Next Article in Journal
Simulation of Water Level and Flow of Catastrophic Flood Based on the CNN-LSTM Coupling Network
Previous Article in Journal
Zooplankton in the Technogenic Aquatic Ecosystem of the North Crimean Canal, the Longest in Europe
Previous Article in Special Issue
Photocatalytic Degradation of Methylene Blue Dye from Wastewater by Using Doped Zinc Oxide Nanoparticles
 
 
Search for Articles:
Title / Keyword
Author / Affiliation / Email
Journal
Article Type
 
 
Advanced Search
 
Section
Special Issue
Volume
Issue
Number
Page
 
Journals
Water
Volume 15
Issue 13
10.3390/w15132328
water-logo
Submit to this Journal Review for this Journal Propose a Special Issue
► Article Menu

Article Menu

share Share announcement Help format_quote Cite question_answer Discuss in SciProfiles thumb_up
...
Endorse textsms
...
Comment
first_page
settings
Order Article Reprints
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Background:
Article

Synergistic Removal of β-Hexachlorocyclohexane from Water via Microorganism–Plant Technology and Analysis of Bacterial Community Characteristics

by
Huijun Shi
1,
Shuang Luo
1,
Yanpeng Liang
2,3,*,
Litang Qin
1,2,
Honghu Zeng
1,3,* and
Xiaohong Song
1,2
1
College of Environmental Science and Engineering, Guilin University of Technology, Guilin 541004, China
2
Guangxi Key Laboratory of Environmental Pollution Control Theory and Technology, Guilin University of Technology, Guilin 541004, China
3
Collaborative Innovation Center for Water Pollution Control and Water Security in Karst Region, Guilin University of Technology, Guilin 541004, China
*
Authors to whom correspondence should be addressed.
Water 2023, 15(13), 2328; https://doi.org/10.3390/w15132328
Submission received: 4 May 2023 / Revised: 13 June 2023 / Accepted: 20 June 2023 / Published: 22 June 2023
(This article belongs to the Special Issue Application of Sustainable Chemical and Biological Methods for Pollutants Removal from Water)
Browse Figures
Review Reports Versions Notes

Abstract

:
In recent years, β-Hexachlorocyclohexane (β-HCH) has been detected frequently in water, seriously threatening human health and ecological balance. To explore the effects of different treatment groups on the removal of β-HCH in experimental water and the response of microbial community structure in the system, three strains of β-HCH-degrading bacteria—Ochrobactrum sp. (Och1, Och2) and Pseudomonas sp. (Pse1)—combined with Canna were selected for microbial, plant, and microbe–plant repair hydroponic experiments, respectively. Solid-phase extraction combined with GC-ECD and high-throughput sequencing determined the β-HCH content and bacterial community in water and Canna tissues. The results showed that when β-HCH stress concentrations were 10 μg·L−1 and 100 μg·L−1, Och1 and Pse1 showed the best degradation performance (33.49% and 60.02%, respectively). Following this, the three degrading strains were combined with Canna. Under the two β-HCH stress concentrations, the combination of Och1–Canna showed the highest β-HCH removal efficiency (96.74% and 99.06%). At the same time, we measured the concentration of β-HCH in Canna tissues and found that Och1 had a better removal effect on β-HCH in water and that the addition of Pse1 may significantly improve the absorption capacity of β-HCH in Canna roots. In addition, the relative abundance of Methophilic bacteria in experimental water and Canna root samples increased significantly after the inoculation of degrading bacteria, suggesting that Methophilic bacteria may be vital in degrading benzene-ring-containing substances. The results of this research can provide a theoretical basis and technical support for the prevention and control of the non-point source pollution of organic pesticides.

1. Introduction

Hexachlorocyclohexanes (HCHs) are specific organochlorine pesticides (OCPs), and China used to be the primary producer and user of HCHs [1]. In 1991, it was found that γ-HCH in technical hexachlorocyclohexane was the main insecticide component; thus, the production and use of HCHs in China gradually decreased [2]. However, due to their characteristics of lipophilicity, high toxicity, persistence, and limited biodegradability, many isomers of hexachlorocyclohexane persist at the production site and migrate through environmental carriers, having harmful effects on human health and the ecological environment [3,4]. The chlorine atom in HCHs makes these organochlorine compounds highly stable in the environment, and of these, β-HCH is the most stable and difficult to degrade [5]. At present, HCHs are still commonly detected in surface water, groundwater, rivers, and oceans, with a detection rate of almost 100%, about 1.1~14.8 ng·L−1 [6]. Studies have shown that HCH concentrations of 4.72~11.19 ng·g−1 have also been detected in aquatic plants [7]. HCHs have caused global environmental pollution, ecological imbalance, and other problems [8]; therefore, efficient and reliable techniques are urgently needed to remove HCHs from water.
The physical and chemical methods used in the application of pesticide removal have shortcomings, as they are difficult to implement, are costly, have poor environmental safety due to secondary pollution, and are unsustainable; thus, it is difficult to employ them for large-scale use [9]. Microbe–plant joint remediation technology overcomes the limitations of traditional treatment processes and makes the remediation process more environmentally and economically beneficial. However, the combination of indigenous microorganisms and plants often has a limited ability to degrade specific pollutants; thus, the injection of exogenous high-efficiency degrading strains can improve the problem of pesticide contamination [10]. This green and sustainable remediation technology can accelerate natural degradation and reduce secondary pollution [11,12]. Specific contaminant-degrading bacteria are crucial for successful remediation, and studies have shown that this method is possible. Kurashvili et al. (2018) [13] used Pseudomonas sp. TBM6 and Pseudomonas sp. 4JL50 combined with soybeans to reduce the amount of DDT in the soil by 80%. Khan et al. (2014) [14] used Pseudomonas PD1-willow to degrade phenanthrene in soil, and the degradation rate of phenanthrene increased from 25% to 40% compared to the control group. This may be attributed to the fact that exogenous high-efficiency degrading bacteria degrade toxic pesticides into simple inorganic substances, and the dehalogenation products formed by microorganisms after pesticide degradation are absorbed by plants and are further oxidized by enzymes involved in degradation to improve the removal efficiency of pollutants in the system [15,16]. Moreover, in aquatic ecosystems, microbial communities originate mainly from environmental water, but vegetation can change the structures of plant rhizosphere microbial communities. Microorganisms colonize the rhizosphere to form biofilms, and they grow and reproduce through the utilization of nutrients, such as oxygen and organic carbon, secreted by the rhizosphere to accelerate the degradation of organic pollutants [17,18], thus forming microbial communities different to those in the water environment.
In previous studies, we determined the enrichment capacity of Canna indica L. for β-HCH and screened three strains of bacteria that can effectively degrade β-HCH in the soil near the roots of Canna [19]. In the inorganic culture medium containing 1 g yeast powder, after 5 days, the average degradation rates of β-HCH for 10 μg·L−1 and 100 μg·L−1 were 66.31%, 67.38, 62.68%, and 61.61%, 64.07%, 59.26%, respectively [20]. Many studies have shown that microorganisms and plants are effective in removing pesticides from soil [14,21], but few studies have determined their effect in removing water pollution. This study established a small-scale hydroponic system of biodegradable bacteria and Canna to determine the optimal combination of different biodegradable bacteria and Canna to remove β-HCH from water. Meanwhile, the changes in the microbial communities in water and Canna rhizosphere under the stress of β-HCH were explored, providing a scientific basis for the ability of functional biodegradable bacteria and plant systems to repair water β-HCH pollution.

2. Materials and Methods

2.1. Experimental Materials

Enrichment medium: peptone, 0.6 g; NaCl, 0.1 g; KH2PO4, 0.1 g; reverse osmosis (RO)-water, 1 L.
Beef extract peptone medium: beef extract, 3 g; peptone, 10 g; AGAR powder, 15 g; NaCl 5 g, reverse osmosis (RO)-water, 1 L.
Inorganic culture medium: KH2PO4, 2 g; NH4Cl, 1 g; K2HPO4, 7.5 g; NaCl, 0.5 g; MgCl, 0.1 g; (RO)-water, 1 L.
Main reagents: β-HCH standard solution (purity > 99.0%) was purchased from the Ministry of Ecology, Agriculture and Environment, China. Ethyl acetate and methylene chloride were purchased from Jingchun Biochemical Technology Co., Ltd., Shanghai, China. Acetone and methanol were purchased from TEDIA Company, Ohio, United States of America. All the chemicals used were chromatographically pure.

2.2. Preparation of β-HCH-Degrading Bacteria

For the strain-screening method, a mixture of mud and water from the rhizosphere of Canna in a β-HCH-contaminated area was cultured in 100 mL of enrichment medium for 48 h, diluted 10−6 times, and coated on beef extract peptone solid medium. After being cultured at 37 °C for 24 h, a single colony was selected to verify the degradation ability. Finally, three strains of β-HCH-degrading bacteria were identified: Och1, Och2, and Pse1. After purification, three types of bacteria were inoculated into beef extract peptone medium for amplification. The temperature was set at 30 °C, and the rotation speed for the shaking incubator was set to 160 rpm for 24 h; after centrifugation at 10,000 rpm, the bacteria were diluted to 0.6 absorbances at 600 nm using PBS and were used as a bacterial suspension for subsequent experiments.

2.3. Water Remediation

All experiments were conducted in a natural environment outside the Guilin University of Technology. Water was added over time to reduce the influence of natural water evaporation on the experiment. The vessels (10 cm × 10 cm × 30 cm) used in the experiment had a plant retainer at the bottom, and each contained 1L of deionized water. Each phytoremediation and microbe–plant remediation unit included two Canna samples that were about 60~70 cm tall. Additional carbon source methanol and β-HCH dissolved in acetone were added to the experimental water at concentrations of 10 μg·L−1 and 100 μg·L−1, and this modified sample was recorded as the control group. To repair the β-HCH found in the experimental water, the methods described in the paragraphs below were tested to determine the repair effect. The experiment was divided into control, microbial remediation, plant remediation, and microbe–plant remediation phases, and all experiments were repeated three times, with an experimental period of 7 days.

2.3.1. Microbial Remediation

Different bacterial suspensions with an absorbance level of 1.0 at 600 nm were inoculated with 1% of the experimental water volume and mixed thoroughly and recorded as Och1, Och2, and Pse1.

2.3.2. Phytoremediation

Canna (Canna indica L.) was used in this study, as it is often used to restore organic pollutants. In this experiment, Canna was taken from the long-term stable operation of the wetland model device at the Guilin University of Technology water station, and β-HCH was not detected in the soil or in the Canna samples. Canna seedlings with similar growth potentials (about 60~70 cm) were collected, washed, and pre-cultured in deionized water without β-HCH.

2.3.3. Microbe–Plant Remediation

Based on the conditions for microbial remediation, the pre-cultured Canna was added for combined remediation, and samples were recorded as Och1-C, Och2-C, and Pse1-C.

2.4. Experimental Method

2.4.1. Sample Collection Method

After 7 days of operation, the experimental water and the plant roots, stems, and leaves were collected. For water sample collection, 100 mL of experimental water was used to detect the β-HCH content and microbial community. For plant collection, after collecting the plant samples and drying them in a ventilated place, 3 g (dry weight) samples of the roots, stems, and leaves were taken to determine the β-HCH content, and 10 g (fresh weight) root, stem, and leaf samples were placed into a sterilized 50 mL centrifuge tube for microbial community detection.

2.4.2. Sample Pretreatment Method

After filtering the water sample, 1% methanol was added by volume, followed by the addition of nitric acid, to adjust the pH value of the water sample to lower than 2. Finally, using the automatic solid-phase extraction device, the water sample concentration was extracted, and the re-dissolution of nitrogen after drying was measured.
For the plant samples, 3 g of dried root, stem, and leaf samples was ground. Then, 1 g of anhydrous sodium sulfate and 20 mL of ethyl acetate were added in turn, and the extracted solution containing β-HCH was poured into a 50 mL centrifuge tube after the completion of an ultrasound. Finally, 15 mL and 10 mL of ethyl acetate were added to continue the extraction. The resulting extract was centrifuged for 10 min at 10,000 rpm, and 15 mL of the liquid in the tube was removed for filtration. The filtrate was blown below 2 mL of nitrogen in a 40 °C water bath, the water sample was extracted and concentrated using an automatic solid phase extraction device, and the eluent was collected. A GC-ECD was used to determine the β-HCH content in the samples.

2.4.3. Microbial Sequencing Method

The DNA and PCR amplification of the genome were carried out first, followed by the aliquoting and purification of PCR products, and finally, library construction was performed using the Truseq DNA PCR-free Sample Preparation Kit Library Kit (Illumina, San Diego, CA, USA), which was sequenced online using NovaSeq6000, following library eligibility. This study used high-throughput sequencing technology to detect microbial communities in water and the Canna rhizosphere by testing by the Novomagic company (Tempe, AZ, USA).

2.5. Chromatographic Conditions

The sample was injected using an autosampler with a splitting ratio of 10:1 and an injection volume of 0.8 μL. Column Elite-5MS (30 m × 0.25 mm × 0.25 μm) columns were used, and the chromatographic conditions were as follows: initial temperature of 80 °C; temperature was increased to 210 °C at 8 °C/min, held for 2 min, then increased to 220 °C at 2 °C/min, held for 2 min without split-flow injection, and finally increased to an inlet temperature of 280 °C. The detector (ECD) temperature was 320 °C.

2.6. Statistical Analyses

The values are expressed as mean ± standard deviation (SD). A one-way analysis of variance was used to demonstrate differences among values. Regression analyses and graph processing were performed using Origin 8.0 for Windows.

3. Results and Discussion

3.1. Identification of Strains

After incubation, three bacteria that could effectively degrade β-HCH were identified. Strain identification was carried out by Bioengineering Co., Ltd. (Shanghai, China). The 16S rDNA sequence analysis showed that the bacteria Och1 and Och2 were Ochrobactrum sp. and that bacterium Pse1 was Pseudomonas sp. (Figure 1). The sequence information is given in the Supplementary Materials.

3.2. The Effectiveness of Microbial Remediation for Removal of β-HCH from Water

In order to verify the effectiveness of microbial remediation in removing β-HCH from water, the degradation rates of β-HCH by different degrading bacteria were compared through experiments (Figure 2). The results showed that under 10 μg·L−1 β-HCH stress, the β-HCH degradation rate for Och1 was 33.49%, which was 3.18 and 1.18 times that of Och2 and Pse1, respectively. Compared with Och2, the degradation rate of β-HCH was significantly increased by Och1 and Pse1 (p < 0.05); these results indicated that Och1 and Pse1 could effectively degrade β-HCH at low concentrations. Under 100 μg·L−1 β-HCH stress, Och1 and Och2 showed poor tolerance of the β-HCH concentration, and the degradation rates were only 5.72% and 0.62%. However, Pse1 showed strong degradation ability, and the removal rate of β-HCH reached 60.02%, which indicated that the degrading bacteria were susceptible to the external environment. The tolerance and degradation ability of β-HCH was different among different species of bacteria. It was worth noting that different concentrations of β-HCH had a more significant effect on different strains. When the concentration of β-HCH increased from 10 μg·L−1 to 100 μg·L−1, the degradation rate of β-HCH was significantly decreased by Och1 and Och2, which may be because the proliferation of Och1 and Och2 was inhibited, and the cell viability was reduced at this concentration [22]. The advantage of microbial remediation is that it can consume organic pollutants in the environment as a carbon source to maintain its metabolic growth, completely removing them from the water by mineralizing them into harmless end products [23]. In practical engineering, the strains used in this study can be made into biological agents and introduced into polluted waters. However, Ochrobactrum may contain pathogens that are harmful to humans and animals. Therefore, it is important to remember that access to sites with polluted water in which this bacterial suspension has been used should be restricted for 6 days. Previous experiments have confirmed that these three strains stop growing after 6 days and then lyse and die [20]. However, the obligate degrading bacteria are susceptible to the external environment, their survival rate is not high, and they are often at a disadvantage in competition with indigenous microorganisms. Therefore, the single addition effect of degrading bacteria is not ideal in repairing organically polluted water, and the existence of plants may make up for this defect, providing a good living environment for obligate degrading bacteria [24].

3.3. The Effectiveness of Microbe–Plant Remediation for Removal of β-HCH from Water

Overall, compared with microbial remediation (Och1, Och2, and Pse1), plant group C and the microbe–plant treatment (Och1-C, Och2-C, and Pse1-C) had significantly better removal effects on β-HCH in water (Figure 3). Canna roots provide a habitat for the growth and development of microorganisms that proliferate and form biofilms on the roots [25]. In aquatic ecosystems, plant roots are an essential carrier for biofilm attachment. Plants secrete nutrients and release oxygen through roots to improve the living environment of microorganisms and provide a larger surface area for degrading pollutants in the water environment [26,27]. However, external factors affect the bioremediation process in a very complex way. The types of degrading bacteria and the concentration of pollutants affect the activity of microorganisms and produce different interactions, thus promoting or inhibiting the degradation of organic pollutants [28,29]. It can be concluded that an effective interaction between aquatic plants and bacteria is the key to improving the removal effect of organic pollutants in water [30,31]. When the stress concentration of β-HCH was 10 μg·L−1, there was a synergic effect between degrading Och1 and Och2 and Canna. The removal rates of β-HCH from water in Och1-C and Och2-C were 96.74% and 67.86%, respectively, which were 40.96% and 12.08% higher than the rate in C and 63.25% and 57.33% higher than those in Och1 and Och2, respectively. However, there was no synergistic effect between degrading bacteria Pse1 and Canna. The removal rate of β-HCH in Pse1-C was only 8.9%, much lower than in other treatment groups. When the β-HCH stress concentration was 100 μg·L−1, the removal rates of β-HCH in Och1-C and Pse1-C were 99.06% and 93.69%, which were increased by 5.38% and 0.01% compared to C and 93.34% and 33.67% compared to Och1 and Pse1, respectively. However, the antagonism between Och2 and Canna resulted in the removal rate of β-HCH in Och2-C decreasing by 24.57% compared to C but still increasing by 68.49% compared to Och2.
In low-concentration β-HCH-polluted water, inoculating biodegradable bacteria that produce synergistic effects with plants plays a vital role in the remediation of water environment pollution, which is consistent with the study of Shahid et al., (2020) [28]. In this study, under different stress concentrations of β-HCH, Och1-C showed the synergistic removal of β-HCH from water, with the highest removal rate. Och2-C showed synergism at low concentrations (10 μg·L−1) but antagonism at high concentrations (100 μg·L−1). In contrast, Pse1-C showed antagonism under low-concentration stress and synergism under high-concentration stress. Therefore, it is critical to effectively remove organic pesticides, such as β-HCH, from water by screening the microbial plant combination that can produce synergistic effects according to different pollution levels. In the polluted water with a high concentration of β-HCH, the removal effect of C on β-HCH was not significantly different from that of Och1-C and Pse1-C. However, this did not mean that inoculation with degrading bacterial agents was useless. Plants can absorb pesticides from the environment and transport them to various areas [16]. However, since there is no mineralization path for organic pollutants in plants, they diffuse to tissues through root absorption and cannot wholly degrade pesticides. Therefore, combining obligate degrading bacteria to degrade β-HCH is a better choice [28]. Therefore, the β-HCH concentration in Canna tissues was detected to explore the role of degrading bacteria.

3.4. The Effectiveness of Microbe–Plant Remediation for Removal of β-HCH from Water

The concentrations of β-HCH in the Canna root, stem, and leaf tissues were measured to assess the effect of degrading bacteria on the accumulation of β-HCH in Canna tissues and whether they played a role in the removal efficiency of β-HCH (Figure 4). When the β-HCH stress concentration was 10 μg·L−1, Och1, Och2, and Canna showed a strong synergic effect, which enhanced the tolerance and absorption capacity of Canna to β-HCH stress. The β-HCH content in the whole plant of Canna was increased by 13.34 μg·kg−1 and 10.36 μg·kg−1 compared to C, respectively. In the treatment group inoculated with Pse1, the content of β-HCH was similar to that of C, but none of the concentrations were transferred upward in the roots. In the treatment group inoculated with Och1 and Och2, a higher content of β-HCH was detected in the stems and leaves. When the stress concentration of β-HCH was 100 μg·L−1, the content of β-HCH in Canna plants treated with Och2 was slightly increased, while that in the other treatment groups was significantly increased (90~457%). The concentration of β-HCH in the roots and in transshipment to the stem and leaves increased significantly in C and Pse1 (287% and 580%, respectively). Interestingly, the concentration in the roots increased significantly (279%) under Och1 treatment, but the translocations to the stems and leaves showed no significant change. These results indicated that the ability of Canna to absorb and transport β-HCH was affected by its stress concentration and the inoculated degrading strains. Compared with the stems and leaves, β-HCH mainly accumulated in the roots of C, Och1, and Pse1, and the maximum accumulation was 60.02 μg·kg−1.
A comprehensive analysis of the removal rate of β-HCH in each treatment group (Figure 3) and the content of β-HCH in Canna (Figure 4) showed that during the purification and removal of β-HCH in water using a combination of degrading bacteria and Canna, the two interact with each other and contribute differently to the removal of β-HCH in different concentrations. Under 10 μg·L−1 β-HCH stress, Och1 and Och2 enhanced the accumulation and transport of β-HCH in Canna. The removal rates of Och1-C and Och2-C were significantly higher than those of C, indicating that the main pathway of β-HCH removal was Canna absorption and accumulation. Under 100 μg·L−1 β-HCH stress, the removal rate and β-HCH content in Canna in Pse1-C had no change compared with that in C, and the removal rate in Och1-C was higher, but the β-HCH content in Canna was significantly reduced. The β-HCH content in Och2-C was similar to that in the 10 μg·L−1 β-HCH treatment, but the removal rate was still improved. The above results indicate that Och1 and Och2 significantly increased the contribution rate of β-HCH removal and degraded a large proportion of β-HCH in Och1-C and Och2-C, while Canna absorption and accumulation were mainly present in Pse1-C. Therefore, in the process of environmental pollution bioremediation, the mutual promotion or inhibition between microorganisms and plants directly affected the method of and contribution to pollutant removal. At the same time, the degradation and removal effect of microorganisms and plants on organic pollutants in water also depends on the environmental temperature, the function and type of strains, and the durability of survival in water after inoculation [32,33]. Among all the treatments in this study, the β-HCH content in the Canna roots in Pse1-C was the highest, reaching 55.05 μg·kg−1, while the content in the stems and leaves was the lowest, indicating that Pse1 improved the ability to absorb and accumulate β-HCH in the Canna roots and inhibited its translocation upward. Related studies have also shown HCHs to mainly accumulate in the environment through root absorption [28].

3.5. Analysis of Bacterial Community Characteristics

The microbial community plays an essential role in the removal of organic pollutants. In aquatic systems, the bacterial community of the biofilm attached to plant roots differs from that in the water environment and its host-specific composition [34,35]. Therefore, in this study, the diversity of a 16S rRNA gene MiSeq sequence was used to analyze the characteristics of microbial community composition in the rhizosphere of Canna and in the experimental water system.

3.5.1. Alpha Analysis

A total of 1653 OTUs were obtained by comparing the results with the Silva132 database and annotating species. Richness estimators (Chao 1 and Ace) and diversity indices (Shannon) were obtained. Pesticides have been reported to reduce the abundance and diversity of soil microbial communities [36], consistent with the conclusion of this study. The OTU, Shannon, Chao1, and ACE indices of deionized water of pre-cultured Canna were 734, 6.82, 755.69, and 759.75, respectively. Table 1 shows the bacterial alpha diversity index in the experimental water for different treatment groups under two stress concentrations of β-HCH, from which it can be seen that each index decreased significantly after the addition of β-HCH. The OTU, Chao 1, and Ace of Och1 and Pse1 were all greater than that of the control, while the indices of Och2 were all lower than that of the control, Och1, and Pse1. In the microbe–plant treatment, the OTU, Chao 1, and Ace indices of Och1-C were all higher than the control, Och2-C, and Pse1-C but slightly lower than Och1. When the β-HCH stress concentration was 10 μg·L−1, the Shannon index of microbial plant treatment was significantly lower than that of the unplanted treatment. However, when the β-HCH stress concentration was 100 μg·L−1, there was no significant difference in the Shannon index among all groups, and Pse1 and Och1-C had the highest values. This may be due to the addition of degrading bacteria and the generation of Canna rhizosphere actions, which changed the microbial community structure in the water, and the microbial response conducive to β-HCH degradation was thus strong [37]. Table 2 shows the alpha diversity analysis of Canna rhizosphere bacteria, comprising 755 OTUs. Under two stress concentrations of β-HCH, the OTU, Shannon, Chao1, and Ace indices in the treatment group treated with degraded bacteria were significantly higher than those in the treatment group treated with non-degraded bacteria. Under 10 μg·L−1 β-HCH stress, the OTU value and Shannon index in Och1-C were higher than in other groups. In contrast, the indices in Pse1-C were slightly higher than those in the control group. Under 100 μg·L−1 β-HCH stress, the indices in the treatment group inoculated with degraded bacteria were significantly higher than those in the control group. However, there was no significant difference in the indices in the treatment group inoculated with different degraded bacteria. This might be due to the complex reaction between water and plant roots, and the growth and development of rhizosphere bacteria were affected by environmental and plant organ niche factors. At the same time, the removal of pollutants in water not only depends on a certain type of highly degradable bacteria, but it also depends on the interaction between bacteria groups [38].

3.5.2. Analysis of Species Composition of Bacterial Community at Genus Level

The bacterial community structure characteristics of the experimental water and Canna rhizosphere in each treatment group are shown in Figure 5. In the water samples of the control10, control100, C10, and C100 treatment groups, the total bacteria that accounted for more than 1% were Methylophilus (38.02%), Bradyrhizobium (11.86%), Paracoccus (2.15%), and Methylobacillus (1.36%); the bacteria genera that accounted for more than 1% in the Canna root samples of C10 and C100 included Methylophilus (46.72%), Simplicispira (8.84%), Niveibacterium (1.67%), and Streptomyces (1.09%). Compared with the treatments that were not inoculated with degrading bacteria under 10 μg·L−1 and 100 μg·L−1 β-HCH stress, the total proportion of Methylophilus in Canna-degrading bacteria treatments increased by 78.59% and 48.04%, respectively. The total proportion of Paracoccus and Methylobacillus increased by 3.28% and 1.92%, 0.48%, and 4.34%, respectively. The total proportion of Methylophilus in the Canna root samples of the Canna-degrading bacteria treatments increased by 40.88% and 36.61%, respectively. After inoculation with degraded bacteria, Methylophilus was the dominant bacterium in both the experimental water and Canna root samples, indicating that Methylophilus may play an essential role in removing β-HCH, showing high activity. Methylophilus has been implicated in the degradation of organic pollutants such as PAHs and benzophenone-3 in many studies [39,40]. Paracoccus and Methylobacillus have also been reported to degrade pesticides [41,42]. Although no degraded bacteria were directly found in the experimental water and the Canna rhizosphere; according to the experimental results, this may be due to the degradation bacteria promoting the growth of biodegradable β-HCH bacteria, such as Methylophilus, which were more involved in the detoxification of β-HCH before their disappearance. Additionally, this also affected the accumulation and transport of β-HCH in Canna.

4. Conclusions

Three strains, Och1, Och2, and Pse1, which were found to be able to degrade β-HCH, were used to repair experimental water with Canna. Interestingly, under the two β-HCH stress concentrations, different degrading bacteria and their combinations with Canna showed different removal efficiencies and interactions. At low concentrations, Och1 had the highest degradation rate for β-HCH in water, while at high concentrations, Pse1 had the highest degradation rate. The Och2-C and Pse1-C treatments showed different interactions for the removal of β-HCH from water. In contrast, Och1-C showed a synergic removal of β-HCH in water, and Och1-C thus demonstrated better repair potential. Overall, the removal effects of different degrading bacteria/Canna treatments on β-HCH in water were as follows: Och1-C > Pse1-C > Och2-C. Moreover, after inoculation with degrading bacteria, the relative abundance of Methylophilus bacteria increased significantly in both experimental water and Canna root samples, suggesting that Methylophilus bacteria might be an essential bacteria involved in the degradation of benzene ring materials. The high-efficiency bacterial degrading agents for target pollutants are potentially available natural biological agents, simple to operate, and have incredibly high environmental and economic benefits in the large-scale removal of organic pollutants in sewage. Thus, they represent a promising alternative for treating β-HCH-contaminated water in combination with plants.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/w15132328/s1, Figure S1: Experimental process diagram; Figure S2: Chromatogram of β-HCH detection; Figure S3: Standard curve of β-HCH.

Author Contributions

Conceptualization, H.S. and Y.L.; methodology, H.Z.; validation, S.L., H.S. and Y.L.; formal analysis, H.S.; investigation, Y.L.; resources, L.Q.; data curation, H.S. and S.L.; writing—original draft preparation, H.S.; writing—review and editing, H.Z.; visualization, X.S.; supervision, L.Q.; project administration, X.S.; funding acquisition, Y.L. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the National Natural Science Foundation of China, grant numbers 51868012 and 51578171. The APC was funded by the Natural Science Foundation of Guangxi Province, China, grant number 2018GXNSFAA281186, and by Guilin University of Technology, China, grant number GUTQDJJ2003041.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Not applicable.

Acknowledgments

The authors would like to acknowledge the members of the Zeng lab for their assistance throughout the study and also acknowledge Sze-Mun Lam for reviewing and improving the manuscript.

Conflicts of Interest

The authors declare no conflict of interest.

References

  1. Ma, Y.; Yun, X.T.; Ruan, Z.Y.; Lu, C.J.; Shi, Y.; Qin, Q.; Men, Z.M.; Zou, D.Z.; Du, X.M.; Xing, B.S.; et al. Review of hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) contamination in Chinese soils. Sci. Total. Environ. 2020, 749, 141212. [Google Scholar] [CrossRef]
  2. Vijgen, J.; de Borst, B.; Weber, R.; Stobiecki, T.; Forter, M. HCH and lindane contaminated sites: European and global need for a permanent solution for a long-time neglected issue. Environ. Pollut. 2019, 248, 696–705. [Google Scholar] [CrossRef] [PubMed]
  3. Srivastava, V.; Srivastava, T.; Kumar, M.S. Fate of the persistent organic pollutant (POP) Hexachlorocyclohexane (HCH) and remediation challenges. Int. Biodeterior. Biodegrad. 2019, 140, 43–56. [Google Scholar] [CrossRef]
  4. Lozowicka, B.; Jankowska, M.; Hrynko, I.; Kaczynski, P. Removal of 16 pesticide residues from strawberries by washing with tap and ozone water, ultrasonic cleaning and boiling. Environ. Monit. Assess. 2016, 188, 1–19. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  5. Wacławek, S.; Silvestri, D.; Hrabák, P.; Padil, V.V.; Torres-Mendieta, R.; Wacławek, M.; Dionysiou, D.D. Chemical oxidation and reduction of hexachlorocyclohexanes: A review. Water Res. 2019, 162, 302–319. [Google Scholar] [CrossRef]
  6. Zaghden, H.; Barhoumi, B.; Jlaiel, L.; Guigue, C.; Chouba, L.; Touil, S.; Sayadi, S.; Tedetti, M. Occurrence, origin and potential ecological risk of dissolved polycyclic aromatic hydrocarbons and organochlorines in surface waters of the gulf of gabès (Tunisia, southern mediterranean sea). Mar. Pollut. Bull. 2022, 180, 113737. [Google Scholar] [CrossRef] [PubMed]
  7. Guo, W.; Zhang, H.; Huo, S. Organochlorine pesticides in aquatic hydrophyte tissues and surrounding sediments in Baiyangdian wetland, China. Ecol. Eng. 2014, 67, 150–155. [Google Scholar] [CrossRef]
  8. Vazquez, N.D.; Chierichetti, M.A.; Acuña, F.H.; Miglioranza, K.S. Organochlorine pesticides and chlorpyrifos in the sea anemone Bunodosoma zamponii (Actiniaria: Actiniidae) from Argentina’s southeastern coast. Sci. Total. Environ. 2022, 806, 150824. [Google Scholar] [CrossRef]
  9. Saleh, I.A.; Zouari, N.; Al-Ghouti, M.A. Removal of pesticides from water and wastewater: Chemical, physical and biological treatment approaches. Environ. Technol. Innov. 2020, 19, 101026. [Google Scholar] [CrossRef]
  10. Verma, J.P.; Jaiswal, D.K.; Sagar, R. Pesticide relevance and their microbial degradation: A-state-of-art. Rev. Environ. Sci. Bio./Technol. 2014, 13, 429–466. [Google Scholar] [CrossRef]
  11. Finley, S.D.; Broadbelt, L.J.; Hatzimanikatis, V. In silico feasibility of novel biodegradation pathways for 1, 2, 4-trichlorobenzene. BMC Syst. Biol. 2010, 4, 7. [Google Scholar] [CrossRef] [Green Version]
  12. Li, Q.; Xing, Y.; Fu, X.; Ji, L.; Li, T.; Wang, J.; Zhang, Q. Biochemical mechanisms of rhizospheric Bacillus subtilis-facilitated phytoextraction by alfalfa under cadmium stress–Microbial diversity and metabolomics analyses. Ecotox. Environ. Saf. 2021, 212, 112016. [Google Scholar] [CrossRef]
  13. Kurasvili, M.V.; Adamia, G.S.; Amiranashvili, L.L.; Ananiasvili, T.I.; Khatisashvili, G.A. Targeting of detoxification potential of microorganisms and plants for cleaning environment polluted by organochlorine pesticides. Ann. Agr. Sci. 2016, 14, 222–226. [Google Scholar]
  14. Khan, Z.; Roman, D.; Kintz, T.; delas Alas, M.; Yap, R.; Doty, S. Degradation, phytoprotection and phytoremediation of phenanthrene by endophyte Pseudomonas putida, PD1. Environ. Sci. Technol. 2014, 48, 12221–12228. [Google Scholar] [CrossRef] [PubMed]
  15. Vergani, L.; Mapelli, F.; Zanardini, E.; Terzaghi, E.; Di Guardo, A.; Morosini, C.; Borin, S. Phyto-rhizoremediation of polychlorinated biphenyl contaminated soils: An outlook on plant-microbe beneficial interactions. Sci. Total. Environ. 2017, 575, 1395–1406. [Google Scholar] [CrossRef] [PubMed]
  16. Hussain, F.; Tahseen, R.; Arslan, M.; Iqbal, S.; Afzal, M. Removal of hexadecane by hydroponic root mats in partnership with alkane-degrading bacteria: Bacterial augmentation enhances system’s performance. Int. J. Environ. Sci. Technol. 2019, 16, 4611–4620. [Google Scholar] [CrossRef]
  17. Mori, K.; Toyama, T.; Sei, K. Surfactants degrading activities in the rhizosphere of giant duckweed (Spirodela polyrrhiza). Jpn. J. Water Treat. Biol. 2005, 41, 129–140. [Google Scholar] [CrossRef] [Green Version]
  18. Tanner, C.C.; Headley, T.R. Components of floating emergent macrophyte treatment wetlands influencing removal of stormwater pollutants. Ecol. Eng. 2011, 37, 474–486. [Google Scholar] [CrossRef]
  19. Peng, G.S.; Lu, Y.Q.; Zeng, H.H.; Qin, L.T. Purification Effect of β-HCH by Vertical Flow Constructed Wetlands with Different Plant. Technol. Water Treat. 2020, 46, 34–38. [Google Scholar]
  20. Luo, S.; Liang, Y.P.; Zeng, H.H.; Qin, L.T. Screening and identification of bacteria β-HCH rhizosphere degradation in constructed wetland plants. J. Henan Univ. Sci. Technol. Nat. Sci. 2021, 42, 78–87. [Google Scholar]
  21. Wang, W.; Liu, A.; Fu, W.; Peng, D.; Wang, G.; Ji, J.; Guan, C. Tobacco-associated with Methylophilus sp. FP-6 enhances phytoremediation of benzophenone-3 through regulating soil microbial community, increasing photosynthetic capacity and maintaining redox homeostasis of plant. J. Hazard. Mater. 2022, 431, 128588. [Google Scholar] [CrossRef]
  22. Song, F.H.; Zhang, G.L.; Li, H.H.; Ma, L.L.; Yang, N. Comparative transcriptomic analysis of Stenotrophomonas sp. MNB17 revealed mechanisms of manganese tolerance at different concentrations and the role of histidine biosynthesis in manganese removal. Ecotox. Environ. Saf. 2022, 244, 114056. [Google Scholar] [CrossRef] [PubMed]
  23. Arslan, M.; Imran, A.; Khan, Q.M.; Afzal, M. Plant–bacteria partnerships for the remediation of persistent organic pollutants. Environ. Sci. Pollut. Res. 2017, 24, 4322–4336. [Google Scholar] [CrossRef]
  24. Zhang, L.; Zhao, J.; Cui, N.; Dai, Y.; Kong, L.; Wu, J.; Cheng, S. Enhancing the water purification efficiency of a floating treatment wetland using a biofilm carrier. Environ. Sci. Pollut. Res. 2016, 23, 7437–7443. [Google Scholar] [CrossRef] [PubMed]
  25. Shahid, M.J.; Arslan, M.; Siddique, M.; Ali, S.; Tahseen, R.; Afzal, M. Potentialities of floating wetlands for the treatment of polluted water of river Ravi, Pakistan. Ecol. Eng. 2019, 133, 167–176. [Google Scholar] [CrossRef]
  26. Thorén, A.-K. Urea transformation of wetland microbial communities. Microb. Ecol. 2007, 53, 221–232. [Google Scholar] [CrossRef] [PubMed]
  27. Pang, S.; Zhang, S.; Lv, X.; Han, B.; Liu, K.; Qiu, C.; Wang, C.; Wang, P.; Toland, H.; He, Z. Characterization of bacterial community in biofilm and sediments of wetlands dominated by aquatic macrophytes. Ecol. Eng. 2016, 97, 242–250. [Google Scholar] [CrossRef]
  28. Shahid, M.J.; Al-Surhanee, A.A.; Kouadri, F.; Ali, S.; Nawaz, N.; Afzal, M. Role of microorganisms in the remediation of wastewater in floating treatment wetlands: A review. Sustainability 2020, 12, 5559. [Google Scholar] [CrossRef]
  29. Ali, M.; Kazmi, A.A.; Ahmed, N. Study on effects of temperature, moisture and pH in degradation and degradation kinetics of aldrin, endosulfan, lindane pesticides during full-scale continuous rotary drum composting. Chemosphere 2014, 102, 68–75. [Google Scholar] [CrossRef]
  30. Hardoim, P.R.; van Overbeek, L.S.; Berg, G.; Pirttilä, A.M.; Compant, S.; Campisano, A.; Sessitsch, A. The hidden world within plants: Ecological and evolutionary considerations for defining functioning of microbial endophytes. Microbiol. Mol. Biol. R. 2015, 79, 293–320. [Google Scholar] [CrossRef] [Green Version]
  31. Pavan, F.; Breschigliaro, S.; Borin, M. Screening of 18 species for digestate phytodepuration. Environ. Sci. Pollut. R. 2015, 22, 2455–2466. [Google Scholar] [CrossRef] [PubMed]
  32. Santoyo, G.; Guzmán-Guzmán, P.; Parra-Cota, F.I.; Santos-Villalobos, S.D.L.; Orozco-Mosqueda, M.D.C.; Glick, B.R. Plant growth stimulation by microbial consortia. Agronomy 2021, 11, 219. [Google Scholar] [CrossRef]
  33. Carrión, V.J.; Perez-Jaramillo, J.; Cordovez, V.; Tracanna, V.; de Hollander, M.; Ruiz-Buck, D.; Raaijmakers, J.M. Pathogen-induced activation of disease-suppressive functions in the endophytic root microbiome. Science 2019, 366, 606–612. [Google Scholar] [CrossRef]
  34. He, D.; Ren, L.; Wu, Q. Epiphytic bacterial communities on two common submerged macrophytes in Taihu Lake: Diversity and host-specificity. Chin. J. Oceanol. Limnol. 2012, 30, 237–247. [Google Scholar] [CrossRef]
  35. Haichar, F.Z.; Marol, C.; Berge, O.; Rangel-Castro, J.I.; Prosser, J.I.; Balesdent, J.; Heulin, T.; Achouak, W. Plant host habitat and root exudates shape soil bacterial community structure. ISME J. 2008, 2, 1221. [Google Scholar] [CrossRef]
  36. Chen, Q.; Shi, H.J.; Liang, Y.; Qin, L.T.; Zeng, H.H.; Song, X.H. Degradation Characteristics and Remediation Ability of Contaminated Soils by Using β-HCH Degrading Bacteria. Int. J. Env. Res. Public Health 2023, 20, 2767. [Google Scholar] [CrossRef]
  37. Egbe, C.C.; Oyetibo, G.O.; Ilori, M.O. Ecological impact of organochlorine pesticides consortium on autochthonous microbial community in agricultural soil. Ecotoxicol. Environ. Saf. 2021, 207, 111319. [Google Scholar] [CrossRef]
  38. Zhang, Z.; Xiao, Y.S.; Zhan, Y.; Zhang, Z.; Liu, Y.; Wei, Y.; Li, J. Tomato microbiome under long-term organic and conventional farming. iMeta 2022, 1, e48. [Google Scholar] [CrossRef]
  39. Martin, F.; Torelli, S.; Le Paslier, D.; Barbance, A.; Martin-Laurent, F.; Bru, D.; Jouanneau, Y. Betaproteobacteria dominance and diversity shifts in the bacterial community of a PAH-contaminated soil exposed to phenanthrene. Environ. Pollut. 2012, 162, 345–353. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  40. Jin, C.; Geng, Z.; Pang, X.; Zhang, Y.; Wang, G.; Ji, J.; Guan, C. Isolation and characterization of a novel benzophenone-3-degrading bacterium Methylophilus sp. strain FP-6. Ecotox. Environ. Saf. 2019, 186, 109780. [Google Scholar] [CrossRef]
  41. Zhang, J.; Yin, J.G.; Hang, B.J.; Cai, S.; He, J.; Zhou, S.G.; Li, S.P. Cloning of a novel arylamidase gene from Paracoccus sp. strain FLN-7 that hydrolyzes amide pesticides. Appl. Environ. Microb. 2012, 78, 4848–4855. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  42. Kumar, V.; Maitra, S.S. Biodegradation of endocrine disruptor dibutyl phthalate (DBP) by a newly isolated Methylobacillus sp. V29b and the DBP degradation pathway. 3 Biotech 2016, 6, 200. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Water 15 02328 g001 550
Figure 1. Phylogenetic trees of strains.
Figure 1. Phylogenetic trees of strains.
Water 15 02328 g001
Water 15 02328 g002 550
Figure 2. Degradation of β-HCH at different concentrations in water under different degrading bacteria treatments. Values are expressed as mean ± standard deviation (SD). Different lowercase letters (a, b) indicate significant (p < 0.05) differences in the concentration of β-HCH among conditions (alike colors should be compared with each other).
Figure 2. Degradation of β-HCH at different concentrations in water under different degrading bacteria treatments. Values are expressed as mean ± standard deviation (SD). Different lowercase letters (a, b) indicate significant (p < 0.05) differences in the concentration of β-HCH among conditions (alike colors should be compared with each other).
Water 15 02328 g002
Water 15 02328 g003 550
Figure 3. Degradation of β-HCH at different concentrations in water by degrading bacteria combined with Canna. Values are expressed as mean ± standard deviation (SD). Different lowercase letters (a–d) indicate significant (p < 0.05) differences in the concentration of β-HCH among conditions (alike colors should be compared with each other).
Figure 3. Degradation of β-HCH at different concentrations in water by degrading bacteria combined with Canna. Values are expressed as mean ± standard deviation (SD). Different lowercase letters (a–d) indicate significant (p < 0.05) differences in the concentration of β-HCH among conditions (alike colors should be compared with each other).
Water 15 02328 g003
Water 15 02328 g004 550
Figure 4. Effects of different degrading bacteria treatments on β-HCH concentrations in different parts of Canna. Values are expressed as mean ± standard deviation (SD). *, p < 0.05; **, p < 0.01; -, p > 0.05. Alike colors are compared to each other.
Figure 4. Effects of different degrading bacteria treatments on β-HCH concentrations in different parts of Canna. Values are expressed as mean ± standard deviation (SD). *, p < 0.05; **, p < 0.01; -, p > 0.05. Alike colors are compared to each other.
Water 15 02328 g004
Water 15 02328 g005 550
Figure 5. Under different stress concentrations of β-HCH, the top 10 bacterial communities with horizontal abundance are listed in the histogram. Water sample (a) and Canna root sample (b).
Figure 5. Under different stress concentrations of β-HCH, the top 10 bacterial communities with horizontal abundance are listed in the histogram. Water sample (a) and Canna root sample (b).
Water 15 02328 g005
Table
Table 1. The analysis of bacterial alpha diversity in the experimental water body under different β-HCH stress concentrations.
Table 1. The analysis of bacterial alpha diversity in the experimental water body under different β-HCH stress concentrations.
Treatment10 μg·L−1 β-HCH100 μg·L−1 β-HCH
OTUShannonChao1AceOTUShannonChao1Ace
Control2043.962332572632.91288294
C3213.833553631862.56210222
Och13163.083603853152.60349362
Och21422.18152160962.30107105
Pse12972.833253462733.68294310
Och1-C2801.163273574053.61457468
Och2-C2481.993043153012.38345358
Pse1-C2381.432753032692.14299324
Table
Table 2. The analysis of bacterial alpha diversity in Canna rhizosphere under different β-HCH stress concentrations.
Table 2. The analysis of bacterial alpha diversity in Canna rhizosphere under different β-HCH stress concentrations.
Treatment10 μg·L−1 β-HCH100 μg·L−1 β-HCH
OTUShannonChao1AceOTUShannonChao1Ace
C1660.942012041380.85181183
Och1-C2383.072632692371.90271269
Och2-C2482.273393192482.91275280
Pse1-C1871.792132312372.82283287
Disclaimer/Publisher’s Note: The statements, opinions and data contained in all publications are solely those of the individual author(s) and contributor(s) and not of MDPI and/or the editor(s). MDPI and/or the editor(s) disclaim responsibility for any injury to people or property resulting from any ideas, methods, instructions or products referred to in the content.

Share and Cite

MDPI and ACS Style

Shi, H.; Luo, S.; Liang, Y.; Qin, L.; Zeng, H.; Song, X. Synergistic Removal of β-Hexachlorocyclohexane from Water via Microorganism–Plant Technology and Analysis of Bacterial Community Characteristics. Water 2023, 15, 2328. https://doi.org/10.3390/w15132328

AMA Style

Shi H, Luo S, Liang Y, Qin L, Zeng H, Song X. Synergistic Removal of β-Hexachlorocyclohexane from Water via Microorganism–Plant Technology and Analysis of Bacterial Community Characteristics. Water. 2023; 15(13):2328. https://doi.org/10.3390/w15132328

Chicago/Turabian Style

Shi, Huijun, Shuang Luo, Yanpeng Liang, Litang Qin, Honghu Zeng, and Xiaohong Song. 2023. "Synergistic Removal of β-Hexachlorocyclohexane from Water via Microorganism–Plant Technology and Analysis of Bacterial Community Characteristics" Water 15, no. 13: 2328. https://doi.org/10.3390/w15132328

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop