Microbiology Research 2017; volume 8:7302 In vitro growth and heavy metal contamination.1 indoleacetic acid production Bioremediation consists on a group of Correspondence: Valdir Marcos Stefenon, Av. applications, which involves the detoxifica- Antonio Trilha, 1847, São Gabriel, RS, ZIP by Mesorhizobium loti tion of hazardous substances by means of 97300-000, Brazil. SEMIA806 and SEMIA816 microbes and plants, instead of transferring Tel.: +55.55.3237.0851 under the influence the pollutants from one substrate to another. E-mail: [email protected] Rhizoremediation is a bioremediation of copper ions Key words: Bacterial growth, bioremediation, method that consists in the enhancement of IAA, rhizobia, rhizoremediation. the metal extraction process through the 1 Jéssica Dutra Vieira, root system of plants by its association Acknowledgements: Authors acknowledge 2 1,2 Paulo Roberto Diniz da Silva, with microbes. FAPERGS by the PROBIC grant to JDV, 1-3 Valdir Marcos Stefenon For the effectiveness of rhizoremedia- UNIPAMPA by financial support and labora- 1Núcleo de Microbiologia Aplicada à tion, an efficient microbe-plant interaction torial facilities, and Dr. Jeferson Luiz Franco is needed. The indole-3-acetic acid (IAA) who kindly provided technical support for the Biorremediação, 2Núcleo de Cultura de produced by plant-associated microbes is an spectrophotometric analyses. Tecidos Vegetais, 3Programa de Pós important phytohormone signaling this Graduação em Ciências Biológicas, interaction. This phytohormone is involved Contributions: This work was carried out in Universidade Federal do Pampa, in many processes of nodule formation, collaboration among all authors. JDV and PRDS performed the laboratory experiments Campus São Gabriel, Brazil such as founder cell specification, nodule and wrote the first draft of the manuscript. initiation and differentiation, vascular bun- VMS designed the study, performed the data dle formation, and nodule numbers. analysis, supported the laboratory activities Therefore, IAA is essential for the forma- and wrote the last draft of the manuscript. All 3 Abstract tion of the nodules in the roots. However, authors read and approved the final version of sub-lethal levels of copper nanoparticles in The indoleacetic acid produced by sym- the manuscript. the growth medium have been shown to biotic bacteria is an important phytohor- reduce the secretion of secondary com- Conflict of interest: the authors declare no mone signaling microbe-plant interaction, pounds by bacteria, including IAA.4 potential conflict of interest. being therefore essential for rhizoremedia- Copper is an essential element for all tion. In this study, the effect of different living beings, since it acts as co-factor in Received for publication: 5 July 2017. concentrations of copper ions on the bacter- Revision received: 24 July 2017. several enzymatic reactions. However, ial growth and indoleacetic acid production Accepted for publication: 8 August 2017. exposition to excessive levels of this metal was investigated in two strains of may cause different metabolic problems. In Mesorhizobium loti in in vitro conditions, This work is licensed under a Creative humans, short-term exposure to drinking aiming to determine critical concentrations Commons Attribution NonCommercial 4.0 water contaminated with copper ions may License (CC BY-NC 4.0). of this heavy metal for rhizoremediation of cause gastrointestinal distress, while long- contaminated soils using this bacterium. term consumption may cause liver or kid- ©Copyright J. Dutra Vieira et al., 2017 The experiment consisted on a control cul- ney damage.5 Therefore, the bioaccumula- Licensee PAGEPress, Italy ture without copper and three treatments Microbiology Research 2017; 8:7302 tion of copper causing toxicity in human, supplemented with 10 mg.L-1, 20 mg.L-1 or doi:10.4081/mr.2017.7302 -1 animals and plants is an important issue for 50 mg.L of CuSO4. For both strains, the environmental health and safety, and rhi- growth stopped after 48h and no significant zoremediation is an important alternative difference was observed across treatments. vitro conditions, aiming to determine criti- to diminish this problem.1 The production of indoleacetic acid by the cal concentrations of this heavy metal for Mesorhizobium loti is a soil and rhizos- control treatment without copper was sig- rhizoremediation of contaminated soils phere bacteria of agronomic importance due nificantly higher in comparison to the cop- using this bacterium. to the nitrogen-fixing symbioses formed per-containing treatments. Mesorhizobium with leguminous plants. This species is able loti SEMIA806 and SEMIA816 are resist- -1 to form determinant-type globular nodules ant to up to 50 mg.L of CuSO4 in the cul- and to perform nitrogen fixation on several ture medium, presenting effective growth. Materials and Methods Lotus species.6 In vitro investigations have The synthesis of indoleacetic acid was shown the capacity of this bacterial species Turfs of Mesorhizobium loti strains strongly reduced but not excluded by ions for growing in culture conditions with high SEMIA806 and SEMIA816 were kindly copper in the medium. So, it is expected that concentrations of copper (Roll et al., provided by the State Foundation of environmental copper found in the soil up unpublished data), suggesting potential for Agricultural Research of the Rio Grande do to the concentration of 50 mg.L-1 will not bioremediation of soils contaminated with Sul (FEPAGRO), São Gabriel, Brazil. The preclude the symbiotic interaction between this metal. In association with fast-growing M. loti strains were firstly cultured in Petri M. loti and leguminous host plant in rhizo- -1 legume tree species as Mimosa scabrella dishes with solid YM broth (1.0 g.L yeast remediation enterprises. -1 -1 (Leguminosae), M. loti may be a quite extract, 0.5 g.L K2HPO4, 0.2 g.L MgSO4, -1 -1 - promissory option for environmental and 0.1 g.L NaCl, 1.0 g.L CaCO3 and 10 g.L landscape recuperation in areas polluted 1 of D-mannitol). Before bacterial inocula- with copper, using rhizoremediation. In this tion, the YM broth was solidified with bac- Introduction study, the interference of different concen- teriological agar (15 g.L-1) and sterilized at In recent years, several approaches for trations of copper ions on the bacterial 1.2 kg.cm−1 at 121°C for 15 min. bioremediation have been proposed as growth and IAA production was investigat- For each strain independently, 1.0 g of alternatives for diminishing environmental ed in two strains of Mesorhizobium loti in in turf was diluted in 100 mL of saline solution [Microbiology Research 2017; 8:7302] [page 55] Article (0.8% NaCl dissolved in ultrapure water). Reagents were purchased by Sigma- Subsequently, 100 µL of the dilution from Aldrich (St. Louis, USA). All experiments Results and Discussion each strain was inoculated in independent were performed in quadruplicate and the Bacterial growth Petri dishes with solidified YM broth and inoculation and sample transfer steps were For both bacterial strains, the growth cultivated at 28°C during 48 h. Actively performed within a sterilized airflow cham- stopped after 48h (Figure 1) and no statisti- growing M. loti colonies were isolated ber Trox model TLF (Trox Technik, cally significant difference (P>0.27) was from this cultures and employed in the Germany). The statistical significance of observed across treatments in the evaluated analysis of bacterial growth and IAA the difference among the estimated means periods (0, 24, 48 or 120 h of culture). synthesis in liquid YM broth with copper. for bacterial growth and IAA synthesis was However, the bacterial growth revealed dif- The experiment consisted on a control determined using the Mann-Whitney test in 8 ferent patterns for strains SEMIA806 and culture without copper and three treat- the software PAST 3.04. SEMIA816 (Table 1 and Figure 1). After ments: i) YM broth supplemented with 10 -1 mg.L of CuSO4; ii) YM broth supplemen- -1 ted with 20 mg.L of CuSO4; and iii) YM broth supplemented with 50 mg.L-1 of CuSO4. Before bacteria inoculation, 20 mL of the YM broth was dispensed in 300 mL glass flasks and sterilized at 1.2 kg.cm−1 and 121°C for 15 min. Single colonies were added to the YM broth and incubated for five days at room temperature under 100 RPM shaking. Bacterial growth was evaluated in each treatment by reading the absorbance of the broth at 560 nm (OD560 nm) after 0, 24, 48 and 120 h of culture, using a plate reader EnSpireTM 2300 Multilabel Reader (Perkin-Elmer Inc., USA). The IAA pro- duction was estimated after five days of culture. For the estimation of IAA produc- tion, the broth was centrifuged at 12,000 RPM for 15 min, the cell-free supernatant reserved and the IAA was estimated spec- trophotometrically by mixing 1.0 mL of the supernatant with 1.5 mL of the modified ferric chloride-sulphuric acid reagent (1.0 mL of 0.5M FeCl3, 30 mL of H2SO4 and 50 mL H2O) as described by Gordon and Weber (1951)7. The optical density was Figure 1. Bacterial growth of M. loti strains SEMIA806 and SEMIA816 cultivated in YM broth without and with addition of copper (YM = YM broth without copper addition; recorded at 530 nm (OD530 nm) after 75 –1 YM+Cu10 = YM broth with addition of 10 mg.L of CuSO4; YM+Cu20 = YM broth with min of incubation at room temperature in a addition of 20 mg.L–1 of CuSO ; YM+Cu50 = YM broth with addition of 50 mg.L–1 of dark environment.7 4 CuSO4). Values are means over four replicates. Table 1. Spectrophotometric lectures of bacterial growth at 560 nm and amount of IAA produced by M. loti strains SEMIA806 and SEMIA816. Values are means over four replicates. The polynomial regression and the corresponding R2 values for the growth curve are given for each treatment.