Microbial Concrete- A Sustainable Solution for Concrete Crack Repair and RehabilitationDr M V Seshagiri Rao, FIE, Professor in Civil Engineering, JNTUH College of Engineering, Hyderbad-85

V Srinivasa Reddy, MIE, Associate Professor in Civil Engineering, GRIET, Hyderbad-90

Abstract:

IntroductionModern man-made engineering materials demonstrate excellent mechanical properties, but the lack of the ability of self healing, i.e. the ability to remove or neutralize microcracks without (much) intentional human interaction, which is typical for most materials as encountered in nature. In contrast, natural materials, such as skin, wood, bones and skeletons, grass, etc, often have the special ability that they can, more or less autonomously, heal cracks and other forms of accidental damage.

Routes and mechanisms towards self healing behavior in engineering materials

In this paper the focus is on the physical or chemical mechanism used to obtain autonomous or stimulated healing. In the routes described above all healing actions were due to physico chemical reactions, not involving intermediate agents to control or accelerate the healingreaction. However, in one route the use of calcium carbonate-producing bacteria, as agent to control the healing action inconcrete, has been demonstrated successfully. Efficient sealingof surface cracks by mineral precipitation was observed whenbacteria-based solutions where externally applied by sprayingonto damaged surfaces or by direct injection into cracks.These treatments resulted in regained material strength and reductionof surface permeability [48, 49]. However, in the latterstudies bacteria or their derived enzymes were not applied asa truly self healing system but rather as an alternative, moreenvironmental friendly, repair system. To create autogenousself healing behaviour, at the Technical University Delft theoption of using viable bacteria as a matrix-embedded healingagent was explored [50, 51]. Major challenge in the latterapproach was to identify bacteria and their needed metaboliccomponents which are not only sustainable, but which alsodo not negatively influence other concrete characteristics. Moreover, this biological system must also feature a longtermself healing functionality, preferably in agreement withthe constructions service life. In the latter studies a specializedgroup of alkali resistant spore-forming bacteria affiliatedto the genus Bacillus was identified as an ideal self healingagent as the spores of these bacteria appeared particularly resistantto concrete incorporation and, moreover, feature lifespans of over 100 years [52]. Furthermore, once incorporatedin concrete, these bacteria appeared able to convert various

natural organic substances to copious amounts of large, over100-μm sized, calcium carbonate-based crystalline precipitates

Such a bacteria-based self-healing mechanism thus appearsto be a promising alternative to non-sustainable cementbasedhealing systems particularly because the formation oflarge crystalline precipitates potentially enables sealing oflarger than 100 μm-wide cracks. The formation of large precipitatescan be explained by the high local bacterial CO2production rates. Due to conversion of CO2 into carbonateions under alkaline conditions and subsequent reaction withfree calcium ions leached from the concrete matrix calciumcarbonate-based precipitates are formed. Furthermore, locallyproduced CO2 directly reacts with matrix portlandite (calciumhydroxide) crystals which contributes to calcium carbonatebasedmineral formation. The intrinsic CO2-producing capacitywith the concomitant production of large-sized precipitatesin the bacteria-based self healing concrete may result in a superiorself healing rate and capacity compared to traditional orengineered non-sustainable self healing cementitious systems.The beneficial effect of bacterial closure of the deformationinduced cracks has been demonstrated in water permeabilitytests. Even for quite substantial cracking the bacterial selfhealing concrete showed no residual permeability after allowingtwo weeks for crack healing. Such a restoration ofwater impermeability may be as important as the restorationof mechanical properties, in particular for underground constructions(tunnels, basements and underground garages) insloppy water carrying soils. While several technical issuesyet need to be solved, the route of bacteria supported selfhealing concrete seems very promising and is now enteringa pre-commericalisation stage.

To turn these experimental approachesunder laboratory conditions into commercially availablematerials and products, it is necessary to initiate research intothe quantification of the healing behaviour as a function ofthe damage characteristics and the healing mechanisms. Suchan extension is now becoming overdue.

Effect of ureolytic bacteria on concrete properties

Recently, microbial mineral precipitation resulting from metabolic activities of some specificmicroorganisms in concrete to improve the overall behavior of concrete has become an important areaof research. It has been hypothesized that almost all bacteria are capable of CaCO3 production because

precipitation occurs as a byproduct of common metabolic processes such as photosynthesis, sulfatereduction, and urea hydrolysis.

The application of concrete is rapidly increasing worldwide andtherefore the development of bacterial mediated concrete isurgently needed for environmental reasons. As presently, about8% of atmospheric carbon dioxide emission is due to cement production,mechanisms that would contribute to longer service lifeof concrete structures would make the material not only moredurable but also self repair, i.e., the autonomous healing of cracksin concrete. The potential of bacteria to act as self healing agent inconcrete has proven to be a promising future. This field appears tobe more beneficial as bacterial concrete appears to produce moresubstantially more crack plugging minerals than control specimens(without bacteria).

Introduction

The applicability of specifically mineral producing bacteria for sand consolidation and limestone

monument repair [4–8] and filling of pores and cracks inconcrete have been recently investigated [9–12]. In all these studiesso far, bacteria or derived ureolytic enzymes were externallyapplied on cracked concrete structures or test specimens, i.e., assurface treatment or repair system. An integrated healing agentwould save manual inspection and repair and moreover increasestructure durability. Addition of such an agent to the concrete mixturewould thus save both money and the environment as lessmaintenance and use of environmental friendly repair material isneeded.Microbial carbonate precipitation (biodeposition) decreases thepermeation properties of concrete. Hence, a deposition of a layer ofcalcium carbonate on the surface of concrete resulted in a decreaseof water absorption and porosity.

In the laboratory, bacteria are usually grown using solid orliquid media. Solid growth media such as agar plates are used toisolate pure cultures of a bacterial strain. However, liquid growthmedia are used when measurement of growth or large volumesof cells are required. Growth in stirred liquid media occurs as aneven cell suspension, making the cultures easy to divide and transfer,although isolating single bacteria from liquid media is difficult.The use of selective media (media with specific nutrients added ordeficient or with antibiotics added) can help identify specificorganisms.Most laboratory techniques for growing bacteria use high levelsof nutrients to produce large amounts of cells cheaply and quickly.

However, in natural environments nutrients are limited, meaningthat bacteria cannot continue to reproduce indefinitely. This nutrientlimitation has led the evolution of different growth strategies.

carbonate biomineralization

Biomineralization is defined as a biologically induced precipitationin which an organism creates a local micro-environment withconditions that allow optimal extracellular chemical precipitationof mineral phases

Anovel technique for the remediation of damaged structural formationshas been developed by employing a selective microbial pluggingprocess, in which metabolic activities promote precipitationof calcium carbonate in the form of calcite. Biomineralization ofcalcium carbonate is one of the strategies to remediate cracks inbuilding materials.Recently, microbial mineral precipitation resulting from metabolicactivities of some specific microorganisms in concrete toimprove the overall behavior of concrete has become an importantarea of research. It has been hypothesized that almost all bacteriaare capable of CaCO3 production because precipitation occurs as abyproduct of common metabolic processes such as photosynthesis,sulfate reduction, and urea hydrolysis [15].

When this hydrolysis occurs in a calcium-richenvironment, calcite (calcium carbonate) precipitates from solutionforming a solid-crystalline material. The binding strength of theprecipitated crystals is highly dependent on the rate of carbonateformation and under suitable conditions it is possible to controlthe reaction to generate hard binding calcite cement (or biocement).

Members of the genus Bacillus are Gram-positive,rod-shaped, endosporeforming bacteria commonly found in soil[26]. B. pasteurii, a member of this genus, converts urea to ammoniumcarbonate more actively than any other known bacterium.Therefore, B. pasteurii and other members of the Bacillus genusare pincorporated into studies to determine their influence on calciumcarbonate precipitation in various environments.

Microbiologically induced (also called ‘‘bacteriogenic’’) calciumcarbonate precipitation is comprised of a series of complex biochemicalreactions

The ammonia increasesthe pH in surroundings, which in turn induces precipitation ofCaCO3, mainly as a form of calcite.

In aqueous environments, the overall chemical equilibriumreaction of calcite precipitation can be described as [27]:

The solubility of CaCO3 is a function of pH and affected by ionicstrength in the aqueous medium.

Ca2+ is not likely utilized by microbial metabolic processes;rather it accumulates outside the cell. In medium, it is possible thatindividual microorganisms produce ammonia as a result of enzymaticurea hydrolysis to create an alkaline micro-environmentaround the cell. The high pH of these localized areas, without aninitial increase in pH in the entire medium, commences the growthof CaCO3 crystals around the cell. Possible biochemical reactions inurea–CaCl2 medium to precipitate CaCO3 at the cell surface

Bacteria-based self-healing concrete

Crack formation in concrete is common, but a typical phenomenonrelated to durability. Percolation of cracks may lead to leakage problems or ingressof deleterious materials, causing deterioration of the concrete matrix or corrosion ofembedded steel reinforcement. Durability can be enhanced by preventing furtheringress of water and other substances. In recent years a bacteria-based selfhealingconcrete is being developed to extend the service life. A two componenthealing agent is added to the concrete mixture. The agent consists of bacteria andan organic mineral precursor compound. Whenever cracks occur and water ispresent the bacteria become active and convert the incorporated organic compoundsinto calcium carbonate, which precipitates and is able to seal and blockcracks. This paper aims to review the development of bacteria-based self-healingconcrete, introducing the proposed healing system. Different stages in the developmentare discussed, and some recommendations for further research are given.

Self-healing is characterized by regaining performance after a defect occurs.Damage targeted in bacteria-based self-healing concrete particularly relates toincreased durability and leakage prevention and extending service life of concretestructures. Jonkers (2007) introduced a two-component healing agent to be addedto the concrete mixture, consisting of bacteria and a mineral precursor compound.Upon cracking the system is activated by ingress water. Bacteria convert the mineral

precursor compound into the mineral calcium carbonate, better known aslimestone. Precipitation of the limestone on the crack surface enables sealing andplugging of the cracks, making the matrix less accessible to water and other deleteriousmaterials. New studies will focus on further developmentof the system in order to make practical application of the material feasible.

Target for selfhealingconcrete is to reduce matrix permeability by sealing or blocking cracks.Healing agent is incorporated in the concrete matrix and acts without human intervention. To make the material technically and economically competitive, healingagent should be cheap in relation to the low price of concrete, remain potentiallyactive for long periods of time and be concrete compatible to not negatively affectmaterial characteristics.

Microbial healing

Microbial Concrete – Sustainable Solution to enhance concrete durability

Microbial concrete is a new type of sustainable, eco-friendly material that enhances concrete performance by the microbial deposition of calcium carbonate within concrete pores through molecular reactions reducing fissures and cracks. The use of these reactions to improve concrete performance at the microscopic and macroscopic level is investigated in this paper in terms of increase in strength and durability, environmental impacts, and economical aspects. Experimental results have shown a 27% increase in compressive strength at 28 days, a 13% reduction in volume of pores, and a 12% decrease in water absorption in concrete specimens. These outcomes were attributed to the activity of the bacteria, as observed by scanning electron microscopy (SEM). The cost analysis showed an increase in cost relative to conventional concrete. Finally, the environmental analysis showed a 26.8% reduction in carbon dioxide produced per cubic meter of mix concrete projecting to a 28.6% decrease of carbon dioxide emissions by the cement industry on a global scale.

INTRODUCTION Concrete is one of the most widely used construction materials in the world, and is actually the second most consumable product after water

Microbial concrete is produced by incorporating a certain species of calcium depositing bacteria, Sporosarcina pasteurii, formerly known as Bacillus pasteurii , into concrete. This bacterial species has also been investigated as a remedy for the liquefaction of soils under buildings, to replace potentially harmful epoxy chemicals [4]. However, the advantages of using this bacterium in concrete have only been recently discovered; and it is expected to have a positive impact on concrete properties, particularly permeability, strength, cost, and the effect on environment. Sporosarcina pasteurii is used to deposit calcite, a sealing agent, in the pores in concrete by the process of microbiologically induced calcium carbonate precipitation,

MICCP. The structure, distribution, and connectivity of these pores have a great influence on concrete strength and durability.

Inside the bacterial cell, urease hydrolyses urea into ammonia and carbonate. The latter directly decomposes, producing ammonia and carbonic acid. The resulting ammonia and carbonic acid equilibrate in water, inducing an increase in pH. According to Le Chatellier principle the equilibrium will shift in such away as to reduce the effect of the induced change. In this case the bicarbonate equilibrium is shifted to produce carbonate ions at the vicinity of the cell, and eventually in the entire solution as shown in (2).

From equation 1, the increase in the concentration of carbonate ions, finally leads to the deposition of calcium carbonate crystals. Several experiments have been conducted to investigate the effect of adding the bacteria to concrete. Achal et Al.(2010) mixed samples of mortar cement with a culture of bacteria. The compressive strength of the samples mixed with bacteria was increased compared to the control samples. Ramachandran et Al. (2001) studied the remediation of cracks and fissures by MICCP in concrete beams. Results showed better resistance to harsh environments in microbial concrete beams compared to control beams. In this paper, experimental work has been performed to test the qualities of microbial concrete, including compressive strength, permeability, and absorption. In addition, samples of microbial concrete and conventional concrete were observed under a scanning electron microscope. Furthermore, the implementation of this technology was examined in terms of cost and environmental effects, specifically its effect on the global carbon footprint of the cement industry.

MATERIALS AND METHODOLOGY A. Concrete mix The Portland cement bags were purchased locally from Holcim Liban, and were in accordance to ISO 9001:2008. The coarse aggregates used were mainly composed of crushed angular limestone purchased from a local quarry. Natural river sand with a fineness modulus of 2.82 was used as fine aggregates.

B. Bacterial suspension Bacteria known as Sporosarcina Pasteurii ATCC 11859 were used in this research. It was purchased from the American Type Culture Collection, USA through the Environmental Engineering Research Center at the American University of Beirut. In order to preserve the bacterial culture, a subculture was performed every 48 hours on Ammonia-Yeast Extract agar plates and incubated with no agitation at 30°C.

As for the bacterial solution, colonies of Sporosarcina pasteurii were inoculated in an Nutrient Broth-Urea (NBU) solution containing Nutrient Broth (0.8%), Urea (2%), Sodium Chloride (0.5%) and Calcium Chloride (2.775gr. per Lit). The bacteria-NBU solution was agitated for 24 hours at 37°C. Several experiments in the structural laboratory were conducted showing that the highest 28 day compressive strength increase is obtained with a concentration of 105 cells/ml. The concentration of bacteria was validated using miles and misra- serial dilution.

C. Concrete Mixing and Curing Two types of Concrete mixes were prepared for this research. The mixes had similar proportions of sand, coarse aggregates, cement and water. However, the main difference was in the water component: Tap Water was used for specimens without bacteria (Mix A), while NBU-Bacteria solution was used for specimens with bacteria (Mix B).

D. Compressive Strength Test Three specimens of each type were tested under compression at 7, 14 and 28 days after casting.

Research done by Achal (2011) and Chattopadhvav (2010) showed that no increase in strength will occur using a NBU solution without bacteria hence this research did not include specimens of that type.

E. Water Absorption and Volume of Voids Test To determine the total water absorption and measure the volume of voids in hardened concrete cylinders, a series of tests were conducted according to the ASTM C642-13.The following steps were performed: . 1. 28 days after casting, the specimens were oven dried at around 110°C for 24 hours, and the oven dried sample mass was measured. 2. The specimens were immersed in water at 21 oC for 52 hours, until two successive values of the surface-dried specimen’s mass showed an increase of less than 0.5% of the larger value. The final surface dry mass after immersion was obtained. 3. The specimens were boiled for 5 hours, cooled for 15 hours, and the surface-dried specimen was weighed. 4. The apparent mass of the specimen in water after boiling was found.

F. Scanning electron microscope (SEM) To prove presence of bacteria in the microbial concrete, the samples were observed under a scanning electron microscope (SEM) and compared to concrete samples without bacteria. Broken pieces of 28 day old concrete from the compression test that had been dried at room temperature for 48 hours were crushed to a maximum size of 1mm and attached to SEM stubs using carbon tape. Two stubs of each type of concrete were prepared.

G. Cost analysis The cost of using microbial concrete compared to conventional concrete is critical in determining the economic feasibility of the technology. To compare cost, the compressive strength of concrete samples was fixed at 38MPa. The microbial concrete mix (B38) gave a compressive strength of 37.85 MPa when tested at 28 days. The concrete mix design and corresponding prices are given in table 1 and those of the NBU-bacterial solution are given in table 2. A conventional

concrete mix (mix A38) was designed for a compressive strength of 38 MPa according to Mamlouk and Zaniewski (2011). The mix design and prices are given in table 3.

H. Environmental analysis

One of the major advantages of microbial concrete is the possible reduction of the carbon footprint of concrete production. The production of cement, particularly clinker production, is the major contributor to carbon dioxide emissions [10]. The carbon dioxide producing reaction in

cement manufacturing is given in (3)

Portland cement consists of 95% clinker of which 64% to 67% is Calcium Carbonate CaCO3 [10]; According to equation (3) this results in approximately 0.264 Kg of carbon dioxide emissions per kilogram of cement produced. Since microbial concrete yields the same compressive strength with decreased cement content, the amount of carbon dioxide emissions during the production of cement for 38MPa concrete was obtained for Mix A38 and Mix B38 and compared. Consequently, the ratio of carbon dioxide reduction was used to obtain the projected global decrease in emissions.

I. Calcium carbonate quantification A chemical experiment is being performed in the Environmental Engineering Research Center in order to track the deposition of Calcium Carbonate by the Sporosarcina Pasteurii in mortar cement. Calcium carbonate CaCO3 reacts with hydrochloric acid (HCl) to produce Carbon Dioxide gas through the following equation (4): HCl (aq) + CaCO3 (s) CaCl2(aq) + H2O(l) + CO2 (g) (4) Two types of cement specimens were casted: For the first type, 1 Kg of cement was mixed with 360 ml of water and for the second type; 1 Kg of cement was mixed with 360 ml of NBU-bacteria solution. 1 gram of each oven-dried sample was crushed to powder and tested at the 1st and 2nd days. Tests will continue on the 3rd, 4th,5th,7th , 14th , and 28th days respectively. The cementeous powder was led to a complete reaction with 200ml of a 1.2M HCl solution with agitation. The volume of gas produced was collected under water in an inverted measuring cylinder. The experimental set up is shown in Fig. 3. When bubbled into lime water, the gas

produced turned it from colorless to milky white, hence confirming the nature of the gas as

carbon dioxide.

II. RESULTS AND DISCUSSION A. Compressive strength test The following bar chart (Fig. 4) shows the compressive strength results at 7, 14 and 28 days for concrete cylinders of types A and B.

At 7 days-The average compressive strength of both types is comparable, marked by a 0.92% increase. Provided that the cylinders were somehow wet and the concrete did not completely harden, the compressive strength is mainly associated to the weakness of humid concrete during the early stages. The following results confirm that during the beginning of the curing period, the precipitation of calcite is slow and Sporosarcina pasteurii is being gradually activated. Furthermore, the results confirm that the same mixing process and design was adopted for both sets of specimens. Therefore, the comparative analysis can be accurately validated based on this set of specimens. At 14 days- The 14th day strength results show an increase of 12.48% in the specimens containing NBU-bacterial cells. This improvement is mainly due to the strengthening of concrete material and deposition of CaCO3 filling small sized pores. During bacterial growth, the calcium precipitation process occurs continuously, clogging the internal pores with calcium precipitate. The calcium carbonate precipitate prevents oxygen from reaching the bacterial cells leading to their death. The cells are transformed into endospores that act as organic solid fibers. The results are considered to be consistent since all obtained values are close within a range of 1.1 Mpa, and are within 60%-70% of maximum strength. At 28 days- At this stage, the concrete has completely hardened, small and big size pores are filled with bacteria. CaCO3 crystals were formed and developed on the external bacterial surface within the pore structure. The 27% strength increase is mainly due to the calcium deposition within the pores of the sample. The values obtained vary within a range of 1.35 Mpa, hence the results are considered properly accurate. Fig. 5 summarizes the compression test results showing an increase in compressive strength compared to control samples.

B. Water absorption and volume of voids test Water absorption experiment was conducted on three cylinders of each type (A and B). After being dried at 110 oC, the cylinders went through three 52-hour cycles of immersion and weighing. Fig. 6 shows the mass of water absorbed in each cylinder after the last cycle. It can be clearly seen that specimens of type B containing bacteria, absorbed 12.30% less water than the ones of type A. The reduction in mass of water absorbed is consistent throughout the samples and varies between 13g and 20g.

The volume of permeable pores was also calculated after boiling 3 cylinders of each type. The results are presented in Fig. 7 showing an average of 13.1% decrease in the volume of permeable pores.

The experimental results are associated to the decrease in the number of pores within the concrete internal structure due to the calcium carbonate precipitation. C. Scanning electron microscope (SEM) Rod shaped structures, which were perceived to be Sporosarcina pasteurii, were observed in both specimens with bacteria (type B). On the other hand, no such structures were found in the samples without bacteria (type A). Fig. 8 shows a microbial concrete micrograph and Fig. 9

shows conventional concrete micrograph. Since the only difference between the micrographs is the presence of the rod like structures, it is most likely that the activity of these structures has caused the 27% increase in compressive strength that was observed at 28 days.

D. Cost Analysis The cost analysis showed an increase in cost of 4.9 times between microbial concrete and conventional concrete (mix B38 and A38 respectively). The major contributor to the cost of the B38 mix is the nutrient broth, amounting to over 70% of the cost per cubic meter of concrete. Therefore, further research needs to be devoted to decrease the amount of the nutrient broth used or to find a cheaper alternative source for bacteria nutrition in order to make the technology financially feasible. Nevertheless, it is believed that microbial concrete will yield cost reductions on the long run through decreased need for rehabilitation and maintenance. Furthermore, the high cost of the technology is outweighed by its positive environmental impact, which is discussed in the following section. E. Environmental Analysis According to the calculations explained in the methodology, Mix A38 yields 125.4 kg of carbon dioxide during cement production while Mix B38 yields 89.5 kg of carbon dioxide per cubic meter of concrete, amounting to a 26.8 % decrease in carbon dioxide emissions per cubic meter of concrete. Using the percent reduction obtained above when using the NBU-bacterial solutions, the reduction in carbon dioxide emissions was projected to the global scale yielding the data given in Fig. 10. There is a 28.6% reduction of cement related carbon dioxide emissions on a global scale, specifically from 219.7 to 156.8 megatons of carbon dioxide by the cement industry.

F. Calcium carbonate quantification The results for the experiments are presented in table 4. Over all, there is an increase in the amount of gas produced in the samples containing bacteria showing that there is more Calcium Carbonate in these samples. Since the samples have the same proportions of cement and water the results are attributed to the presence of Sporosarcina pasteurii. Furthermore, to make sure that the nutrient broth solution does not produce carbon dioxide when reacted with HCl, the same test was done using NBU-bacterial solution and acid only; no gas was produced.

III. WORK IN PROGRESS In addition to the experiments and results described earlier, this section outlines further experiments that will be conducted, namely freeze and thaw, the effect of the bacterial solution on steel, and splitting tensile strength test.

A freeze and thaw experiment will be conducted according to ASTM C67. The experiment requires 15 freeze and thaw cycles for 15 days. In each cycle, the concrete will be placed in a plate that contains 0.5 inch of water and then subjected to 16 hours of freezing at -4± 1o C, followed by 8 hours of thawing at room temperature. After the 15 cycles have been completed, the compressive strength of the concrete will be determined. Furthermore, the effect of microbial concrete on steel will be examined to ensure the feasibility of the technology in reinforced concrete. Steel will be placed in the medium containing bacteria and cement powder for a certain time period. The steel will be removed at regular time intervals and any resulting rust will be analyzed. The results obtained will be compared to two control experiments, in the first, the steel is placed in water and cement powder and in the second, the steel is placed in cement and NBU-solution. Finally, a tensile split test will be performed on concrete cylinders in order to assess the effect of microbial calcium carbonate precipitation on tensile capacity IV. CONCLUSION

The purpose of this study is to examine the properties of microbial concrete in addition to its cost and its effect on the environment compared to conventional concrete. Experimental results have shown increased compressive strength and decreased absorption and porosity which were attributed to the precipitation of Calcite by the Sporosarcina pasteurii that was observed under the scanning electron microscope. In addition, reacting the microbial mortar with hydrochloric acid yielded more carbon dioxide than with control samples, indicating the presence of more calcium carbonate. Furthermore, analysis has shown an increase in the cost of production and a significant decrease in carbon footprint compared to conventional concrete. Microbial concrete is thought to be a promising innovation. Nevertheless, research has to be devoted to finding methods to decrease the cost of production and to implement microbial concrete on an industrial scale to ensure the success of the technology.

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[8] B. Chattopadhyay, "Bioremediase a unique protein from a novel bacterium BKH1, ushering a new hope in concrete technology," Enzyme and Microbial Technology, Vol. 46, Issue 7, 581–587, 2010. [9] M.S. Mamlouk & J.P. Zaniewski, Materials for Civil and Construction Engineers. 3rd ed., New Jersey: Pearson, 2011, pp.264-326 [10] Worrell, L. Price, N. Martin, C. Hendriks, L. Meida, N. Martin, "Carbon dioxide emissions from the global cement industry," Annual Review of Energy and the Environment, Vol. 26, pp. 303-329, 2001.

Bio-based Crack repair and Performance enhancements of Microbial Concrete

Application of bacteria-based repair system to damaged concrete structuresThe goal of this study is to present the development of bacteria-based repair system whichfeatures improved durability and sustainability characteristics compared to currently commercially availablesystems.

Concrete is strong and relatively cheap, but it is alsosubjected to a number of degradation processeswhich hamper the structure to reach its required servicelife. To anticipate durability problems duringthe lifetime of a structure, costly measures ofmaintenance and repair have to be undertaken.Currently available concrete curing and repairsystem aiming to decrease porosity and repair ofcracks in aged concrete structures are largely basedon environmental unfriendly materials systems.Moreover, periodic maintenance operations for concretestructures are generally focused on repairingconcrete damages while not considering the relevantdurability issues of the repair system itself (Robery,2011; Tilly and Jacobs, 2007).

Working principle of bacteria-based repair system

The bacteria used are alkaliphilicspecies from the genus Bacillus whichgrow in alkaline environment such as in concrete.Different pathways appear to be involved incalcium carbonate precipitation.The first pathway involves the sulphur cycle, inparticular sulphate reduction, which is carried outby Advanced Topics in Bio mineralization sulphatereducing bacteria under anoxic conditions.A second pathway involves the nitrogen cycle,and more specifically,

(1) The oxidative deamination of amino acids inaerobiosis,(2) The reduction of nitrate in anaerobiosis ormicroaerophily and(3) The degradation of urea or uric acid inaerobiosis (by ureolytic bacteria).

Finding Right Bacteria:The starting point of the research is to find bacteria capable ofsurviving in an extreme alkaline environment. Cement andwater have a pH value of up to 13 when mixed together,usually a hostile environment for life most organisms die in anenvironment with a pH value of 10 or above [13]. The searchconcentrated on microbes that thrive in alkaline environmentswhich can be found in natural environments, Samples ofendolithic bacteria (bacteria that can live inside stones) will becollected along with bacteria found in sediments in the lakes.Strains of the bacteria genus Bacillus will be found to thrive inthis high-alkaline environment. Different types of bacteriawhich can survive in such a high Ph environment is mentionedin Table 1. It is found that the only group of bacteria that willbe able to survive is the ones that produced spores comparableto plant seeds. Such spores have extremely thick cell walls thatenable them to remain intact for up to 200 years while waitingfor a better environment to germinate. They would becomeactivated when the concrete starts to crack, food is available,and water seeps into the structure [19]. This process lowers thepH of the highly alkaline concrete to values in the range (pH10 to 11.5) where the bacterial spores become activated.

Preparation of Microbial Cement Paste andMortar Cubes:5cm3 moulds were used. Cement paste and mortar (cement:sand, 1:3) cubes were casted by mixing grown bacterialcultures of concentration 105, 106, 107, and 108 cells/ml ofwater, at a W/C ratio of 0.4. Conventional cement pastespecimens with regular water are also casted parallel. Thecubes were cured under tap water at room temperature andtested at 1, 7, and 28 days.

Preparation of Concrete Samples with Bacteria:Bacterial concrete casted by using ordinary Portland cementmixed with bacterial concentration 106 cells/ml of water.Conventional concrete samples were also casted in parallel.The specimens were cured under tap water at roomtemperature and tested at 7, and 28 days.

Introduction1Self-healing concrete is defined as the ability ofconcrete to repair its small cracks autonomously [1]. Ideaof self-healing concrete is inspired from naturalphenomenon at organisms such as trees or animals.Damaged skin of trees and animals can be repairedautonomously [2]. Remediating cracks in concretestructure is necessary because cracks not only influencethe service durability, but also harmful for the structuresafety [3].Recently developing of self-healing concretetechnology has been becomes an important objective ofresearches in biotechnology and civil engineering areaSeveral processes have been suggested for selfhealingconcrete design. Self-healing processes areclassified into natural and man-made processes. Amongof proposed natural processes, formation of calciumcarbonate and calcium hydroxide are the most importantreason to heal concrete naturally [11-18]. Although,Corresponding author: Muhd Zaimi Abd Majid,Construction Research Alliance, Faculty of Civilengineering, Universiti Teknologi Malaysia, UTMSkudai, 81310 Johor Bahru, Malaysia, e-mail:mzaimi@utm.my.concrete has naturally ability of healing itself, only smallcracks can be naturally healed [19].Chemical and biological processes (as man-madeprocesses) are useable to design self-healing concrete.Many articles have been published about chemical andbiological self-healing concrete development. However,number of articles on biological methods to design selfhealingconcrete is not considerable. Gollapudi et al.introduced biological self-healing concrete as anenvironmentally friendly process at the mid-1990s [20].Especial strains of bacteria that are able to precipitateespecial chemicals such as polymorphic iron-aluminumsilicate((Fe5AI3)(SiAl)Ol0(OH)5) and calcium carbonate(CaCO3) are used to design a biological self-healingconcrete. Precipitation of these especial chemicals on theconcrete using microorganisms can reduce permeabilitytowards gas and capillary water uptake.

Although using of biological methods is anenvironmental friendly, pollution free and natural way todesign self-healing concretes, these methods have some

disadvantages such as many prerequisites to be met,measures should be taken to protect bacteria in concrete,and mechanical properties recovery and effectivenessunder multiple damage events could be concerns [12].

Some researchers have been carried out by addingmicroorganisms directly into fresh concrete. It is cheapestway to conduct a research on biological self-healingconcrete [33]. pH of fresh concrete is between 10 to 13.The temperature of fresh concrete can be also near 70degree centigrade. After drying of concrete, there is notenough water. Therefore, suitable bacteria have to have ahigh resistance against high pH, temperature, and seriouslimitation of water. Usually mesophilic microorganismscannot have a normal growing in this condition.Dislike of bacteria, spore of bacteria is veryresistance against inappropriate condition and somebacterial spores can live more than 60 years. Then insome studies instead of direct using of microorganisms infresh concrete, spores were used. To avoidmicroorganisms from inappropriate condition,encapsulated microorganisms can be used. Encapsulationof microorganisms is an expensive and complex way.Using of vascular or microvascular networks to distributeof a liquid contain microorganisms throughout ofconcrete are other ideas to avoid microorganisms frominappropriate condition. However, these methods areextremely complex and they do not have constructabilityusing present technology.The use of immobilized microorganisms onto silica gel oractivated carbon is a suitable way from aspect of cost.However, effect of using these materials on thestrengthening of concrete is not completely clear.

Fig1.Shows direct stereomicroscopic observation of cracksfrom control and bacteria-based specimens before and after100 days of immersion in tap water. Width of completelyhealed cracks was significantly larger in bacteria-basedspecimens (0.46 mm) compared to control specimens(0.18 mm).

ADVANTAGES AND DISADVASNTAGESThe Self Healing Concrete has comparatively very lesspermeability, more durability and strain bearing capacity thanthe conventional concrete.A potential drawback of this reaction mechanism isthat for each carbonate ion two Ammonium ions aresimultaneously produced which may result in excessiveenvironmental nitrogen loading.

CONCLUSIONS While most healing agents are chemically based,more recently the possible application of bacteriaas self-healing agent has also been considered. Ina number of published studies the potential ofcalcite precipitating bacteria for concrete orlimestone surface remediation or durabilityimprovement has been investigated. Metabolically active bacteria consume oxygen;the healing agent may act as an oxygen diffusionbarrier protecting the steel reinforcement againstcorrosion. So far, bacteria have never been used toremove oxygen from the concrete matrix to inhibitreinforcement corrosion and further studies areneeded to quantify this potentially additional

beneficial process. While, in this study many features of Self HealingConcrete have been quantified but the life ofbacteria, cost of construction and efficiency stillneeds a separate study.

Problem Statement

It was an eminent fact that concrete structures are extremely susceptible to microcracking which allows water, gases and other potential harmful liquids enter and degrade the concrete, reducing the performance of the structure in terms of strength and durability aspects. To defeat this disadvantage it requires expensive continuous maintenance in the form of micro crack repairs. When these microcracks propagate further deep, not only the concrete itself will be damaged, but also leads to corrosion in the steel reinforced concrete structures. Microcracks are therefore the major cause in reducing the durability of concrete structures. There are many crack repair techniques available to surmount this problem but each technique has its own advantages and disadvantages. Methods currently used for crack remediation often use synthetic polymers which are expensive, incompatible, doubtful long-term performance, needs skilled human assistance and aesthetically unpleasant (especially in repairing historic monuments) most importantly these methods are not environment friendly and are very expensive. The idea of self–crack healing mechanism in concrete is developed to circumvent the above stated disadvantages. One such alternative crack-repair mechanism is application of biominerilization of bacteria to seal and heal cracks in concrete. Synthetic polymers such as epoxy treatment etc, at present being used for repair of concrete, are harmful to the environment; therefore the use of a biological repair technique in concrete was focused upon. The principle of self crack healing mechanism is that certain kinds of healing agents will be released from the concrete when cracks occur. Every repair method follows the procedure of detection, monitoring and repair of cracks but in the self crack healing method the procedure of detection, monitoring and repair is autogeneous throughout the structure’s life-cycle thus reducing the repair maintenance significantly. Repairs can be particularly time consuming and expensive because it is often very difficult to gain access to the structure to make repairs, especially if they are underground or at a great height. Currently available concrete repair systems are largely based on environmental unfriendly material systems. So research is focused on the biotechnology based crack remediation in concrete to study the crack healing process of concrete with less or no human intervention and also examine the effect of biogenic calcite precipitation on the mechanical and durability aspects of concrete structures.

2.2.2 Research Significance

In the recent past, investigations attempted to study about the application of biomineralization in civil engineering. As a part of those studies, researchers around the world started working on the use of specific bacteria in cementitious materials to self-heal and seal cracks without human intervention. Available literature has not reported any such suitable self-healing system which has features such as long-term compatibility, eco-friendliness, good bonding with surrounding cement matrix, less human intervention, inexpensive and organic in nature. Though it is reported that the use of specific alkaliphilic mineral forming bacteria enhances the properties of cement mortar but there exists little understanding of the effect of bacteria on the mechanical and

durability properties of concrete. In the present research work, studies related to characterization of mineral precipitation, permeation properties, resistance to aggressive environment, resistance to corrosion, behaviour at elevated temperature etc of bacteria incorporated concrete has been reported.

Gaps in the Literature

Not much work has been reported on the use of microorganism Bacillus Subtilis JC3, isolated and cultured at JNTUH Hyderabad, and its calcite mineral precipitation efficiency using the nitrogen cycle as its microbial pathway in enhancing the mechanical and durability characteristics of different grades of concrete for Indian conditions. An exhaustive comparative study of mechanical and durability characteristics of ordinary grade (M20), standard grade (M40) and high strength grade (M60 and M80) concrete with and without addition of bacteria Bacillus Subtilis JC3 are made to understand the microstructure of bacteria incorporated concrete at micro and macro level. Hence to address the gaps available in the research, investigations are planned to study the effect of calcite mineral producing bacteria Bacillus subtilis JC3 on the microstructure of various grades of concrete and its impact on its mechanical and durability properties. Once the gaps available in the literature are identified, the main research objectives of the present research work are outlined for obtaining detailed experimental data to address these gaps, which will help to understand the bacteria incorporated concrete and its characteristics in terms of strength and durability aspects under Indian conditions.

ASSESSMENT OF CRACK REPAIR EFFICIENCY

Studies on Crack Remediation and Strength Regain

IntroductionThe aim of this test is to evaluate the efficiency of crack remediation technique using bacteria Bacillus subtilis JC3 and determine the degree of strength regain in cracked concrete specimens. Test methodologyTo demonstrate the crack healing efficiency and strength regain, cracked cement mortar samples were prepared in two different ways. The first method resulted in samples with standardized cracks while the second method gave rise to more realistic cracked samples. (a) Standardized cracksStandardized cracks were made in twelve number of cement mortar cubes of size 70.6x70.6x70.6mm by making a cut to simulate a crack. The width of cut is kept at an average of 3mm width and a 20mm depth as shown in Fig 9.1. Cracks in the three cement mortar specimens are filled up with Indian standard grade II sand (1mm to 0.5 mm) mixed with water and specimens are cured in distilled water after air dried for one hr. The cracks in another set of three specimens are closed with Indian standard grade II sand mixed with 105 cell concentration of bacteria Bacillus subtilis JC3 and after air dried for one hr, cubes are cured in bacteria-nutrient medium. The medium is changed after 14 days. Similarly another set of three cut cement mortar specimens containing no filling were kept exposed to air. All the three sets of cement mortar specimens were tested for the compressive strengths after 28 days.

(a) No filling in cut (b) with filling in cutFig 9.1: Simulated standard cracks made in cement mortar cubes

(b) Realistic cracksRealistic cracks were obtained in cement mortar specimens by applying at least 60 % of ultimate load until a crack was visible with the naked eye. Then the cracked samples were placed in bacteria Bacillus subtilis JC3 and nutrient medium for 28 days. During the period of immersion, bacteria started to precipitate CaCO3 resulting in a compete filling of the crack. The deposition of calcium carbonate was visually monitored periodically. At the end of the 28 day exposure, the cubes were tested for compressive strength.

Test results and DiscussionThe table 9.1 tabulates the compressive strength values of all three sets of cement mortar specimens with standardized cracks.

Table Compressive strengths of cement mortar specimens with standard simulated cracks

Specimen Type Compressive strength at 28 days (MPa)

Control specimen(with no crack made) 52.6

Specimen with simulated Crack(crack not filled up) 28.91

Specimen with simulated crack (crack filled up with standard sand mixed with distilled water) 34.56

Specimen with simulated crack (crack filled up with standard sand mixed with bacterial culture)

42.36

The above studies showed that the simulated cracked cube filled up with standard sand mixed with bacterial culture has regained the strength of about 46%, when compared to the cut and non remediated cube (cut with no fill up). Strength loss due to simulated crack left untreated (no fill

up) is 45%. Strength loss due to simulated crack treated with standard sand mixed with distilled water is 34.3%. Strength loss due to simulated crack filled with standard sand mixed with bacterial culture is 19.5 %. Strength gain due to biogenic treatment is 22.6% this is mainly due to chemical bonding between CaCO3 precipitated by bacterial cells and sand particles which consolidate the crack space.

Table Compressive strengths of cement mortar specimens with realistic cracks Type of specimen Compressive strength at 28 days (MPa)Control cement mortar specimen 51.68Cracked cement mortar specimen(immersed in water) 44.99

Cracked cement mortar specimen(immersed in bacterial culture) (Bio-remediated)

49.17

Strength loss for cracked cement mortar specimens immersed in water is 13 %. Strength loss for cracked cement mortar specimens treated in bacterial culture is 4.9 %. Strength gain due to biogenic treatment is 9.3 %

ConclusionsThe visual examination of crack surface of the cement mortar samples reveals the fully grown calcite crystals, with distinct and sharp edges all over the surface of the crack, acts as an agent for an eventual plugging and crack remediation. Bacteriogenic mineral precipitation contributed to the bonding and regaining strength of the already cracked cubes. This microbial mixture with sand filled in the cracks was found to remain intact after five days treatment confirming the microbial calcite precipitation. Higher strength regain was obtained because of the bacterial CaCO3 precipitation inside the simulated cracks of cement mortar specimens. This strength recovery can be attributed to chemical bonding between CaCO3 precipitated by bacterial cells and sand particles which consolidate the crack space.

Preliminary Cost EvaluationThe cost/benefit analysis of bacterial concrete balances the increased cost of the concrete against substantial repair material costs, enhanced durability and aesthetic benefits. The benefits are apparent at strength and performance of the finished product. Only expensive component in the development of bacterial concrete is nutrients. In the market bacteria is available is lyophilized state. So cost depends on the surface treatment area or volume of concrete used. Nutrients used for this study are laboratory nutrients which are quite expensive so other inexpensive nutrient sources can also be tried to reduce the commercial production cost of bacterial concrete. However, any nutrients such as inexpensive, high-protein-containing industrial wastes such as corn steep liquor (CSL) or lactose mother liquor (LML) effluent from starch industry can also be used so that overall process cost reduces dramatically. These industrial effluents which are potential environmental pollutants and also available locally with a price of nearly Rs 100 per liter, which is very economic compared with standard laboratory nutrient medium. In this project, to prepare one liter of nutrients mixed bacterial culture, it required 13 grams of nutrients broth powder. The cost of 500 grams of HIMEDIA M002 nutrient broth powder costs about

2300 rupees so to prepare one litre of nutrients mixed bacterial culture costs Rs 60. In this project nearly 125 liters of nutrients mixed bacterial culture was used costing nearly 7500 rupees.

[48] S.S. Bang, J.K. Galinat, and V. Ramakrishnan, “Calcite precipitationinduced by polyurethane-immobilized Bacillus pasteurii”,Enzyme Microb. Tech. 28, 404–409 (2001).[49] W. De Muynck, D. Debrouwer, N. De Belie, and W. Verstraete,“Bacterial carbonate precipitation improves the durabilityof cementitious materials”, Cem. Concr. Res. 38, 1005–1014 (2008).[50] H.M. Jonkers, “Self healing concrete: a biological approach”,in Self Healing Materials – an Alternative Approach to 20 Centuriesof Materials Science pp. 195–204, ed. S. van der Zwaag,Springer, Dordrecht, 2007.[51] H.M. Jonkers and E. Schlangen, “Development of a bacteriabasedself healing concrete”, Proc. Int. FIB Symposium 1, 425–430 (2008).[52] H.M. Jonkers and E. Schlangen, “A two component bacteriabased self healing concrete”, Concr. Repair, Rehab. and Retrofit.1, 119–120 (2009).

J. Aizenberg and P. Fratzl, “Biological and biomimetic materials”,Adv. Mater. 21, 387–388 (2009).

Bio-data of Authors

Author 1:Dr M V Seshagiri Rao , FIE (FIE No: 015739/9)Professor in Civil EngineeringJNTUH College of EngineeringHyderbad-85Email: rao_vs_meduri@yahoo.comPh: 944 036 1817

Awards/ medals received: 1. Brij Mohan Lal best paper Award for the paper published in Vol.80; August 1999, IEI Journal

2. Best paper award for the paper presented at the 67th Annual technical session of The Institution of Engineers awarded in October 2005. 3. Out standing Concrete technologist 2006 Award by Indian Concrete Institute(AP, Hyderabad )4. Best paper award for the paper presented at the 70th Annual technical session of

The Institution of Engineers awarded in October 2008.5. Best Teacher Award for the year 2009 by the Govt. of Andhra Pradesh

Research Publications : 175 Teaching & Research Experience: 38 yrs

Memberships : FIE (Life Member), MISTE (Life Member), MICI (Life Member)M.I.W.R.S (life Member)

Number of PhD Scholars Guided: 18

Author 2 :

Mr. V Srinivasa Reddy, MIE (M-146335-1)Associate Professor in Civil EngineeringGokaraju Rangaraju Institute of Engineering and Technology Email: vempada@gmail.comPh: 970 468 3149

Research Publications : 50

Memberships : MIE (Life Member), MISTE (Life Member), MICI (Life Member)