1.

INTRODUCTION

Textile demand is steadily increasing worldwide following world population

growth and stimulated by a growing gross domestic product (GDP) in many

countries. Cotton and polyester account for 78% of the world’s textile demand.

Following this trend, reactive and disperse dyes are the most utilized dyes for

colouring cellulosic fibres. Azo dyes that account for 60 – 80% of the dyes

consumed in textile processing [1] are characterized by a typical double azo bond

linkage (-N=N-), which is the most common chromophore of reactive dyes. The

delivery of colour onto fabrics is not an efficient process and up to 40% of the dyes

are lost during the dyeing process [2-4]. Dyeing, desizing and scouring are the

major sources of water pollution in textile effluent. Textile industry is one of the

oldest industries in Nigeria. In the last few years, environmental legislation about

the appearance of colour in discharges, combined with increasing cost of water in

the industrial sector has made treatment and re-use of dyeing effluents increasingly

attractive to the industry [5,6]. Effluents from textiles and dye stuff industries

discharge into water bodies is currently causing significant health concerns to

environmental

regulatory agencies. Government legislation is increasingly becoming more

stringent especially in the more developed countries regarding the removal of dyes

from industrial effluents. However, in developing countries like Nigeria, there is

lack of legal enforcement of environmental legislation, mainly as a result of limited

financial resources. This results in significant contamination of the environment by

large industries. Protection and preservation of the environment in Nigeria is

increasingly becoming a national concern. Textile and dyestuffs wastewaters are

characterized by their highly visible colour, high chemical oxygen demand (COD),

suspended solids and alkaline pH (9 – 11) [5,7] thus, effluent discharge from these

industries into the environment is a major cause for concern. As dyes are designed

to colour various substances and solutions indefinitely, there is a great potential for

these dyes to accumulate in the environment as many of them are

recalcitrant to normal bioremediation [8]. Several chemical and physical

decolourization methods that are available include: adsorption, precipitation,

coagulation/flocculation, oxidation, electrolysis and membrane extraction. These

techniques are effective for colour removal but are energy intensive and introduce

chemicals which are not wanted in the first place. Dye wastewaters enter the

environment from manufacturers and consumers (i.e. textile, leather and food

industries) usually in the form of dispersion or a true solution [5,8,9] and often in

the presence of other organic compounds originating from operational processes.

Colour is the first contaminant to be recognized in textile wastewater and has to be

removed before discharging into water bodies or onto land [5,10,11]. The presence

of small amounts of dyes in water (even < 1 ppm) is highly visible and it affects
the aesthetic merit, causes significant loss of luminosity and any increase in the

temperature will greatly deplete the dissolved oxygen concentration in waste water.

This results in subsequent alteration of the aquatic ecosystem [4]. The removal of

colour from wastewater is often more important than the removal of soluble

colourless organic substances which usually contribute to the major fraction of

biochemical oxygen demand (BOD). Methods for the removal of BOD from most

of these effluents are fairly well established [5,10,12]. On the other hand, textile

wastewaters exhibit low BOD to COD ratios (< 0.1) indicating there difficulty to

bioremediate or breakdown [13]. On the whole, textile wastewater is characterized

by unfixed dyes, organic pollutants (much higher than regular domestic

wastewater), large amounts of COD (organic compounds), high conductivity due

to salts, high amounts of sulphide and heavy metals due to chlorinated bleaching

agents and halogen, sulphur or heavy metal dyes [14]. Currently, much research

has been focused on the biodegradation of the industrial effluents [1517]. It mainly

shows interest towards the pollution control using bacteria, fungi in combination

with other biological methods involving plants [18,19]. In situ degradation of the

effluent is a novel method under the biodegradation process. In this method, the

microorganisms isolated from the site of pollution and the same microorganism

can be used for the treatment of such site [5]. Improvement in the ability of

microorganisms to degrade a pollutant could be achieved through modification of

the environment or the organism. The organism can be modified through

mutagenesis. This study was therefore, aimed at determining the effects of varying

environmental factors on textile effluents biodecolourization ability of

mutagenised strains of Bacillus and Pseudomonas species isolated from dye

contaminated soil.

2. MATERIALS AND METHODS

2.1 Study Area

The study was carried out at the industrial area of Kano metropolis-Challawa.

Kano is located in Kano State, it occupies a central position in Northern Nigeria.

River Challawa (Lat 11° 52 m 41s N, Long 08° 28 m 08° 09s E) is 515 metres

above sea level, originates from the Challawa Gorge dam in Challawa village and

stretches to River Kano that empties into Lake Chad [5].

2.2 Sample Collection Effluent samples and soil contaminated by untreated textile

wastewater were collected in accordance with the methods reported by Roy et al.

[20]. Samples were collected during dry season from November 2014 to April
2015. Effluent samples were collected at a point of discharge using clean 2-littre

polyethylene container. Dye contaminated soil samples at different locations (1 km

interval) from the stream into which effluent was discharged was collected from

top 4 cm soil profile (approximately 20 g) using clean, dry and sterile polythene

bags along with sterile spatula. The samples was labeled and transported to the

laboratory using ice pack chest and stored in the refrigerator at about 4°C prior to

analysis.

2.3 Isolation of Dye Degrading Bacillus and Pseudomonas Species

Dye degrading bacteria were isolated from dye contaminated soil samples using

the method reported by Saraswathi and Balakumar, [21]. Minimal basal medium

which is composed of (NH4)2SO4 1.24 g, K2HPO4, 1.36 g, NaCl, 5.0 g, and

MgSO4, 0.1 g dissolved in 1000 ml distilled water, and trace elements CuSO4(0.2

mg/l), MnSO4 1.1 mg/l, FeSO4(0.14 mg/l) and ZnSO4 (0.2 mg/l) and textile

effluent (1, 1.5, 2, 2.5 and 5%) as a sole source of carbon was prepared. The

medium (100 ml) in 250 ml Erlenmeyer flask was inoculated with 10 ml soil

suspension (water diluted soil samples) in 100 ml Erlenmeyer flask and incubated

at room temperature 37°C on orbital shaker (150 rpm). After 48 hrs of incubation,
1.0 ml of the culture broth was appropriately diluted and plated on Nutrient Agar

and King’s B Agar using dilution 10-5 for the isolation of Bacillus and

Pseudomonas species respectively. After incubation, the isolates were sub-cultured

on nutrient agar and pure culture stocks stored at 4°C on nutrient agar slopes until

needed.

2.4 Characterization of Colonies

Individual colonies were characterized using Microgen biochemical test-kits and

used according to the manufacturer’s instructions to identify the isolates to species

level. Finally, comparison was made between the biochemical profiles obtained

and tabulated characteristics in Bergey’s Manual of systematic bacteriology to

further identify the isolates.

2.5 Cultural Characteristic

The growth of the organisms on nutrient agar and centrimide agar was observed

including pigment formation and odour of the colonies. Gram staining and motility

test was also conducted using standard procedures.

2.6 Mutation with UV Irradiation at 254 nm

This was carried out by using a procedure reported by Idise [22]. The organisms

were incubated in nutrient broth for 24 hours at 37°C and their microbial count

determined by calculating the colony forming unit. Ten milliliters of broth of each

organism was aseptically transferred into separate sterile Petri dishes and exposed

to UV irradiation at 6 cm from the source of UV light, the organisms were treated

at intervals of 5, 10, 15, 20, 25 and 30 minutes in a dark room. The UV irradiated

organisms were then transferred into a sterile twenty milliliter test tube in a dark

room and treated with 0.2% (w/v) caffeine and allowed to stand at room

temperature in the dark for 5 hours. The irradiated cells were then centrifuged at

1500 rpm for five minutes, re-suspended in normal saline and re-centrifuged and

the supernatant used. The treated organism suspensions were incubated at 18°C for

16 hours.

2.7 Mutation with Nitrous Acid

This was carried out by using the procedure reported by Iftikar et al. [23]. The

bacterial growth was harvested from an overnight culture on nutrient broth and

their microbial counts determined. The cell suspensions was mixed with the

prepared nitrous acid (HNO2) (0.2 M sodium nitrate in acetate buffer, pH 4.5) and

treated at interval of 10, 15, 20, 25 and 30 min by incubating the mixture at 30°C.

Treated cells

were then centrifuged at 1500 rpm for three to five minutes, re-suspended in

normal saline and re-centrifuged to remove traces of mutagen and supernatant

discarded. The mutagenised cells were then plated on nutrient agar, incubated at

37°C for 24 hours.

2.8 Screening for Amino Acid Requirement of Mutant This was carried out using

the procedure reported by Idise [22]. A minimal basal medium was used in the

screening of mutated organisms. The medium was dispensed into sterile 20 ml test

tube and inoculated with wild strains and selected mutant strains pre- grown on

nutrient agar using sterile inoculating needle. Seven essential amino acids

(histidine, leucine, lysine, methionine, phenylalanine, tryptophan and valine) were

used due to their availability. To a set of seven test tubes, an amino acid were

added and incubated with one test organism. A control was prepared containing the

different amino acids without the test organism. The inoculated tubes were
incubated at 37°C for 24 hrs and observe for growth. Cloudy broths showed

utilization of the test amino acid and were recorded as positive utilization of amino

acid. 2.9 Biodecolourization Test

This was carried out in accordance with the method reported by Kalyanee, [24].

100 ml flask containing 50 ml of the effluent with 2 g of yeast extract and 2 g of

glucose added as a cosubstrate was prepared. The pH of the medium was adjusted

to 7±0.2 using phosphate buffer. The flask was sterilized by autoclaving for

15 mins at 121°C. The sterilized flask containing the effluent was inoculated with

3 ml inoculums (3.6x109CFUml) of each test micro-organism and incubated for 10

days at room temperature 37°C on rotary shaker at the speed of 200 rpm. This was

replicated thrice. Optical density (OD) at 560 nm was determined. After

incubation, 10 ml of degraded effluent was centrifuged at 4000 rpm for 15 minutes.

An un-inoculated flask containing textile effluent, which was sterilized by

autoclaving served as control.

2.10 Decolourization Efficiency Calculation

Colour reduction % was determined using colorimeter at wavelength of 540 nm

after 10 days intervals. The decolourization efficiency of the different isolates was

calculated using the equation below.

(I-F) x 100 I Where I is the initial absorbance and F

is the absorbance of decolorized medium.

2.11 Effects of Environmental Factors on Biodecolourization Abilities of Strains

This was carried out by a modification of procedure reported by Kalyanee, [24].

Environmental condition such as pH, temperature, incubation time, agitation,

presence and absence of co-substrate were varied and reported as follows:

2.11.1 Determination of effects of temperature

To determine the effects of temperature on the decolourization potential of the

mutagenised isolates, modification of procedure reported by Kalyanee [24] was

repeated with the following temperature ranges 30°C, 35°C, 40°C and 45°C in

water bath. All other parameters were kept constant.

2.11.2 Determination of effects of pH

To determine the effects of pH on the decolourization potential of the mutagenised

isolates, modification of procedure reported by Kalyanee, [24] was repeated with

the following pH ranges 7,8,9 and 10. All other parameters were kept constant. 1N

HCl buffered was used to adjust the pH towards acidity while 1N NaOH buffered

was to adjust the pH towards alkalinity. Temperature maintained at room

temperature 37°C and all other parameters remains the same.

2.11.3 Determination of effects of incubation period

To determine the effects of incubation period on the decolourization potential of

the mutagenised isolates, modification of procedure reported by Kalyanee [24] was

repeated, but the incubation periods was extended to 14 days and reading taken at 2

days interval. All other parameters kept constant.

2.11.4 Determination of effect of agitation

To determine the effects of agitation and static condition on the decolourization

potential of the

mutagenised isolates, modification of procedure reported by Kalyanee [24] was

repeated, the biodegradation test was carried out on a plane bench under static

condition while under agitation the biodegradation test was carried out on rotary

shaker at 150 rpm. All other parameters kept constant.

2.11.5 Determination of effects of cosubstrate

To determine the effects of presence and absence of co-substrates on the

decolourization potential of the mutagenised isolates, modification of procedure

reported by Kalyanee [24] was repeated. In the absence of cosubstrates, the

biodegradation test was carried out without yeast extract and glucose. All other

parameters kept constant.

2.12 Descriptive Statistic

Data were entered into Microsoft excel 2007 spread sheet and means and standard

deviations were calculated.

3. RESULTS

3.1 Characterization of the Isolates

The isolates were identified as Bacillus cereus, Bacillus firmus, Pseudomonas

aeruginosa and Pseudomonas fluorescens as shown in Table 1 and 2. Bacillus

cereus and Bacillus firmus showed positive Gram reaction, Oxidase, Motility and

indole test. Bacillus cereus showed positive Arginine test and negative ONPG test.

Bacillus firmus showed negative Arginine test and positive ONPG. Pseudomonas

aeruginosa and Pseudomonas fluorescens showed negative Gram reaction and

positive Motility and Oxidase test. Pseudomonas fluorescens showed positive

Citrate and urease test while Pseudomonas aeruginosa showed negative citrate and

urease test.
3.2 Screening for Abilities of the Wild Strain Isolates to Grow in Different

Concentrations of Textile Effluent

This is presented in Table 3. The organisms possessed the ability to grow in the

presence of textile effluent as they metabolize dyes in the Minimal basal medium

containing textile effluent leading to isolates multiplication. Bacillus cereus and

Pseudomonas aeruginosa possessed a higher ability as they multiple in Minimal

basal medium containing 5% textile effluent while Pseudomonas fluorescens and

Bacillus firmus grew in Minimal basal medium containing 2.5 and 2.0% textile

effluent respectively.

3.3 Mutagenic Treatment of the Wild Strain Isolates

The effects of the mutagen treatment are presented in Fig 1 and 2. The effects of

Ultraviolet (UV) irradiation (254 nm) on parent strains at different time 5, 10, 25,

20, 25 and 30 min, showed percentage survival that range from 3.04% Bacillus

cereus (B8UV30) to 56.15% Pseudomonas fluorescens (P23UV5). The effect of

nitrous acid (HNO2 2.0 M) treatment after exposure of the parent strains (B6WS,

B8WS, P2WS and P23WS) at different time interval (10, 15, 20, 25, 30 min)
presented in Fig. 2 showed percentage survival ranging from 10.37% Pseudomonas

fluorescens (P23NA(10)) to 58.76% Bacillus cereus ((B8NA(10)). The result in

Figs. 1 and 2 showed that nitrous acid treatment of strains had a higher survival

percentage and the result also showed that percentage survival is inversely

proportional to time.

3.4 Screening for Amino Acid Requirement by Parent and Modified Strains

The requirement of essential amino acids – leucine, lysine, tryptophan,

methionnine, histidine, valine and phenylalaine for growth by the parent and

modified strains is shown in Table 4. It was observed that most of the organisms

required all the tested amino acids for growth except B8UV30, P23NA30 and

P2UV20 which do not require histidine, valine and phenylalanine respectively.

This showed the mutagenized strains having amino acid requirement different from

their parents, hence, their selection for further analysis.

3.5 Biodecolourization of Textile Effluent

This is presented in Tables 5 and 6. It was observed that parent Bacillus cereus

(B8WS) performed better than the parent Pseudomonas aeruginosa (P2WS),

Bacillus firmus (B6WS) and Pseudomonas fluorescence (P23WS). The parent

strains showed the following order of biodecolorization activities of 42.56±0.12,

40.43±0.62, 39.31±0.28 and 36.01±1.36% as shown in Table 5. The parents do not

show any appreciable level of textile effluent

biodecolourization. The mutagenised strains were better decolourizer than parent

strains and also UV-irradiated strains performed better than the nitrous acid treated

strains. Bacillus cereus (B8UV30) irradiated at 30 mins and Pseudomonas

aeruginosa (P2UV20) irradiated at 20mins showed the highest percentage

biodecolorization of 60.30±0.02% and 55.13±0.51% respectively while

Pseudomonas fluorescens (P23NA30) treated with nitrous acid at 30mins showed

53.09±0.37%. The results in Table 6 also showed that increase in time of treatment

is directly proportional to percentage biodecolorization. The control samples

showed no biodecolourization.

3.6 Effects of Temperature on Biodecolourization of Textile Effluent

The results in Fig. 3 showed the effects of temperature on textile effluent

biodecolourization by the parent and mutant strains. The biodecolourization

activities increased steadily with increase in temperature from 30°C to 35°C but

later reduce at 40°C to 45°C. Fig. 3 recorded the highest biodecolorization

activities of 53.92±0.79% at 35°C and the highest activity of 42.98±0.98% at 35°C

by parent. The mutant strains showed better biodecolourization than the parent at

the different temperature range. Bacillus cereus (B8UV30) and Pseudomonas

fluorescens (P23NA30) showed the highest biodecolourization activity. Mutant

strains were more thermostable than the parent strains.

3.7 Effects of pH on Biodecolorization of Textile Effluent

The result of the effects of different pH values on biodecolourization activities are

shown in Fig. 4. They activities of the parent and mutant strains decreases with

increase in pH values except in some instances. The mutant strains showed highest

biodecolourization of 58.82±1.02% while the parent strains showed highest

biodecolourization of 43.20±0.8% at pH7. Fig. 4 also shows that higher pH were

detrimental to the strains. The biodecolourization activity began to decline at pH8.

Fig. 4. also shows that the activity of the mutant strains were affected at pH 8-10
but still did better than the parent strains. At pH10 Bacillus cereus (B8UV30) has

this highest percentage biodecolourization of 26.12±1.04% while parent Bacillus

cereus (B8WS) shows the highest biodecolourization of 7.21±0.76% among the

parent strains.

3.8 Effects of Agitation on Biodecolourization of Textile Effluent

The results of the effects of agitation (shaking) and static condition on

biodecolourization potential of the parent and modified strain is shown on the Fig.

5.The modified strains were better decolourizer on both static and shaking

condition than the parent strains. The Fig. 5 result showed slight differences

between the shaking and static condition. The highest biodecolourization activity

(58.38±0.10) was shown by Pseudomonas fluorescens (P23NA30) on state

condition. The highest biodecolourization activities (54.83±1.05) on shaking

condition were recorded by Bacillus cereus (B8UV30). It’s shown in Fig. 5 that

both parent and mutant strains performed slightly better under static than shaking

conditions.

3.9 Effects of Co-substrate on Biodecolorization of Textile Effluent

The results of the effects of co-substrate (glucose and yeast extract) on

biodecolourization activity of both parent and mutant strains are shown in Fig. 6

The parent and mutant strains performed better in the presence of the cosubstrate.

The highest result in the absence of co- substrate was obtained by Bacillus cereus

(B8UV30) at percentage biodecolourization of 26.56±1.20 while the highest

biodecolourization percentage of 56.42±0.11 in the presence of cosubstrate was

performed by the same organism. The parent strains showed the highest

biodecolourization percentage of 13.92±0.69 and 43.74±0.54 in the absence and

presence of cosubtrates respectively, which was achieved by Bacillus cereus

(B8WS) and Pseudomonas aeruginosa (P2WS) respectively.

 Table 1. Characterization of Pseudomonas
Test isolates using
After 12 h incubation microgen
After test kit
12 h incubation
Growth on CA green coloured colonies creamy coloured colony
Gram reaction - -
Motility + +
Key: + = Utilization; - = non-utilization; ND+= Not Determined; ONPG = O-nitrophenyl-β-D-galactopyranoside;
Oxidase +
Nitrate VP = Vogas proskauer; + +
TDA = Tyrosine-D-arginine; CA= Centrimide Agar
Lysin + +
Ornithine - -
H2S - -
Glucose + -
Mannitol - -
Xylose + +
ONPG - -
Indole - -
Urease + -
VP - -
Citrate + -
TDA + +
Gelatine - -
Malonate - +
Inositol - -
Sorbitol - -
Rhemnos - -
Sucrose - -
Atabinose + -
Adonitol - -
Raffinos - -
Salicin - -
Arginine + +
0
Growth at 25 C + ND
Isolate identified Pseudomonas fluorescens Pseudomonas
aeruginosa

3.10 Effects of Incubation Period on Biodecolourization of Textile Effluent

The results of the effects of incubation period on biodecolourization activity of

both parent and mutant strain are shown in Fig. 7. These showed progressive

increase in biodecolourization activities of the organisms as the days increase. The

biodecolourization activities showed a

decline at the day 10 with few progressing slowly to day 14. All the parent strains

reached their peaks at day 10 with Pseudomonas aeruginosa (P2WS) showing the

highest activity of 51.57±0.70%. The mutant strains progressed steadily to day 14.

Bacillus cereus (B8UV30) showed the highest activity of 66.16±1.16% at day 14.

Table 2. Characterization of Bacillus isolates using microgen test kit

 Test After 12 h After 24 h After 12 h After 24 h
Growth on nutrient agar White dry colonies Gray dry colonies
Gram reaction + + + +
Catalase + + + +
Motility + + + +
Arabinose - - - -
Cellebrose - - - -
Inositol - - - -
Mannitol - - - -
Mannose - - - -
Raffinose - - - -
Rhamnose - - - -
Salicin - - + +
Sorbitol - - - - Table
Sucrose - - - -
Trehalose - - + +
Xylose - - - - 3.
Adonitol - - - -
Galactose - - - -
MON - - - -
MDG - - - -
Inulin - - - -
Melezitose - - - -
Indole ND + ND -
ONPG + + + +
Arginine - - - -
Citrate - - - -
Voges proskauer ND + ND +
Nitrate ND + ND +
Isolates identified Bacillus firmus Bacillus cereus

Growth of isolates on minimal basal medium containing textile effluent

Isolate 1% v/v textile 1.5% v/v textile 2% v/v textile .5% v/v textile 5% v/v textile
B8WS + + + + +
B6WS + + + - - P2WS + + + + +
P23WS + + + + -
Key: Ws = Wild strain; B8 = Bacillus cereus; P2 = Pseudomonas aeruginosa; B6 = Bacillus firmus;
P23 = Pseudomonas fluorescens, + = growth, - = No growth
 Fig. 1. Survival percentage of the UV-light exposed wild strains (ws) of Pseudomonas and Bacillus sp.
% = Percentage Table 4. Screening for amino acid requirement of wild and mutagenised strains

Amino B8WS B8UV30 B8NA30 B6WS B6UV20 B6NA30 P2WS P2UV20 P2NA30 P23WS P23UV25
Histidine + - + + + + + + + + +
Lysine + + + + + + + + + + +
Methionine + + + + + + + + + + +
Phenylalanine + + + + + + + - + + +
Valine + + + + + + + + + + +
Leucine + + + + + + + + + + +
Tryptophan + + + + + + + + + + +

Key: B8UV = UV-irradiated Bacillus cereus; P2UV = UV- irradiated Pseudomonas aeruginosa; B6UV= UV- irradiated
Bacillus firmus; P23UV= UV- irradiated Pseudomonas fluorescens; B8NA = Nitrous acid treated Bacillus cereus; P2NA = Nitrous
acid treated Pseudomonas aeruginosa; B6NA= Nitrous acid treated Bacillus firmus; P23NA= Nitrous acid treated
Pseudomonas fluorescens; WS = Wild strains; + = Utilization ; - Non utilization
 70
NAB6 %
60 NAB8 %
PERCENTAGE SURVIVAL

NAP2 %
50
NAP23 %
40

30

20

10

0
TM-10 TM-15 TM-20 TM-25 TM-30
TREATMENT TIME (min)

Table 5. Mean percentage biodecolourization performance by wild strains of the isolates

Strains % biodecolourization of textile

effluents
B8WS 42.56±0.12
P2WS 40.43±0.62
B6WS 39.31±0.28
36.01±1.36
P23WS
Key: Ws = Wild strain; B8 = Bacillus cereus; P2 =
Pseudomonas aeruginosa; B6 = Bacillus firmus; P23 = Pseudomonas fluorescens.

4. DISCUSSION

The role of some bacterial species in the biodecolourization of textile effluents has

been reported [24-26]. Chen et al. [27] also reported isolation and screening of
bacteria capable of decolorizing various dye from industrial effluent samples

collected from wastewater treatment site contaminated by dyes. The isolates

Bacillus species and Pseudomonas species were susceptible to mutation with UV-

light irradiation and nitrous acid treatment. Hence they were UV

light and nitrous acid sensitive. The treatment of the wild strains with ultraviolet

irradiation and the nitrous acid treatment indicated that the increase in duration of

the exposure of the organisms to the mutagen is directly proportion to the increase

in rate of biodecolourization activities of the organisms. Mutant strains B8UV30

obtained after 30 minutes irradiation gave highest biodegradation of 60.30±0.02%.

This study disagrees with the previous work by Ado et al. [28] which state that it is

not directly proportional.

Table 6. Mean percentage biodecolourization performance by mutagenized strains

Strains % biodecolourization of textile effluent B8UV30 60.30±0.02 P2UV20

55.13±0.51 P23NA30 53.09±0.37 Keys: B8UV30 = UV-irradiated Bacillus

cereus, P2UV20 = UV-irradiated Pseudomonas aeruginosa, P23NA30 = Nitrous

acid treated Pseudomonas fluoresens. 30 and 20 = Time of exposure to mutagens,

± = Standard error
Fig. 3. The effects of temperature on biodecolourization of textile effluent Key:

B8UV = UV-irradiated Bacillus cereus; P2UV = UV- irradiated Pseudomonas

aeruginosa; P23NA= Nitrous acid treated Pseudomonas fluorescens; WS = Wild

strains; Time of treatments = (20 and 30 minutes

Results revealed that wild strains of Bacillus cereus, Pseudomonas aeruginosa,

Bacillus firmus and Pseudomonas fluorescens achieved 42.56±0.12, 40.43±0.62,

39.31±0.28 and 36.01±1.36% decolourization of textile effluents respectively. The

results agreed with work done by Husseiny, [29]. These isolates probably had

acquired natural adaptation to survive in the presence of the textile effluent. The

ability of the organisms to degrade textile effluents is as a result of their metabolic

activities.
Biodecolourization activities of the wild strains were enhanced by genetic

improvement of the organisms using mutation technique. The technique has been

widely employed to obtain strains with desired characteristics [30]. This was

achieved by a combination of physical and chemical mutagens using ultraviolet

irradiation and nitrous acids. The selections of B8UV30, P2UV20 and P23NA30

for further studies were based on them having different amino acids requirement

when compared with the parents. The treatment of organisms with mutagenic

substances does not necessarily result in mutants being produced as the organisms

possessed various mechanisms for reversal of such actions through repair of the

damaged DNA. For example photo-reactivation could occur which consist of a

single enzyme that can split pyrimidine dimmers in the presence of light, a process

catalysed by photolyase enzyme which is found in many bacteria [31]. This is

usually avoided by incubation of UV-irradiated organism for 5 hr with 2% caffeine

in the dark [28]. Nitrous acid treatment, base excision repair (BER) could be

employed in the removal of the damaged or inappropriate base from its

sugar with glycosidase enzyme while a new base is incorporated by DNA

polymerase using the other strand as a template [31,32].

Mutant strains B8UV30 obtained after 30 minutes irradiation with a percentage

survival of 3.00% gave the highest biodecolourization of 60.30±0.02%. This

finding is in agreement with the observation of Ado et al. [28] that for a mutagenic

agent to be successful, it should have a high rate of kill. The improved quality of

textile effluents biodecolourization observed during this study after introduction of

mutant strains, is attributed to mutation of parent strains. This corroborates the

reports of previous studies by WHO, [33]; Ado et al. [28]; Idise et al. [22].

Temperature has a significant influence on biodecolourization of textile effluent by

micro

organisms [19]. The effects of temperature revealed the maximum percentage

biodegradation of 53.92±0.79% at 35°C. This result supported the work done by

Bisht et al. [34]. The mutagenised strains showed higher performance than the

parent isolates at a higher temperature of 45°C. This could be as a result of

mutation which made the mutant strains biodecolourization enzyme more resistant

to temperature.

The effects of pH results revealed that the maximum percentage of decolourization

was 58.82±1.02 at pH7. This result supported the concept of Pandey et al. [30] that

better bacteria growths usually occur at pH7. However B8UV30 at pH10 resulted

slightly higher than at pH9 in biodecolourization. It is possible that pH10 was

detrimental to the bacteria and caused the release of enzyme or redox mediators to

cause dye reduction and the dye could also be reduced by alkaline hydrolysis [24].

The comparative study between the efficiency of static and shaking condition (150

rpm) for performance of the wild strains and parent strains on the decolourization

of textile effluent revealed that the highest percentage biodecolourization

(58.37±0.10%) by P23NA30 was obtained under static condition. It was found that

static conditions were more efficient than the shaking condition for both parent and

mutant strains. This results are similar to those obtained by Daneshrar et al. [35]

using another type of microorganism. High rate of agitation decrease bacterial

growth and the activities of some biological substance such as enzymes which play

important roles in the decolourization of the dye [36,37].

The effects of co-substrates on the activities of the organisms indicated low

biodecolourization activities of the organisms in the absence of glucose and yeast

extracts. This was in agreement with the work conducted by Sarawasthi and

Balakumar [21]. This implies that the organisms could not metabolize the dye as

sole source of carbon and energy to appreciable level.

This work contradicted the previous work done by Murugalatha et al. [38] that

organisms were able to show maximum level of textile effluent biodecolourization

within 90 days of incubation as compared to highest obtained at 14 day.

The effects of incubation period showed decolourization rate of 66.16±0.12% was

achieved at a period of 14 days. In this study, biodecolorization proceeded

gradually up 14th days in the mutated strains whereas in the wild strains treatment,

there was rapid decolourization during the initial days of exposure by the parent

strains that slowed down abruptly on day 5th to 7th day of treatment. This is in

agreement with work done by Mutambanengwe [39].

5. CONCLUSION

Environmental factors from this study proved to have an effect on the

biodecolourization activities of the mutagenised strains of Bacillus cereus,

Pseudomonas aeruginosa and Pseudomonas fluorescens which resulted in

increased decolourization efficiency of textile effluents as compared to the parent

strains. They were able to biodecolourize appreciable level of textile effluent

within a shorter period of 14 days. The mutant of organisms showed high tolerance

to temperature and pH. The result obtained from this work shows, adequate

manipulation of environmental condition could result to greater textile effluent

decolourization by these organism. Hence, the introduction of these strains

especially their enzymes in the wastewater treatment plant could be a plausible

solution to the environment pollution problem therein in the discharge of the textile

effluent currently.

6. RECOMMENDATION

Further investigations using other forms of mutagens should be conducted on other

species of bacteria and fungi to build up a data bank of biodecolourization

organisms. It should also be interesting to identify, isolate and concentrate the

active enzymes of biodecolourization in the organism studied.

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