Abstract

Acid orange 7 (AO7) is an azo dye widely used in the dyeing and direct printing industry. AO7 is an environmental pollutant because the cleavage of azo bonds produces aromatic amines, which are considered mutagenic and carcinogenic. Microbial degradation is one of the most effective methods to remove environmental pollutants. A bacterium strain L-15 was isolated from the wastewater treatment system of a dye manufacturer. This strain is capable of decolorizing AO7. The strain was identified as Flavobacterium mizutaii based on its morphological, physiological and biochemical characteristics, and the sequence of 16S rDNA. The AO7-degrading characteristics and the effects of culture condition on the degrading efficiency of the strain were investigated by shake-flask culturing. The optimal degradation condition of L-15 was 30 °C and pH 7.0. After culturing at 30 °C for 3 days with the initial AO7 concentration of 20 mg/L, the degradation rate of AO7 was 60.45%. The optimal salt concentration was lower than 2%.

INTRODUCTION

Dyes are substances capable of colouring fabrics, as they cannot be removed by rubbing or washing. Azo dyes are the largest and the most versatile class of synthetic dyes used in textile, food, printing, and cosmetic industries. About 60–70% of dyes used in the textile industry are azo dyes (Tony et al. 2009). Azo dyes are characterized by nitrogen to nitrogen double bonds (-N=N-). The colour of azo dyes is determined by the azo bonds and their associated chromophores and auxochromes. Acid orange 7 (AO7) is a typical azo dye with orange-yellow colour and widely used in dyeing and direct printing of wool, silk and nylon fabric, and colorizing of leather and paper. The chemical structure of AO7 is shown in Figure 1. AO7 causes severe water pollution such as colour increasing and quality deterioration if direct discharged without treatment. The anaerobic degradation of AO7 by microorganisms in the water body produces aromatic amines, which cause carcinogenic and mutagenic harm to humans and organisms. Therefore, research on the biodegradation of AO7 is very necessary and important.

Figure 1

Chemical structure of Acid Orange 7.

Figure 1

Chemical structure of Acid Orange 7.

Studies indicated that approximately 10% of dyes produced per year enter the environment during manufacturing and processing operations (Sandhya et al. 2005; Forgacs et al. 2004). A relatively high loss of azo dyes in aqueous effluents during manufacture and textile colorization processes as well as insufficient treatment of the wastewaters from dyestuff industries leads to environment contamination (Pearce et al. 2003; Deng et al. 2008; Hao et al. 2000). Therefore, various treatment methods including physical, chemical, photocatalysis and biological processes have been studied for removal of azo dyes from effluents before being discharged into the environment (García-Montaño et al. 2006; Mezohegyi et al. 2012; Wang et al. 2019; Zhang et al. 2019). Comparatively, biological methods have been considered as priority choice due to them being cost-effective, environmental-friendly, and efficient in completely mineralizing organic pollutants (Tan et al. 2013; Huang et al. 2015).

Researches on microorganisms decolorizing azo dyes are mainly focused on the use of bacteria and fungi (Miranda et al. 2013). Many fungal strains capable of decolorizing azo dyes through bio-absorption and bio-degradation have been reported (Kalmiş et al. 2008; Gou et al. 2009; Qu et al. 2010; Arora et al. 2011; Liu et al. 2011). However, little literature on AO7 biodegradation has been found (Mutafov et al. 2007; Fernando et al. 2012). In the present study, we describe a newly isolated bacterium strain Flavobacterium mizutaii L-15 capable of decolorizing azo dye AO7. The environmental factors affecting AO7 decolorizing by this strain were also examined. The work in this study can enrich the azo-dye bio-decolorizing literature and provide a useful functional strain for the remediation of AO7-contaminated environments or bioaugmentation of AO7-containing wastewater treatment.

METHODS

Chemicals and media

AO7 selected as a model azo dye in this study was purchased from Sigma Aldrich. All chemicals used in this study were analytical grade. Minimal salt medium (MSM), prepared at pH 7.0, contained (g/L) NaCl 1.0, NH4NO3 1.0, K2HPO4 1.5, KH2PO4 0.5, MgSO4•7H2O 0.2. Luria-Bertani (LB) medium contained (g/L) tryptone 10.0, yeast extract 5.0 and NaCl 5.0.

Enrichment and isolation of AO7-decolorizing bacteria

To isolate AO7-decolorizing bacteria, 5 mL activated sludge sampled from a dye wastewater treatment system was added into 100 mL MSM supplemented with 50 mg/L AO7 and 500 mg/L glucose as carbon and energy source. The mixture was incubated on a 180 rpm rotary shaker at 30 °C for 3 days, then 5 mL cultured mixture was transferred to 100 mL fresh medium for subculturing. After three subculturing processes, the isolation was done by conventional plating method. The final enrichment culture was plated on LB agar plates. Colonies were picked up and purified using streaking method. Degradation capacity of the isolates was estimated by observation of the absorbance value at 484 nm of the liquid medium.

Identification of the strain L-15

The physiological and biochemical identification of L-15 was carried out according to Kang et al. (2013). Identification of the isolated bacterium strain L-15 was based on 16S rDNA sequence and phylogenetic analysis. The 16S rRNA gene of the strain L-15 was amplified with the bacteria universal primers 27F and 1525R and then sequenced. Polymerase chain reaction primer synthesis and DNA sequencing (Applied Biosystems) were conducted at Sangon Biotech Co. Ltd. (Shanghai, China). The resulting nucleotide sequences were compared to those in GenBank using a BLAST search. A phylogenetic tree was constructed using MEGA version 6.0 software with the neighbour-joining method, based on 16S rDNA sequences with high sequence identity in the alignments results of NCBI blast. The dataset was bootstrapped 1,000 times.

Construction of the AO7-decolorizing reaction system

One loop of cells was inoculated into liquid LB medium and incubated overnight on a 180 rpm rotary shaker at 30 °C. Cells were harvested by centrifugation after overnight pre-culturing, washed twice and resuspended in LB medium to OD600 = 1.0. The AO7 stock solution was added into the cell suspension to reach the final concentration of 20 mg/L to build the basic reaction system. Unless otherwise indicated, all of the decolorizing reaction systems in this work were built based on this basic system.

Effects of other environmental factors on AO7 degradation

To examine the effect of incubation temperature on AO7 degradation, decolorizing mixtures were incubated at gradient temperature of 18 °C, 28 °C, 30 °C and 37 °C, respectively. To observe the effect of initial pH on AO7 degradation, the LB medium initial pH value was adjusted to 3.0, 4.0, 5.0, 6.0, 7.0, 8.0 and 9.0, respectively, with either HCl or NaOH solution. Sodium chloride was added into the decolorizing mixtures to make the final concentration of 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5 and 4.0%, respectively, to determine the effect of salinity on AO7 degradation. After incubation under certain conditions for 24 h, the remaining AO7 of all samples was detected in 484 nm.

RESULTS AND DISCUSSION

Isolation and characterization of the strain L-15

A bacterium strain, named as L-15, capable of degrading over 60% of total AO7 at a concentration of 50 mg/L in LB medium in 24 hours was isolated. After 2 days of incubation on the LB agar plate, a weak-yellow colony was formed (Figure 2(a)). Under the microscope, the bacterium cells were short rod-shaped and Gram-stain negative (Figure 2(b)). In physiological and biochemical tests for strain L-15, the strain tested positive for aesculin hydrolase and β-galactosidase, and glucose, arabinose, mannose, N-acetyl-glucosamine and maltose can be assimilated as carbon sources (Table 1). Carbon sources aesculin, D-xylose, galactose, raffinose, fructose, rhamnose and amygdalin can be used to produce acid (Table 2) by L-15. Also, several carbon sources including D-arabinose, L-arabinose, adonol, D-turanose, D-laisuose and L-fucose were detected with a weakly positive reaction in the acid-producing tests (Table 2). The strain tested negative for other carbon sources in this test. The 16S rDNA sequence of L-15 was 99.5% similar to Flavobacterium mizutaii (Figure 3). Finally, strain L-15 was identified as Flavobacterium mizutaii based on physiological and biochemical characteristics and 16S rDNA sequence.

Table 1

Physiological and biochemical characteristics of strain orange L-15 – enzyme activity, carbon source assimilation

Test itemTest result
NO3 (nitrate reduction) − 
TRP (indole production) − 
GLU (d-glucose fermentation) − 
ADH (arginine dihydrolase) − 
URE (urease) − 
ESC (aesculin hydrolase) 
GEL (protease) − 
PNPG (β-galactosidase) 
GLU (assimilation of glucose) 
ARA (assimilated arabinose) 
MNE (assimilated mannose) 
MAN (assimilation of mannitol) − 
NAG (assimilation of N-acetyl-glucosamine) 
MAL (assimilation of maltose) 
GNT (assimilation gluconate) − 
CAP (assimilation of niacin) − 
ADI (assimilation of adipic acid) − 
MLT (assimilation of malic acid) − 
CIT (assimilation of citric acid) − 
PAC (assimilation of phenylacetic acid) − 
Test itemTest result
NO3 (nitrate reduction) − 
TRP (indole production) − 
GLU (d-glucose fermentation) − 
ADH (arginine dihydrolase) − 
URE (urease) − 
ESC (aesculin hydrolase) 
GEL (protease) − 
PNPG (β-galactosidase) 
GLU (assimilation of glucose) 
ARA (assimilated arabinose) 
MNE (assimilated mannose) 
MAN (assimilation of mannitol) − 
NAG (assimilation of N-acetyl-glucosamine) 
MAL (assimilation of maltose) 
GNT (assimilation gluconate) − 
CAP (assimilation of niacin) − 
ADI (assimilation of adipic acid) − 
MLT (assimilation of malic acid) − 
CIT (assimilation of citric acid) − 
PAC (assimilation of phenylacetic acid) − 

+: positive reaction; −: negative reaction.

Table 2

Physiological and biochemical characteristics of strain orange L-15 – using carbon source to produce acid

Reagent strip corresponding tubeSubstrate detection resultReagent strip corresponding tubeSubstrate detection result
0 Control − 25 Aesculin 
1 Glycerol − 26 Salicin − 
2 Erythritol − 27 Cellobiose − 
3 D-arabinose 28 Maltose − 
4 L-arabinose 29 Lactose − 
5 Ribose − 30 Honey disaccharide − 
6 D-xylose 31 Sucrose − 
7 L-xylose − 32 Trehalose − 
8 Adonol 33 Inulin − 
9 β-Methyl-D-xylose − 34 Pine syrup − 
10 Galactose 35 Raffinose 
11 Glucose − 36 Starch − 
12 Fructose 37 Glycogen − 
13 Mannose − 38 Xylitol − 
14 Sorbose − 39 Geraniol − 
15 Rhamnose 40 D- turanose 
16 Guardianol − 41 D-laisuose 
17 Myo-inositol − 42 D-tagatose − 
18 Mannitol − 43 D-fucose − 
19 Sorbitol − 44 L-fucose 
20 α-Methyl-D-mannose − 45 D-arabitol − 
21 α-Methyl-D-glucoside − 46 L-arabitol − 
22 N-acetyl-glucosamine − 47 Gluconate − 
23 Amygdalin 48 2-keto-gluconate − 
24 Abutin − 49 5-keto-gluconate − 
Reagent strip corresponding tubeSubstrate detection resultReagent strip corresponding tubeSubstrate detection result
0 Control − 25 Aesculin 
1 Glycerol − 26 Salicin − 
2 Erythritol − 27 Cellobiose − 
3 D-arabinose 28 Maltose − 
4 L-arabinose 29 Lactose − 
5 Ribose − 30 Honey disaccharide − 
6 D-xylose 31 Sucrose − 
7 L-xylose − 32 Trehalose − 
8 Adonol 33 Inulin − 
9 β-Methyl-D-xylose − 34 Pine syrup − 
10 Galactose 35 Raffinose 
11 Glucose − 36 Starch − 
12 Fructose 37 Glycogen − 
13 Mannose − 38 Xylitol − 
14 Sorbose − 39 Geraniol − 
15 Rhamnose 40 D- turanose 
16 Guardianol − 41 D-laisuose 
17 Myo-inositol − 42 D-tagatose − 
18 Mannitol − 43 D-fucose − 
19 Sorbitol − 44 L-fucose 
20 α-Methyl-D-mannose − 45 D-arabitol − 
21 α-Methyl-D-glucoside − 46 L-arabitol − 
22 N-acetyl-glucosamine − 47 Gluconate − 
23 Amygdalin 48 2-keto-gluconate − 
24 Abutin − 49 5-keto-gluconate − 

+: positive reaction; −: negative reaction; w: weak positive reaction.

Figure 2

Colony morphology and cell morphology of strain L-15. (a) Single colony morphology of strain L-15 on LB agar plate after 2 days of incubation at 30 °C; (b) cell morphology of L-15 under the microscope after Gram-staining.

Figure 2

Colony morphology and cell morphology of strain L-15. (a) Single colony morphology of strain L-15 on LB agar plate after 2 days of incubation at 30 °C; (b) cell morphology of L-15 under the microscope after Gram-staining.

Figure 3

The phylogenetic tree of strain L-15 by the neighbour-joining approach. Bootstrap values obtained with 1,000 repetitions are indicated as percentages at all branches. Scale bar represents 0.02 substitution/site. The accession numbers are in parentheses.

Figure 3

The phylogenetic tree of strain L-15 by the neighbour-joining approach. Bootstrap values obtained with 1,000 repetitions are indicated as percentages at all branches. Scale bar represents 0.02 substitution/site. The accession numbers are in parentheses.

Effect of temperature on degradation ability

Temperature is an important factor affecting microbial growth and degradation. To investigate the effect of temperature on degradation, the reaction mixtures were incubated at different temperature. After 24 h of incubation, the remaining AO7 in all samples was detected. The results in this test indicate that low temperature and high temperature can obviously inhibit the degradation efficiency. As shown in Figure 4, the degradation rates of AO7 were very low at both low temperature of 18 °C and high temperature of 37 °C. However, significantly higher degradation rates were obtained when the reaction temperature is around 28–30 °C. Therefore, we inferred the suitable temperature for AO7 degradation by strain L-15 is around 28–30 °C. In this temperature range, more than 50% of AO7 was degraded. The maximum degradation rate (65.21%) was obtained at 30 °C (Figure 4). The optimum temperature for stain L-15 to degrade AO7 was at 30 °C.

Figure 4

Effect of temperature on degradation ability of strain L-15.

Figure 4

Effect of temperature on degradation ability of strain L-15.

The effect of initial pH on AO7 degradation

Initial pH is a very important environmental factor that remarkably affects pollutant biodegradation. Therefore, the degradation rates of AO7 under different initial pH were also investigated in this work. As shown in Figure 5, the degradation rates of AO7 by strain L-15 were higher than 30% at the initial pH of 7–9. We got the highest degradation rate when the initial pH was set as 8 in this test. The degradation rate reached 60.50% at this condition. Based on this result, we estimate the optimum initial pH was at 8 under the experimental conditions in this work. In addition, it should be noted that the degradation rates of AO7 were only 7.80% and 10.20%, respectively, when the initial pH was at 5 and 6 (Figure 5). These results indicated that an acid environment was an environmental stress for strain L-15 and obviously inhibited the AO7 degradation. Azo-dye decolorization by the bacterium strain was mainly catalysed by the azoreductase enzyme. Azoreductase was reported to be stable and, high efficiency of decolorization is often at a neutral or slightly alkaline pH value. Strongly acid and strongly alkaline conditions can rapidly decrease the colour removal of azo dyes (Pearce et al. 2003). Obviously, the decolorizing response of strain L-15 to different initial pH values was consistent with the literature.

Figure 5

Effect of initial pH on AO7 degradation.

Figure 5

Effect of initial pH on AO7 degradation.

Effect of initial dye concentration on strain L-15 AO7 degradation and strain L-15 growth

The initial concentration of the dye also affected the degradation rate of the AO7. To investigate the suitable initial dye concentration for degradation, several reaction systems with initial dye concentration ranging from 10 mg/L to 100 mg/L were constructed and incubated for 24 hours at 30 °C. The tests results showed that the highest degradation rate of AO7 by stain L-15 was obtained when the initial dye concentration was 20 mg/L (Figure 6). The degradation rates were significantly lower with low or high initial dye concentrations (Figure 6). It is noteworthy that the L-15 cell concentration (OD600) showed a downward trend with the increase of initial dye concentration, which means that the presence of high concentration dyes inhibited the growth and proliferation of stain L-15 (Figure 7). It is possible that high dye concentration is highly toxic to strain L-15, resulting in the reduction of degradation efficiency.

Figure 6

Effect of initial dye concentration on degradation of AO7 by stain L-15.

Figure 6

Effect of initial dye concentration on degradation of AO7 by stain L-15.

Figure 7

Effect of initial dye concentration on the growth of stain L-15.

Figure 7

Effect of initial dye concentration on the growth of stain L-15.

Effect of salt concentration on degradation efficiency of L-15

The effects of salt concentration on the growth and the degradation efficiency of L-15 were also investigated in this work. Reaction mixtures with different salt concentration were set as described in the ‘Methods’ section. Azo dye degradation rates and cell concentrations were detected after 24 hours' incubation at 30 °C. As shown in Figure 8, increasing of the salt concentration significantly inhibited the survival and growth of strain L-15. However, the degradation efficiency of L-15 was not remarkably affected by the increasing salt concentration: there was only slightly decrease of degradation rates when the salt concentration was higher than 2% (Figure 8).

Figure 8

Effect of salt concentration on degradation efficiency of L-15. The degradation rates are the means of results for triplicated samples.

Figure 8

Effect of salt concentration on degradation efficiency of L-15. The degradation rates are the means of results for triplicated samples.

CONCLUSIONS

In this study, we obtained a high-efficiency AO7-decolorizing bacterium strain, L-15. According to physiological and biochemical characteristics and 16S rDNA sequence, strain L-15 was identified as Flavobacterium mizutaii. The environmental condition tests indicated that the reaction temperature can significantly affect the decolorization and 30 °C was the optimum temperature for AO7 decolorizing. Strong acid and strong alkaline conditions can rapidly inhibit AO7 decolorizing, and the optimum initial pH was 8.0. Suitable initial AO7 concentration for decolorization was at 20 mg/L. High concentration of AO7 showed strong toxicity to strain L-15 and can remarkably inhibit decolorization. High salt concentration can slightly influence AO7 decolorization, but it can significantly affect the survival of strain L-15.

FUNDING

This work was supported by Key Laboratory for Water Pollution Control and Environmental Safety of Zhejiang Province, China.

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