Nigerian Journal of Science Vol 51 - No 1 (2017) 15-22Date Received 16/06/2016

Corrected & Acceptance Date 03/06/2017

Abstract

Many bacteria strains have been used in dye decolorization including Klebsiella pneumoniae, however limited data are available on dye decolorization by Klebsiella oxytoca ITK4. This study reported various conditions (Temperature, pH, Time and Inoculum density) for Black WNN dye decolorisation by K. oxytoca. Decolorization experiment was analyzed using a five-level-four-factor Response Surface Central Composite Design (RSCCD) (SAS, 9.3) Temperature (25, 30, 35, 40 and 45 ºC), pH (5.6,7,8 and 9), Time (24, 48,72, 96 and 120 h), and inoculum density (0.5, 0.6, 0.7, 0.8 and 0.9) were the variables considered during the study. Results showed that temperature, inoculum density and time were significant at P<0.05. Optimal decolorization of black WNN dye by Klebsiella oxytoca was at temperature of 35 ºC, pH 7, and after 83 h with inoculum density of 0.9. This study showed that Klebsiella oxytoca ITK 4 could be used to remediate black WNN dye effluent.

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Keywords: Decolorisation, Reactive Dye, Klebsiella oxytoca, dye effluent, response surface

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S.A. Balogun et al.: Response Surface Optimization of Black WNN Dye Decolorization by Klebsiella oxytoca ITK

Figure 1: Decolorisation of black WNN reactive dye by selected bacterial isolates

Nigerian Journal of Science Vol 51- No 1 (2017): 15-22

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After different independent variables were combined (X = pH, X = temperature, X = time and 1 2 3

X = inoculum density). Analysis of the RSCCD 4

result using ANOVA showed that inoculum density, temperature and time were significant (P < 0.05). RSCCD showed that linear effect (X X and X ), 1, 3 4

interactive effect (X X , X X X X and X X ) and 1 2 1 4, 2 3 3 42 2 2quadratic effect (X , X and X ) have significant 1 2 4

effect on reactive black WNN dye decolourisation.

2Regression co-efficient (R ) was 0.9190 (i.e less than 1), which showed that RSCCD analysis is suitable for prediction purpose of black WNN decolourization using Klebsiella oxytoca.

3D plots showed the interaction and response of the variables to dye decolorisation by K . oxytoca. Optimization of reactive black WNN decolourization using RSCCD prediction showed that the best combination of the independent variables (temperature, pH, inoculum density and time) in black WNN decolourisation was inoculum density of 0.9, temperature at 35º C, pH 7 at 83 hours with its desirability of 88 % (Fig 2-4).

Figure 2 shows 3D plot of the interactive effect of pH and temperature on black WNN decolorisation by K. oxytoca ITK 4. It showed that increase in temperature and pH gave higher absorbance value (i.e lower decolorisation). Highest decolorisation

o(i.e lowest absorbance) was observed at 35 C and pH 7.0 (Figure 2). Dye decolorisation was positive until

o35 C and after 72 hours, decolorisation declined. Increase in temperature and time showed increase in absorbance indicating lower decolorisation rate (Figure 3). Figure 4 showed increase in decolorisation (i.e reduction in absorbance) as pH increase from 5.0 to 9.0. Highest decolorisation was at pH 7.0. For optical density (OD), decolorisation increase with increase in OD from 0.5 to 0.9. Optimal decolorisation was at inoculum density of 0.9 Interactive effect exists between inoculum density and time As the inoculum density increases from 0.5 to 0.9 decolorisation increases. Also increase in time had a positive effect on decolorisation. Optimal decolorisation due to inoculum density and time was at 0.9 and 83 hours (Figure 5).

. .

Y1: ABSORBANCE

25.00

30.00

35.00

40.00

45.00

5.00

6.00

7.00

8.00

9.00

0.14723

0.175423

0.203615

0.231808

0.26

X1: TEMPERATURE

X2: pH

Figure 2: 3D surface Plot of Temperature versus pH on black WNN dye decolorization by Klebsiella oxytoca ITK 4

S.A. Balogun et al.: Response Surface Optimization of Black WNN Dye Decolorization by Klebsiella oxytoca ITK

Y1:ABSORBANCE

25.00 30.00

35.00 40.00

45.00

24.00

48.00

72.00

96.00

120.00

0.144529

0.173397

0.202264

0.231132

0.26

X1:TEMPERATURE

X4:TIME

Figure 3: 3D surface plot of temperature versus time on black WNN dye decolorisation by Klebsiella oxytoca ITK 4

Y1: ABSORBANCE

5.00

6.00

7.00

8.00

9.00

0.50

0.60

0.70

0.80

0.90

0.12

0.135846

0.151693

0.167539

0.183385

X2: pH

X3: INOCULUM-DENSITY

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Figure 4: 3D plot of inoculum density and pH on black WNN dye decolorization by Klebsiella oxytoca ITK 4

Nigerian Journal of Science Vol 51- No 1 (2017): 15-22

Y1: ABSORBANCE

0.50

0.60

0.70

0.80 0.90

24.00

48.00

72.00

96.00

120.00

0.12

0.15

0.18

0.21

0.24

X3: INOCULUM DENSITY

X4: TIME

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Figure 5: 3D plot of inoculum density and time on black WNN decolorisation by Klebsiella oxytoca ITK 4

Discussion

The bacterial genera isolated have been reported to be associated with dye effluent by many workers. Bacillus sp (Wuhrman et al., 1980; Olukanni et al., 2009; Chaube et al., 2010; Rajesh and Arun 2010; and Gurav et al., 2011), Staphylococcus. sp (Hsueh and Chen 2007; Hsueh et al., 2009 and Olukanni et al., 2009) and Klebsiella sp (Chaube et al.,2010).

Decolorisation potential of these isolates suggests the adaptability of the strains to the severe conditions of the effluent and their survival in highly contaminated water. In the decolorisation assay, K. oxytoca was the only Gram negative bacterium that was able to compete with other Gram positive isolates in the screening phase. Sirianuntapiboon et al., 2004 posited that cell wall of microorganisms are indispensable in the decolorisation of textiles dyes because they have ability to hold or fix dyes in their cell wall. Cell wall configuration showed that Gram positive cell

wall components are tightly packed while Gram negative cell wall have periplasmic space in between (Prescott et al.,2002).This suggests why Gram positive bacteria are able to retain dyes more than the Gram negative negative ones. Also three out of the four isolates used for the decolorisation experiments were isolated from a local tie and dye industry.

Removal of colour from the dye (i.e decolorisation) by isolates and subsequent increase in turbidity of the culture medium indicates that certain nutritive compounds present in the dye supported the growth of the isolate. (Barakat et al., 2009). Studies have shown that organic nitrogen sources are considered as an essential medium supplement for the generation of NADH that act as an electron donor for the reduction of azo dyes by microorganism (Bibi et al., 2012).

Seventy per cent (70%) decolorisation of black WNN by K. oxytoca ITK 4 is lower compared to 97% by Staphylococcus epidermidis on the same dye (Anamika and Sarabjeet, 2012). Though S.

S.A. Balogun et al.: Response Surface Optimization of Black WNN Dye Decolorization by Klebsiella oxytoca ITK

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epidermidis is Gram positive while K. oxytoca is Gram negative. Black WNN decolorisation was further optimized to ascertain the best condition for the decolorisation using RSCCD. RSCCD is a useful tool that gives information about the optimum conditions in a few trials, to reduce experimental errors, to enhance efficiency and reproducibility of the laboratory experiments (Krishna et al., 2005).

This work corroborates previous findings that favourable pH for dye decolourisation by bacterial strains has been found to be between 7 - 8 (Rajesh and Arun, 2010).

Black WNN decolorisation at 35 ºC is similar to the findings of Anamika and Sarabjeet, (2016) who reported optimal decolorisation at 35 ºC. Also Bibi et al., 2012 stated that higher temperature above 40 ºC have negative effect on decolorisation of dyes, which may be due to thermal deactivation of the enzyme responsible for decolorisation.

Increase in decolorisation of black WNN dye was achieved with rise in inoculum concentration. This is similar to the report of Telke et al., 2008.This suggests higher transmittance of light through the effluent solution and hence, higher decolorisation of dye solution (Seesuriyanchan et al., 2011).

Time was an important variable in black WNN decolorisation such that decolorisation decline after 83 hours. Anamika and Sarabjeet, 2012 reported decline in black WNN decolorisation by Staphylococcus epidermis after 72 hours.

Although, Klebsiella oxytoca ITK 4 have not been used extensively in dye decolorisation. This study revealed that pH, temperature, time and inoculum density was significant in WNN dye decolorisation by K. oxytoca. Optimal decolorisation of black WNN by K oxtoca was at pH 7, temperature of 35° C, inoculum density of 0.9 and after 83 h of incubation. It can be concluded that K. oxytoca ITK 4 has great potential to decolorize dyes especially black WNN dye effluent.

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S.A. Balogun, S.O. Adebajo, A.K. Akintokun and A.A. LasisiNigerian Journal of Science Vol. 51- No 1 (2017): 15-22ISSN 0029 0114www.sciencenigeria.org

S.A. Balogun et al.: Response Surface Optimization of Black WNN Dye Decolorization by Klebsiella oxytoca ITK