Pseudomonas fluorescens

[Full title: Pseudomonas fluorescens: A Bioaugmentation Strategy for Oil-Contaminated and Nutrient-Poor Soil]

International Journal of Environmental Research and Public Health Article Pseudomonas fluorescens : A Bioaugmentation Strategy for Oil-Contaminated and Nutrient-Poor Soil Eduardo Jahir Guti é rrez 1 , Mar í a del Rosario Abraham 2 , Juan Carlos Baltazar 3 , Guadalupe V á zquez 4 , Eladio Delgadillo 4 and David Tirado 4, * 1 Facultad de Ciencias Qu í mico-Biol ó gicas, Universidad Aut ó noma de Campeche, Av. Agust í n Melgar S / N, Buena Vista, Campeche 24039, Mexico; ejgutier@uacam.mx 2 Divisi ó n de Ciencias de la Vida, Departamento de Ingenier í a en Alimentos, Universidad de Guanajuato, Hacienda El Copal Km 9, Carretera Irapuato-Silao; AP 311, Gto. Irapuato 36500, Mexico; mrosarioabdiciva@gmail.com 3 Divisi ó n de Ingenier í as, Departamento de Ingenier í a en Minas, Universidad de Guanajuato, Metalurgia y Geolog í a, Ex Had. De San Matias S / N. Col. San Javier, Gto. Guanajuato 36020, Mexico; jc.baltazarvera@ugto.mx 4 Divisi ó n de Ingenier í as, Departmento de Ingenier í a Civil y Ambiental, Universidad de Guanajuato, Av. Ju á rez N 77, Col. Centro, Guanajuato Gto. 36000, Mexico; vazquez.g@ugto.mx (G.V.); e.delgadillo@ugto.mx (E.D.) * Correspondence: d.tirado@ugto.mx; Tel.: + 52-473-181-2370 Received: 12 August 2020; Accepted: 15 September 2020; Published: 23 September 2020 Abstract: Bioremediation technology is one of the most profitable and sustainable strategies for remediating soils contaminated with hydrocarbons. This study focuses on assessing the influence of biostimulation and bioaugmentation with Pseudomonas fluorescens to contribute to the removal of total petroleum hydrocarbons (TPHs) of a soil. Laboratory studies were carried out (measurements of emitted CO 2 , surface tension, and residual TPH) to select the best bioaugmentation and biostimulation treatment. The sources of C, N, and P were glucose–yeast extract, NH 4 Cl–NaNO 3 , and K 2 HPO 4 –K 3 PO 4 , respectively. The e ff ect of culture conditions on the reduction of TPH and respiratory activity was evaluated through a factorial design, 2 3 , in a solid culture system After 80 days of incubation, it was observed that treatments of yeast extract–NH 4 Cl–K 2 HPO 4 (Y 4) and glucose–NaNO 3 –K 3 PO 4 (Y 5) presented a higher level of TPH removal (20.91% and 20.00% degradation of TPH, respectively). Biostimulation favors the production of biosurfactants, indirectly measured by the change in surface tension in the soil extracts. The treatments Y 4 and Y 5 showed a lower change value of the surface tension (23.15 and 23.30 mN · m − 1 at 25 ◦ C). A positive correlation was determined between the change in surface tension and the removal of TPH; hence there was a contribution of the biosurfactants produced to the removal of hydrocarbons Keywords: TPH; Pseudomonas fluorescens ; biodegradation; bioavailability; biosurfactant; surface tension 1. Introduction In the process of extracting fossil fuels, the industry dedicated to the extraction and refinement of petroleum has generated, per year, hundreds of thousands of tons of soil contaminated with petroleum hydrocarbons [ 1 ], which comprise a complex mix of nonaqueous components. Their hydrophobic characteristics make their degradation by the soil microbiota less e ffi cient, resulting in persistent and recalcitrant pollutants in the ecosystem [ 2 ]. They are, in rank, number nine on the priority list of substances of the Agency for Toxic Substances and Disease Registry 2017 (ATSDR) [ 3 ]. The natural attenuation of hydrocarbon is a ff ected by soil properties like organic matter content, particle size, and the abundance of microbiota [ 4 ]. Of the many treatments used for dealing with this type of pollutant, Int. J. Environ. Res. Public Health 2020 , 17 , 6959; doi:10.3390 / ijerph 17196959 www.mdpi.com / journal / ijerph

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 2 of 14 bioremediation technology is one of the most profitable and sustainable strategies for remediating soils contaminated with hydrocarbons [ 5 ] and accelerating the natural biodegradation process [ 6 ]. Within bioremediation, the two most successful remediation technologies are bioaugmentation and biostimulation, with the former consisting of the addition of a microorganism suspension to the soil The suspension is taken from the same indigenous microorganisms or from exogenous microorganisms with previously studied degradative capabilities [ 7 – 9 ]. Biostimulation involves the addition of nutrients to remove the metabolic limitation of the microbial community and, thus, stimulate its ability to degrade the pollutant [ 10 , 11 ]. Prior research has proven that these two techniques improve the degree to which hydrocarbons can be eliminated from the soil [ 9 , 12 , 13 ]. Various groups of microorganisms, such as sulfate, nitrate, and iron-reducing archaea or fermentative, syntrophic, and methanogenic archaea, directly or indirectly contribute to the degradation of petroleum hydrocarbons [ 14 ]. Several factors need to be taken into account for adequate bioremediation of the polluted soil. First, there are pollutant properties such as polarity, solubility, volatility, and ability to react with other substances. The pollutant concentration defines whether another technology needs to be employed [ 15 ]. Second, the following soil properties define the chemical and physical environments: hydrophobicity, pH, moisture content, and organic matter content. The pH a ff ects the solubility, mobility, availability, and ionic forms of a pollutant. Soil moisture is important for oxygen di ff usion, but after 60% moisture content, the relative rates of respiration of microbiota decrease [ 16 ]. A greater organic matter content can decrease the mobility of organic pollutants [ 17 , 18 ]. Surfactants can be used to increase the bioavailability of petroleum hydrocarbons that are hydrophobic to the aqueous system, reducing the surface tension of the medium and creating two phases with di ff erent polarities in the pollutant [ 19 ]. The biosurfactants produced by various microorganisms present lower toxicity and, depending on the type of microorganism, a higher level of bioavailability than chemical surfactants [ 20 , 21 ]. Among the most studied are the rhamnolipids, lipopeptides, and phospholipids [ 19 , 22 ]. Biosurfactants have, therefore, been shown to have a wider application for use in the degradation of petroleum hydrocarbons [ 21 , 23 – 25 ]. Various studies can be found in the scientific literature on the segregation and characterization of biosurfactants produced by the Pseudomonas genus [ 26 , 27 ]. While some studies have reported the production of biosurfactants in culture medium [ 28 , 29 ], it is important to generate more information on the e ffi cacy of microorganisms to produce biosurfactants in the complex mixture of petroleum hydrocarbons. The objective of this study is to evaluate both the e ffi ciency of total petroleum hydrocarbon (TPH) degradation, applying biostimulation and bioaugmentation strategies with Pseudomonas fluorescens ATCC 49642 and the production of biosurfactants in the soil via the simultaneous study of both strategies 2. Materials and Methods 2.1. Selection of the Study Area and Soil Sample For the selection of a sampling site, a place that was contaminated by a hydrocarbon spill and had not received treatment to remediate the soil was located; the site had had a year since a hydrocarbon spill occurred. The soil used in this study was taken from a field belonging to the Escolin petrochemical plant, in the municipality of Poza Rica, Veracruz, M é xico, located in the center of the state at 20 ◦ 32” N and 97 ◦ 27” E at an altitude of 50 m.a.s.l. This region is considered to have conglomerate soil. The climate is warm, with an annual average temperature of 24.2 ◦ C, annual average precipitation of 1010 mm, and relative humidity of 76% to 80% (INEGI, 2015). Twenty-five soil samples were taken, with a depth range of 0–50 cm and an approximate weight of 4 kg each; all samples were mixed to obtain a composited sample 2.2. Culture Medium and Growth Kinetics of P. Fluorescens The basal medium for the P. fluorescens cell culture was selected in accordance with that implemented by Liu et al. [ 19 ], with some modifications, to inoculate Pseudomonas taiwanensis and

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 3 of 14 degrade crude oil. The media, contained in milliequivalent water, was prepared with the following nutrients (g · L − 1 ): 13.6 glucose 0.01 yeast extract, 1 NH 4 Cl, 5.0 NaNO 3 , 0.2 Fe 2 SO 4 .7 H 2 0, 0.2 K 3 PO 4 , 2.0 K 2 HPO 4 , 0.2 MgSO 4 .7 H 2 0, 5.0 NaCl, 0.2 KCl, 0.2 CaCl.2 H 2 0, 0.006 ZnSO 4 .7 H 2 0, 0.1 MnSO 4 .7 H 2 0, 0.0006 CoCl 2 .6 H 2 0, and 0.0006 CuSO 4 .5 H 2 0. The pH of the media was adjusted to 7.0 with NaOH 7 M and then sterilized for 15 min at 121 ◦ C in an autoclave. For the growth kinetics, 100 mL of culture medium was prepared in 250 mL Erlenmeyer flasks and inoculated with the biomass obtained from the washing of two Petri dishes. The sample was incubated at a temperature of 35 ◦ C at 220 rpm for 24 hrs. Three replicates were carried out per sample. Absorbance at 550 nm (VELAB UV / visible spectrophotometer, VE-5100 UV) was measured at distinct incubation times in order to determine growth kinetics (hours, and 1, 2, 3, and 4 d). Once the log phase of microbial growth had been studied, the researchers opted to prepare and inoculate the culture medium again, this time taking 10% of the culture medium to inoculate the treatments from the experimental design. In the growth kinetics of P. fluorescens, a maximum exponential growth was observed at 48 h of incubation, and it stabilized from 72 h onwards, entering into its stationary phase from the third day onwards 2.3. Physicochemical Characterization of the Soil The physicochemical and biological properties of the soil were investigated. Samples were crushed with a mortar and pestle after being air-dried and passed through a 2-mm sieve. Soil moisture was determined by gravimetry. The pH of the soil suspension was measured by potentiometry. The density was made by the pycnometer method. For nitrogen and total phosphorus, they were measured by micro-Kjeldahl and Bray I. Organic matter content was obtained by oxidation with chromic and sulfuric acid. The concentration of TPH present in the soil was calculated by gravimetry. For the concentration of the microbiota present in the soil, the colony-forming units (CFUs) on a plate were counted 2.4. Experimental Design The maximum and minimum concentrations of the nutrients were selected based on a review of the literature [ 30 , 31 ]. The information on P. fluorescens regarding the production of biosurfactants is very limited; for this reason, the selection was based on the nutrients that influence the production of biosurfactants in other microorganisms of the same genus [ 20 , 21 , 30 – 33 ]. To determine the response variables, a 2 3 factorial design was undertaken and is presented in Table 1 . There were two controls: the first containing soil contaminated with TPH and native microbiota, without the addition of nutrients (C 1), and the second containing soil contaminated with TPH, P. fluorescens inoculum, and native microbiota, without the addition of nutrients (C 2). The following continuous variables were in the experimental design: C / N = 100:5; C / P = 100:5; 80 days of incubation; environmental temperature (30–33 ◦ C); 30% humidity, moisture content was monitored every 5 days with a Mengshen dew point temperature and humidity meter; 10% inoculum of P. fluorescens at a concentration of 3.2 × 10 6 CFU with 24 h incubation; each sample contained 3 kg of soil contaminated, the concentration of TPH in soil was 50,000 mg · kg − 1 . Three replicates per treatment were manipulated. The independent variables were the addition of sources of C, N, and P (Table 1 ). Finally, the response variables measured in this experimental design were residual TPH, surface tension, respiratory activity, and colony-forming units for fungi (CFU f ) and colony-forming units for bacteria (CFU b ).

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 4 of 14 Table 1. Representation of factors and levels, the experimental matrix for the 2 3 factorial design, the experimentation plan, and the responses measured Factors (Independent Variables) Levels Sources of − 1 (g · kg − 1 of soil) + 1 (g · kg − 1 of soil) X 1 : Source of carbon 13.6 (Glucose) 0.01 (Yeast extract) X 2 : Source of nitrogen 5.0 (NaNO 3 ) 1.0 (NH 4 Cl) X 3 : Source of phosphorous 2.0 (K 2 HPO 4 ) 0.2 (K 3 PO 4 ) Experiment matrix Experimentation plan Response Treatment X 1 X 2 X 3 Source of carbon Source of nitrogen Source of phosphorous 1 − − − Glucose NaNO 3 K 2 HPO 4 Y 1 2 + − − Yeast extract NaNO 3 K 2 HPO 4 Y 2 3 − + − Glucose NH 4 Cl K 2 HPO 4 Y 3 4 + + − Yeast extract NH 4 Cl K 2 HPO 4 Y 4 5 − − + Glucose NaNO 3 K 3 PO 4 Y 5 6 + − + Yeast extract NaNO 3 K 3 PO 4 Y 6 7 − + + Glucose NH 4 Cl K 3 PO 4 Y 7 8 + + + Yeast extract NH 4 Cl K 3 PO 4 Y 8 2.5. Analytical Methods for the Measurement of the Response Variables 2.5.1. Extraction of Residual TPH from the Soil The extraction and determination of TPH were carried out according to Mishra et al. [ 34 ], with some modifications; 10.0 g of dry soil sample was separately collected from every treatment, mixed with 50 mL dichloromethane (DCM) and 2 g of Na 2 SO 4 . The mixture was shaken in a vortex for a period of 45 s and extracted after 15 min using an ultrasonic processor (JY 96-IIN, Ningbo Science Biotechnology CO., LTD, Zhejiang, China), and then centrifuged for 10 min at 6000 rpm. The extraction procedure was repeated three times. The combined extracts were dried using a rotary evaporator at 45 ◦ C, and TPH was then quantified gravimetrically 2.5.2. Surface Tension The surface tension was measured using the hanging drop method in a Model 200–00 Standard Goniometer with DROPimage Standard software (ram é -hart instrument co., Succasunna, New Jersey, EEUU) and Young–Laplace software, which is used to describe the shape of the drop under equilibrium conditions [ 35 ]. 2.5.3. Respiratory Activity (CO 2 ) Gas (CO 2 ) analysis was measured using the Gow-Mac Series 580 gas chromatograph, which is equipped with a flame ionization detector (FID) and a capillary column (30 m × 0.32 mm × 40 um) Hydrogen was used as the carrier gas; the oven was at 250 ◦ C, the injector at 250 ◦ C, and the flame ionization detector at 350 ◦ C. A calibration curve was constructed with CO 2 as a reference standard All readings were done in duplicate. To carry out the dilutions of the CO 2 standard, a propylene bag was used, and compressed air was used as a solvent. To inject the samples, a 5 mL SOCOREX syringe for gases was used [ 36 ]. 2.5.4. Microbial Count Two groups of microorganisms were evaluated—bacteria and fungi. Nutrient agar (Bioxon, Cuautitl á n, Izcalli, Mexico State, Mexico) and rose bengal agar (Difco, Franklin Lakes, New Jersey, USA) were used for the microbial count and for the fungi, respectively. The microorganism count was

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 5 of 14 undertaken using the plate count method for the colony-forming units (CFUs), in which 1 g of soil was weighed and then diluted in 9 mL of sterile saline water (NaCl at 0.9% w / v ). A serial dilution of 1 mL in 9 mL of sterile distilled water was undertaken on the colloidal suspension obtained, with each dilution shaken prior to the next. Once the dilutions (10 − 1 to 10 − 8 ) had been undertaken, 100 µ l was taken from the 10 − 2 –10 − 4 solutions to obtain the heterotrophic microorganisms, while 100 µ l was taken from the 10 − 4 –10 − 6 solutions to obtain the fungi. This was then poured, in sterile conditions, into Petri dishes previously prepared with the medium. Each dilution was inoculated in triplicate. The samples inoculated for the growth of bacteria were incubated at 37 ◦ C for 48 to 72 h, while the Petri dishes inoculated for the growth of fungi were incubated at 28 ◦ C for 7 days in darkness 2.6. Statistical Analysis The experiments of TPH degradation were carried out in triplicate, results of which represent the mean ± standard deviation. Two-way analysis of variance (ANOVA) and multiple comparisons of means with the LSD and Tukey tests were performed with Minitab Version 18 software. Di ff erences between means at the 5% level ( p < 0.05) were considered significant. The analysis of the comparison of treatments with the controls was carried out with Dunnett’s tests, HSU, and a response optimizer 3. Results and Discussion 3.1. Soil Properties Characterization of the contaminated soil showed that there was an unfavorable ratio among the principal nutrients, which were carbon, nitrogen, and phosphorous. The phosphorous was not detected (Table 2 ). The TPH content shows that this was a highly contaminated soil with a slightly alkaline pH, with a moisture content of close to 30% [ 37 ], which explains the relationship reported between the moisture content and the degradation of TPH [ 38 ]. The indigenous microorganism content in the soil was low (1.04 × 10 4 CFU); as reported by Kumari et al. [ 39 ], the concentration of P. fluorescens for the bioaugmentation treatment was 1.2 × 10 7 colony forming units (CFUs) Table 2. The di ff erent analytical methods applied to determine the physicochemical parameters of the soil sampled, with each parameter carried out in triplicate Parameter Value Method Moisture (%) 32.64 ± 0.46 Gravimetry pH 7.85 ± 0.01 Potentiometric Density (kg.cm 3 ) 1.09 ± 0.03 Pycnometer Total nitrogen (%) 0.25 ± 0.00 Micro-Kjeldahl Total phosphorous (mg · kg − 1 ) n / d Bray I Organic matter (%) 11.14 ± 0.26 Oxidation Texture Sandy-clay Hydrometer TPH (mg · kg − 1 ) 50,000 ± 852 Gravimetry Total bacteria (CFU b ) 1.04 × 10 4 ± 3.21 × 10 2 Plate count Total fungi (CFU f ) 1.06 × 10 3 ± 3.06 × 10 1 Plate count The data were mean of triplicates; n / d: not detected, ± : standard deviation. CFU b : colony-forming units for bacteria; CFU f : colony-forming units for fungi The presence of hydrocarbons results in high levels of organic matter content (C), which causes a low concentration of indigenous microorganisms in the soil [ 40 ]. A deficiency in both N and P has been reported as one of the limiting factors for the growth of microorganisms capable of degrading pollutants [ 41 ] due to the fact that they cannot synthesize compounds such as proteins and nucleic acids, which are essential for microorganism growth, reproduction, and metabolic activity. The low values in the microbial count are due to the fact that not all microorganisms are capable of growing in the presence of hydrocarbons, owing to their structural complexity and their toxicity, so they cannot be

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 6 of 14 degraded by the action of the microbiota. This leads to a decrease in the microbial count compared to noncontaminated soils [ 40 ]. Therefore, considering the low microbial concentration identified in the soil featured in this study, researchers opted to apply bioaugmentation using P. fluorescens to degrade the hydrocarbons more actively and help the soil recover its properties. Additionally, the biostimulation of the indigenous microorganisms by applying a 2 k experimental design to obtain the best response treatment and whether there is synergistic or antagonistic interaction between these two remediation strategies was studied 3.2. Residual TPH The analysis of variance undertaken on this experimental design revealed that the nutrient sources and their respective interactions presented significant di ff erences in the response variable, which, in this case, is TPH. The values corresponded to a homogeneous distribution and were independent, for which reason they did not depend on a covariate. The nutrients of the soil used were adjusted by applying inorganic fertilizer compounds. According to the test statistics used (LSD and Tukey), the best treatments were Y 4, followed by Y 5. Reviewing Table 1 , where the treatments of the experimental design can be observed, treatment Y 4 is composed of the factors yeast extract, NH 4 Cl, and K 2 HPO 4 , and treatment Y 5 is composed of glucose, NaNO 3 , and K 3 PO 4 . The Dunnett test results showed that all treatments were significant compared to the control (natural attenuation), with treatments Y 4 and Y 5 having a greater e ff ect on TPH, with no significant di ff erence found between them. In order to select the best treatment, HSU and response optimizer tests were applied; it was found that Y 4 treatment was still the best option (yeast extract–NH 4 Cl–K 2 HPO 4 ). It is thus necessary to balance the cost of the glucose, the nutrients, and the quantities added when taking into account the nutrient concentrations These ratios of C:N:P were used as they are known to provide the required quantity to convert 100% of petroleum C to cellular biomass [ 41 ]. Di ff erent behavior was observed in the majority of the biostimulation treatments using di ff erent nutrients for the removal of TPH. Figure 1 presents the removal of TPH in the system augmented with P. fluorescens , biostimulated with di ff erent sources of nutrients, along with their two respective controls (C 1—natural attenuation and C 2—bioaugmented system without biostimulation). A low degradation rate was observed in C 1, which is due to the low concentration of indigenous microorganisms in the soil and this suggests that the application of biostimulation improves the biodegradation of the pollutant in the soil [ 42 ]. A low level of TPH removal was observed in Control 2 (C 2), a treatment representative of bioaugmentation with P. fluorescens in contaminated soil. This is because more than one bacterial species is required to e ffi ciently degrade TPH, as no single microbial species can metabolize the complex mixture of petroleum [ 8 ]. A possible option to improve soil bioaugmentation is to add a substrate that allows P. fluorescens to form biofilms; in previous studies, it has been observed that the implementation of substrates increases the formation of biofilms of P. putida , improving the bioremediation of polluted soil [ 43 , 44 ]. The initial concentration of TPH for all treatments was 50,000 mg · kg − 1 . After an incubation period of 80 days, it was observed that treatments Y 4 and Y 5 presented a higher level of TPH removal (8667 and 8334 mg · kg − 1 , respectively). Bioremediation depends mainly on soil characteristics and pollutant aging [ 45 ]. The presence of high organic matter content and clay may a ff ect the extent of biodegradation due to a priming e ff ect on microbial communities and a decrease in accessibility to microorganisms [ 46 ]. The bioavailability of hydrophobic compounds could be reduced by entrapping the compounds into the solid phase of the organic matter [ 47 ].

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 7 of 14 Int. J. Environ. Res. Public Health 2020 , 17 , x 7 of 15 comparing them with control 2 (C 2), which corresponds to the bioaugmentation without adding nutrients, the difference in degradation efficiencies in favor of nutrient treatment can be observed. Another observation that can be deduced from these results is that, in reality, the soil studied lacked nutrients, which has prevented the natural attenuation of the pollutant in the soil and resulted in the low degradation rate of the bioaugmentation treatment with P. fluorescens [51]. The presence of biosurfactants produced using P. fluorescens increases the rates at which pollutants are degraded. Previous studies have quantified biosurfactants in bacteria of the same genus, which improved the availability of the pollutant, as is the case with Pseudomonas putida and aeruginosa [52,53]. In another study, bioaugmentation of engine-oil-contaminated soil samples with P. aeruginosa TPHK-4 resulted in 22% more degradation of hydrocarbons than that occurring in naturally-attenuated samples within 90 days; P. putida TPHK-1 also performed better by effecting 18% more degradation of hydrocarbons when compared with natural attenuation [54]. In several studies, the observed increase in TPH removal when soil inoculation was performed was due to different species of Pseudomonas ( brassicacearum, mandelii, frederiksbergensis, putida ) with a hydrocarbon-degrading ability [55–58]. Therefore, the obtained data seems to be consistent with a previous comparative study of Agnello et al. [56] that demonstrated that bioaugmentation was more effective than natural attenuation on the degradation of total petroleum hydrocarbon. Figure 1. Removal of total petroleum hydrocarbons (TPHs), applying different biostimulation treatments. Standard deviation of the means ( Ι ). “a,A”: The lower case and uppercase letters in parentheses correspond to the differences between means of the LSD and Tukey tests, respectively. Statistically, the source of nitrogen is the factor that presents the greatest effect on TPH. Nitrogen is considered one of the most important factors due to both its essential role as a cellular constituent and the conservation of metabolic energy [59]. The source of phosphorous has a less significant effect on TPH. The best effect on TPH is achieved with glucose–NH 4 Cl–K 3 PO 4 . It can be deduced that the effects of the interaction among the three sources of biostimulation in relation to TPH are statistically significant. As a source of carbon, glucose presents a higher level of interaction with NaNO 3 and K 3 PO 4 , while the yeast extract with NH 4 Cl and K 2 HPO 4 presents a higher level of interaction with TPH. As a source of nitrogen, NaNO 3 has a higher level of interaction with glucose and K 3 PO 4 , while NH 4 Cl has a higher level of interaction with yeast extract and K 2 HPO 4 . The addition of nutrients suggests that this is a key strategy for facilitating the degradation process [60] The three factors, with the respective interactions, influence TPH, with the nitrogen source and carbon source (BC) interactions exercising the greatest effect on TPH. It is observed that the interaction between the three biostimulation sources presents the lowest effect. The source of phosphorous can be eliminated from the treatment and can, thus, reduce the remediation costs Figure 1. Removal of total petroleum hydrocarbons (TPHs), applying di ff erent biostimulation treatments. Standard deviation of the means (I). “a,A”: The lower case and uppercase letters in parentheses correspond to the di ff erences between means of the LSD and Tukey tests, respectively Using a system of biostimulation with inorganic nutrients and bioaugmentation with the Acinetobacter strain KF 453955, the authors of [ 48 ] achieved degradation like that obtained in this investigation, with an initial TPH concentration of 46,000 mg · kg − 1 . Wu et al. showed that biostimulation is more e ffi cient in degrading TPH than bioaugmentation used separately, as inorganic nutrients stimulate microbial growth and, therefore, increase degradative activity [ 49 ]. These results agreed with previous studies that concluded that biostimulation is more e ffi cient than bioaugmentation for remediating soils contaminated with hydrocarbons [ 10 , 11 , 50 ]. In previous studies, the combination of biostimulation and bioaugmentation treatments increased the degradation of TPH in soils [ 9 , 13 ]. Figure 1 shows that the biostimulation process is the most important since the eight treatments received the same amount of P. fluorescens inoculum, and, comparing them with control 2 (C 2), which corresponds to the bioaugmentation without adding nutrients, the di ff erence in degradation e ffi ciencies in favor of nutrient treatment can be observed. Another observation that can be deduced from these results is that, in reality, the soil studied lacked nutrients, which has prevented the natural attenuation of the pollutant in the soil and resulted in the low degradation rate of the bioaugmentation treatment with P. fluorescens [ 51 ]. The presence of biosurfactants produced using P. fluorescens increases the rates at which pollutants are degraded. Previous studies have quantified biosurfactants in bacteria of the same genus, which improved the availability of the pollutant, as is the case with Pseudomonas putida and aeruginosa [ 52 , 53 ]. In another study, bioaugmentation of engine-oil-contaminated soil samples with P. aeruginosa TPHK-4 resulted in 22% more degradation of hydrocarbons than that occurring in naturally-attenuated samples within 90 days; P. putida TPHK-1 also performed better by e ff ecting 18% more degradation of hydrocarbons when compared with natural attenuation [ 54 ]. In several studies, the observed increase in TPH removal when soil inoculation was performed was due to di ff erent species of Pseudomonas ( brassicacearum, mandelii, frederiksbergensis, putida ) with a hydrocarbon-degrading ability [ 55 – 58 ]. Therefore, the obtained data seems to be consistent with a previous comparative study of Agnello et al. [ 56 ] that demonstrated that bioaugmentation was more e ff ective than natural attenuation on the degradation of total petroleum hydrocarbon Statistically, the source of nitrogen is the factor that presents the greatest e ff ect on TPH. Nitrogen is considered one of the most important factors due to both its essential role as a cellular constituent and the conservation of metabolic energy [ 59 ]. The source of phosphorous has a less significant e ff ect on TPH. The best e ff ect on TPH is achieved with glucose–NH 4 Cl–K 3 PO 4 . It can be deduced that the

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 8 of 14 e ff ects of the interaction among the three sources of biostimulation in relation to TPH are statistically significant. As a source of carbon, glucose presents a higher level of interaction with NaNO 3 and K 3 PO 4 , while the yeast extract with NH 4 Cl and K 2 HPO 4 presents a higher level of interaction with TPH As a source of nitrogen, NaNO 3 has a higher level of interaction with glucose and K 3 PO 4 , while NH 4 Cl has a higher level of interaction with yeast extract and K 2 HPO 4 . The addition of nutrients suggests that this is a key strategy for facilitating the degradation process [ 60 ]. The three factors, with the respective interactions, influence TPH, with the nitrogen source and carbon source (BC) interactions exercising the greatest e ff ect on TPH. It is observed that the interaction between the three biostimulation sources presents the lowest e ff ect. The source of phosphorous can be eliminated from the treatment and can, thus, reduce the remediation costs 3.3. Surface Tension Surface tension for the C and N sources were statistically di ff erent. Nitrogen had the greatest e ff ect on TPH attenuation. The phosphorous source was less significant in response to the attenuation of TPH. Therefore, the Y 4 treatment, which corresponds to yeast extract–NH 4 Cl–K 2 HPO 4 , presented lower surface tension, which translates to the treatment generating the greatest amount of biosurfactant Yeast extract, as a carbon source, has a greater level of interaction with NH 4 Cl and K 2 HPO 4 . For glucose, no statistical significance was found between nitrogen and phosphorus sources in terms of surface tension. As a source of N, NH 4 Cl has a higher level of interaction with yeast extract and K 2 HPO 4 Yeast extract–NH 4 Cl–K 2 HPO 4 had the greatest e ff ect on the study of surface tension To prove the presence of biosurfactants in the previous systems, an extractant solution was used to measure the changes in the surface tension of the extract in the soils. The initial tension of the systems was considered to be 71.79 mN · m − 1 , while the tension of the culture medium was considered to be 71.93 mN · m − 1 at a temperature of 25 ◦ C, in accordance with [ 32 ]. Figure 2 shows the surface tensions for the di ff erent treatments. The treatments that presented lower surface tension were Y 4, Y 5, and Y 3 (23.16, 23.30, and 29.51 mN · m − 1 , respectively). These results are greater than those reported in another study conducted with P. fluorescens , which presented a surface tension of 30 to 35 mN · m − 1 using olive oil as a carbon source and NaNO 3 as a nitrogen source [ 39 ]. The results obtained in this research are less than those reported by Patowary et al. [ 53 ], who obtained a surface tension of 25.6 mN · m − 1 with P. aeruginosa SR 17 culture, using glucose as a carbon source with an incubation period of 5 days. As can be seen in Figure 2 , treatments Y 4 and Y 5 present a greater level of biosurfactant production as they present values less than the reference values for the electrolyte solution. For this reason, the lower the surface tension, the higher the level of biosurfactant production, with reference values for the electrolyte solution and the culture medium. These surface tension changes confirm the production of biosurfactants by P. fluorescens , principally lipoproteins and polymers [ 61 ]. The low surface tension has a direct relationship with the removal of TPH, given that previous studies, reporting low surface tension, have found both a higher level of biosurfactant related to increased pollutant removal [ 38 ]. Thus, the biosurfactants that may have been produced by P. fluorescens could facilitate the solubilization and uptake of insoluble hydrocarbons [ 62 ]. These results tend to be similar to those obtained with the response to TPH / TPH, from which it can be deduced that the production of surfactants decreases the surface tension of the pollutant and enables it to degrade at a higher level. Furthermore, the use of biosurfactants to improve the degradation of TPH has a more positive environmental e ff ect than synthetic surfactants [ 38 ]. Within the Pseudomonas species that degrade hydrocarbons, P. aeruginosa and P. putida are the best known for hydrocarbon utilization as sources of carbon and energy and for biosurfactant (glycolipid type) production [ 63 ]. Biosurfactants improve the desorption of hydrophobic compounds from polluted soil [ 64 ]. In the presence of the biosurfactants, there is an increase in the solubilization and desorption of the petroleum hydrophobic hydrocarbons from the soil particles into water [ 65 ], facilitating the solubilization and mobilization of the oil compounds [ 66 ].

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 9 of 14 Int. J. Environ. Res. Public Health 2020 , 17 , x 9 of 15 Figure 2. Surface tension values for the different treatments. Standard deviation of the means ( Ι ). “a,A”: The lower case and upper case letters in parentheses correspond to the differences between means of the LSD and Tukey tests, respectively. Electrolyte solution γ = 71.79 mN/m − 1 at 25 °C; culture medium γ = 71.93 mN·m − 1 at 25 °C. 3.4. Soil Respiration The microbial activity was measured using the production of CO 2 (Figure 3). CO 2 production in this response was measured over the course of the entire incubation period, with production as a variable across all treatments. The results showed an active microbial population [63]. The treatments presenting the highest levels of CO 2 production were Y 4 > Y 5 > Y 7 > Y 3. The CO 2 production at different treatments changed in time. The selected nutrients presented an increase in the growth of indigenous microorganisms and P. fluorescens . These results suggest that the increase in CO 2 may be related to the degradation efficiency of TPH. Figure 3. Mean CO 2 release over 80 days. 3.5. Population Growth of Bacteria and Fungi At the end of the experiment, the microorganisms were enumerated. It was observed that the cultivatable bacteria were reactivated across all the treatments (as occurred in other studies reported by [67]), in that microorganism growth was observed under culture conditions of 34–37 °C for 48 hrs in nutrient agar for bacteria and at 28–30 °C for 7 days in rose bengal agar for fungi. This indicates Figure 2. Surface tension values for the di ff erent treatments. Standard deviation of the means (I). “a,A”: The lower case and upper case letters in parentheses correspond to the di ff erences between means of the LSD and Tukey tests, respectively. Electrolyte solution γ = 71.79 mN / m − 1 at 25 ◦ C; culture medium γ = 71.93 mN · m − 1 at 25 ◦ C 3.4. Soil Respiration The microbial activity was measured using the production of CO 2 (Figure 3 ). CO 2 production in this response was measured over the course of the entire incubation period, with production as a variable across all treatments. The results showed an active microbial population [ 63 ]. The treatments presenting the highest levels of CO 2 production were Y 4 > Y 5 > Y 7 > Y 3. The CO 2 production at di ff erent treatments changed in time. The selected nutrients presented an increase in the growth of indigenous microorganisms and P. fluorescens . These results suggest that the increase in CO 2 may be related to the degradation e ffi ciency of TPH Int. J. Environ. Res. Public Health 2020 , 17 , x 9 of 15 Figure 2. Surface tension values for the different treatments. Standard deviation of the means ( Ι ). “a,A”: The lower case and upper case letters in parentheses correspond to the differences between means of the LSD and Tukey tests, respectively. Electrolyte solution γ = 71.79 mN/m − 1 at 25 °C; culture medium γ = 71.93 mN·m − 1 at 25 °C. 3.4. Soil Respiration The microbial activity was measured using the production of CO 2 (Figure 3). CO 2 production in this response was measured over the course of the entire incubation period, with production as a variable across all treatments. The results showed an active microbial population [63]. The treatments presenting the highest levels of CO 2 production were Y 4 > Y 5 > Y 7 > Y 3. The CO 2 production at different treatments changed in time. The selected nutrients presented an increase in the growth of indigenous microorganisms and P. fluorescens . These results suggest that the increase in CO 2 may be related to the degradation efficiency of TPH. Figure 3. Mean CO 2 release over 80 days. 3.5. Population Growth of Bacteria and Fungi At the end of the experiment, the microorganisms were enumerated. It was observed that the cultivatable bacteria were reactivated across all the treatments (as occurred in other studies reported by [67]), in that microorganism growth was observed under culture conditions of 34–37 °C for 48 hrs in nutrient agar for bacteria and at 28–30 °C for 7 days in rose bengal agar for fungi. This indicates Figure 3. Mean CO 2 release over 80 days 3.5. Population Growth of Bacteria and Fungi At the end of the experiment, the microorganisms were enumerated. It was observed that the cultivatable bacteria were reactivated across all the treatments (as occurred in other studies reported by [ 67 ]), in that microorganism growth was observed under culture conditions of 34–37 ◦ C for 48 hrs in nutrient agar for bacteria and at 28–30 ◦ C for 7 days in rose bengal agar for fungi. This indicates that similar behavior is observed for microbiological latency. The presence of P. fluorescens was prominent in

Int. J. Environ. Res. Public Health 2020 , 17 , 6959 10 of 14 comparison to the other colonies by its morphological characteristics (small cocci colonies with a creamy texture and a yellow-beige color). The growth of round transparent bacteria was also observed Figure 4 shows that the bacteria population is larger for treatments Y 4 > Y 5 > Y 3, which could be due to the addition of P. fluorescens as a bioaugmentation system. Various studies have shown that the significant increase in viable bacterial counts is due to the enrichment of the soil because of the nutrient biostimulation [ 68 – 72 ]. Int. J. Environ. Res. Public Health 2020 , 17 , x 10 of 15 that similar behavior is observed for microbiological latency. The presence of P. fluorescens was prominent in comparison to the other colonies by its morphological characteristics (small cocci colonies with a creamy texture and a yellow-beige color). The growth of round transparent bacteria was also observed. Figure 4 shows that the bacteria population is larger for treatments Y 4 > Y 5 > Y 3, which could be due to the addition of P. fluorescens as a bioaugmentation system. Various studies have shown that the significant increase in viable bacterial counts is due to the enrichment of the soil because of the nutrient biostimulation [68–72]. Figure 4. Colony-forming units ( CFUs) of fungi and bacteria in the biostimulation system. “ Ι ” indicates the standard deviation of the means. However, the fungi population was larger for treatments Y 5 > Y 4 > Y 3, possibly due to the addition of nutrients via the biostimulation strategy, which not only benefitted the bacteria populations but also caused the fungi populations to present higher growth levels. Above all, a synergy effect was observed among the bacterial populations, fungi, and P. fluorescens. With no statistical significance found for microbial growth between C 1 (natural attenuation) and C 2 (addition of P. fluorescens ), the results of this study reveal that biostimulation is the most effective strategy for the remediation of the soil. From this, it can be concluded that the concentration of nutrients in the attenuated soil is not sufficient to successfully carry out the remediation. 4. Conclusions In this study, we assessed the bioremediation potential of soil contaminated with oil, following different approaches such as natural attenuation, biostimulation, and bioaugmentation. The soil contained high concentrations of hydrocarbons, and natural attenuation was not appreciable; however, soil treatment with CNP nutrients at the recommended levels for biostimulation resulted in a significant increase in natural attenuation, which was even greater for bioaugmentation with P. fluorescens . The introduction of a hydrocarbon-degrading bacterial strain, P. fluorescens , into hydrocarbon-contaminated soil samples resulted in pronounced bioaugmentation. A combination of both bioaugmentation and biostimulation significantly improved the bioremediation of weathered TPH. Author Contributions: Conceptualization, E.J.G. and D.T.; methodology, D.T. and M.d.R.A.; software, E.D.; validation, D.T., M.d.R.A., and G.V.; formal análisis, E.J.G. and D.T.; investigation, E.J.G.; resources, D.T. and J.C.B.; data curation, J.C.B. and E.D.; writing—original draft preparatión, E.J.G.; writing—review and editing, D.T. and G.V.; funding acquisition, D.T. All authors have read and agreed to the published version of the manuscript Funding: This research was funded by Programa para el Desarrollo Profesional Docente (PRODEP), grant number UGTO-PTC-670, and the APC was funded by Secretaría de Educación Pública (SEP). Figure 4. Colony-forming units (CFUs) of fungi and bacteria in the biostimulation system. “I” indicates the standard deviation of the means However, the fungi population was larger for treatments Y 5 > Y 4 > Y 3, possibly due to the addition of nutrients via the biostimulation strategy, which not only benefitted the bacteria populations but also caused the fungi populations to present higher growth levels. Above all, a synergy e ff ect was observed among the bacterial populations, fungi, and P. fluorescens With no statistical significance found for microbial growth between C 1 (natural attenuation) and C 2 (addition of P. fluorescens ), the results of this study reveal that biostimulation is the most e ff ective strategy for the remediation of the soil From this, it can be concluded that the concentration of nutrients in the attenuated soil is not su ffi cient to successfully carry out the remediation 4. Conclusions In this study, we assessed the bioremediation potential of soil contaminated with oil, following di ff erent approaches such as natural attenuation, biostimulation, and bioaugmentation. The soil contained high concentrations of hydrocarbons, and natural attenuation was not appreciable; however, soil treatment with CNP nutrients at the recommended levels for biostimulation resulted in a significant increase in natural attenuation, which was even greater for bioaugmentation with P. fluorescens . The introduction of a hydrocarbon-degrading bacterial strain, P. fluorescens , into hydrocarbon-contaminated soil samples resulted in pronounced bioaugmentation. A combination of both bioaugmentation and biostimulation significantly improved the bioremediation of weathered TPH Author Contributions: Conceptualization, E.J.G. and D.T.; methodology, D.T. and M.d.R.A.; software, E.D.; validation, D.T., M.d.R.A., and G.V.; formal an á lisis, E.J.G. and D.T.; investigation, E.J.G.; resources, D.T. and J.C.B.; data curation, J.C.B. and E.D.; writing—original draft preparati ó n, E.J.G.; writing—review and editing, D.T. and G.V.; funding acquisition, D.T. All authors have read and agreed to the published version of the manuscript Funding: This research was funded by Programa para el Desarrollo Profesional Docente (PRODEP), grant number UGTO-PTC-670, and the APC was funded by Secretar í a de Educaci ó n P ú blica (SEP).

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