Microbial activity of soil with sulfentrazone associated with phytoremediator species and inoculation with a bacterial consortium

Phytostimulation plays a key role in the process of rhizodegradation of herbicides in soil. Additionally, bio-enhancement associated with phytoremediation may increase the efficiency of the decontamination process of soils with herbicides. Therefore, the objective of this study was to evaluate the biomass and microbial activity of soil contaminated with sulfentrazone and cultivated with phytoremediator species plus a bacterial consortium. The experiment was conducted in a greenhouse, carried out with a 2 × 4 × 4 completely randomized factorial design with 4 replications. The first factor consisted of the presence or absence of bio-enhancement with a bacterial consortium composed of Pseudomonas bacteria; the second factor consisted of a monoculture or mixed cultivation of 2 phytoremediator species Canavalia ensiformis and Helianthus annuus, besides the absence of cultivation; the third factor was made up by the bio-remediation time (25, 45, 65, and 85 days after thinning). soil And PlAnt nutrition Article Microbial activity of soil with sulfentrazone associated with phytoremediator species and inoculation with a bacterial consortium Christiane Augusta Diniz Melo*, Wendel Magno de Souza, Felipe Paolinelli de Carvalho, André Marcos Massenssini, Antonio Alberto da Silva, Lino Roberto Ferreira, Maurício Dutra Costa Universidade Federal de Viçosa Fitotecnia Viçosa (MG), Brazil. *Corresponding author: chrisadinizmelo@yahoo.com.br Received: May 22, 2016 – Accepted: Oct. 3, 2016 Uncultivated soils displayed low values of microbial biomass carbon and microbial quotient as well as high values of metabolic quotient throughout the bio-remediation time, indicating the importance of cultivating phytoremediator species for the stimulation of soil microbiota. Bio-enhancement with the bacterial consortium, in general, promoted an increase in the microbial biomass and activity of soil contaminated with sulfentrazone. In the presence of the bacterial consortium, Canavalia ensiformis stimulated a greater activity of associated microbiota and supported a higher microbial biomass. Phytoremediation associated with microbial bio-enhancement are thus promising techniques for the bio-remediation for soils contaminated with sulfentrazone. This technique enhances the biomass and activity of soil microorganisms.


introduction
Soil is a complex and dynamic ecosystem, where biological activity is determined mainly by microorganisms (Zhou et al. 2011).Its microbes play an important role in nutrient cycling, pest control, maintenance of the soil structure and quality as well as in crop productivity (Gil et al. 2011;Lupwayi et al. 2012).Moreover, microorganisms influence the behaviour of herbicides in the soil since they have the capacity to obtain energy and nutrients for their survival from the transformation of these compounds into less toxic substances for the environment.
Many studies have shown the impact of herbicides on the soil microbial community.The results are varied, with direct or indirect effects, null (Zilli et al. 2008;Nakatani et al. 2014) or negative effects (Santos et al. 2006;Lane et al. 2012), depending on the products and their physical and chemical properties, dosage, type of application, and time after application.However, there are few reports on the microbial activity of soils contaminated with herbicides, and in particular with sulfentrazone, submitted to bio-remediation.
The application of sulfentrazone adversely affects soil microbial activity and biomass (Vivian et al. 2006;Silva et al. 2014).On the other hand, following the application of herbicides, a natural process of adaptation of existing native microbiota in the soil tends to occur, selecting for microorganisms capable of tolerating and or degrading the molecule, therefore, reducing the concentrations of those substances over time.
Sulfentrazone has high residual activity and can persist for years in the soil.Due to its persistence, it can cause injury of sensitive crops grown in succession (Artuzi and Contiero 2006) as well as increase the risk of leaching and environmental contamination.To decontaminate soils with a history of application of this herbicide, bio-remediation techniques involving phytoremediator plants and microorganisms with remedial potential can be used.
Plants contribute significantly to the process of bioremediation of soil contaminated with herbicides.Through the phytoremediation process, Helianthus annuus and Canavalia ensiformis are able to reduce toxic levels of sulfentrazone in soils (Belo et al. 2011;Madalão et al. 2012).Phytoremediation may occur by extraction, stabilization, volatilization, accumulation or degradation of the herbicide in a plant tissue or by stimulating associated indigenous microbiota, known as phytostimulation.Plants are able to support large microbial populations in the rhizosphere by exuding substances through the roots, such as carbohydrates and amino acids (Turpault et al. 2007), which supply important sources of nutrients for the microorganisms in the soil-root interface with a consequent stimulus to rhizodegradation.
Phytoremediation associated with bio-enhancement, which is the introduction of microorganisms or consortia to accelerate and to enhance the removal efficiency of in situ toxic compounds (Martin-Hernandez et al. 2012), promotes faster soil decontamination, allowing for a faster planting of sensitive crops in previously contaminated fields.Studies confirm that the increase in initial biomass level improves the biodegration rate of pollutants and increases the treatment efficiency (Pimmata et al. 2013;Szulc et al. 2014).
Soil respiration and enzyme activity are soil biological and biochemical processes regarded as useful indicators of quality and health of this ecosystem (Xiong et al. 2013).In addition, the metabolic and microbial quotients are widely studied as they reflect the microbiota efficiency in the use of carbon and in the quality of soil organic matter (Thirukkumaran and Parkinson 2000;Santos et al. 2006).
The study of such indicators in sulfentrazone contaminated soil can help in the understanding of the process of bio-remediation of this herbicide by soil microorganisms and remediator plants.
Thus, the objective of this study was to evaluate the microbial biomass and the microbial activity of sulfentrazone contaminated soil and cultivated with phytoremediator plants as well as a bacterial consortium.

MAtEriAl And MEtHods
The experiment was conducted in a greenhouse at the Federal University of Viçosa, in Viçosa (lat 20°45′S; long 42°52′W), Minas Gerais State.During the experiment, the average maximum and minimum temperatures were 27.6 and 15.5 °C, with 78% of average relative humidity.
The experiment was carried out in a 2 × 4 × 4 completely randomized factorial design with 4 replications.The first factor consisted on the presence or absence of inoculation with a previously selected bacterial consortium; the second factor consisted of a monoculture or a mixed cultivation of 2 phytoremediator species and the absence of cultivation; the third factor was composed of bio-remediation time: 25; 45; 65 and 85 days after thinning (DAT).
Pots with 12.0 dm 3 capacity were coated with a plastic bag and filled with 10.0 dm 3 of medium texture soil, without herbicide application history, with the following chemical characteristics: pH (water) of 6.1; organic matter content = 4.12 dag•kg −1 ; P = 6.9 and K = 200 mg•dm −3 ; Ca = 3.7; Mg = 1.0;Al = 0.0; H + Al = 1.15; and CTC effective = 5.21 cmol c •dm −3 .The soil was fertilized with ammonium sulphate (0.20 g•dm −3 of N) and simple superphosphate (1.80 g•dm −3 of P 2 O 5 ).After that, sulfentrazone was applied in all pots using a backpack sprayer with constant pressure maintained by CO 2 , bar coupled with 2 fan type tips TT110 02, spaced by 0.5 m, and working pressure of 250 kPa with spray volume of approximately 140 L•ha −1 at a dose of 1,000 g•ha −1 a.i.The environmental conditions at the time of application were T = 27 °C, RH = 72%, and wind speed of 1.9 km•h −1 .
One day following herbicide application, the remedial species Canavalia ensiformis (Madalão et al. 2012) and Helianthus annuus (cultivar Tera 860 HO) (Belo et al. 2011) were sown.Sowing was performed at 5 cm depth using 6 seeds per pot.After 15 days of sowing, thinning was conducted, leaving 2 plants of the same species or 1 of each (mixed cultivation) in each experimental unit.
The bacterial consortium used was selected based on the degradation potential presented by the isolates in a previous study (Melo et al. 2016).The consortium consisted of 6 isolates identified as Pseudomonas putida, P. lutea, and P. plecoglossicida, as well as 3 isolates of Pseudomonas sp.They were grown separately on nutrient broth medium (1.0 L water; 2.0 g Na 2 HPO 4 ; 3.0 g NaCl; 3.0 g of meat extract; 5.0 g peptone; 6.8 pH) for about 10 h at 30 °C and 150 rpm until reaching an optical density of 0.6.The medium was centrifuged (5,000 rpm; 5.0 min; 4 °C) and bacterial cells, suspended in saline solution (NaCl 0.85%).The consortium was established by pipetting volumes of solution of each of the 6 isolates to assure equal amount of colony forming units (CFU•mL −1 ) of each species, totalling 12.0 mL of solution, and inoculating 4.5 × 10 4 CFU•g −1 of soil in the corresponding treatments, right after the thinning.
The phytoremediator species were grown for 25; 45; 65 and 85 DAT, at which they were cut close to the ground.At the harvest dates, Canavalia ensiformis plants were at the vegetative stages V2 to V3; V4 to V5; V6 to V7 and V8 to V9 and Helianthus annuus plants were at the stages V16 to V18; R1 to R3; R5.1 to R5.8 and 5.10 to R6, respectively.The root system of the plants was removed, and all the soil was homogenized to take samples from each pot.The samples were placed in plastic bags and kept under refrigeration for 24 h until moisture determination.Respiratory rate, microbial biomass carbon, soil metabolic quotient, and microbial quotient were all estimated.
To assess respiratory rate, the respirometric method was used to determine C-CO 2 evolved from the soil in which samples of 100.0 g of sieved soil (2 mm mesh) and humidity equal to 60% of field capacity, in duplicate, were incubated for 15 days in airtight bottles at room temperature.The C-CO 2 released from soil was carried by continuous flow of free CO 2 air into another bottle containing 100.0 mL of NaOH 0.5 mol•L −1 .Fifteen days after incubation, the C-CO 2 evolved was estimated from the titration of 10.0 mL of NaOH (0.5 mol•L −1 ) solution, added with 5.0 mL of BaCl 2 (0.5 mol•L −1 ) with solution of HCl 0.5 mol•L −1 and 3 drops of phenolphthalein (1%).
Following the incubation period, 18.0 g of soil from each jar were weighed for determination of microbial biomass carbon (MBC) according to a methodology modified from Islam and Weil (1998).From the obtained values of C-CO 2 evolution and MBC, the metabolic quotient (qCO 2 , in µg•µg −1 •d −1 ) was calculated by dividing the daily average of soil evolved C-CO 2 by MBC determined in the soil (Anderson and Domsch 1993).The microbial quotient (qMIC, in %) was calculated as the ratio between the MBC and organic carbon (OC) (Anderson and Domsch 1989) of each experimental unit obtained through the content of soil organic matter (OM = OC × 1.724) estimated by the Walkley-Black method.
Data were submitted to analysis of variance by the F-test at 5% probability.Significant interactions were evaluated and the means, compared by Tukey's multiple comparisons test (p < 0.05).For bio-remediation times, simple linear models, Y = α + β 1 •X + ε, and also the quadratic ones, Y = α + β 1 •X + β 2 •X 2 + ε, were adjusted.The models were adjusted and compared for equality of the parameters α, β1, and β2 (Littell et al. 2006).

rEsults And discussion
The triple interaction among cultivation, inoculation, and bio-remediation time was significant for all assessed variables.The respiratory rate found in soils cultivated simultaneously with the 2 phytoremediator species was 42% higher than that of soils cultivated with C. ensiformis in the absence of inoculation, on the 25 th DAT.On the 65 th DAT, in treatments with inoculation of the bacterial consortium, the soil cultivated with H. annuus had a higher respiratory rate when compared to the other soils (Table 1).
Soil inoculated with the bacterial consortium promoted specific effects depending on the species cultivated and on the bio-remediation time.On the 25 th DAT, inoculation provided an increase of 44% in the respiratory rate of soil cultivated with C. ensiformis.The same behavior was observed for soils with H. annuus and mixed cultivation on the 85 th DAT.On the other hand, the lowest respiration rate was found in soil cultivated with C. ensiformis, in the presence of inoculation on the 65 th DAT (Table 1).
The respiratory rate of the soil cultivated with H. annuus and inoculation linearly increased over bio-remediation time, as well as uncultivated soil, regardless of the presence of bacterial consortium.Those treatments presented higher evolution of C-CO 2 (Figure 1a).
Soil cultivated with H. annuus and mixed cultivation, both without inoculation, also presented a linear behavior over time; however, lower microbial activity was observed (Figure 1a).Treatments with C. ensiformis and mixed cultures, both in the presence of inoculation, did not differ from each other and were adjusted into a common curve with quadratic effect and increase in microbial respiration from the 40 th DAT.
Uncultivated soil, regardless of inoculation and bioremediation time, had lower microbial biomass carbon than cultivated soils (Table 2).Regarding the presence of cultivation, C. ensiformis, on the 65 th and 85 th DAT in the presence and absence of microbial consortium, respectively, supported higher microbial biomass than H. annuus and mixed cultivation.On the 45 th and 85 th DAT, soils inoculated and solely cultivated with C. ensiformis and H. annuus exhibited greater MBC (Table 2).
Inoculation provided an increase in MBC in soils cultivated with the 2 species together on the 25 th DAT, by C. ensiformis on the 65 th and the 85 th DAT and by H. annuus on the 85 th DAT (Table 2).
Uncultivated soils, inoculated and non-inoculated showed a decreased MBC from the 63 th DAT and, in general, lower microbial biomass in relation to the cultivated ones.For the other treatments, an increase in the MBC was found over bio-remediation time (Figure 1b).In the presence of the bacterial consortium, C. ensiformis favoured the establishment of high microbial biomass in the soil over the 85 days of cultivation (Figure 1b).
Soil metabolic quotient with no cultivation was the highest, regardless of inoculation on the 65 th and 85 th DAT (Table 3).On the 85 th DAT, the average difference between cultivated and uncultivated soils reached 66% in the presence of inoculation and 71% in the absence of the bacterial consortium (Table 3).
On the 25 th DAT, soil without inoculation cultivated with C. ensiformis exhibited lower qCO 2 (Table 3) in an inverse manner and at the same proportion as on the 65 th DAT.A significant difference caused by the presence of the bacterial inoculum was also observed in uncultivated soil on the 65 th DAT (Table 3).
Over bio-remediation time, uncultivated soils displayed increasing values of metabolic quotient from the 50 th DAT (Figure 1c).Soil cultivated with C. ensiformis without inoculation and cultivated with H. annuus with inoculation presented an average reduction in qCO 2 from the 55 th DAT (Figure 1c).The mixed cultivation, regardless of inoculation, as well as C. ensiformis with inoculation and H. annuus without inoculation, did not have metabolic quotient changes over the cultivation period, especially with low values close to 0.20 µg•µg −1 •d −1 (Figure 1c).
The microbial quotient was influenced by all studied factors, being lower in uncultivated soils, despite the season and inoculation (Table 4).Monocultures of C. ensiformis and H. annuus, in the presence of inoculation, had higher qMIC values on the 45 th and 85 th DAT.The qMIC associated with C. ensiformis was higher on the 65 th and 85 th DAT, with the presence and absence of inoculum, respectively (Table 4).Inoculation promoted beneficial effects as qMIC increased in soils cultivated with H. annuus and C. ensiformis simultaneously on the 25 th DAT, with C. ensiformis and uncultivated soil on the 65 th DAT and with C. ensiformis and H. annuus separately on the 85 th DAT (Table 4).
Cultivation of C. ensiformis added with microbial consortium inoculation provided an increase in qMIC over bio-remediation time, compared to the other treatments (Figure 1d).The qMIC of soil cultivated with both phytoremediator species simultaneously without inoculation increased linearly over time as well.In uncultivated soils, regardless of inoculation, the qMIC presented decreasing values from the 54 th DAT (Figure 1d).
Microbial degradation of herbicides in the soil depends on the presence and activity of microorganisms able of (co)metabolizing them.The presence of herbicide in the soil, and particularly the cultivation of phytoremediator species, may support microbial growth and induce the release of increasing C-CO 2 over bio-remediation time, as observed in Figure 1a.The respiratory rate of the soil has been widely used in studies on waste decomposition and bio-remediation, associated with the quantification of the pollutant levels in the soil (Thirukkumaran and Parkinson 2000;Lamy et al. 2013).The evolution of CO 2 in the soil, alone, does not allow consistent interpretations since a high respiratory rate may indicate an active and degrading population in an ecosystem with a high level of productivity as well as an ecological disorder or disturbance.The latter may explain the high respiratory rates observed over time in uncultivated soils (Figure 1a).
MB C was ver y s ensit ive to t he pres ence of phytoremediator species, reducing dramatically in the absence of cultivation (Table 2, Figure 1b).In uncultivated soils, it is very common to find fewer microorganisms and lower metabolic activity compared to cultivated soils because there is no supply of carbon and energy via plant root exudation (Sandmann and Loos 1984).Cultivated soils support higher microbial biomass by means of the root exudates, which influence the growth of bacteria and fungi that colonize the rhizosphere with environmental changes in the surrounding soil serving as a substrate for selective growth of microorganisms (Cardoso and Nogueira 2007).Greater activity and microbial biomass in the rhizosphere soils treated with herbicides, in relation to nonvegetated soils, were also found in other studies (Pires et al. 2005;Santos et al. 2007;Santos et al. 2010), and this effect is attributed to phytostimulation of the associated microbiota.
This study failed to draw conclusions on the reduction of sulfentrazone concentration in the soil upon the action of microorganisms.However, there is a relationship between the size of soil microbial biomass and herbicide degradation capacity and contaminants in this ecosystem (Voos and Groffman 1997;Lamy et al. 2013).In the context of bioremediation, high and active microbial biomass associated with phytoremediator species C. ensiformis and H. annuus is of great interest because it may enhance the soil decontamination process with sulfentrazone.
Given the complexity of the soil ecosystem, the variety of compounds exuded by the roots, and the countless interactions established between plants and microorganisms, there are many possibilities for the transformation of xenobiotic compounds by rhizodegradation, a mechanism that contributes, in an integrated manner, to phytostimulation (Santos et al. 2007).Moreover, the variety of exudate compounds as well as the phenological growth stage of plants may influence their effectiveness in the bio-remediation process (Santos et al. 2010).The species Helianthus annuus and Canavalia ensiformis, previously identified as sulfentrazone phytoremediators (Belo et al. 2011;Madalão et al. 2012), display quite different morphophysiological and growth characteristics, which may result in distinct capacities for phytostimulation, as found in this study, and, therefore, for herbicide rhizodegradation in the soil.
Overall, the bio-enhancement provided positive results, improving the activity and microbial biomass associated with certain phytoremediator species at certain times.The effects observed over time suggest the existence of tolerance and possible degradation of sulfentrazone by the most adapted microbiota and selected after application of the herbicide.According to Wang et al. (2014), one of the limitations of bio-enhancement is that, in many occasions, contaminated sites may be deficient in nutrients that do not support the rapid growth of the added bacteria.However, in this study, although the survival of the bacterial consortium was not evaluated, it is believed that the presence of cultivation was crucial to ensure favorable conditions for growth and viability of these inoculated microorganisms.There is not much information in the literature regarding bioenhancement in the bio-remediation of soils contaminated with herbicides.
The treatment consisting of C. ensiformis added with the bacterial consortium stood out among the others in relation to MBC and qMIC over bioremediation time.Such evidence reinforces that C. ensiformis is effective in stimulating microbiota and very promising for the remediation of soils contaminated with sulfentrazone.Pires et al. (2005), evaluating the rhizospheric activity of species with potential for phytoremediation of the herbicide tebuthiuron, found that the microbial community associated with roots of C. ensiformis was the largest and the most active among the treatments studied.
The low qCO 2 values observed during bio-remediation in soils cultivated with C. ensiformis added with bacterial consortium, H. annuus without inoculation, and mixed cultivation indicate savings in the use of energy by microorganisms and presumably reflect a more stable environment or closer to its equilibrium state.This result indicates a lower C-CO 2 loss and higher incorporation of carbon in the microbial cells, which may evidence an active and more efficient microbiota in nutrient acquisition.On the other hand, the high qCO 2 values found in uncultivated soils are indicative of ecosystems that underwent stress or disturbance (Tótola and Chaer 2002), which may be related to the presence of herbicide and exhaustion of the nutrients in these soils.Such conditions stimulate the oxidation of organic matter by microorganisms resulting in lower soil conservation.These achievements thus indicate the long-term carbon losses that may occur if soils contaminated with sulfentrazone are not submitted to any bio-remediation technique as well as the importance of cultivation to encourage microorganisms to promote rhizodegradation and to improve the efficiency of soil herbicide decontamination.
A similar b ehaviour of the c ur ves adjusted to MBC and qMIC evidences the great influence of the MBC on the results of qMIC and a small influence of the soil organic carbon that has shown low variation among treatments.Carbon utilization capacity was increased (> qMIC) over time in inoculated soil, cultivated with C. ensiformis; however, in uncultivated soils, regardless of the inoculation, the microorganisms showed less economical metabolism (< qMIC).Overall, less organic carbon (C) was channelled into energy metabolism and more C was fixed in the microbial cells in cultivated soils.These results indicate a microbiota efficient in the use and incorporation of carbon into biomass and an active population in the soil, as well as desirable features that may reflect on the degradation of the herbicide.

conclusion
By means of microbiological indicators, it was obser ved that uncultivated soils displayed lower microbial biomass and activity than the cultivated ones.The phytoremediator species, in monoculture or mixed cultivation, have different capacities of stimulating the rhizosphere microorganisms.Canavalia ensiformis in the presence of a bacterial consortium is able to provide greater activity of associated microbiota and to support a higher microbial biomass.Phytoremediation associated with microbial bio-increase is a promising technique for the bio-remediation of soils contaminated with sulfentrazone.

table 1 .
Respiratory rate of soil with sulfentrazone cultivated with the phytoremediator species Canavalia ensiformis and Helianthus annuus in monoculture or mixed cultivation, in the absence or presence of bacterial consortium.
¹Means followed by the same lowercase letters in the column and uppercase letters on the row for each season do not differ by the Tukey's test (p > 0.05).DAT = Days after thinning; CV = Coefficient of variation.Figure 1.Respiratory rate (a), microbial biomass carbon (b), metabolic quotient (c), and microbial quotient (d) of soil contaminated with sulfentrazone and cultivated with phytoremediator species Canavalia ensiformis and Helianthus annuus, in monoculture or mixed cultivation, for different times, in the absence or presence of bacteria consortium.Models that did not differ from each other were presented in a common curve by the Model Identity Test (p ≤ 0.05).** , * and °Significant at 1, 5 and 10%, respectively, by the t-test.

table 2 .
Microbial biomass carbon of soil with sulfentrazone cultivated with the phytoremediator species Canavalia ensiformis and Helianthus annuus in monoculture or mixed cultivation, in the absence or presence of a bacterial consortium.Means followed by the same lowercase letters in the column and uppercase letters on the row for each season do not differ by the Tukey's test (p > 0.05).DAT = Days after thinning; CV = Coefficient of variation.

table 3 .
Metabolic quotient of soil with sulfentrazone cultivated with the phytoremediator species Canavalia ensiformis and Helianthus annuus, in monoculture or mixed cultivation, and in the absence or presence of a bacterial consortium.

table 4 .
Microbial quotient of soil with sulfentrazone cultivated with phytoremediator species, in monoculture or mixed cultivation, and in the presence or absence of a bacterial consortium.