Investigation of bacterial activity on compressive strength of cement mortar in different curing mediD Hemalatha Thiyagarajan , SrinivasaQ MaheswarDQ, Maitri Mapa , Sarayu Krishnamoorthy , Bhuvaneshwari Balasubramanian, Avadhanam Ramachandra Murthy, and Nagesh R. Iyer

Journal of Advanced Concrete Technology, volume 14 ( 2016 ), pp. 125-133

Crack Self-healing Behavior of Cementitious Composites incorporating Various Mineral Admixtures

Tae-Ho Ahn , Toshiharu Kishi Journal of Advanced Concrete Technology, volume 8

( 2010 ), pp. 171-186

Development of Engineered Self-Healing and Self-Repairing Concrete−State-of-the-Art Report

Hirozo Mihashi , Tomoya Nishiwaki Journal of Advanced Concrete Technology, volume 10 ( 2012 ), pp. 170-184 Robust Self-Healing Concrete for Sustainable Infrastructure

Victor C. Li , Emily Herbert Journal of Advanced Concrete Technology, volume 10 ( 2012 ), pp. 207-218

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Journal of Advanced Concrete Technology Vol. 14, 125-133 April 2016 / Copyright © 2016 Japan Concrete Institute

Scientific paper

Investigation of Bacterial Activity on Compressive Strength of Cement Mortar in Different Curing Media *

Hemalatha Thiyagarajan1, Srinivasan Maheswaran2, Maitri Mapa3 , Sarayu Krishnamoorthy4, Bhuvaneshwari Balasubramanian5, Avadhanam Ramachandra Murthy6 and Nagesh R. Iyer7 Received 15 July 2015, accepted 22 March 2016

doi:10.3151/jact.14.125

Abstract Recently, bacterial concrete is gaining popularity due to the advantages such as self healing property, increased durability, enhanced strength etc. When living organisms are introduced into cementitious materials, many factors are expected to influence their activity. A conducive environment such as temperature, nutrients, pH etc. is necessary for the survival and intended activity of bacteria in concrete. Among all the influential factors, nutrients for the survival of bacteria in cement mortar is considered in this study. In order to supply nutrients for the bacterial growth, three different curing media such as normal water, Luria Bertania broth (controlled nutrients) and wastewater (uncontrolled nutrients) are chosen. Compressive strength are determined at various stages of curing to study the influence of bacterial activity in cement mortar. Further, X ray diffraction and thermo gravimetric analysis are carried out to understand the transformation in hydration phases with the incorporation of bacteria. From the strength study conducted, cubes cured in waste water showed more strength which indicates that the bacterial activity is more in waste water.

1. Introduction The emerging technologies towards the evolution of concrete from ordinary concrete to different types of concrete such as high strength (HS), high performance (HPC), ultra high performance (UHPC), self compacting (SCC), fibre reinforced (FRC) concrete etc. is developing at the faster rate to achieve the goal in terms of strength and durability. In the past few decades enormous work has been carried out in order to improve the performance by means of application of various materials and technologies. Microbial mineral precipitation resulting from metabolic activities of some specific microorganisms in concrete to improve the overall behaviour of concrete has become an important area of research. In the recent past, researchers found that compressive strength and durability of cementitious system

1

Scientist, CSIR-Structural Engineering Research Centre, Chennai, India. 2 Principal Scientist, CSIR-Structural Engineering Research Centre, Chennai, India. 3 Formerly Quick Hire Fellow, CSIR-Structural Engineering Research Centre, Chennai, India. * Corresponding author, E-mail: [email protected], [email protected] 4 Formerly Quick Hire Fellow, CSIR-Structural Engineering Research Centre, Chennai, India. 5 Formerly Senior project Fellow, CSIR-Structural Engineering Research Centre, Chennai, India. 6 Principal Scientist, CSIR-Structural Engineering Research Centre, Chennai, India. 7 Former Director, CSIR-Structural Engineering Research Centre, Chennai, India.

can be improved with the inclusion of micro organisms (Ramachandran et al. 2001; Ramakrishnan et al. 2001; Ghosh et al. 2005; Siddique and Chahal 2011). Extensive research has been carried out with different species of bacteria in improving the strength and durability of concrete (De Muynck et al. 2008; Ghosh et al. 2005; Ghosh et al. 2009; Yang and Cheng 2013; Jonkers and Schlangen 2007; Jonkers and Schlangen 2008; Wang et al. 2012; Chahal et al. 2012). Microbiologically induced mineral precipitation improves the strength by about 25% (Ghosh et al. 2005) to 33% (Abo-El-Enein et al. 2013) according to the reported literature. The reasons for strength improvement as reported (Ghosh et al. 2009) are due to a) the growth of filler material within the pores of the cement mortar b) the formation of new phases of silicates and c) uniform distribution of silicate phases and increased Ca/Si ratio within the C-S-H gel of the matrices due to bacterial treatment. The effects of carbonate precipitation by bacteria on the durability of mortar specimens are widely studied and reported (De Muynck et al. 2008). It is found that the surface deposition of calcium carbonate crystals decreased the water absorption by 65 to 90% depending upon the filler capacity of the specimens. As a result, the carbonation rate and chloride migration reduced by about 25 to 30% and 10 to 40%, respectively. Water absorption of cement sand mortar is decreased through the calcium carbonate precipitation by microbiologically induced process (Abo-El-Enein et al. 2013). De Muynck et al. (2008) found the reduction in capillary water uptake and permeability towards gas by the bacterial deposition of calcite on the surface of the specimens. Further, they found that the type of bacterial culture and medium composition had an impact on CaCO3 crystal

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morphology. Numerous work has been carried out to investigate the possibility of application of bacteria in self healing of the concrete to extend the lifetime of concrete. Jonkers and Schlangen (2008) studied the self healing property of bacterial concrete and found that bacteria mediate the production of minerals which seal the freshly formed cracks which in due course decreases the concrete permeability. Also, they found 100 µm sized calcite particles can possibly seal the micro to macro sized cracks. Hence, this bacterial concrete represent a new type of durable and sustainable concrete with a wide range of potential to solve the problems. Meanwhile, the application of bacteria for the repair or maintenance of various materials is not new and in previous studies, the potential of bacteria to clean concrete surfaces (DeGraef et al. 2005), improve the strength of cement-sand mortar (Ghosh et al. 2005), repair of degraded limestone and ornament stone surfaces and crack repair on surfaces of concrete structures were also investigated. When cement reacts with water, the alkalinity of cement paste increases due to the formation of hydration products, this causes a problem for the survival of bacteria in bacteria incorporated cement. Numerous attempts have been made to protect the bacteria from the alkalinity of cement. Wang et al. (2012) investigated the possibility of using silica gel or polyurethane as the carrier for bacteria to protect the bacterial activity as it is affected by high pH environment in concrete. It was found that the silica gel immobilized bacteria showed higher activity than polyurethane immobilized bacteria. However, cracked mortar specimens healed by polyurethane immobilized bacteria had a higher strength regain and lower water permeability coefficient compared with specimens healed by silica gel immobilized bacteria. Another function of bacteria was explored by Grabiec et al. (2012) in which they studied the surface modification of recycled aggregate concrete by bio-deposition involving a method of employing Sporosarcina pasteurii (Bacillus pasteurii) bacteria. It was possible to obtain reduction in water absorption of aggregate and the effect was more visible for finer fractions and the aggregates originating from inferior quality concrete. In their work, calcium chloride was used for precipitation of calcium carbonate, while culture medium consisting of beef extract, peptone and urea was used for cultivation of microorganisms. In addition, whey which is an ecologically dangerous by-product from dairy industry was found to be effective as a culture medium. Presence of calcium carbonate crystals covering aggregate grains as confirmed by observations under scanning electron microscope. In this perspective, the proposed method, upon appropriate improvements, seems worthwhile due to ecological and technological reasons. The influence of bacterial count was studied by Jonkers and Schlangen (Jonkers and Schlangen 2007). They studied the incorporation of high numbers of bac-

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teria (109 cell/ml) as well as some suitable organic growth substrates in concrete and found that the immobilized cells have the potential of self healing property though it did not affect the compressive and flexural tensile strength. Kim et al. (2013) investigated the characteristics of microbiological precipitation of calcium carbonate on normal and lightweight concrete by two types of bacteria, Sporosarcina pasteurii and Bacillus sphaericus. It was found that B:sphaericus precipitated denser calcium carbonate crystals than S:pasteurii. Moreover, the concrete specimens treated by the medium with B:sphaericus showed the lowest weight increase per unit area. From the various studies reported, it is understood that for the survival of bacteria, like all other living organisms, food, water, pH and appropriate temperature is essential. Most bacteria can decompose organic materials and receive nourishment in this process. Here, in this study such nutrients are supplied to bacteria by controlled amount of nutrients by preparing Luria Bertania Broth (LB) and by wastewater (uncontrolled amount of nutrients). In general, LB has also been used as a general-purpose bacterial culture medium for a variety of facultative organisms. As waste water itself is full of organic material and nutrients, bacteria can break down this organic material to obtain energy and utilize for its cell growth in a similar way that humans gain energy and material for growth from their food. Numerous studies have been reported in the recent past with the incorporation of bacteria in concrete. Although strength improvement, self healing property and permeability control due to incorporation of bacteria are widely reported, influence of bacterial growth in enhancing all these properties are rarely addressed. In this study, supply of nutrients is considered as a crucial parameter for the survival of bacteria among the various factors affecting the bacterial growth.

2. Experimental details 2.1 Preparation of culture Bacteria isolated from ant hill and identified as Bacillus licheniformis is used in this work. Bacteria are inoculated into autoclaved liquid culture medium and grew in a incubating shaker for 48 hours. After growing for two days, the bacterial cell concentration is determined by optical density test. The bacteria culture medium is then centrifuged at 10,000 rpm for 30 mins to obtain highly concentrated bacterial cells. The concentrated bacteria cells obtained from centrifuge are diluted to get two concentrations such as 106 and 108 cells/ml. Serial dilution is followed in obtaining the required concentration of cells. A serial dilution is the stepwise dilution of a substance in solution. Usually the dilution factor at each step is constant, resulting in a geometric progression of the concentration in a logarithmic fashion. A ten-fold serial dilution could be 1M, 0.1 M, 0.01 M, 0.001 M, etc. Serial dilutions are used to accurately create highly di-

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2.2 Preparation and casting of test specimens Cement mortar cubes of size 70.6 mm are prepared for measuring compressive strength. The specimens are cast in accordance with Indian Standard IS: 516-1959 (IS 516. 1959). In this study, the bacteria isolated from dry ant hill is used with two different concentrations of 106cells/ml and 108cells/ml in the cement mortars and cement pastes. For incorporating bacteria in cement mortar, the calculated water is entirely replaced by bacterial culture. Cement mortar cubes with cement to sand ratio of 1:3 are cast and subjected to different types of curing such as normal (NC), Luria Bertania Broth (LBC) and waste water curing (WWC) in order to supply controlled and uncontrolled nutrients for the growth of microorganisms. Three cubes without any bacterial culture are also cast and subjected to normal curing (C). The sequence of procedure adopted in mixing of mortar and additive rate for bacteria are provided as follows: 1. Materials (cement and sand) are dry mixed for first 30 seconds at very slow speed to ensure homogeneous mixing of cement and sand. 2. Then, 80% of the total bacterial culture required to get adequate consistency is added and mixed thoroughly for 60 seconds at a medium speed. 3. After that, mixer is stopped and sides of mixer basin is scrapped and again mixed for 30 seconds. 4. Then, remaining 20% of bacterial culture is added and mixing is continued for another 60 seconds and finally, the paste is mixed for 30 seconds at higher speed before poured in the respective moulds. Three numbers of cubes each for 3, 7, 28, 56 and 90 days accounting for the total of 15 cubes for each type of curing are cast towards compressive strength studies. After casting, the specimens are allowed to remain in iron molds for first 24 h at room temperature (27± 2)°C and demoulded and cured in different curing media till the day of testing. Another set of cubes made of same mix without bacteria is also prepared for waste water curing to study the influence of curing media.

3. Results and discussion 3.1 Consistency and setting time The standard consistency test and initial and final setting time tests have been carried out for the mixes with two different concentrations of culture and also one with water without any bacteria and results are shown in Table 1. The bacterial culture required for standard consistency of cement is found to be 29% and for one without bacterial culture the water requirement is found to be 30.5%. It is noticed that with bacterial culture, the water requirement is lesser than the control for obtaining the

Table 1 Results of Consistency, Initial and Final setting time test.

Consistency Initial setting Final setting

Fresh properties Control cement Cultured cement 30.5% 29% 215 mins 230 mins 420 mins 450 mins

standard consistency. The initial and final setting time values for both the medium with cell count 106 and 108 are found to be the same and obtained as around 230 minutes and 450 minutes, respectively. It is noted that the bacterial concentration is not influencing the setting times. The initial and final setting time of control without culture is obtained as 215 and 420 minutes, respectively. The initial setting time of cultured cement is delayed by 15 minutes in comparison with control cement. Besides determining the time available for transportation and placement, it is necessary to have the control of on set of hydration, hence, initial setting time of mixes are useful in understanding the effectiveness of addition of bacterial culture into cement. The results show that the initial and final setting time of the mixes with bacterial culture is slightly higher than that of control. 3.2 Compressive strength To study the effect of addition of bacteria in the cement mortar, mortar cubes are prepared and cured in different curing media. It is necessary to probe, how long the bacteria are survived in the cement for its intended purpose, hence, compressive strength has been measured at 3, 7, 28, 56 and 90 days of curing in this study. As mortar cubes are prepared with same composition, only variation being curing media here, any change observed in the compressive strength is exclusively due to the bacterial activity in the cement environment. The compressive strength results of bacteria (108) incorporated mortar cubes along with the control specimens are shown in Fig. 1. It is observed that there is an improvement in the strength from 3rd to 28th day in all the 60

3 days 7 days 28 days 56 days 90 days

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Compressive strength (MPa)

luted solutions as well as solutions for experiments resulting in concentration curves with a logarithmic scale. Serial dilution is also a cheaper and simpler method for preparing cultures from a single cell than optical tweezers and micromanipulators.

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Fig. 1 Compressive strength of control and bacterial ce8 ment mortar cubes under different curing media (10 cell/ml).

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specimens ranging from 15% to 37%. However, there is no significant improvement after 28 days in control and bacterial cement mortar cubes cured in normal water (NC). This may be attributed to the fact that there is no bacterial action after 28 days in NC specimens due to inadequate nutrients. Whereas in LBC and WWC, there is a gradual improvement in strength of about 35% is noticed over a period of 90 days probably due to sufficient supply of nutrients to bacteria. WWC specimens show higher compressive strength than LBC that may be attributed to the presence of active bacteria in cement mortar as a result of presence of rich source of organic material in waste water. Further, to study the influence of concentration of cell on the strength of cement mortar, another set of cement mortar cubes with 106 cell/ml concentration is prepared and compressive strength is measured at 3, 7, 28, 56 and 90 days of curing. Figure 2 shows the compressive strength results of cement mortar cubes cured at various curing media at all ages upto 90 days. From the plot, it is clearly shown that there is a gradual increase in strength upto 28 days, after that it become stagnant upto 56 days. However, there is steep increase in strength is noticed after 56 days. Whereas in control mortar cubes there is no significant improvement in the strength after 28 days. In WWC, there is no significant improvement in strength after 7 days and upto 56 days whereas it suddenly shoots up at 90 days. The reason for this may be explained with the help of bacterial growth phase diagram as shown in Fig. 3. According to the reported literature (Skarstad et al. 1983; Zwietering et al. 1990; Novick 1955), the first phase of the bacterial growth is the lag phase which represents the period of time the organisms need to adapt to the new environment and this is the immediate phase after microbes have been added and population growth during this period is very close to zero. The second phase is log phase which represents the period of optimal population growth. During this phase, the microbes multiplies at the faster rate and approach the upper limit to their continued growth called the carrying capacity (k). Once the popu-

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Fig. 2 Compressive strength of control and bacterial ce6 ment mortar cubes under different curing media (10 ).

lation grows beyond k, it will run out of nutrients and space and may crash. Hence, beyond this point, bacterial population levels out and population growth nears zero again and is indicated by stationary phase and this stage may last for a long period of time. Finally, waste and dead cells begins to accumulate, causing the death phase. Though it does not completely crash, the population declines during this phase. Spore formers can persist beyond this stage and can regenerate a population if conditions once again become favorable. This growth phase of bacteria is directly reflected in strength improvement in WWC specimens. Gradual increase in strength is noticed from 3 to 28 days due to significant bacterial activity. However, there is no noticeable improvement from 28 to 56 days as interpreted by stationary phase. Whereas after 56 days a sudden increase in strength is observed which can be related to the growth phase of the bacteria. During exponential phase, due to the faster multiplication of bacteria, the bacterial activity is high that results in the gradual increase of strength. When bacteria multiplied exponentially, all the bacteria will not get nutrients sufficiently for its survival and starts dying. Due to that reason the

Fig. 3 Schematic diagram of bacterial growth phase (Skarstad et al. 1983; Zwietering et al. 1990; Novick 1955).

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Table 2 Analysis of waste water. Parameters Colour and appearance Total suspended solids (TSS) (mg/l) Total dissolved solids (TDS) (mg/l) pH Oxygen consumed from N/8 KMnO4 in 3 minutes (mg/l) 4 hours (mg/l) Bio-chemical oxygen demand (5th day at 20°C) (mg/l) Chemical oxygen demand (mg/l) Chlorides as Cl- (mg/l) Oil & grease (mg/l)

Results Grayish and turbid 205 985 8.2 26 32 60 121 248 Nil

and not due to any other outsourcing bacteria. Further, improvement in strength is not noticed from 28 to 90 days in cement mortar cured in normal water (C) whereas strength of mortar without bacteria cured in waste water showed strength improvement from 28 to 90 days. In conventional concrete cured in normal water, the hydration is expected to complete within 28 days and due to this reason, strength improvement is not significant after 28 days in cement mortar cubes cured in normal water in this study. Whereas, marginal increase in strength is noticed in cement mortar (without bacteria) cured in waste water. This may be due to the interference of characteristics of waste water in cement hydration process. Furthermore, tests (mechanical properties) have been repeated with the same kind of waste water for reproducibility, authors are able to get the similar results with about 5% error. Hence, the waste water of similar characteristics can provide reproducible results. 3.3 X-ray diffraction (XRD) XRD is defined as the analysis technique through which the compounds present in the sample can be identified based on the crystal structure of the compound. This method of analysis allows the identification of various compounds present in the sample using x-ray diffraction. 50 Compressive strength (MPa)

strength improvement is not so significant from 28 to 56 days. Later on, when bacterial count reduces to such number that, all the remaining bacteria gets sufficient nutrients and bacterial activity is resumed. Because of that there is an improvement in the strength again from 56 to 90 days. Considering the influence of bacterial counts, there is only a marginal difference in the strength obtained, when cell count is increased from 106 to 108. In WWC, upto 28 days at lower concentration the strength is high but at 90 days the strength improvement is more at higher concentration. In LBC, the variation in strength is only minimal. In NC, there exist a marginal improvement from 106 to 108. In mortars with bacteria cured in normal water (NC), there is no notable changes in the strength after 28 days compared to control mortar (C) and this may be due to the decrease in bacterial count owing to the lack of nutrients for its survival. Further, it is observed that the mortars cured in different curing media showed the significant improvement in compressive strength even after 28 days of curing. Mortars cured in LBC and WWC, there is a continual improvement in strength after 28 days upto 90 days. This increase in strength may be attributed to the presence of active bacteria in the cubes which results in the continuous precipitation of calcium carbonate for its strength improvement. The bacteria incorporated in cement mortar receives nutrients from Luria Bertania Broth and waste water for its survival. Further, it is noticed that among LBC and WWC, strength improvement in WWC specimens are considerably more than that of LBC cured mortars. This may be due to the presence of rich source of nutrients contained in waste water than in LBC. In order to confirm this statement, waste water is characterized through chemical analysis and results are presented in Table 2. Significance of waste water in curing can be explained from the waste water analysis. From the test results, it is found that considerable amount of total suspended solids and total dissolved solids are present in waste water which are helping bacteria for its survival by supplying adequate nutrients. Further, it is observed that COD value is very low which shows oxygen demand for oxidation of chemical constituents is less, hence, more oxygen may be available for the bacteria present in the mortar for its existence. When the conclusion of WWC provides better strength is drawn, there arises a question that the waste water itself has numerous bacteria, hence, the strength improvement is due to the incorporated bacteria? or bacteria present in the waste water?. To clarify this, another set of mortar cubes without bacteria is cured in waste water. The compressive strength results of mortar cubes with and without bacteria in waste water are shown in Fig. 4. This figure shows that the strength improvement in mortar cubes with incorporated bacteria is showing higher strength than without bacteria. This indicates that the strength improvement is mainly due to the incorporated bacteria

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With bacteria Without bacteria

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28 56 90 Mixes Fig. 4 Comparison of waste water cured mortar cubes with and without bacteria.

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The compressive strength results are best interpreted with the support of XRD results. XRD analysis is carried out to understand the influence of bacteria in forming the hydration products. X-ray diffraction (XRD) data of prepared materials is collected on a D2 PHASER Ni- filtered Cu K α radiation ( λ=1.542 ) equipped with one-dimensional LYNXEYE detector. It employs a Cu target radiation at 30 kV and 10 mA. Each sample is crushed and ground before mounting onto a glass fiber stub to examine the phase change. The components of the sample are identified by comparing them with standards established by the International Center for Diffraction Data (ICDD). XRD is carried out on cement samples with and without bacterial culture to identify the contribution of bacteria in the formation of hydration products. The powder crystal X-ray analysis of the samples with and without bacteria (Fig. 5) shows that there are some extra peaks in the XRD spectra of control samples which are absent in the bacterial samples. In bacterial cement pastes, the major peaks such as portlandite, C3S and C2S peaks are only dominant. From the typical XRD pattern of bacterial cement paste with 106 concentration, it is noticed that the background is more in control than in bacterial cement paste which indicates that more formation of calcium silicate hydrate gel at 28 days in control specimens. This is reflected in strength results too. The background of the typical XRD pattern at 28 days for two concentrations of bacterial culture have been given in Fig. 6. All the major peaks, porlandite (CH), C3S and C2S, carbonate (CC) are identified in all the XRD patterns however the bacterial incorporation does not form any new compound. Further, it is noticed that at lower concentration higher intensity peaks of portlandite are present. This indicates lesser conversion of portlandite to calcium silicate hydrate. The XRD spectrum obtained from the cement paste cured at 28th day of hydration reaction (Figs. 7 and 8) also reveals that the strain is efficient in increasing faster hydration to obtain C-S-H. Lesser intensity peaks at 108 concentration shows more formation of C-S-H and is reflected in the compressive strength results. Further, it is noticed that there is no extra peak and no characteristic changes in 2θ value of respective peak position in the XRD spectrum of powdered cement sample due to different curing media. This proves the absence of any inorganic material in different curing media and effectiveness of the different curing media to continue the pozzolanic reactions during hydration process. The considerable decrease in the intensity at 2θ value of 18.2° and 34.0° at 28 days in waste water curing compared to normal water curing confirms the utilization of portlandite to form C-S-H. XRD of the cement sample prepared with culture concentration of 108 cured in different curing media shows completely different trend in terms of CH formation compared to culture concentration of 106. With 108 concentration, considerable decrease in intensity of 2θ

Fig. 5 X ray diffraction of cement mortar with and without bacteria at 28 days.

Fig. 6 X ray diffraction of bacterial cement mortar with 8 6 two concentrations (10 and 10 cell concentration) cured in waste water.

Fig. 7 X ray diffraction of bacterial cement mortar (10 cell concentration) cured in different media.

8

value at 18.2° and 34.0° is noticed at 28 days in WC compared to samples in other two curing media. This reduction in intensity of CH may be attributed to the

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CH to C-S-H or calcite. As described by Ramachandran et al. (Ramachandran et al. 2001) the ettringite formation is supported by providing a favorable environment which is generally attributed to the free hydroxyl ions present in the cement matrix cured in LB. Strain is capable of precipitating high amount of portlandite at 18.1, 28.7, 34.1 and 47.1 degrees in presence of LB which indicates the normal reactive chemistry that prevailed in the cement paste with the presence of sufficient buffer that is required for further hydration. Furthermore, it indicates probable efficiency in long term bio- healing/strengthening when compared to other curing media.

Fig. 8 X ray diffraction of bacterial cement mortar (10 cell concentration) cured in different media.

6

formation of calcium silicate hydrate or calcium carbonate or both. Generally, compressive strength is enhanced either with the formation of hydration products (C-S-H, CASH) or by pore filling (CC). In this line, in the present study, the improved strength in mortar cubes cured in waste water may be due to the conversion of calcium hydroxide to calcium silicate hydrate or calcium carbonate. Formation of C-S-H is due to simple pozzolanic reaction whereas calcium carbonate forms mainly because of bacterial activity. LB and NC samples show more CH compared to waste water confirming the lesser formation of C-S-H. The principal peak at 15.8° and 23.0° (Fig. 7) confirms the formation of ettringite which is generally due to the combination of C3A with gypsum in the presence of calcium carbonate during the early stage of setting. Ettringite formation is observed in all the samples but a prominent peak is noticed in the cement paste sample cured with LB. The peak is comparatively weaker in the samples cured in waste water and normal water. Same trend in the peak intensity of ettringite is observed in 106 concentration also. XRD spectra characterized further for calcite peaks have shown the presence of major principal peaks at 2Ɵ of 29.41°, 35.5° and 56.4°. Considerable increase in the intensity of peaks due to calcite for the cement matrix cured in LB has demonstrated the efficiency of the strain in precipitating calcium carbonate by generating carbonate ions and utilizing calcium ions. Among the samples containing 108 cell counts scanned for the XRD spectra, the waste water cured sample is found to have the least calcite precipitation when compared to that of the sample cured in normal water followed by LB. However, samples containing 106 cell counts scanned for the XRD spectra shows minimum calcite precipitation and CH formation for normal water curing as well. The XRD patterns of two concentrations show slight difference in the intensity of peaks which indicates that strain concentration plays a major role in the cement paste which seems to be more effective in converting

3.4 Thermogravimetric analysis (TGA) TGA is defined as the analysis technique through which mass loss of the substance in a heated environment is recorded. This method of analysis allows the estimation of various compounds present in the sample using weight loss. Thermogravimetric analysis is carried out to quantify the compositional change in the control and bacterial specimens when subjected to various curing media. The bacterial activity which is reflected in the formation of hydration compounds can be obtained through TGA analysis. Typical TGA plots showing the weight loss of various compounds for control and WWC specimens at 28 days are shown in Figs. 9 and 10. In general, the weight loss at 25° to 200 °C is due to chemically bound water. Weight loss associated with dehydroxylation of CH takes place at the temperature between 420° and 540 °C. Further, the carbonate decomposition due to loss of carbon di oxide happens when specimen heated over 600 °C. In Figs. 9 and 10, it is noticed that the weight loss due to dehydroxylation of CH is observed between 420° and 520° C in both control and WWC. The weight loss of CH in WWC is lesser than control which indicates more quantity of CH is transformed to calcium silicate hydrate or calcium carbonate or both. Further, comparing the weight loss above 600 °C for control and bacterial samples, the bacterial sample showed more carbonate decomposition confirming more carbonate precipitation by bacterial activity. The combined effect of more C-S-H and carbonate precipitation in WWC may be responsible for the higher compressive strength as indicated by the improvement in strength in WWC specimens.

4. Discussion To study the influence of cell concentrations on the compressive strength of cement mortar, the compressive strength obtained at various stages of curing for two different concentrations are compared and typical plot at 28 days showing the variation in strength with cell concentration is presented in Fig. 11. This figure shows that at lower concentrations, the strength improvement is more in NC and LBC. This may be due to the reason that at higher concentrations, all the bacteria may not be able to survive due to the lack of nutrients in NC and

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LBC and thus the decrease in strength is noticed with increase in bacterial concentrations. Whereas in WWC, due to the presence of rich organic source, the bacteria may get adequate nutrients for bacterial activity and hence the higher strength. However, this is not the case with all the curing periods, as bacterial growth phase vary with the period. At 90 days, the strength is slightly higher in NC and LBC at higher concentration. Hence, it is difficult to generalize the capability of concentrations in the enhancement of the strength. Further, it is understood that during the initial curing period, microbial cells obtain good nourishment, because the cement mortar is still porous; but its growth might not be proper due to the completely new environment for microbes as mentioned earlier. This is indicated by the lag phase in Fig. 3. It may also be possible that as the pH of the cement remains high, cells are at inactive condition and as the curing period is increased, it starts growing slowly. This is happening during the exponential phase. Upon cell growth, calcite would have precipitated on the cell surface as well as within the cement mortar matrix which causes the cement mortar to gain strength. Once many of the pores in the matrix are plugged, the flow of the nutrients and oxygen to the bacterial cells stops, eventually the cells either die or turn into endospores and act as an organic fibers; this is associated with the increase of compressive strength of the mortar cubes at later stages. This explains the behavior of the relatively higher compressive strength value after 28 days in case of cement mortar cubes prepared with microbial cells. There is a measurable increase in compressive strength of cement mortar cubes prepared with Bacillus licheniformis in this study which is in agreement with the result reported in previous studies (Ramakrishnan et al. 2001; Bang et al. 2001).

5. Conclusion The bacterial activity in the presence of two different types of nutrients (LBC and WWC) have been studied with two different concentrations of bacterial cells. The

Fig. 10 Thermo gravimetric analysis of WWC sample at 28 days. 45 10

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40 35 Compressive strength (MPa)

Fig. 9 Thermo gravimetric analysis of control sample at 28 days.

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Fig. 11 Comparison of compressive strength with two different cell concentrations.

following conclusions are made. 1. Supply of nutrients play a significant role in the bacterial activity in cement mortar. In LBC where controlled amount of nutrients are present, the bacterial growth is limited compared to WWC which provides uncontrolled nutrients. 2. It is understood that waste water rich in organic sources supply sufficient nutrients for the survival of bacteria. From the results obtained with and without bacterial concentrations in cement mortar cured in waste water, it is revealed that the incorporated bacteria is playing a major role in strength improvement not the outsourcing bacteria present in the waste water. 3. The non uniformity of the strength gain over the period indicates that bacterial activity is highly dependent on the period of curing as well as type of curing medium.

H. Thiyagarajan, S. Maheswaran, M. Mapa, S. Krishnamoorthy, et al. / Journal of Advanced Concrete Technology Vol. 14, 125-133, 2016

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