Immobilization of P. stutzeri on Activated Carbons for Degradation of Hydrocarbons from Oil-in-Saltwater Emulsions

nanomaterials Article Immobilization of P. stutzeri on Activated Carbons for Degradation of Hydrocarbons from Oil-in-Saltwater Emulsions Karol Zapata Acosta 1,2,*, Francisco Carrasco-Marin 3, Farid B. Cortés1, Camilo A. Franco 1, Sergio H. Lopera 2and Benjamín A. Rojano 4 1Grupo de Investigación en Fenómenos de Superficie—Michael Polanyi, Departamento de Procesos y Energía, Facultad de Minas, Universidad Nacional de Colombia, Sede Medellín, Medellín 050034, Colombia; [email protected] (F.B.C.); [email protected] (C.A.F.) 2Grupo de Investigación Yacimientos de Hidrocarburos, Departamento de Procesos y Energía, Facultad de Minas, Universidad Nacional de Colombia, Sede Medellín, Medellín 050034, Colombia; [email protected] 3 Grupo de Investigación Materiales de Carbón, Departamento de Química Inorgánica, Facultad de Ciencias, Universidad de Granada, 18071 Granada, Spain; [email protected] 4Grupo de Investigación Química de los Productos Naturales y los Alimentos, Facultad de Ciencias, Universidad Nacional de Colombia Sede Medellín, Medellín 050034, Colombia; [email protected] *Correspondence: [email protected]; Tel.: +57-3104960256 Received: 21 February 2019; Accepted: 23 March 2019; Published: 1 April 2019   Abstract: Production water is the largest byproduct of the oil industry and must be treated before disposal, either by reinjection or shedding processes, with the purpose of eliminating emulsified crude oil and avoiding the operational and toxic problems associated with it. The objective of this work was to immobilize a hydrocarbon-degrading strain on activated carbons, to evaluate the biocomplex’s capacity for catalyzing hydrocarbons from Oil in Brine emulsions (O/W) simulating produced waters. Activated carbons were prepared and their chemical and porous properties were estimated by XPS, pH PZC and SEM, N 2 adsorption, and mercury porosimetry. Biomaterials were synthesized and hydrocarbon removal tests were performed. The basic and neutral carbons immobilized Pseudomonas stutzeri by physisorption in the macroporous space and electrostatic interactions (10 8 –10 9 UFC · g −1 ), while acid materials inhibited bacterial growth. Removal of aromatic hydrocarbons was more efficient using materials (60%–93%) and biomaterials (16%–84%) than using free P. stutzeri (1%–47%), and the removal efficiencies of crude oil were 22%, 48% and 37% for P. stutzeri and two biomaterials, respectively. The presence of minor hydrocarbons only when P. stutzeri was present confirmed the biotransformation process. Keywords: biomaterials; catalysis; crude oil; immobilization; hydrocarbons; produced water 1. Introduction One of the main products of the oil industry is production water, i.e., water that is produced along with the oil, and water that is injected during recovery processes. Production water is usually contaminated with hydrocarbons, which must be removed before either reinjection or disposal [ 1 ]. For each barrel of oil, approximately five barrels of water are produced, and, in mature fields, this ratio could reach 1:15 [2]. This situation has required the development of technologies to ensure control of the production water in accordance with industry standards for its disposal or reinjection (National Association of Corrosion Engineers NACE). Currently, there are different mechanisms for the removal of oil from the production water including adsorption [ 3 ], filtration [ 4 ], reverse osmosis [ 5 ], flotation [ 6 ] Nanomaterials 2019,9, 500; doi:10.3390/nano9040500 www.mdpi.com/journal/nanomaterials Nanomaterials 2019,9, 500 2 of 15 and coagulation [ 7 ]. Adsorption is the most commonly employed technique in the tertiary stage of oil removal (“dissolved” oil) due to its simplicity and effectiveness. Typical adsorbents used in the treatment of produced water are activated carbon, organoclay, copolymers, aerogels, zeolites, resins and, lately, nanomaterials. The combination of absorbents as carbons and organoclays is efficient for total oil removal (TPH) up to 85% [ 8 ], and the use of nanomaterials in hydrocarbon adsorption processes in the aqueous phase has been 100% efficient [ 9 ]. Regardless of the positive results, the adsorptive approaches have limitations for the desorption and final elimination of the contaminant. Biological processes are an emerging technology capable of solving these issues due to the capacity of specific microorganisms to catalyze “miscible” hydrocarbons in water [ 10 ]. While the physical treatments and many of the chemicals are based on transferring the contamination between gaseous, liquid and solid media, in the biological processes the contaminating compound is transformed into another more oxidized and less toxic state. Likewise, biological developments are less invasive methods and generally do not require the structural or mechanical components that pose a threat to the environment. Biological processes are also cost-effective and, because they are natural, are usually accepted by the public [ 10 – 12 ]. Some researchers have demonstrated the early disappearance of microbial consortium introduced in an environment contaminated with hydrocarbons [13,14]. The reason is that there are stress factors, high temperatures, water activity, pH, dissolved oxygen content, nutritional limitations, and toxicity of contaminants, which limit cell viability. Consequently, the development of efficient biological technologies for the removal of hydrocarbons is of great ecological and economic value. Strategies such as the immobilization of microbial cells in materials are being used [15]. The support for microbial immobilization provides a niche in microbial inoculants through the provision of a protective surface or porous space. The contaminants are first absorbed on the surface of the material in which the microorganism is immobilized and then gradually pass through the pores; the participation of these three types of adsorbates generate a biofilm that favors the transformation of the contaminant [16]. Therefore, organisms have sufficient contact time for their catalytic action. Several synthetic polymers, polyacrylamide, polyethylene glycol and polyurethane [ 17 , 18 ], carrageenan, agar, collagen, chitin and chitosan [ 19 ], have been used as matrices for the immobilization of microorganisms. They have also been used as a wide range of carriers prepared from natural materials, clays [ 16 ], activated carbons [20], and other materials [11,21,22]. However, to the best of our knowledge, no research has yet been conducted on the immobilization of microorganisms on activated carbon for the removal of hydrocarbons in O/W emulsified systems. Activated carbons are structured materials with high porosity and large surface chemistry, controllably obtained from the pyrolysis of organic matter, which can be used as important adsorbent materials of bacteria and hydrocarbons [23–25]. This research aims to understand the effects of immobilizing hydrocarbon-degrading bacteria on activated carbon and evaluating the biomaterial capacity to remove aromatic hydrocarbons and crude oil from simulated produced water. 2. Materials and Methods 2.1. Materials Olive stones were harvested in Granada, Spain, and were used as the raw material for the activated carbon preparation. The olive stones were dried at 80 ◦ C until constant weight and sieved to 1.6–2.4 mm size. Potassium hydroxide (reagent grade ≥ 90%, Sigma-Aldrich, St. Louis, MO, USA) and phosphoric acid (mass fraction of 85% in H 2 O, Sigma-Aldrich, St. Louis, MO, USA) were used for the chemical activation of the olive stones before carbonization, while melamine (99%, Sigma Aldrich, Nanomaterials 2019,9, 500 3 of 15 St. Louis, MO, USA) and phosphoric acid (mass fraction ≥ 85% in H 2 O, Sigma-Aldrich, St. Louis, MO, USA) were used for the modification of surface chemistry. Chlorihydric acid was used for the washing protocol (mass fraction of 37% in H 2 O, Sigma-Aldrich, St. Louis, MO, USA). Acetonitrile (mass fraction of 99.8% in water, Sigma-Aldrich, St. Louis, MO, USA) was employed for rhamnolipid quantification. Tryptone glucose extract agar (TGEA) and bushnell haas broth (BH, Sigma Aldrich, St. Louis, MO, USA) were used for the bacterial culture. A Colombian light crude oil (33 ◦ API), deionized water and NaCl (99%, Merck KGaA, Darmstadt, DE, Germany) were used for preparing oil in brine emulsions. Benzene, toluene, and phenol ( ≥ 98%, CARLO ERBA Reactive-SDS, Barcelona, ES, Spain) were used for the assays of the removal of aromatics hydrocarbons. 2.2. Methods 2.2.1. Preparation of Activated Carbons Activated carbons were prepared by chemical activation of olive stones with phosphoric acid (CP) and potassium hydroxide (CK) following the procedure previously published by Bautista et al. [ 26 ]. A portion of olive stones was combined separately with potassium hydroxide and phosphoric acid in a mass ratio of 1:2. The blend was macerated and dried under IR lamps. It was then carbonized by heating at 10 ◦ C · min −1 to 840 ◦ C with a soak time of 2 h under a nitrogen flow of 300 cm 3· min −1 using a tubular furnace (Heraeus, Hanau, Germany). The CK was washed with HCl 0.1 N and ultrapure water until no chloride ions were present in the washing water. The CP was cleaned with distilled water until pH = 7. 2.2.2. Chemical Modification of Activated Carbons An incipient impregnation method was used for surface modifications [ 23 ]. The CK and CP materials were dried at 110 ◦ C for 24 h. An appropriate amount of modifying agents, phosphoric acid and melamine were prepared with a minimum amount of water or ethanol, according to their solubility for including heteroatoms of P or N, respectively. The solutions were then slowly dripped on each material. Impregnated materials were dried under IR lamp for 24 h and were later heated at 700 ◦ C for 1 h under a nitrogen flow of 300 cm 3· min −1 using a tubular furnace (Heraeus). Finally, four materials were synthesized, CK and CP with melamine, labeled as CKN and CPN, respectively, and CK and CP with phosphoric acid, to obtain CKP and CPP, respectively. 2.2.3. Characterization of Activated Carbons The material morphology was studied by scanning electronic microscopy (SEM) using a LEO (Carl Zeiss, Oberkochen, Germany) GEMINI-1530 microscope. Textural characterization was carried out by N 2 adsorption at − 196 ◦ C using Quadrasorb SI equipment (Quantachrome instruments, Boynton beach, FL, USA). Prior, the carbon samples were degassed overnight at 100 ◦ C. The Brunauer–Emmett–Teller (BET) equation was applied to the N 2 -adsorption isotherms to determine the surface area (S BET ). The Dubinin–Radushkevich equation was used to determine the micropore volume (V mic ) and the mean micropore width (L mic ). Furthermore, the Barrette-Joynere-Halenda (BJH) method was used to calculate the mesopore volume (V meso ) and the mean mesopore width (L meso ). The total pore volume (V 0.95 ) was considered to be the volume of N 2 adsorbed at P/P 0 = 0.95 [ 23 ]. Mercury porosimetry was carried out using Micromeritics (Norcross, GA, USA) AutoPore IV 9510 equipment up to a pressure of 60,000 psi to obtain the mesopore volume V 2 (dpore 6 to 50 nm), macropore volume V 3 (dpore 50 to 10,000 nm) and mean macropore width (L macro ). Pore size distributions (PSD) were obtained by coupling the BJH and mercury porosimetry results. The material chemical characterization was analyzed by X-ray photoelectron spectroscopy (XPS), and information about the material acidity was obtained by measuring the pHPZC, according to the methodology described by Perez et al. [26]. Nanomaterials 2019,9, 500 4 of 15 2.2.4. Immobilization Kinetics The method proposed by Sekaran et al. [ 20 ], with some modifications, was employed. For this, 0.01 g of sterile materials were mixed with 5 mL of P. stutzeri (previously isolated and identified) and suspended (10 10 u.f.c) in NaCl 0.9% and a mass fraction of 0.33% of BH ® . The mixture was shaken at 100 rpm and 35 ◦ C. Every three hours, the material was retired and washed with K 2 HPO 4 0.15 M to eliminate weakly adhered cells. Lastly, the number of cells on the material was analyzed by plate count in Tryptone Glucose Extract Agar (48 h/35 ◦ C). The results were expressed as the number of cells immobilized per gram of material. Carbon sterilization was carried out following the method described by Sekaran et al. [ 20 ]. For this, approximately 1 g of carbon was placed in an Erlenmeyer flask of 200 mL, slightly closed, and autoclaved (WestLab, Surrey, UK) at 121 ◦ C and 15 psi for 15 min. The manipulation of the materials was always carried out in a laminar flow chamber (DSS, New Delhi) to guarantee asepsis. Sterilization was based on the denaturation of the proteins of the microorganisms due to the moist heat supplied by the autoclave. 2.2.5. Preparation of Oil-Brine Emulsions (O/W) Saltwater (brine) was prepared by mixing NaCl and deionized water to obtain a mass fraction of 2% in H 2 O, based on a procedure previously reported by our group [ 27 ]. Oil in brine (O/W) emulsions were prepared by mixing 8000 mg · L −1 of crude oil and brine with a mass fraction of 0.33% Bushnell Haas Broth BH ® (nitrogen, phosphates and minerals source) at 10,000 rpm for 20 min. The stability of the emulsions was monitored for 120 h by observing the size of the oil drops, using an optical microscope, and the changes in absorbance, using UV-VIS spectrophotometer Genesys 10S (Thermo Scientific, Massachusetts, US) [ 28 ]. In parallel, brines with specific hydrocarbons were prepared by mixing benzene, toluene and phenol 600 mg · L −1 at 600 rpm for 10 min in deionized water with a mass fraction of 0.33% of Bushnell Haas Broth BH®. 2.2.6. Hydrocarbon Degradability Test Crude oil and aromatic hydrocarbon degradability tests were performed following the protocol proposed by Juang and Tsai [ 29 ], with some modifications. The biomaterials were inoculated into 15 mL of W/O emulsions. After, the flasks were cultured at 150 rpm and 35 ◦ C for 24 h. Free cells (10 6 u.f.c) were used as a control. Crude oil and aromatic hydrocarbon content was analyzed by Gas chromatography–mass spectrometry (GC-MS) and High performance liquid chromatography (HPLC), respectively, according to the method reported by Galazka [30]. After the isolation and identification of P. stutzeri, the biomass was obtained according to the method described by Juan and Tsai [ 29 ]. For this, the pure culture of P. stutzeri was cultured in Nutrient Broth (Sigma-Aldrich, St. Louis, MO, USA) for 60 h at 150 rpm and 35 ◦ C, until it reached the stationary state. Then, the biomass was filtered and stored under asepsis. 2.2.7. Rhamnolipid Quantification For the rhamnolipid quantification, standard solutions were prepared (CAS Standard: 0869062420 Sigma-Aldrich, St. Louis, MO, USA) and the calibration curve was elaborated using HPLC (Prominence, Shimadzu, Kioto, Japan). A LiChrospher ® 100 RP-18 column (5 µ m; 250 × 4 mm) was used as the stationary phase and water/acetonitrile 1:1 as the mobile phase. The experimental conditions were an absorption wavelength of 254 nm, an injection volume of 70 µ L, 27 ◦ C of temperature, a flow rate of 0.8 mL·min−1and a retention time of 2.5 min. 2.2.8. Statistical Analysis Immobilization kinetics and bio-removal experiments were made in triplicate. The reported data correspond to the arithmetic mean ± standard deviation. In all cases, deviations ( ± SD) were Nanomaterials 2019,9, 500 5 of 15 lower than 10% of the mean (coefficient of variation). To determine statistical differences between measurements, a unidirectional analysis of variance was performed in the 95% confidence interval using Statgraphics Centurion V (Statgraphics, Seattle, WA, USA). Comparisons of the means were made with a Duncan’s multiple range test (p< 0.05). 3. Results and Discussion 3.1. Characteristics of Activated Carbons The morphologies of (a) CK and (b) CP samples are shown in Figure 1. CK and CP are a three-dimensional network of amorphous particles. CP, in particular, presents bigger and wider units. Visually, the CK pores are smaller than CP, which is in agreement with the results reported by Moreno et al. [ 31 ]. Table 1summarizes the chemical composition of CK and CP samples. Overall, the surfaces of the materials are constituted mainly of C (>85%) and O (7.7%) atoms. The CP material showed residual phosphorus due to the H3PO4employed for activation (mass fraction of 6.7%). Nanomaterials 2019, 9, x FOR PEER REVIEW 5 of 16 3. Results and Discussion 3.1. Characteristics of Activated Carbons The morphologies of (a) CK and (b) CP samples are shown in Figure 1. CK and CP are a threedimensional network of amorphous particles. CP, in particular, presents bigger and wider units. Visually, the CK pores are smaller than CP, which is in agreement with the results reported by Moreno et al. [31]. Table 1 summarizes the chemical composition of CK and CP samples. Overall, the surfaces of the materials are constituted mainly of C (>85%) and O (7.7%) atoms. The CP material showed residual phosphorus due to the H3PO4 employed for activation (mass fraction of 6.7%). (a) (b) Figure 1. The microstructure of activated carbons prepared by chemical activation of olive stones with (a) potassium hydroxide (CK) and (b) phosphoric acid (CP); estimated by SEM analysis. Table 1. Chemical composition of activated carbons prepared by chemical activation of olive stones with potassium hydroxide (CK) and phosphoric acid (CP); estimated by XPS analysis. Sample C O P (mass fraction %) (mass fraction %) (mass fraction %) CK 92.3 7.7 - CP 85.5 7.7 6.7 The specific chemical bonds in the surfaces of CK and CP were assessed by deconvolution of XPS signals, as shown in Table 2. The carbon spectra showed six peaks for CK and CP related to carbon atoms bonded to carbon and oxygen. The C=C bound the most frequently (≥70% of carbon bonds) for both samples were C=C, C–O, C=O, O=C–OR, 𝐶𝑂  and 𝐶𝑂. Studying the Oxygen spectrum, CK and CP presented two peaks, which corresponded to the presence of i) double bonded oxygen, O=C and/or O=P (50% oxygen bonds), and ii) single bonded oxygen, O–C and/or P–O–C [32– 34]. Regarding the P2p region of the CP sample, three different peaks were found: reduced phosphorus (C–P; 40% phosphorous bonds), Pyrophosphate (C–PO3; 40% phosphorous bonds), and polyphosphates and/or phosphates (C–O–PO3; 20% phosphorous bonds). Hasegawa et al. [35] observed that P atoms are introduced into carbon principally in reduced states and these unstable groups are gradually oxidized by oxygen, leading to oxidized P-containing functional groups. In the CP sample, the 60% phosphorus group was oxidized. Figure 1. The microstructure of activated carbons prepared by chemical activation of olive stones with (a) potassium hydroxide (CK) and (b) phosphoric acid (CP); estimated by SEM analysis. Table 1. Chemical composition of activated carbons prepared by chemical activation of olive stones with potassium hydroxide (CK) and phosphoric acid (CP); estimated by XPS analysis. Sample C O P (Mass Fraction %) (Mass Fraction %) (Mass Fraction %) CK 92.3 7.7 - CP 85.5 7.7 6.7 The specific chemical bonds in the surfaces of CK and CP were assessed by deconvolution of XPS signals, as shown in Table 2. The carbon spectra showed six peaks for CK and CP related to carbon atoms bonded to carbon and oxygen. The C=C bound the most frequently ( ≥ 70% of carbon bonds) for both samples were C=C, C–O, C=O, O=C–OR, CO−2 3 and CO2 . Studying the Oxygen spectrum, CK and CP presented two peaks, which corresponded to the presence of i) double bonded oxygen, O=C and/or O=P (50% oxygen bonds), and ii) single bonded oxygen, O–C and/or P–O–C [ 32 – 34 ]. Regarding the P 2 p region of the CP sample, three different peaks were found: reduced phosphorus (C–P; 40% phosphorous bonds), Pyrophosphate (C–PO 3 ; 40% phosphorous bonds), and polyphosphates and/or phosphates (C–O–PO 3 ; 20% phosphorous bonds). Hasegawa et al. [ 35 ] observed that P atoms are introduced into carbon principally in reduced states and these unstable groups are gradually oxidized by oxygen, leading to oxidized P-containing functional groups. In the CP sample, the 60% phosphorus group was oxidized. Nanomaterials 2019,9, 500 6 of 15 Table 2. Chemical bonds of activated carbons prepared by chemical activation of olive stones with potassium hydroxide (CK) and phosphoric acid (CP); estimated by deconvolution of XPS signals. Peak Binding Energy Assignment % Peak CK CP O1s 531.3 C=O/P=O* 48 40 533.0 C-O/C-O-P-O/C-PO* 52 60 C1s 284.5–284.6 C=C/C-O-P* 73 72 285.5–286.4 C-O 17 17 287.4–287.6 C=O 4 5 288.4–288.48 O=C-OR 3 4 290.4–290.8 CO− 3CO242 P2p 132.3 Reduced phosphorus compound C-P (P-1) - 39 133.1 Pyrophosphate, C-PO3 (P-2) - 39 134.1 Polyphosphates and/or phosphates, C–O–PO3 (P-3) - 22 All isotherms are represented in Figure 2. The results show that the CK isotherm was type I, while the other materials exhibited type II isotherms. The CK isotherm was concave in terms of the relative pressure axis (P/P 0 ), increased rapidly at low pressures (P/P 0 < 1 × 10 −3 ) and later reached horizontal saturation. This class of isotherm is characteristic of highly microporous materials. The high adsorption energy of the micropores allows the gas to adsorb at low pressures. Once the entire volume of the micropores has been completed, the curve remains almost constant over a wide range of pressures, producing the so-called plateau. CKN, CKP, CP, CPN and CPP isotherms were also concave at low pressures (P/P 0 < 1 × 10 −3 ) with respect to the relative pressure axis (P/P 0 ) but later increased linearly and finally became convex. This phenomenon assumes monolayer adsorption at low pressures (micropores), as well the formation of multimolecular layers at medium and high ranges of relative pressures (mesopores) [36] The material’s textural properties are summarized in Table 3. CK materials were exclusively microporous, their micropore volume (V micro ) represented approximately 80% of V 0.95 , while CP materials were micro and mesoporous; V micro and V meso each represented 50% of V 0.95 . The CP samples exhibited a significantly higher V 0.95 than the CK samples. The difference between mesoporous volume by BHJ method (V meso ) and mesoporous volume by porosimetry method (V 2 ) indicated that, for all materials, the mesopores were between 2 and 6.5 nm. Nanomaterials 2019, 9, x FOR PEER REVIEW 7 of 16 Figure 2. Nitrogen isotherms of activated carbons prepared by the chemical activation of olive stones with (a) potassium hydroxide (CK) and (b) phosphoric acid (CP). Adsorption curve—filled symbols; desorption curve—unfilled symbols. Table 3. Textural proprieties and point of zero charge (pHPZC) of activated carbons prepared by chemical activation of olive stones with potassium hydroxide (CK) and phosphoric acid (CP). Sampl e pHPZ C Bulk Densit y SBET N2-ADSORPTION MERCURY POROSIMETRY Vmicro (0.5–2 nm) Lmicr o Vmeso (2–30 nm) Lmes o V0.95 V2 (6.5–50 nm) V3 (50–10,000 nm) Lmacr o g·cm−3 m2·g −1 cm3·g−1 nm cm3·g−1 nm cm3·g −1 cm3·g−1 cm3·g−1 nm CK 6.69 0.113 1395 0.496 1.48 0.119 2.20 0.638 0.054 4.906 4456 CKN 8.34 0.114 879 0.312 0.95 0.286 2.17 0.600 0.114 4.420 3992 CKP 4.62 0.206 1176 0.418 0.68 0.193 2.19 0.614 0.017 0.629 4533 CP 2.44 0.584 1202 0.430 1,33 0.482 2.46 0.839 0.206 0.138 4251 CPN 2.96 0.546 1062 0.377 1,36 0.512 2.44 0.864 0.182 0.125 4433 CPP 2.03 0.614 1229 0.437 1,40 0.609 2.44 0.971 0.199 0.123 4341 Vmicro: microporous volume; Lmicro: average micropore size; Vmeso: mesoporous volume by BJH method; Lmeso: average mesopore size; V0.95: volume until P/Po equal a 0.95; V2: mesoporous volume by mercury porosimetry method; V3: macroporous volume by mercury porosimetry method; Lmacro: average macropore size. V3 obtained by mercury porosimetry showed that CK samples, besides being micro, were prominently macroporous materials, especially CK and CKN, and confirmed that CP samples were exclusively micro and mesoporous materials. Pore size distributions (PSD) are plotted in Figure 3. Both distributions showed multimodal behaviors; the mean pore width in the CK samples was centered on d, 1.03 ± 0.41 nm and 4533 ± 293 nm, with final percentages of micro and macroporosity of 18% and 76%, respectively. CP samples exhibited a mean pore width centered on d, 2.25 ± 0.11 nm and 4342 ± 91 nm, with final percentages of meso and macroporosity 50% and 12%, respectively. CK samples exhibited significant macropore volume because of the large space located between the fine primary particles (low bulk density), unlike the CP samples that had less interparticle space (high bulk density); similar results were previously reported for microporous carbons [20,37]. The pHPZC values are also included in Table 3. All CP materials were strong acids due to the use of acid activation that promoted cleavage reactions and the formation of crosslinks with the phosphorous atom (Lewis acids), as verified by XPS. CK and CKP materials were slightly acidic or Figure 2. Nitrogen isotherms of activated carbons prepared by the chemical activation of olive stones with ( a ) potassium hydroxide (CK) and ( b ) phosphoric acid (CP). Adsorption curve—filled symbols; desorption curve—unfilled symbols. Nanomaterials 2019,9, 500 7 of 15 The surface area (S BET ) for CK and CP decreased with the surface modifications, which was to be expected considering that the heteroatoms block the pore by deposition in the microporous space. However, the modification with phosphoric acid (P) of CP had a double activating effect, increasing Vmicro and SBET. Table 3. Textural proprieties and point of zero charge (pH PZC ) of activated carbons prepared by chemical activation of olive stones with potassium hydroxide (CK) and phosphoric acid (CP). Sample pHPZC Bulk Density SBET N2-ADSORPTION MERCURY POROSIMETRY Vmicro (0.5–2 nm) Lmicro Vmeso (2–30 nm) Lmeso V0.95 V2 (6.5–50 nm) V3 (50–10,000 nm) Lmacro g·cm−3m2·g−1cm3·g−1nm cm3·g−1nm cm3·g−1cm3·g−1cm3·g−1nm CK 6.69 0.113 1395 0.496 1.48 0.119 2.20 0.638 0.054 4.906 4456 CKN 8.34 0.114 879 0.312 0.95 0.286 2.17 0.600 0.114 4.420 3992 CKP 4.62 0.206 1176 0.418 0.68 0.193 2.19 0.614 0.017 0.629 4533 CP 2.44 0.584 1202 0.430 1,33 0.482 2.46 0.839 0.206 0.138 4251 CPN 2.96 0.546 1062 0.377 1,36 0.512 2.44 0.864 0.182 0.125 4433 CPP 2.03 0.614 1229 0.437 1,40 0.609 2.44 0.971 0.199 0.123 4341 V micro : microporous volume; L micro : average micropore size; V meso : mesoporous volume by BJH method; L meso : average mesopore size; V 0.95 : volume until P/Po equal a 0.95; V 2 : mesoporous volume by mercury porosimetry method; V3: macroporous volume by mercury porosimetry method; Lmacro: average macropore size. V 3 obtained by mercury porosimetry showed that CK samples, besides being micro, were prominently macroporous materials, especially CK and CKN, and confirmed that CP samples were exclusively micro and mesoporous materials. Pore size distributions (PSD) are plotted in Figure 3. Both distributions showed multimodal behaviors; the mean pore width in the CK samples was centered on d, 1.03 ± 0.41 nm and 4533 ± 293 nm, with final percentages of micro and macroporosity of 18% and 76%, respectively. CP samples exhibited a mean pore width centered on d, 2.25 ± 0.11 nm and 4342 ±91 nm, with final percentages of meso and macroporosity 50% and 12%, respectively. CK samples exhibited significant macropore volume because of the large space located between the fine primary particles (low bulk density), unlike the CP samples that had less interparticle space (high bulk density); similar results were previously reported for microporous carbons [20,37]. The pH PZC values are also included in Table 3. All CP materials were strong acids due to the use of acid activation that promoted cleavage reactions and the formation of crosslinks with the phosphorous atom (Lewis acids), as verified by XPS. CK and CKP materials were slightly acidic or almost neutral, while CKN was a basic material for the presence of amino groups on the surface (Lewis basis); similar data were obtained by Moreno [38]. Nanomaterials 2019, 9, x FOR PEER REVIEW 8 of 16 almost neutral, while CKN was a basic material for the presence of amino groups on the surface (Lewis basis); similar data were obtained by Moreno [38]. Figure 3. Pore size distribution of activated carbons prepared by chemical activation of olive stones with (a) potassium hydroxide (CK) and (b) phosphoric acid (CP) functionalized with melamine (CKN and CPN), and phosphoric acid (CKP and CPP). 3.2. Immobilization Kinetics The immobilization kinetics of P. stutzeri on CK and CP materials are shown in Figure 4. The results indicate that for all CK materials the immobilization process advanced linearly with the interaction time until the third hour and equilibrium was reached after. The linear zone was attributed to the migration of the cells from the bulk cell suspension to the particle surfaces, while the plateau region corresponded to the diffusion of P. stutzeri from the surface to the interior of the pores [20]. Figure 4. Immobilization kinetics of P. stutzeri on activated carbons prepared by chemical activation of olive stones with (a) potassium hydroxide (CK) and (b) phosphoric acid (CP); functionalized with melamine (CKN and CPN) and phosphoric acid (CKP and CPP). The ability to immobilize cells is given in the following order: CK > CKN > CKP. The results were based in the bacterial–material interaction. The cationic and neutral surface of CKN and CK Figure 3. Pore size distribution of activated carbons prepared by chemical activation of olive stones with ( a ) potassium hydroxide (CK) and ( b ) phosphoric acid (CP) functionalized with melamine (CKN and CPN), and phosphoric acid (CKP and CPP). Nanomaterials 2019,9, 500 8 of 15 3.2. Immobilization Kinetics The immobilization kinetics of P. stutzeri on CK and CP materials are shown in Figure 4. The results indicate that for all CK materials the immobilization process advanced linearly with the interaction time until the third hour and equilibrium was reached after. The linear zone was attributed to the migration of the cells from the bulk cell suspension to the particle surfaces, while the plateau region corresponded to the diffusion of P. stutzeri from the surface to the interior of the pores [20]. Nanomaterials 2019, 9, x FOR PEER REVIEW 8 of 16 almost neutral, while CKN was a basic material for the presence of amino groups on the surface (Lewis basis); similar data were obtained by Moreno [38]. Figure 3. Pore size distribution of activated carbons prepared by chemical activation of olive stones with (a) potassium hydroxide (CK) and (b) phosphoric acid (CP) functionalized with melamine (CKN and CPN), and phosphoric acid (CKP and CPP). 3.2. Immobilization Kinetics The immobilization kinetics of P. stutzeri on CK and CP materials are shown in Figure 4. The results indicate that for all CK materials the immobilization process advanced linearly with the interaction time until the third hour and equilibrium was reached after. The linear zone was attributed to the migration of the cells from the bulk cell suspension to the particle surfaces, while the plateau region corresponded to the diffusion of P. stutzeri from the surface to the interior of the pores [20]. Figure 4. Immobilization kinetics of P. stutzeri on activated carbons prepared by chemical activation of olive stones with (a) potassium hydroxide (CK) and (b) phosphoric acid (CP); functionalized with melamine (CKN and CPN) and phosphoric acid (CKP and CPP). The ability to immobilize cells is given in the following order: CK > CKN > CKP. The results were based in the bacterial–material interaction. The cationic and neutral surface of CKN and CK Figure 4. Immobilization kinetics of P. stutzeri on activated carbons prepared by chemical activation of olive stones with ( a ) potassium hydroxide (CK) and ( b ) phosphoric acid (CP); functionalized with melamine (CKN and CPN) and phosphoric acid (CKP and CPP). The ability to immobilize cells is given in the following order: CK > CKN > CKP. The results were based in the bacterial–material interaction. The cationic and neutral surface of CKN and CK under experimental pH attracted the negative charge of the cell wall. Both Gram-negative and positive bacteria possessed a relatively negative charge on their surface due to the presence of active proton functional groups, such as hydroxyl, carboxyl, and phosphoryl deprotonated to pH = 7.0 [ 39 ]. CK and CKN materials exhibited remarkable macroporous volume where bacteria could be hosted. Similar results were previously reported [20]. In CP materials, after 6 h of contact, no bacterial cells remained attached to the materials. The results can be explained by considering that the CP materials exhibited low pH PZC and were very negative in neutral media, presenting strong repulsions with the bacterial exocellular membrane that is also negatively charged under the same conditions. In addition to the low adsorption of microorganisms, the absence of a bacterial count in the final suspension established the antimicrobial effect of CP materials. When acid materials were exposed to a pH of 7 they were negatively charged due to the hydrogen release that came from the dissociation of the surface chemical groups such as hydroxyl and carboxyl [ 39 ]. Subsequently, the proton concentration in the medium increased and the pH was reduced, leading to the antimicrobial effect. The acid media affected the bacterial membrane H+-ATPase and denaturalized the bacterial enzymes, causing cell death. CK and CKN were chosen as suitable materials for the immobilization of P. stutzeri. The cells adsorbed on CK and CKN were 1.0 × 10 9 and 6.6 × 10 8 UFC · g −1 , respectively (biomaterials). The results of this investigation are in accordance with other reports [ 40 ]. The SEM micrographs confirmed biomaterial formation, as shown in Figure 5. The presence of P. stutzeri on the materials was evidenced by the formation of a continuous layer (biofilm) that was absent in CK materials. Nanomaterials 2019,9, 500 9 of 15 Nanomaterials 2019, 9, x FOR PEER REVIEW 9 of 16 under experimental pH attracted the negative charge of the cell wall. Both Gram-negative and positive bacteria possessed a relatively negative charge on their surface due to the presence of active proton functional groups, such as hydroxyl, carboxyl, and phosphoryl deprotonated to pH = 7.0 [39]. CK and CKN materials exhibited remarkable macroporous volume where bacteria could be hosted. Similar results were previously reported [20]. In CP materials, after 6 h of contact, no bacterial cells remained attached to the materials. The results can be explained by considering that the CP materials exhibited low pHPZC and were very negative in neutral media, presenting strong repulsions with the bacterial exocellular membrane that is also negatively charged under the same conditions. In addition to the low adsorption of microorganisms, the absence of a bacterial count in the final suspension established the antimicrobial effect of CP materials. When acid materials were exposed to a pH of 7 they were negatively charged due to the hydrogen release that came from the dissociation of the surface chemical groups such as hydroxyl and carboxyl [39]. Subsequently, the proton concentration in the medium increased and the pH was reduced, leading to the antimicrobial effect. The acid media affected the bacterial membrane H+-ATPase and denaturalized the bacterial enzymes, causing cell death. CK and CKN were chosen as suitable materials for the immobilization of P. stutzeri. The cells adsorbed on CK and CKN were 1.0 × 109 and 6.6 × 108 UFC·g−1, respectively (biomaterials). The results of this investigation are in accordance with other reports [40]. The SEM micrographs confirmed biomaterial formation, as shown in Figure 5. The presence of P. stutzeri on the materials was evidenced by the formation of a continuous layer (biofilm) that was absent in CK materials. (a) (b) (c) Figure 5. The microstructure of activated carbons prepared by chemical activation of olive stones with potassium hydroxide (CK), (a) without P. stutzeri, (b) P. stutzeri on CK surface, and (c) P. stutzeri on melamine-functionalized CK (CKN); surface estimated by SEM analysis. Figure 5. The microstructure of activated carbons prepared by chemical activation of olive stones with potassium hydroxide (CK), ( a ) without P. stutzeri, ( b )P. stutzeri on CK surface, and ( c )P. stutzeri on melamine-functionalized CK (CKN); surface estimated by SEM analysis. 3.3. Hydrocarbon Degradability Test The biomaterial’s capacity to remove aromatic hydrocarbons from oil-brine emulsions (O/W) is displayed in the Figure 6.P. stutzeri, CK and CKN materials were used as the controls. In particular, the adsorption efficiencies of benzene and toluene, using CK and CKN materials, were greater than 90%, while their capacity to adsorb phenol was less than 70%. These results can be explained by considering the difference in polarity between the adsorbates. The capacity of P. stutzeri to metabolize aromatic hydrocarbons can be understood as follows: benzene > toluene > phenol. P. stutzeri’s ability to consume phenol from a simulated medium was almost nonexistent. In particular, the adsorption efficiencies of benzene and toluene using CK and CKN materials, were greater than 90%, while their capacity to adsorb phenol was less than 70%; these results can be explained by considering the difference in polarity between the adsorbates. The capacity of P. stutzeri to metabolize aromatic hydrocarbons is as follows: benzene > toluene > phenol. P. stutzeri’s ability to consume phenol from a simulated medium was almost nonexistent. The use of CK and CKN biomaterials for phenol removal produced a drop in the removal efficiency, originally greater than 50%, of CK and CKN materials. These results could be attributed to the loss of porosity when P. stutzeri was immobilized, as well as the absence of any P. stutzeri affinity for phenol. The experiments in the presence of phenol were useful to interpret the ways in which the cells altered the porosity of CK and CKN. The comparison between the phenol removal efficiencies using materials and biomaterials shows the way the pore space was modified by bacterial immobilization. Due to the low ability of P. stutzeri to consume phenol, the removal was strictly limited to the adsorptive process.