Study of biodegradation of chloramphenicol by endophytic fungi isolated from Bertholletia excelsa (Brazil nuts)

https://doi.org/10.1016/j.bcab.2019.101200Get rights and content

Highlights

  • Endophytic fungi were isolated from Bertholletia excelsa collected at Amazon rainforest.

  • Chloramphenicol (CAP) biodegradation by endophytic fungi was observed.

  • A total of 30% biodegradation was obtained (24 h, pH 7.0 and 150 mg.L−1 of CAP).

  • The metabolite 4-nitrobenzaldehyde was identified as main biodegradation product.

Abstract

Chloramphenicol (CAP) is a micropollutant that resists to conventional residual water treatment. Therefore, the aim of this study was to assess CAP biodegradability by five endophytic fungi strains isolated from Bertholletia excelsa collected in the Brazilian Amazonia. The fungi strains were screened in solid and liquid medium, and then experimental design was performed to optimize culture conditions. In addition, an environmental toxicology assessment was carried out using the algae Chlorella vulgaris. Results from fungi cultures in solid medium demonstrated that CAP affected the strains growth and interfered in the development of conidia and spores. The biodegradation in liquid culture medium showed that all strains increased the degradation of this antibiotic. The most efficient strain Trichoderma sp. (BIORG 7) was subjected to an experimental design (Box-behnken) consisted of 15 experiments, having as variables: pH, time, and CAP concentration, and reaching 30% biodegradation determined by HPLC-UV analyses (24 h, pH 7.0 and 150 mg L−1). The main metabolite 4-nitrobenzaldehyde was identified by GC-MS analyses and presented a higher ecotoxicity to green algae than CAP.

Introduction

Pharmaceutical compounds constitute a very important category of emerging micropollutants, which are considered a major risk to ecosystems due to their harmful biological effects (Miran et al., 2018; Thelusmond et al., 2018). In addition, the most frequently microcontaminants detected in aquatic environments are drugs such as analgesics, antibiotics, lipid regulators, anti-inflammatories and synthetic hormones (Santos et al., 2010).

Antibiotics have been investigated as emerging environmental contaminants, mainly because these compounds can contribute for the development of resistant bacteria, which are a major issue of public health due to the increased occurrence of clinical infections (Yang et al., 2016). The antibiotic chloramphenicol (CAP), the target of this study, has been recurrently found in aquatic environments (Xiong et al., 2019).

CAP is a broad-spectrum antibiotic that has been commonly used to treat meningitis, plague, cholera, and typhoid fever, but it has limited use due to its carcinogenic, genotoxic and hepatotoxic effects in humans (Epaulard and Brion, 2010; Liang et al., 2013). This drug is a liposoluble compound that diffuses through the cell membrane and reversibly binds to the 50S protein subunit of the prokaryote cell ribosomes, preventing the transfer of amino acids to the peptide chains in formation and consequently inhibiting the synthesis of proteins (Martins et al., 2018). The biotoxicity of the nitro and chlorine groups present in this compound are responsible for the resistance to bacterial biodegradation, resulting in a persistence of CAP in conventional processes of biological wastewater treatment (Guo et al., 2017).

Many physical-chemical methods has been reported in the literature to the degradation of CAP, such as thermal (Tian and Bayen, 2018), photocatalytic (Amildon Ricardo et al., 2018; Chatzitakis et al., 2008) and electrochemical (Sun et al., 2017) processes. However, there are few reports of aerobic biological processes (biodegradation) by fungus (Navada and Kulal, 2019), bacteria (Ma et al., 2019) or microalga species.

Biodegradation processes are environmentally friendly and represent a low cost option for micropollutants treatment (Alvarenga et al., 2014; Birolli et al., 2018). Generally, the employed microorganisms consume the substrate by the action of their enzymes, converting pollutants into nutrients and energy source for their survival (Le Borgne et al., 2008; Mouele et al., 2015).

The total biodegradation or mineralization involves the consumption of intermediate compounds, which can be more or less toxic substances than the starting compound (Serrano-González et al., 2018). These processes have several advantages, including low energy employment and green catalysis. Moreover, the biodegradation of different organic pollutants by fungi has been successfully employed (Alvarenga et al., 2014; Vacondio et al., 2015).

Endophytic fungi are promising biocatalysts that survive in the tissues of healthy plants without causing any infection in the host (Afzal et al., 2014). Additionally, studies have proven that these fungi can also be used in the degradation of organic compounds (Potin et al., 2004), i.e., phenanthrene by Ceratobasidum stevensii isolated from a Eupharbiaceae plant (Dai et al., 2010) and 4-hydroxybenzoic acid, ferulic acid, cinnamic acid and sinapic acid by Phomopsis liquidambari, isolated from Bischofia polycarpam (Chen et al., 2013; Fu et al., 2018).

Thus, the objective of this study was the exploration of the biodegradation of the CAP antibiotic by endophytic fungi isolated from Bertholletia excelsa, collected in the Brazilian Amazon rainforest for the first time. Additionally, the influence of the pH, time and concentration of the antibiotic in the rates of biodegradation were also assessed employing an experimental design.

Section snippets

Reagents, solvents and culture media

The antibiotic chloramphenicol (98%) was obtained from Vetec. Salts, reagents and solvents were obtained from Synth and AppliChen Panreac. Malt extract and Agar were purchased from Kasvi (Brazil), isopropanol (HPLC grade) and acetonitrile (HPLC grade) were obtained from PANREAC and TEDIA, respectively.

Isolation and identification of endophytic fungi from Bertholletia excelsa

The seeds of Bertholletia excelsa (Brazil nuts) were collected and assigned by the Brazilian Agricultural Research Corporation – Amapá, Brazil, at the localization area 1 - W 52°18′20,976″ and S

Fungal growth on solid medium

Radial growth tests with the strains isolated from Brazil nut tree containing CAP at 100 mg L−1 or only Malt 2% medium as control experiment were performed for a preliminary assessment of the CAP effects on the endophytic fungi strains. The experiments were carried out for 9 days and the growth of the colonies were evaluated at every 24 h. The results are shown in Table 2.

For the strain Aspergillus bertholletiae BIORG 4, the presence of CAP induced a slight increase of the colony diameter, when

Conclusion

This was the first study that showed the use of endophytic fungi in the biodegradation of the micropollutant CAP. The strains Aspergillus sp. BIORG 9 and Trichoderma sp. BIORG 7 presented the best results of biodegradation, 29.3% and 25.2% of biodegradation for 9 days, respectively. The experimental design applied to the strain Trichoderma sp. BIORG 7 was essential for the optimization of the experimental conditions. Reducing the employed resources as proposed by the green chemistry principles,

Acknowledgements

The authors would like to acknowledge Fundação de Amparo à Pesquisa do Estado do Amapá (FAPEAP, grant no. 34568.515.22257.28052017) for financial support. WGB thanks to Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, grant no. 2017/19721-0) for his postdoctoral researcher grant.

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