Synergistic effect of Klebsiella sp. FH-1 and Arthrobacter sp. NJ-1 on the growth of the microbiota in the black soil of Northeast China

https://doi.org/10.1016/j.ecoenv.2019.110079Get rights and content

Highlights

  • Grain chaff as the optimal carrier material for making microbial inoculum.

  • Microbial inoculum made up of Klebsiella sp. FH-1 and Arthrobacter sp. NJ-1.

  • Remediation of Atrazine contaminated soil with the microbial inoculum.

  • Recovering effect of microbial inoculum on the bacteria and fungi in soil.

  • Utility of microorganisms in the assessment of soil contamination by Atrazine.

Abstract

The application of Atrazine in soil has always been a main problem in agriculture because its residuals may maintain in the soil for a long term. In this paper, two strains of Atrazine degrading bacteria (Klebsiella sp. FH-1 and Arthrobacter sp. NJ-1) were used to make biological compound microbial inoculum to repair the Atrazine contaminated typical black soil in Northeast China. Grain chaff was chosen as the optimal carrier material for microbial inoculum. The dynamic changes of Atrazine were detected by gas chromatography. The half-life of Atrazine in soil containing microbial inoculum was shortened from 9.8 d to 4.2 d. The Atrazine sensitive crops grown in the repaired soil showed increased stem length, root length, and emergence rate. The effects of microbial remediation on the original bacterial and fungal biota in the typical black soil in Northeast China were analyzed using the metagenomic approach. Results showed that Atrazine inhibited the original bacteria and fungi populations. The total numbers of bacterial and fungal species in the soil were partially recovered by adding the microbial inoculum. Two genera (Sphingosinicella and Sphingomonas) were the dominant bacteria. The beneficial bacterial biota was recovered and the number of species of the beneficial bacteria was higher than that in the original soil after adding the microbial inoculum. The dominant fungi included genera Guehomyces and Chaetomella. There was a total of 113 unclassified fungal genera (22.6% of 499), indicating the potential utility of the unclassified fungal species in the assessment of the soil contamination by Atrazine.

Introduction

Atrazine (2-chloro-4-ethylamino-6-isopropylamino-1,3,5-triazine) is a type of herbicides widely used in corn and other crop fields to control most of the weeds worldwide. Its extensive use has led to severe pollutions in both soil and water (Albright et al., 2013). Atrazine also causes damages in many species of animals. For example, Atrazine is considered as an endocrine disruptor (Loughlin et al., 2016) causing the endocrine disorders in the hormone system responsible for sex differentiation in freshwater crayfish (Cherax quadricarinatus). It has also been reported that Atrazine shortens the maturation process of Xenopus oocytes, reduces the Emi2 level of oocytes, and affects the division of fertilized eggs, leading to a high mortality rate during early embryogenesis (Ji et al., 2016). Northeast China is the main corn producing area, accounting for 29% of the total corn production in China (Liu et al., 2017). The amount of Atrazine has been extensively used by the farmers in order to control the weeds in the field, worsening the soil pollution by Atrazine in Northeast China. Therefore, it is important to repair the Atrazine pollution in the black soil (categorized as Mollisols; Wei et al., 2008) in Northeast China. At present, increasing attentions have been paid to the restoration of the soil environment polluted by Atrazine, and various types of microbial inoculum with effective degradation of Atrazine are investigated to repair the soil polluted by Atrazine (Zhao et al., 2019).

Plants, invertebrates, and microorganisms are generally served as biological indicators for the ecotoxicity assessment of the environmental contaminations (Ramadass et al., 2016), while microorganisms have been proved to be the most sensitive, effective, and reliable (Richardson et al., 2015). In recent years, microorganisms in the soil environments have been identified to assess the degree of soil pollution (Thavamani et al., 2015). However, the search for the appropriate microorganism indicators remains a challenge because of the varied physical properties of different types of soil and the uneven individual biological reactions of the microorganisms to the pollutants (Baiamonte et al., 2019). Bacteria with a large diversity of genera and populations have been identified as the potential microorganism indicators, while studies on fungi to identify the potential indicators for ecotoxicity assessment in soil are sparsely reported (Zhang et al., 2019a). To date, a variety of biological models have been established to study the toxicity of Atrazine. For example, in the algal species Raphidocelis subcapitata, the toxicity caused by Atrazine is not significantly repaired by the environmental combination of increased nutrition and decreased intensity of light and temperature under the standard conditions equivalent to the simulated aquatic systems in the Midwestern United States (Baxter et al., 2016). Earthworm was used to detect the combined toxicity of Atrazine and Butachlor (Chen et al., 2014; Lin et al., 2018). The toxicity of Atrazine was also studied using the sex glands of the female experimental mice and the young mice at their early stages of embryonic development (Mansour et al., 2014). The short-term reproductive experiments using fathead minnow (Pimephales promelas) and Japanese medaka (Oryzias latipes) were conducted to evaluate the effects of Atrazine on the environments (de Paiva et al., 2017; Brain et al., 2018). In a species of planktonic crustacean (Daphnia magna), Atrazine was demonstrated to act as a potent activator in vitro of the xenobiotic-sensing nuclear receptor, HR96, related to vertebrate constitutive androstane receptor (CAR) and pregnane X-receptor (PXR) (Schmidt et al., 2017). In frog, the risk of exposing Atrazine on the heart was revealed to be closely related to the critical functions of its rhythmic contractions, which is the foundation of the optimal circulation and stability (Saka et al., 2018; Johnson et al., 2019). However, little information has been reported on the effects of Atrazine degrading microbial inoculum on the soil microorganisms after the remediation of the polluted soils.

In the past, the effects on the primary microbiota in the soil by the foreign materials were generally studied by dilution plate method and the denatured gradient gel electrophoresis (DGGE) (Bao et al., 2019). In recent years, due to its capability of simultaneously sequencing numerous DNA molecules from plentiful samples, the high throughput sequencing technology has been successfully applied to the analysis of microbiota in soil, sludge, food, water, and other samples (Reddy and Dubey, 2019; Xia et al., 2019) and has become an important tool to analyze the structure and relative abundance of microbial communities in complex environments. The restoration of the soil environment is generally accompanied by the change of microbiota in the soil. It is reported that there is a correlation between the soil organic carbon (SOC) and the abundance of bacterial biota in the soil (Khan et al., 2018). The abundance changes of some abundant microorganisms in soil samples are also related to some of the soil properties. For example, the number of bacteria in Desulfitobacterium changed following the changes of the anodes in the soil (Wang et al., 2018b). Therefore, the correlations between the soil properties and the bacterial biota could be used directly to evaluate the degree of changes in the soil environment.

The objectives of this study were to: (1) evaluate the efficiency of the microbial inoculum, composed of two strains of Atrazine degrading bacteria (Klebsiella sp. FH-1 and Arthrobacter sp. NJ-1), in repairing the microbial environments of the typical black soil in Northeast China contaminated by Atrazine, (2) assess the changes in abundance of both the bacterial and fungal communities caused by the microbial inoculum using the metagenomic method, and (3) investigate the correlation between the abundance of microbes in soil and soil properties in order to explore the biological indices of the microbiota which can be used quantitatively to assess the degree of contamination caused by Atrazine. We hypothesized that beneficial microbes could be recovered by the application of microbial inoculum and potentially used as microbial indicators in assessing the environmental pollutions caused by Atrazine.

Section snippets

Samples and media

Powder of Atrazine (purity 80%) was purchased from Shandong Kesaijinong Biotechnology Co., Ltd., China. All the other chemicals used in this study were of the analytical grade or better. The liquid inorganic salt medium contained sucrose 2.0 g, NaCl 1.0 g, K2HPO4 1.5 g, KH2PO4 0.5 g, MgSO4⋅7H2O 0.2 g, FeSO4·7H2O 0.099 g, and ZnCl2 0.003 g. The Luria-Bertani medium (LB) contained tryptone 10 g, yeast extract 5 g, and NaCl 10 g per liter. The liquid nutrient medium contained NaCl 1.0 g, K2HPO4

Preparation of microbial inoculum

In 11 days, the amount of Atrazine degraded by the mixed bacterial strains FH-1 and NJ-1 at the ratio of 1:1, 1:2, 2:1, 2:3, and 3:2 was 78.9%, 82.7%, 84.1%, 88.8%, and 98.1%, respectively (Fig. S1). Therefore, the optimal ratio of mixing strains FH-1 and NJ-1 was set to 3:2 for further experiments.

Among the four types of carrier materials, wheat bran and grain chaff were the most optimal with two of the highest absorption capacities for bacteria of 45% and 41%, respectively (Table S1). The

Preparation of the microbial inoculum and the remediation effect

The overall remediation effect in soil produced by the microbial inoculum composed of Klebsiella sp. FH-1 and Arthrobacter sp. NJ-1 with a ratio of 3:2 (V:V) meets the agricultural requirements. This is demonstrated by our results of Atrazine sensitive crops (cucumber and soybean) grown in the soil repaired by the microbial inoculum (Fig. 1B and C). It has been reported that high porosity essentially promotes cell adhesion, survival, and proliferation (Jakus et al., 2018), while the adsorption

Conclusions

Using grain chaff as the optimal carrier materials, two bacterial strains (Klebsiella sp. FH-1 and Arthrobacter sp. NJ-1) were combined with the ratio of 3:2 (V:V) to make the microbial inoculum used to repair the soil contaminated by Atrazine. The concentrations of Atrazine in the repaired soil were decreased and subsequently reducing the toxic effect of Atrazine on the Atrazine sensitive plants (i.e., soybean and cucumber). The microbial inoculum also repaired the beneficial bacteria and

Author contribution section

J.Z., H.Z., F.S., Conceptualization; J.Z., H.Z., Y.X., S.L., X.M., Z.L., P.S., Data curation; J.Z., P.S., H.Z., F.S., Formal analysis; H.Z., Funding acquisition; J.Z., H.Z., F.S., Investigation; J.Z., H.Z., F.S., Methodology; H.Z., Project administration; H.Z., Resource; J.Z., H.Z., P.S., F.S., Software; H.Z., F.S., Supervision; J.Z., H.Z., P.S., F.S., Validation; J.Z., P.S., H.Z., F.S., Visualization; J.Z., H.Z., F.S., Writing – original draft; J.Z., H.Z., F.S., Writing – review & editing.

Declarations of competing interest interest

None.

Acknowledgements

This study is supported by the National Key Research and Development Program of China (2016YFD0200203), the Science and Technology Development Program of Jilin Province, China (20190301063NY), and the National Natural Science Foundation of China (31672051).

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