Application of calcium carbonate forming bacteria on the durability of cement : 시멘트 내구성에 대한 탄산칼슘형성세균의 응용 :

박성진

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자료유형: 학위논문

저자정보: 박성진 (경북대학교, 경북대학교 대학원)

지도교수: 김사열.

발행연도: 2015

저작권: 경북대학교 논문은 저작권에 의해 보호받습니다.

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이 논문의 연구 히스토리 (2)

2015

Application of calcium carbonate forming bacteria on the durability of cement : 시멘트 내구성에 대한 탄산칼슘형성세균의 응용

박성진 생명과학부 2015.01 학위논문

2012

건축공학분야에서 탄산칼슘형성세균의 응용과 전망

박성진 , 김사열 한국미생물·생명공학회지 2012.01 학술저널

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시멘트 구조물과 건물은 많은 물리·화학적 인자들에 의해 풍화·침식 되는데, 그러한 환경요인들의 침식 작용에 의해 구조물 내부 공극은 계속적으로 확장된다. 이러한 공극 내부로 물과 이산화탄소, 염소이온 등이 스며들 게 되고 침투한 이온들에 의해 시멘트 구조물은 내구성에 치명적인 영향을 받는다. 이러한 외부 이온침투를 방지하기 위해 아크릴, 에폭시수지, Ba(OH)2 같은 많은 합성 케미칼들이 사용되어지고 있다. 하지만 이러한 케미칼들은 시멘트 구조물 표면에 적용성이 떨어지고, 원재료의 2차부식에 영향을 주기도 하며, 많은 휘발성 유독물질을 배출하기 때문에 인간과 환경에 해로운 영향을 준다.
이 문제를 해결하기 위해 비교적 친환경적이고, 내구성 증진과 보수에 효과적인 세균 탄산칼슘형성 작용이 응용되고 있다. 합성케미칼과 비교해 미세한 크기의 세균은 미세 균열부위에 적용이 가능하고 원재료와 결합력이 뛰어난 장점이 있다. 또한, 석회용액에 혼입해서 사용할 시 석회용액 단일로 사용할 때보다 훨씬 견고하게 원재료와 결합되는 효과가 있다. 현재까지 시멘트 표면의 균열부위를 충진 하거나, 시멘트 모르타르 제작시에 혼입하여 압축강도를 증가시킬 수 있음이 알려지고 있다.
세균 탄산칼슘형성 작용은 유레아 가수분해에 의해 유도된다. 유레아가 세균의 유레아제에 의해 분해되면 암모니아 이온을 방출 하는데 이것이 세균 주변 환경의 pH를 올린다. 이렇게 높아진 pH는 세균 세포벽이나 생물막 속 음극전하 부위에 결합된 칼슘이온과 탄산의 결합 mortive force로 작용한다. 세균 탄산칼슘형성작용은 크게 유도탄산칼슘형성작용과 제어탄산칼슘형성작용으로 나눌 수 있다. 세균 유도탄산칼슘형성작용은 세균의 대사과정중에 우연히 탄산칼슘결정 형성이 유도되어지는 것을 말하며 환경에 의존적인 현상이고, 제어탄산칼슘형성작용은 특정 유전자가 관여하여 탄산칼슘을 형성하는 것을 말하며 환경에 독립적인 현상이다.
이전 보고에 따르면, 시멘트 모르타르 표면에 형성된 Bacillus sphaericus의 탄산칼슘형성은 염소이온의 침투를 막고 냉해방지에도 효과가 있음을 보고 했다. 게다가 Micrococcus spp.에 의한 탄산칼슘형성 작용은 시멘트 모르타르 내부로 수분침투를 차단하고, 그 모르타르의 압축강도를 증가시켜 내구성을 증대 시켰다. 또한 다양한 탄산칼슘형성세균의 적용을 통해 종특이적으로 시멘트 구조물 내구성 증대에 영향을 줄 수 있어 다양한 세균의 분리 및 적용에 대한 가능성이 넓혀지기도 했다.
본 연구에서는, 첫 번째, 알려지지 않은 탄산칼슘형성세균의 분리 및 특징화를 위해 독도로부터 7종의 탄산칼슘형성세균을 분리, 동정 하였으며, 탄산칼슘결정형성을 정성 분석 하였다. 분리된 세균들은 시멘트 페이스트 균열부위에 적용하여 균열보수 효과를 검증했다. 시멘트 모르타르 제작시 세균을 혼입하여 압축강도 증가 효과를 검증했다. 두 번째, 시멘트 구조물 내부에서 높은 활성이 기대되는 호염성세균을 분리 및 특징화 하였다. 13종의 호염성 세균을 분리, 동정하고 그중, 생물막을 다량 형성하는 E. marinum KNUC513의 생체막 형성에 따른 압축강도 증진 효과를 검증했다. 시멘트 압축강도 증진에 생체막 형성이 연관된다는 것을 처음으로 확인 했다. 세 번째, B. subtilis 168의 탄산칼슘형성을 시멘트 페이스트 표면에 적용하는 것 만으로 압축강도를 증진 시킬 수 있음을 확인 했다. 168 생물침전에 의해 균열이 수복되고, 칼슘막이 형성 되었으며, 수분 침투가 방지됨을 확인 했다. 마지막으로 P. polymyxa E681의 항진균 활성 효과를 시멘트 페이스에 부과할 수 있는지 여부를 확인 했다. E681이 포함된 중성화된 시멘트 페이스는 멸균 증류수만 처리한 대조군과 달리 시멘트 표면 오염균인 A. niger의 생장을 막는 것을 확인 했다.
이 연구를 통해 새로운 탄산칼슘형성세균 및 호염성세균들이 시멘트 내구성 증진에 효과가 있음을 확인 했고, 시멘트 구조물 표면에 처리하는 방법을 통해 수분침투 방지, 균열보수, 압축강도 증진 효과를 볼수 있음을 확인 했다. 무엇보다도 세균의 항진균 활성효과를 시멘트 페이스트에 부여할 수 있음을 처음으로 확인 했다. 그래서 시멘트 내구성에 대한 세균의 응용범위가 확대되어 졌다.

Cement structures and buildings are damaged by several physical, chemical and biological factors (Shirakawa et al., 2011; Park et al., 2012a). In addition, continuous weathering and erosion of environmental factors causes dissolution of the mineral matrix in concrete material, resulting in increased porosity and decreased mechanical features (De Muynck et al., 2008; Dick et al., 2006; Park et al., 2012ab; Rodriguez-Navarro et al., 2003). To prevent the degradation of cement materials, several conservative treatments such as acrylic or epoxy resins and Ba(OH)2 solutions that polymerize the inside of the cement pores are used; however, these treatments commonly result in the formation of incompatible, often harmful surface films (De Muynck et al., 2010; Le. Me´tayer-Levrel et al., 1999; Park et al., 2012a; Rodriguez-Navarro et al., 1994). These treatments generally release noxious volatile compounds that are harmful to humans and environment (Sarda et al., 2009; Tiano et al., 1999; Park et al., 2012a, Wakefield et al., 1998).
To solve this problem, microbiologically calcium carbonate precipitation (MCCP) has been proposed as an eco-friendly and long lasting solution for the repairing and enhancement of the durability of cement materials (Clifton et al., 1982; De Muynck et al., 2008, 2010; Park et al., 2012a). In this method, the protective effects rely on the bacterially induced formation of acompatible carbonate precipitate on limestone, which, unlike the lime-water treatment, appears to be highly coherent (Le. Me´tayer-Levrel et al., 1999; Lewin et al., 1974; Park et al., 2012a).
MCCP is being applied for the aesthetic restoration of cement building destroyed by physi-cochemical attacks and to prevent the water penetration into the cement mortar (Park et al., 2012c). After determining the advantages of this technique, many related studies on architecture engineering filed about the application of microorganisms to increase cement material were reported in America and Europe (Park et al., 2012c). Research to date involving the use of MCCP in crack repair and protection have focused on filling of pours and cracks in cement paste (De Muynck et al., 2008, 2010; Park et al., 2012c), enhancement of compressive strength of cement mortar (Ghosh et al., 2005, 2009, Park et al., 2012c), and as a biodeposition on the surface of cement material to increase durability (Tiano et al., 1999, 2006; Park et al., 2012c, 2012).
MCCP is induced by bacterial urea hydrolysis, which increases the alkalinity of the environment (Park et al., 2010a). The induced alkalinity effect as a motive force on calcium carbonate precipitation and the negatively charged bacterial cell wall favors binding of divalent cations such as Ca2+ and Mg2+ as a nucleation site of crystal maturation (Douglas et al., 2003; Hammes et al., 2003, Myszka et al., 2009; Park et al., 2010ab). Biomineralization induced by bacteria occurs via two different mechanisms: biologically controlled and biologically induced biomineralization (Lewin et al., 1974; Park et al., 2010ab, 2012a). In biologically controlled mineralization, the nucleation and growth of an organic-inorganic mineral is controlled by microorganisms. Specifically, the microorganism synthesizes minerals in a form that is specific to that species, independent of environmental conditions (De Muynck et al., 2010; Park et al., 2010ab).
Conversely, biologically induced mineralization is largely dependent on environmental conditions (Rivadeneyra et al., 1994; Park et al., 2012a) with no specialized structures or specific molecular mechanisms involved (Chiara et al., 2007; Park et al., 2012a). An example of biologically induced mineralization is the induction of favorable mineralization in the environment by ureolytic calcite-forming bacteria (Ghosh et al., 2005; Park et al., 2012a).
Previous studies have shown the potential for the application of calcinogenic bacteria to improve the durability of concrete using different treatment methods (Park et al., 2010b; 2012a). De Muynck et al. (2008), investigated the biodeposition of Bacillus sphaericus for re-establishment of the calcium layer in different porosities (Park et al., 2012a). The authors found that biodeposition resulted in an increased resistance of mortar specimens toward carbonation, chloride penetration and freezing. Moreover, bacterial treatment showed a protection against degradation processes similar to those of conventional surface treatments such silanes, siloxanes, silicates and acrylates (De Muynck et al., 2010; Park et al., 2012a). Tiano et al. (1999) also reported decreased water penetration after biodeposition treatment of limestone with Micrococcus spp. and B. subtilis strains (Tiano et al., 2006). Unlike the surface treatment, Ramachandran et al. (2001) found that the addition of calcium carbonate forming bacteria could increase the compressive strength of cement mortar (18% increased) (Park et al., 2010a).
According to Ghosh et al. (2005), improvement of the compressive strength of mortar specimens is induced by the addition of the non-calcite forming bacteria, Shewanella, suggesting that the choice of the microorganism plays an important role in improvement of the compressive strength. More specifically, the production of extracellular polymeric substance (EPS) by the bacteria appeared to be of importance (Dreesen et al., 2004).
An exposed cement surface acquires a protective layer through the dissolution of carbonate within pore water, evaporation, and precipitation of calcite (De Muynck et al., 2010; Dreesen et al., 2004; Park et al., 2014). This layer is characterized by increased hardness and density compared to underlying layers (De Muynck et al., 2010; Dreesen et al., 2004; Park et al., 2014). However, the calcin layer is slowly degraded by atmospheric pollutants, losing its protective role (De Muynck et al., 2010; Park et al., 2014). According to Willem et al., (2010) the use of bacteria can contribute to the formation of new calcin layers on exposed cement surfaces, and this biodeposition should be regarded as an environmentally friendly method for the protection of decayed ornamental stone (Park et al., 2014).
The aims of this study is to investigate the application of the MCCP for improvement of the durability of cement material. Results showed that the MCCP could repair artificial forced cracks and enhance the compressive strength of cement mortar. Additionally, we suggest that the surface treatment of B. subtilis 168 could induce positive effects on cement block against water penetration, remediation of cracks on cement surface and improvement of compressive strength of cement structures. Above all, we indicated that the fungal inhibition by P. polymyxa E681 in cement paste is the first application and evaluation research that incorporates calcite formation with antifungal capabilities of microorganisms for an environment-friendly and more effective protection of cement materials. In this research, the conception of smart cement was expanded.

#Biomineralization #calcium carbonate precipitation #cement durability

CONTENTS
Contents ······················································································································· ⅰ
List of figures ············································································································· ⅴ
List of tables ·············································································································· ⅷ
1. General Introduction ································································································ 1
2. Isolation and characterization of calcium carbonate forming bacteria from Dok-do for improving the durability of cement ······················································ 6
2.1 Introduction ······························································································· 6
2.2 Materials and Methods ············································································ 7
2.2.1 Isolation of calcium carbonate forming bacteria ······················· 7
2.2.2 PCR amplification of 16S rRNA Genes ···································· 7
2.2.3 Crack remediation test ·································································· 8
2.2.4 Compressive strength test ····························································· 9
2.3 Results and Discussion ·········································································· 10
2.3.1 Isolation and characterization of CaCO3 induced by Dok-do isolates ························································································· 10
2.3.2 Validation of crack remediation ··············································· 13
2.3.3 Strength improvement of cement-sand mortar ························· 15
3. Application of alkaliphilic biofilm-forming bacteria to improve compressive strength of cement-sand mortar ················································································ 17
3.1 Introduction ····························································································· 17
3.2 Materials and Methods ·········································································· 19
3.2.1 Isolation of ABB ········································································ 19
3.2.2 PCR amplification of 16S rRNA genes ··································· 19
3.2.3 Bacterial growth in alkaline environment ····························· 20
3.2.4 Adhesion assay ············································································ 20
3.2.5 Microbiologically induced CaCO3 precipitation ······················· 21
3.2.6 Compressive strength test ··························································· 21
3.2.7 Inhibition of biofilm formation ················································· 22
3.2.8 Effect of biofilm formation of E. marinum KNUC513 on compressive strength ··································································· 23
3.2.9 Field emission scanning electron microscopy (FE-SEM) ······· 23
3.2.10 Nucleotide sequence accession numbers ································· 24
3.3 Results and Discussion ·········································································· 25
3.3.1 Isolation and characterization of alkaliphilic bacteria ············· 25
3.3.2 Compressive strength improvement ··········································· 30
3.3.3 Degradation of biofilm used Dnase I ····································· 33
3.3.4 Effect of biofilm forming on compressive strength of cement ·· 35
4. Application of Bacillus subtilis 168 as a multifuntional agent for improvement of the durability of cement mortar ·········································································· 38
4.1 Introduction ····························································································· 38
4.2 Materials and Methods ·········································································· 40
4.2.1 Bacterial strains and culture media ·········································· 40
4.2.2 Microbiologically controlled CaCO3 precipitation ··················· 40
4.2.3 Biodeposition on the surface of the cement paste ················· 41
4.2.4 Water permeability test ······························································ 41
4.2.5 Crack remediation test ······························································· 42
4.2.6 Compressive strength test ·························································· 43
4.2.7 Field-emission scanning electron microscopy images ············· 43
4.3 Results and Discussion ·········································································· 45
4.3.1 Microbiologically controlled CaCO3 precipitation ·················· 45
4.3.2 Biodeposition on the surface of cement paste ························ 49
4.3.3 Improvement of compressive strength ······································ 54
4.3.4 Remediation of cracks in cement paste ··································· 57
5. The effects of Paenibacillus polymyxa E681 on antifungal and crack remediation of cement paste ····················································································· 61
5.1 Introduction ····························································································· 61
5.2 Materials and Methods ·········································································· 64
5.2.1 Bacterial strains and culture media ·········································· 64
5.2.2 Microbiologically-controlled calcium carbonate precipitation ··· 64
5.2.3 Preparation of fungal spore suspension ···································· 65
5.2.4 MCCP on surfaces of cement specimens ································ 66
5.2.5 Crack remediation test ································································ 67
5.2.6 Compressive strength test ··························································· 67
5.2.7 Neutralization of cement paste containing microorganisms ··· 68
5.3 Results and Discussion ·········································································· 70
5.3.1 Microbiologically-controlled CaCO3 precipitation ···················· 70
5.3.2 Bacterial calcium deposition on cement surfaces ···················· 76
5.3.3 Effects of bacterial biodeposition on compressive strength and crack remediation ········································································ 78
5.3.4 Antifungal effects of bacteria-containing cement mortar ········ 81
5.3.5 Effects of bacterial cement pastes on fungal growth ············· 83
6. Summary ················································································································· 85
7. References ·············································································································· 86
국문 초록 ··················································································································· 95
감사의 글 ··················································································································· 97
Curriculum vitae ········································································································· 99

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