Microbial Activation of Bacillus subtilis-Immobilized Microgel Particles

Subscriber access provided by UNIV OF CAMBRIDGE

Article

Microbial activation of Bacillus subtilis-immobilized microgel particles for enhanced oil recovery Han AM Son, Sang Koo Choi, Eun Sook Jeong, Bohyun Kim, Hyun Tae Kim, Wonmo Sung, and Jin Woong Kim Langmuir, Just Accepted Manuscript • DOI: 10.1021/acs.langmuir.6b02010 • Publication Date (Web): 09 Aug 2016 Downloaded from http://pubs.acs.org on August 15, 2016

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Langmuir is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

Microbial activation of Bacillus subtilis-immobilized microgel

2

particles for enhanced oil recovery

3 4

Han Am Son,†,‡ Sang Koo Choi,§ Eun Sook Jeong,§ Bohyun Kim,§ Hyun Tae Kim,†,*

5

Won Mo Sung,‡ Jin Woong Kim §,∥,*

6 7

†

Korea Institute of Geoscience and Mineral Resources, Daejeon 34132, Republic of Korea

8

‡

Department of Natural Resources and Environmental Engineering, Hanyang University,

9

Seoul 04763, Republic of Korea

10 11

§

Department of Bionano Technology, Hanyang University, Ansan 15588, Republic of Korea

∥

Department of Applied Chemistry, Hanyang University, Ansan 15588, Republic of Korea

12 13 14 15

ABSTRACT: Microbially enhanced oil recovery involves the use of microorganisms to

16

extract oil remaining in reservoirs. Here, we report fabrication of microgel particles with

17

immobilized Bacillus subtilis for application to microbially enhanced oil recovery. Using B.

18

subtilis isolated from oil-contaminated soils in Myanmar, we evaluated this microbe’s ability

19

to reduce the interfacial tension at the oil–water interface via production of biosurfactant

20

molecules, which eventually yield excellent emulsification across a broad range of the

21

medium pH and ionic strength. To safely deliver B. subtilis into a permeable porous medium,

22

in this study, these bacteria were physically immobilized in a hydrogel mesh of microgel

23

particles. In a core flooding experiment, in which the microgel particles were injected into a

24

column packed with silica beads, we found that these particles significantly increased oil

25

recovery in a concentration-dependent manner. This result shows that a mesh of microgel

26

particles encapsulating biosurfactant-producing microorganisms holds promise for recovery

27

of oil from porous media.

28 29

Keywords: microbially enhanced oil recovery, microgel particles, biosurfactants

30 1

ACS Paragon Plus Environment

Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

1. INTRODUCTION

2

Microbes have been used as an injected agent to enhance oil recovery from petroleum

3

reservoirs because microbes are environmentally friendly and viable under harsh reservoir

4

conditions, such as high temperatures and salinity.1–3 Some of them can produce surface-

5

active compounds, which are usually called “biosurfactants.” These compounds lower the

6

oil–water interface tension, thus facilitating the release of the remaining oil from rock

7

pores.4–6 In terms of enhancement of oil recovery, utilization of microbes is indeed

8

advantageous. Nevertheless, direct injection often causes accumulation of the microbes on

9

the inner wall of the nozzle before arrival at the reservoir. If there is any microbial growth,

10

then the injection nozzle can be completely clogged; this situation eventually hampers further

11

injection of other materials into the reservoir.7,8 To get rid of the microbial plug, application

12

of unsafe and undesirable high pressure to the nozzle is required. As a consequence, for

13

practically useful microbially enhanced oil recovery, it is important to develop a

14

straightforward approach that allows for efficient transport of the microbes into the oil

15

reservoir.9

16

To transport living species, such as microbes, to the target area, they first should be

17

reliably protected from the harsh environmental factors. As a practical solution to this issue,

18

the use of the microencapsulation technology has been widely evaluated in a variety of fields,

19

including pharmaceuticals,10 biochemical sensors,11,12 cosmetics,13 and chemical catalyst

20

reactions.14,15 Recent advances in the microencapsulation technology have been made by

21

means of the microfluidic method.16 This protocol enables encapsulation of living species,

22

such as living cells and microorganisms, in a mesh of microgel particles with high

23

encapsulation efficiency without any harmful chemicals.17–19 The living species physically

24

immobilized in a biocompatible hydrogel phase can adsorb and release biocompounds

25

through the hydrogel mesh. This approach allows researchers to design a practical

26

microcapsule system in which microbes are immobilized in the hydrogel phase while

27

producing biosurfactants through the hydrogel mesh. The biosurfactants are expected to be

28

readily released through the hydrogel mesh because their molecular size is smaller than the

29

size of the mesh pores. This approach has advantages over conventional encapsulation

30

methods because it does not require any specific stimuli to trigger the release of microbes

31

from the capsule.20–22 2


Page 2 of 23

Page 3 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

The ultimate goals of this study were to fabricate microgel particles with immobilized

2

microbes by the microfluidic method and to determine whether they can be useful for

3

microbial enhancement of oil recovery as a recovery agent. The microfluidic method allowed

4

us to produce microgel particles with excellent size monodispersity. The particle size is

5

determined by the shear forces and is adjustable by simply regulating the microfluidic device

6

geometry and flow rate conditions. The microgel particles contain a Bacillus strain in their

7

hydrogel phase. The Bacillus strain was collected from an oil reservoir in Myanmar. To

8

experimentally demonstrate that the B. subtilis strain selected in this study produces

9

biosurfactants, we evaluated the interfacial activity of B. subtilis strain at different pH and

10

salinity levels. Finally, we carried out an oil recovery experiment using the microgel particles

11

with B. subtilis immobilized in a porous medium. The microgel particles were injected into a

12

column packed with silica beads filled with n-decane and their oil recovery capacity was

13

evaluated quantitatively.

14 15

2. EXPERIMENTAL SECTION

16

2.1. Materials. Poly(ethylene glycol) diacrylate (PEGDA, Mw = 700 g/mol), sodium 4-

17

vinylbenzenesulfonate

18

poly(diallyldimethylammonium chloride) (average Mw = 20,000–350,000 g/mol, 20 wt% in

19

H2O), poly(sodium 4-styrenesulfonate) (average Mw = ~70,000 g/mol), glycerin, and paraffin

20

oil were purchased from Sigma-Aldrich (USA) and used without further purification. Cetyl

21

PEG/PPG-10/1 dimethicone (Abil EM 90, Evonik, Germany) was used as received.

(NaSS),

2-hydroxy-2-methylpropiophenone

(Darocur

1173),

22 23

2.2. Extraction of B. subtilis and incubation for biosurfactant production. The candidate

24

organism for microbially enhanced oil recovery was isolated from the oil-contaminated soils

25

collected in the Shwezetaw Formation (depth 460–550 m) in the Salin fore-arc basin,

26

Myanmar. The microorganism, which grew on the minimal salt medium (MSM) containing a

27

carbon source (glucose, 2 wt%) and a trace element solution (TES; 0.2 wt%), was isolated

28

and identified as a strain of B. subtilis by means of 16S ribosomal RNA sequencing. Using

29

the microorganism, we produced biosurfactant. The Erlenmeyer flasks containing MSM broth

30

was sterilized by autoclaving at 120 °C for 20 minutes. Next, B. subtilis strain was

31

incorporated and carbon source (glucose, 2 wt%) was added into the medium. The culture 3


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

was incubated in a shaker incubator with a rotation speed of 120 rpm for 96 h at 34°C. In

2

order to experimentally confirm the effective production of biosurfactants in incubated stock

3

solution, the interfacial tension was measured with varying the concentration of B. subtilis in

4

the stock solution at pH 8.0. Additionally, to evaluate whether the bacterial species really

5

made a difference, Escherichia coli as a non-oil-tolerant bacterial species was incubated with

6

the carbon source. Then, we investigated the changes of interfacial tension in the presence of

7

E. coli under the same conditions as the case of using B. subtilis.

8 9

2.3. Determination of interfacial tension between oil and water. The interfacial tension

10

was measured to characterize the B. subtilis’s behavior at the oil–water interface at pH 4–10

11

without NaCl and at pH 8.0 with 0–10 wt% NaCl. The interfacial-tension measurements were

12

done with an optical tensionmeter (Biolin Scientific, Sweden) by reversed pendant drop

13

shape analysis at room temperature. Incubated B. subtilis (0.9 g/L) was present in the aqueous

14

phase at different pH levels and NaCl concentrations in each transparent rectangular cell.

15

Then, a hooked needle was immersed in the aqueous phase in a rectangular cell, and the n-

16

decane drop was hanging on the hooked needle. The shape of the n-decane drop is

17

determined by means of the balance of forces including the interfacial tension between n-

18

decane and the aqueous phase. The interfacial tension can be calculated by means of the

19

Young-Laplace equation in software (One Attension, Biolin Scientific).

20 21

2.4. Production of n-decane emulsions. Before emulsification test, B. subtilis was incubated

22

with carbon source for production of biosurfactant. An n-decane emulsion was prepared with

23

incubated aqueous solution containing B. subtilis (0.9 g/L). We controlled pH of the solution

24

at 4 or 8, while changing the salinity by adding NaCl up to 10 wt%. To induce rapid phase

25

separation, the volume of n-decane was set to 50% of the aqueous phase volume. Then, the B.

26

subtilis suspension and n-decane were mixed for 2 min at 3000 rpm with an ultrasonic

27

homogenizer. After sonication, phase separation of the emulsion samples was observed

28

during storage at room temperature

29 30

2.5. Fabrication of microcapillary microfluidic devices. To fabricate the devices, a

31

cylindrical glass capillary (outer diameter 1.0 mm, World Precision Instruments, USA) was

32

heated and pulled using a pipette puller (Model P-97, Sutter Instruments, USA). The thin tip 4


Page 4 of 23

Page 5 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

of the tapered glass capillary was cut to the desired diameter using an MF 830 Micro Forge

2

(Narishige, Japan). To make the surface of the round capillary tubes hydrophobic, 1 wt%

3

hexyl-trimethoxysilane in toluene was circulated through the glass capillaries. Then, a

4

tapered cylindrical glass capillary was inserted into a square capillary (inner diameter 1.0

5

mm, Atlantic International Technology, USA). Each end of the square capillary was

6

connected to a needle and pasted with an epoxy resin. The needle was connected to a

7

polyethylene tube (PE-5, outer diameter 1.32 mm, Scientific Commodities, USA), which was

8

again connected to a glass syringe (Hamilton Gastight, USA).

9 10

2.6. Microfluidic synthesis of microgel particles. The dispersion fluid and outer fluid were

11

injected through the interstices between the round injection capillary and the square

12

collection capillary. The flow rates were precisely adjusted using the syringe pumps (Pump

13

11 Elite, Harvard Apparatus, USA). The dispersion fluid was an aqueous monomer solution

14

containing the incubated B. subtilis suspension (48 wt%), PEGDA (20 wt%), NaSS (10 wt%),

15

glycerin (20 wt%), and Darocur 1173 (a photoinitiator, 2 wt%). The outer fluid consisted of

16

paraffin oil and 0.5 wt% Abil EM 90. Formation of emulsion drops was monitored under an

17

inverted microscope equipped with a high-speed camera (Phantom Miro eX2, Vision

18

Research Inc., USA). Then, the emulsion drops were solidified by photo-polymerization

19

under ultraviolet (UV) at a wavelength of 365 nm for 1 min (UV light, A&D Co., Korea), and

20

the emulsion drops were transferred to microgel particles. The paraffin oil, remnant

21

monomers, and other additives were thoroughly removed with isopropanol by repeated

22

centrifugation at 5000 rpm. Finally, the microgel particles with immobilized B. subtilis could

23

be recovered from the continuous phase of paraffin oil without the use of chemical solvents.

24 25

2.7. Apparatus for core flooding experiment. We conducted the flooding experiment to

26

quantitatively evaluate the microbial activity of the microgel particles with immobilized B.

27

subtilis as a recovery agent. The outline of the flooding process is shown in Fig. 1. The

28

apparatus for the core flooding experiment was fabricated by assembling an injection pump,

29

an accumulator, a core holder, and a measuring cylinder. The piston plate was installed in the

30

cylindrical accumulator, and the dispersion of the microgel particles filled the container

31

located above the piston of the accumulator. When water was injected into bottom of the

32

cylindrical accumulator by injection pump, the piston plate was pushed up. Then, the 5


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

dispersion of the microgel particles was flowed into a column filled with silica glass beads 1

2

mm in diameter. The fluid that flowed through the interstice of silica beads was collected in a

3

volume-readable cylinder. Oil reservoir contained oil and water in the rock pore. Before the

4

flooding experiment, water was injected into the silica beads, and then n-decane was injected

5

into the water-saturated medium until no more water drained off. Then, the initial volume

6

fraction of water and oil in the silica beads was calculated from the volume of water

7

collected.

8 9

3. RESULTS AND DISCUSSION

10

B. subtilis is known as a bacterium that produces biosurfactants. A good example is

11

surfactin.23 Analogous to typical surfactants, those biosurfactants have an amphiphilic

12

molecular structure, so that they can assemble at the interface of fluids of different polarity,

13

such as water and oil phases. There have been debates on how they manifest the surface

14

activities. For instance, the biosurfactants may increase bioavailability of a hydrophobic

15

substrate by increasing their apparent solubility in hydrocarbon compounds, thus easily

16

desorbing them from the surface. Aside from that, they can regulate adhesion of

17

microorganisms to a surface.24 Studies have shown that B. subtilis biosurfactants enhance

18

aqueous solubility of hydrophobic substances; this change reduces the interfacial tension

19

between oil and water. To elucidate in detail how the presence of B. subtilis biosurfactants in

20

the aqueous phase affects the surface activity, in this study, we tried to directly measure

21

changes in the interfacial tension between n-decane and water in various solution

22

environments. Before the measurement of interfacial tension, B. subtilis solution was

23

incubated for production of biosurfactant. To identify the production of biosurfactant in the

24

B. subtilis solution, the interfacial tension was measured with varying dilution of B. Subtilis

25

solution at pH 8 (Fig. 2).

26

Here, the initial concentration of of B. Subtilis in solution before dilution was 0.9 g/L, and

27

the declined concentration of B. Subtilis means the decreasing concentration of biosurfactant.

28

As the aqueous phase is concentrated with B. subtilis, the interfacial tension sharply dropped

29

from 49 dyne/cm to 18 dyne/cm. However, in the case of using the non-oil-tolerant bacterial

30

species, E. coli, the interfacial tension retained unchanged irrespective of the concentration of

31

E. coli, as shown in Fig. 2. This means that biosurfactant was effectively produced by the 6


Page 6 of 23

Page 7 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

activity of B. subtilis. Also, we analysed the effect of pH and salinity on the B. subtilis

2

biosurfactants. The result is shown in Fig.3. The interfacial tension between n-decane and the

3

aqueous solution containing B. subtilis (0.9 g/L) decreased as the pH of the aqueous solution

4

increased (Fig. 3A).The interfacial tension decreased from 32 to 18 dyne/cm as pH increased

5

from 4 to 8. Across the pH range from 8 to 12, little or no change in the interfacial tension

6

was observed. This is because the biosurfactants produced by B. subtilis strain have a

7

tendency toward precipitation at low pH due to their low solubility under highly acidic

8

conditions.25-27 Therefore, improved interfacial activity may be attained under neutral and

9

alkaline conditions. The effect of salinity on the interfacial activity of B. subtilis was also

10

evaluated by addition of NaCl (Fig. 3B). The interfacial tension gradually increased from 18

11

to 23 dyne/cm as the concentration of NaCl reached 10 wt%. Even though there was some

12

deterioration in the interfacial tension, this was an inspiring result because B. subtilis

13

biosurfactants still showed interfacial activity even under such conditions of harsh salinity.

14

Having confirmed that the presence of B. subtilis in the aqueous phase lowered the

15

interfacial tension between oil and water by the formation of biosurfactants, we decided to

16

incorporate B. subtilis solution, including produced biosurfactants, into an emulsification

17

system. As shown in Fig. 4, the emulsion that was produced in the absence of solution

18

containing B. subtilis (pure water) was easily separated into upper and lower phases (Sample

19

1). The emulsions that were produced at pH 4 were also readily broken up within minutes

20

(Samples 2–5). Even after 7-day storage, no improvement in the emulsion stability could be

21

achieved. This finding implies that biosurfactants produced by B. subtilis couldn’t retain their

22

own interfacial activity, because they tend to aggregate at pH 4. In contrast to the cases at low

23

pH, when the emulsions were produced at pH 8, the emulsion stability was significantly

24

enhanced (Samples 6–9). Even after long-term storage, the emulsion drops remained

25

unchanged. This result indicates that B. subtilis biosurfactants have excellent interfacial

26

activity at pH 8, in agreement with the results in Fig.3A. To assess the effect of salinity, we

27

added various concentrations of NaCl to the aqueous solution containing B. subtilis. The

28

emulsion index, i.e., the height of the emulsified layer as a percentage of the total height of

29

the liquid column,28 was determined for ~20% to 5 wt% NaCl. The emulsion index

30

remarkably increased to approximately 55% at a high NaCl concentration (10 wt% NaCl,

31

Sample 9). This unexpected result may be attributed to the migration of biosurfactants to the 7


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

oil–water interface from the aqueous phase because of the presence of excess electrolytes in

2

the aqueous phase.29

3

To transport B. subtilis to the target oil reservoir, we physically immobilized these

4

bacteria in a hydrogel mesh of microgel particles. For production of microgel particles with

5

uniformly immobilized B. subtilis, monodisperse emulsion drops were generated in a

6

microcapillary-based microfluidic device (Fig. 5A). The aqueous mixture consisting of B.

7

subtilis, PEGDA, NaSS, and photoinitiator was injected into the paraffin oil containing a

8

nonionic surfactant (Abil EM 90) by means of the coaxial jetting fluid phenomenon.

9

Typically, the fluid thread that formed near the collection capillary tip pinched off into small

10

drops because of hydrodynamic instability (Fig. 5B). The stability of dropping frequency led

11

to formation of monodisperse emulsion drops. The coefficient of variation for our emulsion

12

drops was less than 10%. After collection of the W/O emulsions, they were irradiated with

13

UV light to polymerize them. Because B. subtilis is a few micrometers long, these bacteria

14

can be immobilized within the hydrogel mesh whose mesh size is 5–10 nm in a swollen state

15

in water19 (Fig. 6A–C). The efficiency of encapsulation of B. subtilis in the mesh of microgel

16

particles was almost 100%, which is a key advantage of the microfluidic method. We found

17

that the particle diameter of microgel particles in water increased by ~40% in comparison

18

with the diameter of emulsion drops in paraffin oil (Fig. 6D). This finding can be attributed to

19

the fact that the confined polymer network in the emulsion drop freely uncoiled in water, thus

20

resulting in volume expansion.

21

A core flooding experiment was performed on microgel particles with immobilized B.

22

subtilis in a silica bead-filled model column. One pore volume (PV) of the microgel

23

dispersion was injected into the glass beads after 2 PV of water was injected. Here, pore

24

volume (PV) means total volume of void space in silica beads. 1 PV unit was calculated to be

25

140 mL in our experiments. On the basis of the cumulative oil recovery with the PV of the

26

injected fluid in the core flooding experiment, we could confirm that injection of the microgel

27

particles significantly increased the oil recovery (Fig. 7A). Oil recovery was determined by

28

measuring the amount of extracted oil from containing oil (original oil in place, OOIP; 119.8

29

mL) in silica beads. Injection of water up to 2 PV led to 83.9-85.5 vol% (100.5-102.4 mL)

30

release of OOIP from the silica glass beads. Additional 1 PV injection of the microgel fluid

31

containing the B. subtilis-immobilized microgel particles increased the recovery of n-decane

32

with the concentration-dependent manner. When the microgel particles were concentrated to 8


Page 8 of 23

Page 9 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

5.0 wt%, the recovery of n-decane could be additionally enhanced over 7.1 vol % of OOIP

2

(Fig. 7B). This means that 45.2 % (8.5 mL) of residual n-decane (18.8 mL) after water

3

injection was released.

4

Additionally, to evaluate whether the viability of B. subtilis actually affect the oil

5

recovery, we conducted the core flooding experiment using the microgel particles containing

6

UV-killed B. subtilis cells. After UV-irradiation (254 nm) for over 1 h, the number of CFU of

7

B. subtilis cells was decreased by 92%. Injection of 5 wt% microgel particles containing UV-

8

killed B. subtilis into silica bead led to the recovery of n-decane below 0.6 vol%. This

9

recovery efficiency is quite comparable to the case of injecting the microgel particles

10

containing living-B. subtilis. These results show that the use of microgel particles with

11

immobilized B. subtilis successfully enhanced the oil recovery. Thus, the B. subtilis that was

12

reliably protected by the hydrogel phase could be delivered to n-decane-filled porous media.

13

Then, the biosurfactants produced by B. subtilis readily diffused out through the hydrogel

14

mesh because their molecular size is several fold smaller than that of the hydrogel mesh pores

15

in water. Under such reservoir conditions, the biosurfactants should preferably adhere to the

16

n-decane captured in the silica glass beads to reduce the interfacial tension, thus eventually

17

facilitating recovery of the residual n-decane.

18

There have been reports showing that a surfactant-producing strain of a Bacillus species

19

can enhance oil recovery.26,30,31 For studies on applicability of these bacteria to microbially

20

enhanced oil recovery, the challenge is the issue of clogging during flooding.8,32 From the

21

practical standpoint, the use of the microencapsulation technology not only enables protection

22

of microbes from the concentration reduction during flow in the medium but also allows

23

researchers to control the release rate of the incorporated microbes.10 For example, for active

24

delivery of capsules into an oil-bearing environment, a delivery protocol combining polymer

25

microcapsules with an organic-solvent stimulus has been developed.22 This approach

26

convincingly showed that the release of microbes from polystyrene microcapsules can be

27

triggered by solvation-induced capsule rupturing for oil recovery in porous media. Compared

28

to microcapsule-based microbe delivery, the method developed in the present study has an

29

advantage: we do not need to use such a trigger to induce the release of microbes from the

30

particles. As can be seen from the model diffusion experiments in Fig. 8, the small molecules

31

that physically trapped in the mesh of microgel particles have a tendency toward slowly

32

diffusing out with no aids of external stresses. In fact, the diffusion kinetics are mainly 9


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

determined by the mesh size. In the flooding experiment, therefore, we can expect that the

2

biosurfactants produced by microbes would simply diffuse out of the microgel particles and

3

show the ability to recover additional oil from oil-bearing structures.

4 5

4. CONCLUSION

6

Conventional methods of microbially enhanced oil recovery involve simple injection of

7

microbes or nutrients into the oil reservoir. In contrast, as proof of concept, we physically

8

immobilized microbesin microgel particles by the microfluidic method and tried to transport

9

these bacteria to the target reservoir. In this study, B. subtilis served as a model microbe that

10

can produce biosurfactants. We confirmed empirically that B. subtilis reduces the interfacial

11

tension between oil and water. In the flooding experiment, we demonstrated that injection of

12

microgel particles (on which B. subtilis is immobilized) after the water flooding may enhance

13

oil recovery over 7 vol%, which corresponds to more than 45 vol% recovery of the residual

14

oil from a model reservoir column packed with silica beads. This finding is comparable to the

15

result of flooding water only. Such improved oil recovery is possible because B. subtilis

16

produces biosurfactants that readily diffuse out of the microgel particles through the hydrogel

17

mesh in the reservoir, thus facilitating emulsification of oil in water during oil recovery.

18

Consequently, the proposed technology for fabrication of microgel particles with

19

immobilized microbes is expected to be applied to microbially enhanced oil recovery in

20

permeable porous media.

21 22

AUTHOR INFORMATION

23

Corresponding Author: Prof. Jin Woong Kim

24

*E-mail: [email protected]

25

*Telephone: +82-31-400-5499

26

*Address: Department of Bionano Technology and Department of Applied Chemistry,

27

Hanyang University, Ansan 15588, Republic of Korea.

28

Corresponding Author: Dr. Hyun-Tae Kim

29

*E-mail: [email protected]

30

*Telephone: +82-42-868-3216.

31

*Address: Korea Institute of Geoscience and Mineral Resources, Daejeon 34132, Republic of 10


Page 10 of 23

Page 11 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Langmuir

Korea.

2 3

The authors declare no competing financial interest.

4 5

ACKNOWLEDGMENTS

6

The work was carried out with financial support from the Korea Institute of Geosciences and

7

Mineral Resources and by the National Research Foundation of Korea (NRF) grant funded by

8

the Korea government (MSIP) (No. 2008-0061891 and No. 2016R1A2B2016148). H. A. Son

9

and S. K. Choi equally contributed to this work.

10 11

REFERENCES

12

(1) Desai, J. D.; Banant, I. M. Microbial production of surfactants and their commercial

13

potential. Microbial. Mol. Biol. Rev.1997, 61, 47-64.

14

(2) Lin, J.; Hao, B.; Cao, G.; Wang, J.; Feng, Y.; Tan, X.; Wang, W. A study on the microbial

15

community structure in oil reservoirs developed by water flooding.J. Petroleum Sci.

16

Eng.2014, 122, 354-359.

17

(3) Ni’matuzahroh; Nurmalasari, R.; Silvia, R. A.; Nurhariyati, T.; Surtiningsih, T. Microbial

18

enhanced heavy crude oil recovery through biodegradation using bacterial isolates from an

19

Omani oil field. J. Appl. Environ. Biol. Sci.2015, 5, 83-87.

20

(4) Li, D.; Lu, S.; Liu, Y.; Wang, D. The effect of biosurfactant on the interfacial tension and

21

adsorption loss of surfactant in ASP flooding.colloids surf. B2004, 244, 53-60.

22

(5) Al-Sulaimani, H.; Al-Wahaibi, Y.; Al-Bahry, S.; Elshafie, A.; Al-Bemani, A.; Joshi, S.;

23

Zargari, S. Optimization and partial characterization of biosurfactants produced by Bacillus

24

species and their potential for ex-situ enhanced oil recovery. SPE Journal2011, 16, 672-682.

25

(6)Biria, D.; Maghsoudi, E.; Roostaazad, R. Application of biosurfactants to wettability

26

alteration and IFT reductionin enhanced oil recovery from oil–wet carbonates. Pet. Sci.

27

Technol.2013, 31, 1259-1267.

11


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

(7) Sarkar, K.; Georgiou, G.; Sharrna, M. M. Transport of bacteria in porous media: 1. An

2

experimental investigation. Biotechnol.Bioeng.1994, 44, 489-497.

3

(8) Lazar, I.; Petrisor, I. G.; Yen, T. F. Microbial enhanced oil recovery (MEOR). Pet. Sci.

4

Technol.2007, 25, 1353-1366.

5

(9) Jang, L. K.; Chang, P. W.; Findley, J. E.; Yen, T. F. Selection of bacteria with favorable

6

transport properties through porous rock for the application of microbial enhanced oil

7

recovery. Appl. Environ. Microbiol.1983, 46, 1066-1072.

8

(10) Singh, M. N.; Hemant, K. S. Y.; Ram, M.; Shivakumar, H. G. Microencapsulation: A

9

promising technique for controlled drug delivery. Res. Pharm. Sci.2010, 5, 65-77.

10

(11) McShane, M. J.; Brown, J. Q.; Guice, K. B.; Lvov, Y. M. Polyelectrolyte microshells as

11

carriers for fluorescent sensors: loading and sensing properties of a ruthenium-based oxygen

12

indicator. J. Nanosci. Nanotechnol.2002, 2, 411-416.

13

(12) Kazakova L. I.; Shabarchina, L. I.; G. B. Sukhorukov, Co-encapsulation of enzyme and

14

sensitive dye as a tool for fabrication of microcapsule based sensor for urea measuring. Phys.

15

Chem.Chem. Phys.2011, 13, 11110-11117.

16

(13) Karnik, R; Gu, F.; Basto, P.; Cannizzaro, C.; Dean, L.; Kyeimanu, W.; Langer, R.;

17

Farokhzad, O. C. Microfluidic platform for controlled synthesis of polymeric nanoparticles.

18

Nano Lett.2008, 8, 2906-2912.

19

(14) Ley, S. V.; Ramarao, C.; Gordon, R. S.; Holmes, A. B.; Morrison, A. J.; McConvey, I.

20

F.; Shirley, I. M.; Smith, S. C.; Smith M. D. Polyurea-encapsulated palladium(II) acetate: a

21

robust and recyclable catalyst for use in conventional and supercritical media. Chem.

22

Commun.2002, 13, 1134-1135.

23

(15) Poe, S. L.; Kobaslija, M.; McQuade, D. T.; Mechanism and application of a

24

microcapsule enabled multicatalyst reaction. J. Am.Chem. Soc. 2007, 129, 9216-9221.

25

(16) Whitesides, G.M. The origins and the future of microfluidics.Nature2006, 442, 368-373.

12


Page 12 of 23

Page 13 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1

(17) Kim, J. W.; Utada, A. S.; Fernandez-Nieves, A.; Hu, Z.; Weitz, D. A. Fabrication of

2

monodisperse gel shells and functional microgels in microfluidic devices, Angew. Chem. Int.

3

Ed.2007, 46, 1819-1822.

4

(18) Martinez, C. J; Kim, J. W; Ye, C; Ortiz, I; Rowat, A. C; Marquez, M; Weitz, D. A. A

5

microfluidic approach to encapsulate living cells in uniform alginate hydrogel

6

microparticles.Macromol.Biosci.2012, 12, 946-951.

7

(19) Park, J.; Byun, A.; Kim, D. H.; Shin, S. S.; Kim, J. H.; Kim, J. W. Microfluidic

8

fabrication and permeation behaviors of uniform zwitterionic hydrogel microparticles and

9

shells. J. ColloidInterface Sci.2014, 426, 162-169.

10

(20) Kim, S. H.; Kim, J. W.; Cho, J. C.; Weitz, D. A. Double-emulsion drops with ultra- thin

11

shells for capsule templates.Lab Chip.2011, 11, 3162-3166.

12

(21) Zhou, S.; Fan, J.; Datta, S. S.; Guo, M.; Guo, X.; Weitz, D. A. Thermally switched

13

release from nanoparticle colloidosomes. Adv. Func.Mater.2013, 23, 5925-5929.

14

(22) Abbaspourrad, A.; Carroll, N. J.; Kim, S. H.; Weitz, D. A. Polymer microcapsules with

15

programmable active release.J. Am. Chem. Soc.2013, 135, 7744-7750.

16

(23) Arima, K.; Kakinuma, A.; Tamura, G. Surfactin, a crystalline peptide lipid surfactant

17

produced by Bacillus subtilis: isolation, characterixation and its inhibition of fibrin clot

18

formation. Biochem.Biophys. Res. Commun.1968, 31, 488-494.

19

(24) Rosenberg , E.; Ron, E. Z. High- and low-molecular-mass microbial surfactants. Appl.

20

Microbiol. Biotechnol.1999, 52, 154-62.

21

(25) Mabrouk, M. E. M.; Youssif, E. M.; Sabry, S. A. Biosurfactant production by a newly

22

isolated soft coral-associated marine Bacillus sp.E34: Statistical optimization and

23

characterization. Life. Sci. J.2014, 11, 756-768.

24

(26) Al-Wahaibi, Y.; Joshi, S.; Al-Bahry, S.; Elshafie, A.; Al-Bemani, A.; Shibulal, B.

25

Biosurfactant production by Bacillus subtilis B30 and its application in enhancing oil

26

recovery. colloids surf. B2014, 114, 324-333.

13


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

(27) Amani, H. Study of enhanced oil recovery by rhamnolipids in a homogeneous 2D

2

micromodel. J. Petroleum Sci. Eng.2015, 128, 212-219.

3

(28) Cooper, D. G.; Goldenberg, B. G. Surface-active agents from two Bacillus species.Appl.

4

Environ. Microbiol.1987, 53, 224-229.

5

(29) Binks, B. P. Particles as surfactants’ similarities and differences. Curr.Opin. Colloid

6

Interface Sci.2002, 7, 21-41.

7

(30) Gudina, E. J.; Pereira, J. F. B.; Costa, R.; Coutinho, J. A. P.; Teixeira, J. A.; Rodrigues,

8

L. R. Biosurfactant-producing and oil-degrading Bacillus subtilis strains enhance oil recovery

9

in laboratory sand-pack columns. J. Hazard. Mater.2013, 261, 106-113.

10

(31) Pereira, J. F.; Gudiña, E. J.; Costa, R.; Vitorino, R.; Teixeira, J. A.; Coutinho, J. A.;

11

Rodrigues, L. R. Optimization and characterization of biosurfactant production by Bacillus

12

subtilis isolates towards microbial enhanced oil recovery applications. Fuel2013, 111, 259-

13

268.

14

(32) Luef, B.; Fakra, S. C.; Csencsits, R.; Wrighton, K. C.; Williams, K. H.; Wilkins, M.

15

J.; Downing, K. H.; Long, P. E.; Comolli, L. R.; Banfield, J. F. Iron-reducing bacteria

16

accumulate ferric oxyhydroxide nanoparticle aggregates that may support planktonic growth.

17

ISME J.2013, 7, 338-350.

18 19 20 21

14


Page 14 of 23

Page 15 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1

Langmuir

Graphical Abstract

2 3 4

15


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 1. A schematic diagram of the experimental apparatus for microbially enhanced oil

3

recovery.

16


Page 16 of 23

Page 17 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2 3

Figure 2. Changes in the interfacial tension of n-decane-water with changing the

4

concentration of B. subtilis and E. coli in the aqueous phase. The initial concentration of B.

5

subtilis and E. coli in the aqueous phase was tuned to be 0.9 g/L. The deviation in the values

6

of interfacial tensions came from pendant drop profiles.

17


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 3. Changes in the interfacial tension of n-decane–water interfaces as a function of (A)

3

pH and (B) NaCl concentration in the presence of B. subtilis in the aqueous phase. The

4

concentration of B. subtilis in the aqueous phase was set to 0.9 g/L in (A) and (B), and pH

5

was adjusted to 8 in (B).

6

18


Page 18 of 23

Page 19 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2

Figure 4. Interfacial activation of Bacillus subtilis in n-decane emulsions. (A) Immediately

3

after emulsification and (B) after 7-day storage at room temperature. Sample 1 corresponds to

4

the n-decane emulsion without B. subtilis (pure water). Samples 2–5 correspond to n-decane

5

emulsions containing B. subtilis: 0, 2, 5, or 10 wt% NaCl, respectively, at pH 4. Samples 6–9

6

are the n-decane emulsions containing B. subtilis: 0, 2, 5, and 10 wt% NaCl, respectively, at

7

pH 8.

8

19


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 5. (A) A schematic of a coaxial jetting microfluidic capillary device. (B) A bright-

3

field micrograph showing formation of monodisperse microgel precursor emulsion drops

4

containing B. subtilis.

5

20


Page 20 of 23

Page 21 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2

Figure 6. (A) A bright-field micrograph of B. subtilis in the aqueous phase. Bright-field

3

micrographs of (B) microgel precursor emulsion drops and (C) microgel particles with

4

immobilized B. subtilis. (D) Particle size distributions before and after microgel formation.

5

21


Langmuir

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

1 2

Figure 7. (A) Overall oil recovery as a function of pore volume for injection of water and

3

microgel particles with immobilized B. subtilis. (B) Cumulative oil recovery immediately

4

after injection of microgel particles with B. subtilis. The injection fluid contained 0.05 wt%

5

(), 1.5 wt% (), 5.0 wt% (◆) microgel particles with living B. subtilis and 5.0 wt% (▲)

6

microgel particles with UV-killed B. subtilis.

7

22


Page 22 of 23

Page 23 of 23

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Langmuir

1 2

Figure 8. (A) Releasing kinetics of fluorescein sodium salt (FSS, 376.27 g/mol) from

3

microgel particles at 25°C. FSS was physically encapsulated in PEGDA microgel particles:

4

700 g/mol PEGDA (~75 Å calculated mesh size, --), 6000 g/mol PEGDA (~290 Å

5

calculated mesh size, --). Fluorescence microscopy images of FSS-loaded PEGDA

6

microgel particles: 700 g/mol PEGDA after storage for 1 day (B) and 11 days (C), 6000

7

g/mol PEGDA after storage for 1 day (D) and 11 days (E) in water.

23


Microbial Activation of Bacillus subtilis-Immobilized Microgel Particles

Recommend Documents