Bacterial Biodegradation of Congo Red Dye Using Local Bacterial Isolates

: The current study aims to get local bacterial isolates isolated from wastewater samples, w the highest ability to ediouoloced one selected Azo dye (Congo red as a model), and then test its ability to decompose Congo red dye, to obtain the most efficient bacterial isolate Four samples of wastewater collected from sewage transport pipes were used for the bacterial isolation. yoooF-two bacterial isolates were obtained after inoculating these samples in the liquid MS medium, pH 7 with 1% glucose, and then on solid MS medium supplemented with50 ppm of Congo red dye. Results from primary tests showed that only eighteen bacterial isolates own varying abilities to ediouoloced Congo red dye and the isolates WR7, WR18 and WR30 give high clear zones (20 mm). The secondary screening was achieved to determine the most efficient isolates to degrade the Congo red dye. The results indicated that the isolates WR7, WR18, and WR30 appeared to have the highest ability to decolorize Congo red dye reaching 88.6%, 83.9%, and 92.8%, respectively. The results from the optimum conditions experiment revealed that the isolates WR7, WR18 and WR30 appeared to have the highest ability to degrade dye reached 88.7%, 83.9%, and 92.9% respectively when 100 ppm of Congo red dye was used after 3 days of incubation as compared with other studied concentrations. While the second step in this experiment confirmed that the isolate WR30 has the highest percentage to degrade the Congo red dye during all incubation periods, which reached 14.6%, 42.7%, 92.9%, 92.4% and 91.8% after 1, 2, 3, 4, and 5 days of incubation when 100 ppm of Congo red dye was used respectively as compared with WR7 and WR18 isolates which showed lower values of degradation for all studied periods. The three isolates which have the highest capability to decolorize Congo red dye were identified according to morphological, physiological and biochemical tests. The results indicated that the isolates WR7, WR18 and WR30 were Bacillus subtilis, Pseudomonas aeruginosa , and Bacillus cereus respectively.


Introduction:
The dyes were extracted and manufactured from vegetable sources at the start of their use. Thereafter with their increasing needs in industries, they were industrially produced to cover the increasing market demand [1]. ehd expansion of industries and consequently the increase in the types and quantities of dyes led to the creation of many environmental problems, especially for the aquatic environment as sewage water containing these industrial dyes, was released into the rivers and seas [2].
Bioremediation is the process that ludu ooro cusu such as bacteria, molds, yeasts, algae and others to clean up sites contaminated with pollutants. This process has been applied to degrade many pollutants in soil and wastewater. It is superior when compared oo the chemical and physical methods because of its lower cost and good efficiency in biological treating units to remove contaminants [3]. All synthetic dyes own complex chemical structures and are highly resistant to natural degradation. Around 280,000 tons of textile dyes are discharged into the water bodies every year worldwide [4].
Azo are one of the textile dyes widely used in industries. These dyes contain one or more Azo bonds (-N=N-) in their structure, about 70% of dyestuff used in textile processing are Azo dyes because of ohdco low price, good stability and availability of different iouolou as compared to the other dyes, ohd textile industry uses around 70% Azo dyes. Therefore, about 10 -15 % of them are discharged into the water bodies as effluent causing toxicity problems to the water ecosystem [5,6].
Using microorganisms such as bacteria and fungi, many studies have successfully achieved to decolorize or degraded different types of Azo dyes. With the aim of developing wastewater treatment techniques by safe method, other researchers have also proven that the study of the biodegradation process of Azo dyes has many benefits [7,8,9].. Various microorganisms, including bacterial and fungal isolates, have been used to treat Azo dyes as their widespread use in the textile industry has badly affected the aquatic environment. Many researchers have used different species of bacteria such as Bacillus, E. coli, Klebsiella, Enterobacter, and Pseudomonas to decolorize Congo red dye [10][11][12][13][14][15].
The present study focuses on obtaining local bacterial isolates capable of decolorizing Congo red dye as a model for the biodegradation process of Azo dyes, in order to select the most active isolate and study some factors affecting the biodegradation of this dye.

Materials and methods: Samples collection:
Clean and autoclave containers were used to collect four samples of wastewater and sludge from different sites of pipelines that transport wastewaterin Al-Ghazaliya citysituated on the outskirts of Baghdad in Iraq.

Bacterial isolation and screening:
The medium used for bacterial isolation, characterization, and screening for dye biodegradation is solid mineral salts medium (MSM) with 1% glucose, pH 7 with some modification according to the purpose of the research.
Subsequently, 1 ml from each sample was added to flasks containing 50 ml sterilized liquid MS medium. The flasks were incubated at 37 °C for 2 days in a shaker incubator at 150 rpm, and thereafter 1 ml from each flask was spread on solid MS medium and incubated at 37 °C for 3 days. The bacterial colonies were purified by transferring the samples to new plates of solid MS medium.
To perform an initial examination and to determine the ability of bacterial isolates to breakdown the dye, the poured bacterial colonies were re-culturing in liquid MS medium with 1% glucose at 37 °C for 2 days., Then 1 ml from each flask was re-spread on solid MS medium supplemented with 50 ppm Congo red dye and the plates were incubated at 37 °C for 3 days. Subsequently, the colonies that caused a clear zone on the medium were selected [16].
The second step of screening to decolorize Congo red dye by bacterial isolates was achieved in liquid MS medium, pH 7 supplemented with 50 ppm. Congo red dye, flasks were inoculated with 1 ml bacterial growth from each bacterial isolate (triplicate for each) and incubated at 37 °C for 3 days in a shaker incubator at 150 rpm. Later on 5 ml from each flask was withdrawn and was centrifuged at 8000 rpm for 10 min to get the supernatant of the free cells. The absorbance of test samples and control were measured at 490 nm using a UV-Visible spectrophotometer and the percentage of decolorization was determined using the below equation [17].

Optimum conditions to decolorize Congo red dye:
The effect of dye concentration on Congo red dye decolorization was evaluated in the liquid MS medium, pH7.upplemented with different concentrations (25,50,100,150,200, and 250 ppm), flasks were inoculated with 1 ml of bacterial growth from the most active bacterial isolates (triplicate for each) and incubated at 37 °C for 3 days in a shaker incubator with 150 rpm, 5 ml from each flask was later withdrawn and centrifuged at 8000 rpm for 10 min to get the supernatant of the free cells. The absorbance of test samples and control, prepared as the sample test without bacterial inoculums, was measured at 490 nm using a UV-Visible spectrophotometer and the percentage of decolorization was determined using the above equation.
Also, the effect of incubation periods on Congo red decolorization was investigated in liquid MS medium, pH 7 supplemented with 100 ppm Congo red dye. Flasks were inoculated with 1 ml of bacterial growth from each isolate (triplicate for each) and incubated at 37 °C at different periods (1, 2, 3, 4 and 5 days) in a shaker incubator at 150 rpm. After each period, 5 ml from each flask was withdrawn and centrifuged at 8000 rpm for 10 min to get the supernatant of the free cells. The absorbance of test samples and control were measured at 490 nm using a UV-Visible spectrophotometer and the percentage of decolorization was determined using the same previous equation [17,18].

Identification of decolorizing bacterial isolates:
The highest decolorized bacterial isolates were identified based upon macroscopic characteristics (such as the color, shape, texture, and transparency of colonies) and microscopic characteristics (Gram staining, capsule staining, and endospore staining), and biochemical tests (motility, catalase, oxidase, indole, and others). All the identification procedures were performed by methodologies reported in the Bergey's manual of determinative bacteriology [19].

Results and discussions: Bacterial isolation and, screening of dye degradation:
Using mineral salts medium with 1% glucose, fourty-two bacterial isolates were obtained from the four wastewater samples, as shown in the Table 1 below. The wastewater samples are considered a good source for obtaining different types of bacterial isolates that are capable of degradation or consuming many organic pollutants, including industrial dyes [20]. The primary results showed that only 18 isolates were able to degrade the Congo red dye when using the solid medium of mineral salts supplemented with 1% Congo red dye, which gave a clear zone ranging from 8-20 mm, Table 2.The biological processes to decolorize and degrade the industrial dyes are considered a friendly method for the environment and have shown many advantages when compared with chemical and physical methods [21]. Therefore, much research has been conducted to collect the bacterial isolates which decolorize the dyes from different environmental samples [22,23]. When [18], the spread plate method was used to obtain 14 bacterial isolates, results revelaed that the AMS-XIII isolate exhibited maximum decolorization with 96.81% [24]. Results were qualified to isolate twenty-two bacterial isolates from soil samples contaminated with Azo dyes, where isolate SKB16 showed the highest ability to decolorize and degrade two types of Azo dyes reaching up to more than 91% [25]. Also, the results from the second step of screening revealed that the best dissociation of the Congo red dye was obtained by isolates WR7, WR18, and WR30 respectively, where the percentage of decolorization values reached 88.6%, 83.9% and 92.8% respectively, as compared with the results of other studied isolates, Table 3. isolates to decolorize and degrade six different types of Azo dyes., The results obtained from this study indicated that all studied isolates were able to decolorize the six Azo dyes with high rates of decolorization that were higher than 98% [26].

Optimum conditions to decolorize Congo red dye:
The results of studying the effect of the concentration of Congo red dye on the efficiency of decolorization indicate that the studied isolates gave a clear variation in the decolorization susceptibility to all studied concentrations, where isolate WR30 showed the best decolorization results at all the studied concentrations, where the decolorization reached 96.8% and 92.8% at the concentrations 25 and 50 ppm respectively. While the isolates WR7 and WR18 gave 94.3% and 92.6% at 25 ppm respectively, and 88.6% and 83.9% at 50 ppm. The results also showed that the rate of decolorization decreases gradually with increasing the concentration of the dye to a concentration of 100 ppm and then the stability occurs at the rate of decolorization for other concentrations, Figure 1. The results in Figure 2 indicate that the decolorization rate of Congo red dye was low on the first and second day of incubation by all studied isolates. The best decolorization rate was obtained on the third day of incubation for all isolates, Whereas incubation. Whereas, upon the other incubation periods (after 4 and 5 days), the rate of decolorization was similar to the third day in all of the studied isolates. Furthermore, the WR30 isolate gave the highest decolorization rate that reached 92.9% as compared to isolates WR7 and WR18, which gave 88.7% and 83.9% respectively. The rate of decolorization and degradation of dyes can be influenced by many factors such as temperature, pH, time and dye concentration. The type of bacteria also influences the rate of degradation. Therefore many previous studies have shown that the rate of decolorization decreased gradually by increasing the dye concentration, this can either be because high concentrations of dyes lead to toxicity in living cells, or that the high concentrations of dyes require the use of higher biomass as an inoculum for growth medium [27,28]. Sciences were studied [29] the effect of dye concentration on the rate of decolorization using isolate Escherichia coli NG188, the results showed that the rate of decolorization of the studied dye decreased with increasing concentration and that the highest decolorization was 59%, while the lowest decolorization was 35% when using concentrations 50 and 1000 ppm respectively [29]. In a previous study, the bacterial isolate Acinetobacter baumannii YNWH 226 was used to decolorize and degrade the Congo red dye using a liquid growth medium containing three different concentrations of Congo red dye (100, 200, and 500 ppm), where the bacterial isolate showed a good ability to decolorize the two concentrations (100 and 200 ppm) at a high decolorization rate reached to 98.62% and 96.31% respectively after two days of incubation, while the third concentration (500 ppm) was decolorized with 80.73% only after the same period [30].

Identification of best isolates:
According to results obtained from macroscopic characteristics, biochemical tests and microscopic characteristics, for the isolates WR7, WR18, and WR30, the three Isolates with the highest efficiency were identified as Bacillus subtilis, Pseudomonas aeruginosa, and Bacillus cereus respectively.

Conclusions:
The results of the current study indicate that wastewater samples are a good source in obtaining local bacterial isolates capable of decolorizing the Congo red dye. These isolates showed a discrepancy in their ability to consume the Congo red dye. This may be due to the toxic effect of this dye.