ABSTRACT
Biodegradability and resistance from indigenous bacterial communities to dyes were tested using samples from both polluted and unpolluted surface waters in Buenos Aires. Five dyes were selected for the study: Acid Black 210, Direct Orange 39, Malachite Green, Gentian Violet, and Alizarin Red. Water quality was assessed by measuring chemical oxygen demand, biochemical oxygen demand, and both Escherichia coli and enterococci counts. Biodegradability was tested using a respirometric method, while resistance was assessed by determining the minimum inhibitory concentration (MIC). No bacterial strains capable of degrading the dyes as the sole carbon source were isolated from the respirometric tests. However, from the MIC tests, 28 strains capable of dye discolouration were identified, using nutrient broth as a supplement. Two of them were able to degrade Malachite Green and Acid Black 210 at a concentration of 50 mg L−1 in less than 24 h and with an efficiency greater than 87%. These strains were identified as Aeromonas sp. and Shewanella sp. through MALDI-TOF/MS and 16S rRNA gene sequencing. The determination of biodegradability and resistance can be used to enhance the characterization of watercourses. Furthermore, this methodology provides a means to isolate biodegrading bacteria that could be applied in effluent treatment processes.
HIGHLIGHTS
Biodegradability to five dyes and resistance of indigenous bacteria to them were tested in polluted and unpolluted surface waters.
An attempt was made to correlate the presence of degrading bacteria and their resistance with contamination.
Indigenous strains able to degrade Malachite Green and Acid Black 210 were isolated.
These strains could be employed for the treatment of effluents containing the aforementioned dyes.
INTRODUCTION
Water pollution remains one of the most pressing environmental challenges today. Despite widespread recognition of its impact, the scale of pollution continues to grow. Every year, large volumes of untreated liquid effluents are discharged into surface waters, exacerbating the problem (Zhou et al. 2019). Globally, it is estimated that over 80% of all wastewater is released without treatment, with significant disparities between countries based on income levels (WWAP 2017).
Colourants are among the most significant pollutants in water bodies worldwide (Teo et al. 2022). However, their role as pollutants of the aquatic environment is often underestimated, as other groups of water contaminants have been comparatively more thoroughly studied (Tkaczyk et al. 2020). Colourants degrade the aesthetic quality of the aquatic resource not only by colouring it but also by increasing the organic matter present, which leads to consumption of dissolved oxygen. It must be considered that oxygen production is also reduced due to the greater opacity of the coloured waters, which restricts the light available for photosynthetic processes (Holkar et al. 2016). Moreover, colourants are often persistent in the environment, bioaccumulative, and can have toxic, mutagenic, or carcinogenic effects on aquatic life (Zhou et al. 2019).
Historically, colourants were derived from natural sources, such as plants and insects. Today, however, the majority of dyes used in industry are synthetic (Hagan & Poulin 2021). Colourants are generally classified into two categories: dyes, which are soluble in water, and pigments, which are insoluble (Grace Pavithra et al. 2019). Dyes can further be classified based on their chemical structure or the methods used for their industrial application (Benkhaya et al. 2020). More than 100,000 variants of synthetic dyes have been developed to date, with an estimated annual production rate of 7.0 million tons (Katheresan et al. 2018).
Synthetic dyes are a common pollutant in the liquid effluents from various industries, including textile, tannery, cosmetic, and food industries (Katheresan et al. 2018; Tkaczyk et al. 2020). The discharge of coloured effluents from these industries can easily attract public attention (Hassan et al. 2018). However, it is not uncommon for liquid effluents to be discharged directly into water courses, without any treatment (Sanu Ogundairo et al. 2024). The textile industry, in particular, discharges large quantities of coloured wastewater due to poor dye uptake by fabrics (Holkar et al. 2016).
Synthetic dyes are more resistant to physical, chemical, or biological degradation than natural ones (Varjani et al. 2020), and therefore, conventional treatment is usually insufficient for the removal of these persistent organic pollutants from wastewater. For this reason, different treatment technologies have been proposed, including physical methods (adsorption, coagulation/flocculation, and filtering), chemical methods (Electro-Fenton, photocatalysis, ozonation), and biological methods (use of enzymes or microbes for biosorption or biodegradation) (Bal & Thakur 2022). The latter offers potential advantages over physical and chemical technologies (Varjani et al. 2020), one of the most important being their ability to achieve, in some cases, the total mineralization of the compound (Alexander 1999). In contrast, many other methods generate secondary pollution, which complicates environmental management (Katheresan et al. 2018).
Dyes are xenobiotic compounds that are resistant to biodegradation. One approach to mitigate their environmental impact is the design of engineered bacteria. However, the release of transgenic bacteria for field application is still a matter of controversy (Kumar et al. 2020). Another alternative is the use of indigenous bacteria adapted to the degradation of these compounds. Bacterial pre-exposure to toxic and persistent pollutants like dyes can enhance the selection of indigenous strains capable of tolerating their toxic effects or even metabolizing them (Alexander 1999). As such, more contaminated environments often harbour microorganisms that can degrade or tolerate the pollutants present (Gallego et al. 2018).
When standardized inocula are used in biodegradation tests, biodegradability could be considered an inherent property of a compound. However, this property is not fixed, as biodegradability results from the interaction between the compound and the environment. Similarly, the inhibitory effect of a certain substance on microorganisms can be understood as a property of the compound when a standardized inoculum is used, or of the environment, when the populations exposed are the variable.
In this context, testing the biodegradability and toxicity of a compound against indigenous microbial communities from different watercourses can provide valuable information for characterizing the environmental conditions of those water bodies (Gallego et al. 2018). Furthermore, the analyses of biodegradability and resistance can be employed as methodologies to isolate bacteria that are both tolerant and capable of biodegrading pollutants (Fortunato et al. 2018).
The aim of this investigation was (a) to evaluate bacterial capacity to tolerate or degrade dyes in natural environments with different degrees of contamination and (b) to select from these assays indigenous bacteria capable of degrading dyes, with the potential application for effluent treatment processes.
METHODS
Sampling points
Location of sampling points. 1. La Plata river; 2. Medrano stream; 3. La Boca; 4. Lanús; 5. Morón stream.
Location of sampling points. 1. La Plata river; 2. Medrano stream; 3. La Boca; 4. Lanús; 5. Morón stream.
Sampling points are shown in Figure 1.
One of the sites is located in a presumably uncontaminated place, La Plata river, near the water intake of the plant that provides drinking water to the city of Buenos Aires (34°32′49.4″S, 58°25′48.5″W). The other sites correspond to places that receive discharges of different types of effluents.
The Medrano stream crosses an important textile industry hub located in the district of San Martín (Morandeira et al. 2019). The sampling point is located at the outfall of the basin in La Plata river (34°32′14.1″S, 58°27′31.0″W). La Boca is the outfall of the Matanza-Riachuelo basin, a paradigm of pollution in the country (34°38′26.8″S, 58°21′38.5″W). More than four million people and a number of industries are located on the banks of the basin, in some cases, in precarious settlements that lack sewers and therefore discharge their effluents into the river (ACUMAR 2016). The point named ‘Lanús’ is also in the Matanza-Riachuelo basin, in this case, in the vicinity of an important tanning centre located in the district of Lanús (34°40′51.4″S, 58°26′22.5″W). Finally, the Morón stream (34°33′45.6″S, 58°37′36.4″W) is one of the most polluted points of another emblematic contaminated basin, the Reconquista River basin, also characterized by the dumping of untreated domestic and industrial effluents (Salibián 2006; Tufo et al. 2021).
Characterization of samples
The presence of organic matter was evaluated by measuring the chemical oxygen demand (COD) and biochemical oxygen demand (BOD). COD was determined by using the closed reflux method (HACH Loveland, USA). BOD was performed by the respirometric method, using a BODtrack apparatus (HACH Loveland, USA). To establish the degree of contamination of the samples, the following microbial parameters were evaluated: total heterotrophic bacteria (THB), Escherichia coli, and enterococci. All determinations were carried out according to APHA (2023).
Chemicals and culture mediums
Five dyes belonging to different chemical groups were selected. Two azo dyes, Acid Black 210 (AB) and Direct Orange 39 (DO); two triphenylmethane derivatives, Malachite Green (MG) and Gentian Violet (GV), and Alizarin Red (AR), an anthraquinone. Azo dyes were provided by a tanning industry and are the bulk products used for dyeing leather. Acid black 210 was provided by EUROCOLOR (Castel Bolognese, Ravenna, Italy), Direct Orange 39 by TCL (Thirumalai Chemicals Limited. Mumbai, Maharashtra, India). MG and GV were provided by Biopack (C.A.B.A., Argentine) and AR by Mallinckrodt (St. Louis, USA). The stock solutions were prepared in sterile distilled water according to the solubility of each dye. The final concentrations were 10,000 mg L−1 for AB, DO, and MG; 5,000 mg L−1 for AR; and 1,000 mg L−1 for GV. Vitamins were supplied from Sigma-Aldrich (St Louis, USA). Nutrient Broth, Mac Conkey Broth, Glucose Azide Broth, Triptone Soy Broth, Triptone Soy Agar, Slanetz Bartley Agar, and Brain Infusion Agar were provided by Biokar (Pantin, France). Chromagar™ was provided by Kanto Chemical Co (Tokyo, Japan). All other chemicals were of analytical reagent grade and purchased from Mallinckrodt Chemical (St. Louis, USA) and Merck (Darmstadt, Germany).
Biodegradability of dyes in surface waters
Samples from the different sampling points were added with 20 mg L−1 of each dye separately and incubated for 10 days at 20 °C in a BODtrak Hach™ respirometric equipment. Each assay was performed in duplicate. Surface water without the addition of any dye was used as a control. The final volume in each bottle was 350 mL, which, according to the manufacturer's instructions, allows the recording of up to 70 mg L−1 of oxygen. The oxygen consumption of each dye was compared with that of the control. A lower oxygen consumption than the control would indicate the inhibition of the existing population due to the presence of the dye, while a higher oxygen consumption allows us to assume a probable biodegradation of the compound under study. In this last case, bacterial communities were preserved for subsequent degradation studies.
Evaluation of bacterial resistance to dyes in surface water samples (MIC assay)
An assay was carried out to determine the minimum necessary concentration of each dye to inhibit the bacterial population present in the water sample. The test consists of exposing the surface water in assay tubes at decreasing concentrations of each dye and determining the minimum inhibitory concentration (MIC) that prevents bacterial growth in a culture medium. The medium employed was the nutrient broth. It was supplemented with each dye at the following concentrations (mg L−1): 500, 250, 125, 62.5, and 31.25. The medium was finally inoculated with 0.1 mL of surface water and incubated for 7 days at 28 °C. Growth was determined visually by turbidity. From the tubes that showed bacterial development, resistant communities were preserved for subsequent dye degradation studies.
Selection of degrading bacteria
Bacterial communities obtained from the biodegradability assay were tested for the degradation of the corresponding dye as the sole carbon source, as follows. Erlenmeyer flasks containing 100 mL of minimal mineral medium (Fortunato et al. 2018) supplemented with the dye (20 mg L−1) as the sole carbon source were inoculated with 1 mL of the bacterial community. The composition of the synthetic minimal medium was (g L−1) 1.73 g K2HPO4; 0.68 g KH2PO4; 0.83 g (NH4)2SO4; and 0.1 g MgSO4·7H2O (final pH 7.4), 0.5 mL of a trace elements solution, and 1 mL of a stock vitamin solution which were sterilized separately by filtration and added aseptically to the autoclaved synthetic minimal medium. The trace elements solution (L−1) contained 1 g CaCl2·2H2O; 0.3 g MnSO4·H2O; 0.5 g FeSO4·7H2O, and 0.2 g disodium salt of EDTA dihydrated. The stock vitamin solution was prepared by dissolving 40 mg calcium D-pantothenate, 2 mg folic acid, 200 mg inositol, 40 mg nicotinic acid, 20 mg p-aminobenzoic acid, 40 mg pyridoxine hydrochloride, 20 mg riboflavin, and 40 mg thiamine hydrochloride in 100 mL of distilled water. The cultures were incubated at 28 °C in a rotatory shaker (200 rpm). Discolouration was measured daily.
Bacterial communities obtained for the MIC assay were tested for the degradation of the corresponding dye in the presence of a supplementary carbon source as follows. Erlenmeyer flasks containing 100 mL of nutrient broth supplemented with the dye (20 mg L−1) were inoculated with 1 mL of the bacterial community. The cultures were incubated also at 28 °C in a rotatory shaker (200 rpm) and discolouration was measured daily.
When discolouration occurred in either of the two assays, the communities were subcultured in the same media (minimal medium or nutrient broth, according to the assay) with increasing concentrations of dye with the objective of selecting degrading bacteria. After repeated subcultures, individual strains were isolated on nutrient agar and tested for their degradation capability in a minimal medium with the addition of dye (assay for the use as the sole carbon source), or in nutrient broth with the addition of dye (assay for discolouration in the presence of a supplementary carbon source).
Isolated bacterial strains for both assays were preserved for identification.
Evaluation of discolouration
Discolouration was evaluated by spectrophotometric measurement (Metrolab 1700) of the dyes. Previously, spectral scans were carried out to determine the absorption maximum for each dye, and a calibration curve was made for each compound. The wavelengths were 625 nm for AB, 425 nm for DO, 615 nm for MG, 575 nm for GV, and 515 nm for AR. To determine the remaining dye concentration in the cultures, bacterial cells were separated by centrifugation (4,000 rpm) and the filtered supernatant fluid was analysed at the corresponding wavelength according to the experience. The corresponding culture medium without inoculation was used as a blank. Discolouration can be expressed directly as a percentage, in terms of the decrease in absorbance (Agrawal et al. 2014; Jamee & Siddique 2019; El-Bendary et al. 2023). Using the calibration curve, the result can also be expressed in terms of concentration.
Identification of bacterial strains
Bacterial strains were identified through conventional biochemical tests, as described by Procop et al. (2017), MALDI-TOF mass spectrometry and 16S rDNA gene sequencing.
Mass spectra were acquired using a Microflex LT mass spectrometer (Bruker Daltonics, Germany) equipped with the MALDI-Biotyper Software package (version 3.4). Spectra were analysed within the m/z range of 2,000–20,000 Da. Calibration of the mass spectrometer was performed using the Bruker Bacterial Test Standard (BTS) following manufacturer instructions.
The spectra obtained were matched against the Bruker Daltonics MALDI-Biotyper Library database. This comparison yields a score (SCORE) that indicates the reliability of identification at either the genus or species level, following the manufacturer's criteria: log scores ≥ 2.0 indicate probable identification at the species level; log scores between 1.7 and 1.99 indicate probable identification at the genus level; log scores < 1.7 indicate unreliability for identification purposes.
Genomic DNA was extracted by heat lysis. The 16S rRNA gene was amplified by PCR using the primers 27F (5′-AGAGTTTGATCMTGGCTCAG) and 1492R (5′-GYTACCTTGTTACGACTT) (Marchesi et al. 1998). The reaction was performed in a final 25 μL volume, containing 12.9 μL of nuclease-free ddH2O, 1.0 μL of dNTP mix (10 μM) (Invitrogen), 2.5 μL (10 μM) of forward primer, 2.5 μL (10 μM) of reverse primer, 1.0 μL of DNA template, 2.4 μL of MgCl2 (25 mM) (Invitrogen), 2.5 μL of Taq buffer 10X (Invitrogen), and 0.2 μL of Taq polymerase (5 U/μL) (Invitrogen). The thermal cycling conditions consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 40 s, annealing at 54 °C for 1 min, and extension at 72 °C for 2 min, with a final extension step at 72 °C for 10 min. Amplified fragments were purified with the QIAquick PCR purification Kit (QIAGEN) and sequenced in both strands using an ABI Prism DNA 3700 sequencer. Nucleotide sequences were compared with databases using the NCBIs' Basic Local Alignment Search Tool (Blast).
Degradation assays
Bacterial strains were adapted by inoculation in nutrient broth supplemented with 50 mg L−1 of the corresponding dye and incubated in a rotary shaker at 28 °C for 24 h to provide stock culture. Biodegradation assays were performed in 250 mL Erlenmeyer flasks containing 100 mL of the same culture medium. The system was inoculated with 1 mL of the stock culture and stored in an incubator at 28 °C. In the experiments with agitation, the Erlenmeyer flasks were incubated in a rotary shaker (200 rpm) at the same temperature. During incubation, 5 mL samples were removed aseptically at appropriate intervals in order to determine the amount of remaining MG or AB, according to the assay, and to evaluate microbial growth by the plate count method, using nutrient agar as a culture medium. Abiotic loss of the compound was estimated in control assays containing the dyes without inoculation. The specific growth rate was calculated by plotting the natural logarithm of bacterial number as a function of time and obtaining the slope for the exponential growth phase.
RESULTS AND DISCUSSION
Characterization of sampling points
Table 1 shows the results obtained at the sampling points. With the exception of point 1, the results show high faecal contamination in all sampled locations.
Characterization of sampling points
Sampling point . | COD (mg O2 L−1) . | BOD5 (mg O2 L−1) . | THB (CFU mL−1) . | Escherichia coli (CFU mL−1) . | Enterococci (CFU mL−1) . |
---|---|---|---|---|---|
1. La Plata river | 42 | 14 | 1.3 × 104 | 1.6 × 102 | <10 |
2. Medrano stream | 88 | 53 | 1.5 × 105 | 1.6 × 104 | 1.3 × 103 |
3. La Boca | 82 | 23 | 4.8 × 105 | 3.5 × 103 | 3.4 × 102 |
4. Lanús | 76 | 19 | 2.1 × 106 | 2.5 × 103 | 5.4 × 102 |
5. Morón stream | 51 | 16 | 8.1 × 105 | 4.4 × 102 | 2.9 × 102 |
Sampling point . | COD (mg O2 L−1) . | BOD5 (mg O2 L−1) . | THB (CFU mL−1) . | Escherichia coli (CFU mL−1) . | Enterococci (CFU mL−1) . |
---|---|---|---|---|---|
1. La Plata river | 42 | 14 | 1.3 × 104 | 1.6 × 102 | <10 |
2. Medrano stream | 88 | 53 | 1.5 × 105 | 1.6 × 104 | 1.3 × 103 |
3. La Boca | 82 | 23 | 4.8 × 105 | 3.5 × 103 | 3.4 × 102 |
4. Lanús | 76 | 19 | 2.1 × 106 | 2.5 × 103 | 5.4 × 102 |
5. Morón stream | 51 | 16 | 8.1 × 105 | 4.4 × 102 | 2.9 × 102 |
COD, chemical oxygen demand; BOD5, biochemical oxygen demand; THB, total heterotrophic bacteria; CFU, colony-forming units.
It should be taken into account that the current legislation considers a maximum permissible value for effluent discharges into a sewage conduit of 200 mL−1, expressed in terms of thermotolerant coliforms. The values for organic matter also follow a similar distribution pattern, especially in terms of COD. The data obtained confirms contamination of water courses with sewage effluents and is in accordance with the data reported by the different basin committees that carry out monitoring in the different areas (ACUMAR 2016; COMIREC 2022).
Analysis of biodegradability and resistance
Table 2 shows oxygen consumption after 10 days of incubation of the surface waters, to which 20 mg L−1 of each dye was added, compared to a control.
Oxygen consumption of water rivers after 10 days of incubation at 20 °C expressed in concentration values for the control and as percentage of the corresponding control for the river samples supplemented with 20 mg L−1 of each dye
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aSurface water without any dye.
b% of the corresponding control.
An oxygen consumption value higher than the control could indicate that aerobic biodegradation of the dye is taking place, while lower values, in contrast, could be due to inhibition of the bacterial populations in the presence of the compound. A decrease in oxygen consumption greater than 25% was considered inhibitory (OECD 1992). Likewise, an increase of 25% was taken as significant, in order to continue with the investigation of degrading bacteria in the sample. Although it would be strictly more appropriate to express this increase in terms of percentage of the theoretical oxygen consumption necessary for the degradation of the added dye, since sometimes the oxygen consumed by the control is very low, we considered it preferable to analyse more samples than to miss the opportunity to isolate strains that could possibly degrade the compound as the sole carbon source.
There were only two situations in which oxygen consumption was greater than the control, both corresponding to the Medrano stream. The dyes involved were AB and DO, from the azo group. Bacterial communities involved were selected for subsequent evaluation of dye degradation as the sole carbon source. The inhibition of populations, however, was much more common. With the exception of the Medrano stream and Lanús, in all other sampling points, inhibition was the most frequent result obtained. This happened for all the dyes being tested in Morón stream and for three of them in the rest of the points. The results obtained are in accordance with what has been reported in the literature on the toxicity and persistence of these compounds (Dissanayake et al. 2021; Das et al. 2023).
The inhibition of bacterial populations was analysed in more detail with the MIC assay, which allows to establish the inhibitory threshold concentrations. Azo dyes (AB, DO) were the least inhibitory. In all cases, there was growth even with concentrations as high as 500 mg L−1. Triphenylmethane derivatives (MG; GV), on the other hand, were the most toxic. At one of the sampling points, Gentian violet caused total growth inhibition at only a concentration of 31.25 mg L−1 (Table 3). In most cases, growth was accompanied by total or partial discolouration of the compound, so subcultures were carried out for the subsequent evaluation of the selected strains.
Minimal inhibitory concentration for each dye in the sampling points
Sampling point . | MIC (mg L−1) . | ||||
---|---|---|---|---|---|
AB . | DO . | MG . | GV . | AR . | |
1. La Plata river | >500 | >500 | 125 | 31.25 | 250 |
2. Medrano stream | >500 | >500 | >500 | 125 | >500 |
3. La Boca | >500 | >500 | 125 | 62.5 | >500 |
4. Lanús | >500 | >500 | 500 | 125 | 250 |
5. Morón stream | >500 | >500 | 125 | 125 | >500 |
Sampling point . | MIC (mg L−1) . | ||||
---|---|---|---|---|---|
AB . | DO . | MG . | GV . | AR . | |
1. La Plata river | >500 | >500 | 125 | 31.25 | 250 |
2. Medrano stream | >500 | >500 | >500 | 125 | >500 |
3. La Boca | >500 | >500 | 125 | 62.5 | >500 |
4. Lanús | >500 | >500 | 500 | 125 | 250 |
5. Morón stream | >500 | >500 | 125 | 125 | >500 |
As a general rule, the more toxic a compound is, the greater the environmental impact that could be expected as a result of its discharge. However, in practice, the environmental concentrations that the compounds can reach must also be taken into account. There are two factors that will determine the environmental concentration of a given pollutant, its persistence and the amount that is discharged into the environment. It may happen that very toxic but more degradable compounds could be eliminated from the environment without reaching problematic concentrations (Polo et al. 2011). On the other hand, compounds that show lower toxicity but are poorly degradable, or that are discharged in large quantities, exceeding the rate at which they can be purified, can become an environmental issue (Escher et al. 2023).
All dyes under study have been reported both as persistent and toxic, and, in some cases, specifically for azo compounds or MG, as genotoxic (Heath Canada 2020; Cristovam et al. 2022). Even though the information on environmental concentrations is scarce, their environmental presence is expected due to their widespread use. AB is one of the most used black dyes by the leather industry (Pelegrino Rocha et al. 2017). DO is another dye from the azo group widely used industrially (Health Canada 2015). Although, in both cases, the reported environmental concentrations are low in relation to their toxicity, it must be taken into account that some byproducts of their degradation, such as amines, are also toxic (Heath Canada 2020; Christovam et al. 2022). In addition to human toxicity, environmental toxicity must also be considered, since in many cases wastewater can end up in contact with crops. Dye wastes, particularly, have been shown to be toxic for agriculture and marine environments (Jadhaw et al. 2010). MG, abundantly present in industrial effluents, persists in the environment for long periods and has genotoxic, mutagenic, and carcinogenic effects. MG has been found widely in surface water, soil, and groundwater and can also infiltrate the food chain, particularly in fish, where it has been shown to accumulate (Sharma et al. 2023). GV, from the same chemical group, shares its toxicity, although environmental concentrations reported are relatively lower (Heath Canada 2020; IARC 2022). Finally, there is also data in the literature on the toxicity and persistence of anthraquinone dyes, such as AR, widely used for dyeing wool and nylon, which report it to be toxic, carcinogenic and can cause a serious hazard to aquatic ecosystems (Ramavandi et al. 2019).
As mentioned in the introduction, the results obtained for biodegradability and MIC have to be analysed not only based on the compounds but also on the environment. Paradoxically, a more contaminated environment can exhibit a greater capacity to purify contaminants than a pristine site, in which microorganisms have never been exposed to the compounds, and therefore, degrading populations have not been selected. One of the classic problems of biodegradability tests, not yet overcome, is precisely how to standardize an inoculum so that results are reproducible (Howard & Banerjee 1984).
In the present work, the analysis of biodegradability assays shows a lower percentage of inhibition of the bacterial communities in Arroyo Medrano and Lanús, two points located near textile industries and tanneries. These results could be due to pre-exposure and adaptation of the communities in those sites to the tested dyes. Comparatively, in other points, the percentage of inhibition is higher.
This analysis is not intended to be conclusive: the presence of an industry does not guarantee that a certain compound is present in the environment. In order to be more categorical, the presence of individual chemical compounds in the watercourse should be studied to establish the relationship between a given pollutant and growth inhibition, thus ensuring that prior exposure has occurred. In this case, such determinations were not possible. However, knowing environmental concentrations would not be conclusive either: it would still be possible that adaptation had taken place due to pre-exposure to structural analogues, even in the absence of the compounds under study (Chopra et al. 1997; Gallego et al. 2018).
A similar analysis can be done for the resistance of the communities found, with the same limitations. Pre-exposure can be understood as a reason for the development of resistance, although naturally some dyes may be more toxic to microorganisms than others. Furthermore, structural analogues, or even unrelated compounds, have been associated with resistance development processes. This has been much more studied for antimicrobials, where increased resistance is not only an environmental problem but a matter of life and death (Chopra et al. 1997). The concepts of resistance and biodegradability are closely related: being able to degrade a toxic compound is also a resistance mechanism (Perri et al. 2020).
Despite the limitations listed, these tests would allow the evaluation of watercourse characteristics that other analyses do not reveal, such as pre-exposure to certain compounds. In addition, their implementation provides the opportunity to select strains capable of degrading the compounds under study for their subsequent use in biodegradation tests.
Selection of dye-degrading bacteria
Samples selected from the biodegradability assay were tested for the biodegradation of AB and DO as the sole carbon source by subculturing in a minimal mineral medium supplemented with the dyes. Despite the results obtained in the respirometric test, bacteria capable of degrading the compounds could not be recovered. No discolouration was obtained in the Erlenmeyer flasks inoculated with the samples in the presence of 20 mg L−1 of the respective dyes after 7 days of incubation at 28 °C with shaking. Although there are some examples in the literature (Du et al. 2018; Song et al. 2020), the use of dyes as the sole source of carbon and nitrogen is uncommon (Jamee & Siddique 2019). According to Das et al. (2023), the majority of dyes have low carbon levels, and this hinders bacterial growth in the absence of an additional carbon source.
In the MIC assays, however, several strains capable of carrying out discolouration in the presence of an additional carbon source were obtained. A summary of the results is presented in Table 4.
Two strains were selected for further studies, strain MG-Me2 capable of degrading MG and strain AB-Me2, involved in the degradation of AB. Both microorganisms came from the Medrano stream, as indicated in Table 4.
Identification of bacterial strains
Strain MG-Me2 was initially identified through conventional biochemical tests as belonging to the genus Aeromonas. The strain was a Gram-negative motile bacillus, oxidase positive and capable of using glucose by a fermentative metabolism (Procop et al. 2017). Other biochemical tests are shown in Table 5. Subsequent MALDI-TOF/MS analysis identified Aeromonas hydrophila with a SCORE of 2.331. However, it has been reported that this methodology cannot achieve accurate discrimination between different species within the genus, and some species are not even represented in the database (Rocca et al. 2020). Therefore, it is recommended to report the isolate as Aeromonas sp. or as part of the Aeromonas hydrophila complex, which encompasses species such as Aeromonas hydrophila, Aeromonas bestiarum, and Aeromonas salmonicida. 16S rRNA gene sequence confirmed the genus but did not allow for differentiation among A. encheleia, A. rivipollensis, and A. hydrophila, as all sequences showed 100% identity and 100% coverage when compared to the query sequence. Consequently, only the genus could be reliably identified (Supplementary material). The sequence was deposited in the GenBank database and is available under accession number PQ834549.
Biochemical test
Test . | MG-Me2 . | AB-Me2 . |
---|---|---|
Gram staining | − | − |
Oxidase | + | + |
Catalase | + | + |
OF glucose | F | O |
Motility | + | + |
Nitrate reduction | + | + |
Indole production | + | − |
Arginine dyhidrolase | + | − |
Lysine decarboxylase | + | − |
Ornithine decarboxylase | + | + |
H2S production | − | + |
Esculine hydrolysis | + | − |
DNAse | + | + |
Pigment production | − | Pink |
Test . | MG-Me2 . | AB-Me2 . |
---|---|---|
Gram staining | − | − |
Oxidase | + | + |
Catalase | + | + |
OF glucose | F | O |
Motility | + | + |
Nitrate reduction | + | + |
Indole production | + | − |
Arginine dyhidrolase | + | − |
Lysine decarboxylase | + | − |
Ornithine decarboxylase | + | + |
H2S production | − | + |
Esculine hydrolysis | + | − |
DNAse | + | + |
Pigment production | − | Pink |
F, fermentative; O, oxidative.
Strain AB-Me2 could not be definitively identified by conventional biochemical tests; however, certain characteristics were observed. The strain was a Gram-negative motile bacillus. The fact that it was a non-fermenting, sulfide-producing bacterium suggested that it might belong to the Shewanella genus (Procop et al. 2017). MALDI-TOF/MS analysis identified Shewanella putrefaciens with a SCORE of 2.104. The 16S rRNA gene sequence confirmed the genus but did not allow for differentiation between S. xiamanensis and S. putrefaciens. Consequently, only the genus could be reliably identified (Supplementary material). The sequence was deposited in the GenBank database and is available under accession number PQ834528.
Degradation of Malachite Green by Aeromonas sp.
Degradation of MG by Aeromonas sp. Agitation (200 rpm): (- -▪- -) Bacterial growth and (- -o- -) % Remaining MG. Without agitation: (- -□- -) Bacterial growth and (- -o- -) % Remaining MG.
Degradation of MG by Aeromonas sp. Agitation (200 rpm): (- -▪- -) Bacterial growth and (- -o- -) % Remaining MG. Without agitation: (- -□- -) Bacterial growth and (- -o- -) % Remaining MG.
Aeromonas is a genus frequently found in surface water. There are numerous reports in the literature of the use of Aeromonas sp. for the degradation of dyes in textile and industrial effluents (Ji et al. 2020; Srinivasan & Sadasivam 2021). Additionally, Du et al. (2018) isolated a strain of Aeromonas sp. capable of degrading MG as the sole carbon source. It proved capable to maintain MG degradation efficiency even in static cultures, without agitation because it is a facultative organism (Liu 2015). Nevertheless, the time required for degradation under stirring conditions was shorter, possibly due to the greater efficiency of aerobic processes (Berg et al. 2022). The efficiency is comparable to that reported for other microorganisms, as well as the time to complete the degradation. For similar concentrations, a strain of Achromobacter xylosoxidans reached 90% efficiency within 17 h, using the same carbon supplement, nutrient broth (Wang et al. 2011). Faster processes have been described using other carbon sources, such as glucose and yeast extract (Kabeer et al. 2019) or molasses (Parshetti et al. 2006). In the few cases where the use of MG as the sole source was described, the processes were slower but with comparable efficiency. Thus, Song et al. (2020) report for Pseudomonas veroni 168 h for the degradation of 50 mg L−1 with an efficiency of 93.5%. Du et al. (2018) describe a strain of Aeromonas sp. that takes 48 h to degrade 100 mg L−1 with an efficiency of 94%.
Degradation of Acid Black 210 by Shewanella sp.
Degradation of AB by Shewanella sp. Agitation (200 rpm): (- -▪- -) Bacterial growth and (- -o- -) % Remaining AB. Without agitation: (- -□- -) Bacterial growth and (- -o- -) % Remaining AB.
Degradation of AB by Shewanella sp. Agitation (200 rpm): (- -▪- -) Bacterial growth and (- -o- -) % Remaining AB. Without agitation: (- -□- -) Bacterial growth and (- -o- -) % Remaining AB.
Shewanella is also a genus related to the bio-discolouration of dyes. Originally isolated from ocean water, this facultative anaerobic bacterium is also present in polluted wastewater and its ability to reduce azo and anthraquinone compounds has been a subject of study (Li et al. 2019; Liu et al. 2023). The Shewanella sp. strain isolated by Liu et al. (2023) was capable of degrading Reactive Black 5, an azo dye. For a concentration of 100 mg L−1, the efficiency on degradation was comparable to the results obtained in this work, 87.6%. The addition of an extra carbon source was necessary for the process to take place, as discolouration with the dye as the sole carbon source was of only 5%. There are also reports in the literature of AB discolouration by other microorganisms such as Providencia sp. (Agrawal et al. 2014) or Bacillus thuringiensis (Dave & Dave 2009). In both cases, the microorganisms described are capable of degrading not only AB but also other dyes. Likewise, the indigenous Shewanella strain isolated in this work was also able to degrade DO, the other azo dye tested, with a similar efficiency.
CONCLUSIONS
The Hill of Seven Colors is one of the most emblematic landscapes of Argentina. Less known, however, is the stream of seven colours, the popular name by which a polluted watercourse is known in the vicinity of a textile hub on the outskirts of Buenos Aires. From a similar watercourse, two native strains were isolated, MG-Me2 and AB-Me2, respectively, identified as Aeromonas sp. and Shewanella sp., that show 96.7% efficiency in the degradation of 50 mg L−1 of MG and 87.6% of AB, so they could potentially be used for the treatment of effluents that contain them. Currently, the influence of different factors on the degradation process is being evaluated, such as the most convenient supplementary carbon source, pH, temperature, salinity, the maximum concentration of dye that can be degraded, and its application in continuous processes for the treatment of synthetic effluents.
But apart from the microorganisms selected in particular for this study, the techniques used to determine biodegradability and MIC are simple and can be used in other situations, not only to be applied in the selection of new indigenous degrading strains, but also to complete the characterization of water courses. Resistance and the ability to degrade a xenobiotic compound are the result of long processes of adaptation to the compounds. The presence of resistant or degrading bacteria can then be considered as an indicator of past contamination, which may even be longer lasting than that of the contaminant itself.
FUNDING
We thank the Universidad de Buenos Aires for the grant given for this study, supported by UBACYT Program-Project 20020220200148BA 2023-2027.
DATA AVAILABILITY STATEMENT
All relevant data are included in the paper or its Supplementary Information.
CONFLICT OF INTEREST
The authors declare there is no conflict.