done by using microorganisms to stabilize the organic and inorganic load of the effluent. The aim of the present work is to study the dairy wastewater micro biota and to identify some new active strains which can bring about fast biodegradation of the organic compounds. Study Design: Isolation and determination of bacteriological characteristics of the dairy effluents. Methodology: Studies were carried out to isolate the microorganisms from collected effluent sample from the dairies under studies. Isolation of microorganisms was done by primary screening, Cultural Characterization, Biochemical characterization and Identified by using Bergey’s Manuals of Systematic Bacteriology. Results: During 2011-2013 from two different districts of Maharashtra (India), dairy industry effluents were collected for the isolation of micro organisms. Effluent samples were collected as per Jacksch and piper method and primary screening was done and totally 7 Isolates were screened out. These isolates were characterized on nutrient agar at room temperature for 24 hrs. Isolates were observed for the cultural characters like size, shape, colour, margin, elevation, opacity and consistency and morphological characters like Gram nature, sporulation, shape and arrangement of cells, motility etc. and were recorded. Physio-biochemical characterization was followed by biochemical tests for enzymatic activities like catalase, oxidase, nitrate reduction, urease, caseinase etc and carbohydrates utilization tests for lactose, maltose, inositol, xylose etc. performed to check their ability for Research Article British Microbiology Research Journal, 3(3): 346-354, 2013 347 metabolization. On the basis of these characteristics, isolates were identified by using Bergey’s Manuals of Systematic Bacteriology. The identified Bacterial Isolates were of Genus Lactobacillus, Bacillus, Staphylococcus, Enterococcus, Listeria etc. Conclusion: These Bacterial isolates have the ability to utilize the components like nitrate, starch, gelatin, sugars like sucrose, maltose, lactose etc. which was confirmed by the biochemical tests. Bacterial flora from the effluents can be identified and efficiently applied for the biological treatment of the dairy effluents.


INTRODUCTION
Environmental pollution is one of the major ecological challenge, for the last few decades all over the world, in which developing countries are most affected. Major cause of the pollution is rapid industrialization, which is progressive at breakneck speed, and is suffocating the earth with the common problems of pollution. A 2007 study reports that the discharge of untreated sewage is single most important cause for pollution of surface and ground water in India. Among the major industries in India, dairy is one of the industries producing waste water rich in organic matter and thus leading to creation of odorous and high COD containing water [1]. The problem is not only that India lacks sufficient treatment capacity but also that the sewage treatment plants that exist do not operate and are not maintained [2]. The dairy industry on an average has been reported to generate 6-10 liters of waste water per liter of the milk processed [3]. It is estimated that about 2% of the total milk processed is wasted into drains [4]. Due to the high pollution load of dairy waste water, the milk-processing industries discharging untreated/partially treated waste water cause serious environmental problems [5][6]. In recent years, urban people are facing many problems and water pollution is one among them. Environmentalists and government are looking for efficient, cheap and long lasting solutions for waste treatment and recycling. Physico-chemical methods of waste water treatment are inevitably cost intensive and cannot be employed in all industries especially in developing countries like India. Hence, in recent years, the importance of biological treatment systems has attracted the attention of workers all over the world and has helped in developing relatively efficient, low cost waste treatment systems.
In order to design an efficient biological waste water treatment it is important to know the waste water microbiota composition and the biochemical properties correlated to the origin of pollutants, as well as the optimum metabolic activity and the physical-chemical conditions [7][8].The study of wastewater microbiota and to identify some new active strains adapted to the wastewater physical-chemical conditions, which metabolize organic compounds, similar to those which determine the pollution of wastewaters such as starch, casein, basic carbohydrates and lactic acid [9]. Microbial strains were identified for the rapid biodegradation of the organic compounds. In the present work bacteriological studies were carried out to isolate the microorganisms from dairy effluent samples.

MATERIALS AND METHODS
The designed experimental studies followed the methodology which represented in the Fig.  1.

Primary Screening for Isolation of Micro Organisms
The effluent samples were collected from dairy 1 from Solapur at 75º East and 17º North and dairy 2 from Kolhapur at 74º East and 19º North of Maharashtra state in India. The samples were collected, in duplicate, in a clean sterile plastic container and stored at 4ºC until the analysis was carried out according to the standard methods of APHA [10] and Trivedy and Goel [11]. The experiments were carried out in five replicates and the mean value was considered. For primary screening, the dilutions of effluent samples were prepared in distilled water. Selective dilutions were spread on nutrient agar and incubated for 24 hours at room temperature. Isolated colonies were selected for the further studies.

Characterization of the Bacterial Isolates
Isolates were incubated on nutrient agar for 24 hours at room temperature and was characterized. Each of the isolates were observed for the cultural characters like size, shape, colour, margin, elevation ,opacity and consistency and morphological characters like Gram nature, sporulation, shape and arrangement of cells, motility etc. Biochemical characterization was followed by tests for enzymatic activities like catalase, oxidase, nitrate reductase, urease, caseinase etc. Carbohydrates utilization tests for various sugars like lactose, maltose, inositol and xylose were carried out. For characterization the methodologies were followed as per stander methods [12][13][14][15][16] as given in the Table 1. Microbial isolates were identified according to Bergey's Manual of Determinative Bacteriology [17,18]. Bacterial isolates were to be inoculated in tubes containing sterile peptone nitrate broth and incubated for 24hours at room temperature.
Positive test show red colour formation after addition of sulphanilic acid -naphthyl-amine.

Urease
Slants of sterile Christensen's urea agar (1946) were inoculated with the bacterial isolates and incubated at 28ºC for 24 hours Hydrolysis of urea was detected by the appearance of the pink colour in the medium and the absence of pink colour indicate negative test.

Oxidase
Colonies of the bacterial isolates are transferred by a glass rod on the filter paper strip moistened with freshly prepared 1% tetramethyl-1-pphenylene diamine (TAPA) solution and observed.
Appearance of deep blue colour on the strip indicates the positive result for oxidase.

Starch hydrolysis
Spot inoculation of bacterial isolates on sterile starch agar containing 1% starch and incubated at 28ºC for 24 hours.
Lugols iodine when poured on the plates show zone of starch hydrolysis around the colonies for positive test.

Hugh and Leifsons (H&L)Test
The bacterial isolates were inoculated in to the two tubes of Hugh and Leifsons Medium (Aerobic and Anaerobic) in order to check oxidative and fermentative metabolism of sugar and incubated at 28ºC for 24 hours.
Observe for acid (yellow colour) and gas (bubble in durhum's tube) production on incubation of tubes for positive test.

Hydrogen Sulphide Production Test
The bacterial isolates were inoculated in to sterile standard thiosulphate iron agar stab medium and incubated at 28ºC for 24 hours.
Formation of black ferrous Sulphide precipitate in the medium indicated thiosulphate reduction and hydrogen sulphide production.

Carbohydr ates Utilization
Peptone water is used as a basal medium for the tests. 5ml aliquots each to be taken to test the utilization of different carbohydrates. Tests were carried out for hexose sugar glucose; disaccharides like lactose, maltose and sucrose; sugar alcoholsmannitol and sorbitol; Sugar Vitamin -Inositol; Peptose -Xylose.
After incubation observe the tubes for acid and gas production for positive test.

RESULTS AND DISCUSSION
Characteristics of industrial wastewater vary greatly from industry to industry and within industries also. In the present studies effluent samples collected from dairy1 (Table 2)   Totally 7 Isolates were screened out from the effluents and were characterized on nutrient agar by incubating for 24 hours at room temperature. Each of the isolates was observed for the cultural characters like size, shape, colour, margin, elevation, opacity and consistency (Table 3). Similar results were reported by Vijayakumar et. al. [19]. They isolated different bacterial genera from dairy wastes for the biodegradation of dairy effluent and observed different cyanobacteria with different effluents. The colonies of the isolates on nutrient agar were circular to irregular shaped and of the size ranging from <1mm to 3mm. The colour of the colonies was dirty white and yellowish mostly. The colonies of all the isolates exhibited regular to irregular margins with mostly flat to convex elevation and moist, opaque, transparent, translucent consistency in nature.
The bacterial isolates on nutrient agar were stained for their morphological characters and the results are presented (Table 4). All seven bacteria isolated were Gram-positive in nature. Among that isolate number 2, 5, 6 and 7 were Gram-positive cocci and isolates 1, 3 and 4 were observed as Gram-positive rods. Isolates 1, 2 and 4 were observed as motile, while 3, 5, 6 and 7 were non-motile. Endospore production was showed by isolates 1, 4 and 6.
Sreekumar et.al [20] have successfully isolated a new strain of spore forming Bacilli that is capable of fermenting lactose from dairy effluents.
Enzymatic activities were checked for catalase, oxidase, nitrate reduction, urease and caseinase (Table 5). These tests confirmed the ability of bacterial isolates to degrade various substrates. Studies on enzymatic activities indicated the degrading capacity of these microbes and thus they can be utilized for the further treatment of dairy waste water. All isolates showed positive results for Hugh and Leifsons (H&L) test and nitrate reduction, except isolate 7. Isolate 1, 2, 3 and 7 showed positive test for catalase. Except isolate 2, other isolates indicated negative test for hydrogen sulphide production. Six of the isolates showed caseinase activity. Isolate 2 and 6 showed negative test for oxidase. Starch hydrolysis or amylase test indicated negative for isolate 4 while all others gave positive test. Urease tested positive for isolates 1, 3, 4, 5 and 7. Isolates 2 and 4 showed absence of gelatinase. Carbohydrates utilization tests for lactose, maltose, inositol, mannitol, xylose, glucose and sucrose were carried out ( Table 6). Prakashveni and Jagadeesan [21] reported the isolation of Lactobacillus from dairy effluent. Where " +": Spore producer ; " -" : Non-Spore producer.   -+ --+ + + Table 7 represents the tentative identification of bacterial isolates by Bergey's Mannuals of Systematic Bacteriology. Phanse et al. [22] collected and studied soil samples, waste water samples (including industrial effluents, dairy waste, domestic sewage) and activated sludge samples from various sources and used for the isolation of bacteria and (PHA) Polyhydroxyalkanoic acids producing strains belonged to the genera-Bacillus, Pseudomonas, Azotobacter and Staphylococcus.

CONCLUSION
In any dairy plant, the quantity and characteristics of effluent is depending upon the extent of production activities. The present study indicates that isolated bacterial cultures from dairy effluent have the ability to utilize the components like nitrate, starch, gelatin, sugars like sucrose, maltose, lactose etc. These micro organisms can be efficiently applied for the biological treatment of the dairy effluent treatments. Micro organisms uses the organics in waste water as a food supply so that water pollution threats due to dairy effluents can be reduced through biological methods and this will enable the recycling of water.