Influence of kaolinite and montmorillonite on benzo[a]pyrene biodegradation by Paracoccus aminovorans HPD-2 and the underlying interface interaction mechanisms

Xinhong GANYing TENG*Jian XUNing ZHANGWenjie RENLing ZHAOPeter CHRISTIE and Yongming LUO

1 Key Laborator y of Soil Environment and Pollution Remediation,Institute of Soil Science,Chinese Academy of Sciences,Nanjing210008(China)

2 State Environmental Protection Key Laborator y of Soil Environmental Management and Pollution Control,NanjingInstitute of Environmental Science,Ministr y of Ecology and Environment(MEE)of China,Nanjing210042(China)

3 University of Chinese Academy of Sciences,Beijing100049(China)

ABSTRACT Clay minerals play an important role in biogeochemical cycling.Here,kaolinite and montmorillonite,the two most abundant and widespread clay minerals with typical layered structures,were selected to investigate and compare their effects on the biodegradation of benzo[a]pyrene(BaP)by Paracoccus aminovorans HPD-2 and to investigate the underlying interface mechanisms.Overall,the BaP degradation efficiency was significantly higher 7 d after montmorillonite addition,reaching 68.9%(P＜0.05),when compared with that of the control without addition of clay minerals(CK,61.4%);however,the addition of kaolinite significantly reduced the BaP degradation efficiency to 45.8%.This suggests that kaolinite inhibits BaP degradation by inhibiting the growth of strain HPD-2,or its strong hydrophobicity and readily agglomerates in the degradation system,resulting in a decrease in the bio-accessibility of BaP to strain HPD-2.Montmorillonite may buffer some unfavorable factors,and cells may be fixed on the surface of montmorillonite colloidal particles across energy barriers.Furthermore,the adsorption of BaP on montmorillonite may be weakened after swelling,reducing the effect on the bio-accessibility of BaP,thus promoting the biodegradation of BaP by strain HPD-2.The experimental results indicate that differential bacterial growth,BaP bio-accessibility,interface interaction,and the buffering effect may explain the differential effects of the different minerals on polycyclic aromatic hydrocarbon biodegradation.These observations improve our understanding of the mechanisms by which clay minerals,organic pollutants,and degrading bacteria interact during the biodegradation process and provide a theoretical basis for increasing the biodegradation of soil pollutants by native microorganisms under field conditions.

Key Words: bio-accessibility,cell viability,clay mineral,degrading bacteria,nano-scale secondary ion mass spectrometry,polycyclic aromatic hydrocarbon

INTRODUCTION

Polycyclic aromatic hydrocarbons(PAHs)are a class of compounds that are of environmental and public health concern because of their known or suspected toxicity and genotoxicity and their frequent occurrence at contaminated sites(Witt and Trost,1999).Polycyclic aromatic hydrocarbon pollution in soil endangers the safety of agricultural products and the health of the soil ecosystem.Therefore,remediation of soil contaminated by PAHs has become a topic of great concern(Ganet al.,2009).Microbial remediation has become the main means of remediation of contaminated farmland due to its economic and environmentally friendly characteristics(Vilaet al.,2015).However,the ability of microorganisms to degrade PAHs in soil may be influenced by many factors that can affect microbial remediation(Megharajet al.,2011).

We have previously isolated a highly efficient PAHdegrading bacterium,identified asParacoccus aminovoransHPD-2,from highly PAH-contaminated soil,which can utilize benzo[a]pyrene(BaP,exhibiting the highest carcinogenicity among the 16priority control PAHs(Nisbet and Lagoy,1992))as the sole carbon and energy source for growth(Tenget al.,2010;Ganet al.,2018).The isolate could degrade 89.7%of BaP after incubation for 5 d in mineral salt medium(MSM)solution containing 3.0 mg L-1BaP.However,only 30%of BaP in the contaminated soil was degraded by the isolate after 14 d.Furthermore,our preliminary studies on PAH biodegradation in soil suggested that the pyrene dissipation ratio was considerably lower in red soil(Argi-Udic Ferrosols)than in black soil(Black-Lithomorphic Isohumosols)throughout the incubation period(42 d)(Renet al.,2016),with a decline of more than 90%in pyrene by day 21 in the black soil,whereas no such decline could be observed in the red soil(Renet al.,2015).Clay minerals are soil components exhibiting high specific surface area,cation exchange capacity,and surface charge density(Churchmanet al.,2006).They play an important role in biogeochemical cycling and have a significant impact on the environmental fate of pollutants(Liuet al.,2013;Gonget al.,2016).It is therefore important to investigate soil clay minerals(Ruanet al.,2018a).

Unfortunately,clay mineral-microbial interactions may result in either positive or negative outcomes in the removal of organic contaminants(Dong and Lu,2012;Biswaset al.,2015).Numerous studies have indicated that clay minerals can promote the biodegradation of organic pollutants through the following mechanisms:i)providing a carrier for microbial growth and biofilm formation(Chaerunet al.,2005;Chaerun and Tazaki,2005;Sarkaret al.,2012),ii)maintaining enzyme activity or protecting microorganisms from adverse conditions(Sarkaret al.,2012;Quintelaset al.,2013;Cébronet al.,2015),iii)enriching pollutants for biodegradation(Baglieriet al.,2013),and iv)optimizing and adjusting the medium(Govarthananet al.,2017,2020).However,inhibitory effects of clay minerals on biodegradation have also been reported(Dong and Lu,2012).Clay minerals may suppress biodegradation by reducing enzyme activities(Sarkaret al.,2013)and organic pollutant bioaccessibility(Chenget al.,2012;Dong and Lu,2012),or by inhibiting microbial growth(Morrisonet al.,2016;Londonoet al.,2017).

Montmorillonite(2:1 type expandable clay mineral)and kaolinite(1:1 type non-expandable clay mineral)are the most abundant and widespread clay minerals with typical layered structures(Tombácz and Szekeres,2006).Kaolinite dominates the clay minerals in red soil,whereas montmorillonite is the dominant clay mineral in black soil(Sánchez and Tavani,1994).However,it is not yet known if kaolinite and montmorillonite promote or inhibit BaP degradation by HPD-2,or if the effects of these clay minerals can explain the different degradation effects of pyrene in different types of red and black soils.Thus,it is important to illuminate the interaction mechanisms among soil,pollutant-degrading bacteria,and organic pollutants(Biswaset al.,2015).Here,we selcted kaolinite and montmorillonite to illustrate their effects on BaP biodegradation byP.aminovoransHPD-2.The objective was to investigate the interactions among clay minerals,HPD-2,and BaP during biodegradation.

MATERIALS AND METHODS

Reagents

Benzo[a]pyrene(≥96%purity)was purchased from Sigma—Aldrich(St.Louis,USA).High performance liquid chromatography(HPLC)-gradeN,N-dimethylformamide(DMF)and acetonitrile were purchased from Merck KGaA(Darmstadt,Germany).Montmorillonite,kaolinite,and other analytical grade reagents were purchased from Sinopharm Chemical Reagent Co.Ltd.(Shanghai,China).A stock solution of BaP(5 000 mg L-1)was prepared in DMF.All other solutions were prepared using ultrapure water.The composition of the MSMand Luria-Bertani(LB)broth is described in Section S1(See Supplementary Material for Section S1).

Preparation and characterization of clay mineral colloids and strain HPD-2 cells

Colloidal particles(＜2μm)were obtained from the purchased kaolinite and montmorillonite by a sedimentation siphon method(Xiong,1985).The sediment was washed with ultrapure water until the electrical conductivity of the washing liquid was＜20μS cm-1,and then with 95%alcohol,followed by drying at 110°C for 24 h.The prepared mineral was ground to pass through a 100-mesh sieve for subsequent use.The clay minerals were characterized by X-ray diffraction(XRD)using an Ultima IV(Rigaku Corporation,Tokyo,Japan).The clay mineral content was obtained by comparison with the International Center of Diffraction Data(ICDD).The Brunauer-Emmett-Teller specic surface area of the clay minerals was determined using a V-Sorb 2800P analyzer(Gold APP Instrument Co.,China).The contact angles of the clay minerals were measured using a drop shape analyzer(DSA100,Krüss GmbH,Hamburg,Germany)with three types of liquid(H2O,CH3NO,and CH2I2),and the zeta potential was determined using a zeta potential and particle size analyzer(ZetaPlus 90,Brookhaven Instruments Corporation,Holtsville,USA)at pH 7.0(1 mmol L-1KNO3as background solution)(Honget al.,2012).These data determined above are shown in Table SI(See Supplementary Material for Table SI).Fourier transform infrared spectra(FTIR)was obtained in the transmission mode over the range of 4 000—400 cm-1(Vertex 70,Bruker Optics,Ettlingen,Germany).

HPD-2 was isolated from a historically PAH-contaminated soil from Wuxi,Jiangsu Province,East China.Colonies of the bacterium were inoculated in 10 mL LB and incubated in an orbital shaker for 12 h(150 r min-1,30°C).The isolate was amplified in a 250-mL flask containing 100 mL LB,cultivated for 24 h under the same incubation condition and then centrifuged.The cells were retained,washed thrice with MSM solution,and re-suspended to obtain the bacterial suspension.Optical density at 600 nm(OD600)of the bacterial suspension was determined(Bio-Tek μQuant,Winooski,USA)and adjusted to OD600=1.0(signifying about 2.8×108colony forming units(CFUs)mL-1).The details of preparation of kaolinite,montmorillonite,and bacteria are given in Section S2(See Supplementary Material for Section S2).Inter face interaction of strain HPD-2 and BaP on clay minerals

Interaction at the interface of clay particles and bacterial cells was determined as follows.i)Bacterial suspensions(0.25—10 mL)were added to 50-mL flasks containing 200 mg kaolinite or montmorillonite to evaluate the adsorption of HPD-2 on clay minerals.The BaP stock solutions(5—200 μL)were added to 50-mL flasks containing 200 mg kaolinite or montmorillonite,and 200 mg L-1NaN3was used as a biological inhibitor.To avoid co-solvent effects,the amount of DMFadded in all treatments was 200μL.ii)Bacterial suspensions(0.25—10 mL)were added to 50-mL flasks containing 200 mg clay minerals,adjusted to pH 7 by addition of KOH and HNO3solutions,and KNO3electrolyte solution was then supplemented to achieve a final volume of 20 mL.The mixture was then shaken at 25°C for 240 min.After the injection of 1 mL of 60%(weight/weight)sucrose solution,the suspension was centrifuged at 3 000×gfor 20 min.The supernatant containing free bacteria was pipetted out and analyzed at 600 nm.The percentage of bacteria adsorbed on clay minerals was determined by subtracting the amount of free bacteria from the initial amount of bacteria added.For BaP adsorption on clay minerals,a series of volumes of BaP stock solution(5—200μL)was added to 50-mL flasks containing 200 mg clay minerals,and 200 mg L-1NaN3was used as a biological inhibitor.The final volume was brought to 20 mL,and the suspension was shaken at 25°C for 7 d in the dark,centrifuged at 3 000×gfor 20 min,and then subjected to HPLC(Class-VP HPLC system,Shimadzu,Kyoto,Japan)analysis.The BaP extraction and determination were improved following the method of Huanget al.(2013b).For the HPLC system,a fluorescence detector(RF-20 A)was used,equipped with a C18 reverse-phase column(VP-ODS,250 mm×4.6mm),and the mobile phase was water and acetonitrile binary mobile phase(1:9,volume/volume),with column temperature of the C18 reversed-phase column set to 35°C and the flow rate set to 1 mL min-1.Excitation and emission wavelengths of the detector were 290 and 410 nm,respectively,for BaP separation.iii)Two clay mineral colloids(0.40 g)were added to 100-mL conical flasks containing 40 mL MSMand sterilized by autoclaving at 121°C for 30 min.The reaction flask was spiked with BaP stock solution to achieve an initial BaP concentration of 10 mg L-1,and 200 mg L-1NaN3was used as a biological inhibitor.All samples were placed in a shaker at 150 r min-1and 30°C in the dark for 0,15,and 30 d,and then extracted with 50 mmol L-1hydroxypropyl-β-cyclodextrin(HPCD)(Cramponet al.,2016).The specific implementation details of interface interaction experiment are shown in Section S3(See Supplementary Material for Section S3)

The Ex-DLVO theory was used to calculate the interaction energy between HPD-2 and clay mineral surfaces,and the calculation is presented in Section S4(See Supplementary Material for Section S4).

Effects of kaolinite and montmorillonite on BaP degradation by strain HPD-2 and its cell viability

The clay mineral colloids(0.40 g)were added to 100-mL conical flasks containing 36mL MSM and sterilized by autoclaving at 121°C for 30 min.The reaction flask was spiked with BaP stock solution to achieve an initial BaP concentration of 10 mg L-1.As in a previous study(Mao,2008),negligible effect of DMFon the growth of theP.aminovoransHPD-2 was found.Then,2 mL bacterial suspension and 2 mL MSM were added to the flask,to achieve a final volume of 40 mL.Samples were mixed in a shaker at 150 r min-1and 30°C in the dark for 7 d.Two control treatments,control treatment without any clay minerals(CK)and CK without bioaugmentation(CK-P),were set up in this experiment.

After culturing for 7 d,all suspension cultures was centrifuged at 3 000×gfor 20 min(Mao,2008).The supernatant was then extracted with ethyl acetate,and the precipitate was lyophilized and extracted with 70 mL dichloromethane by Soxhlet extraction.The two BaP fractions were purified and analyzed.To evaluate the effect of kaolinite and montmorillonite on cell viability,the flask was shaken evenly upon completion of growth kinetics(from the stationary phase,about 60 h),and 100μL suspension was collected at random and spread on LB plates after 104-and 105-fold dilution with sterile water in sterile conditions.The LB plates were incubated overnight at 30°C.Colony forming units were quantified and compared with two control treatments(CK and CK-P)to check the viability of the bacterial cells following treatment with different clay minerals.According to the instructions provided by the Bac-Light Bacterial Viability Kit(L7012,molecular probes,Invitrogen,Thermo Fisher Scientific,Waltham,USA),cells treated with kaolinite or montmorillonite for different durations were stained with SYTO green-fluorescent nucleic acid stain(SYTO-9,Thermo Fisher Scientific,Waltham,USA)and propidium iodide(PI).After washing out the extracellular nucleic acid dye,the cells were observed using a confocal laser scanning microscope(CLSM,LSM710,Carl Zeiss,Jena,Germany)with a 40×objective lens.The excitation wavelength was 470 nm,and integrated intensities of the green(510—540 nm)and red(620—650 nm)emissions were acquired.The specific information of materials and operations in the experiment affecting cell activity are shown in Section S5(See Supplementary Material for Section S5).

Cell mor phology and contribution of kaolinite and montmorillonite to cell aluminum

Scanning electron microscopy(SEM)was used to evaluate the effect of the presence of clay minerals on the morphology of HPD-2.Free aluminum ions were quantified by inductively coupled plasma-mass spectrometry(ICP-MS).Nano-scale secondary ion mass spectrometry(Nano-SIMS)(Liet al.,2008)was used to examine the aluminum distribution on HPD-2 treated with kaolinite and montmorillonite.Two milliliters ofP.aminovoransHPD-2(OD600=1.0)was mixed with clay minerals(200 mg)in 20 mL MSMsolution for 2 and 6h.The samples were then centrifuged,washed twice with MSMsolution,and fixed in 2.5%glutaraldehyde at 25°Cfor 2 h.The complex was dehydrated with 50%,75%,and 100%ethanol onto silicon wafers.Surface morphology,shape,and size of the bacterial cells were examined using SEM(S-3400 N II,Hitachi,Tokyo,Japan).The contribution of the clay minerals to cellular aluminum was evaluated by mixing 2 mLP.aminovoransHPD-2(OD600=1.0)suspension and kaolinite or montmorillonite(20 mg)with 10 mg L-1BaP in 20 mL sterile MSM solution for 48 h.No clay mineral was used as a controlled experiment.The bacterial cells were collected by centrifugation at 10 000×gfor 5 min and then washed thrice with 1.0%citric acid-citrate buffer in 0.1%Tween80(pH 7.0)to remove aluminum from the cell surface(Arakhaet al.,2015).Five milliliters of 60%sucrose solution was placed at the bottom of the centrifuge tube and centrifuged at low speed(1 500×g)for 30 min,and the supernatant was centrifuged at high speed(10 000×g)for 5 min.The collected cells were washed twice with MSM solution and divided into two parts.One part was diluted to about 0.70 of its OD value with the MSMsolution.One milliliter of the cell suspension and 1 mLaqua regiawere bath-digested in boiling water for 2 h.The mixture was diluted to 10 mL for aluminum determination by ICP-MS.The other part was used for observation by Nano-SIMS.A 1μL aliquot of the bacterial suspension was dried onto a silicon wafer for Nano-SIMS imaging.This beam was rastered over a 16μm2region containing cells,with a dwell time of 10 ms pixel-1(256×256pixels)for 8 layers(1.5 h measurement).Secondary negative ions of12C-and27Alwere collected simultaneously using electron multiplier detectors.The details of this chapter are seen in Section S6(See Supplementary Material for Section S6).

Adsorption of BaP onto kaolinite and montmorillonite

Isothermal adsorption experiments of BaP on kaolinite and montmorillonite were conducted to compare the adsorption strength of BaP between the two clay minerals.Hydroxypropyl-β-cyclodextrin(50 mmol L-1)extraction(Cramponet al.,2016)was used to evaluate the effects of kaolinite and montmorillonite on BaP bio-accessibility.Twophoton confocal laser scanning microscopy(TP-CLSM,Neon 40 cross-beam system,M/S Carl Zeiss GmbH,Oberkochen,Germany)was used to characterize the BaP distribution between the two clay minerals.The TP-CLSM indirectly monitored the amount of BaP adsorbed by comparing the fluorescence intensity.Excitation and emission wavelengths of the laser were set at 760 and 410 nm,respectively.All mixtures were shaken at 25°C for 12 h(initial pH of 7,with 0.1 mg kg-1BaP in 20 mL MSM solution),and the suspension was then centrifuged at 8 000×gfor 5 min.The precipitate was washed thrice with the MSMsolution to remove free BaP.Finally,1 mL of MSMsolution was added to re-suspend the precipitate,and 5μL of the suspension was dripped onto a glass slide for TP-CLSManalysis.The details of TP-CLSM analysis are described in Section S7(See Supplementary Material for Section S7).

Data analysis and statistics

All experiments were conducted in triplicate to ensure reproducibility.Data were presented as means±standard errors.Statistical differences between mean values were analyzed using one-way analysis of variance.ThePvalue≥0.05 suggests that there is no statistically significant difference(within 95%confidence intervals).

RESULTS AND DISCUSSION

Effects of kaolinite and montmorillonite on BaP degradation efficiency and cell viability of strain HPD-2

Addition of kaolinite significantly inhibited the BaP degradation efficiency(P＜0.05)when compared with CK after 3,5,and 7 d(Fig.1a).The BaP degradation efficiency under montmorillonite amendment was not significantly different from that of CK after 3 and 5 d;however,it was significantly higher than that of CK when the degradation proceeded to 7 d,reaching 68.9%.The cell viability of degrading microorganisms should be initially investigated for the study of the factors affecting biodegradation.The CFU in each treatment was analyzed during the degradation process(Fig.1b).It could be seen that the cell viability peaked after 3 d of degradation,and then decreased continuously.This may be due to the reduction of substrates and the production of intermediate metabolites during the degradation process(Carroquinoet al.,1992).Compared with CK,the addition of kaolinite significantly inhibited the cell viability of degrading microorganisms,whereas montmorillonite addition had no significant effect on cell viability at the early stages,but it significantly increased cell viability after 7 d(P＜0.05).To clarify the differential effects of the two clay minerals on cell viability,the interaction at the interface between microbial cells and clay mineral particles should be considered.

Fig.1 Effects of kaolinite(Kao)and montmorillonite(Mont)on benzo[a]pyrene(BaP)degradation efficiency(a)and Paracoccus aminovorans strain HPD-2 colony forming unit(CFU)(b).CK=control treatment without any clay minerals;CK-P=CK without bioaugmentation.Different letters above the bars indicate significant difference(P＜0.05).The initial bioaugmentation was approximately 2.8×107 CFU mL-1.

Inter face interaction between clay mineral colloids and strain HPD-2

Results of isothermal adsorption(Fig.S1,See Supplementary Material for Fig.S1)showed that the maximum theoretical adsorption capacity of montmorillonite to strain HPD-2 was considerably higher than that of kaolinite,which might be related to the larger specific surface area and surface potential of montmorillonite(Table SI).The interaction energy was further calculated and analyzed by the Ex-DLVO theory(Fig.2a,b)to explain the difference in adsorption of strain HPD-2.From the theory,electrostatic force played a major role in the interaction between strain HPD-2 and mineral colloidal particles.To enable cells to adsorb on the surface of mineral particles,energy barriers must be crossed.Bacteria could cross such energy barriers through following ways:i)kinetic energy generated by bacterial self-movement and ii)special structures on the surface of cells(such as flagellum or hairs)or polymers secreted by cells,which could span the distance between cells and surfaces(Van Loosdrechtet al.,1989).The Ex-DLVO theory indicated that the energy barrier value needed for adsorption on kaolinite was larger than that for montmorillonite.The FTIR was used to characterize the binding mechanism.The results showed that the absorption peaks of kaolinite and montmorillonite changed at the amide group(—CO—NH—),indicating that the interaction between clay minerals and cells was mainly caused by the extracellular protein of strain HPD-2.Cells were bound loosely to the colloid particles.The degradation process was characterized by SEM 3 d after the addition of kaolinite or montmorillonite(Fig.2c,d).It could be seen from the SEMimages that strain HPD-2 treated with kaolinite were obviously fewer than those treated with montmorillonite,and their combination with mineral particles was looser.For the treatment with montmorillonite,it was found that strain HPD-2 grew well in the degradation process,and more cells adhered to the surface of montmorillonite colloidal particles.This phenomenon directly confirmed the results of the CFU test(Fig.1b).Cells could cross the energy barrier and adhere to montmorillonite colloidal particles,because the energy barrier between montmorillonite colloidal particles and cells was less than that of kaolinite(Van Loosdrechtet al.,1989).Therefore,montmorillonite provides a carrier for cell,which was conducive to cell growth.To better explain the effect of clay minerals on cell viability,the release of dissolved Al and its contribution to cellular Al in the MSMmedium were measured and characterized.It was found that kaolinite could release a certain amount of dissolved Al in MSM compared with montmorillonite(Fig.3a).Nano-SIMS was further used to directly characterize the contribution of clay minerals to cellular Al during the degradation process.The distribution maps of carbon(C)and aluminum(Al)elements were processed by image-Jto obtain the ratio of the relative strength of the two elements(27Al/12C ratio).The different contribution of the two clay minerals to cellular Al could be indirectly compared(Fig.3b,c).It can be seen from Fig.3c that kaolinite addition significantly increased the content of Al in cells.Furthermore,it has been reported that the presence of dissolved Al can inhibit the permeability of cell membrane,thus inhibiting cell viability(Londonoet al.,2017).In addition,a bio-imaging technique was used to visually characterize the differences in strain HPD-2 viability under treatment with the two clay minerals.Figure S2 shows the CLSM images of strain HPD-2 adsorbed on kaolinite and montmorillonite for 6h by live/dead staining.Ratio of dead/live cells(D/L)of strain HPD-2 was calculated on the basis of red/green fluorescence intensity,which roughly reflected the proportion of dead and live cells in the system.After interaction with strain HPD-2 for 6h,the ratio of D/L under treatment with montmorillonite was much lower than that under kaolinite.The results proved that kaolinite exerted a direct lethal effect on strain HPD-2,whereas montmorillonite did not exhibit such a lethal effect.

Fig.2 Ex-DLVO theory for the interaction energy between Paracoccus aminovorans strain HPD-2 and clay mineral colloids of kaolinite(a)and montmorillonite(b)and scanning electron microscopy images(day 3)in the biodegradation process under treatment with kaolinite(c)and montmorillonite(d).LW=van der Waals attractive force;AB=acid-base interaction;EL=attractive or repulsive electrostatic force;Tot=LW+AB+EL.

Fig.3 Release of soluble Al from kaolinite(Kao)and montmorillonite(Mont)in mineral salt medium(a),Nano-scale secondary ion mass spectrometry images of 27Al and 12C distribution in Paracoccus aminovorans strain HPD-2 in the biodegradation process after 96h(b),and the ratio of the relative strength of 27Al and 12C(27Al/12C ratio)(c).

Inter facial interaction of clay mineral colloids with BaP

Isothermal adsorption experiments were used to visually compare the adsorption capacity of the two clay minerals on BaP.The results showed that kaolinite has stronger adsorption capacity for BaP compared with montmorillonite(Fig.4a).This may be due to the high octanol-water partition coefficient and strong hydrophobicity of BaP.The hydrophilic hard cations,e.g.,Na+,mixed into the interlayer and diffusion layer during metal ion saturation act as a barrier on the surface of montmorillonite due to their large hydration radius.Compared with the surface of kaolinite,the hydrophobic organic pollutant-BaP is not more easily adsorbed on montmorillonite.On the contrary,kaolinite is attacked by only a small amount of hydration ions,and it has low hydration characteristics than the charged mineral montmorillonite,resulting in a hydrophobic surface(Laird,1999).Therefore,kaolinite has a stronger adsorption capacity for BaP than montmorillonite(Jiaet al.,2012).Bio-accessibility is another important factor in microbial degradation(Martinset al.,2003).Our results(Fig.4b)demonstrated that kaolinite significantly reduced the bio-accessibility of BaP,when compared with montmorillonite,after aging for 15 d.Addition of kaolinite inhibited the biodegradation of BaP by strain HPD-2.In order to further explain this phenomenon,XRD spectra of inorganic colloids were analyzed before and after degradation(Fig.5).After degradation for 7 d,the crystal structure of kaolinite did not change significantly,whereas that of montmorillonite had degraded,and the specific layered structure was disordered(Fig.2c,d).Although the interlayer spacing decreases,water molecules are more likely to enter the interlayer structure,resulting in water absorption and expansion.In previous studies on the modification of montmorillonite,Na+was often used to completely replace Ca2+between layers of montmorillonite,and organic or other modification treatment was carried out to enhance the adsorption performance of montmorillonite.The interlayer spacing of kaolinite remains unchanged,much smaller than that of expanded montmorillonite.Considering the strong hydrophobicity of BaP,the absorbed montmorillonite makes it difficult for BaP molecules to enter the expanded interlayer of montmorillonite.Structural peaks of calcium phosphate(Fig.5)were also detected in the XRD spectra of montmorillonite.This is due to the Ca2+between layers of montmorillonite combined with sodium phosphate in MSMto form calcium phosphate precipitation.The BaP is a highly hydrophobic organic molecule.When montmorillonite swells,the adsorption and bio-accessibility of BaP will be reduced naturally.In order to verify this,clay minerals and BaP were added to inorganic salt medium for 24 h,and the suspension was then destabilized to form glass slides for two-photon laser confocal microscopy(TP-CLSM)observation(Fig.4c).The TP-CLSMimage was processed by image-J software,and the fluorescence intensity of the two colloids was compared.From the TP-CLSM image,kaolinite particles aggregated because of their hydrophobicity,while montmorillonite expanded by water absorption was dispersed in the inorganic salt system.Furthermore,the fluorescence intensity of BaP adsorption on kaolinite was higher than that on montmorillonite,indicating stronger adsorption of BaP on kaolinite.

Fig.4 Isothermal adsorption of benzo[a]pyrene(BaP)on kaolinite(Kao)and montmorillonite(Mont)(a),effects of these two clay minerals on BaP bioavailability(b),and observation of BaP distribution by two-photon laser confocal microscopy(c).Fluorescence intensity in Fig.4c represents the amount of BaP.

Fig.5 X-ray diffraction spectra of kaolinite(Kao,a)and montmorillonite(Mont,b)before and after the biodegradation process.Treated Kao=kaolinite after benzo[a]pyrene(BaP)biodegradation process with Paracoccus aminovorans strain HPD-2;treated Mont without and with HPD-2=montmorillonite after BaP degradation process without and with strain HPD-2,respectively.d=layer-spacing.

Effects of kaolinite and montmorillonite on pH and Eh during the biodegradation process

In order to better explain the influence of clay minerals on the degradation process of BaP,the changes in pH and Eh of the clay minerals are shown in Fig.6.As the degradation proceeds,the pH of the system increases(initial pH 7.0),whereas the Eh decreases.This may be due to the formation of alkaline substances,such as hydroxyl-phenanthrene,during the degradation process,which increases the pHof the system(Liu,2009).The change in pH was the largest in CK,where pH increased rapidly from days 1 to 3 and then exhibited a slower increase.This is because the strain HPD-2 was added to the degradation system for a period of stability,and then continued to grow,and BaP was degraded.After 3 d,nutrient consumption and harmful substances increased.The growth environment in the system was not conducive to the growth of strain HPD-2,making the viability of strain HPD-2 decline,and the degradation efficiency of BaP gradually decreased.However,the degradation efficiency of BaP with montmorillonite was the highest,but the pH maintained in a relatively stable and suitable range for microbial growth(Fig.6).This is due to the buffer effect of the amphoteric functional group Al-O-on the surface of montmorillonite on pH(Liet al.,2014).The XRD analysis showed that the crystal structure and interlayer spacing of the minerals changed after the interaction of montmorillonite with strain HPD-2,and the stacking order in the direction of the C axis decreased.When montmorillonite was added into the degradation system,it could buffer the unfavorable factors in the degradation process and promote the degradation of BaP by strain HPD-2(Ruanet al.,2018b).

Fig.6 Changes in pH and Eh during the degradation process under control treatments without any clay minerals(CK)and with kaolinite(Kao)or montmorillonite(Mont).

Inter face interaction mechanisms of k aolinite and montmo-rillonite duringdegradation of BaP by strain HPD-2

Two types of common clay minerals(montmorillonite and kaolinite)were selected and compared with CK,and the possible mechanisms of the interface effect were outlined.The specific interface effect was summarized in Fig.7.There was a huge energy barrier between kaolinite and strain HPD-2,which prevented the cells from easily adsorbing onto kaolinite colloidal particles.Kaolinite can release a certain amount of Al3+,so that hydrogen peroxide produced in the catalytic degradation system can produce low levels of persistent free radicals,which could not support the growth of cells,and has a certain inhibitory effect on cell viability.Furthermore,due to its strong hydrophobicity,kaolinite can adsorb a large number of BaP molecules(the contact angle data was shown in Table SI),which reduces the bioavailability,and subsequently the degradation of BaP.

Fig.7 Interface interaction mechanisms of montmorillonite and kaolinite during benzo[a]pyrene biodegradation by Paracoccus aminovorans strain HPD-2.

After adding montmorillonite to inorganic salt medium,Na+in the inorganic salt can completely replace Ca2+between the layers of montmorillonite layers,making water molecules enter into montmorillonite and swell water,thus weakening the adsorption of montmorillonite on BaP and increasing the effect on the bioavailability of BaP.Moreover,the crystal structure of montmorillonite was weakened after swelling water absorption.Strain HPD-2 can easily adhere to the surface of montmorillonite colloidal particles due to the lower energy barrier,and montmorillonite does not exhibit bacteriostasis,allowing cells to grow on its surface to form a biofilm.The investigation of environmental factors showed that montmorillonite had a buffering effect on the pH of the system during degradation,which maintained the viability of strain HPD-2 and promoted BaP degradation by microorganisms.

CONCLUSIONS

The BaP degradation efficiency was significantly higher 7 d after montmorillonite addition,reaching 68.9%(P＜0.05),when compared with that of the control without addition of clay(61.4%).Conversely,kaolinite addition significantly reduced the BaP degradation efficiency,yielding a value of 45.8%.Addition of kaolinite markedly inhibited BaP degradation by strain HPD-2 from two sides:i)inhibiting the growth of strain HPD-2 and ii)decreasing the bio-accessibility of BaP to strain HPD-2,which may be attributed to the strong hydrophobicity and easy agglomeration of kaolinite in the degradation system.The amphoteric functional group Al—O—on the surface of montmorillonite can buffer some unfavorable factors in the system,intermediate harmful products,e.g.,pH,Eh,etc.,so that the environmental factors are maintained in a range suitable for the growth of microorganisms.Cells can attach on the surface of montmorillonite colloidal particles across energy barriers,thus colonizing on the mineral surface and forming biofilms.Adsorption of BaP on montmorillonite was weakened after swelling,which reduced the effect of strain HPD-2 on the bio-accessibility of BaP,thus promoting the biodegradation of BaP by strain HPD-2.It could thus be concluded that the addition of montmorillonite to the degradation system can increase the degradation of BaP by HPD-2 strain and enhance the potential of BaP biodegradation.This work provided an enhanced understanding of the interface mechanisms influencing clay minerals in the biodegradation of organic pollutants by degrading bacteria and had a certain theoretical guiding significance for improving the biodegradation of soil pollutants by soil microorganisms under field conditions.

ACKNOWLEDGEMENT

This study was supported by the grants from Chinese Academy of Sciences(CAS)Key Laboratory of Soil Environment and Pollution Remediation,Institute of Soil Science,CAS,the Natural Science Foundation of Jiangsu Province,China(No.BK20150049),and the Chinese National Key Research and Development Program(Nos.2017YFA0207001 and 2019YFC1803700).

SUPPLEMENTARY MATERIAL

Supplementary material for this article can be found in the online version.