Colloids and Surfaces B: Biointerfaces 105 (2013) 223– 229
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Colloids and Surfaces B: Biointerfaces
jou rn al h om epage: www.elsev ier .com/ locate /co lsur fb
nterobacter cloacae as biosurfactant producing bacterium: Differentiating itsffects on interfacial tension and wettability alteration Mechanisms for oilecovery during MEOR process
egah Sarafzadeha,1, Ali Zeinolabedini Hezavea,1, Moosa Ravanbakhsha,1, Ali Niazib,hahab Ayatollahia,∗,1
EOR Research Centre, Shiraz University, Shiraz, IranInstitute of Biotechnology, Shiraz University, Shiraz, Iran
r t i c l e i n f o
rticle history:eceived 6 July 2012eceived in revised form1 December 2012ccepted 17 December 2012vailable online xxx
eywords:icrobial core flooding
a b s t r a c t
Microbial enhanced oil recovery (MEOR) process utilizes microorganisms or their metabolites to mobilizethe trapped oil in the oil formation after primary and secondary oil recovery stages. MEOR technique isconsidered as more environmentally friendly and low cost process. There are several identified mecha-nisms for more oil recovery using MEOR processes however; wettability alteration and interfacial tension(IFT) reduction are the important ones. Enterobacter Cloacae, a facultative bio-surfactant producer bac-terium, was selected as a bacterial formulation due to its known performance on IFT reduction andwettability alteration. To quantify the effects of these two mechanisms, different tests including oilspreading, in situ and ex situ core flooding, wettability measurement (Amott), IFT, viscosity and pH
nterobacter Cloacaeettability alteration
nterfacial tension reductionarbonate rocks
measurements were performed. The obtained results revealed that the experimental procedure used inthis study was able to quantitatively identify the individual effects of both mechanisms on the ultimatemicrobial oil recovery. The results demonstrated considerable effects of both mechanisms on the ter-tiary oil recovery; however after a proper shut in time period, more tertiary oil was recovered becauseof wettability alteration mechanism. Finally, SEM images taken from the treated cores showed biofilmformation on the rock pore surfaces, which is responsible for rock surface wettability alteration.
. Introduction
During the first stage of oil production, natural reservoir pres-ure declines and considerable amount of the original oil in places left behind depending on the characteristics of the rocks and theuids in place. Water flooding has been used widely to maintainhe reservoir pressure, pushing more oil out of the rock toward theroducing wells. After all, more than half of the oil is trapped inhe reservoir rock after primary oil recovery and secondary (waterooding) process because of pore structure heterogeneities, flu-
ds/fluids and rock/fluids surface forces.In the past three decades, applications of new technologies
alled enhanced oil recovery (EOR) processes to produce therapped oil in the oil reservoirs to meet the increasing world’s oilemand have gained special attentions [1,2]. The most widely used
∗ Corresponding author at: Enhanced Oil Recovery Research Center, School ofhemical and Petroleum Engineering, Shiraz University, P.O. Box 71346-1719,ehran, Iran. Tel.: +98 711 6474602.
EOR techniques are namely carbon dioxide injection [3], surfactantflooding [4–6], polymer flooding [7], alkaline–surfactant–polymerflooding [8,9], steam injection and microbial enhanced oil recov-ery (MEOR) [10–13]. The results from the past field experiencesrevealed that the application of these techniques, typically leads to5–15% more oil recovery [2].
MEOR has been examined both in the laboratories and fields lessoften although it was initiated more than fifty years ago mostlybecause of the challenges remained for the acting mechanisms[14–17]. This biological based technology utilizes bio-products torecover the trapped oil with many known advantages comparedto the other techniques [18–24]. It utilizes low cost microbes andnutrition’s, being environmentally friendly and it is also inde-pendent of oil price fluctuations. Besides, the existing productionunits need only slight modifications to be used for this novelprocess. It is recently confirmed that MEOR is especially more effi-cient than current EOR processes for the carbonated reservoirs[14,15,18,19,21,25].
During MEOR processes, bacteria use reservoir crude oil orinjected nutrients for their metabolic processes and excrete somenon-toxic chemicals such as biosurfactants, biopolymers, biosol-vents, bioacids and gases [26,27]. These bio products increase
he oil sweep efficiency by changing reservoir’s physicochem-cal characteristics [27]. Biosurfactants, known as one of the
ost important bioproducts reduce the capillary forces in theeservoir rock by the reduction of oil/water interfacial ten-ion and wettability alteration [28–38]. Previous studies havehown that several effective mechanisms including interfacialension reduction [11,12,18,19,22,39–41], wettability alteration18,19,22,39–47], flow pattern variation [48,49], gas production50] and selective plugging [51] control MEOR oil recovery effi-iency. However, the recent studies have shown that IFT reductionnd wettability alteration are the most dominant mechanisms39,40,42].
Kowalewski et al. [41] have reported recently that bacterialctivities are able to both reduce the IFT and change the wett-bility. In addition, the performed simulation by Kowalewskit al. [41] demonstrated that gradual and significant reductionn IFT as well as wettability alteration was among the major
echanisms affect MEOR tertiary oil recovery. Besides, Cres-ente et al. [39] investigated the influence of two variants,urfactant producing and non-surfactant producing of Rhodococ-us sp.094 bacterium, on the efficiency of tertiary oil recovery.hey have also found that IFT reduction and wettability alter-tion were the most important active mechanisms in thatnvestigation.
Especial type of laboratory MEOR mechanism must be imple-ented to identify individual effects of different mechanisms on
ltimate oil recovery efficiency. However, because of the difficul-ies associated with MEOR process, no quantitative differentiationsetween the effective mechanisms were obtained and reported
n the literature. A simulation study by Sidsel Marie Nielsen [42]howed a rather successful separation of different mechanismsncluding IFT reduction and microscopic fluid diversion due to sur-actant production and biofilm formation, respectively. However,imulation technique used in this study adopted simplificationshich lead to low level of accuracy compared to the experimental
esults.In this study, a new injection protocol was designed to system-
tically investigate the individual effects of the IFT reduction andettability alteration on the ultimate oil recovery during in situ and
x situ microbial core flooding tests. In addition, Amott wettabil-ty tests and interfacial tension measurements as well as viscositynd pH measurements were performed to check the other effec-ive mechanisms. Enterobacter Cloacae – a facultative biosurfactantroducing strain, isolated from southern heavy oil contami-ated soil in Iran, was used as the microbial formulation in thistudy.
. Materials and methods
.1. Bacteria, brine and crude oil characteristics
E. cloacae isolated from southern heavy crude oil contaminatedoil of Iran, was carefully screened and utilized for this experimen-al study. This strain tolerates the harsh oil reservoir conditionsuch as high salinity, pressure and temperature (up to 60 ◦C) [23].esides, it can excrete large amounts of biosurfactant suitable foril enhancement purposes through IFT reduction and wettabilitylteration [39].
The used crude oil in this study was supplied from one of theouthern semi-heavy oil reservoir of Iran with the API◦ of 24.2.
Supplementary material related to this article found, in the
nline version, at http://dx.doi.org/10.1016/j.colsurfb.2012.12.042.
In addition, all the tests were carried out using the formationrine with the composition of (g/lit) MgSO4: 1.26, NaHCO3: 0.051,Cl: 0.61, NaCl: 50, MgCl2: 76, CaCl2: 9.2.
: Biointerfaces 105 (2013) 223– 229
2.2. Rock
The cores used in the core flooding tests to mimic MEOR pro-cess in the laboratory were obtained from outcrop rock samples inthe south of Iran almost from the same formation. This formationstructure was recognized to be dolomite based on the XRD testsanalysis. The used sample rocks were found to be mostly vuggy.
2.3. Bacterial medium preparation
2.3.1. Bacterial solution preparation for in situ experimentsIn the first stage, the bacterium was pre-cultured on brain
heart infusion (BHI) broth and incubated for 24 h in the rotaryshaker incubator at the temperature of 37 ◦C and shaking rate of150 rpm. Then, it was used as inoculum for cultivation on mineralsalt solution (MSSO) with following composition (g/lit) KH2PO4:2.7, K2HPO4: 13.9, NaCl: 1, NaNO3: 1, yeast extract: 0.5, carbonsource: 1. Furthermore, different carbon sources were examined inthis medium which finally leads to the selection of sun flower oilas the best carbon source. Finally, the prepared 24-h-old culturedbacterial solution was utilized for the in situ tests.
2.3.2. Crude biosurfactant solution preparation for ex situexperiments
The crude biosurfactant solution (cell-free supernatant) wasobtained by centrifuging the prepared bacterial solution at the 25 ◦Cfor 25 min with the rate of 4000 rpm.
2.4. Core flooding procedure
The core flooding apparatus used in this study was describedalready in more details in the previously published papers [23,52].In brief, after, porosity and permeability measurements, the coreplugs were placed in the core-holder. Then, the cores were satu-rated by injecting several pore volumes (PV) of brine followed by2–3 PV oil injection to mimic the original oil in place at connatewater condition.
Commonly, brine injection is utilized as the water flooding pro-cess however, in this study it was replaced by nutrition injectionin order to observe any possible effects of the nutrition and bac-terial activities on the oil recovery to eliminate any side effects ofnutrition in the subsequent steps (preparing the reference case).Then the prepared core plugs, which are at their residual oil sat-uration, were considered for MEOR process. In the next step, thebacterial medium flooding (either bacterial solution for in situ orcrude biosurfactant solution for ex situ experiments) was carriedout and continued until no further oil was produced. The amountof recovered oil in this stage was considered as first stage of micro-bial oil recovery. Then, the system was shut-in for 1-week, followedby formation brine flooding. The obtained oil recovery in this stageis known as secondary microbial recovery in this study. The ulti-mate microbial oil recovery efficiency was summation of these twoincremental oil recovery efficiencies based on the original oil inplace.
The rate of injected solutions (brine, nutrition, microbial andbiosurfactant solution) and oil phase were 0.03 and 0.05 ml/min,respectively, and the temperature was held constant at 37 ◦C. Tocheck the exact effect of this process, a control test was performedalongside the original test using similar condition without any bac-
terial colonies or bioproducts present in the injected solutions.
The point must be mentioned is that all of the core floodingexperiments were repeated at least 3 times and then the averagevalues were reported as the obtained results for each experiments.
Amott Harvey wettability test, which is considered as the vali-ated average wettability measurement for the core plugs [53] wassed to check the possible wettability changes for the microbialreated cores. This method combines two spontaneous imbibitionsnd two forced displacement measurements described as follow54]:
o = Vwsp
Vwt(1)
w = Vosp
Vot(2)
= Iw − Io = Vosp
Vot− Vwsp
Vwt(3)
here, Iw is the Amott water index, Io is the Amott oil index, Vwsp
s the displaced brine due to the spontaneous oil imbibitions, Vwt
s the overall displaced brine due to spontaneous and forced oilmbibitions. Similarly, Vosp and Vot are the displaced oil due to thepontaneous brine imbibitions and the overall displaced oil dueo spontaneous and forced brine imbibitions, respectively. In thistudy, the preliminary test revealed no uptake of crude oil into theore plugs even after long period of time (3 weeks). Therefore, theo index was assumed to be zero for the calculation of overall Amottarvey wettability index (Eq. (3)).
.6. Dynamic IFT measurement
Different techniques to measure the interfacial tension areescribed in the literature [55–57]. The dynamic behavior of
nterfacial tension for the microbial mediums and crude oil waseasured using spinning drop tensiometer (SITE 100HS, KRUSS)
58].
.7. Viscosity and pH measurements
In the present study, the viscosity of the aqueous phases andil were measured using CANNON-FENSKE type viscometer, using5 and 200 sizes for aqueous and oil phase, respectively. Besides,he pH of the solutions used in this study was measured using anccurate pH meter.
.8. Scanning electron microscopy (SEM)
Scanning electron microscopy (SEM) (S360-CAMBRIDGE) wassed to observe the possible biofilm formation for the microbialreated core plugs.
. Results and discussions
In the first stage of this study, different tests were conducted toelect the proper bacterial formulation and nutrition. Then, twoifferent core flooding experiments namely, ex situ and in situsee Table 1) along with their control tests were carried out. Thesexperiments were performed using a specific designed injectionrotocol to quantify the effects of IFT reduction and wettabilitylteration separately. Then, different tests including Amott Har-ey wettability tests, dynamic IFT, viscosity and pH measurementsere carried out to check if these two are the most dominant mech-
nisms and how their effects on oil recovery could be monitored.
.1. Bacteria screening
In the first stage of this study, proper bacteria formulation wascreened from 12 available strains. The criterion to select the proper
: Biointerfaces 105 (2013) 223– 229 225
bacteria formulation was the biosurfactant production and conse-quent IFT reduction examined by the oil spreading method [59].Among the available bacteria, E. cloacae showed the lowest IFT,thereby it was considered as the proper bacteria formulation.
Supplementary material related to this article found, in theonline version, at http://dx.doi.org/10.1016/j.colsurfb.2012.12.042.
3.2. Nutrient selection
In the next stage, MSSO was selected as the proper nutrientcompared to other nutrients e.g. nutrient broth (NB), Luria-Bertani(LB), brain hearth infusion (BHI) due to its ability to incite biosur-factant production. Subsequently, 27 different species from fourdifferent types, namely miscible and immiscible carbon sources aswell as vegetable and non-vegetable oils were utilized as the car-bon sources. The results of oil spreading tests for these nutritionand carbon sources are shown in Fig. 1 as the height of the bar forthe amounts of IFT reduction. In other words, higher the heightof the bar, the higher IFT is achieved. In addition, in Fig. 1, theparts in red and blue show the effect of bacterial activities, mainlybiosurfactant production, and the blank culture medium on ulti-mate IFT reduction, respectively. It was observed that vegetableand some non-vegetable oils lead to the maximum level of biosur-factant production, therefore, sun flower oil was selected as theefficient carbon source. This selection was based on the facts that,besides its high potential of biosurfactant production, it must bemore available and also the side effects of nutrient on the reductionof interfacial tension should be negligible.
Supplementary material related to this article found, in theonline version, at http://dx.doi.org/10.1016/j.colsurfb.2012.12.042.
3.3. Core flooding tests
In this section, the results of two different tests including in situad ex situ experiments are presented and discussed in details.
3.3.1. In situ experimentsIn the first stage, in situ experiments were carried out to inves-
tigate the effect of different mechanisms on the microbial oilrecovery. Fig. 2a presents the ultimate microbial oil recovery effi-ciency obtained for two different stages including the first period(before shut in) and second microbial recovery stage (after oneweek shut in).
The obtained results of the main test showed that using thisspecific injection protocol leads to 4.0% and 6.3% more oil recoveryfor the first and second stages based on the original oil in place(OOIP), respectively. The results clearly indicated that 4.0% increasein oil recovery was directly related to the metabolic activities ofbacterial solution replacing nutrition as the displacing medium. Inaddition, one week shut-in time resulted in 6.3% of OOIP more oilrecovery efficiency because of microbial incubation process.
Traditionally the secondary oil recovery process is referred tothe technique using brine as the flooding agent, however in thisstudy the nutrition was used instead of brine during the secondarystage to consider its effect on oil recovery for the sole measure-ments of microbial effects during the next stage.
Our hypothesis behind the possible mechanisms is that, dur-ing the first microbial recovery stage as a short period, not enoughtime is given the surface for wettability changes. Thus by consider-ing “IFT reduction” and “wettability alteration” as the dominantmechanisms, IFT reduction seems to be more dominant in thisstage. In contrast, the shut-in period allows for the wettability to be
altered in the second stage of microbial recovery. In other words, itseems that IFT reduction shows its ultimate effect in the first stageof recovery while in the second stage wettability alteration is themost important mechanism. To verify the results, different IFT and
a All of the above data are the average of at least three dependent experiments with maximum deviation of 3.2%.
micr
we3rvspit
F
Fig. 1. The obtained oil spreading results using MSSO
ettability tests were carried out to examine the suggested hypoth-sis. IFT measurements showed that the original IFT of oil/brine was2 mN/m while the IFT for oil/bacterial solutions was significantlyeduced to 2.75 mN/m after 25 min and reached to its equilibriumalue. The dynamic interfacial tension behavior of the oil/bacterialolution is shown in Fig. 3. This figure illustrates that in a time
eriod lower than 30 min the IFT reached its ultimate value which
s one sixth of the total time period needed for a core floodingest. It seems that during the first stage of oil recovery the IFT has
ig. 2. Results of core flooding experiments, (a) in situ ones, and (b) ex situ ones.
obial mediums prepared by different carbon sources.
been reduced to its final value, by which the ultimate effects of IFTreduction on the oil recovery was achieved in this stage.
Besides, the Amott wettability tests were carried out to examinethe possible wettability alteration at different stages of core flood-ing tests including the fresh rock, after the first stage and secondstage of microbial treatment processes. The obtained results tabu-lated in Table 2 show that the wettability alteration was negligibleduring the first stage of microbial recovery, compared to the sec-ond stage. Therefore, the wettability has been changed significantlyduring the second microbial recovery stage.
It can be concluded that IFT reduction and wettability alter-ation are the effective mechanisms in the first and second stages ofmicrobial recovery, respectively.
Additionally, other experiments including pH and viscositymeasurements were conducted to check their possible effectson more oil recovery. The obtained results revealed that the oiland medium viscosities were 29.79 cp and 0.90 cp for oil/blankmedium solution, while the oil and medium viscosities were
25.70 cp and 0.98 cp for oil/microbial medium, respectively. In addi-tion, the measurements showed that the oil/blank medium pHand oil/microbial medium pH were 7.27 and 7.29, respectively.The obtained results which showed no significant changes in the
Fig. 3. Dynamic interfacial tension behavior between the bacterial solution and oilused in in situ core flooding experiments.
P. Sarafzadeh et al. / Colloids and Surfaces B: Biointerfaces 105 (2013) 223– 229 227
Table 2The results of the wettability alteration during different in situ core flooding stages.
Status of tested core Spontaneous imbibitions Forced imbibitions Amott wettability indexa
In situAt the beginning of MEOR process 1.17 0.15 0.89After 1st microbial recovery 0.45 0.15 0.87After 2nd microbial recovery 1 0.48 0.67
Ex situAt the beginning of MEOR process 1.17 0.15 0.89
0.1 0.750.75 0.11
occaimtiinct
3reif
R
R
ua
ifiitIwsat
TIi
After 1st microbial recovery 0.65
After 2nd microbial recovery 0.09
a The maximum error limitation for all the above measurements is ±1.7%.
il/solutions viscosities and solutions pH, indicated that oil vis-osity reduction, bio-polymer and bio-acid production cannot beonsidered as the effective mechanisms in MEOR efficiency. Inddition, the results of monitored pressure drop during the wholenjection process indicated that plugging as one of the known
echanisms of oil recovery is not effective during these labora-ory tests. In other words, microorganisms can lead to a reductionn permeability (enhancing the pressure drop during the core flood-ng experiments) by multiplying and plugging the pores. So, sinceo significant increase in the pressure drop was observed it can beoncluded that the plugging was not a dominant mechanism duringhe core flooding experiments.
.3.1.1. Quantitative evaluation of wettability alteration and IFTeduction effects on oil recovery efficiency. In order to quantify theffects of the main mechanisms on the oil recovery efficiency dur-ng the first and second microbial enhanced oil recovery stages,ollowing concepts were proposed:
ole of IFT reduction = 1st microbial recovery1st microbial recovery + 2nd microbial recovery
(1)
ole of wettabilty alteration = 2nd microbial recovery1st microbial recovery + 2nd microbial recovery
(2)
sing this procedure, the quantitative effects of these mechanismsre calculated and tabulated in Table 3.
Te results already presented here showed that the reductionn IFT from 32 to 2.75 mN/m and Amott wettability index changesrom 0.87 to 0.68 resulted in 38.5% and 61.6% more oil recovery dur-ng in situ process. In other words, although a significant reductionn IFT was observed, however the effect of wettability alteration onhe total microbial oil recovery was found to be more significant.t has been shown in the literature that during MEOR processes,
ettability alteration occurs due to the biofilm formation or bio-urfactant adsorption [22,60]. Scanning electron microscopy (SEM)nalyzing was also used to check biofilm formation by this bac-erium in the cores as shown in Fig. 4. These images clearly revealed
able 3ndividual effects of IFT reduction and wettability alteration on oil recovery duringn situ and ex situ experiments.
Mechanism Individual effects on total oil recovery
Oil recovery (% OOIP) Mechanisms portion
In situIFT reduction 4. 0 38.45%Wettability alteration 6.3 61.55%
Ex situIFT reduction 4.0 22.34%Wettability alteration 14.0 77.66%
Fig. 4. Scanning electron microscopy image of biofilm generated by E. cloacae, (a)intact core with magnification of 1000×, (b) formed biofilm after microbial core
flooding (magnification 2000×) and (c) formed biofilm after microbial core flooding(magnification of 10,000×).
that the core surfaces of the microbial treated plugs was signifi-cantly different compared to the non-treated cores as they are fullycovered by biofilm.
It had been previously reported by Gray et al. [22] that althoughwettability alteration of the surface plays the less dominant rolein the oil recovery process from sandstone reservoirs, however it
is much more dominant in the carbonated formations. Similarly,Ayirala [61] reported that wettability alteration is more dominantcompared to IFT reduction in oil recovery from carbonated rocks
228 P. Sarafzadeh et al. / Colloids and Surfaces B
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ig. 5. The Amott-Harvey index residual oil dependence in the carbonates [62].
uring chemical core flooding processes. Besides, the obtainedesults in this study were found to be in a good agreement withhe results of Nielsen et al. [42] simulating the separate effects ofhese two mechanisms on microbial enhanced oil recovery.
.3.2. Ex situ experimentsThe results of ex situ core flooding test compared to its control
est shown in Fig. 2b, revealed that the first and second microbialil recovery efficiencies were 4.0% and 14.0%, respectively. Veryimilar to the in situ tests, the Amott wettability tests and IFT mea-urements show that these mechanisms are the most dominantnes.
Comparing the obtained results for in situ and ex situ exper-ments showed that although, the first microbial oil recoveryfficiencies are relatively similar for both in situ and ex situ coreooding tests, the oil recovery of second stage during ex situ was
mproved compared to the in situ test (see Table 3). The IFT mea-urements showed that the IFT was reduced to 2.95 mN/m fromhe original value (32 mN/m) very close to the in situ test. In addi-ion, the wettability test showed significant Amott Harvey indexeduction from 0.89 to 0.11. Anderson [62] documented a graph-cal scheme relating the residual oil saturation (Sor) to the Amottarvey index (Ia–h) for the carbonates (see Fig. 5). This illustrates
hat as the Amott Harvey index decrease from 0.05 to 0.25, theowest residual oil saturation; i.e. maximum oil recovery efficiencyecause of wettability alteration will be achieved. Thus, the highil recovery efficiency during the second recovery stage of ex situest is due to the more significant wettability alteration.
Finally, the individual effects of each mechanism were quan-itatively evaluated as previously described. The obtained resultssee Table 3) show higher impact of wettability alteration (77.66%)ompared to IFT reduction (22.34%) based on the total microbial oilecovery efficiency during ex situ tests. The mechanisms of wett-bility alteration for this stage as it could be simulated to surfactantooding were already discussed elsewhere [6].
. Conclusion
In the present experimental study, several different tests werearried out to quantify the individual effects of the IFT reductionnd wettability alteration mechanisms for MEOR process using E.loacae. A novel experimental protocol was used to examine theseechanisms in core flooding tests. The in situ and ex situ tests
howed ultimate oil recoveries of 10.3% and 18.0% based on theOIP, respectively, revealed the feasibility of using this strain for
EOR processes. Besides, the obtained results showed more signif-
cant role of wettability alteration to improve oil recovery efficiencyuring tertiary MEOR process for both ex situ and in situ scenarios.
n addition, the results showed that wettability critically changes
[
[
[
: Biointerfaces 105 (2013) 223– 229
during shut-in period for both in situ and ex situ processes in whichthe bacteria have more chance to adhere and form bio-films or bybiosurfactant adsorption on the rock surfaces. Besides, the higherwettability alteration obtained in ex situ test shows that biosurfac-tant adsorption has more potential to alter the rock wettability thanbiofilm formation. Besides, the results showed that the optimumwettability alteration based on the Anderson graphical scheme wasobtained during the ex situ microbial flooding experiments leadsto the minimum residual oil saturation. Hence, the biosurfactantproduced from E. cloacae is proposed as a good candidate for thetertiary oil recovery process because of its high capability to reduceIFT and changing rock wettability.
Acknowledgements
The authors are grateful to thank Shiraz University EOR ResearchCenter and Shiraz University Biotechnology Center for their sup-port.
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