In this work, we evaluate the ability of a novel isolated bacteria identified as Bacillus thuringiensis RI16 (KM111604) to decolorize methyl red. Results showed its efficiency for the treatment of azo-dye-contaminated wastewaters under static conditions at pH values ranging from 7 to 9. The isolated bacteria could tolerate higher doses of dyes as it was able to decolorize up to 1000 mg/L. For better methyl red decolorization, we evaluate the potential use of Bacillus subtilis SPB1-derived lipopeptide biosurfactant. The study showed its ability to accelerate the decolorization rate and slightly maximized the decolorization efficiency at an optimal concentration of 0.025%. Compared with synthetic surfactants, the bacterial surfactant was more efficient as we observe a drastic decrease and an inhibition of methyl red decolorization by cetyl trimethylammonium bromide (CTAB) and sodium dodecyl sulfate (SDS) additions. Also, studies ensured that methyl red removal by this strain could be due to extracellular enzymatic activities. SPB1 biosurfactant could improve enzyme activities and/or enzyme synthesis and diffusion.
B. thuringiensis RI16 for methyl red biodecolorization under static conditions and neutral and alkaline pH values.
B. subtilis SPB1-derived lipopeptide-enhanced methyl red decolorization.
Inhibition of methyl red decolorization by CTAB and SDS additions.
Tween 80 enhanced methyl red decolorization.
Improvement of color removal-related enzymes’ activities and/or enzymes’ synthesis and diffusion by SPB1 biosurfactant.
A wide variety of compounds can be found in textile effluent. In the Color Index, there are more than 8000 chemical products associated with the dyeing process ranging from inorganic compounds and elements to polymers and organic products (Priyanka et al. 2022). The most abundant are non-biodegradable synthetic dyes, pigments, hydrocarbons and heavy metals (Ardila-Leal et al. 2021; Al-Tohamy et al. 2022). Synthetic dyes include several structural varieties of dyes, such as acidic, reactive, basic, disperse, azo, diazo, anthraquinone-based and metal-complex dyes (Ardila-Leal et al. 2021; Al-Tohamy et al. 2022). These chemicals are used for desizing, scouring, bleaching, dyeing, printing and finishing (Al-Tohamy et al. 2022). Also, in textile industries, during the dyeing process, up to 15% of the total textile dye remains unreacted and is directly lost in the effluents generating huge amounts of wastewaters (Ardila-Leal et al. 2021; Al-Tohamy et al. 2022).
Azo dyes are the largest synthetic chemicals extensively used in textile, food, plastic, printing, leather, cosmetics and pharmaceutical industries (Benkhaya et al. 2020). They are highly used in the industry for dyeing fabrics (Ngo & Tischler 2022). Generally, they are characterized by a nitrogen-to-nitrogen double bond –N = N– (Benkhaya et al. 2020). As they have poor exhaustion properties, nearly 10–15% of the dyestuff used remains unbound to the fiber, synthetic dyes are released into the environment leading to ecotoxicity risk, direct and indirect toxic effects and potential danger of bioaccumulation (Lellis et al. 2019; Ngo & Tischler 2022). First, the presence of dyes in water is highly visible and affects its transparency and esthetics impeding light penetration and reducing dissolved oxygen concentration (Lellis et al. 2019; Al-Tohamy et al. 2022; Ngo & Tischler 2022). Second, synthetic dyes can lead to the formation of tumors, cancers and allergies besides growth inhibition of bacteria, protozoan, algae, plants and different animals including human beings (Ardila-Leal et al. 2021). Having the ability to cause great damage to the environment and living organisms, an urgent need for their detoxification and/or elimination from the environment was developed recently (Ardila-Leal et al. 2021).
Numerous studies were developed including physical methods such as adsorption, ion exchange and membrane filtration and chemical methods such as coagulation-flocculation, electrochemical and advanced oxidation processes are used to treat dye-containing wastewater (Al-Tohamy et al. 2022). However, they are quite expensive and unable to completely remove the recalcitrant azo dyes and/or their organic metabolites (Al-Tohamy et al. 2022). Also, they generate significant amounts of sludge and toxic byproducts and metabolites during the treatment process that may cause secondary pollution problems and involve complicated procedures for their elimination and discharge (Al-Tohamy et al. 2022). So, biological treatment methods using microorganisms appear to be the best alternative to physicochemical methods since they are eco-friendly and cost-competitive (Lellis et al. 2019; Ngo & Tischler 2022). They involve the use of bacteria or fungi capable of dye decolorization either in pure culture or in consortia (Lellis et al. 2019; Abd El-Rahim et al. 2021). Recent studies reported the use of pure bacterial culture (Kalme et al. 2007; Dawkar et al. 2008; Ayed et al. 2010, 2011; Cai et al. 2012; Garg et al. 2012; Ng et al. 2014; Thakur et al. 2014; Nair et al. 2017; Chittal et al. 2019; Khairun et al. 2019; Al-Tohamy et al. 2020), mixed bacterial culture (Fang et al. 2004; Moosvi et al. 2005; Joshi et al. 2008; Lalnunhlimi & Krishnaswamy 2016; Kamal et al. 2022), pure fungi (Chakraborty et al. 2013; Hadibarata et al. 2013; Zuleta-Correa et al. 2016), fungal consortium (Almeida & Corso 2019), yeast (Jadhav et al. 2007; Jafari et al. 2014) and algae (El-Sheekh et al. 2009; Ishchi & Sibi 2020) for dyes decolorization.
The ability of microorganisms to decolorize and metabolize azo dyes has long been known, and the use of bioremediation-based technologies for treating textile wastewater has attracted interest (Ngo & Tischler 2022). Being cost-effective, environmentally friendly and producing low sludge, biological approaches are very advantageous (Ngo & Tischler 2022). Methyl red (2-(N,N-dimethyl-4-aminophenyl)azobenzenecarboxylic acid), also called C.I. Acid Red 2, is one of the most frequently used secondary diazo dyes well known by their ecotoxicity (Benkhaya et al. 2020; Ngo & Tischler 2022). Numerous microbial strains were reported to have the ability to degrade azo dyes especially methyl red, including bacteria (Ayed et al. 2011; Ng et al. 2014; Abd El-Rahim et al. 2021; Ngo & Tischler 2022), fungi (Zuleta-Correa et al. 2016; Abd El-Rahim et al. 2021), cyanobacteria (Dellamatrice et al. 2017), yeast (Al-Tohamy et al. 2020) and algae (El-Sheekh et al. 2009; Ishchi & Sibi 2020). The increasing interest in bacteria is a result of their potential to degrade and mineralize most textile dyes effectively; bacteria also have a rapid growth rate and are straightforward to culture (Ayed et al. 2011). In addition, biological treatment methods involving the use of bacteria belonging to Bacillus strains were highly discussed to treat azo dyes (Thakur et al. 2014; Nair et al. 2017; Khairun et al. 2019; Ngo & Tischler 2022; Pinheiro et al. 2022). In this aim, this study will discuss the ability of a Bacillus thuringiensis RI16 (KM111604) strain to decolorize the azo-dye methyl red. In this regard, the present study aims to investigate, primarily, the potential of a hydrocarbon-degrading bacterium B. thuringiensis RI16 (KM111604) to decolorize the azo-dye methyl red. Subsequently, we studied the efficiency of Bacillus subtilis SPB1 lipopeptide biosurfactant (BioS) in the improvement of the dye bioremoval process.
MATERIALS AND METHODS
A B. thuringiensis RI16 (KM111604) strain was used for the study of the biodecolorization of the azo-dye methyl red. It was isolated and identified in our previous study for its ability to degrade diesel oil (Mnif et al. 2015a). A wild-type strain of B. subtilis SPB1 (HQ392822) was used to produce BioS (Ghribi et al. 2012). As presented in our previous work, the lipopeptide BioS was composed mainly of different Surfactin isoforms with molecular weights of 1007, 1021 and 1035 Da, Iturin isoforms with molecular weights of 1028, 1042 and 1056 Da and Fengycin isoforms with molecular weights of 1432 and 1446 Da along with two new clusters of lipopeptide isoforms having molecular weights of 1410 and 1424 Da and 973 and 987 Da (Mnif et al. 2016). Mass spectroscopic analysis served to identify the lipopeptides BioS after acid precipitation and purification (Mnif et al. 2016).
Culture conditions and preparation of the purified lipopeptide preparation
Culture conditions for BioS production were carried out as described by Mnif et al. (2021a, 2021b, 2021c). They were incubated for 2 days at 37 °C and 150 rpm. At the end of the cultivation, the culture was centrifuged at 10,000 rpm and 4 °C for 20 min to remove bacterial cells. Hence, BioS were extracted from the supernatant-free cells as described in the next section. Lipopeptide BioS were partially purified during three consecutive cycles of acid precipitation–dissolution as described in our previous work (Mnif et al. 2021a, 2021b, 2021c). In fact, each time, the pellet formed by acid precipitation was suspended in alkaline water at a concentration of 4 mg/mL and the pH was readjusted to 8 with NaOH 1 N. The supernatant was collected by centrifugation at 10,000 rpm and 4 °C for 20 min followed by second acid precipitation. The final pellet formed was washed three times with acid water (pH = 2), dissolved in distilled water at a concentration of 10 mg/mL, pH adjusted to 8 with NaOH 1 N and lyophilized. This serves as crude lipopeptide preparation to enhance the decolorization efficiency (Mnif et al. 2015a, 2015b, 2015c).
Growth medium and decolorization of dye solution
The decolorization of methyl red was studied at different concentrations of the mentioned dyes (50, 200, 500, 750 and 1000 mg/L) in 250-mL Erlenmeyer flasks containing 50-mL mineral salt medium (MSM). The pH of the medium was adjusted to 7 as described by Ayed et al. (2011). The inoculum was prepared in an LB medium. B. thuringiensis RI16 strain was streaked on a nutrient agar slant and incubated overnight at 37 °C. After that, one loop of cells was dispensed in 50-mL LB medium (250-mL Erlenmeyer flasks) and incubated in a shaker at 150 rpm and 37 °C ( ± 0.5) overnight. Flasks were inoculated by the newly isolated strain at a final optical density of 600 nm corresponding to 0.2. The culture was incubated under shaking conditions (150 rpm) and at static conditions at 37 °C for 24 h. Color removal was quantified after 4, 8 and 24 h of incubation. The resultant culture was centrifuged for 10 min at 10,000 rpm and the cell pellet was washed twice in sterile saline solution (9‰) and re-suspended in the liquid culture medium (Mnif et al. 2015a).
Effect of SPB1 biosurfactant addition on methyl red biodecolorization
In order to study the effect of SPB1 lipopeptide addition on methyl red removal efficiency by the respective strain, various concentrations (0.025, 0.05, 0.075 and 0.1%) were added in the nutrient growth before incubation. The effect of SPB1 lipopeptide was compared to those of three different chemical surfactants, the anionic surfactant sodium dodecyl sulfate (SDS) (8.2 mM at 25 °C = 0.2364%) (Mukerjee & Mysels 1971); the cationic surfactant cetyl trimethylammonium bromide (CTAB) (0.89–0.93 mM at 25 °C = 0.0324–0.0338%) (Yaofeng 2009) and the nonionic surfactant polyoxyethylene-sorbitan monooleate; polysorbate 80 (Tween 80) (0.012 Mm at 25 °C = 0.00157%) (Chou et al. 2005).
Mechanism of microbial decolorization
In order to elucidate the mechanism of microbial decolorization of methyl red, decolorization tests were realized using living cells, dead cells and two enzymatic preparations of the respective decolorizing strain corresponding to extracellular enzymes in the supernatant and the intracellular enzymes present in the sonication product. Similar experiments were conducted by Jadhav et al. (2011), and Mohanty & Kumar (2021). In fact, B. thuringiensis RI16 was grown in LB medium overnight at 37 °C and 150 rpm. The supernatant was filter-sterilized using 0.2-μm membranes to remove residual cells and serve as extracellular enzyme preparation to measure enzymatic dye biodegradation. The resulting biomass was harvested by centrifugation (10,000 rpm, 20 min) and suspended in 50-mM potassium phosphate buffer (pH 7.4). The first prize of the biomass suspension was sonicated (30 s, 60 amplitude, 10 strokes) at 4 °C (Jadhav et al. 2011). After centrifugation, this extract was used as an intracellular enzyme source to quantify intracellular enzymatic dye biodegradation. A second prize of the biomass suspension was autoclaved and the resultant cellular debris was used as biosorbent material to quantify methyl red removal. A third part serves as active cells to quantify color removal resulting from decolorization and biodegradation. In total, four tests were realized in parallel; one is using living beings’ cells, one using dead cells, one using bacterial supernatant and one using intracellular bacterial content. They were carried out in duplicate with and without the addition of SPB1 lipopeptide BioS at its optimal concentration determined in the result part. Biodecolorization efficiencies were measured for each trial.
RESULTS AND DISCUSSION
Methyl red decolorization under different culture conditions
Effect of initial dye concentration
Effect of static and shaking conditions
Results presented in Figures 1 and 2 indicated that the strain could decolorize methyl red under both static and shaking conditions, but the decolorization under static conditions appeared to be significantly higher. In fact, as shown in Figures 1 and 2, the decolorization of methyl red (100–1000 mg/L) under shaking condition by B. thuringiensis RI12 cannot exceed 76% with very weak biodecolorization at high dye concentration. However, under static conditions, a complete biodecolorization was obtained for doses between 50 and 200 mg/L with 82.41% when working in the presence of 500 mg/L. Results are similar to many literature studies reporting high microbial decolorization of azo dyes under static conditions (Fang et al. 2004; Telke et al. 2010; Garg et al. 2012). Also, Cai et al. (2012) reported efficient azo-dye biodegradation under static conditions. However, several studies reported the efficient biodecolorization of methyl red under shaking conditions (Ayed et al. 2011).
Effect of pH on methyl red decolorization
Effect of SPB1 lipopeptide biosurfactant addition on dye biodecolorization
Mechanism of bacterial decolorization of methyl red
Decolorization of the dye solution may take place in two ways: either adsorption on the microbial biomass or enzymatic biotreatment of the dyes (Pinheiro et al. 2022). When conducting biodecolorization using different preparation, efficient decolorization was observed using living cells of B. thuringiensis RI12 and extracellular supernatant of the strain (data not presented). However, no decolorization was observed by the application of the dead cells and intracellular enzymatic preparation. Therefore, these results indicate that methyl red removal by this strain can be due to extracellular enzymatic activities. Other studies reported the enzymatic biotreatment of certain azo dyes (Liu et al. 2020; Singh et al. 2020; Ardila-Leal et al. 2021; Ngo & Tischler 2022; Vishani & Shrivastav 2022). However, many reports described the adsorptive removal of azo-dye using bacteria, fungi and yeast (Almeida & Corso 2019; Öztürk et al. 2020; Ngo & Tischler 2022; Pinheiro et al. 2022). Studies with and without BioS addition were also conducted when treating methyl red with living cells and extracellular enzymatic preparation. Results indicated the enhancement of methyl red biodecolorization in both cases due to the addition of 0.025% BioS. Regarding the preliminary results showing the enhancement of methyl red biodecolorization by the addition of SPB1 bioemulsifier, we can propose an enhancement of bacterial growth and, therefore, methyl orange biodecolorization due to the fact that this BioS is biodegradable and could be used as a primary and more accessible carbon source to initiate microorganisms' growth (Bautista et al. 2009; Mnif et al. 2015b). Also, we can propose that the BioS improve enzyme production and, therefore, their biological activities. Similar results are described by Liu et al. (2017) and Oliva-Taravilla et al. (2020) and reporting the improvement of enzyme production by the addition of lipopeptides and rhamnolipids, respectively. Moreover, we can assume that the addition of SPB1 BioS may increase bacterial membrane permeability as reported in other studies (Vaidyanathan et al. 2014; Kaczorek et al. 2018) facilitating the enzymes diffusion. Regarding the enhancement of methyl red biodecolorization when using textracellular enzymatic preparation, we can assume that the bioemulsifier can activate decolorizing enzymes. In fact, recent studies reported the promotion of enzyme activities by chemical surfactants addition. As reported by Singh & Singh (2017), laccase activity was improved by Triton X-100 and Tween 20. Similarly, as described by Bento et al. (2020), surfactant-based ionic liquids improve the efficiency of laccase to degrade indigo carmine. Additionally, Britos et al. (2018) proved the improvement of laccase activity by ionic surfactant addition. Also, Tween 80 increased the cellobiohydrolase and endoglucanase activities from 63.0 and 76.7% to 123.2 and 103.1%, respectively (Ying et al. 2021), and Tween 20 enhanced cellulose activity (Wang et al. 2020). Similarly, Jadhav et al. (2011) showed the intensified effects of rhamnolipid on the enzymes involved in the dye degradation process and the stimulatory effect on lignin peroxidase (increased by 324.52%) and Veratryl alcohol oxidase (increased by 100%) were much eminent. Also, Liu et al. (2008) and Liang et al. (2010) reported 161.98 and 86% increase in the activity of lignin peroxidase in the presence of dirhamnolipid, respectively. Moreover, Zhou et al. (2011) and Liu et al. (2012) reported the improvement of laccase production and phenol biodegradation by rhamnolipid addition. Similar findings describing the improvement of diverse enzymes activities through dirhamnolipid addition were reported (Shao et al. 2017; Oliva-Taravilla et al. 2020; Gayathiri et al. 2022).
Also, we can assume a stabilizing effect on enzymes enhancing, therefore, their activities. Similarly, Ji et al. (2009) reported the stabilizing effect of laccase by Triton X-100 addition. Also, P. aeruginosa BSZ-07 rhamnolipid BioS was demonstrated to stimulate rice straw hydrolysis bioprocess by Trichoderma reesei ZM4-F3, a cellulose decomposing fungus, through increasing the activity of β-glucosidase and stabilizing Cel7A activity (Zhang et al. 2009).
B. thuringiensis RI16 showed excellent potential for bioremediation application as it can decolorize up to 1000 mg/L of methyl red dye at neutral and basic pH values. Results were better under static conditions and for lower dye concentrations. When elucidating the mechanism of dye removal, biodecolorization of methyl red can result from enzymatic activities. The addition of SPB1 BioS at 0.025% increased the biotreatment yields significantly. It seems to activate the extracellular enzymes involved in dye biodecolorization. However, chemical surfactants addition seems to have a deleterious effect on biodecolorization efficiency. The addition of SPB1 bioemulsifier reduced energy costs by reducing effective decolorization time, and BioS-stimulated bacterial decolorization method may provide a highly-efficient, inexpensive, and time-saving procedure in the treatment of textile effluents.
This work was supported by grants from the Tunisian Ministry of Higher Education, Scientific Research and Technology. It is a part of a research project on Biosurfactant Production, Characterization and Application.
Funding for this research work was granted by the Ministry of Higher Education and Research of Tunisia.
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All authors directly participated in the planning, execution, or analysis of this study. All authors read and approved the final manuscript.
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CONFLICT OF INTEREST
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