Abstract
2,4-dichloro phenoxy acetic acid (2,4-D) is an ionizable herbicide; its residues are only minimally maintained by soil components, and they readily pollute surface and ground water. For these reasons, microbe-mediated biodegradation is a practical method to remove 2,4-D residues from polluted environments. In the current study, 5 different termite mound soil bacterial isolates capable of utilizing 2,4-D as their sole carbon and energy source were isolated by soil enrichment on minimal salt medium (MSM) containing 2,4-D. A one promising bacterial isolate was selected and identified as Enterobacter cloacae, based on biochemical characteristics and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The growth studies conducted on Enterobacter cloacae reveal that it can thrive within a broad pH range of 2 to 6 and a temperature range of 15 to 45 °C. However, the optimal conditions for its growth over a 24-hour period were observed at a pH of 3.5 and a temperature of 25 °C. Furthermore, adding Enterobacter cloacae with an inoculum size of 1 ml (3 × 108 CFU/ml) to agricultural leachate resulted in a significantly higher degradation rate of 90.4% compared to only 9.4% degradation in uninoculated agricultural leachate. This finding suggests that Enterobacter cloacae has the potential to be utilized for bioremediation through bioaugmentation in the cleanup of water contaminated with the herbicide 2,4-D.
Keywords: Biodegradation, herbicide, 2,4-D; Isolation; Biochemical; Agricultural leachate
Introduction
Pesticides are synthetic or natural compounds used in a variety of agricultural practices to control plant diseases, weeds, and pests [29]. However, only a small amount of the pesticides used on crops actually reach their intended target; the remaining amount often transfers to other areas of the ecosystem and can contaminate surface and groundwater [9].
Herbicides, insecticides, fungicides, rodenticides and nematicides fall under the category of pesticides [6]. Among these, 2,4-Dichlorophenoxyacetic acid (2,4-D), which has been widely used as a broadleaf herbicide for more than 60 years [31]. However, due to its high solubility in water, extensive use of 2,4-D has led to contamination of surface and ground water [7], and the contaminated water results in cancer in humans and destruction of the environment [5]. Like many agricultural chemicals, 2,4-D application can have both positive and negative effects on the environment and human health. 2,4-D impact on soil, microorganisms, crop yield, and human health are examined in detail here.
2, 4-D impact on soil
Like many herbicides, 2, 4-D can affect soil quality in a variety of ways based on environmental factors, application frequency, and concentration. The possible effects are:
Soil toxicity and residual effects
Depending on variables such as soil texture, temperature, and moisture, 2,4-D can persist in the soil for a few days to several months. 2,4-D generally degrades more quickly in warm, humid circumstances, but it can also decompose more slowly in dry, cold ones. If the herbicide leaches or remains in the soil, persistent residues may have an impact on the growth of non-target plants [18].
Soil pH and nutrient availability
High levels of 2,4-D can change the pH of the soil and influence the availability of some nutrients [1], its impact on soil nutrient cycles and microbial populations can influence overall soil fertility.
2,4-D impact on soil microorganisms
The decomposition of organic matter, the cycling of nutrients and the general health of the soil depend on soil microbes. The effects of 2, 4-D on soil microbiota depend on factors such as dosage, soil type, and microbial diversity. Here are the key impacts:
Inhibition of microbial growth
Higher concentrations of 2, 4-D have the potential to disrupt the nitrogen cycle and decrease soil microbial diversity by inhibiting the growth of soil bacteria and fungi. Some research indicates that bacteria involved in nitrogen fixation, such as Rhizobium species, may be sensitive to 2,4-D, which could lower soil fertility [23].
Effect on decomposition and organic matter
2,4-D can slow down the decomposition of soil organic matter and lower the general health of the soil ecosystem by influencing microbial populations that decompose plant residues [32].
2, 4-D impact on crop yield
Depending on the particular crop being treated and the method of herbicide application, 2, 4-D has various effects on crop production. Among the important points are:
Target crop damage
2,4-D can harm crops by drift or overapplication when applied at inappropriate concentrations or during highly susceptible growth stages, resulting in lower yields [20].
Impact on human health
Because of worries about its possible health effects, 2,4-D has been the focus of a great deal of research. Possible risks include:
Carcinogenicity
The International Agency for Research on Cancer (IARC) classified 2,4-D as a Group 2B carcinogen (possibly carcinogenic to human [27].
Acute toxicity
Symptoms of acute 2,4-D exposure include headaches, nausea, vomiting, dizziness, and in rare instances, severe poisoning. However, inappropriate handling or severe overexposure such as spills or disregard for safety precautions during application are typically the cause of these consequences [15].
Therefore, highly efficient methods for cleaning up contaminated environments must be developed. A number of techniques, such as chemical precipitation, oxidation-reduction, filtration, ion exchange, and electrochemical treatment, can eliminate 2,4-D from soil, water, and air [18], but their high cost and hazardous nature have become major constraints [4]. Thus, biodegradation by the microbes is a more cost-effective and environmentally friendly method for the clean-up of sites contaminated with pesticides [2].
Numerous studies on 2,4-D bioremediation have used microbes [22], but no bacteria strains from termite mound soil have been characterized or identified that may be able to promote its degradation. Therefore, the primary objective of the current study was to isolate, characterize, and identify 2,4-D-degrading bacteria strains from termite mound soil and their contribution to the field of bioremediation.
Materials and methods
Pesticide used
In this investigation, technical-grade 2,4-D (Sigma-Aldrich, Malaysia) that was 98% pure was utilised. The physical properties of the 2,4-D are reported in Table 1. All other chemicals used in this study were of high purity and used without further purification.
Table 1.
Physicochemical properties of 2,4-D
| Molecular formula | C8H6Cl2O3 |
| Molecular weight | 221.03 g |
| Appearance | Colorless or white solid |
| Water solubility | 900 mg/L at 25 °C:500 mg/L at 20 °C |
| pKa | 2.73 |
|
Log Kowa of 0.001 M solution at 25 °C (unbuffered solution) |
pH 5:2.14; pH 7: 0.177; pH 9: 0.102 |
KowaOctanol -water partition coefficient
Termite mound soil sample collection and description of the study sites
TMS samples were collected from Meki town, East Shewa zone, and Oromia region, Ethiopia (Fig. 1). Termite mound soil samples were collected in plastic bags and correctly labeled, then transported in aseptic conditions to the Microbial Biotechnology Laboratory at the National Agricultural Biotechnology Research Center (NABRC) for further processing and microbial isolation.
Fig. 1.
The geographical location of TMS sample collections
Isolation of 2,4-D degrading bacteria by soil enrichment
In order to isolate 2,4-D-degrading bacteria, the minimal salt medium (MSM) was made using the following components: MgSO4 0.2 gm/L, (NH4)2SO4 (0.5 gm/L), KH2PO4 (0.5 gm/L), NaOH (0.02 gm/L), ZnSO4 (0.004 gm/L), CuSO4 (0.001 gm/L), Na2SO4 (0.0001 gm/L), Na2MoO4 (0.001 gm/L), CoCl2 (0.0001 gm/L), MnSO4 (0.0004 gm/L), H2SO4 (0.5 mL) at pH (7) and as carbon and energy source, 500 mg/L 2,4-D was added [13]. All the nutrients were sterilized by autoclaving at 121 °C for 15 min. After sterilization, 10 g of termite mound soil samples were added to 100 ml of MSM with 500 mg/L of 2,4-D herbicide and incubated on a rotary shaker at 150 revolutions per minute at 30 °C for 7 days. Then, 10 mL of the enrichment culture from turbid flasks (indicating 2,4-D degradation) was transferred to 100 mL of fresh MSM containing 500 mg/L of 2,4-D and incubated for 7 days. After successive subculturing, 0.1 ml of culture broth was pipetted and spread on 2,4-D supplemented agar media, then incubated at 30 °C for three days. Several colonies had been picked considering the colony characteristics (size, shape, colour, texture, etc.) [19]. Then subsequent plating was conducted until the colonies and cells were found to be uniform and pure, respectively [8, 17]. The pure colonies were kept at 4 °C for further investigation.
Morphological and biochemical characterization of bacterial isolates
Morphological and biochemical characterization was done according to Bergey’s Manual of Systematic Bacteriology [11, 12, 16]. Biochemical tests done on the isolates included catalase, oxidase, indole, urease, citrate, MR and lactose.
Screening of 2,4-D degrading bacteria
To screen promising isolates, five isolates were grown in triplicate in 100-ml Erlenmeyer flasks containing a mixture of 10 ml of MSM and 2,4-D. The inoculum preparations were conducted in 1:50 inoculum-to-medium ratios. Then the cultures were incubated at 37 °C for three days. After incubation, to obtain the supernatants, the aliquots were centrifuged for 5 min at 6000 rpm [14], and the amount of 2,4-D that remained in the solution was determined using UV-Vis spectrophotometers. For expected biodegradation testing, the cell pellet was maintained at 4 °C.
Total degradation (%) was estimated using Eq. (1) [24].
 |
where AO is the value of the initial absorbance of 2,4-D acquired at 282 nm, while A is referred to as the final absorbance denoting the absorbance of 2,4-D after biodegradation.
Effects of temperature and initial pH on the selected bacterial growth
The effect of pH on bacterial growth was studied with 10 µL of isolated bacteria in 100 µL of nutrient broth at varying pH (2, 3.5, 4.5, and 6) and incubated at 37 °C for 24 h. Similarly, for temperature optimization, bacterial isolates were grown in the nutrient agar at various temperatures (15 °C, 25 °C, 35 °C, and 45 °C) at pH 7 and kept under static conditions along with controls for 24 h. After the completion of the incubation period, bacterial growth was measured in terms of optical density (OD) using a UV/Vis spectrophotometer (BMS UV-160) at 600 nm [21].
Identifications of the selected bacterial isolate
The bacterial isolate showing a higher percentage of 2,4-D degradation was subjected to morphological and biochemical characterization as mentioned in Bergey’s Manual of Systematic Bacteriology [11, 16]. Furthermore, the bacterial isolate was identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Samples for the MALDI-TOF instrument were generated using the direct transfer (DT) approach, in which a single colony of biological material was transferred directly as a thin film on a MALDI spot plate derived from an aqueous PCA petri dish that had been pre-incubated for an overnight period. The standard solvent was made up of 2.5% trifluoroacetic acid, 47.5% water, and 50% acetonitrile. The matrix utilised was HCCA (α-cyano-4-hydroxycinnamic acid). The biological material was coated with one microliter of the HCCA matrix solution. At RT, the spotted layers were allowed to air dry. The target layer was then placed onto a MALDI-TOF device, and an AutoFlex mass spectrometer that was attached to the MALDI device was used to examine the mass spectrum that had been automatically created from the samples [25].
Biodegradation activities of the selected isolates
Agricultural leachate containing 2,4-D was collected from a near-teff farm in the Legedadi area, Sendafa sub-catchment, Dabe Muda Godo kebele, where the 2,4-D content was 52.1 mg/L, with a pH of 6.67. 100 ml of MSM with 52.1 mg/L of 2,4-D herbicide inoculated with an inoculum size of 1 ml (3 × 108 CFU/ml) in standard conditions. The inoculum was kept under standard circumstances for 16 days, with a temperature of 25 degrees Celsius and a rotating speed of 150 revolutions per minute. A control flask was kept under the same conditions but was not inoculated. 5 mL sample volumes were centrifuged at 6000 rpm for 5 min to detect 2,4-D residues at various intervals [14]. From the supernatant, the residual 2,4-D herbicide concentration was then determined at 282 nm with an “Agilent Cary 60 UV/Visible spectrophotometer.” Similar experimental methods and procedures were used with synthetic wastewater.
Data analysis
Using SAS version 9, a one-way analysis of variance (ANOVA) was performed. The Tukey’s test was used to determine a significant difference between the treatment means at the P˂0.05 level of significance.
Results and discussion
Termite mound soil characterization
The collected termite mound soil samples were characterized, and the results are presented in Table 2.
Table 2.
Termite mound soil characteristics
| Parameter | Values |
|---|---|
| pH | 6.5 |
| P2O5 (%) | 0.33 |
| Total C (%) | 2.3 |
| Total S (%) | ND |
| Total N (%) | 0.13 |
ND Indicate not detected
Table 2 illustrates how the termite mound soil sample offers a rich substrate for bacterial isolation, with a potentially diversified bacterial community supported by high organic carbon content. The most prevalent bacterial groups will depend on the low amounts of nitrogen and sulfur, moderate phosphorus, and minor acidity. A variety of microbial species can be predicted by knowing these chemical characteristics.
Isolation of 2,4-D degrading bacteria by soil enrichment and the most promising bacteria selection
Using a soil enrichment procedure [21], five bacterial isolates (GT7, GT3, GT1R2, GT1, and GT8) were selected on the basis of morphological and biochemical characterization (Table 3), and the five isolates were tested to screen the most promising isolates for 2,4-D degradation. Figure 2 displays the degradation capability of the five bacterial isolates (GT7, GT3, GT1R2, GT1, and GT8) tested for the removal of 2,4-D from an aqueous solution. Among these isolates, GT7 exhibited the highest degradation ability, achieving a removal rate of 62.1%. This result indicates that GT7 is the most promising isolate for the degradation of 2,4-D.
Table 3.
Morphological and biochemical characteristics of 2,4-D-degrading isolates
| Characteristics | 2,4-D degrading isolates | ||||
|---|---|---|---|---|---|
| Morphological | GT7 | GT3 | GT1R2 | GT1 | GT8 |
| Colony color | White | Grayish-white | White | Grayish-white | White |
| Gram nature | - | - | - | - | - |
| Cell morphology | Rod Shape | plump rod -shape | Straight | plump rod –shape | Rod Shape |
| Biochemical | |||||
| Catalase | + | + | + | + | + |
| Oxidase | - | - | + | - | - |
| Indole | - | - | - | - | - |
| Urease | - | - | - | - | - |
| Citrate | + | - | + | - | + |
| MR | - | - | - | - | - |
| Lactose | + | - | - | - | + |
-, and + means Negative and Positive respectively
Fig. 2.
Different bacteria isolates used for the degradation of 2, 4-D
These results are in line with earlier research that identified and isolated bacterial strains that can break down herbicides like 2,4-D. For instance, similar screening methods for bacterial isolates were described by [21], who found potential candidates for herbicide breakdown. Similar to the methods used here, isolates were evaluated in that study based on their capacity to use 2,4-D as a sole carbon source. Although [21] found a number of strains with significant degrading potential, the maximum removal rates attained were less than the 62.1% found in our GT7 investigation. This implies that, in comparison to other strains found in earlier studies, GT7 might have distinct enzymatic pathways or higher metabolic efficiency for 2,4-D breakdown.
As shown in Table 3, the present study identified five distinct isolates (GT7, GT3, GT1R2, GT1, and GT8) capable of degrading the herbicide 2,4-D, which is an important characteristic given the environmental persistence of this compound. Our morphological and biochemical results showed notable diversity among these isolates, which aligns with previous findings in the field, where microbial degradation of 2,4-D has been attributed to various bacterial species with different phenotypic traits.
Morphological characteristics
The isolates exhibited a range of colony colors, including white and grayish-white. This variation in colony morphology could be indicative of the diversity in the metabolic pathways utilized by these microorganisms to degrade 2,4-D. For instance, isolates GT7, GT1R2, and GT8 formed white colonies, while GT3 and GT1 produced grayish-white colonies. Such variability is not uncommon in studies investigating pesticide-degrading bacteria. For instance, a study found that several 2,4-D-degrading bacteria produced colonies with unique color profiles, which they linked to the synthesis of various pigments or secondary metabolites connected to degradation pathways [28].
Biochemical properties
All the isolates in our study tested positive for catalase, suggesting that they may possess protective mechanisms against oxidative stress, a common trait in organisms exposed to environmental pollutants like 2,4-D. Catalase activity has been commonly linked to the ability of bacteria to survive in environments with high oxidative conditions [26]. However, the variation in other biochemical traits, such as oxidase and citrate utilization, points to metabolic diversity among the isolates.
For example, GT3 and GT1R2 were oxidase-negative, while GT1R2 and GT8 were citrate-positive, which may suggest differences in their metabolic flexibility and potential for adapting to various substrates beyond 2,4-D. These findings are consistent with those of [3], who observed that 2,4-D degrading strains showed varied patterns of biochemical activity, which they attributed to different degradation pathways being utilized.
pH and temperature effect on the growth range of Gt7
At different pH levels and temperatures, the (GT7) culture density was` examined. The findings revealed that the optimal pH was 3.5, but that appropriate growth densities were also seen over a wide pH range of 2 to 6 (Table 4; Fig. 3). Maximum OD (Optical Density) was seen for GT7 at a temperature of 25 °C (0.5107), whereas culture density was observed at 15, 35, and 45 °C (Table 5; Fig. 4).
Table 4.
Effect of pH on GT7 bacterial growth
| Treatments (pH) | OD (Optical Density) |
|---|---|
| T1 (pH 2) | 0.3350B |
| T2 (pH 3.5) | 0.5724A |
| T3 (pH 4.5) | 0.4045B |
| T4 (pH 6) | 0.3650B |
| LSD (0.05) | 0.1118 |
| CV (%) | 13.34 |
| R2 | 0.86 |
Means with the same letter are not significantly different at 5% probability level
Fig. 3.
Effect of pH on GT7 growth at A600 nm. Results represent the means of three experiments
Table 5.
Effect of temperature on GT7 bacterial growth
| Treatments (Temperature) | OD (Optical Density) |
|---|---|
| T1 (15°c) | 0.3027B |
| T2 (25 °c) | 0.5107A |
| T3 (35 °c) | 0.3197B |
| T4 (45 °c) | 0.3137B |
| LSD (0.05) | 0.02 |
| CV (%) | 2.8 |
| R2 | 0.9937 |
Means with the same letter are not significantly different at 5% probability level
Fig. 4.
Effect of temperature on GT7 growth at A600 nm. Results represent the means of three experiments
Identification of the isolate (GT7)
The isolate showing a higher percentage of degradation in UV/Vis Spectrophotometer analysis (GT7) was identified using matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). The results revealed that the test isolate (GT7) is Enterobacter cloacae. Some bacteria, such as Bacillus, Pseudomonas, Nocardia, and Streptomyces nitrospirae, Cyanobacteria, Bacteroidetes, Spirochaetes, Actinobacteria, and Fibrobacteres, Candidate division TM7, Chloroflexi, Proteobacteria, WCHB1-60, Elusimicrobia, Planctomycetes, Spirochaetes, Chlorobi, Gemmatimonadetes, Armatimonadetes, Acidobacteria, SM2F11, Firmicutes, Candidate division WS3 [10], were identified from termite mound soil in the previous report, but there is no Enterobacter clocea report yet.
2,4-D biodegradation by (GT7) Enterobacter cloacae
With and without Enterobacter cloacae, we assessed the patterns of degradation in 2,4-D-contaminated distilled water and actual waste water (Figs. 5 and 6). In 2,4-D-contaminated distilled water and real waste water with Enterobacter cloacae incubated for 14 days, 86% and 90.4% of 2,4-D were degraded in 2,4-D-contaminated distilled water and real waste water, respectively. Moreover, the incubation was done without Enterobacter cloacae, and the result revealed that no 2,4-D degradation occurred in 2,4-D-contaminated distilled water, but in real water, 9.4% of 2,4-D degraded. 2,4-D degradation in real waste water without Enterobacter cloacae indicated that other indigenous microorganisms showed some 2,4-D degradation ability. Many microorganisms have been isolated from nature that have the capacity to degrade 2,4-D, which include Acinetobacter sp., Stenothrophomonas maltophilia, and Flavobacterium sp [30]. In this study, Enterobacter clocea was isolated from termite mound soil and had never been reported as being able to degrade 2,4-D before.
Fig. 5.
2,4-D degradation percentage in synthetic waste water with Enterobacter cloacae and (control) without Enterobacter cloacae
Fig. 6.
2,4-D degradation percentage in real waste water (agricultural leachate) with Enterobacter cloacae and (control) without Enterobacter cloacae
Conclusion
The present study has found that isolate GT7, identified as Enterobacter cloacae, has the maximum potential for degrading the 2,4-D herbicide. This particular isolate demonstrated a 90.4% degradation pattern of 2,4-D at a temperature of 25 °C and a pH of 3.5. These findings suggest that Enterobacter cloacae could be a strong candidate for degrading 2,4-D found in agricultural leachate. Therefore, this isolate has the potential to be used in the biodegradation of agricultural leachate contaminated with 2,4-D. The strain isolated from termite mound soil shows promise and can be further explored for potential industrial-scale applications.
Acknowledgements
We would like to thank Ethiopian Institute of Agricultural Research (EIAR), National Agricultural Biotechnology Research Center, Microbial Biotechnology Research Program and Wudassie Diagnostic Center for providing us with support and their laboratory facilities.
Authors’ contributions
Yalemtsehay Debebe (the first author) investigated the research, wrote up the manuscript, and conducted experiments in the field and laboratory. Zemene Worku (second author) supervised the experiment and read, edited, and approved the final manuscript. Adaba Tilahun (third author) conducts the experiment in laboratory. Esayas Alemayehu (the fourth author) supervised the experiment, read, edited, and approved the final manuscript. All the authors made significant contributions to the document and agreed to its publication.
Funding
This research received no external funding.
Data availability
The data used to support the findings of this study are included within the article.
Declarations
Ethics approval and consent to participate
Not applicable.
Consent for publication
Not applicable.
Competing interests
The authors declare no competing interests.
Footnotes
Publisher’s note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
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Data Availability Statement
The data used to support the findings of this study are included within the article.