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Characterization and immobilization of bacterial consortium for its application in degradation of dairy effluent.

The food industry is one of them who have highest consumptions of water and so it became the biggest producer of effluent per unit of production, additionally, these industries generate a large volume of sludge as well during the biological treatment (1). In effluent the organic content is constituted of milk which is used as the raw material for dairy products that reflects as high chemical oxygen demand (COD) effluent with high levels of biochemical oxygen demand (BOD), oils and grease, nitrogen and phosphorus. The cleaning in place (CIP), automatic cleaning system, discard rinse waters with pH varying between 1.0 and 13.0, ultimately making the effluent treatment complicated (1).

In the dairy effluent, dissolved sugars, proteins and fats with residues of additives are present. The parameters for untreated effluent are BOD, with an average ranging from 0.8 to 2.5 kilograms per metric ton (kg/t) of milk in the untreated effluent; COD, which is normally about 1.5 times the BOD level; total suspended solids, at 100-1,000 milligrams per litre (mg/L); total dissolved solids: phosphorus (10-100 mg/L), and nitrogen (about 6% of the BOD level). Cream, butter, cheese, and whey production are major sources of BOD in dairy wastewater. The waste load equivalents of specific milk constituents are: 1 kg of milk fat is equal to 3 kg of COD; 1 kg of lactose equals to1.13 kg of COD; and 1 kg protein makes around 1.36 kg of COD (2-3).

Dairy waste water with all the impurities decompose rapidly and deplete the dissolved oxygen (DO) level of the receiving streams which results in anaerobic conditions and release strong foul odor due to nuisance conditions. The casein precipitation from dairy waste decomposes further into a highly odorous black sludge (4). The dairy waste water contains soluble organics, and suspended solids which degrade to promote release of various gases, bad odor, imparts dark grey or black color, turbidity, and promotes eutrophication (5). However, the dairy industry produces different products, such as milk, butter, yogurt, ice-cream, and various types of desserts and cheese, the characteristics of these effluents also vary widely both in quantity as well as in quality, depending on the type of system and the methods of operation used for the production. Dairy effluent consists of organics soluble in water, suspended solids and trace organics. These components contribute in high BOD and COD. The main characteristics of dairy waste water are temperature, color, pH, DO, BOD, COD, dissolved solids, suspended solids, chlorides, sulphates and oil & grease. The dairy effluent contains abundance of milk constituents like casein, inorganic salts, besides detergents and disinfectants used for washing. The dairy effluent has high sodium content due to the use of caustic soda for cleaning (6).

The common techniques from various techniques used for dairy industry effluent treatment includes, oil and grease traps, oil water separators to separate the floatable solids, flow equalization, and some clarifiers to remove suspended solids. The biological method of effluent treatment is said to be aerobic and anaerobic treatment. Sometimes anaerobic treatment followed by aerobic treatment is employed for the reduction of soluble organic matter and biological nutrient removal is also employed for the reduction of nitrogen and phosphorus. Aerobic biological treatment involves use of microbes for degradation and oxidation of waste in the presence of oxygen. Conventional treatment of dairy wastewater by aerobic processes includes activated sludge, trickling filters, aerated lagoons, or a combination of these (7). A comparison between aerobic and anaerobic treatment are summarized in Table 1.

Bioremediation is found to be the most efficient method for degradation of hazardous pollutants because it is natural, economic and eco-friendly. The immobilized cells of microbes may be useful to treat the waste to convert the toxicant into nutrient, biomass and C[O.sub.2] via biodegradation (7). The microbial cells which are immobilized within a suitable matrix provide a physical support for bacterial cells, ideal size, mechanical strength, rigidity and porous characteristics to the biological material which is immobilized (8). The biggest advantage of complete cell immobilization is that the enzymes remain active and stable for longer period of time due to their natural environment they are in (9-10).

The aim of the present study is to identify efficient bacterial isolates from waste water that could degrade dairy effluent rapidly either individually or in combination. In addition, a comparative study has been done to evaluate the bioremediation efficiency of bacterial isolates. An experimental observation is carried out using immobilized bacterial consortium, to find the potential method of bioremediation of dairy effluent that would be simple, economic and easy to use.

MATERIALS AND METHOD

Sample collection

Fresh dairy effluent sample was obtained from three different dairy industry effluent treatment plants located in Delhi-NCR (28[degrees]402 N 77[degrees]132 E). The samples were collected in a plastic container. The containers used for sample collection were pre-treated by washing with alcohol and later rinsed with distilled water, dried in an oven for 1h at 30[degrees]C and allowed to cool to room temperature. At the collection point, container was rinsed with the sample thrice and then filled, corked tightly and taken to the laboratory for further analysis. The sample was stored at a temperature below 4[degrees]C to avoid any physicochemical changes in the effluent. Physicochemical analysis of effluent

The dairy effluent from all three industries were taken and analysis was performed by following APHA, 2005 (11). The Physical parameter-Temperature, pH, TS, TDS and SS and Chemical parameter- Chloride, Dissolved Oxygen (DO), Chemical oxygen Demand (COD), Biological Oxygen Demand (BOD), Sulphate, Chloride, Oil and Grease were studied. The pH of samples was determined at the site itself by using portable hand held pH meter and temperature in Degree Celsius on scientific thermometer.

Isolation and characterization of bacteria

For the isolation of bacteria from effluent sample, tenfold serial dilution was done with the effluents. After dilution the diluted samples were spread over nutrient agar plates and kept overnight for incubation at 37[degrees]C. Further, the single colonies of bacteria were identified on spread plate and streaked on nutrient agar plates to obtain the pure culture of isolated bacteria. For gram staining, Smear was prepared and heat fixed. The smear was treated with crystal violet for 1min and was then removed by Gram's iodine to react for 1min. The smear was then washed with water and treated with 95% ethanol for 15 sec. Smear was washed with water and again treated with safranin for 30 sec. The smear was then washed, dried and examined under microscope.

Maintenance of culture

The bacterial isolates were maintained on nutrient agar slants at 4[degrees]C and glycerol stocks of pure culture were also prepared.

Biochemical Characterization

Biochemical activities of the isolated bacteria were analyzed by different biochemical tests such as amylase production, cellulase production, degradation of pectin, hydrolysis of gelatin, casein hydrolysis, urease test, hydrogen sulfide production, carbohydrate catabolism, IMVic tests, citrate utilization, catalaste test and oxidase test. Estimation of protein and reducing sugars were done by the methods described by Lowry et al., (12) and Miller et al., (13)

Mutual Antagonistic effect

The isolated strains were tested for their mutual antagonistic activities in order not to influence each other. Different strains were cross-streaked but did not intersect on a Luria-Bertani (10g peptone, 5g yeast extract, 10g NaCl and 20g Agar in 1L distilled water) plate. The antagonistic phenomenon was observed after incubation at 30[degrees]C for 48 hours.

Identification of bacteria

Isolated bacteria were identified in the present study by routine morphological, microbiological and biochemical methods followed by 16S rRNA Gene Sequencing.

Development of Bacterial Consortium

Bacterial consortium was developed by inoculating more than one strain (added in equal ratios; 100 [micro]l in 100 ml) in Luria-Bertani Broth (10g peptone, 5g yeast extract and 10g NaCl in 1L distilled water). Effect of pH and temperature influencing bacterial growth were also observed.

Immobilization of bacterial consortium in beads

The bacterial consortium (individual strains and consortium raiment ISD1 & ISD4 strain) was immobilized as beads according to the procedure of Leung et al (14) 100 ml of 2% sodium alginate solution is prepared in sterile distilled water by heating it to 60[degrees]C and mixing it thoroughly on a magnetic stirrer. Later sodium alginate solution is cooled to room temperature and 10% (10ml culture in 100ml sodium alginate solution) of the cell culture is added. The contents were mixed well by vigorous shaking to get a homogenized mixture. The sodium alginate containing cell culture suspension was extruded drop wise through a syringe and allowed to fall in a separate beaker containing 100ml of 0.1M calcium chloride solution. The beads of sodium alginate gel formed are left in the beaker overnight for hardening. Then beads were washed and stored in distilled water at 28 [+ or -] 2[degrees]C.

Biodegradation study of dairy industry effluent

10% of the inoculum comprised of immobilized beads of consortium made up of ISD1 and ISD4 were added to the dairy effluent in 500ml conical flask and kept on a rotator shaker. The degradation studies of the treated effluent were evaluated at intervals of 12 hrs.

RESULTS AND DISCUSSION

This study was undertaken to detection of the important pollution parameters in dairy industry waste water. The mean values of physicochemical parameters of collected samples from different industries are shown in Table 2. The study revealed that dairy effluent is alkaline in nature. The high BOD and COD values of the analyzed dairy effluent indicate the presence of heavy load of organic matter. The discharge of waste water to the environment without any treatment plays significant risk to public health and environmental pollution. The industrial wastes leads of the water, soil and air when they are discharged without being subjected to treatment or when they are treated using inappropriate methods.

Mean values of physical characteristics such as pH, TDS and TSS are 9.2, 1580mg/L and 252mg/L, respectively, and mean values of chemical characteristics such as DO, BOD, COD and O&G are 1.2mg/L, 787mg/L, 1528mg/L, and 13mg/L, respectively. Mean value for total solids were found 1832mg/L. However, alkalinity (as CaC[O.sub.3]) and temperature were calculated 600mg/L and 36[degrees]C, respectively.

In the present study, total seven (ISD 1, ISD 2, ISD 3, ISD 4, ISD 5, ISD 6, ISD 7) bacterial isolates were obtained as pure culture. After Gram method and microscopic observation characteristic specification of bacteria isolated from dairy effluent was carried out (Table 3). The isolates ISD 1, ISD 2, ISD 3, ISD 4, ISD 5 and ISD 7 were found Gram Negative whereas ISD 6 was found Gram Positive. The colony appearance on agar plate and shape of the isolates were microscopically observed. ISD 1 was thick, white, round and opaque on agar plate while its shape was found cocci, ISD 2 was thin, white and clear with limited growth and was found rod shaped, ISD 3 was thick, white, viscous and opaque and its shape was found cocci, ISD 4 was off-white, thick, granular and translucent on plate while it was rod shaped, ISD 5 on agar plate was thin, white, granular and translucent while its shape was observed cocci, ISD 6 was thin, golden grey, shiny and transparent and it was observed as cocci and ISD 7 was off-white, thick, round and transparent while microscopic observation showed its rod shaped.

All the seven isolated bacterial strains were also characterized by biochemical tests (Table 4 & Figure 1). In the amylase production test; ISD1, ISD3, ISD4, ISD5 and ISD7 were found positive where as ISD2 and ISD6 were found negative. In carbohydrate catabolism all seven (ISD1, ISD2, ISD3, ISD4, ISD5, ISD6, ISD7) isolated strains were found positive. In casein hydrolysis test; ISD1, ISD4 and ISD7 were found positive while ISD2, ISD3, ISD5 and ISD6 were found negative. Catalase test showed all seven isolated strains positive. In citrate utilization; ISD4 and ISD6 was found positive while ISD1, ISD2, ISD3, ISD5 and ISD7 were observed negative. Gelatin hydrolysis was found positive for ISD1, ISD2, ISD3 and ISD4 while it was found negative for ISD5, ISD6 and ISD7. In hydrogen sulphide production; ISD1, ISD4 and ISD7 was positive while ISD2, ISD3, ISD5 and ISD6 were found negative. Indole test was positive for ISD1, ISD2, ISD3, ISD4 and ISD7 while negative results were found for ISD5 and ISD6. ISD2, ISD4 and ISD5 were positive for MR test and ISD1, ISD3, ISD6 and ISD7 were negative. All the seven isolates were positive for oxidase test. In urease test only strain ISD7 was found positive, all other strains ISD1, ISD2, ISD3, ISD4, ISD5 and ISD6 were found negative. For VP test all the seven isolated strains showed no result and all of them were found negative.

Figure 2 shows the result of antagonistic phenomenon. The isolated strains were tested to test their tendency to influence each other by streaking them on Luria-Bertani agar plates. No antagonistic phenomenon was observed after incubation at 30[degrees]C for 48 hours.

16S rRNA gene cloning and phylogenetic analysis

Two bacterial isolates ISD 1 and ISD4 from the seven isolates were selected for the identification on the basis of the result of the biochemical assay. These strains were chosen for further studies because of their maximum degradation and moderate degradation activity towards different substrate observed during the biochemical assay.

For ISD 1, 1227bp 16S rRNA gene was sequenced. ISD1 was found to be most similar to Bacillus sp. RD_DARAB_02 16S ribosomal RNA gene, partial sequence [Sequence ID: gb|KU597549.1]. The next closest homologue was found to be Bacillus thuringiensis strain VKK-FCI-3 16S ribosomal RNA gene, partial sequence [Sequence ID: gb|KT714037.1].

For ISD4, 1376bp 16S rRNA gene was sequenced. ISD4 was found to be most similar to Citrobacter freundii strain LCJY-002 16S ribosomal RNA gene, partial sequence [Sequence ID: gb|KC691177.1|]. The next closest homologue was found to be Citrobacter sp. SJH-004 16S ribosomal RNA gene, partial sequence [Sequence ID: gb|KC335138.1|].

The identified strains ISD1&ISD4 were further studied for their growth parameters at different conditions. Under the significant variation of pH and temperature, the growth of these two strains was studied after 24hours of incubation. Both the strains ISD1 and ISD4 were acting same and showing similar results at different range of pH and temperature. Optimum pH range for growth was found 6.5 to 7.5 for both the strain (Figure 5). Optimum temperature range for growth of both the strain was found between 3 5[degrees]C to 40[degrees]C (Figure 6).

Immobilized Bacterial Consortium as shown in figure 7 was prepared by inoculating equal amount (100pl in 100 ml) of each single strain culture (ISD1 & ISD4) in the same Luria Bertani Broth and further immobilized in sodium alginate beads (Figure 7).

Initially, comparative biodegradation potentials of ISD1 & ISD4 strains in different forms were studied in terms of percent decrease in highly concentrated BOD levels of dairy effluent. The different forms were ISD1 & ISD4 strains as individual, mixed ISD1 & ISD4 strains as consortium and ISD1 & ISD4 strains consortium as immobilized beads and their BOD degradation efficiency were shown in Figure 8.

BOD is the amount of dissolved oxygen needed by aerobic organisms present in a water body to breakdown the organic materials at certain temperature over a specific time period. The decrease in BOD was rapid when the consortium was used than the individual organisms. The immobilized consortium showed efficient reduction in comparison to the free cell consortium. There was a considerable decrease in BOD on the third day itself, when immobilized consortium was used for the treatment. The BOD reduced to 91.2% at the end of the treatment.

The untreated dairy effluent was analyzed for pH, total solids, dissolved oxygen, biological oxygen demand, chemical oxygen demand and oil & grease and it was 9.4, 2156, 0.9, 830, 1670 and 19, respectively. The untreated effluent was then treated with free cell consortium and immobilized consortium. After 72 hrs of incubation, the treated effluent was analyzed to check the changes in the physico-chemical parameters. The pH, TS, DO, BOD, COD and oil and grease were measured as 7.8, 383, 1.9, 147, 395 and 9.6, respectively for the effluent treated with free cell consortium where as the pH was 7.3, TS was 357, DO was 2.4, BOD was 73, COD 247 and oil and grease was measured as 7.3 for the effluent treated with immobilized consortium (Table 5). The estimation of proteins and reducing sugar present in the dairy effluent was done by methods described by Lowry et al., and Miller et al.,. The total reduction in the amount of protein and reducing sugar were estimated after treating the effluent upto 72hours by individual bacteria, free cell consortium and immobilized consortium. The maximum percent reduction in protein and reducing sugars after treatment was calculated as 67.3% for proteins (Figure 9) and 74.6% for reducing sugars (Figure 10), when treated with immobilized consortium.

The results obtained in present study are consistent to those reported by Dharmsthiti and Kuhasuntisook (15). Prasad and Manjunath (16) have reported the use of B.subtilis, B.licheniformis, B.amyloliquefaciens, S.marsescens, P.aeruginosaand S.aureusin for waste water treatment. The average BOD value was reduced from 3200 mg/L to less than 40 mg/ L and lipid content was reduced from 25,000 mg/L to 80 mg/L, respectively within 12 days of incubation. P.aeruginosa showed reduction of BOD value from the day one in palm oil refinery effluent, dairy effluent and domestic water effluent.

CONCLUSION

In the present study, the dairy industry effluents were characterized and resistant bacteria were isolated from the effluent. A broad spectrum of behavior in growth of various bacterial isolates was observed. The biodegradation ability of isolated bacterial strains was studied and consortium was made by selecting the two most efficient strains (ISD1-Bacillus sp RD_DARAB_02 & ISD4-Citrobacter freundii LCJY-002) on the basis of result obtained in their biodegradation ability. The selected strains were studied for their growth on different pH and temperature to find out the optimum pH and temperature for their growth. A comparative study was done in between the immobilized consortium and free cells bacterial consortium by inoculating them into dairy effluent. Immobilized bacterial consortium has shown maximum reduction in physicochemical parameters. Present study revealed that immobilized consortium was better and efficient in removal of organic pollutants in comparison to the free cell consortium or individual bacterial strains. Results concluded that immobilized consortium of selected isolated strain (ISD 1 & ISD4) is competent to treat dairy wastewater holistically.

ACKNOWLEDGEMENTS

The authors are thankful to the dairy industries located in Delhi-NCR for providing waste water samples as and when required. The authors are also thankful to Amity Institute of Biotechnology, Amity University Uttar Pradesh for providing infrastructure and scientific support to conduct this study.

REFERENCES

(1.) Balannec, B., Gesan-Guiziou, G., Chaufer, B., Rabiller-Baudry, M., & Daufin, G. Treatment of dairy process waters by membrane operations for water reuse and milk Constituents Concentration. Desalination, 2002; 147(1), 8994.

(2.) Rao M.N., Datta A. K., "Waste water treatment", third edition--Oxford & IBH Publishing Co. Pvt Ltd, 2012

(3.) Shete, B. S., & Shinkar, N. P. Comparative study of various treatments for dairy industry wastewater. IOSR J Eng, 2013; 3, 42-47.

(4.) Kolhe, A. S., Ingale, S. R., & Bhole, R. V. Effluent of dairy technology. Shodh, Samiksha aur Mulyankan Int Res J, 2009; 2(5), 459-461.

(5.) Mrs. Bharati S. Shete, Dr. N. P. Shinkar "Comparative Study of Various Treatments for Dairy Industry Wastewater" IOSR Journal of Engineering 2013; 3(8): 42-47. e-ISSN: 2250-3021, p-ISSN: 2278-8719.

(6.) Dr. A. S. Kolhe, S. R. Ingale, Dr. R. V. Bhole, "Effluent of Dairy Technology" Shodh, Samiksha aur Mulyankan (International Research Journal) 2009; 2(5): (Nov.08-Jan.09). ISSN-0974-2832.

(7.) Suzana Claudia; Silveira Martins, "Immobilization of microbial cells: A proising tool for treatment of toxic pollutants in industrial waste water", African Journal of Biotechnology, 2013; 12(28): 4412-4418.

(8.) T Sivaruban, "Bioremediaton of Tannery Effluent Using Lyngbya Sp. with Coir Pith" Res. J. Pharmaceutical, Biological and Chemical Sciences, 2014; 5(4): 217-224.

(9.) Rakesh Kumar; Ritika Vats, "Protease Production by Bacillus subtilis Immobilized on Different Matrices" New York Science Journal, 2010; 3(7): 20-24.

(10.) Abida Anwar, "Calcium Alginate: A Support Material for Immobilization of Proteases from Newly Isolated Strain of Bacillus subtilis KIBGE-HAS" World applied sciences journal, 2009; 7(10): 1281-1286.

(11.) APHA, "Standard methods for the examination of water and wastewater", Lenore S. C., Greenberg A. E., Eaton A. D. 21st Edition, American Public Health Association, NW, Washington, DC, 2005.

(12.) O. H. Lowry, N. J. Rosebrough, A. L.Fori, and R. J. Randall, Protein determination, J Biol. Chem. 1951; 193: 265-275.

(13.) G. L. Miller, Use of DNSA reagent for the determination of reducing sugar, Anal Chem 1959; 31: 426.

(14.) Leung, W.C., M.F. Wong and C.K. Leung. Removal and recovery of heavy metals by bacteria isolated from activated sludge treating industrial effluents and municipal waste water. Water Science Technology, 2000; 12: 233-240.

(15.) S. Dharmasthiti and B. Kuhasuntisook, Lipase from Pseudomonas aeruginosa LP602 : Biochemical properties and application for wastewater treatment, Industrial Microbiol & Biotech. 1998; 21: 75-80.

(16.) M. P. Prasad and K. Manjunath, Comparative study on biodegradation of lipid rich wastewater using lipase producing species, Indian.J Biotech. 2010; 10: 121-124.

Abhinav K. Srivastava [1], S.VS. Rana [2], Tithi Mehrotra [1] and Rachana Singh [1] *

[1] Water Quality Monitoring and Bioremediation Lab, Amity Institute of Biotechnology, Amity University Uttar Pradesh--201 313, India.

[2] Chaudhary Charan Singh University, Meerut, Uttar Pradesh--250 004, India.

(Received: 10 April 2016; accepted: 21 May 2016)

* To whom all correspondence should be addressed.

Mob: +91-9810557475; E-mail- rsingh2@amity.edu

Caption: Fig. 1. Result of biochemical characterization (A) Amylase production test (B) Carbohydrate catabolism test (C) Casein hydrolysis test (D) Catalase test (E) Citrate utilization test (F) Gelatin hydrolysis test (G) H2S production test (H) Indole test (I) MR test (J) Oxidase test (K) Urease test (L) VP test

Caption: Fig. 2. LB Agar plate showing no antagonistic effect

Caption: Fig. 3. Phylogram obtained after 16S rRNA sequencing for isolated strain ISD1

Caption: Fig. 4. Phylogram obtained after 16S rRNA sequencing for isolated strain ISD4

Caption: Fig. 5. Bacterial growth at different pH range (OD at 600nm, incubation time 24hours)

Caption: Fig. 6. Bacterial growth at different temperature range (OD at 600nm, incubation time 24hours)

Caption: Fig. 7. Immobilized consortium as beads

Caption: Fig. 8. % BOD Reduction of dairy effluent with time

Caption: Fig. 9. %BOD Reduction of dairy effluent with time

Caption: Fig. 10. Percent reduction of protein in dairy effluent after treatment
Table 1. Comparison of aerobic and anaerobic treatment of dairy
industry wastewaters

Factors            Aerobic process            Anaerobic process

Reactors           Aerated lagoons,           UASB, Anaerobic filter,
                   oxidation ditches,         Upflow packed bed
                   Stabilization ponds,       reactor, CSTR, Down
                   Trickling filters and      flow fixed-film
                   Biological discs           reactor, Buoyant Filter
                                              Bioreactor,

Reactor size       Aerated lagoons,           Smaller reactor size is
                   oxidation ditches,         required
                   Stabilization ponds,
                   Trickling filters and
                   Biological discs
                   requires larger land
                   area but SBR needs
                   comparatively lower
                   area.

Effluent Quality   Excellent effluent         Effluent quality in
                   quality in terms of        terms of COD is fair
                   COD, BOD and nutrient      but further treatment
                   removal is achieved.       is required. Nutrient
                                              removal is very poor.

Energy             High energy is required.   These processes produce
                                              energy in the form of
                                              methane

Biomass yield      In comparison to           Lower biomass is
                   anaerobic process, 6-8     produced.
                   times greater biomass is
                   produced

Loading rate       Maximum 9000 g COD/m3 d    Very high Loading rate
                   is reported in             of 31 kg COD/m3 d has
                   literature                 been reported. This is
                                              the reason for smaller
                                              reactor volume and
                                              lesser area.

Oil and grease     These do not cause         Fats in wastewater
removal            serious problems in        shows the inhibitory
                   aerobic processes          action during anaerobic
                                              treatment of dairy
                                              wastewaters

Shock loading      Excellent performance in   Anaerobic processes
                   this regard.               showed not good
                                              responses to this shock
                                              loading.

Source: Mrs. Bharati S. Shete, Dr. N. P. Shinkar (2013)

Table 2. Characteristics of dairy industry effluent before treatment

Sample   pH        Temp        Alkalinity     TDS      TSS       TS
               ([degrees]C)    (mg/L) as     (mg/L)   (mg/L)   (mg/L)
                              CaC[O.sub.3]

Site 1   9.6        38            614         1680     239      1919
Site 2   8.6        36            556         1225     195      1420
Site 3   9.4        34            630         1835     321      2156
Mean     9.2        36            600         1580     252      1832

Sample     DO      BOD      COD     Oil and
         (mg/L)   (mg/L)   (mg/L)   Grease
                                    (mg/L)

Site 1    1.2      810      1595      12
Site 2    1.5      720      1320       8
Site 3    0.9      830      1670      19
Mean      1.2      787      1528      13

Table 3. Characteristics of bacterial strains isolated from dairy
waste water sample

S.No   Strain   Colony appearance on plate                 Shape

1      ISD 1    Thick, white, round, opaque                Cocci
2      ISD 2    Thin, white, limited growth, clear         Rod
3      ISD 3    Thick, white, viscous, opaque              Cocci
4      ISD 4    Thick, off-white, granular, translucent    Rod
5      ISD 5    Thin, white, granular, translucent         Cocci
6      ISD 6    Thin, golden-grey, shiny, transparent      Cocci
7      ISD 7    Thick, off-white, round, transparent       Rod

S.No   Gram Test

1      Negative
2      Negative
3      Negative
4      Negative
5      Negative
6      Positive
7      Negative

Table 4. Biochemical Characterization of bacterial isolates from
dairy industry effluent

S.No   Strain   Amy   Car   Cas   Cat   Cit   Gel   h2s   Indole   MR

1      ISD 1    +++   +++   ++     +     -    ++     +      +      -
2      ISD 2     -     +     -     +     -     +     -      +      +
3      ISD 3     +     +     -     +     -     +     -      +      -
4      ISD 4    +++   +++   +++    +     +    +++    +      +      +
5      ISD 5     +     +     -     +     -     -     -      -      +
6      ISD 6     -     +     -     +     +     -     -      -      -
7      ISD 7     +     +     +     +     -     -     +      +      -

S.No   Oxi   Ure   VP

1       +     -     -
2       +     -     -
3       +     -     -
4       +     -     -
5       +     -     -
6       +     -     -
7       +     +     -

(+++ = Maximum activity,++ = Moderate activity; Amy--Amylase
prduction, Car--Carbohydrate catabolism, Cas--Casein hydrolysis,
Cat--Catalse test, Cit--Citrate utilization, Gel--Gelatin hydrolysis,
[H.sub.2]S--Hydrogen sulphide production,  MR--Methyl Red test,
Oxi--Oxidase test, Ure--Urease test, VP--Voges-Proskauer test)

Table 5. Dairy effluent treatment with free cell consortium and
immobilized consortium after 72 hours

Sample                      pH      TS       DO      BOD      COD
                                  (mg/L)   (mg/L)   (mg/L)   (mg/L)

Untreated Effluent Sample   9.4    2156     0.9      830      1670
Treated with Free Cell      7.8    383      1.9      147      395
  Consortium
Treated with Immobilized    7.3    357      2.4       73      247
  consortium

Sample                         Oil and
                            Grease (mg/L)

Untreated Effluent Sample        19
Treated with Free Cell           9.6
  Consortium
Treated with Immobilized         7.3
  consortium
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Author:Srivastava, Abhinav K.; Rana, S.Vs.; Mehrotra, Tithi; Singh, Rachana
Publication:Journal of Pure and Applied Microbiology
Article Type:Report
Date:Sep 1, 2016
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