Atrazine Bioremediation and Its Influence on Soil Microbial Diversity by Metagenomics Analysis

Abstract

Pesticide accumulation in agricultural soils is an environmental concern, often addressed through distinct bioremediation strategies. This study has tried to analyze various soil bioremediation options viz., biostimulation, bioaugmentation, and natural attenuation in terms of efficiency and the response of autochthonous microbial flora by using atrazine as a model contaminant. Soil mesocosms were established with 100 kg of soil simulating the field conditions. The soil previously exposed to the herbicide was used for the bioaugmentation strategy undertaken in this study. We have tried to analyze how the microbial community responds to a foreign compound, both in terms of taxonomic and functional capacities? To answer this, we have analyzed metagenome of the mesocosms at a time point when 90% atrazine was degraded. Bioaugmentation for bioremediation proved to be efficient with a DT90 value of 15.48 ± 0.79 days, in comparison to the natural attenuation where the DT90 value was observed to be 41.20 ± 1.95 days. Metagenomic analysis revealed the abundance of orders Erysipelotrichales, Selemonadales, Clostridiales, and Thermoanaerobacterales exclusively in SBS mesocosm. Besides Pseudomonas, bacterial genera such as Achromobacter, Xanthomonas, Stenotrophomonas, and Cupriavidus have emerged as the dominant members in various bioremediation strategies tested in this study. Inclusive results suggest that inherent microbial flora adjust their community and metabolic machinery upon exposure to the pollutant. The site under pollutant stress showed efficient microbial communities to bio-remediate the newly polluted terrestrial ecologies in relatively less time and by economic means.

Electronic supplementary material

The online version of this article (10.1007/s12088-020-00877-4) contains supplementary material, which is available to authorized users.

Artificially synthesized chemicals such as pesticides have brought a revolution in agricultural production but at the same time raised various health and environmental concerns [1]. Among the man-made pesticides, atrazine was chosen as a model compound for this study, as it is an extensively used herbicide in the cultivation of sugarcane, corn, and cotton in several countries [2]. It is known to cause damaging effects on the organelle, mitochondria in different mammalian cells including human [3, 4]. Indigenous microorganisms that inhabit the soil play key roles in processes such as nutrient cycling, bioremediation, bioleaching, etc. [5]. Owing to the efficiency and versatility of the microbial network, a polluted environment could be restored effectively to its original form via bioremediation [6, 7]. However, the information regarding factors controlling bioremediation is limited, which impairs its effective implementation. Advancements in molecular and genomic research have provided tools and techniques to understand and improve the bioremediation processes [2, 8]. Recently, high throughput DNA sequencing techniques (HTS) have been extensively used to understand the microbiome in polluted soils. Through metagenomic studies, soil microbiome in different niches and the genetic mechanism responsible for bioremediation of polluted soils were studied greatly [9].

In the present study, the shift in microbial diversity via metagenome analysis under the impact of atrazine was examined. The work plan undertaken in this study is summarized in Fig. S1. Five mesocosms were established to analyze three different bioremediation strategies for their atrazine biodegradation efficiency; (1) bioaugmentation (BA), (2) biostimulation using mustard seed meal (SBS), biostimulation using molasses (MBS), biostimulation using citrate (CBS) and (3) natural attenuation (NA). Degradation kinetics and metagenomic analysis were performed for all mesocosms along with the control soil sample. Details of the five mesocosms set-up and subsequent methods followed in this study were enclosed in the Supplementary data.

The bioaugmentation strategy made use of soil, previously exposed to the target herbicide as inoculum instead of using traditional bacterial inoculant. The hypothesis behind this strategy comes from combining the experience of previous studies, where we have demonstrated that soil acquires atrazine metabolizing potential on exposure to the herbicide aiding natural attenuation [2]. Atrazine degradation pathways/genes have been recently assembled since, atrazine is a relatively new pesticide and pathways for its degradation are still evolving [10].

Biodegradation efficiency of three bioremediation options such as bioaugmentation, biostimulation, and natural attenuation was analyzed in five different mesocosms. Biodegradation of atrazine was monitored by assessing two parameters in mesocosm studies; (1) removal of atrazine, and (2) levels of the intermediate cyanuric acid. Figure 1 demonstrates the levels of atrazine and its intermediate, cyanuric acid in all mesocosms over time. As seen in the figure, bioaugmentation with soil having a history of atrazine use, proved to be an efficient treatment strategy, with 60% atrazine removal in the first 7 days and complete removal of atrazine by 21 days. Biostimulation with molasses also proved to be efficient, with 95% atrazine removal in 21 days. However, in the case of biostimulation with molasses, both target compounds, atrazine, and cyanuric acid, could not be detected at all in 35 days.

Fig. 1.

Degradation profile of atrazine and cyanuric acid with respect to time in the mesocosms

All biostimulation treatments and bioaugmentation demonstrated complete removal. Natural attenuation indicated that the soil microbiome possesses an inherent potential for atrazine biodegradation but the natural process was slow and hence, some treatment options were required to speed up the process. Of all the mesocosms, the biostimulation with mustard seed meal (SBS) took the longest time (more than 35 days) for complete removal of atrazine. Kinetic analysis indicated that atrazine removal rate followed the single first-order kinetics (Table 1). The degradation rate for 90% atrazine removal, DT90 was observed 12.14 ± 0.51 days for MBS; 15.48 ± 0.79 days for BA; 17.75 ± 0.89 days for CBS; 25.90 ± 0.48 days for SBS and 41.20 ± 1.95 days for NA mesocosms. C:H:N:S analysis results in Table S1 may give us a clue about the efficiency pattern in the biostimulation experiments. While the carbon content of both additives is similar, molasses had lower nitrogen content. We hypothesize that since atrazine is mainly utilized as a nitrogen source, the seed meal supplement could have hindered the utilization of atrazine, while molasses promoted atrazine uptake in soil microbes. These results are in alignment with our previous studies where a three-membered consortium consisting of Arthrobacter sp. AK_YN10, Pseudomonas sp. AK_AAN5, and Pseudomonas sp. AK_CAN1 was used for bioaugmentation, that exhibited 90% atrazine degradation within the first six days [11].

Table 1.

Degradation kinetics values obtained by single first-order rate reaction

Sample	Rate constant (k) day−1	DT50	DT90
SBS	0.089 ± 0.0017	7.79 ± 0.14	25.90 ± 0.48
CBS	0.1297 ± 0.0064	5.34 ± 0.27	17.75 ± 0.89
MBS	0.1896 ± 0.0083	3.66 ± 0.15	12.14 ± 0.51
BA	0.1487 ± 0.0077	4.66 ± 0.23	15.48 ± 0.79
NA	0.0559 ± 0.0027	12.40 ± 0.59	41.20 ± 1.95

k Rate constant of decline 1/days

Illumina platform-based whole metagenome sequencing resulted in six metagenomic datasets for the mesocosms set up in this study. The metagenomic reads were examined for their phylogenetic and functional genes [12]. Taxonomic assignments revealed the dominating bacterial domain across all the metagenomes as seen in Table S2 and Fig. S2a. 99.83%, 99.75%, 99.88%, 99.48%, 99.43% and 99.87% of bacterial annotations were observed for samples SBS, CBS, MBS, BA, NA, and control sample respectively. Archaea sequences were completely absent in CBS, MBS and control samples. Proteobacteria were the most abundant phyla observed for all samples ranging from 84 to 96% abundance, followed by Firmicutes with 1–13% abundance and a few unclassified bacteria with 1–2% abundance (Fig. S2b). In comparison to the control sample, there was a significant decrease in Proteobacteria abundance in BA, and NA samples, whereas Firmicutes exhibited increased abundance in MBS, BA, and NA mesocosm samples. The rarefaction curve and PCoA plot for the metagenomic datasets are depicted in Figs. S3 and S4, which reveal the diversity in mesocosm microbial populations.

The change in fold abundance of bacterial orders categorized under Firmicutes and Proteobacteria phyla with respect to the control sample is depicted in Fig. S5a and b. In SBS mesocosm, with respect to the control sample, order Bacillales decreased whereas, orders Erysipelotricales and Clostridiales exhibited remarkably increased fold abundance (Fig. S5). This could probably be attributed to additional natural organics which enter the mesocosm.

Variations in the microbial populations of mesocosms were analyzed using the word cloud charts in comparison to the control mesocosm dataset (no perturbations by any addition, neither of herbicide nor nutrient) at the genus level (Fig. S6). Top 30 dominating bacterial genera of all mesocosm samples were retrieved and analyzed with reference to the control sample. In the control sample, Pseudomonas, Acinetobacter, and Ochrobactrum constituted 74.7% of the total microbial community, but distinct changes were observed in other treatments owing to external substrate received as a carbon source by the soil system (Fig. S6). Bacterial genera such as Achromobacter in MBS and SBS samples, Xanthomonas in SBS and CBS samples, Stenotrophomonas and Cupriavidus in BA sample were observed as dominating members in the microbial community. In the control sample, the 30 dominating genera constituted majority of the control sample (92.9%) whereas the top 30 dominating genera accounted for approximately 56-77% of the total microbial population in mesocosm samples, indicating a diversified microbial population in these samples.

The presence of catabolic genes involved in the degradation of benzoate, chlorocyclohexane, chlorobenzene, atrazine, nitrotoluene, aminobenzoate, dioxin, drug metabolism-other enzymes, caprolactam, steroid, chloroalkane, and chloroalkene compounds was confirmed for garden soil sample (Fig. S7). The mesocosms samples with different treatments revealed new degradation pathways which were previously absent before atrazine application (control sample), namely 1,1,1-Trichloro-2,2-bis(4-chlorophenyl) ethane, xylene, and toluene degradation pathway in MBS mesocosm, polycyclic aromatic hydrocarbon degradation pathway in SBS and BA mesocosms, and styrene and fluorobenzoate degradation pathway BA and NA mesocosms (Table S3). Metagenomic sequences were also analyzed for aromatic compound degrading; peripheral, central and anaerobic pathways using subsystem database and percentage abundance of the genes and pathways present in the six metagenomes can be viewed in Fig. S8.

The number of hits obtained and the diversity of the genes reported for atrazine degradation pathways were listed in Table S4. Figure S9 depicts the abundance of atrazine degrading pathways genes of the six metagenome samples along with the intermediate molecule of the degradation pathways. Bacterial species reported to carry atrazine degrading genes and the number of hits obtained in the six metagenome samples was enlisted in Table S5. It was observed that atzC, atzD, and thc genes were not detected in control samples although the genera reported were present in the sample. The genes responsible for the conversion of N-isopropylammelide to cyanuric acid and ammelide to cyanuric acid are present only in sample BA and NA (Fig. S9). Moreover, the genes for the initial steps of atrazine conversion to deisopropylatrazine or deethylatrazine were absent in the control sample. These genes were present in all other five treatment samples in equal proportions which indicates that microbial adaptation is an integral trait, and it can be successfully exploited in bioremediation of pesticide-contaminated niches. Certain genes could not be detected in sequence data (atzC), but all mesocosms exhibited efficient atrazine degradation.

The bioaugmentation strategy made use of soil having a history of pollutant, which accelerated the process in terms of time and also led to a reduction in the monitoring events. The results of this study recommends the method of bioaugmentation for efficient atrazine degradation. Low nitrogen supplement (molasses) exhibited better removal efficiency than a rich nitrogen supplement (seed meal). These degradation studies demonstrated that nitrogen levels dictate atrazine utilization efficiency. The application of microbes, which are capable of metabolizing the xenobiotic compound can prove to be very feasible, especially in cases requiring in situ soil bioremediation. Bioaugmentation strategy has proved to be an economic and rapid path to pollutant removal especially when the degradation of pollutant followed first-order kinetics as observed in this study.

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Funding

Funding from Department of Biotechnology, Ministry of Science and Technology, New Delhi is acknowledged.

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

The study does not involve any human participants or animals in this study.