research article pores and microanalysis of microbe...

Hindawi Publishing CorporationJournal of NanomaterialsVolume 2013 Article ID 826816 5 pageshttpdxdoiorg1011552013826816

Research ArticlePores and Microanalysis of Microbe-Inspired Nano-CaCO3Cementing Sand Columns

Li Li1 Qian Chunxiang2 and Zhao Yonghao3

1 School of Materials Science and Engineering Nanjing University of Science and Technology Street of Xiao Ling Wei 200Nanjing Jiangsu 210009 China

2 East of Ji Yin Da Dao School of Materials Science and Engineering Southeast University Jiangning Nanjing Jiangsu 211189 China3Nano-Structural Materials Center School of Materials Science and Engineering Nanjing University of Science and TechnologyStreet of Xiao Ling Wei 200 Nanjing Jiangsu 210009 China

Correspondence should be addressed to Li Li lili smsenjusteducn

Received 5 September 2013 Revised 4 November 2013 Accepted 4 November 2013

Academic Editor Hui Xia

Copyright copy 2013 Li Li et al This is an open access article distributed under the Creative Commons Attribution License whichpermits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

This paper introduced an innovative bioengineering method of consolidating incompact sand by urea hydrolysis producing calcitecementation under the inducement of reproductive urease microbe The result of mercury intrusion porosimetry showed the porevolume fractions of sand columns cemented by microbe-inspired nano-CaCO

3on an accelerating reduction during the time of

cementation the process of which represented the bulk density of nano-CaCO3cementing sand columns that reinforced themselves

positively all the time The white precipitate particles exhibited a uniformed distribution of about 15 120583m size and of a hexahedralshape mostly under SEM Based on the Scherrer equation the crystallite size of the white precipitate was calculated for 185 nmThe diffraction pattern of the white precipitate under TEMwas taken in alignment with the 52 inorganic crystal composed of threeelements of Ca C and O in PCPDFWIN-2000 and the precipitate was identified as ID86-2334 calcite calcium carbonate

1 Introduction

Biomineralization is discovered and applied in geologicalmaterial consolidation [1 2] With the application of thisdiscovery to sand biocementation as the in situmicroinspiredcalcite precipitation technique (MCP technique for abbrevi-ation) a solution of urease-productive bacteria and urea wasmixed and calcium chloride was first injected in sand andthen in bacteria-producing urease dehydrates urea shown in(1) and (2) and consequently calcium carbonate precipitatesand fills in the spacing of sand shown in (3) and (4)

CO(NH2)2+H2O bacteria997888997888997888997888997888rarr NH

2COOH +NH

3(1)

NH2COOH +H

2O 997888rarr NH

3+H2CO3 (2)

H2CO3larrrarr 2H+ + CO

3

2minus (3)

Ca2+ + CO3

2minuslarrrarr CaCO

3darr (4)

Microstructure analysis was made by the formulas of XRDTEM and SEM combinedwithmercury intrusion porosime-try method to investigate the CaCO

3particles size distribu-

tions and locations This paper focused on the characteri-zation of nano-CaCO

3precipitate itself and its influence on

sand-based material properties

2 Materials and Methods

21 Microorganism and Growth Conditions Liquid culturemedia consisting of 3 gL nutrient broth 5 gL peptone and24 gL urea were adjusted to pH 70 and sterilized for 25minat 121∘C 2mL (OD600 nm=08) Bacillus pasteurii (isolatedand preserved appropriately in our lab) were added into100mL culture medium and incubated at 30∘C at 170 rpm for24 hours in the shaker (XinZhi China) [3]

22 Microbe-Inspired Nano-CaCO3-Sand Cementation 15 g

of sand in 200120583m diameter was added with appropriate

2 Journal of Nanomaterials

Table 1 Calculated porosity structure factors of MIP data report of sand columns

No Daysd Grades Permeable area10minus3 120583m2 Total porosity Pore volumemLg Specific aream2m Pore throatporeA1 5 Super low 008356 40466 02500 00166 38286A9 10 Super low 011084 35724 02119 00169 41489A13 15 Super low 003961 35538 02137 00186 28909A20 20 Super low 001479 27855 01437 00236 38554A27 28 Super low 000044 27305 01394 01170 04865B1 28 Super low 001210 18183 01008 00079 46848

Table 2 Permeability grades of rocks

Grades 1 2 3 4 5 6 7Permeability Super high Very high High Medium Low Very low Super lowPermeable area10minus3 120583m2

gt1000 1000sim500 500sim100 100sim50 50sim10 10sim1 lt1

Inlet

OutletSand column

Figure 1 Photos of microbe-inspired CaCO3cementation

vibrations into a plastic injection tube 22mm in diameterand 95mm in length Those tubes filled with sand wereprepared for microbe-inspired CaCO

3cementation [4] This

endeavor was a method of reaction fluid gravitational per-mease and external pressure [5] It was to make the sandcompact as much as possible under 01MPa axial pressuresduring the whole cementation An iron frame was designedto allow tubes to locate in it steadily and properly (Figure 1)

23 Mercury Intrusion Porosimetry Method Porosimetrysuch as pore diameter total pore volume superficial areaand pore throat ratio was measured to demonstrate nano-CaCO

3-sand structure of samples taken as Figure 2 under

PoreMaster-60 of Quantachrome USA The sizes and thesphere of distribution of pores and throats and their arrange-ment relating to each other were determined by mercuryintrusion-extrusion capillary pressure curves And the porethroat aspect ratio was that of pore radius to the relevantthroat radius which indicated their dimensional arrange-ment in material (Figure 3)

24 X-Ray Diffraction Samples were taken from calcite pre-cipitate in sand columns to make X-ray diffraction analysisGrain size phase retrieval and lattice constant refinementwere conducted on MDI JADE 5

25 Transmission Electron Microscope Calcite precipitatemorphology was observed under Tecnai T20 The latticediffraction pattern was taken to identify the phase of theprecipitate with powder diffraction database of PCPDFWIN-2002

26 Scanning Electron Microscope SEM samples were takenfrom both ends and also from the middle part of the sandcolumns shown in Figure 5 to observe the amount and thelocation of the calcite precipitate under JSM-5900 of SirenJapan

3 Results and Discussion

The characterizations of the white precipitate in cementingthe loose sand were discussed focused on porosity and per-meability morphological observation phase identificationgrain size calculation and crystal type determination

31 Porosity As shown in Figure 2 from which MIP sampleswere taken plenty of data were obtained from the porositystandard report ofMIP such as permeable area total porosityvolume total porosity volume fraction specific superficialarea and pore throat aspect ratio all of which were listedin Table 1 Compared with the permeable area of grades inTable 2 permeability of all the sand columns fell into thesuper low grade

The pore throat aspect ratio of sand columns at 672hours sharply decreased to 049 from 383 at 120 hours(Figure 4)Thepore throat aspect ratio indicated dimensionalarrangement of pores and throats and it explained thereduction of pore radius when more and more precipitationformed and filled in the pores with increasing cementationtime

Journal of Nanomaterials 3

15 mm 15 m

m15

mm

Inlet

Outlet

(a)

Inlet

Outlet10 mm 10 mm10 mm

(b)

Figure 2 Sample taken places demonstration (a) MIP samples (b) SEM samples

Sand radius

Pore radius

Pore throat radius

Figure 3 2D sand pore and pore throat radius

008

010

012

014

016

018

020

022

024

026

Pore

thro

at-p

ore r

atio

Porosity volumePore throat-pore ratio

5

4

3

2

1

0

Sand column types

28-d

ayno

-pre

ssin

gpl

uggi

ng

5-da

ypr

essin

g

28-d

aypr

essin

g

20-d

aypr

essin

g

15-d

aypr

essin

g

Poro

sity

volu

me (

mL

g)

10-d

aypr

essin

g

Figure 4 Porosity volume and pore throat aspect ratio of sandcolumns at different ages

32 Morphological Observation

321 Precipitate Distribution SEM samples were taken fromthe inlet the middle and the outlet part of white precipitateand sand Figure 4 As shown in Figure 4 the amount

of precipitate particles reduced constantly along the inletthe middle and the outlet which explained why microbe-inspired precipitate reaction flew from the inlet to the outletand quite amount of the calcite precipitate accumulated in theinlet area

322 Particle Size White precipitate particles exhibited auniform distribution of about 15 120583m size and of a hexahedralshape mostly in Figure 5 Since crystal growth is affectedby various constraints the irregular shape of the precipitateparticles was due to the unequal compression in differentparts of the sand column

33 Phase Identification and Quantity Analysis

331 Phase Identification X-ray diffraction pattern of themicrobe-inspired precipitate was shown in Figure 5 indi-cating that the precipitate was calcium carbonate in calcitecrystals

332 Grain Size Scherrer equation was introduced to cal-culate the average crystallite size of the calcite precipitate asshown in

120573 =119870120582

119871 cos 120579+ 4119890 tg 120579 (5)

or

120573 cos 120579120582=119870

119871+4119890 sin 120579120582

(6)

wherein 120573 is the full width of the Bragg peak as measuredat half height rad 120579 the Bragg peak angle degree 120582 thewavelength of the radiation A 119871 the average crystallite sizeA 119890 the average crystallite size of distortion A and 119870 aconstant The value depends on the method used and variesbetween 05 and 1 but is usually taken as around 09

Calculation steps were as follows

(a) determine 120582 as 15406 A(b) set 119884 = 120573 cos 120579120582 119883 = sin 120579120582 and the linear

function was 119884 = 119860 + 119861119883 in which 119860 = 119870119871 and119861 = 4119890

(c) while119870 = 09 119871 = 185 nm

Calcium carbonate crystallite size based on the Scherrerequation was calculated for 185 nm


(a) The inlet

(b) The middle

(c) The outlet

(d) White precipitate particles

Figure 5 SEM photos white spots calcite particles and greyirregular solids sand particles

34 Crystal Structure Analysis TEM morphological photoswere taken as shown in Figure 6 and the flaky outward growthof the nano-CaCO

3was observed The diffraction pattern of

the nano-CaCO3was also taken to be analyzed as follows

(a) Input the diffraction pattern into Photoshop CS5 andread data as follows 119909

1= 516 pixels 119910

1= 1237 pixels

1199092= 1968 pixels 119910

2= 915 pixels 119899

1= 7 119909

3= 725

pixels 1199103= 380 pixels 119909

4= 1595 pixels 119910

4= 1802

pixels and 1198992= 3 as shown in Figure 7

1200

800

400

0

Lin

(cou

nts)

Calcite

15 30 45 602120579

Figure 6 Microbe-inspired precipitate XRD

200 nm

(a)

50 nm

(b)

Figure 7 TEMmorphological photos of white precipitate (a) times250(b) times1000

(b) Input the above data into a self-edited calculationprogram in Matlab The results were 119889

1= 30651 A

1198892= 30358 A and 120579 = 745448∘

(c) Since 11988911198892 asymp 1 120579 = 745448∘ 1198891119886 = 09934 and119886 = 30450 A were deduced from the 119891

119888119888structure

with the general error scope of 10 as 119886 and 5as 120579


R2

R1

h1k1l1

h2k2l2(x3 y3)(x2 y2)

(x4 y4)(x1 y1)

500 nmminus1

(a) (b)

Figure 8 (a) TEM diffraction pattern of CaCO3precipitate and (b) PCPDFWIN-2002 search result ID 86-2334 calcite

(d) In alignment with the 52 inorganic crystal composedof three elements of Ca C and O in PCPDFWIN-2002 the precipitate was identified as ID86-2334calcite calcium carbonate as shown in Figure 8

4 Conclusion

This paper introduced an innovative bioengineering methodof microbial carbonate precipitation in porous media [6]The whole material preparation procedure incorporates thereaction fluid flow solute transport crystal growth anddeposition on sand grains The final objective is an industry-scale application for geological material consolidation andstrengthening such asmineral plugging immobilizing heavymetal ions in ground water and strengthening of concrete orsand [7ndash11]

References

[1] P Cacchio C Ercole G Cappuccio and A Lepidi ldquoCalciumcarbonate precipitation by bacterial strains isolated from alimestone cave and from a loamy soilrdquoGeomicrobiology Journalvol 20 no 2 pp 85ndash98 2003

[2] R P Reid P T Visscher A W Decho et al ldquoThe role ofmicrobes in accretion lamination and early lithification ofmodern marine stromatolitesrdquo Nature vol 406 no 6799 pp989ndash992 2000

[3] A Sharma andA Bhattacharya ldquoEnhanced biomimetic seques-tration of CO

2into CaCO

3using purified carbonic anhydrase

from indigenous bacterial strainsrdquo Journal of Molecular Cataly-sis B vol 67 no 1-2 pp 122ndash128 2010

[4] J Geotech and E Geoenvir ldquoUrease effects on urea hydrolysisrdquoBiological Considerations in Geotechnical Engineering vol 131no 10 pp 1222ndash1233 2005

[5] S Stocks-Fischer J K Galinat and S S Bang ldquoMicrobiologicalprecipitation of CaCO

3rdquo Soil Biology and Biochemistry vol 31

no 11 pp 1563ndash1571 1999

[6] H Eccles ldquoTreatment ofmetal-contaminatedwastes why selecta biological processrdquo Trends in Biotechnology vol 17 no 12 pp462ndash465 1999

[7] S-W Lee S-B Park S-K Jeong K-S Lim S-H Lee andM C Trachtenberg ldquoOn carbon dioxide storage based onbiomineralization strategiesrdquoMicron vol 41 no 4 pp 273ndash2822010

[8] JDejong ldquoMicrobially inducedCaCO3cementation to improve

soil behaviorrdquo Sedimentary Geology vol 2 pp 769ndash773 2007[9] V S Whiffin M P Harkes and L A van Paassen ldquoMicro-

bial carbonate precipitation as a soil improvement techniquerdquoGeomicrobiology Journal vol 24 no 5 pp 417ndash423 2007

[10] L Li C X Qian and R X Wang ldquoA lab investigation of MIPmethod used in Cd++ contaminated soilrdquo Journal of Soil andSediments vol 25 no 2 pp 331ndash335 2010

[11] J K Stolaroff G V Lowry and D W Keith ldquoUsing CaO- andMgO-rich industrial waste streams for carbon sequestrationrdquoEnergy Conversion andManagement vol 46 no 5 pp 687ndash6992005

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Advances in

Materials Science and EngineeringHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Smart Materials Research



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Table 1 Calculated porosity structure factors of MIP data report of sand columns

No Daysd Grades Permeable area10minus3 120583m2 Total porosity Pore volumemLg Specific aream2m Pore throatporeA1 5 Super low 008356 40466 02500 00166 38286A9 10 Super low 011084 35724 02119 00169 41489A13 15 Super low 003961 35538 02137 00186 28909A20 20 Super low 001479 27855 01437 00236 38554A27 28 Super low 000044 27305 01394 01170 04865B1 28 Super low 001210 18183 01008 00079 46848

Table 2 Permeability grades of rocks

Grades 1 2 3 4 5 6 7Permeability Super high Very high High Medium Low Very low Super lowPermeable area10minus3 120583m2

gt1000 1000sim500 500sim100 100sim50 50sim10 10sim1 lt1

Inlet

OutletSand column

Figure 1 Photos of microbe-inspired CaCO3cementation

vibrations into a plastic injection tube 22mm in diameterand 95mm in length Those tubes filled with sand wereprepared for microbe-inspired CaCO

3cementation [4] This

endeavor was a method of reaction fluid gravitational per-mease and external pressure [5] It was to make the sandcompact as much as possible under 01MPa axial pressuresduring the whole cementation An iron frame was designedto allow tubes to locate in it steadily and properly (Figure 1)

23 Mercury Intrusion Porosimetry Method Porosimetrysuch as pore diameter total pore volume superficial areaand pore throat ratio was measured to demonstrate nano-CaCO

3-sand structure of samples taken as Figure 2 under

PoreMaster-60 of Quantachrome USA The sizes and thesphere of distribution of pores and throats and their arrange-ment relating to each other were determined by mercuryintrusion-extrusion capillary pressure curves And the porethroat aspect ratio was that of pore radius to the relevantthroat radius which indicated their dimensional arrange-ment in material (Figure 3)

24 X-Ray Diffraction Samples were taken from calcite pre-cipitate in sand columns to make X-ray diffraction analysisGrain size phase retrieval and lattice constant refinementwere conducted on MDI JADE 5

25 Transmission Electron Microscope Calcite precipitatemorphology was observed under Tecnai T20 The latticediffraction pattern was taken to identify the phase of theprecipitate with powder diffraction database of PCPDFWIN-2002

26 Scanning Electron Microscope SEM samples were takenfrom both ends and also from the middle part of the sandcolumns shown in Figure 5 to observe the amount and thelocation of the calcite precipitate under JSM-5900 of SirenJapan

3 Results and Discussion

The characterizations of the white precipitate in cementingthe loose sand were discussed focused on porosity and per-meability morphological observation phase identificationgrain size calculation and crystal type determination

31 Porosity As shown in Figure 2 from which MIP sampleswere taken plenty of data were obtained from the porositystandard report ofMIP such as permeable area total porosityvolume total porosity volume fraction specific superficialarea and pore throat aspect ratio all of which were listedin Table 1 Compared with the permeable area of grades inTable 2 permeability of all the sand columns fell into thesuper low grade

The pore throat aspect ratio of sand columns at 672hours sharply decreased to 049 from 383 at 120 hours(Figure 4)Thepore throat aspect ratio indicated dimensionalarrangement of pores and throats and it explained thereduction of pore radius when more and more precipitationformed and filled in the pores with increasing cementationtime


15 mm 15 m

m15

mm

Inlet

Outlet

(a)

Inlet


(b)


Sand radius

Pore radius

Pore throat radius


008

010

012

014

016

018

020

022

024

026

Pore

thro

at-p

ore r

atio


5

4

3

2

1

0

Sand column types

28-d

ayno

-pre

ssin

gpl

uggi

ng

5-da

ypr

essin

g

28-d

aypr

essin

g

20-d

aypr

essin

g

15-d

aypr

essin

g

Poro

sity

volu

me (

mL

g)

10-d

aypr

essin

g









120573 =119870120582

119871 cos 120579+ 4119890 tg 120579 (5)

or

120573 cos 120579120582=119870

119871+4119890 sin 120579120582

(6)





(c) while119870 = 09 119871 = 185 nm



(a) The inlet

(b) The middle

(c) The outlet







1= 516 pixels 119910

1= 1237 pixels

1199092= 1968 pixels 119910

2= 915 pixels 119899

1= 7 119909

3= 725

pixels 1199103= 380 pixels 119909

4= 1595 pixels 119910

4= 1802


1200

800

400

0

Lin

(cou

nts)

Calcite

15 30 45 602120579


200 nm

(a)

50 nm

(b)



1= 30651 A

1198892= 30358 A and 120579 = 745448∘





R2

R1

h1k1l1

h2k2l2(x3 y3)(x2 y2)

(x4 y4)(x1 y1)

500 nmminus1

(a) (b)



4 Conclusion


References




2into CaCO






no 11 pp 1563ndash1571 1999















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




15 mm 15 m

m15

mm

Inlet

Outlet

(a)

Inlet


(b)


Sand radius

Pore radius

Pore throat radius


008

010

012

014

016

018

020

022

024

026

Pore

thro

at-p

ore r

atio


5

4

3

2

1

0

Sand column types

28-d

ayno

-pre

ssin

gpl

uggi

ng

5-da

ypr

essin

g

28-d

aypr

essin

g

20-d

aypr

essin

g

15-d

aypr

essin

g

Poro

sity

volu

me (

mL

g)

10-d

aypr

essin

g









120573 =119870120582

119871 cos 120579+ 4119890 tg 120579 (5)

or

120573 cos 120579120582=119870

119871+4119890 sin 120579120582

(6)





(c) while119870 = 09 119871 = 185 nm



(a) The inlet

(b) The middle

(c) The outlet







1= 516 pixels 119910

1= 1237 pixels

1199092= 1968 pixels 119910

2= 915 pixels 119899

1= 7 119909

3= 725

pixels 1199103= 380 pixels 119909

4= 1595 pixels 119910

4= 1802


1200

800

400

0

Lin

(cou

nts)

Calcite

15 30 45 602120579


200 nm

(a)

50 nm

(b)



1= 30651 A

1198892= 30358 A and 120579 = 745448∘





R2

R1

h1k1l1

h2k2l2(x3 y3)(x2 y2)

(x4 y4)(x1 y1)

500 nmminus1

(a) (b)



4 Conclusion


References




2into CaCO






no 11 pp 1563ndash1571 1999















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




(a) The inlet

(b) The middle

(c) The outlet







1= 516 pixels 119910

1= 1237 pixels

1199092= 1968 pixels 119910

2= 915 pixels 119899

1= 7 119909

3= 725

pixels 1199103= 380 pixels 119909

4= 1595 pixels 119910

4= 1802


1200

800

400

0

Lin

(cou

nts)

Calcite

15 30 45 602120579


200 nm

(a)

50 nm

(b)



1= 30651 A

1198892= 30358 A and 120579 = 745448∘





R2

R1

h1k1l1

h2k2l2(x3 y3)(x2 y2)

(x4 y4)(x1 y1)

500 nmminus1

(a) (b)



4 Conclusion


References




2into CaCO






no 11 pp 1563ndash1571 1999















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials




R2

R1

h1k1l1

h2k2l2(x3 y3)(x2 y2)

(x4 y4)(x1 y1)

500 nmminus1

(a) (b)



4 Conclusion


References




2into CaCO






no 11 pp 1563ndash1571 1999















CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials










CeramicsJournal of







Biomaterials




Journal of


Journal of





CoatingsJournal of

Advances in








MaterialsJournal of


Nano

materials



research article pores and microanalysis of microbe...

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