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 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, , 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 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 on several ture medium, presenting effective growth. Materials and Methods 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

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(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. Bacterial growth Amount of IAA produced after 120h of culture ( g.mL-1) 2 OD560 Regression R 0h 24h 48h 120h SEMIA806 YM* 1.034 1.107 1.158 1.158 y = -0.018x2 + 0.134x + 0.917 0.99 2.42 YM+10Cu 1.048 1.078 1.099 1.099 y = -0.008x2 + 0.056x + 0.999 0.99 0.11 YM+20Cu 1.034 1.153 1.072 1.072 y = -0.029x2 + 0.152x + 0.926 0.47 0.13 YM+50Cu 1.036 1.177 1.072 1.072 y = -0.035x2 + 0.176x + 0.913 0.45 0.28 SEMIA816 YM 1.048 1.020 1.124 1.124 y = 0.007x2 − 0.003x + 1.032 0.67 2.48 YM+10Cu 1.075 1.011 1.146 1.146 y = 0.016x2 − 0.045x + 1.087 0.56 0.12 YM+20Cu 1.062 1.087 1.186 1.186 y = -0.006x2 + 0.078x +0.981 0.88 0.14 YM+50Cu 1.048 1.103 1.199 1.186 y = -0.017x2 + 0.135x +0.922 0.93 0.28 -1 -1 –1 *YM = YM broth without copper addition; YM+Cu10 = YM broth with addition of 10 mg.L of CuSO4; YM+Cu20 = YM broth with addition of 20 mg.L of CuSO4; YM+Cu50 = YM broth with addition of 50 mg.L of CuSO4.

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-1 24h of culture, strain SEMIA806 revealed CuSO4) to 2.42 µg.mL (control treatment there are no universally accepted metal con- higher growth for treatments with 20 mg.L-1 without copper) for SEMIA806, and from centrations to define bacterial tolerance or -1 -1 -1 21 and 50 mg.L of CuSO4, in comparison to 0.12 µg.mL (treatment with 10 mg.L of resistance. However, it is of extreme -1 the control without copper and to the treat- CuSO4) to 2.48 µg.mL (control treatment importance to determine whether the bacte- -1 ment with 10 mg.L of CuSO4. At 48h, without copper) for SEMIA816 (Table 1). rial isolates intended to be used for biore- treatments with 20 mg.L-1 and 50 mg.L-1 of Concerning the copper-containing treat- mediation are able to grow under environ- CuSO4 revealed higher decline in growth. ments, the highest production of IAA was mental contamination. -1 Differently, strain SEMIA816 shown bacte- observed for the 50 mg.L of CuSO4, fol- The present study demonstrated that M. -1 - rial growth after 24h and 48h of culture in lowed by 20 mg.L of CuSO4 and 10 mg.L loti SEMIA806 and SEMIA816 are resist- -1 -1 1 -1 the treatments with 20 mg.L and 50 mg.L of CuSO4 for both bacterial strains (Table ant to up to 50 mg.L of CuSO4 in the cul- of CuSO4, while the treatment with 10 1). In plants, IAA is involved in multiple ture medium, presenting effective growth -1 14,15 mg.L of CuSO4 revealed decrease in growth processes and stress responses. not significantly different from the control growth after 24h and increased growth after The higher amount of IAA produced by experiment without copper. In addition, it 48h of culture (Table 1 and Figure 1). bacteria cultivated under the higher concen- was shown that the synthesis of IAA was Former in vitro studies9-12 have demon- tration of copper in this study may be strongly reduced but not eradicated by ions strated that copper is more toxic than zinc response of the to stress. copper in the medium. Even though this and cadmium, inhibiting the growth of Higher amount of exopolysaccharides pro- heavy metal strongly reduced the IAA pro- Mesorhizobium at concentrations lower duction by M. loti strains SEMIA806 and duction for both studied strains of M. loti, than 20 mg.L-1. The resistance of SEMIA816 under a more stressful environ- the amount of hormone synthesis was simi- Rhizobium, Bradyrhizobium, Sinorhizobium ment concerning pH of the culture media lar to the quantity produced by different and Azorhizobium to copper reached con- was previously reported.16 Similar result bacterial species able to form nodules in centrations ranging mostly from 20 to 40 concerning exopolysaccharides production host plants. Therefore, it is expected that mg.L-1.12 Many microorganisms demon- under stressful conditions was reported for environmental copper found in the soil up -1 strate resistance to metals in water, soil and Bradyrhizobium, Rhizobium, to the concentration of 50 mg.L will not industrial waste. Genes located on chromo- Mesorhizobium, Sinorhizobium and preclude the symbiotic interaction between 12 somes, plasmids, or transposons encode Azorhizobium. Like the IAA, exopolysac- M. loti and leguminous host plant species. specific resistance to a variety of metal charides are symbiont derived signals As thryptophan is an efficient physio- 22 ions.13 The cop operon responsible by cop- essentials for nodulation in legume-rhizobia logical precursor of auxins, the presence 17 per resistance has been recorded in symbiosis. A positive effect of ion copper of this compound in the culture medium Enterococcus hirae, while an operon in the production of IAA by Pseudomonas improves the bacterial IAA synthesis in 4 3 responsible by copper sequestration with chlororaphis O6 was also demonstrated. vitro. Therefore, experiments including the similar nomenclature was described in With the addition of thryptophan to the cul- amendment of thryptophan to the culture Pseudomonas.13 Such genes can be natural- ture medium, the levels of accumulated medium should be performed to evaluate -1 ly transferred among bacteria through con- IAA after 48h was higher than 30 µg.mL the improvement of IAA synthesis by M. jugation and transduction, disseminating in cultures with a copper concentration of loti SEMIA806 and SEMIA816 cultivated -1 this characteristic, which can be quite 43 mg.L . with copper. In addition, controlled experi- important for biotechnological purposes. In The amount of IAA synthesized by M. ments of the interaction plant-bacteria in addition, genetic manipulation can be used loti SEMIA806 and SEMIA816 in this contaminated soils are needed in order to to take advantage of metal resistance mech- study is similar to the extent of IAA pro- evaluate the roots’ nodulation in such envi- anisms.1 duced by different rhizosphere bacteria as ronments, considering also the interaction 18 The use of microorganisms for the Burkholderia phytofirmans (0.84±0.33 copper-soil particles as well as the plant -1 recovery of contaminated areas is an µg.mL ), Rahnella sp, Burkholderia sp, synthesis of IAA. 19 -1 extremely important strategy and has been Pseudomonas sp. (0.57 to 2.43 µg.mL ; widely recognized as a very promissory Khan et al. 2016) and 35 unidentified bacte- 20 bioremediation method1. In that sense, the ria isolated from soybean in Indonesia References (0.051 to 3.208 µg.mL-1). Considering that capacity of growing in a copper-rich envi- 1. Vieira, JD, Stefenon VM. Soil bioreme- all these bacteria were able to interact with ronment demonstrated from M. loti strains diation in heavy metal contaminated the host-plants, the low amount of IAA syn- SEMIA806 and SEMIA816 has a signifi- mining areas: a thesized by M. loti SEMIA806 and cant meaning for the bioremediation of cop- microbiological/biotechnological point SEMIA816 may be sufficient for the estab- per-contaminated areas using the rhizore- of view. J Advances Microbiol 2017; lishment of nodules in roots of selected mediation approach. 4:33782 plant species in soils contaminated with 2. Mani D, Kumar C. Biotechnological copper within the range tested in this study. IAA synthesis advances in bioremediation of heavy Different from the bacterial growth, the metals contaminated ecosystems: an production of IAA was strongly affected by overview with special reference to phy- the copper added to the YM broth. The pro- toremediation. Int J Environ Sci duction of IAA by the control treatment Conclusions and perspectives Technol 2014;11:843-72. without copper was significantly higher Factors such as the culture media 3. Spaepen S, Vanderleyden, J. Auxin and (P<0.005) in comparison to the copper-con- employed, growth conditions, and incuba- plant-microbe interactions. Cold Spring taining treatments. The amount of IAA syn- tion period, besides the various possible Harb Perspect Biol 2011;3:a001438. thesized by the control treatment was from forms and concentrations of metals used in 4. Dimkpa CO, Calder A, Britt DW, et al. eight to 20-fold higher than the copper-con- the tests of tolerance may difficult their Responses of a soil bacterium, taining treatments, ranging from 0.11 standardization and influence the in vitro Pseudomonas Chlororaphis O6 to com- µg.mL-1 (treatment with 10 mg.L-1 of toxicity of the metals. Due to these facts mercial metal oxide nanoparticles com-

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