A Newly Isolated Stress-Resistant Bacterial Strain with Potential Use in Bioremediation of Dyeing Effluents

Abstract

The issue of water pollution is one of the hot topics of global concern, which requires us to efficiently treat pollutants in water, especially printing and dyeing sewage. There are varieties of dyestuffs and intermediates, which are complex and difficult to degrade, and they even contain heavy metals. In this study, a bacterial strain named Q3-6 with potential for sewage treatment was isolated and its physiological, biochemical, and genomic characteristics, and potential application value, were further investigated. The genome sequence confirmed that it belongs to Bacillus thuringiensis. Strain Q3-6 has a significant decolorization effect on the dyes. The decolorization rate for Brilliant blue G-250 (0.1 g/L) and Congo Red (0.1 g/L) can reach 93.9% and 91.9%, respectively. In addition, strain Q3-6 is resistant to many kinds of antibiotics and heavy metals. Further, it has strong heat resistance, and heating at 80 °C can promote the biomass of the strain. Genomic analysis revealed the presence of genes related to heat shock proteins (GroES, GrpE, DnaJ, GroEL, DnaK, ClpB, and ClpA) in strain Q3-6. These results suggest the strain’s exceptional resilience and adaptability to intricate environments with heavy metals, antibiotics, or high-temperature environments, suggesting its pivotal role in the bioremediation of complex contaminated effluents.

1. Introduction

The rapid development of various industries around the world has brought convenience to our lives, but many environmental pollution problems due to the excessive discharge of pollutants have become increasingly prominent. In the textile industry, large quantities of printing and dyeing sewage and its pollutants are often discharged from factories [1]. According to statistics, the annual discharge of dye sewage in the world reaches 30 to 50 billion cubic meters. This discharge accounts for about 80% of the total textile industry and is the main source of sewage in the textile industry [2]. The sewage generated by printing and dyeing processes frequently contains a high concentration of pollutants, characterized by a complex chemical makeup comprising dyes, heavy metals, and a diverse array of organic compounds [3].

Textile dyeing sludge (TDS) [4], a byproduct generated during sewage treatment processes in sewage treatment plants, contains a significant quantity of toxic and hazardous substances, notably heavy metals, particularly Zn (II). The pollutants in the printing and dyeing sewage may cause carcinogenic or mutagenic effects in humans, seriously endangering human life and health [5,6]. Complex and toxic dye sewage can also have an impact on the survival of microorganisms [7,8]. Wastewater treatment methods currently used in the industry vary depending on the components of the wastewater and other factors. However, whether physical or biochemical treatment is used, the process not only consumes a large amount of resources and energy but also generates a large number of additional pollutants [9]. In the traditional sewage treatment process, the drying and incineration of sludge were used, which resulted in the release of a large amount of carbon dioxide [10]; therefore, in the context of the dual-carbon policy, the issue of sewage treatment has become a global concern [11,12]. Subsequently, the efficient treatment of sewage is particularly important. Currently, a multitude of methods exist for treating dye sewage, encompassing physical adsorption, filtration, and various other techniques. Physical adsorption can adsorb the dye in water by a specific adsorbent such as activated carbon and nanoparticles [13,14], and photocatalysis can be combined with the adsorbent to generate OH free radicals to remove the dye in the adsorption process. However, this method has a single effect and it is not applicable to sewage with complex compositions [15]. Furthermore, membrane filtration remains an effective technique; however, it is prone to membrane fouling [16], where the accumulation of impurities on the membrane surface can swiftly lead to clogging [17]. This necessitates frequent membrane replacements, ultimately driving up operational costs [7]. To address this, recent advancements have focused on developing more durable and self-cleaning membrane materials [18], as well as optimizing cleaning protocols to prolong membrane life and reduce replacement frequency, thereby mitigating cost concerns. In general, the current situation of water pollution treatment is not optimistic due to the insufficient treatment capacity, a wide variety of pollutants, and high operation and maintenance costs.

Nowadays, more and more attempts are made to adopt bioremediation techniques for sewage treatment [19,20,21]. Bioremediation, which uses the adsorption or degrading ability of microorganisms, is an environmentally friendly, cost-effective, and more sustainable and efficient option to treat diverse contaminated media [22]. Various microorganisms can decolorize dyes by degradation, mineralization, adsorption, and so on [23]. Degradation of azo dyes by a fungus has been reported in a study by Spadaro et al. [24]. Some previous studies [25,26,27] have also shown that bacteria have a strong decolorizing effect on different dyes. But in practical applications, microorganisms may be easily affected by environmental factors. The process of bioremediation necessitates the presence of optimal levels of nutrients, contaminants, and metabolically active microbial populations, coupled with suitable environmental conditions conducive to microbial proliferation [23].

Therefore, there is still an urgent need to research and discover more microbial resources that have the potential for dye removal in order to cope with the impacts of the increasing volume of effluent from textile dyeing and printing, as well as meet the needs of treating dye sewage with complex composition. Thus, the aim of this study was to explore the removal potential of bacterial strains for dyeing and printing sewage and to provide an inexpensive and efficient treatment solution.

In this study, we isolated and purified the bacterial strains from sewage samples in order to obtain the potential strains with dye treatment ability, and their abilities to remove pollutants were verified. Strain Q3-6 was selected and its physiological, biochemical, molecular biology, and genomic characteristics were determined. The most important characteristic is that strain Q3-6 showed great ability to remove Brilliant blue G-250 and Congo Red, which provide a promising strain for the sewage treatment industry.

2. Materials and Methods

2.1. Sample Collection and Bacteria Isolation

The sewage samples were collected from Tower Lake, Linqing, China (115°42′46″ N, 36°51′46″ E). The sample was diluted and spread on the Beef extract Peptone Medium (BPM) agar. After 30 h of incubation at 37 °C, 32 different single colonies were selected, isolated, and purified. These strains were stored in 15% (v/v) glycerol with sterile 1% (w/v) saline at −80 °C.

In order to screen out the strains that have the potential to remove the pollutants in the sewage for the subsequent research, we evaluated the growth status and selected 12 well-grown strains from the initial 32 strains. Then, the 12 isolates were enriched and re-inoculated into the sewage samples, and the turbidity of the sewage samples with different strains was roughly observed and compared after 10 days. The decrease in turbidity of effluent samples inoculated with strain Q3-6 was more pronounced. So, strain Q3-6 showed the best sewage treatment potential and was selected for further study.

2.2. Bacterial Identification and Phylogenetic Analysis

The 16S rRNA gene sequences of these strains were amplified by polymerase chain reaction (PCR) using two universal primers, 27F and 1492R [28]: 27F (59-AGTTTTGATCMTGGCMTGG-CTCAG-39) and 1492R (59-TACGGGYTACCTTGTTTTTACGA-C-39). The amplified 16S rRNA gene sequences were sequenced at BGI Genomics Ltd. (Shenzhen, China) and aligned via the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 20 October 2023).

The purified PCR product of strain Q3-6 was further ligated into the pMD18-T vector and cloned according to the manufacturer’s instructions to obtain the complete 16S rRNA gene sequence [29] (GenBank accession number: PP567308). The 16S rRNA gene sequence was submitted to the NCBI database (https://www.ncbi.nlm.nih.gov/, accessed on 17 February 2024) and EzBioCloud database (http://www.ezbiocloud.net, accessed on 17 February 2024) to conduct the comparative analysis. The comparison of the 16S rRNA gene sequence of strain Q3-6 and its close relatives was performed using the MUSCLE service [30].

2.3. Genome Sequencing and Analysis

The genomic DNA of strain Q3-6 was extracted by using a genomic DNA extraction kit (Takara Biomedical Technology Co., Ltd., Beijing, China) according to the manufacturer’s recommendations. The genomes of strains Q3-6 were sequenced on the Illumina sequencer (X PLUS) at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). The raw data were filtered with fastp software v0.23.0 (HaploX Biotechnology Co., Ltd., Shenzhen, China) [31] and assembled by SOAPdenovo v2.04 (BGI Genomics Co., Ltd., Shenzhen, China) [32]. Quality assessment of the genomes was conducted using CheckM v1.1.6 [33]. Related strain genomes were downloaded from the NCBI Prokaryotic reference database, and the draft genome content was annotated via the NCBI Prokaryotic Genome Annotation Pipeline PGAP. Further phylogenetic and taxonomic studies were conducted to further confirm the taxonomic status of strain Q3-6. The average nucleotide identity (ANI) values were calculated by using online tools of the EzBioCloud database (https://www.ezbiocloud.net/tools/ani, accessed on 3 June 2024) [34], digital DNA–DNA hybridization (dDDH) values were calculated by using the Genome-to-Genome Distance Calculator v3.0 (ggdc.dsmz.de/ggdc.php, accessed on 7 August 2024) [35]. The concatenated alignment sequences of 120 ubiquitous single-copy proteins were obtained by GTDB-Tk (v1.3.0) [36], and the phylogenetic tree was reconstructed by FastTree using JTT + CAT parameters and IQ-TREE using the LG + F + I + G4 model with 1000 bootstrap replicates. The genome sequence of strain Q3-6 was deposited in GenBank under accession number PRJNA1093269.

To investigate the metabolic pathways and potential functions of bacteria, genomes were annotated using the KEGG database (v3.0) [37]. Bacterial resistance genes were annotated through RAST databases (v2.0). Secondary metabolite prediction was performed using the antiSMASH database 6.0 [38].

2.4. Characteristics of Strain Q3-6

2.4.1. Morphological, Physiological, and Biochemical Characteristics

The strain was inoculated into the liquid BPM at 37 °C for 24 h. Subsequently, it was visualized with SEM (model Nova NanoSEM450, FEI, Portland, OR, USA) [39].

The strain was inoculated onto the BPM agar and incubated at temperatures of 4 °C, 15 °C, 25 °C, 28 °C, 30 °C, 33 °C, 37 °C, 40 °C, 45 °C, and 50 °C for 2 days, respectively (growth was recorded at 4 h intervals). The growth of the strain in different pH ranges (pH 4–9.5, at intervals of 0.5) was tested in the liquid BPM. After 2 days of incubation on a rotary shaker (ZWY-200D, Zhicheng Analytical Instrument Manufacturing Co., Ltd., Shanghai, China) at 37 °C, the growth of the strain was quantified by using a microplate reader (VersaMax, MDC, Sunnyvale, CA, USA) at 600 nm. The pH of mediums was adjusted by using commercial additional buffers at a concentration of 20 mM: MES (pH 5.5 and 6.0), PIPES (pH 6.5 and 7.0), HEPES (pH 7.5 and 8.0), Tricine (pH 8.5), and CAPSO (pH 9.0 and 9.5) [40]. Salt tolerance was determined with different salinity gradients of liquid BPM (0–12% at 1% intervals, w/v).

Biochemical tests were performed on the strain. Enzyme production assays were carried out with substrates including starch (0.2%, w/v), Tweens (20, 40, 60, and 80, 1%, v/v), cellulose (0.5%, w/v), alginate (1%, w/v), and caseins (1%, w/v). Other physiological or biochemical characteristics were performed by applying the API ZYM, API 50CH, and API 20E strips (BioMérieux China Ltd., Shanghai, China) for enzyme activity and substrate fermentation assays, and all reagent strip tests were performed according to the instructions except for setting the optimal NaCl concentration values.

2.4.2. Antibiotic Susceptibility Testing

Drug resistance was determined by the disk-diffusion method [41] and the following twenty kinds of antibiotics were included: Polymyxin B (PB), lincomycin, ceftriaxone, Cefotaxime Sodium, ampicillin (AMP), penicillin (PNC), carbenicillin, neomycin, kanamycin (KAN), Rifampin (RA), norfloxacin (NOR), gentamycin (CN), ofloxacin (OF), streptomycin (STR), vancomycin (VA), tetracycline (TE), tobramycin (TOB), chloramphenicol (CL), erythromycin (EM), clarithromycin (CLR). The colonies cultured for 24 h were picked into sterile purified water containing 1% NaCl to make a suspension, which was then inoculated onto BPM, and different disks containing the above 20 antibiotics were placed on the surface of the agar medium, respectively. After incubation at 40 °C for 48 h, the diameters of the inhibition circles were measured to determine the resistance of the strain to different antibiotics.

2.4.3. Heavy Metal Susceptibility Testing

Minimum Inhibitory Concentrations (MICs) were measured for seven heavy metals: Mn (II), Cu (II), Pb (II), Cr (III), Cd (II), Ni (II), and Zn (II) [42] according to a previous study (Zhu et al., 2023). The bacterial solution that had been incubated for 24 h was transferred into a 96-microtiter well plate (BKMAM BIOTECHNOLOGY Co., Ltd., Changsha, China) that contained the liquid BPM with different concentration gradients of heavy metal compounds, and the concentration of each heavy metal ion solution was 0.1 g/L, 0.5 g/L, and 1.5 g/L [43]. A liquid medium without a bacterial solution was used as a blank control. After incubation at 40 °C for 4 days, the optical density was measured at the wavelength of 600 nm by using a microplate reader to determine the corresponding MIC.

2.5. Media Optimization for Industrial Scale Mass Production and Total Bacterial Community Enhancement

For small-scale refined microbial cultures in the laboratory, using BPM for bacterial culture is common. For industrial-scale mass production, factories prefer to use a low-cost medium that can increase the colony-forming units (CFUs) [44]. Molasses medium was used in this study with the following composition [45]: molasses (15 g/L), soy peptone (10 g/L), KH₂PO₄ (5 g/L), NaCl (2 g/L), and MgSO₄ (1 g/L).

The seed solution in the logarithmic phase was inoculated into the liquid molasses medium with 1% inoculum amount. The solution was incubated at 40 °C, and 1 mL of the bacterial solution was taken and diluted 10⁷ times at 12 h, 24 h, and 36 h, and then 0.1 mL of the diluted bacterial solution was spread on the BPM agar. After incubation at 40 °C for 30 h, the colonies on the plate were counted and the CFU curve was plotted.

2.6. Evaluating Heat Resistance of Strain Q3-6

Strain Q3-6 was inoculated into the liquid molasses medium mentioned above and after incubation at 40 °C for 30 h, 1 mL of the bacterial solution was taken and diluted 10⁸ times in a gradient. Amounts of 0.1 mL of the dilutions of 10⁻⁶, 10⁻⁷, and 10⁻⁸ were coated on the BPM agar, respectively. After incubation at 40 °C for 24 h, a single colony on the medium was counted and cellular concentration in CFU/mL (colony forming units per mL) was quantified. At the same time, the bacterial solution was heated at 80 °C for 15 min, and cellular concentration was detected as mentioned above to assess the heat resistance of strain Q3-6. Further, the bacterial solution, which had been heated at 80 °C for 15 min, was reheated at 90 °C for 15 min, and the cellular concentration in CFU/mL was quantified to assess the spore production rate of strain Q3-6. The experiment was carried out in triplicate and statistical analysis of data were conducted using one-way ANOVA.

2.7. Decolorization of Brilliant Blue G-250 and Congo Red by Strain Q3-6

2.7.1. Decolorization Test of Brilliant Blue G-250

In order to study the decolorization effect of the strain, it was inoculated into Trypticase Soy Broth (TSB) containing 0.15 g/L Brilliant blue G-250 at 30 °C. An amount of 2 mL of medium was taken out at regular intervals and centrifuged at 8000 rpm for 4 min. The OD₅₈₀ of the supernatant was measured. The change in absorbance compared to the initial absorbance was calculated by using the following equation [46]:

$$\mathrm{Decolorization} (\mathrm{\%})={\displaystyle \frac{(\mathrm{Initial} \mathrm{absorbance}-\mathrm{Final} \mathrm{absorbance})}{\mathrm{Initial} \mathrm{absorbance}}}\times 100$$

(1)

Decolorization efficiency was expressed as a percentage of decolorization. The experiments were conducted in triplicate.

2.7.2. Effect of Initial Concentration of Brilliant Blue G-250 on Decolorization Rate

In order to deeply study the factors affecting the decolorization efficiency of the dye by strain Q3-6 as well as the applicability in different environments, the medium was simplified and the ingredients were clarified. The composition of the decolorization medium included the following: glucose 2 g/L, NaH₂PO₄ 0.5 g/L, K₂HPO₄ 2 g/L, MnSO₄-7H₂O 0.02 g/L, FeSO₄-7H₂O 0.01 g/L, MgSO₄-7H₂O 0.2 g/L, CaCl₂ 0.02 g/L, NH₄Cl 1 g/L, and peptone 13 g/L.

The effects of different concentrations of Brilliant blue G-250 on the decolorization efficiency of the strain were studied in depth. Different concentrations of Brilliant blue G-250 (100, 200, 300, 400, and 500 mg/L) were added to the decolorization medium. The procedure described in Section 2.7.1 was followed, the absorbance was measured after a 10-fold dilution of the supernatant, and the change in absorbance after 1 d, 3 d, and 5 d from the initial absorbance was calculated.

2.7.3. Effect of Salinity, Pondus Hydrogenii, and Temperature of Brilliant Blue G-250 on Decolorization Rate

Different concentrations of NaCl were added to the decolorization medium containing 200 mg/L Brilliant blue G-250, the concentration gradients of which were limited to the salinity range of strain growth (0, 1%, 2%, 3%, and 4%) based on previous experiments. In addition, to study the effect of pH, the pH values (5.5, 6.5, and 7.5) of the decolorization medium containing 0.2 g/mL of Brilliant blue G-250 were adjusted according to the optimal growth pH range of strain Q3-6. The effect of different temperatures (16 °C, 30 °C, 37 °C, and 45 °C) on the decolorization of the strains was also studied. The procedure described in Section 2.7.1 was followed, the absorbance was measured, and the change in absorbance after 5 d from the initial absorbance was calculated. Statistical analysis of data was conducted using one-way ANOVA.

2.7.4. Decolorization Test of Congo Red

In order to further investigate the decolorization ability of strain Q3-6 on other dyes, Congo Red dye was also selected for the study of decolorization. An amount of 0.2 g/L of Congo Red dye was added to the decolorization medium as described previously. To measure the decolorization rate, the supernatant after centrifugation was directly used and the absorbance at 480 nm was measured.

2.8. Assessment of Biofilm Formation

Strain Q3-6 was inoculated into 100 mL of the previously described decolorization medium and incubated overnight at 30 °C. The bacterial solution was transferred at 2% inoculum to a new decolorization medium with the foregoing range of concentration of Brilliant blue G-250 in the 96-microtiter well plate, and incubated at 30 °C under stationary conditions to promote biofilm formation. After 2 days of incubation, the liquid portion was discarded and washed gently. Next, the solution was fixed with methanol solution for 15 min and then discarded and air-dried. An amount of 200 μL (0.1% w/v) of crystal violet solution was added to stain for 10 min, then rinsed with sterile water. Next, ethanol was added to dissolve the crystal violet, and then the absorbance was measured at a wavelength of 590 nm. There were 5 parallel samples in each experiment, and the experiment was carried out in triplicate. Statistical analysis of data was conducted using one-way ANOVA.

3. Results and Discussion

3.1. Analysis of Isolates

These strains, which were obtained from the initial screening, were identified by the comparison results of the 16S rRNA gene. They belong to Phylum Bacillota, Class Bacilli, Order Bacillales, Family Bacillaceae, and genus Bacillus. Bacillus can control pests and diseases [47] and plays a key role in crop production [48]. It can be used as an edible probiotic for humans [49], while its spores are often used as feed additives due to their resistance to heat, drying, and acids [50]. Moreover, Bacillus isolated by Zhao et al. [51] was also found to be effective in reducing total nitrogen (TN), chemical oxygen demand (COD), etc., in landscape water in their study. Soltani et al. [52] also discussed that Bacillus sp. can produce degrading enzymes and thus it can be used in the bioremediation of aquaculture-polluted water. These studies suggest that the genus Bacillus can be effective in degrading certain pollutants in sewage.

The turbidity of sewage samples decreased significantly after the twelve strains were incubated into sewage samples separately for a period of time, with the effect of strain Q3-6 being more pronounced than that of the remaining eleven. Strain Q3-6 was selected for further study.

3.2. 16S rRNA Gene Sequence and Phylogenetic Analysis of Strain Q3-6

An almost complete 16S rRNA gene sequence of strain Q3-6, with a length of 1538 bp, was obtained by PCR amplification. After 16S rRNA gene sequence comparison, strain Q3-6 was identified as belonging to the Phylum Firmicutes, Class Bacilli, Order Bacillales, Family Bacillaceae, and genus Bacillus, with the same highest sequence similarity to Bacillus mobilis (99.7%), Bacillus thuringiensis (99.7%), and Bacillus pacificus (99.7%). Further phylogenetic analysis conducted by genome comparisons is described in the next section.

3.3. Genome Sequencing and Annotation

3.3.1. Genome Sequencing

The genome of strain Q3-6 was sequenced and assembled (Figure S1 and

Table 1. Genomic characterization of strain Q3-6.

Features	Numerical Value
Genome size (bp)	5,776,368
G + C content (mol%)	34.92
Number of CDS	5804
CDS as a proportion of the genome (%)	98.31

). The genome of Q3-6 had 5904 genes, of which 5668 had protein-coding functions. It contains 86 tRNA genes, 4 5S rRNAs, 2 16S rRNAs, and 3 23S rRNAs.

The ANI value (

Table 2. Determination of the taxonomic status of strain Q3-6.

Strain	Similarity (%)	ANI (%)	dDDH (%)
Bacillus mobilis 0711P9-1	99.7	91.0	42.8
Bacillus thuringiensis ATCC 10792	99.7	98.1	82.8
Bacillus pacificus EB422	99.7	91.4	43.9

) between Q3-6 and Bacillus thuringiensis ATCC 10792^T was highest (98.1%), and the dDDH value (Table 2) between Q3-6 and Bacillus thuringiensis ATCC 10792^T was also highest (82.8%). ANI and dDDH values were higher than the species delineation thresholds (70% for DDH and 95–96% for ANI). The genomic phylogenetic tree indicated that strain Q3-6 belongs to the species Bacillus thuringiensis (Figure 1).

3.3.2. Prediction of Gene Clusters for Secondary Metabolite Synthesis in Strain Q3-6

By using the antiSMASH software 6.0, 13 clusters of secondary metabolite synthesis genes were predicted (Figure S2), including RiPP-like, three NRPS, Nl-siderophore, NRP-metallophore, NRPS-like, terpene, LAP, T1PKS, lanthipeptide-class-ii, betalactone, and two RRE-containing genes (Table S1). The enzymes and proteins encoded by these gene clusters are responsible for the synthesis of a wide range of structurally complex and functionally diverse secondary metabolites. The diversity of these products enables the strains to respond to environmental changes. One of them with 100% similarity is cluster3, which is petrobactin synthesized from Nl-siderophore. And seven others could not be matched to known gene clusters, suggesting that they are unknown gene clusters with a high probability of having the potential to produce novel compounds.

3.4. Analysis of Strain Characteristics

3.4.1. Physiological, Biochemical, and Phenotypic Characterizations of Strain Q3-6

Physiological and biochemical characterizations of the isolate Q3-6 were detected in this study. It was Gram-stain-positive, rod-shaped (1.0–2.5 μm long and 0.5–1.0 μm wide) (Figure S3). After 24 h of growth at 37 °C, the colonies were creamy white or yellowish, raised, smooth, rounded, with intact margins, and 1.0–5.0 mm in diameter on the BPM agar.

The strain can grow at 15–45 °C, and its optimal growth temperature is 30–40 °C. The growth temperature of Bacillus spp. studied by Gao et al. [53] ranged from 25 °C to 50 °C. The growth temperature of Bacillus spp. isolated from drinking water treatment plants by Vaz-Moreira et al. [54] ranged from 15 °C to 37 °C. The strain grows at pH 4–7.5 (optimal pH 5.5–7.5) range. Compared with others in the same genus [55,56,57,58], strain Q3-6 has a significantly wider optimal growth temperature range as well as an optimal pH range, and it is better adapted to low temperatures, which also suggests that strain Q3-6 has stronger environmental adaptability in treating sewage pollutants. Strain Q3-6 can tolerate NaCl in the range of 0–5%, and the optimal salinity conditions are 0–2%.

Hydrolysis of casein was positive. The result suggests that strain Q3-6 possesses a high-activity tyrosinase enzyme, rendering it proficient in treating effluents with organic wastes. Some other strains [59] with high-activity tyrosinase enzyme were also used in treating dairy processing plant effluents and restaurant effluents. And hydrolysis of Tween 20, 40, and 60 were weakly positive.

By using the API 20E, Arginine dihydrolase, Lysine decarboxylase, Ornithine decarboxylation, Citrate, Tryptophan deaminase, and the Voges–Proskauer reaction were positive for Q3-6, which confirms the utilization of gelatin and glucose. By using the API ZYM, alkaline phosphatase, Esterase (C4), Lipoid esterase (C8), Leucine Arylaminase, Valine arylaminase, pancreatic rennet, acid phosphatase, Naphthol-AS-BI-phosphate hydrolase, and α-Glucosidase were positive for Q3-6. Strain Q3-6 produces pancreatic rennet, which can selectively cleave amino acid fragments from protein chains, indicating that this strain can effectively treat protein-rich sewage. The alkaline phosphatase and acid phosphatase produced by this strain catalyze the hydrolysis of organic phosphorus in sewage [60]. By using the API 50CH, acid production from glycol, d-ribose, d-glucose, d-fructose, d-mannose, N-Acetylglucosamine, amygdalin, ARBULIN, Hepatitis C7H7 and Iron citrate, salicin, d-cellobiose, d-maltose, d-Sucrose, d-Alginose, d-Gentian disaccharide, d-turanose, d-tagatose, Potassium gluconate, potassium 2-ketogluconate, and potassium 5-ketogluconate were positive. Detailed results of the enzyme activity of strain Q3-6 are shown in Table S2.

3.4.2. Effects of Antibiotics and Heavy Metals on the Growth of Q3-6

Studying the ability of strain Q3-6 to survive under different growth conditions is helpful to determine the type of sewage they can bioremediate, especially for water with high antibiotic and heavy metal contents. Printing and dyeing sewage also contains an amount of heavy metal ions.

We selected some commonly used antibiotics to assess the resistance of strain Q3-6. The reference standards for the antimicrobial range of the disk method for antibiotic susceptibility testing of resistant bacteria are shown in Table S3.

According to the Performance Standards for Antimicrobial Susceptibility Testing issued by the Clinical and Laboratory Standards Institute (CLSI) of the USA [61], Table S4 lists the resistance of strain Q3-6 to different antibiotics. Strain Q3-6 is resistant to polymyxin B, lincomycin, ceftriaxone, Cefotaxime Sodium, ampicillin, penicillin, and carbenicillin; moreover, it is intermediate to kanamycin. Several studies have shown that antibiotic contamination, especially ceftriaxone [62], penicillin [63], and lincomycin [64], is present in several regions of the world. The resistance of strain Q3-6 to many antibiotics can ensure that it can survive in antibiotic-containing sewage and continue to play the role of sewage treatment.

Printing and dyeing sewage contain amounts of heavy metal ions (e.g., Zn (II), Cu (II), Cr (III), Ni (II), Cd (II), etc.). The experimental data (

Table 3. Minimum inhibitory concentrations of heavy metal ions on the growth of strain Q3-6.

Heavy Metals	MIC (mg/L)
Pb (II)	1500
Ni (II)	100
Zn (II)	100
CDS as a proportion of the genome (%)	98.31

) show that strain Q3-6 was resistant to several heavy metals, such as Pb (II), Ni (II), and Zn (II), with the highest tolerance to Pb (II), with MIC up to 1500 mg/L. The best removal of Zn (II) by Bacillus cereus was also found in the study of Rajivgandhi et al. [65]. Alotaibi et al. [66] reported the mechanism of detoxification of various heavy metals by Bacillus. This result helps to investigate whether strain Q3-6 can survive and degrade organic pollutants in printing and dyeing effluents with a high content of heavy metal ions, and even adsorb and remove heavy metal ions to some extent.

3.5. Plotting Colony-Forming Unit Curves to Visualize the Microbial Population of Strain Q3-6

As shown by the CFU curve (Figure 2), the molasses medium can produce higher microbial concentrations, which can reach 10⁹ CFU/mL. Thus, it can meet the needs of industrial production. The cost of this medium is relatively low, which ensures the industrial application of strain Q3-6 in sewage treatment.

Papizadeh et al. [67] also tested molasses as an effective nitrogen source for the growth of the strain they studied. Molasses medium has been used in some other studies [68,69] to increase colony concentration as well as reduce costs.

3.6. Calculation of Sporulation Rate and Analysis of Heat Resistance of the Strain

Bacillus responds to adverse environments by producing spores [70], which is important for an in-depth study of the strain’s ability to survive in complex sewage environments. In order to further evaluate the resistance of strain Q3-6 to the effects of environmental stresses and to assess its potential for industrial application, the heat excitation effect and spore production rate of this strain were determined in this study. As can be seen in Figure 3, the biomass of bacteria increased by 281.4% after thermal activation at 80 °C. After the second heating at 90 °C, most of the nutrients were killed, and the spore production rate could reach 29.6%. Significant differences (p < 0.05) were found between the results of the three experimental groups. The amount of strain was significantly increased after heating at 80 °C. This result also provides a basis for the industrial mass production of the strain, which can be simulated by controlling the temperature to greatly promote the number of bacteria and effectively improve the efficiency of treating sewage.

Sublethal heat treatment activates dormant spores, thereby enhancing spore conversion to vegetative cells. Germinated spores have lower heat resistance than dormant spores [71]. The response to heat shock is mediated by heat shock proteins (HSPs), which are thermally stimulated to synthesize when a strain is exposed to high temperatures to protect the organism itself. Analyzed from a genomic point of view, the major HSPs involved in prokaryotes are GroES, GrpE, DnaJ, GroEL, DnaK, HtpG, ClpB, ClpA, and ClpX [72]. All of the major heat shock proteins mentioned above are present in the genome of strain Q3-6, except HtpG and ClpX. ClpB repairs heat-induced shock proteins in conjunction with the chaperones DnaJ, DnaK, and GrpE to repair heat-induced protein damage [73]. These proteins are important for strain Q3-6 to be able to survive in adverse environments, especially high temperatures, and are a prerequisite for being able to effectively treat sewage.

3.7. Evaluation of the Ability of Strain Q3-6 to Decolorize Dyes

3.7.1. Decolorization of Brilliant Blue G-250 by Strain Q3-6

As shown in Figure 4, after 5 days, the decolorization rate of Brilliant blue G-250 was up to 76.2%. As evident from the figure, during the initial 0–1 d period, the optical density (OD) value undergoes a more pronounced and rapid decline, signifying an excellent decolorization efficiency. Subsequently, the OD value decreases at a slower pace and to a lesser extent, marking a significant reduction in decolorization efficacy. This phenomenon might be attributed to the robust growth of the strain at the onset, characterized by a high population density and, consequently, a stronger capacity for dye removal. Over time, however, the microbial population in the solution approaches saturation, coupled with the depletion of nutrients and the toxic influence of the dyes [74], which ultimately leads to a decline in the vitality of the strains or even their demise, thereby diminishing the decolorization efficiency.

3.7.2. The Effect of Different Initial Concentrations of Dyes on Decolorization

The dye decolorization ability of strain Q3-6 was investigated at different initial Brilliant blue G-250 concentrations (100–500 mg/L). As shown in Figure 5, the decolorization ability of strain Q3-6 gradually decreased with the increase in dye concentration. When the concentration of dye was 0.1 g/L, the dye decolorization rate reached 93.9%. When the concentration of dye was 0.5 g/L, the decolorization rate also reached 65.3%. One-way ANOVA analysis of the results showed that the dye concentrations of different groups at the same time point were significantly different (p < 0.05), indicating that the initial concentration had a great effect on the decolorization progress. Sani and Banerjee [75] found that the decolorization effect decreased with increasing dye concentration. Paz et al. [76] showed that Bacillus aryabhattai can decolorize CBB G-250 up to 150 mg/L. The results of the present studies clearly indicate that the treatable concentration of Brilliant blue G-250 has been documented to be as high as 500 mg/L for strain Q3-6, and strain Q3-6 has superior decolorization ability, with good results in terms of treatable concentration as well as treatment time.

3.7.3. The Effect of Salinity, Pondus Hydrogenii, and Temperature on the Decolorization of Brilliant Blue G-250 Dye

From the experimental results in Figure 6, it can be seen that the highest decolorization (60.2%) of Brilliant blue G-250 by strain Q3-6 was achieved at 1% salinity, whereas higher salinity (4%) resulted in a significant decrease in decolorization. This phenomenon may be related to the fact that higher salinity will inhibit the growth of the strain, which is consistent with the results of the previous experiments in this study. In addition, this phenomenon may also be due to competition for adsorption sites between Na (I) and the dye, so an increase in salt concentration leads to the removal of the dye [77].

From the experimental results in Figure 7, it can be seen that the decolorization of Brilliant blue G-250 by strain Q3-6 reached its maximum (75.7%) when the pH was 6.5, and the acidic environment caused a significant decrease in the decolorization rate. The possible reason for this is due to the fact that H (I) in the acidic solution competes with the dye for adsorption sites [78]. This result indicates that strain Q3-6 has the best decolorization effect in a neutral environment. Dyeing wastewater is in most cases weakly alkaline, and other studies have shown that neutral environments are more conducive to bacterial decolorization of dyes [25,79].

As shown in Figure 8, in the optimal temperature interval for the growth of strain Q3-6, the decolorization rate was relatively stable from 30 °C to 45 °C, with a small increasing trend with temperature and the decolorization rate up to 56.0% at 45 °C. While at 16 °C, strain Q3-6 could hardly decolorize Brilliant blue G-250. This result suggests that low temperature will significantly inhibit the ability of strain Q3-6 to decolorize Brilliant blue G-250. Within a certain range, the higher the temperature, the higher the surface activity, meaning that higher temperatures favor the biological removal of dyes [77]. The decolorization of Brilliant blue G-250 by strain Q3-6 in the present study was higher at 30–40 °C compared to the study of Paz et al. [76].

3.7.4. Decolorization Study of Congo Red

The change in the color of the solution before and after the treatment is clearly observed in Figure 9. As shown in Figure 10, the decolorization rate of Congo Red solution at a concentration of 0.1 g/L can reach 91.9% after 3 d. Compared to the results of Liu et al. [80] and Hoyos et al. [81], strain Q3-6 is more efficient in removing Congo Red.

3.8. The Effect of Brilliant Blue G-250 Dye on Q3-6 Biofilm Formation

Strain Q3-6 had a high capacity to form a biofilm (Figure 11), but the Brilliant blue G-250 dye inhibited biofilm formation to a certain extent, and the inhibition increased with increasing concentration. A past study has shown that the reason for this phenomenon is due to the toxicity of the dye [82].

Brilliant blue G-250 is an anionic triphenylmethane dye, and some studies have shown that biofilms have the ability to remove anionic dyes [83] as well as triphenylmethane dyes [84], which was reflected in the strong dye-removal potential of strain Q3-6 demonstrated in this study. In addition, some studies have found that biofilms are associated with the tolerance [85] of the strain as well as the adsorption of heavy metals [86], which is linked to the previous part of this study regarding the ability of strain Q3-6 to tolerate some antibiotics and heavy metals. Therefore, it can be concluded that strain Q3-6 can be adapted to sewage with complex compositions for the bioremoval of pollutants.

4. Conclusions

The present study has conclusively demonstrated the exceptional dye decolorization capabilities of strain Q3-6, achieving remarkable efficiencies of 93.9% for Brilliant blue G-250 (0.1 g/L) and 91.9% for Congo Red (0.1 g/L). Furthermore, this strain has exhibited resistance to a wide array of antibiotics and heavy metals, likely due to its robust biofilm adsorption capacity. These findings highlight the broad biotechnological potential of strain Q3-6, particularly in the bioremediation of sewage treatment and the management of complex dyeing and printing effluents.

The encoded heat shock proteins within the genome of strain Q3-6 confer it with notable heat resistance, which is crucial for its survival and functionality in various environmental conditions. Additionally, the strain’s strong spore production ability and heat-resistant properties make it a promising candidate for industrial mass production applications.

Overall, this study underscores the significant contributions of strain Q3-6 to the field of bioremediation and wastewater treatment. Its unique combination of dye decolorization prowess, antibiotic and heavy metal resistance, and heat tolerance warrants further exploration for potential commercial and environmental applications.