Microbial Mineralization of Montmorillonite in Low-Permeability Oil Reservoirs for Microbial Enhanced OilRecovery

Kai Cui,a Shanshan Sun,a Meng Xiao,b Tongjing Liu,c Quanshu Xu,a Honghong Dong,a Di Wang,a Yejing Gong,a Te Sha,a

Jirui Hou,c Zhongzhi Zhang,a Pengcheng Fud

aState Key Laboratory of Heavy Oil Processing, China University of Petroleum, Beijing, People's Republic ofChina

bCollege of Chemical Engineering, Qingdao University of Science and Technology, Qingdao, People's Republicof China

cResearch Institute of Enhanced Oil Recovery, China University of Petroleum, Beijing, People's Republic ofChina

dState Key Laboratory of Marine Resource Utilization in South China Sea, Hainan University, Hainan, People'sRepublic of China

ABSTRACT Microbial mineralization (corrosion, decomposition, and weathering) hasbeen investigated for its role in the extraction and recovery of metals from ores. Herewe report our application of biomineralization for the microbial enhanced oil recov-ery in low-permeability oil reservoirs. It aimed to reveal the etching mechanismof the four Fe(III)-reducing microbial strains under anaerobic growth conditionson Ca-montmorillonite. The mineralogical characterization of Ca-montmorillonitewas performed by Fourier transform infrared spectroscopy, X-ray powder diffraction,scanning electron microscopy, and energy-dispersive spectrometry. Results showedthat the microbial strains could efficiently reduce Fe(III) at an optimal rate of 71%,alter the crystal lattice structure of the lamella to promote interlayer cation ex-change, and efficiently inhibit Ca-montmorillonite swelling at a rate of 48.9%.

IMPORTANCE Microbial mineralization is ubiquitous in the natural environment. Mi-crobes in low-permeability reservoirs are able to facilitate alteration of the structure andphase of the Fe-poor minerals by reducing Fe(III) and inhibiting clay swelling, which isstill poorly studied. This study aimed to reveal the interaction mechanism betweenFe(III)-reducing bacterial strains and Ca-montmorillonite under anaerobic conditions andto investigate the extent and rates of Fe(III) reduction and phase changes with their ac-tivities. Application of Fe(III)-reducing bacteria will provide a new way to inhibit clayswelling, to elevate reservoir permeability, and to reduce pore throat resistance after wa-ter flooding for enhanced oil recovery in low-permeability reservoirs.

KEYWORDS Ca-montmorillonite, Fe(III)-reducing bacteria, microbial mineralization,clay mineral swelling, low-permeability oil reservoirs

Many oil reservoirs worldwide are increasingly depleted every year. These reservoirsfeature low stratum pressure, low permeability, low single well production, and

increased water absorption, which have made further crude oil production moredifficult. Low-permeability reservoirs mainly contain montmorillonite, illite, mixedmontmorillonite/illite, chlorite, and other irregular clay minerals. Among these, mont-morillonite and its mixed layer of minerals may swell with water contents, while illiteand chlorite barely swell. The clay swelling would significantly decrease the reservoirporosity, permeability, and oil recovery in low-permeability reservoirs. There is thus a

Received 22 January 2018 Accepted 21 April2018

Accepted manuscript posted online 11 May2018

Citation Cui K, Sun S, Xiao M, Liu T, Xu Q, DongH, Wang D, Gong Y, Sha T, Hou J, Zhang Z, Fu P.2018. Microbial mineralization ofmontmorillonite in low-permeability oilreservoirs for microbial enhanced oil recovery.Appl Environ Microbiol 84:e00176-18. https://doi.org/10.1128/AEM.00176-18.

Editor Shuang-Jiang Liu, Chinese Academy ofSciences

Copyright © 2018 American Society forMicrobiology. All Rights Reserved.

Address correspondence to Shanshan Sun,sssun33@163.com, or Pengcheng Fu,pcfu@hainu.edu.cn.

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need to develop efficient strategies to inhibit montmorillonite swelling for enhancedreservoir permeability and oil recovery when water flooding exists.

A combination of well arrangement, optimization of water flooding, fracturing, andacidizing is used in low-permeability oilfields (1). Cationic polymers and quaternaryammonium salt are major chemical agents to inhibit clay swelling in low-permeabilityreservoirs. Despite their inhibitory or shrinking effect on montmorillonite in reservoirs,these chemical agents can cause permanent damage to reservoirs and induce adverseeffects on the ecological environment. These technologies are also laborious and entailhigh energetic, economic, and environmental costs (2). In addition, the injection ofchemical agents into low-permeability formations is somewhat challenging and mayproduce secondary environmental pollution. In recent years, the interaction betweenmicrobes and montmorillonite minerals has become a hot spot in the research com-munity of geological biology (3). Microbial enhanced oil recovery (MEOR) is an envi-ronmentally friendly tertiary recovery method that involves the use of microbialcommunities and their metabolic products, including biogas, biosurfactants, biomass,and acids, to extend the production life of oil wells (4–6). Indigenous Fe(III)-reducingmicrobes, such as Shewanella (7), Bacillus (8), Thermoanaerobacter ethanolicus (9), andDesulfovibrio (10), which are highly adaptable to the oil reservoir environment, arewidely used in MEOR (6). These bacteria alter the mineral structure in clay and convertmontmorillonite to illite or other secondary minerals for inhibition of clay swelling inlow-permeability reservoirs.

At present, the mechanism of Fe(III) reduction in low-permeability oil reservoirs isstill poorly understood. By definition, microbial dissimilatory Fe(III) reduction refers tothe process of oxidizing organic substance under anaerobic conditions with Fe(III) asthe terminal electron acceptor. The relevant literature (11) reviewed three kinds ofpossible mechanisms of microbial enzyme to catalyze dissimilatory Fe(III) reduction andtheir molecular regulation by the interaction among the microbes, Fe(III) oxides,siderophores, and electron-shuttling compounds. Many membrane-bound cytochromeproteins are involved in this process, resulting in a complicated regulation network.

The mineralization (corrosion, decomposition, and weathering) of montmorillonitemediated by the microbes represents an emerging opportunity for researchers todesign innovative solutions for the problems associated with MEOR. The mineralizationactivities on montmorillonite by Fe(III) reduction are realized by the catalyzed conver-sion of montmorillonite to illite and quartz (12–14). These activities will improve theseepage environment of oil water fluid, strengthen pore flow resistance, repress clayswelling, and increase oil recovery in low-permeability reservoirs. The mechanism ofmicrobial etching on the structure and phase of Fe-poor minerals in low-permeabilityreservoirs has been poorly studied.

Our novel study involves four Fe(III)-reducing bacterial strains for the structurealteration of Ca-montmorillonite (Ca-10) in low-permeability reservoirs (Fig. 1). Themineralogical characterization of the microbial cell metabolism on the minerals wasperformed using Fourier transform infrared spectroscopy (FTIR), X-ray powder diffrac-tion (XRD), scanning electron microscopy (SEM), and energy-dispersive spectrometry(EDS). It established the interaction between microbes and reservoir minerals from theperspective of biochemistry to improve oil recovery in low-permeability reservoirs.

RESULTS AND DISCUSSIONFe(III) reduction by bacteria. (i) Changes in pH and total protein content. The

physiological activities of the four Fe(III)-reducing bacterial strains—CA, KB, TC, andTD—were measured on the basis of their acid production and the total protein content(15). As shown in Fig. 2a, the pH in the control remained at about 6.9 during the wholeexperimental period, while the pHs of CA, KB, TC, and TD dropped from the initial pHof 6.9 to about 5.8, 4.9, 5.9, and 4.3, respectively. Our measurements have shown thatthe four Fe(III)-reducing bacterial strains could produce organic acids, such as aceticacid, propionic acid, butyric acid, isobutyric acid, and valeric acid (data not shown),which caused the decrease in pH (16). The growth of CA, KB, TC, and TD in acidic

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reservoir environments enables their Ca-10 dissolution to enhance the contact betweenmicrobial cells and Ca-10 surface area and to promote Fe(III) reduction (17). It was foundthat due to elevated oxidation reduction potential in the acidic environment of thereservoir, Fe(III)-reducing bacteria regenerate ferric iron to provide more energy tosustain their growth for increased pore diameter and improved clay permeability.

As shown in Fig. 2b, the total protein in the control remained at about 5.5 mg/literthroughout the whole experimental period. The total protein changes in the experi-mental group can be divided into two stages. At stage 1, the total protein concentra-tion increased rapidly due to the activities of CA, KB, TC, and TD under anaerobicgrowth conditions after inoculation (Fig. 2b). At stage 2, growth of CA, KB, TC, and TDupon the consumption of nutrients and Fe(III) from Ca-10 reached the stable state andthen gradually began to decline, eventually reaching steady state. This phenomenonmay be attributed to bacterial overpopulation, resulting in the lack of space andnutrition (18). From the fifth day to the end of the experiment, the total proteinconcentrations for the four bacterial strains were maintained at 31.5, 33.2, 28.3, and21.2 mg/liter (Fig. 2b).

(ii) Fe(III) reduction and Fe(II) production during Ca-10 alteration. CA, KB, TC,and TD used Fe(III) in the Ca-10 as their electronic receptor to reduce it to Fe(II) in the

FIG 1 Schematic diagram of Ca-montmorillonite phase transformation.

FIG 2 Changes of pH (a) and total protein concentration (b) in the system.

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etching experiment. Fe(II) accumulated in the solution as ions. It was then taken up bythe microorganisms to provide the energy required for their cell metabolism (19).Therefore, the concentrations of Fe(II) and Fe(III) in the suspension were used toevaluate the degree and efficiency of Fe(III) reduction in Ca-10 by CA, KB, TC, and TD.

The concentrations of Fe(II) and Fe(III) in the growth media were measured todetermine the change of Fe(III) content in Ca-10 by microbial erosion versus time (Fig.3a), a similar comparison was made for the reduction of the generated Fe(II) concen-tration (Fig. 3b), ratio of Fe(II) to Fe(III) (Fig. 3c), and ratio of Fe(II) to total iron (Fig. 3d).The Fe(III) and Fe(II) concentrations in the control were measured as 5.8 and 9.1mg/liter, respectively, which may be due to the contents of Fe(II) and Fe(III) ions in theCa-10 during the experimental period (Fig. 3a and b).

After addition of the four bacterial strains into the reactors, the concentration profileof Fe(III) and Fe(II) was divided into two stages. At the first stage, Fe(III) and Fe(II) wererapidly increased from the lower initial concentration. The highest Fe(III) concentrationin the dissolved and reduced Ca-10 and the maximum Fe(II) concentration wereobtained at the 4th day (Fig. 3b). It was seen that the maximum Fe(II) concentrationappeared at the same time as that of the total protein, indicating that the reductionrate of Fe(III) in Ca-10 was closely related to the enzymatic activities of the bacterialstrains. At the second stage, the Fe(III) accumulation rate and the Fe(II) reduction ratedecreased gradually, with the concentrations of Fe(III) stabilizing at 225.7, 322.5, 301.2,and 233.8 mg/liter, respectively, while Fe(II) reductions were at 103.8, 215.8, 263.6, and203.5 mg/liter, respectively. In addition, it was found that the production of Fe(II) inCa-10 via the Fe(III)-reducing bacteria was positively correlated with the reduction ofFe(III), indicating that the cumulative Fe(II) is mainly produced from the free state offerric ion in the reaction system (20).

From Fig. 3c, it is evident that the rates of reduction of Fe(III) in Ca-10 for TC and TD(87.1% and 86.8%, respectively) were higher than those of CA and KB. However, the

FIG 3 Changes of the extent of Fe(II) and the reduction rate of Fe(II) in the system. (a) Fe(III) concentration;(b) Fe(II) concentration; (c) ratio of Fe(II) to Fe(III); (d) ratio of Fe(II) to total Fe.

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microbial reduction time of Fe(III) in Ca-10 by CA and KB was short, and the Fe(III)reduction rates (68.1% and 80.3% by the two microbes, respectively) were reached by48 h; so were the maximum amounts of protein produced by CA and KB (Fig. 2b). Inaddition, as shown in Fig. 3d, the reduction rates of total Fe(III) in Ca-10 graduallydecreased and finally stabilized, reaching the highest levels— 47.1%, 62.9%, 67.1%, and71.1%—at the 4th day. These values were higher than the iron reduction rates ofShewanella putrefaciens CN32 (19% to 40%) and Thermus scotoductus (32% to 44%) inthe literature (21). In addition, the peak Fe(III) reduction rates in Ca-10 by CA, KB, TC,and TD were in accordance with the maximum enzymatic activities of the four microbialstrains. These results suggest that the production and concentration of Fe(II) are closelyrelated to the activities of CA, KB, TC, and TD.

Characteristics of Ca-10 before and after treatment. (i) FTIR analysis of chem-ical bond vibration. Comparing with the infrared spectra of Ca-10 before and afterpitting by the four Fe(III)-reducing bacterial strains, we observed similar characteristicpeak distributions, both for the Ca-10 samples and the control, corresponding to thechemical bond vibration in the crystal structure. After the reduction of Fe(III), thereappeared to be no change in the absorption bands, while there was a slight reductionin intensity (Fig. 4). A previous study (22) has shown that the FTIR spectrum of Ca-10 canbe broken down into four main regions spanning from 4,000 to 400 wavenumbers (percentimeter), that is, 3,800 to 3,200, 1,800 to 1,400, 1,400 to 1,000, and 950 to 700 cm�1

(23). This absorption peak range is characterized to be the �M-OH (M � Mg, Al, or Fe)stretching bands (24), the O-H deformed vibrating belt, the Si-O stretching vibrationzone, and the �M-OH deformation vibration band in the octahedron, respectively, inwhich Si-O peak is a typical characteristic peak of silicate minerals (25).

As shown in Fig. 4, the intensity of the �M-OH stretching vibration at 3,750 cm�1

was sharply weakened after the erosion of Ca-10 by CA, KB, TC, and TD. The O-Hstretching vibration peaks at 3,460 and 3,320 cm�1 were slightly shifted (Fig. 4c and d)

FIG 4 FTIR changes of Ca-10 before and after reduction by strains CA (a), KB (b), TC (c), and TD (d).

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or disappeared (Fig. 4a and b) after the reduction of Ca-10. The water peak at 3,560cm�1 was weakened, due to the loss of crystal water in Ca-10 causing hydrationswelling. The figure shows also that CA and KB became weakened in the �M-OHstretching zone (Fig. 4a and b), which was correlated with the high Fe(III) reduction ratein CA and KB in the experimental period.

In Fig. 4, the O-H deformed vibrational peaks were significantly attenuated between1,570 and 1,360 cm�1 after Ca-10 reduction; this peak disappeared at 1,360 cm�1 dueto the action of KB (Fig. 4b), indicating that the microbes impacted the distribution ofO-H and interlayer H2O in Ca-10. At the same time, the Si-O peaks at 1,120 and 1,043cm�1 were reduced substantially after the microbial mineralization. The results de-scribed above indicate that the bacterial reduction increased the susceptibility ofmineral dissolution, changed in short-range order within the clay crystal, and alteredthe vibrational energies of structural O-H and Si-O (26). This shift to a lower frequencywas consistent with previous studies of bacterially reduced smectites (24). Thus, weobserved changes in the relative positions and the intensities of the parallel layerstretching vibration and vertical layer stretching vibration of Si-O in the crystal structureof Ca-10, as well as changes in the vertical and horizontal vibrations of the vertical layer.

Furthermore, it is evident from Fig. 4 that the symmetry of the silicon tetrahedronand the strength of the Si-O in the Ca-10 crystal structure were altered, with theintensification of Si-O telescopic vibration zone observed for TC and TD cultures. Ingeneral, the shift of the structural OH and Si-O vibration, as well as the loss of intensityof OH groups, indicates that the bonding and/or symmetry properties in the octahedraland tetrahedral sheets change as Fe(III) is reduced to Fe(II). This observation may beinterpreted to mean that as Fe reduction has modified the crystal structures ofFe-bearing smectites, the overall effects are modest and of about the same extent as fordithionite at similar levels of reduction.

For Fig. 4, it should be noted that some �M-OH deformation bands (M � Mg, Al, orFe) at 700 to 950 cm�1 in FTIR spectra were present, which is characteristic of thevibrations of octahedral cations. In addition, there were strong bands observed at 746,812, 845, and 924 cm�1 in the M-OH deformation region, which could be assigned toFeMg-OH, FeFe-OH, AlMg-OH, and AlFe-OH, respectively (27). Moreover, the peakscorresponding to the period after reduction were obviously shortened, indicating thatthe lattice structure of Ca-10 had shrunk. In summary, the oxidation state of Fe insmectite clay minerals results in significantly altered clay structure, including decreasein structural OH content, migration of Fe from cis- to trans-octahedral sites, formationof trioctahedral domains, and defect sites within the octahedral sheet.

In addition, the absorption peaks at 2,892 and 2,364 cm�1 after microbial mineral-ization are seen to be in accordance with the stretching vibration of lipid C-H (28). Thismay be due to the certain organic products adsorbed on the mineral surface. Theseresults showed that the chemical bond vibration of Ca-10 was altered after the mineraletching by CA, KB, TC, and TD at the cost of Si and/or O atom disassociation around theFe element, which also altered the structural Si-O vibrational and nuclear energies. Thisphenomenon resulted in the collapse of the silicon tetrahedron and the aluminumoxide octahedral framework in Ca-10 and led to the deformation of the overall crystalstructure. Additionally, the sensitivity of Al sites to the change in the Fe sites isconsistent with the change in the band at 3,560 cm�1 observed in the OH stretchingregion. It indicates that Fe reduction altered the structure of non-Fe octahedral sites.

(ii) XRD analysis of mineralogical composition. The mineralogical compositionand morphological characteristics of the solid phases, as well as the effects of thebacteria and their products in Ca-10, were investigated by XRD (29–32) for changes ofFe(III) in the form of octahedral and tetrahedral structures caused by the erosion by thefour bacterial strains in Ca-10 (Fig. 5). The resultant XRD data were converted withOrigin software to obtain a diffraction peak spectrum.

In Fig. 5, it can be seen that the d(001) (the interlayer spacing of clay minerals) peaksin Ca-10 were shifted from 14.486 to 11.861, 11.827, 11.7825, and 11.7369 Å during

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structural Fe(III) reduction via the pitting of CA, KB, TC, and TD, respectively. This shiftled to both the decrease in peak intensity and the increase in half-peak width. Thisphenomenon indicated that the crystal structure of Ca-10 was deformed, the crystalproperties changed, and the crystallinity was reduced. The decrease in interlayerspacing also increases the thickness of smectite particles, raising the number of layersper packet from 2 to 4 to more than 50 (33) in bioreduced smectite. This transformationclearly impacts particle size and settling properties of the clay, as well as permeabilityand hydraulic conductivity (34). Additionally, it suggested that the mechanism of thenewly formed illite was closely related to the bioreduction, also implying the reductivedissolution of Ca-10 and the simultaneous precipitation of illite.

In Fig. 5, it can be seen that the peaks at the same time points appeared at d(003)

of 11.01 Å (2� � 8.07°), d(003) of 10.776 Å (2� � 8.23°), d(004) of 10.52 Å (2� � 8.42°),d(004) of 10.28 Å (2� � 8.62°), and d(004) of 10.09 Å (2� � 8.776°). The layer spacingof these peaks was similar to that of illite (10 Å), indicating that a small amount ofCa-10 was converted into illite minerals by microbial mineralization. According tothese phenomena, we found that the crystal size distribution (CSD) of neoformedillite phase is different from that of Ca-10 by microbial reduction. In addition, twoadditional peaks at d(005) of 7.561 Å (2� � 11.638°) (Fig. 5a) and d(005) of 6.413 Å(2� � 11.771°) (Fig. 5d) were observed, which were possibly the Fe(II) formed duringFe(III) reduction in Ca-10. It has been observed that ferrous ions are able to combinewith Na�, CO3

2�, and PO43�, as well as potassium phosphate to form the siderite

[Na3Fe(PO4)(CO3)] (35) and phosphorous ore (FeHPO4) (Fe2� � HPO42�¡FeHPO4)

(36). These results agree with previous studies on minerals such as illite (31),siderite, and carbonate minerals in the literature (32).

In Fig. 5, there appear to be some peaks in the spectra at d(006) of 4.511 Å (2� �

19.96°), d(007) of 3.419 Å (2� � 26.77°), d(008) of 2.923 Å (2� � 31.789°), and d(008) of 2.692Å (2� � 35.12°), with an enhanced peak intensity. This indicates that the crystal structure of

FIG 5 XRD changes of Ca-10 samples before and after reduction by strains CA (a), KB (b), TC (c), andTD (d).

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Fe(III) in Ca-10 octahedral and tetrahedral lamellae was etched by CA, KB, TC, and TD,resulting in its structural deformation. Some of silicon tetrahedron and aluminum oxideoctahedron layers in Ca-10 have even been eroded to form small d-value minerals, such asquartz, calcite, calcite, and dolomite (37). Conversely, the formation of calcite was unex-pected considering the potentially abundant Fe(II) during iron dissolution in the solution,which should yield Fe-rich carbonate before calcium carbonate precipitates (31).

(iii) SEM analysis of morphology changes. The surface morphology of the Ca-10samples was observed by SEM. As shown in Fig. 6a, SEM revealed that the abioticcontrol did not undergo any mineralogical changes, displaying a typical lamellarstructure and similar flaky texture and uniform distribution.

In comparison, the surface of Ca-10 after the mineralization by CA and TC becamedissipated and the large area of the lamellar structure collapsed or disappeared (Fig. 6band d). It is interpreted that Fe(III) reduction in Ca-10 by CA enabled direct contact

FIG 6 SEM images of Ca-10 samples before and after reduction by strains CA (b), KB (c), TC (d), and TD(e), with untreated Ca-10 (a) as a control. The red squares correspond to the EDS scan area in thecorresponding panels in Fig. 7.

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between the cell membrane and the clay surface areas, leading to the production of achemical chelating agent (38). We further hypothesized that the erosion of CA may becaused by a close association between bacterial cells and biofilm which has beenreported elsewhere (39, 40), which enables the enzymatic mediation of electrontransfer to Fe(III) in Ca-10 (Fig. 6b) (41). Similarly, TC may erode and reduce Fe(III) inCa-10 by its cell attachment through the extracellular polymer, using nanowire as theelectron-chelating agent and electron acceptors as the electron transfer for reduction(42). The above assumption was supported by the fact that when using structural Fe(III)as the sole electron acceptor, cells may boost Fe reduction by direct contact with theclay mineral or by releasing organic acids or chelators that facilitate solubilizationof the structural Fe(III) (32, 43). In addition, a decrease in specific surface area and theassociated collapse of superimposed smectite layers clearly will also affect otherimportant clay mineral properties. As the layers collapse, a fraction of the interlayercations also likely will become less exchangeable.

As shown in Fig. 6c and e, the surface of Ca-10 treated with KB and TD shows pittingthat is distorted into loose porous flocs. The surface structure and electrical propertiesof the microorganisms allowed their adherence to the mineral surface (44), generatinga large number of corrosion pits and causing significant corrosion. These corroded pitswould not only increase the contact surface area between the bacteria and Ca-10 (45)but also reduce Ca-10 crystallization. This would promote the Fe(III) reduction, causeserious deformation of the mineral structure, and increase the binding sites for thegeneration of secondary minerals. Figure 6b to e show that erosion broke the chargebalance between the trivalent iron ion and other cations, changed the bond pricestructure and balance among Si, Al, and Fe, and caused the collapse of the silicontetrahedron and aluminum oxide octahedral frame and destruction of the overallcrystal structure, thus enabling the formation of illite and other nonswelling secondaryminerals (46).

(iv) Analysis of the element contents by EDS. To explain in depth the changes inthe element contents in Ca-10 before and after the reduction of bacteria, EDS wasperformed for the Ca-10 samples originating from SEM (Fig. 6).

As shown in Fig. 7, the Si and Al contents in Ca-10 minerals were drastically reduceddue to the microbial mineralization of CA, KB, TC, and TD. It is shown that the Si/Al massratios decreased from 3.04 to 2.67, 1.96, 1.88, and 1.75 and the Fe(III) contents (by masspercent) were reduced from the initial 2.29% to 1.17%, 0.94%, 0.85%, and 0.66%,respectively. These results were consistent with the reduction efficiency of Fe(III) inCa-10, and excess Fe from the reductive dissolution of Ca-10 resulted in the precipita-tion of minerals as by-products. It seems that the decrease in Si, Al, and Fe contents inCa-10 with the aid of CA, KB, TC, and TD caused the distortion and collapse of the silicontetrahedron and aluminum oxide octahedral crystal in the structure in Ca-10 (47). It isfurther deduced that a large amount of Si, Al, and Fe elements was released (suchelements will support bacterial growth in the depletion of nutrients and vitamins) bymicrobial activities and the new minerals were formed. The distortion and collapse ofcrystal structure of Ca-10 thus triggered its conversion into secondary minerals, such asillite and calcite, which reduced hydration expansion.

In addition, it was observed that the P and K contents of Ca-10 were increased. Onone hand, the increase in P content was mainly due to the formation of blue iron orevia the reaction of the phosphate and Fe(II) in the medium (48), which was consistentwith the appearance of blue iron ore in XRD. On the other hand, the increases in Kcontent (by mass percent) from the initial 0.12% to 1.99%, 3.02%, 1.3%, and 3.46%,respectively, might be caused by the exchange of K� and Na� in Ca-10. Additionally,when the positive charge in the octahedral sheet was decreased as a result of reductionof Fe(III), K was taken into the interlayers to balance the charge and for the formationof illite, which was similar to the results of previous studies (31). The bioreductionincreased the cation exchange capacity in Ca-10 (49) and triggered the replacement ofintermolecular Na� by K� in the culture medium.

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Finally, the conversion of montmorillonite to illite and other secondary minerals wasconfirmed by the data shown in Fig. 6 and 7. At first, the microbes reduced themontmorillonite octahedron and tetrahedral structure of Fe(III) by cell metabolism,which promoted the substitution of other ions in the lattice to increase the negativecharge between the layers and allow the entrance of K� into the interlayer structure.Then the smectite-illite transformation occurred and subsequently formed illite andother secondary minerals. Finally, the microbial erosion of Fe(III) in montmorillonite, themicrobial erosion of Fe(III) in montmorillonite caused the distortion and collapse of thesilica tetrahedron and aluminum oxide octahedron structure and released a largeamount of Si and Al and other elements in montmorillonite crystals. These cations wererecrystallized from K� in the culture medium to form the small particles of illite andother secondary minerals.

Changes of swelling properties of Ca-10. The swell inhibition efficiency of Ca-10was determined by the static test method in accordance with the Chinese NationalStandard (SY/T5613-2000) (50). The Ca-10 samples were measured in both water andkerosene before and after microbial reduction by CA, KB, TC, and TD. The differences inthe swelling volume in the blank versus test groups for 24 h were compared and areshown in Table 1. It can be seen that CA, KB, TC, and TD displayed a high efficiency ofinhibition for Ca-10 swelling, while the Fe(III) in Ca-10 was successfully reduced bybacterial mineralization. Among the four bacterial strains, TD showed the best effect,

FIG 7 EDS changes of Ca-10 samples (corresponding to Fig. 6) before and after reduction by strains CA(b), KB (c), TC (d), and TD (e), with untreated Ca-10 (a) as a control.

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with optimal inhibitory rates of 48.9% in water and 92.6% in kerosene, respectively. Theresults showed that the swelling volume of Ca-10 was significantly reduced; in partic-ular, it was of a low degree in kerosene due to Fe(III) reduction in low-permeabilityreservoirs to change the crystalline structure. The process resulted in the mixed layer ofmontmorillonite-illite in minerals or illite and other secondary minerals. The inhibitoryrate was similar to that of the chemical antiswelling agent used in oilfields as describedin the literature (51).

Conclusions. Microbial erosion by four Fe(III)-reducing bacterial strains on theFe-poor Ca-10 was carried out under anaerobic conditions in this study. It was observedthat the microbes could secret organic acids to etch Ca-10 clay for the deformation ofthe surface area to promote Fe(III) reduction, with an optimal reduction rate of71%. The microbial mineralization changed the surface morphology of Ca-10 andinduced the distortion of the lattice and the structure for enhanced interlayer cationexchanges to decrease the interlayer space for reduced clay swelling.

In addition, mechanism underlying the conversion of montmorillonite to illite andother secondary minerals was found. First, the microbial reduction of montmorilloniteoctahedron and tetrahedral structure of the Fe(III) would promote the substitution ofother ions in the lattice to increase the negative charge between the layers and theprocess of K� entering the interlayer structure. This would enhance the smectite-illitemixture to form the illite and other secondary minerals. Second, the microbial reductionof montmorillonite Fe(III) would cause the structure of the silica tetrahedron andaluminum oxide octahedron to be distorted and collapse to release a large amount ofSi and Al, as well as other elements in montmorillonite crystals. As a result, these cationswere recrystallized from K� in the culture medium to form the small particles of illiteand other secondary minerals. Finally, the resultant inhibition efficiency for the Ca-montmorillonite swelling showed an optimal inhibitory rate of 48.9%.

MATERIALS AND METHODSBacteria and clay minerals. The microbes were isolated from samples of (i) production fluids in the

Da Qing oilfield in China, (ii) a circulating drilling fluid collected at a 1,000-m depth in the Chinesecontinental deep drilling, (iii) production fluid from the Liao He oilfield in China, and (iv) production fluidfrom the Da Gang oilfield (52). They were identified by 16S rRNA gene analysis to exhibit a similarity of98% to 99% to Enterobacter aerogenes (NCBI accession number EFJ63628.1), 99% to Pseudomonas (NCBIaccession number KPW20975.1), 98% to 99% to Bacillus cereus (NCBI accession number COB27440.1), and99% to Proteus (NCBI accession number OSB73291.1), respectively, and are referred to here as CA, KB, TC,and TD.

The experimental protocol was as follows. Ten milliliters of production fluids and 90 ml of iron-reducing bacteria enrichment medium were mixed and kept for 5 to 7 days under anaerobic conditionsat 35°C. When the color of the enrichment medium changed from yellow to light green or the mediumbecame colorless, 2.0 ml of the mixture was transferred to a fresh iron-reducing enrichment medium for3 times. It was then stretched on the petri dish to pick a single colony. Iron-reducing bacterial enrichmentmedium was made based on the modification of the medium in the literature (53) as follows:

TABLE 1 Swelling properties of Ca-10 samples in water and kerosene before and afterreduction by CA, KB, TC, and TDa

Strain Solution

Swelling vol (ml)at:

Difference inswelling vol (ml)

Inhibition ofCa-10 (%)0 h 24 h

None (blank) Water 0.50 0.96 0.46 0CA Water 0.64 0.935 0.295 35.87KB Water 0.65 0.925 0.275 40.22TC Water 0.65 0.90 0.25 45.65TD Water 0.68 0.915 0.235 48.91None (blank) Kerosene 0.494 0.63 0.136 0CA Kerosene 0.46 0.48 0.02 85.29KB Kerosene 0.45 0.465 0.018 86.76TC Kerosene 0.43 0.448 0.015 88.97TD Kerosene 0.45 0.46 0.01 92.65aCa-10 was used at 0.1 g.

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C6H12O6·H2O, 10 g/liter; NH4Cl, 1.0 g/liter; KH2PO4, 0.25 g/liter; K2HPO4, 0.72 g/liter; MgSO4·7H2O, 0.5g/liter; CaCl2, 0.1 g/liter; FeC6H5O7·5H2O, 3.3 g/liter; and trace element solution, 1.0 ml.

Ca-10 clay was purchased from the Jian Qing District of Liao Ning Province in China. The total Fecontent in Ca-10 is 3.7%. The chemical composition of Ca-10 as determined by electron microprobeanalysis was (Ca0.19Na0.17K0.08)0.44(Al1.36Mg0.32Fe0.26)1.94(Si3.87Al0.13)4.0 O10 (OH) 2.0·nH2O. The Ca-10 claywas thoroughly soaked and slightly ground or sonicated in an ultrasonic water bath to disperse clayaggregates but not to break individual particles. Bulk clay was size fractionated at 0.5 to 2.0 �m. Afterdrying at 80°C for 2 h, the size fractions of Ca-10 were ready for experiments.

Interaction of Fe(III)-reducing bacteria and clay minerals. The microbial Fe(III) reduction on Ca-10was carried out under anaerobic conditions. Sucrose was used as the main carbon source, sodium acetateas the electron donor, and the Fe(III) in Ca-10 as the final electron acceptor in the culture medium.Growth medium consisted of (per liter of deionized water) sucrose (10 g), NaCl (5 g), NH4Cl (2 g), KH2PO4

(1.4 g), K2HPO4 (3.7 g), MgSO4·7H2O (0.5 g), CaCl2 (0.07 g), NaCH3COOH (10 mM), yeast extract (0.5 g),vitamin solution (1.0 ml), trace element solution (1.0 ml), and 0.1% resazurin (redox indicator; 1.0 ml) (53).The final pH of the medium was adjusted to 7.0 with 0.1 N NaOH. The medium was made anoxic in125-ml serum bottles with O2-free N2-CO2 gas mix (80:20) via passage through a sterile needle. Thebottles were sealed with thick butyl rubber stoppers and then sterilized via autoclaving at 121°C for 15min. The serum bottles contained 0.1 g of Ca-10 and 80 ml of culture medium [CFe(III) � 404 mg/liter][CFe(III) refers to the concentration of total Fe(III) from Ca-10 in the reaction system]. Before inoculation,the bottles were stored at 65°C overnight to allow the sulfur functional group of the yeast extract toremove any residual O2. The experimental group was inoculated in LB medium (10 g/liter of peptone, 10g/liter of yeast powder, and 10 g/liter of NaCl) containing enrichment cultures of CA, KB, TC, and TD for36 h under anaerobic conditions. The final concentration of 107 to 108 cells/ml (54) was based on acridineorange direct counts, whereby acridine orange was injected into the tubes in the experimental groupand the blank group (inoculated with the same amount without bacterial medium). The tubes wereincubated at 35°C with shaking at 150 rpm for 10 days. To obtain reliable data, all treatments wereperformed in duplicate. All solutions and cultures were transferred by using sterile needles and syringes.

Analysis of fluid composition and characterization of Ca-10. Analysis of fluid chemistry includedpH, total protein content, and Fe(II) and Fe(III) concentrations. First, 3.0 ml of the cell-mineral mixedsuspensions was taken from the anaerobic console each day, 0.5 ml of which was collected using a sterilesyringe. It was added to plastic tubes containing 0.1 ml of 5.5 M NaOH, and then 0.5 ml of 10%physiological saline was added. The mixture was lysed in boiling water for 10 min, cooled to roomtemperature, and then centrifuged. The total protein was determined in accordance with the modifiedCoomassie brilliant blue method. Absorbance at 595 nm was measured on a UV-2802 PC UV-visible(UV-VIS) spectrophotometer, and the total protein concentration was calibrated by absorbance. Theviability of the microbes grown in the mixture was evaluated on the basis of change in total proteincontent. Second, the extent of bacterial reduction of Ca-10 was monitored by measuring Fe(II) and Fe(III)concentrations with the use of improved 1,10-phenanthroline spectrophotometry (55). At selected timepoints, 0.5 ml of the cell-mineral mixture suspension was collected using a sterile syringe and added toplastic tubes containing 0.5 ml of 1 N HCl. The mixture suspension was allowed to stand in HCl for 24h before the measurement of Fe(II) and Fe(III) concentrations. Then 1.0 ml of the mixture suspension ofthe solution was filtered through a 0.22-�m filter and then centrifuged at 6,000 rpm for 10 min. Fe(II) andtotal Fe contents of a 0.2-ml suspension were measured. These data were used to calculate the amountof Fe(II) and Fe(III) reduction in the system. Before the remaining 1.0 ml of cell-mineral mixturesuspension was allowed to settle, the pH of the supernatant was measured in an oxygen-free glove bagto avoid artifacts resulting from the reoxidization of sulfides to sulfate.

Finally, after the mineralization was carried out for 10 days, the solid product was collected, dried at60°C, and ground in an agate mortar. FTIR and XRD were conducted to determine the changes inchemical bonds and crystal structures in Ca-10 before and after bacterial erosion. The surface morphol-ogy of the Ca-10 clay was observed by SEM and EDS for mineral chemical composition analysis.

Analytical methods. (i) FTIR. FTIR spectroscopy was used to monitor changes in chemical bondvibrations and changes in the strength of functional groups in Ca-10 mineral structures before and afterbacterial treatment. For this analysis, 200 mg of KBr and 2.0 mg of sample powder were weighed, mixed,and ground to fully disperse Ca-10 clay in KBr. The samples were analyzed in the diffuse reflectance modeusing a Perkin-Elmer Frontier infrared spectrometer. Fifty scans over the range 400 to 4,000 cm�1 witha spectral resolution of 4 cm�1 were accumulated for each spectrum.

(ii) XRD. Both control and Ca-10 samples were examined by XRD to identify the morphological andstructural changes resulting from microbial etching. Samples for XRD analysis were prepared fromspecimens that were dispersed and washed in 95% ethyl alcohol six to eight times to remove growthmedium and salts. The treated sample was dispersed in ethyl alcohol, pipetted onto circular glass slides,and dried in air at room temperature. Powder XRD patterns were collected by a Scintag X-ray powderdiffractometer using Cu-K� (� � 0.15418) radiation, a fixed slit scintillation detector, and 1,200 W ofpower (40 kV; 100 mA). XRD slides were scanned in 0.02 2� steps with a count time of 2 s per step anda scanning range of 5 to 50° 2�.

(iii) SEM and EDS. Mineralogical changes were further studied via SEM. Ca-10 samples wereprepared following a previously published procedure (29). In brief, the cell-mineral mixture suspensionswere fixed in 2.5% glutaraldehyde in a bicarbonate solution and one droplet of fixed suspension wasplaced onto the surface of a glass coverslip that was precleaned with 1.0 mg/ml of polylysine solution.Ca-10 particles were allowed to settle onto the coverslip for 15 min. The sample-coated coverslips weresequentially dehydrated using various proportions of ethanol and distilled water, followed by critical

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point drying. The coverslips were mounted onto SEM stubs and Au coated for observation using a Zeisslow-vacuum SEM instrument, which was operated at an accelerating voltage of 10 to 15 kV. The EDS(equipment model X-Max 50) provided a primary means for mineralogical identification. A certainworking distance (8.5 mm) and higher beam current (50 to 70 �A) were used for qualitative EDS.

(iv) Swell inhibitory efficiency of Ca-10. The swell inhibition efficiency of Ca-10 was determined bythe static test method in accordance with the Chinese National Standard (SY/T5613-2000) by the statictest method (50). Inhibitory efficiency was calculated using the following equation: � � [(V0 � V)/V0] �100, where V0 is the swell volume of Ca-10 without bioreduction in the reaction solution, V is the swellvolume of Ca-10 with bioreduction in the reaction solution, and � is the swell inhibition efficiency.

ACKNOWLEDGMENTSThis research was financially supported by the National Natural Science Foundation

of China (no. 51634008, 51474223) and National Science and Technology Major Project(no. 2017ZX05009-004).

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