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Journal of Microbiology and Biotechnology Research

Scholars Research Library

J. Microbiol. Biotech. Res., 2014, 4 (5):31-37

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ISSN : 2231 –3168

CODEN (USA) : JMBRB4

31 Available online at www.scholarsresearchlibrary.com

Dairy wastewater treatment by free and immobilized fungal isolates

Amanpreet Kaurand Sonia Chaman*

Department of Biotechnology and Bioinformatics, Sri Guru Gobind Singh College, Sector-26, Chandigarh, India

_____________________________________________________________________________________________

ABSTRACT

Dairy industry is one of the major causesof industrial pollution. Dairy wastewater is highly degradable hence

requires specialized treatment to minimize environmental problems. The aim of the study was to find the potential of

free and immobilized fungal isolates to treat the dairy wastewater at different concentrations of dairy effluent. Five

fungal sp. D1W & D4S (Alternaria sp.), D3S (Fusarium sp.), D2W & D5S (Aspergillus sp.) were isolated from dairy

effluent (D.E)sample collected from Verka Milk Plant Industrial Area-1, Chandigarh, India in two different

seasons.The fungal isolates were efficiently immobilized on sodium alginate beads. Free and immobilized spores

were inoculated in different concentrations of D.Eand incubated at 25ºC at 120rpm. After 3rd , 6

th , 9

thand 12

th day of

treatment sample was checked for various physico-chemical parameters. Free cells of different fungal isolates

reduced COD in maximum range of 80.76% (D1W) to 86.27% (D5S) at different concentrations of D.E on different

days of treatment. Maximum COD reduction was reported for immobilized cells of D5S (86.84%) on 12th

day of

treatment. Free cells of D1W had shown maximum TDS reduction of 67.03% at 33.33% D.E, maximum E.C

reduction of 98.21% at 25% D.E and maximum salinity reduction of 68.75% at 33.33% D.E. Free cells of all fungal

isolates of summer season had shown the potential of reducing COD at different concentrations of D.E. Presentstudy suggested that free cells of D1W (Alternaria sp.)at 25% D.E has the potential to be used in dairy wastewater

treatment.

Keywords: Dairy effluent, fungal isolates, COD, immobilization, Alternaria sp.

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INTRODUCTION

In India, Dairy Industry is one of the major industries causing water pollution. During the past few years, many agrobased industries have come up in India. The number of the dairyplants of medium and large size has increased forthe efficient handling and processing of milk. With the exponential increase in the number of industries, there hasbeen a substantial increase in generation of industrial wastewater which is discharged either into open land or nearby

aquatic ecosystems. This activity promotes varying degree of pollution load in water, soil and air [1].The dairyindustry wastewater are generated primarily from the cleaning and washing operations in the milkprocessing plantsand are estimated to be 2.5 times the volume of the milk processed [2].

In general, this residue contains high concentrations of organic compounds (lipids, proteins and carbohydrates),suspended solids, oil-grease and elevated levels of BOD and COD requiring specialized treatments to prevent orminimize environmental problems [3].

Biological treatment is necessary if organic matter is to be removed from water. Nonetheless, biological treatmentoffers an economical alternative to physical and chemical treatment methods as they are highly selective to the rangeof pollutants removed and are expensive. Microbial treatment systems have advantage of being simple in design andlow in cost [4]. The mechanism underlying biological treatment is the decomposition of finely dispersed matter,colloidal and dissolved substances by metabolism of aerobic microorganisms [5]. The rate of biochemical

decomposition of waste depends on the activity of microorganisms [6]. Biological treatments have been alreadydemonstrated that they are among the most advantageous methods for maximising recycle and recovering theircomponents [7].

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Amanpreet Kaurand Sonia Chaman J. Microbiol. Biotech. Res., 2014, 4 (5):31-37

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The mycoremediation is determining the right fungal species to target a specific pollutant. The importance of fungiin the environment is decomposition and transformation of both organic and inorganic substrates [8].Fungi areinvolved in the biodegradation of undesirable materials or compounds and convert them into harmless, tolerable oruseful products [9]. Hence it is apparent that fungi can play an important role in degrading materials in theecosystem and that they have the potential for remediating contaminated soils and waters [10]. Fungiused inwastewater treatment because it appeared to show higher degradation rates of organic matter. Keeping in view the

importance of fungi in removal of pollutants the present study was aimed to find dairy effluent degrading fungalstrains from dairy effluent itself and to explore efficiency of free and immobilized fungal isolates to degrade dairywastewater at different concentrations of dairy effluent.

MATERIALS AND METHODS

Collection of effluent sample

For the present study the dairy effluent was collected from Verka Milk Plant Industrial Area-1, Chandigarh, India indifferent seasons in the months of February, March, April and May’2014 in the sterile plastic containerand wasstored at 4ºC for further investigation.

Isolation and identification of fungal cells The dairy effluent was serially diluted using sterile pipettes from 10-1 to 10-5 dilution. For the enumeration of fungi

Potato Dextrose Agar medium was used. The dairy effluent (0.1mL) was spread on the solidified PDA plates andincubated at 25ºC for 48 hours. To obtain the pure culture, the cultures were repeatedly streaked on PDA mediumand incubated at 25°C for 72 hours. The isolated fungi were identified by colony morphology and microscopicobservation. The isolated fungal cultures were identified using lacto-phenol cotton blue staining method. PotatoDextrose Agar (PDA) and Potato Dextrose Broth (PDB) medium were used for the maintenance of isolated fungalcultures.

Determination of the growth curve Spores from fungal isolates were inoculated in 100mL of PDB and dairy effluent separately in 250mL ofErlenmeyer flasks and incubated at 32ºC at 120rpm for 10 days. For each fungal isolate, 4 flasks were used, whichwere withdrawn from rotary shaker one by one at different time interval i.e on4th, 6th, 8th and 10th day. The cells werefiltered using what-man filter paper no. 4 and their mycelial dry weight was determined after drying at 70ºC for 24hours in the oven. The growth curves of fungal isolates were constructed from the dry cell weights (g/100mL)

plotted versus incubation time (hours) [11].

Preparation of fungal seed cultures

100mL of PDB was prepared by suspending 2.4g in 100mL of distilled water andsterilized by autoclaving. Thebroth was inoculated with the isolated fungal strains and incubated at 25ºC in rotary shaker at 120rpm for 5 days.

Preparation of free cells

100mL of PDB medium was inoculated with 1mL of seed culture and incubated at 25˚C in a rotary shaker for 72hours. Fully grown spores were harvested by centrifuging at 5000rpm for 15 min. Washings were given to the cellpellet with 50mL autoclaved distilled water (D.W) twice. The pellet was suspended in 10mL of autoclaved distilledwater which was used as free cells [12].

Preparation of immobilized cells

Commercial grade sodium alginate was used as the immobilizing agent for bead preparation. Exponentially growingfungal isolates were harvested by centrifugation (5000 x g for 10 min) and re-suspended in 50mL of sterile water.To this 50mL of suspension, 4% sodium alginate solution was mixed thoroughly to get a final alginate concentrationof 2%. The sodium alginate-fungal mixture was then added drop wise into calcium chloride (0.1M) solution. Thebeads were kept in the same solution for 30 min at 4°C for hardening [13] .

Biodegradation using free and immobilized spores in shaker flasks

The free and immobilized spores of each fungal isolates were inoculated in 250mL of Erlenmeyer flasks containingdairy effluent at four different concentrations (100%, 50%, 33.33% and 25%) and were incubated at 25ºC in rotaryshaker at 120 rpm for 12 days [14]. COD, E.C, TDS and salinity of each sample was recorded on3rd, 6th, 9th and 12th day of incubation.

Physico-Chemical analysis of dairy effluent

Chemical Oxygen Demand of the various samples was determined by using titration method [15]. 50mL of samplewas poured in 100mL of conical flasks. Distilled water was used as blank. After addition of potassium dichromate

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solution, flasks were kept in the waterbath at 100ºC for 1 hour. Samples were allowed to cool for 10 min. 5mL ofpotassium iodide was added to it followed by addition of dil.H2SO4. Contents of the flasks were titrated with the0.1M sodium thiosulphate until the appearance of pale yellow colour. The COD was calculated using the followingformula:

COD of sample (mg/L) = 8 × C × (B-A)

SWhere C = Concentration of the titrant (m mol/ litre)A = Volume of titrant used for blank (ml)B = Volume of titrant used for sample (ml)S = Volume of sample taken (ml)

Electrical Conductivity (E.C), Total Dissolved Solids (TDS) and salinity were determined using the Deluxe Water &Soil analysis kit from M. S. Electronics (India Pvt. Ltd.).

RESULTS AND DISCUSSION

Identification of effluent degrading strain from the dairy effluent

The five fungal strains were isolated from dairy effluent. D1W& D2W were isolated in winter season and identified

as Alternaria sp. and Aspergillus sp.respectively whereas D3S, D4S & D5S were isolated in summer seasonandidentified as Fusarium sp, Alternaria sp.& Aspergillus sp.

Determination of the growth curve

Growth curve provide a useful tool to gain insight into growth characteristics of filamentous fungi in differentnutrient media [16]. Several workers stated that PDA and PDB are the best culture media for mycelial growth [17-18] because of its simple formulation and its ability to support mycelial growth of wide range of fungi [19]. In thepresent study a broad spectrum of behaviour in the growth of various fungal isolates in PDB and D.E was observed.For D1W ( Alternaria sp.)log phase occurred from 4th day to 6th day in PDB and 4th to 10th day in D.E and anaccentuated fall in the mycelial mass until 10th day was observed in PDB leading tothe death phase while no declinephase was obtained upto 10th day in D.E. In PDB both D2W and D5S ( Aspergillus sp.)showed a highestmycelialmass from 4th to 6th day. After that decrease in mass of the mycelium was observed until 10th day due todepletion of nutrients and carbon source in the media. While in D.E steady increment was observed for D2W

( Aspergillus sp.)from 4th

to 10th

day and D5S ( Aspergillus sp.) had shown decrease in mycelium massuntil 10th

day.The study on optimum growth of D3S (Fusarium sp.)in PDB and D.Ereported that the fungal biomass increased at aslow rate for the initial 6 days and thereafter increased at an exponential rate and no negative slope was observeduntil 10th day. In PDB D4S ( Alternaria sp.) showed a log phase from 4th to 6th day, after which the growth curve hada negative slope from 8th to 10th day while in D.E it showed a higher mycelial production and decline phase was notobserved until 10th day of incubation. These observations suggested that substrate had influenced the growth offungal sp and mycelial yield vary widely depending upon the fungal sp. and substrates.

Figure1: Growth of fungal isolates in PDB medium at 32ºC for 10 days

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0 4 6 8 10

M y c e l i a l d r y w e i g h t ( g / 1 0 0 m l )

Incubation time (days)

D₁W

D₂W

D₃S

D₄S

D₅S

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Figure2: Growth of fungal isolates in dairy effluent at 32ºC for 10 days

Fungal isolates had shown the maximum growth in PDB rather than in D.E.The study on the optimum growth of thefungi reported the lag phase upto 6 hours in Aspergillus niger growth pattern, as microbes acclimate to food andnutrients into their new habitat and steady increment was observed up to 70 hoursin glucose based PDB medium.After that no increment was observed [20].The study on optimum growth of Fusarium sp. in PDB medium reportedthat the fungal biomass increased at a slow rate for the initial 48 hours and thereafter increased at an exponentialrate. However, after about 8 days the rapid growth stabilized and the trend became more towards the stationaryphase [21].

Table 1: Effect of free and immobilized fungal isolates on Electrical Conductivity (E.C) at different concentrations of dairy effluent

F.I3rd day 6t day 9t day 12t day

Con. IMC FC Con. IMC FC Con. IMC FC Con. IMC FC

100% Dairy effluent

D1W 0.36 4.35 1.32 0.32 4.80 0.06 0.28 5.47 1.41 0.26 5.83 1.50

D2W 0.36 2.81 1.37 0.32 2.75 0.06 0.28 2.93 1.50 0.26 3.09 1.60

D3S 4.41 9.04 2.58 4.31 9.19 2.45 4.6 10.6 2.73 4.44 10.33 2.87

D4S 4.41 16.04 2.20 4.31 15.84 2.24 4.6 16.36 2.38 4.44 16.25 2.44

D5S 4.41 5.25 2.19 4.31 5.68 2.19 4.6 6.50 2.46 4.44 6.24 3.09

50% Dairy effluent

D1W 1.65 4.46 0.71 1.59 4.18 0.03 1.73 4.73 0.79 1.82 5.09 0.85

D2W 1.65 3.20 0.75 1.59 3.15 0.76 1.73 3.40 0.78 1.82 3.59 0.87

D3S 1.48 12.76 1.45 1.49 12.79 1.29 1.68 13.34 1.68 1.74 13.43 2.07

D4S 1.48 13.50 1.51 1.49 1.57 1.33 1.68 2.34 1.85 1.74 2.47 2.36

D5S 1.48 7.56 2.08 1.49 6.62 1.52 1.68 8.10 1.76 1.74 8.05 1.99

33.33% Dairy effluent

D1W 1.32 4.98 0.47 1.31 0.18 0.47 1.40 5.25 0.47 1.43 5.95 0.53

D2W 1.32 2.06 0.52 1.31 2.78 0.53 1.40 3.01 0.59 1.43 3.24 0.62

D3S 0.88 15.19 1.11 0.89 15.52 1.02 2.23 15.83 0.82 4.89 16.03 1.96

D4S 0.88 11.03 2.01 0.89 11.30 1.11 2.23 9.80 1.96 4.89 5.14 2.01

D5S 0.88 6.50 0.99 0.89 8.02 0.99 2.23 8.78 1.27 4.89 8.75 2.29

25% Dairy effluent

D1W 1.10 4.89 0.43 1.12 4.81 0.02 1.20 5.10 0.53 1.19 5.72 0.41

D2W 1.10 3.01 0.44 1.12 3.00 0.02 1.20 3.46 0.50 1.19 5.59 0.54

D3S 0.67 15.20 0.39 0.76 15.82 0.46 0.89 16.20 0.61 1.04 17.16 1.25

D4S 0.67 12.36 1.19 0.76 14.1 1.09 0.89 12.41 2.51 1.04 16.60 2.78

D5S 0.67 4.33 1.37 0.76 4.26 1.02 0.89 4.54 2.52 1.04 5.88 5.97

Note: F.I- fungal isolates; IMC- immobilized cells; FC- free cells; Con. - Control

E.C measured in mS (millisiemens)

0

0.05

0.1

0.15

0.2

0.25

0.3

0.35

0.4

0.45

0.5

0 4 6 8 10

M y c e l i a l d r y w e i g h t ( g / 1

0 0 m l )

Incubation time (days)

D₁W

D₂W

D₃S

D₄S

D₅S

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Effect of free and immobilized fungal isolates on different physico-chemical parameters at different

concentrations of dairy effluent

Free and immobilized cells of D1W, D2W, D3S, D4S& D5S strains had shown variable efficiency in reducingphysico-chemical parameters at different concentrations of D.E.

Free cells of D1W ( Alternaria sp.) and D2W ( Aspergillus sp.) had shown 98.21% and 81.25% E.C reduction with

minimum E.C of 0.06mS and 0.02mS on 6th

dayat 25% D.E and 100% D.E respectively. Free cells of D 1W( Alternaria sp.) at 50% D.E reduced E.C to 98.11% on 6th day with 0.03mS E.C andD1W ( Alternaria sp.)immobilized cells effectively reduced the E.C with 86.25% reduction at 33.33% D.E with E.C recorded as 0.18mSon 6th day (Table 1).

Table 2: Effect of free and immobilized fungal isolates on Total Dissolved Solids (TDS) at different concentrations of dairy effluent

F.I3rd day 6th day 9th day 12th day


100% Dairy effluent

D1W 0.54 2.73 0.86 0.52 3.14 0.88 0.49 3.54 0.91 0.46 3.78 0.94

D2W 0.54 1.82 0.89 0.52 1.77 0.86 0.49 1.90 0.97 0.46 2.01 1.03

D3S 2.7 5.83 1.67 2.81 5.91 1.59 2.93 6.85 1.77 2.90 6.65 1.87

D4S 2.7 11.7 1.43 2.81 10.84 1.45 2.93 11.57 1.54 2.90 11.59 1.58

D5S 3.9 5.0 2.0 4.1 5.3 2.0 4.4 6.5 2.3 4.4 6.30 2.9

50% Dairy effluentD1W 1.08 2.91 0.46 1.02 2.69 0.47 1.12 3.09 0.51 1.18 3.32 0.55

D2W 1.08 2.07 0.48 1.02 2.05 0.48 1.12 2.21 0.51 1.18 2.35 0.56

D3S 0.96 8.22 0.88 0.97 8.19 0.83 1.09 8.64 1.08 1.13 8.65 1.34

D4S 0.96 8.71 0.95 0.97 1.01 0.86 1.09 1.57 1.20 1.13 1.60 1.70

D5S 0.96 4.87 3.06 0.97 4.25 0.98 1.09 5.27 1.14 1.13 5.20 1.29


D1W 0.85 3.22 0.30 0.84 3.28 0.30 0.91 3.14 0.30 0.92 3.88 0.34

D2W 0.85 1.43 0.34 0.84 1.81 0.34 0.91 1.99 0.38 0.92 2.11 0.40

D3S 0.57 10.16 0.71 0.57 10.51 0.66 1.42 11.0 0.51 3.07 11.13 1.27

D4S 0.57 7.50 1.29 0.57 7.01 0.7 1.42 6.54 0.9 3.07 3.32 1.30

D5S 0.57 4.19 0.64 0.57 5.19 0.64 1.42 5.67 0.82 3.07 5.65 1.48

25% Dairy effluent

D1W 0.72 3.16 0.27 0.72 3.14 0.31 0.78 3.37 0.34 0.81 3.71 0.38

D2W 0.72 1.95 0.28 0.72 1.96 0.31 0.78 2.28 0.33 0.81 2.35 0.35

D3S 0.43 10.16 0.27 0.45 10.87 0.30 0.49 11.3 0.53 0.62 11.68 0.81D4S 0.43 7.31 0.77 0.45 6.76 0.7 0.49 6.43 1.64 0.62 6.63 1.83

D5S 0.43 2.81 0.84 0.45 2.77 0.66 0.49 2.95 1.67 6.62 3.79 3.86

Note: TDS measured in ppt (parts per thousand)

Table 3: Effect of free and immobilized fungal isolates on salinity at different concentrations of dairy effluent

F.I 3rd day 6th day 9th day 12th day


100% Dairy effluent

D1W 0.9 5.0 1.4 0.9 5.3 1.5 0.87 6.2 1.5 0.82 6.8 1.6

D2W 0.9 3.2 1.5 0.9 3.1 1.5 0.87 3.3 1.6 0.82 3.40 1.8

D3S 3.9 8.6 2.4 4.1 8.7 2.2 4.4 10.3 2.6 4.4 9.9 2.7

D4S 3.9 15.5 2.0 4.1 15 2.0 4.4 16.6 2.30 4.40 16.6 2.3

D5S 3.9 5.0 2.0 4.1 5.3 2.0 4.4 6.5 2.3 4.4 6.30 2.9

50% Dairy effluent

D1W 1.8 5.0 0.8 1.8 4.8 0.8 1.9 5.5 0.9 0.2 5.9 0.9

D2W 1.8 3.5 0.8 1.8 3.5 0.8 1.9 3.9 0.8 0.2 4.10 0.9

D3S 1.3 11.7 1.3 1.3 11.5 1.2 1.6 12.8 1.6 1.7 12.6 2.0

D4S 1.3 1.10 1.4 1.3 1.4 1.2 1.6 2.30 1.80 1.70 2.30 2.1

D5S 1.3 7.3 1.1 1.3 6.2 1.4 1.6 8.1 1.7 1.7 7.90 1.9


D1W 1.5 5.7 0.5 1.4 6.2 0.5 1.6 5.9 0.5 1.5 7.0 0.5

D2W 1.5 2.5 0.6 1.4 3.1 0.6 1.6 3.4 0.6 1.5 3.60 0.7

D3S 0.8 14.2 1.0 0.8 14.6 0.9 2.0 16.0 0.8 4.6 15.7 1.9

D4S 0.8 11.0 1.9 0.8 10.4 1.2 2.0 9.80 1.5 4.60 5.10 1.9

D5S 0.8 6.3 0.9 0.8 7.7 0.9 2.0 8.7 1.2 4.6 8.60 2.3

25% Dairy effluent

D1W 1.2 5.7 0.4 1.2 5.3 0.5 1.3 6.0 0.6 1.2 6.6 0.7

D2W 1.2 3.3 0.4 1.2 3.3 0.5 1.3 4.0 0.5 1.2 4.10 0.6

D3S 0.6 13.9 0.3 0.6 15.1 0.4 0.7 16.2 0.7 0.83 17.3 1.2

D4S 0.6 12.0 1.1 0.6 13.9 1.0 0.7 15.4 2.40 0.83 16.9 2.8

D5S 0.6 4.1 1.1 0.6 3.9 0.9 0.7 4.5 2.4 0.83 5.70 5.9 Note: Salinity measured in ppt (parts per thousand)

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D1W ( Alternaria sp.) and D2W ( Aspergillus sp.) free cells had not shown any reduction in TDS for 100% D.E butTDS of D1W ( Alternaria sp.) was 0.30ppt with highest reduction potentialof 67.03% at 33.33% D.E and 62.50%reduction at 25% D.E with TDS 0.27mS. Immobilized D5S cells were effective only after 12 th day of treatment at25% D.E with TDS recorded as 3.79ppt (42.74% reduction) (Table 2).

Only D3S (Fusarium sp.), D4S ( Alternaria sp.) and D5S ( Aspergillus sp.) free cells were able to reduce the salinity

effectively throughout the treatment at 100% D.E in range of 34.09% to 51.21% reduction with salinity recordedminimum as 2.0ppt and maximum of 2.9ppt while D1W ( Alternaria sp.) and D2W ( Aspergillus sp.) free cells hadshown better reduction at 50%, 33.33% and 25% D.E in the range of 41.66% to 68.75% on different days oftreatment (Table 3). Free cells of fungal isolates of winter season had reduced salinity, E.C and TDS in maximumrange as compared to fungal isolates of summer.

Table 4: Effect of free and immobilized fungal isolates on Chemical Oxygen Demand (COD) at different concentrations of dairy effluent

F.I3rd day 6th day 9th day 12th day


100% Dairy effluent

D1W 486 340 420 470 496 328 458 448 300 440 380 272

D2W 486 320 296 470 540 544 458 524 340 440 452 284

D3S 520 96 312 472 288 256 440 296 192 416 344 96

D4S 520 120 120 472 368 288 440 152 272 416 96 192

D5S 520 448 248 472 368 224 440 216 176 416 88 520

50% Dairy effluent

D1W 260 428 520 576 436 524 392 296 528 332 316 384

D2W 260 280 456 576 604 348 392 336 592 332 500 396

D3S 408 160 72 368 176 208 320 168 240 304 248 256

D4S 408 112 76 368 176 240 320 208 136 304 200 72

D5S 408 296 56 368 136 96 320 56 144 304 40 88


D1W 532 476 440 240 316 348 460 508 760 276 408 340

D2W 532 336 620 240 304 352 460 652 456 276 380 344

D3S 368 388 520 312 240 424 280 272 288 224 216 248

D4S 368 256 88 312 248 56 280 248 136 224 216 88

D5S 368 104 328 312 296 304 280 272 440 224 208 416

25% Dairy effluent

D1W 416 460 80 204 288 324 860 464 796 340 364 600

D2W 416 356 280 204 300 408 860 652 712 340 364 332D3S 304 132 368 288 224 304 208 136 184 176 88 72

D4S 304 368 168 288 312 88 208 288 280 176 248 200

D5S 304 96 120 288 176 88 208 80 240 176 64 176

Note: COD measured in mg/L

Both immobilized and free fungal cells efficiently reduced COD in the maximum range of 80.76% to 86.27%. Freecells of D1W had reduced COD from 416mg/L (control) to 80mg/L with 80.76% COD reduction on 3rd day oftreatment in 25% D.E. Free cells of D4S reduced COD to 76mg/L as compared to control (408mg/L) with 81.37%COD reduction on 3rd day in 50% D.E. Immobilized cells of D3S reduced COD from 520mg/L (control) to 96mg/Lwith 81.53% reduction on 3rd day in 100% D.E. Free cells of D4S reduced COD to 56mg/L as compared to control(312mg/L) with 82.05% reduction on 6th day in 33.33% D.E. Free cells of D3S had shown 82.35% COD reductionon 3rd day in 50% D.E. Free cells of D5S reduced COD to 56mg/L with 86.27% reduction on 3rd day in 50% D.Efollowed by immobilized cells of D5S that reduced COD to 40mg/L with 86.84% reduction on 12th day in 50% D.Eas compared to control (304mg/L) (Table 4).

Earlier workers studied the immobilization of different fungal cultures on variety of chemical inert syntheticpolymers [22]. The batch biodegradation of the industrial phenol by fungal isolate Aspergillus niger with paper andpulp effluent showed that the immobilized cells had over all better performance as compared to free cells and

Aspergillus niger had the potential to be used in biodegradation of wastewater containing phenol [14]. Immobilized Aspergillus flavuswas favourable for fungal growth and it proved to be the best for colour removal studies. Hencecould be applied to treat wastewater from dyeing industry and in bioremediation of dye contaminated environments[23]. Aspergillus terreus immobilization on bagasse, coconut coir, loofa sponge and gunny bag (Jute) showed CODreduction of 60.25% on bagasse, 56.31% on coconut coir, 69.71% on loofa sponge, 81.25% on gunny bagrespectively and hence it was proved that Aspergillus terreus immobilized on gunny bag (Jute) efficiently decreasedthe pollution level [24].Immobilized Aspergillus treated effluent showed more removal than free cells of Aspergillus

and all the pollutants were reduced in their level in leather industry effluent due to inoculation of Aspergillus

[13].Biodegradation of phenol by using free and encapsulated cells of Aspergillus sp. showed that the encapsulated Aspergillus sp. LEBM2 had promising application in biodegradation [25]. In above studies of different Aspergillus

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sp., immobilized fungal cells had shown promising results in dyeing, jute and paper industry while our resultsshowed that immobilized cells of Aspergillussp. (D5S) had shown maximum COD removal and free cells of

Alternaria sp. (D1W)proved to be the best candidate for removal of organic load in dairy effluent.

CONCLUSION

In the present study the biodegradation ability of immobilized and free cells of fungal isolates at differentconcentrations of D.E was evaluated. A broad spectrum of behaviour in growth of various fungal isolates in PBDand D.E was observed. Different fungal isolates showed variablereduction in physico-chemical parameters. Freecells of all fungal isolates of summer season had shown maximum reduction in COD at different concentrations ofD.E and free cells of fungal isolates of winter season had reduced E.C, TDS and salinity in maximum range whileimmobilized fungal cells had increased these parameters. Present study revealed that free fungal cells showed betterremoval of organic pollutants in D.E as compared to immobilized fungal cells. Results concluded that free cells ofD1W ( Alternaria sp.)is competent to treat dairy wastewater and have the potential to reduce the physico-chemicalparameters at different concentrations of dairy effluent.

Acknowledgement

The authorsgratefully acknowledge the cooperation of Principal and Head of the Department of Biotechnology &Bioinformatics of SGGS College, for providing lab facilities for the commencement of present research work.

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