View of BIOFILM MEDIATED TEXTILE DYE DEGRADATION BY BACTERIAL SPECIES

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Vol.03, Issue 09, Conference (IC-RASEM) Special Issue 01, September 2018 Available Online: www.ajeee.co.in/index.php/AJEEE

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BIOFILM MEDIATED TEXTILE DYE DEGRADATION BY BACTERIAL SPECIES Nisha Kanwar

Assistant Professor, Institute of Sciences, SAGE University, Indore

Abstract - The main objective of this study is to evaluate the interaction between toxic dyes and biofilm formation. Biofilm-grown cells exhibit a growing interest in recent years to use biofilms for biotechnological applications such as the uptake of toxic dyes. The two bacterial strains were confirmed as best biofilm producers after isolation & screening and were identified as Klebsiella & Escherichia species based on microscopic and biochemical analysis according to Bergey’s manual of determinative bacteriology, also subjected to biofilm formation on chitin flakes ,as a supporting material, confirmed by SEM.

Degradation of dyes by biofilm produced by the strains were performed by visible spectrophotometer read at 620 nm. Klebsiella showed decolourization of 92% and Escherichia showed decolourization of 94% at 72 hrs for malachite green dye. EPS, the major component of biofilm was extracted from two strains & its analysis was done by TLC method. Phytotoxicity test performed on two crop plants Vigna unguiculata and Vigna aconitifolia, was found that dye is toxic to the growth of agricultural crops, can treat polluted dye soil through metabolites of micro-organisms, identified as a cost effective and environment friendly.

Keywords: Biofilm, Bioremediation, Phytotoxicity, SEM, EPS 1.INTRODUCTION

Bioremediation is an emerging in situ technology for the cleanup of environmental pollutants using micro organisms.The biological processes for treating toxic effluents are better than chemical and physical methods in terms of their efficiency and economy and the potential of biofilm communities for bioremediation processes has recently been realized (Paul D et.al.,2005).

Bacterial communities have been utilized for the past centuries to neutralize, degrade, and mineralize many xenobiotic compounds in waste water- activated sludge (Byrns 2001; Bertin et al.

2007).

Attached and sessile micro organisms located in biofilm communities provide structure and protection because of their growth in a self-produced and complex polymeric matrix (Vu et al. 2009).

Encased within extracellular polymeric substances (EPS) secreted by the involved microbes, the biofilm structures grow and incorporate water channels, which allow transport of nutrients, electron acceptors such as oxygen or other more reduced compounds to occur(Picioreanu et al.

2000; Chen et al.2013).

Chitin/Chitosan flakes, beads, and composites have been effectively used in the removal of dyes, heavy metals and other recalcitrant compounds (E. Lorenc- Grabowska et.al.,2007) .The general approach of bioremediation is to improve the natural degradation capacity of the

native organisms. There are many variables or factors affecting enzyme production and decolorization that are expressed by different taxa and culture conditions. The ability of microorganisms to carry out dye decolorization has received much attention and several bacteria capable of dye decolorization, either individually or in consortia (Verma and Madamwar, 2003; Mossvi et al., 2007).

They have developed enzyme systems for the decolourization and mineralization of azo dyes under certain environmental conditions (Pandey et al., 2007).In the case of enzymatic remediation of azo dyes, azo reductases and laccases seem to be the most promising enzymes. Laccases have been shown to decolourize a wide range of industrial dyes (Reyes et al., 1999;

Rodriguez et al., 1999). Recent advances in bioremediation have been taking strides to understand and improve the utilization of biofilm communities as the participating microorganisms in biofilms naturally complement each other’s metabolic needs and demonstrate enhanced resistance to environmental stresses (HallStoodley et al. 2004). In heavily contaminated sites, it has been shown that cells predominantly grow in biofilms to manage the harsh environmental conditions (Gross et al.

2007).

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2 The objective of this research is to provide the strategies for bioremediation based on the benefits from the biofilm mode of growth.

2. MATERIALS & METHODS

2.1 Source of microorganisms: Dye contaminated soil samples were collected from dyeing unit of Indore district for isolation of dye degrading bacteria in sterile containers. The dye contaminated soil isolated bacterial strains were maintained on Luria Bertani Agar &

subcultured periodically to maintain their viability. Identification of these bacterial strains was done by microscopic and biochemical analysis according to Bergey’s manual of determinative bacteriology (P. Srinivasan et.al.,2016) 2.2 Screening of biofilm producing bacterial strains-

2.2.1 Tube assay: Primary qualitative biofilm screening was done using tube staining assay. Seven bacterial isolates were subjected for the biofilm producing ability by test tube assay. The overnight cultures (100µl) were inoculated in 10 ml LB broth and incubated for 72 hours at 37°C. The tubes were decanted and washed with Phosphate Buffer Saline (PBS) (pH 7.3) and dried. The tubes were stained with 0.1% crystal violet. Excess stain was removed by washing the tubes with deionized water. Biofilm formation in tubes were then observed (Christensen et al.,1982)

2.2.2 Congo red agar method- This method is based on the characteristic cultural morphology of biofilm-forming bacteria on Congo Red medium.The isolates were streaked on the Muller Hinton Agar supplemented with 0.8g/l of Congo Red Dye & incubated for 48 hrs at 37⁰C.The production of black colonies with a dry crystalline consistency indicated biofilm formation & non-biofilm producing strains develop red colonies (Mathur et.al.,2006)

2.3 Dye Decolorisation Assay of Bacterial Isolates: Bacterial Inoculums was prepared by incubating loopful bacterial suspension in LB broth containing 0.1% Malachite Green Dye for 24- 72 hours at 37°C.At defined intervals of 2nd, 3rd, 4th and 5th day, the culture was with- drawn, centrifuged at 8000g

for 15 min.The supernatant was examined for absorbance at 620 nm under visible light in a UV-VIS spectrophotometer. The extent of decolourization was expressed as percent (%) decolourization and estimated as-(P.

Srinivasan et.al.,2016)

Decolourization (%) D = A0 - A1 x 100

A0

Where, D= decolourization in %, A0=

initial absorbance, A1= final absorbance 2.4 Preparation of Biofilm support material: Two different types of chitin flakes-Shrimp shell chitin (Hi-Media) &

Chitin Flakes (sd fine chemical ltd) were used.The purchased chitin was boiled for 1-2 hr then washed with distilled water for removing the attached dust particles on the surface of chitin. The washed chitins were heat dried in an oven at 100º C for about 48 h.Then they were made into small flakes of about 4-6 mm size and these chips were used as biofilm support materials. Then these chips were thoroughly washed with distilled water.

Sterilized at 15 psi for 30 min for further process. (P. Srinivasan et.al.,2016)

2.5 Dye adsorption efficiency of Biofilm formed by different isolates:

The dyeadsorbing potential of isolated bacterial strains N1, N2, N3, N4, K2, K4 &

K5 were performed by inoculating the respective bacterial strains in each conical flasks containing LB broth supplemented with 30µg/ml of dye and 0.3 % of chitin flakes.All the inoculated culture flasks were incubated at room temperature for 120hrs. The culture was collected from the culture flaks at regular time intervals (12 hrs) intervals and the culture free supernatant was collected by centrifuge at 8000 rpm for 10 min. The adsorption was estimated by measuring the dye concentrations in the culture filtrate at regular time intervals for every 12hrs. The dye adsorbent ability of biofilm was read at 620 nm using a UV- VIS spectrophotometer.The dye uptaking ability of the biofilm was calculated by following formula-(P. Srinivasan et.al.,2016)

Decolorisation% = Initial absorbance- final absorbance ×100

Initial absorbance

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3 2.6 SEM analysis of biofilm on two different types of chitin flakes-

2.6.1 Biofilm formation: The two bacterial isolates N4 & K5,which shows best result in dye decolorisation assay were subjected for its biofilm producing ability in chitin flakes medium. The prepared chitin chips were used as a biofilm supportive material. About 0.3 % of two different chitin chips were taken in six different conical flasks containing 30 ml of LB medium each.The pre-grown exponential cells were fed into each conical flask containing chitin chips.The inoculated conical flasks were incubated at room temperature for 72hr. (P.

Srinivasan et.al.,2016)

2.6.2 Sample preparation for SEM: In this study, six samples are taken for the analysis of biofilm formation produced by the two bacterial strains.Two types of chitin flakes one is shrimp shell (Himedia )and second from chitin flakes (SD fine) are used as supporting material for biofilm formation.The biofilm chitin flakes were washed twice with 50 mM phosphate buffer (pH 7.0) for 20 min. The washed biofilms were subsequently dehydrated in a gradient of ethanol solutions (80%

ethanol) for 10 min each, and stored in 10% ethanol. These bone chips were dried, and used for SEM analysis. The two control chitin flakes lacking the biofilm were also prepared. (P. Srinivasan et.al.,2016)

2.6.3 SEM Analysis: Biofilm formation was confirmed by Scanning electron microscopy (FModel : JEOL JSM 5600)images at 20 kv. (P. Srinivasan et.al.,2016)

2.7 Phytotoxicity analysis-In this experiment, the effect of malachite green dye at the concentration of 0.1 gm was evaluated on germination of seeds of two crops, Black eye beans (Vigna unguiculata) & cowpea (Vigna aconitifolia). The seeds were germinated in disposable flasks containing 10 g of field soil.Different sets of 10 seeds of both were treated every 24 hours with 10 ml of dye solution and degraded product of Malachite green dye by bacterial isolates N4 & K5 and tap water (Control) separately. All pots were kept under shade near sunlight for the period of 10days. Germination of seeds treated with

dye and degraded dye solutions was calculated after comparing with control.At the end of the germination experiment, the shoot length and root length of seedlings was measured separately for dye, degraded dye product and control (Durve et. al., 2012)

2.8 Extraction of Exopolysaccharide (EPS) from Biofilm & its analysis by TLC: A loopful of bacterial culture of N4

& K5, was inoculated into the flask containing 100 ml of LB Broth &

incubated for 72 hrs.After incubation, the culture broth was centrifuged at 5000 rpm for 15 mins at 4⁰C.Cell pellets were discarded & the supernatant was collected & mixed with equal amount of cold absolute ethanol.After one day of incubation, the EPS was collected by centrifuge at 5000 rpm for 15 min, mixed with distilled water & stored at 4⁰C.

Extracted EPS of two bacterial isolates which are used as a sample for TLC, placed on the pre-coated silica gel plate by capillary tube.Benzene, Acetic Acid & Methanol(1:1:3) used as mobile phase. Staining of TLC plate is done by iodine vapours & then Rf value was calculated (G. Balamurugan et. al., 2012).

3. RESULTS

3.1 Source of microorganisms :Two different soil samples are collected from different regions of dye unit of Indore District. Several bacterial colonies having different morphologies are isolated on Luria Bertani Agar medium by spread plate technique, seven isolates from 10^-1&

10^-2 dilutions are isolated from two samples , streaked on fresh LB media as shown in (figure 1A) & (figure 1B):

Figure1. (A) Bacterial colonies of sample 1 (B) Bacterial colonies of sample 2 obtained on Luria Bertani Agar after incubation at 37⁰C

3.2 Screening of biofilm producing bacterial strains :

3.2.1 Tube Assay A

T h e r e f o r e

i

B

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4 The bacterial strains produced blue color ring on side wall of test tubes. The strain N4 showed visually highest production capacity as compared to other tested bacterial strains, as shown in figure2-

Figure 2. Qualitative & Visual identification of biofilm forming bacterial

strains

3.2.2 Congo Red Agar Screening:

Strains named as N4, K2 & K5 show black colonies with a dry crystalline consistency which indicated positive for biofilm formation as shown in figure3(A),figure3(B) & figure3(C)-

Figure 3. (A-C) Formation of black colonies on Congo Red Agar

3.3 Dye decolorisation assay of bacterial isolates: All the seven isolates were evaluated for their dye decolorizing ability. The two isolates, named as N4 and K5 showed decolourization percentage of 92% and 94% at 72 hrs respectively ,as shown in figure 4 & figure 5-

Figure 4.Visual observation of dye decolorisation assay of bacterial strains

Figure 5. Dye decolorisation assay of bacterial strains

3.4 Preparation of Biofilm support material: Two different types of chitin flakes, which act as supporting material were used for biofilm formation as shown in figure 6(A) & 6(B):

Figure 6. (A) shrimp shell chitin flakes from Hi- Media (B)chitin flakes from sd

fine chemical ltd A

B

C

A

B

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5 3.5 Dye adsorption efficiency of Biofilm :Among the seven bacterial strains along with one control, the biofilm of N4 & K5 formed on large chitin flakes showed 94 % & 90% dye adsorption ability, as shown in figure 7 & 9, whereas the biofilm of N4 & K5 isolates formed on small chitin flakes showed 90% & 88%

dye adsorption ability when compared with other biofilm producing strains, as shown in figure 8 & 9 after three days of incubation at 30µg/ml concentrations of dye.

Figure 7-Visual observation of dye adsorption efficiency of biofilm formed on

large chitin flakes after three days

Figure 8-Visual observation of dye adsorption efficiency of biofilm formed on

small chitin flakes after three days

Figure 9. Dye adsorption efficiency of Biofilm of N4 & K5 formed on large &

small chitin flakes & comparative study between large & small chitin flakes

Table 1. The morphological and biochemical characteristization of the selected strains N4 & K5, which shows

best results in screening of dye decolorisation assay were studied:

Characterization N4 strain K5 strain

Morphological

Gram’s Reaction Pink colored cells Pink colored cells

Capsule staining + -

Cell shape Rod shaped Rod shaped

Colony morphology Round &

white Round & white

Pigmentation - -

Motility - +

Biochemical

Catalase + +

Indole - +

Citrate + -

MR - +

VP + -

Oxidase - -

Fermentation for

acid(A) & gas production(+)

Glucose +/A +/A

Sucrose +/A +/A

Mannitol +/A +

Mac-conkey agar + +

EMB agar Pink mucoid colony Green metallic sheen

Starch hydrolysis + +

Urease + +

Probable genus Klebsiella Escherichia

+ for positive,- for negative, A for acid

3.6 SEM analysis of Biofilm: Scanning electron microscopy images revealed that the bacterial biofilm cells are well distributed on the surface of chitin flakes,as shown in figure10(B), figure10(C),figure11(B) & figure 11(C).

They are attached to the chitin flakes surface and developed into a biofilm within 72 h.

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6 Figure 10. Scanning electron microscopy

observation of biofilm formation on shrimp shell chitin flakes (large) after 72 h incubation:(A)Control (B) Klebsiella (C)

Escherichia

Figure 11. Scanning electron microscopy observation of biofilm formation on sd fine

chitin flakes (small) after 72 h incubation:(A)Control (B) Klebsiella (C)

Escherichia

3.7 Result of Phytotoxicity analysis:

Germination (%) of Vigna unguiculata &

Vigna aconitifolia seeds was found to be less with mixed reactive dyes as compared

with its degraded metabolites as shown in graph 4.

The phytotoxicity study showed that length of plumule and radical was affected in case of the dyes whereas with degraded metabolites it showed significant growth, compared to control.

The phytotoxicity of the dye was estimated by measuring the ability of dye and dye degraded product by Escherichia

& Klebsiella to germinate the seeds, Black eye beans(Vigna unguiculata) &

cowpea(Vigna aconitifolia) used as test plants. Phytotoxicity was evaluated after 10 days of study.

Good germination, shoot length ,and root length of the plants were observed for both degraded dye product of Escherichia & Klebsiella after comparing with the dye and control,as shown in table 2, figure 12, 13 & 14.

Table 2 -Phytotoxicity analysis of dye and degraded dye toxicity-

All values are mean ± SD for triplicate

Figure 12. Phytotoxicity analysis for Vigna unguiculata (Black eye bean):

(A)Control (B) Escherichia (C) Klebsiella (D) Dye(malachite green)

A B

C

A

B

C

C D

A B

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7 Figure 13. Phytotoxicity analysis for Vigna aconitifolia (cowpea) :(A)Control (B)

Escherichia (C) Klebsiella (D) Dye(malachite green)

Figure 14. Phytotoxicity analysis for seeds of Vigna unguiculata (Black eye

bean) & Vigna aconitifolia (cowpea).

3.8 Result of Extraction of Exopolysaccharides & analysis of EPS by TLC- Exopolysaccharides(EPS) are the major structural & functional components of microbial biofilms ,it is extracted from biofilm of Klebsiella & Escherichia as shown in figure 15 & analyzed by TLC, as shown in figure 16.

Figure 15. Extracted EPS: (A) Klebsiella (B) Escherichia

Figure 16. TLC plate having spot of EPS through iodine crystals, having Rf

value=0.5

4. CONCLUSION

It can be concluded that, two best dye decolorizer named as N4 & K5 from two different soil samples was identified as Klebsiella & Escherichia species based on microscopic and biochemical analysis according to Bergey’s manual of determinative bacteriology.

Klebsiella & Escherichia showed decolourization percentage of 92% and 94% at 72 hrs & the biofilm formed on large chitin flakes showed 94 % & 90%

dye adsorption ability, whereas the biofilm formed on small chitin flakes showed 90% & 88% dye adsorption ability when compared with other biofilm producing strains, degrading dye at faster rate with an application of good seed germination efficiency.

Results showed that the dye adsorption ability of biofilm formed on large chitin flakes of shrimp shell shows more potential to decolorize the malachite green dye as compared to small chitin flakes.

Dye degraded product of these strains shows more potential to germinate the seeds. Therefore it has been concluded through this study that the degradation potential of these two strains for malachite green dye was found to be significant for further research.

Therefore these properties thus found useful for the bioremediation of various textile industrial effluents, saving the ecosystem from harmful effects of various dyes.

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ACKNOWLEDGEMENT

I would like to express my greatest gratitude to faculty members of School of Biotechnology Dr. Anil Kumar and Mrs.

Shweta Nakul, School of life science &

School of Biochemistry of Devi Ahilya University, Indore (M.P.) for their constant inspiration, encouragement ,guidance & continuous motivation throughout the project. I am also thankful to Centre Director & Director of UGC- DAE-CSR for allowing me to utilize the SEM facility. Thanks Dr. D.M. Phase for allowing the time slot for performing the SEM experiment. Thanks V.K. Ahire for performing SEM/EDAX experiment &

providing us SEM data.