Interaction between Illite and a Pseudomonas stutzeri-Heavy Oil Biodegradation Complex

Illite is a widely distributed clay mineral with huge reserves in Earth’s crust, but its effect on heavy oil biodegradation is rarely reported. This study made an investigation of the interactions between illite and a Pseudomonas stutzeri-heavy oil complex (PstHO). Results showed that, although illite exerted a negative effect on P. stutzeri degrading heavy oil by inhibiting the biodegradation of 64 saturated hydrocarbons (SHs) and 50 aromatic hydrocarbons (AHs), it selectively stimulated the biodegradation of 45 AHs with a specific structure, and its biogenic kaolinization at room temperature (35 °C) and pressure (1 atm) was observed in PstHO for the first time. The finding points out for the first time that, in PstHO, illite may change the quasi-sequential of AHs biodegradation of heavy oil, as well as its kaolinization without clay intermediate.


Introduction
Illite clay minerals account for more than half of the clay minerals in Earth's crust [1]. However, the majority of the studies on the effects of clay minerals on crude oil biodegradation by microorganisms have mainly focused on montmorillonite (Mon; for all abbreviations in this paper, see Table 1), kaolinite (Kao), saponite (Sap), palygorskite (Pal), vermiculite, and nontronite (Table 2). To the best of our knowledge, possibly because illite does not possess a remarkable specific surface area (SSA) [2], cation exchange capacity (CEC) [3], swelling property [4,5], and unique clay mineral microstructure [6], there are no reports on the effects of illite on crude oil biodegradation (Table 2), especially, biodegradation of heavy oil. Heavy oil is an important resource accounting for 21.3% of the global known recoverable oil resources [7]. As research on heavy oil biodegradation is difficult [8] due to the higher viscosity and density of heavy oil [9], there are only a few reports available on the influence of clay minerals on the biodegradation of heavy oil, except those affected by specific environmental conditions at a certain time ( Table 2).
Most of the previous studies had focused on unidentified microbial community easily degradable crude oil systems (Table 2), and only a few identified microbial species were involved, such as Alcanivorax borkumensis and Pseudomonas aeruginosa ( Table 2). The macro phenomenon of crude oil degradation by an unidentified microbial community is a sum of selective degradations of crude oil by all microorganisms in the microbial community and varies with the composition and abundance of microbial species in the microbial community [10,11], which is an obstacle to correctly understand the role of clay minerals in crude oil biodegradation. As a crude oil degrading bacterium [12][13][14][15], Pseudomonas stutzeri has been detected in heavy oil reservoirs [16]; however, there are no studies on heavy oil biodegradation by this bacterium, not to mention the influence of clay minerals.
The present study is the first to investigate the interaction between illite and a P. stutzeriheavy oil biodegradation complex (PstHO). The biodegradation of heavy oil by P. stutzeri Note: In order of appearance in the context.      [41], the composition of illite from Chengde is: 53.5% SiO 2 ; 27.67% Al 2 O 3 ; 1.14% Fe 2 O 3 ; 0.036% FeO;1.25% MgO; 0.64% CaO; 0.75% Na 2 O; 7.77% K 2 O, and 5.25% loss-on-ignition (LOI). This natural illite clay is initially a wet mud cake without particle size screening, and its CEC is 140 meq/kg [4].

Strain Pseudomonas stutzeri L1SHX-3X
Aerobic P. stutzeri strain L1SHX-3X was isolated from a heavy oil-water mixture of the production well in Liaohe Oil Field, China, and was identified [42,43] by the Research Centre for Geomicrobial Resources and Application, China University of Petroleum, Beijing, China. The strain was preserved by converting it into freeze-dried powder [44] and stored in a refrigerator (Thermo Fisher Scientific, Waltham, MA, USA) at −80 • C.

Heavy Oil
The heavy oil L1YJC23 used in this study was collected from JC23 well in the Jin 45 Block of Liaohe Oil Field and preserved in an airtight plastic bucket [42].
All the chemical agents were of analytical grade and supplied by Tianjin Fuchen Chemical Reagents Factory, Tianjin, China.

Experiment on the Interaction between Illite and P. stutzeri-Heavy Oil Complex
Illite was sifted through a 200-mesh sieve (74 µm) and dried in an oven (75 • C, 48 h), and its particle size and SSA were measured.
Reactivation of P. stutzeri was achieved by adding 1 mL of freeze-dried powder of P. stutzeri (obtained with 500 mL of P. stutzeri L1SHX-3X culture medium in logarithmic growth stage) to 4 mL of modified Van Niel culture medium [45] (MVN-R) under sterile conditions and incubating in a shaker (120 rpm, 35 • C) for 2 days [44]. The MVN-R contained 4.50 g/L C 6 H 12 O 6 , 0.10 g/L of NH 4 Cl, 0.04 g/L of MgCl 2 ·6H 2 O, 0.05 g/L of KH 2 PO 4 , 0.50 g/L of Na 2 CO 3 , 0.10 g/L of Na 2 S·9H 2 O, and 0.10 g/L of NaCl. After 2 days of incubation, 4 mL of the culture were mixed with 40 mL MVN-R under the same culture conditions [44] until P. stutzeri concentration reached 10 8 cell/mL under an Eclipse Ni-U upright microscope (Nikon, Tokyo, Japan) ( Figure S1) for standby.
Before starting the experiment, the viscosity and density of heavy oil were measured, and the genes of in situ microorganisms in heavy oil were analyzed. In order not to affect the composition of the heavy oil, it was not sterilized.
The illite and MVN were first placed in 250 mL conical flasks ( Table 3). The MVN was consistent with MVN-R except that it contained no C 6 H 12 O 6 . Then, the flasks were sealed with high-temperature resistant sealing films (BKMAN, Shanghai, China), and placed in a sterilizer (Zealway, Wilmington, DE, USA) for 20 min (121 • C, 1 atm). Then, P. stutzeri culture medium and heavy oil were added into the conical flasks under sterile conditions, and the flasks were sealed using Parafilm ® sealing film (Bemis, Neenah, WI, USA), which is germproof and breathable (150 cm 3 /m 2 /24 h at 22.78 • C for O 2 ). All the flasks were incubated in a shaker (STIK, Shanghai, China) at 35 • C, 120 rpmfor 56 days.
A total of five groups of illite-PstHO experiments were established ( Table 3). The control groups, P0I0, and P0I8 were used to determine the effects of MVN and illite on heavy oil without P. stutzeri ( Table 3). The experimental groups, P2I0, P2I8, and P2I32, contained the constant volume of P. stutzeri culture medium and different masses of illite (Table 3) to determine the effect of illite content. P2I8 and P2I32 contained 8 and 32 g of illite, respectively, simulating two environmental conditions with different solid contents (15.8% and 43.0%, respectively) and partly representing marine sedimentary and humid soil [22,39], respectively. Three parallel replicates were established for each group (Table 3) for error analysis.  Table 1 for PstHO and MVN. # . P represents P. stutzeri, followed by 0 or 2, which denotes the volume of P. stutzeri culture; I indicates illite, followed by 0, 8, or 32, which shows the mass of illite.
After 56 days, gas samples were collected from the conical flask by piercing the sealing film with a syringe and analyzed by gas chromatography (GC). The contents of the conical flask were filtered using 2-µm filter paper to obtain liquid and illite-heavy oil mixtures. The pH, conductivity (σ), and redox potential (Eh) of the liquid were determined, and the illite-heavy oil mixtures were mixed with 50 mL of organic solvent mixture (C 6 H 14 and C 3 H 6 O at a ratio of 1:4) [46] and subjected to ultrasonication (60 min, 45 • C) to extract heavy oil. Further, heavy oil extraction was performed using solvent CH 2 Cl 2 until CH 2 Cl 2 leachate had no absorption in the spectral range of 200-400 nm on an ultraviolet-visible spectrophotometer (Varian Cary 100 UV-Vis, Agilent, Santa Clara, CA, USA) [47]. The heavy oil dissolved in an organic solvent mixture, and CH 2 Cl 2 was collected and concentrated to 4 mL using a rotary evaporator (Yarong, Qingdao, China) at 65 • C, and then, further dried at room temperature (24 • C) to a constant weight [47] (weighed every 4 h, with differences of three consecutive measurements maintained within 0.0010 g) to obtain heavy oil [47]. Fractions analysis of SHs, AHs, resins (Rs), and asphaltenes (Aps) (SARA) in heavy oil [48] was performed, and the fractions of SHs and AHs were subjected to gas chromatography-mass spectrometry (GC-MS). Illite with CH 2 Cl 2 was dried in an oven at 75 • C to a constant weight (as described previously) and further analyzed by X-ray diffraction (XRD) and scanning electron microscopy (SEM).

Measurements of Particle Size and SSA of Illite
To measure the particle size and SSA of illite, 2 g of illite was mixed with deionized water dispersant and examined under a blue light source with a 466 nm wavelength on a Mastersizer 3000 (Malvern Panalytical, Great Malvern, UK) [49]. The measurement was performed in triplicate, and the average value was determined.

Measurements of Viscosity and Density of Heavy Oil
To measure the viscosity of heavy oil, a BS/U tube viscometer (Cannon, Huntington, NY, USA) loaded with heavy oil L1YJC23 was vertically placed into a constant-temperature water bath at 40 • C for 20 min. The time when the heavy oil reached the specific liquid level was recorded to obtain the viscosity [50].
To determine the density of heavy oil, the test temperature was set to 20 • C, and the heavy oil was injected into the U-shaped pipe of the digital density meter (DMA 4501, Anton Paar, Graz, Austria) using a syringe [51].
Measurements of viscosity and density of heavy oil were performed in triplicates individually, and the average values were obtained.

Gene Sequence Analysis of In Situ Microorganisms in Heavy Oil
Biomass from heavy oil L1YJC23 was extracted with isooctane, and the deoxyribonucleic acid was extracted from the biomass using Fast DNA Spin Kit (MP Biomed-ical, Irvine, California, USA) [42]. The genes were sequenced on a MiSeq TM System (Illumina, San Diego, CA, USA) and analyzed using Galaxy Platform [42,43,52] (https://galaxyproject.org/, accessed on 19 January 2022).
The differences in CO 2 , O 2 , and N 2 contents between the gas samples from the illite-PstHO flasks and atmosphere (∆G, %) were calculated using the following Equation (1): where G a is the percentage content of CO 2 , O 2 , or N 2 in gas from the illite-PstHO (%) experiment and G * is the percentage content of CO 2 , O 2 , or N 2 in the atmosphere (%).

Fractions Analysis of Heavy Oil
SARA analysis separates heavy oil components according to their polarizability and polarity by column chromatography [54]. In the present study, 30 mL n-hexane was added to 20.0-50.0 mg of heavy oil, and the Aps were filtered out with absorbent cotton after ultrasonication (5 min). The SHs, AHs, and Rs were obtained by using a chromatographic column (4 g of silica gel and 3 g of activated alumina) with n-hexane, dichloromethane, and ethanol [47,55].
The fraction content (FC, %) of SARA was calculated using the following Equation (2): where M e is the mass of each SARA in heavy oil L1YJC23 or illite-PstHO (g) and M is the mass of heavy oil L1YJC23 used in each group (0.5 g).

Gas Chromatography-Mass Spectrometry Analysis of Saturated and Aromatic Hydrocarbons
The compounds in the SHs and AHs of the heavy oil were characterized and quantified by GC-MS. Deuterated tetracosane (D 50 -nC 24 , 10 µg) and deuterated dibenzothiophene (D-substituted dibenzthiophene, 10 µg) were used as internal standards for SHs and AHs, respectively. Trace-DSQ mass spectrometer (Thermo Finnigan, San Jose, CA, USA) coupled to an HP 6890 gas chromatograph (Agilent, Santa Clara, CA, USA) was used for GC-MS analysis. The column was HP-5MS (30 m × 0.25 mm, ID) with a 0.25-µm coating, and He (99.99%) was used as the carrier gas. The oven temperature of the gas chromatograph was initially set to 50 • C and was, subsequently, increased to 120 • C at a rate of 20 • C/min, 250 • C at a rate of 4 • C/min, and 310 • C at a rate of 3 • C/min and maintained for 30 min. The mass spectrometer was operated in full-scan electron impact mode with an electron energy of 70 eV [56].
The residual mass content (RMC) of the SHs and AHs in the heavy oil L1YJC23 or illite-PstHO was obtained by GC-MS. The RMC (µg/g), the degradation rate of heavy oil by P. stutzeri (DR, %), and the influence degree (IND, %) of illite on biodegradation were calculated using the following Equations (3)-(5): where k is the response coefficient, S a is the peak area of each compound in SHs and AHs, S * is the peak area of the internal standard, m * is the mass of the internal standard (10 µg), m a is the mass of heavy oil used in fractionation (g), M * is the RMC of each compound of SHs or AHs in heavy oil L1YJC23 (µg/g), M p is the RMC of each compound of SHs or AHs in P2I0, P2I8, or P2I32 with P. stutzeri (µg/g), M ip is the RMC of each compound of SHs or AHs in P2I8 and P2I32 with illite clay and P. stutzeri (µg/g), and M op is the RMC of each compound in SHs or AHs in P2I0 without illite and with P. stutzeri (µg/g). To facilitate comparison with previous studies, the IND of other clay minerals on biodegradation was calculated based on Equation (5).
In order to support the scientific and responsible biochemical processes [57] in the illite-PstHO, mass balance and stoichiometry were performed in the data-checking process using the following Equations (6) and (7): where FC SHs is the fraction content of SHs (%), RMC SH is the RMC of each compound in SHs (µg/g), FC AHs is the fraction content of AHs (%), and RMC AH is the RMC of each compound in AHs (µg/g).
2.2.9. X-ray Diffraction Analysis of Illite XRD analysis of illite was performed using D8 Advance X-ray diffractometer (Bruker, Billerica, MA, USA). The tube voltage was 40 kV and the tube current was 25 mA [53].

Scanning Electron Microscopy Analysis of Illite
The illite was sprayed with gold and observed using TESCAN VEGA 3 (Czech, TESCAN, Brno, Czech Republic) SEM with an electron detector at an accelerating voltage of 20 kV [53].

Effect of Illite on Heavy Oil and In Situ Microorganisms in Heavy Oil
Illite was incapable of altering the SARA fractions of heavy oil in the absence of P. stutzeri; however, it modified the existing state of heavy oil. In this study, the recovery efficiency of eluted SARA fractions ranged from 90% to 99%, completely meeting the requirements for its effectiveness (85-115%) [58]. The SARA fractions of P0I0, P0I8, and heavy oil L1YJC23 were consistent (Figure 1). Due to its high viscosity (1967 MPa·s) and density (0.949 g/cm 3 ), heavy oil remained suspended and dispersed in the MVN as droplets of different sizes or adhered to the inner wall of the conical flask in the absence of illite. However, the adsorption of illite particles (8.67 µm; Figure S2) caused the aggregation of oil droplets and the formation of larger illite-heavy oil mixtures. The mixtures were mostly covered with illite due to the relative excess proportion of illite (8/32 g) to heavy oil (0.5 g). stutzeri; however, it modified the existing state of heavy oil. In this study, the recovery efficiency of eluted SARA fractions ranged from 90% to 99%, completely meeting the requirements for its effectiveness (85%-115%) [58]. The SARA fractions of P0I0, P0I8, and heavy oil L1YJC23 were consistent (Figure 1). Due to its high viscosity (1967 MPa•s) and density (0.949 g/cm 3 ), heavy oil remained suspended and dispersed in the MVN as droplets of different sizes or adhered to the inner wall of the conical flask in the absence of illite. However, the adsorption of illite particles (8.67 μm; Figure. S2) caused the aggregation of oil droplets and the formation of larger illite-heavy oil mixtures. The mixtures were mostly covered with illite due to the relative excess proportion of illite (8/32 g) to heavy oil (0.5 g). Figure 1. SARA analysis of heavy oil L1YJC23 and illite-PstHO. Note: FC is fraction content; SHs, AHs, Rs, and Aps are saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes, respectively; see Table 3 for P0I0, P0I8, P2I0, P2I8, and P2I32. Figure 1. SARA analysis of heavy oil L1YJC23 and illite-PstHO. Note: FC is fraction content; SHs, AHs, Rs, and Aps are saturated hydrocarbons, aromatic hydrocarbons, resins, and asphaltenes, respectively; see Table 3 for P0I0, P0I8, P2I0, P2I8, and P2I32.
Illite did not affect the activity of in situ microorganisms in heavy oil. The gene sequence analysis indicated that the in situ microorganisms in heavy oil L1YJC23 included 61.1% aerobic and 14.1% facultative anaerobic microorganisms (anaerobic and unidentified microorganisms accounted for 17.1% and 7.7%, respectively) ( Figure S3). Among the in situ microorganisms in heavy oil, Pseudomonas was the dominant genus with the largest number of reads (20.1%, Figure S3). Aerobic and facultative anaerobic microorganisms ( Figure S3) activated by MVN consumed O 2 and produced CO 2 , which resulted in gas content differences of CO 2 and O 2 between groups of P0I0 and P0I8 without P. stutzeri and atmosphere ( Figure S4). Comparison of P0I0 with P0I8 revealed that the addition of 8 g of illite did not cause any changes in the CO 2 and O 2 contents ( Figure S4), indicating that illite did not affect the activity of in situ microorganisms in heavy oil L1YJC23. Moreover, in situ microorganisms did not alter the SARA fractions of heavy oil, irrespective of the presence or absence of illite ( Figure 1).

Illite Effect on Activity of P. stutzeri
Illite slightly inhibited the activity of P. stutzeri. In the absence of illite, P2I0 presented the highest CO 2 and lowest O 2 contents ( Figure S4) due to the metabolism of the aerobic bacterium P. stutzeri [12]. The pH, σ, and Eh of P2I0 were the lowest (Table S1) because of the consumption of inorganic salts, O 2 , and petroleum hydrocarbons by P. stutzeri to produce CO 2 and acidic compounds [12]. Consequently, P2I0 also showed the highest DR (23.0%) for heavy oil, followed by P2I8 (17.9%) and P2I32 (13.2%) (Figure 1). Comparison of P2I0, P2I8, and P2I32 revealed that the presence of illite reduced the CO 2 content, increased the O 2 content (Figure S4), reduced the changes in pH, σ, and Eh (Table S1), and decreased DR (Figure 1). These results proved that illite had a negative effect on the activity of P. stutzeri, and the degree of the effect was positively related to the illite content (Figures 1 and S4; Table S1). However, the differences in the parameters (O 2 content < 1%, CO 2 content < 0.19%, pH < 1.5%, σ < 5.8%, Eh < 13.5%, and SHs fraction content < 9.8%; Figures 1 and S4; Table S1) were not sufficiently large to confirm any effect of illite on the metabolic pathways of P. stutzeri.
The slight inhibitory effect of illite on the activity of P. stutzeri might have been caused by a decrease in the bioaccessibility of heavy oil due to adsorption onto illite [59]. Illite was more likely to adsorb heavy oil droplets [60] than P. stutzeri [61] because both illite and P. stutzeri have negatively charged surfaces [12,22], and the excess illite coated the surface of the heavy oil and formed a barrier against P. stutzeri. In contrast, the moderate SSA (1.294 m 2 /g) and CEC [3] (140 meq/kg [4]) of illite were insufficient to elicit a positive effect on microbial activity similar to that of Kao with low SSA and CEC [22,25,27,36].

Illite Inhibition of Saturated Hydrocarbons Biodegradation
The GC-MS results revealed that illite inhibited the biodegradation of all 64 SHs in heavy oil L1YJC23 (Table S2 and Figure S5). Hopane was degraded without the formation of 25-norhopane (Table S2; Figures S5 and S6), indicating that the biodegradation level of heavy oil L1YJC23 was 7-8 on the Peters and Moldowan (PM) scale [8]. Furthermore, all n-alkane, alkyl cyclohexane, and isoprenoid components which should have been in the SHs of crude oil, were missing (Table S2; Figures S5 and S6). The RMC of all the 64 SHs showed a trend of P2I0 < P2I8 < P2I32 ( Figure S5), indicating that the IND of illite was positively correlated with its content. In SHs with content >70 µg/g, the DR of P. stutzeri without illite were related to the molecular weight ( Figure 2). A higher molecular weight usually denotes stronger biodegradation resistance [62,63], thus leading to lower DR. The inhibition of illite on the biodegradation of SHs in heavy oil can be a positive protective way during microbial-enhanced oil recovery processes [53]. The adsorption of illite on SHs reduced their bioaccessibility, which was the main reason for the negative effect of illite. However, with an increase in the molecular weight of SHs, the adsorption of illite on SHs weakened, and the degree of reduction in bioaccessibility decreased, which led to a decrease in the inhibitory effect of illite ( Figure 2). Generally, clay minerals with high SSA and CEC, such as Mon, Sap, and Pal, can stimulate biodegradation of SHs [25] (Figure S7); however, Kao and illite do not possess these properties, and hence, exert a negative effect on SHs biodegradation ( Figure S7). Furthermore, the interlayer of illite does not contain divalent cations [60], and the positive effect of the local bridging effect [22,[24][25][26][27][28][29][30]61] formed by the low concentration of divalent cations (Mg 2+ , 7.8710 -6 mol) provided by MVN was weak, which was not sufficient to alter the inhibition caused by adsorption.

Two effects of illite on aromatic hydrocarbons biodegradation
A total of 96 AHs were detected in the heavy oil L1YJC23 by GC-MS (Table S3, Figures 3 and S8), and were classified into five categories for ease of discussion, namely, naphthalene/phenanthrene/fluorene/biphenyl series and high-ring number (≥4) aromatic hydrocarbons (HRAHs) (Figures S9S13). The degradation rate of AHs by P. stutzeri was not affected by illite ( Figure 1); however, the GC-MS results of AHs suggested that illite might have diverse effects on different AHs compounds ( Figures S9S13). After the verification of fractions and GC-MS data in mass balance and stoichiometry (Eqs (6) and (7)), we found that illite inhibited the biodegradation of 50 AHs, stimulated the biodegradation of 45 AHs, and had no obvious effect on biphenyl (Bph, see Table S3 for the abbreviations of AHs used in this study) ( Figure S12).
The effect of illite on the biodegradation of AHs in PstHO was affected by the number of aromatic rings, and illite appeared to inhibit the biodegradation of AHs with a high number of aromatic rings. For example, although both trimethyl naphthalene (TMN) and trimethyl phenanthrene (TMP) have three methyl groups (Figure 3), illite stimulated TMN biodegradation ( Figure S9) but inhibited TMP biodegradation ( Figure S10), which may The adsorption of illite on SHs reduced their bioaccessibility, which was the main reason for the negative effect of illite. However, with an increase in the molecular weight of SHs, the adsorption of illite on SHs weakened, and the degree of reduction in bioaccessibility decreased, which led to a decrease in the inhibitory effect of illite ( Figure 2). Generally, clay minerals with high SSA and CEC, such as Mon, Sap, and Pal, can stimulate biodegradation of SHs [25] (Figure S7); however, Kao and illite do not possess these properties, and hence, exert a negative effect on SHs biodegradation ( Figure S7). Furthermore, the interlayer of illite does not contain divalent cations [60], and the positive effect of the local bridging effect [22,[24][25][26][27][28][29][30]61] formed by the low concentration of divalent cations (Mg 2+ , 7.87 × 10 −6 mol) provided by MVN was weak, which was not sufficient to alter the inhibition caused by adsorption.

Two Effects of Illite on Aromatic Hydrocarbons Biodegradation
A total of 96 AHs were detected in the heavy oil L1YJC23 by GC-MS (Table S3, Figures 3 and S8), and were classified into five categories for ease of discussion, namely, naphthalene/phenanthrene/fluorene/biphenyl series and high-ring number (≥4) aromatic hydrocarbons (HRAHs) (Figures S9-S13). The degradation rate of AHs by P. stutzeri was not affected by illite ( Figure 1); however, the GC-MS results of AHs suggested that illite might have diverse effects on different AHs compounds (Figures S9-S13). After the verification of fractions and GC-MS data in mass balance and stoichiometry (Equations (6) and (7)), we found that illite inhibited the biodegradation of 50 AHs, stimulated the biodegradation of 45 AHs, and had no obvious effect on biphenyl (Bph, see Table S3 for the abbreviations of AHs used in this study) ( Figure S12).
Microorganisms 2023, 11, x FOR PEER REVIEW 15 of 21 have been caused by the addition of an aromatic ring. Furthermore, monomethyl biphenyl (MeBph), monomethyl pyrene (MePyr), and monomethyl chrysene (MeChr) have one methyl substituent ( Figure 3); illite also stimulated MeBph biodegradation ( Figure S12) with two aromatic rings and inhibited biodegradation of MePyr and MeChr ( Figure S13) with four aromatic rings. The more fused the rings of AHs, the stronger the ability of the compounds to resist biodegradation [64], with illite being more inclined to inhibit their biodegradation.  Table S3 for No. of the AHs; blue, red, and black represent inhibition, stimulation, and uncertain effects of illite, respectively.
The effect of illite on the biodegradation of AHs in PstHO was affected by the number of methyl substituents. In the two-ring naphthalene series (Figure 3), as the number of methyl substituents increased from 0 to 5, the effect of illite varied from inhibition (0, 1, 2) to stimulation (3,4) and then, to inhibition (5) ( Figure S9). In general, with an increase in the number of methyl substituents, the steric hindrance, stability, and biodegradation resistance of naphthalene series compounds increase [64][65][66][67]. However, the effect of illite on the biodegradation of naphthalene series compounds was not consistent with this rule, and this phenomenon of altered influence with the increase in the methyl substitution number of naphthalene series was also exhibited by Mon [36] ( Figure S14). The number of methyl substituents had a secondary effect when compared with the number of aromatic rings. For example, in the three-ring phenanthrene series (Figure 3), the number of methyl substituents increased from 0 to 3, and the effect of illite on their biodegradation was consistently negative (Figure S10), which was different from that on the naphthalene series ( Figure S9). In contrast, although the effect of Mon was positive, consistency in the biodegradation of different compounds in the phenanthrene series was also observed [29,30] (Figure S15).  Table S3 for No. of the AHs; blue, red, and black represent inhibition, stimulation, and uncertain effects of illite, respectively.
The effect of illite on the biodegradation of AHs in PstHO was affected by the number of aromatic rings, and illite appeared to inhibit the biodegradation of AHs with a high number of aromatic rings. For example, although both trimethyl naphthalene (TMN) and trimethyl phenanthrene (TMP) have three methyl groups (Figure 3), illite stimulated TMN biodegradation ( Figure S9) but inhibited TMP biodegradation ( Figure S10), which may have been caused by the addition of an aromatic ring. Furthermore, monomethyl biphenyl (MeBph), monomethyl pyrene (MePyr), and monomethyl chrysene (MeChr) have one methyl substituent ( Figure 3); illite also stimulated MeBph biodegradation ( Figure S12) with two aromatic rings and inhibited biodegradation of MePyr and MeChr ( Figure S13) with four aromatic rings. The more fused the rings of AHs, the stronger the ability of the compounds to resist biodegradation [64], with illite being more inclined to inhibit their biodegradation.
The effect of illite on the biodegradation of AHs in PstHO was affected by the number of methyl substituents. In the two-ring naphthalene series (Figure 3), as the number of methyl substituents increased from 0 to 5, the effect of illite varied from inhibition (0, 1, 2) to stimulation (3,4) and then, to inhibition (5) ( Figure S9). In general, with an increase in the number of methyl substituents, the steric hindrance, stability, and biodegradation resistance of naphthalene series compounds increase [64][65][66][67]. However, the effect of illite on the biodegradation of naphthalene series compounds was not consistent with this rule, and this phenomenon of altered influence with the increase in the methyl substitution number of naphthalene series was also exhibited by Mon [36] ( Figure S14). The number of methyl substituents had a secondary effect when compared with the number of aromatic rings. For example, in the three-ring phenanthrene series (Figure 3), the number of methyl substituents increased from 0 to 3, and the effect of illite on their biodegradation was consistently negative ( Figure S10), which was different from that on the naphthalene series ( Figure S9). In contrast, although the effect of Mon was positive, consistency in the biodegradation of different compounds in the phenanthrene series was also observed [29,30] (Figure S15).
Furthermore, the influence of illite on the biodegradation of AHs in PstHO was affected by the connection mode of the aromatic rings. For instance, although both naphthalene series and biphenyl series have two aromatic rings (Figure 3), illite inhibited the biodegradation of monomethyl naphthalene and dimethyl naphthalene ( Figure S9) but stimulated MeBph and dimethyl biphenyl biodegradation ( Figure S12). The key difference between these two series is that the naphthalene series is formed by the fusion of two benzene rings, whereas the biphenyl series is formed by linking two phenyl groups through a single covalent bond (Figure 3).
The impact of illite on the biodegradation of AHs in PstHO was affected by heteroatoms. In the fluorene series, fluorene (Fle), dibenzothiophene (DBT), and dibenzofuran (DBF) have the same structure, except that the atom at position 5 is carbon, sulfur, and oxygen, respectively ( Figure 3). Illite had different effects on the biodegradation of these compounds, and it is inhibiting Fle biodegradation and stimulating DBT and DBF biodegradation (Figures 3 and S11). Similarly, this trend was also noted in the monomethyl substituents of these compounds, monomethyl fluorene, monomethyl dibenzothiophene, and monomethyl dibenzofuran (Figures 3 and S11). However, DBT exhibited the strongest biodegradation resistance, followed by Fle and DBF [64,68]. Thus, the influence of illite on AHs biodegradation was inconsistent with the biodegradation resistance of AHs. Larger sulfur and oxygen atoms, when compared with carbon atoms, can alter the polarity of the molecules, which might be the reason for the different effects of illite on these compounds. Illite had varied effects on compounds containing different heteroatoms in the fluorene series, unlike other clay minerals (modified or not) ( Figure S16). Kao and Sap could inhibit the biodegradation of Fle and DBT (and their methyl or ethyl substituent), whereas Mon and Pal could stimulate their biodegradation [29,30,36] ( Figure S16).
The influence of illite on the biodegradation of AHs in PstHO was affected by the carbon chain length in the substituent. HRAHs have more than four rings (Figure 3 Figure S13) with the longer carbon chain substituents (C2, C3, C8, C9, and C10). It must be noted that it is common for unmodified clay minerals, such as Mon, Kao, Sap, and Pal, to stimulate the biodegradation of TAS [30,36] ( Figure S17).
The effect of illite on the biodegradation of AHs in PstHO was not dominated by inhibition, as in the case of SHs. The positive effects of moderate SSA (and CEC) and weak local bridging effect were not sufficient to counteract the negative effects caused by adsorption, thus suggesting the possible occurrence of other positive mechanisms to explain the selective stimulation of illite to 45 AHs. Considering the facts that illite could not provide divalent cations (to form the local bridging effect) and this positive effect only occurred in AHs with π bonds, we speculated that the selective stimulation of illite was due to the cation-π [69] preferentially formed by monovalent cations and AHs with above five structure characteristics.

Kaolinization of Illite in P. stutzeri-Heavy Oil Complex
This study is the first to report the kaolinization of illite without smectite formation in illite-PstHO. The XRD patterns of illite clay in P0I8 and unused samples were the same as the standard pattern (Figure 4a, ICDD PDF 26-0911 Illite), indicating that MVN and heavy oil L1YJC23 (including in situ microorganisms therein) did not change the crystal characteristics of illite in the absence of P. stutzeri (Figure 4a). Although the main characteristic peaks (I x /I max ≥ 0.5) [70] of illite crystals still remained in the intermediate (formed in the process of kaolinization of illite) of P2I8, they were significantly weakened (e.g., (002),  (Figure 4a). Furthermore, the edges of the illite particles in P0I8 were sharp (Figure 4b), while those of the intermediate particles of P2I8 were round and had attached precipitates of 250-1000 nm (Figure 4c). This phenomenon of rounded edges (Figure 4c) caused by local dissolution is similar to the kaolinization of illite under abiotic condition [5]. After the destruction of the edges of the illite crystals, K + in the interlayers [71] was released to form cation-π [69], which stimulated the biodegradation of specific AHs. The organic products of biodegradation and ligands promoted the transformation of dissolved illite into the granular precipitate-aluminosilicate gel, which is considered the crucial first step in the two-step biological kaolinization [72]. The gel formed was deposited near the edges of the illite particles, which is considered to provide precursor materials and a crystallization environment for Kao. In the second step, biological metabolic activities further changed the local surrounding environment (pH, σ, and Eh) in and around the gel, resulting in the dissolution of the gel or rearrangement of its solid state to form Kao crystals [72]. Although previous studies have detected smectite (under the action of Pseudogulbenkiania sp.) in the process of kaolinization of illite [17,18], characteristic peaks of smectite were not observed in the present study, which may be attributed to the different microorganisms employed and the presence of heavy oil. the edges of the illite particles in P0I8 were sharp (Figure 4b), while those of the intermediate particles of P2I8 were round and had attached precipitates of 2501000 nm ( Figure  4c). This phenomenon of rounded edges (Figure 4c) caused by local dissolution is similar to the kaolinization of illite under abiotic condition [5]. After the destruction of the edges of the illite crystals, K + in the interlayers [71] was released to form cation-π [69], which stimulated the biodegradation of specific AHs. The organic products of biodegradation and ligands promoted the transformation of dissolved illite into the granular precipitatealuminosilicate gel, which is considered the crucial first step in the two-step biological kaolinization [72]. The gel formed was deposited near the edges of the illite particles, which is considered to provide precursor materials and a crystallization environment for Kao. In the second step, biological metabolic activities further changed the local surrounding environment (pH, σ, and Eh) in and around the gel, resulting in the dissolution of the gel or rearrangement of its solid state to form Kao crystals [72]. Although previous studies have detected smectite (under the action of Pseudogulbenkiania sp.) in the process of kaolinization of illite [17,18], characteristic peaks of smectite were not observed in the present study, which may be attributed to the different microorganisms employed and the presence of heavy oil.

Conclusions
To the best of our knowledge, this is the first report on the interaction of illite with the PstHO complex. Although illite clay exerted a negative effect on the total heavy oil degradation ratio, it stimulated the biodegradation of 45 AHs in heavy oil, and its kaolinization in the illite-PstHO was observed for the first time. The selective inhibition/stimulation effects of illite clay on biodegradation, as a supplementary mechanism of quasi-sequential and/or selective degradation, can be applied to protect high-quality SHs during microbial enhanced oil recovery process and remediation of specific AHs pollutant in polluted environments. Kaolinization of illite clay could be further explored to explain clay minerals transformations in oil reservoirs. For future study, new multivariate complex systems, such as illite-PstHO could lead to richer discoveries in the field.
Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/microorganisms11020330/s1, Figure S1: Micrographs of cells of Pseudomonas stutzeri strain L1SHX-3X in an Eclipse Ni-U upright microscope (a) and a VEGA 3 SEM (b) after 6 days of reactivation by MVN-R from freeze-dried powder (storage at −80 • C) in the shaker with 120 rpm at 35 • C; Figure S2: Particle size distribution of illite measured by Mastersizer 3000; Figure S3: Genus-level analysis of in situ microorganisms in heavy oil L1YJC23; Figure S4: Difference in CO 2 , O 2 , and N 2 contents between illite-PstHO and atmosphere; Figure ; Table S1: The pH, σ and Eh of MVN and illite-PstHO; Table S2: Saturated hydrocarbons in heavy oil L1YJC23 and illite-PstHO detected by GC-MS; Table S3: Aromatic hydrocarbons in heavy oil L1YJC23 and illite-PstHO detected by GC-MS. References [23,25,29,30,36] are cited in the supplementary materials.