Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

BACTERIAL CONCRETE: A NEW AGE CONSTRUCTION MATERIAL

Prof. M V Seshagiri Rao Depar !e" of Ci#i$ E"gi"eeri"g %NTU& Co$$ege of E"gi"eeri"g &'(era)a( rao*#s*!e(+ri,'ahoo.-o! ABSTRACT Cracks in concrete are inevitable and are one of the inherent weaknesses of concrete. Water and other salts seep through these cracks, corrosion initiates, and thus reduce the life of concrete. So there was a need to develop an inherent biomaterial, a self-repairing material which can remediate the cracks and fissures in concrete. Bacterial concrete is a material, which can successfully remediate cracks in concrete. This techni ue is highly desirable because the mineral precipitation induced as a result of microbial activities is pollution free and natural. !s the cell wall of bacteria is anionic "negatively charged#, metal accumulation "calcite# on the surface of the wall is substantial, thus the entire cell becomes crystalline and they eventually plug the pores and cracks in concrete. This paper discusses the crack remediation mechanism using Bacillus subtilis $C% bacteria suspended in laboratory medium. &t was found that calcite mineral precipitation by bacteria Bacillus subtilis $C% improves the compressive strength and durability properties of concrete significantly. Scanning electron microscope "S'(# analysis confirms the presence of the microbiologically induced calcite precipitation in concrete. )ods like

impressions were found on the face of calcite crystals indicating the presence of bacteria in those places. 'nergy- dispersive *-ray "'+*# spectra of the microbial precipitation on the surface of the crack indicated the abundance of calcium precipitation which was inferred to be calcite "CaC,%# mineral.

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

INTRODUCTION Self healing materials are a "e. resear-h area ha ge s a $o of a e" io" i" re-e" 'ears. Self healing concrete is a er! ha is +se( for -e!e" /)ase( !a eria$s ha repair he!se$#es af er he !a eria$ or s r+- +re ge s (a!age( (+e o so!e sor of (e eriora io" !e-ha"is!. (icrocracks have a width of .ust /.0-/.1mm, but that2s enough for water to leak in to degrade the concrete and the steel reinforcements embedded within it. 3sing this potentially damaging water to the advantage, a healing agent is added into the concrete, composed of bacterial spores and nutrients to grow so that the incoming water activates the bacterial spores, causing them to convert the nutrients into limestone that seals the cracks4fissures occurred. Tunnels, basements and highway infrastructure are ideal 5wet environments2 which will benefit from this innovation. This is a landmark in developing 5living2 materials6. ! peptone based nutrients supplied along with bacteria in suspension helps in producing calcite crystals. &t is found that this biominerali7ation process will not interfere with the setting time of the concrete. ,nly factor need to be checked is the effect of nutrients media on the setting time of cement. !t present, the biggest challenge is producing large-scale uantities of the self healing agent at affordable costs. The most e8pensive ingredient in developing bacterial concrete is nutrients. So any ine8pensive alternative for laboratory growth media would potentially bring down the cost of the bacteria based self-healing sustainable concrete. MICROBIOLOGICALL0 INDUCED BACTERIAL MINERAL PRECIPITATION )epairing cracks in concrete structures is a time consuming, costly but necessary business. With the hope of long-term savings from the increased life e8pectancy of constructions, research is focused on how the self-healing capacity of concrete structures can be improved by using calciteprecipitating bacteria and what conditions are necessary for these bacteria to thrive. Concrete has

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

a serious flaw9 it can easily crack when under tension. &f these cracks become too large, they will lead to corrosion of the steel reinforcement, which not only results in an unattractive appearance, but also .eopardi7es the structure2s mechanical ualities. That is why engineers often use a larger than necessary amount of steel reinforcement within a concrete structure in order to prevent the cracks from becoming too large. This e8tra steel has no structural use and is an e8pensive solution as steel prices are high. !nother way to deal with cracks is to repair them, but this can be e8tremely difficult in underground or li uid retaining structures. The ultimate solution would be self-healing concrete, which is e8actly what researchers are working on. By embedding calcite-precipitating bacteria in the concrete mi8ture, it is possible to create concrete that has self-healing capacities. !s the p: value of concrete is very high, only the so-called alkaliphilic bacteria are able to survive. So viability of bacterial spores in hostile environment of concrete is important for mechanism of calcite mineral precipitation. O#er#ie. of #ario+s Co"s r+- io" Ma eria$s !a(e +si"g MICP App$i-a io" Mi-roorga"is! Biological mortar Crack in concrete remediation Crack in concrete remediation Bacterial concrete Bacterial concrete Bacillus cereus Me a)o$is! o8idative deamination of amino acids :ydrolysis of urea :ydrolysis of urea :ydrolysis of urea oxidative deamination of amino acids N+ rie" s ;rowth media "peptone, e8tract yeast, <=,%, =aCl# > CaCl0.0:0,, !ctical, =atamycine =utrient broth, urea, CaCl0.0:0,, =:1Cl, =a:C,% '8tract yeast, urea, CaCl0.0:0, =utrient broth, urea, CaCl0.0:0,, =:1Cl, =a:C,% e!tone" # $%lit., &a'l" # $%lit., (east extract" ) $%lit. Reference (Muynck et al., 2010) (Santhosh et al., 2001) (Belie, 2010) (Santhosh et al., 2001) #M V Seshgiri Rao et al., $%&%' JNTU Hy!era"a!

Bacillus subtilis Bacillus sphaericus Bacillus subtilis

Bacillus subtilis

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

WOR1ING PRINCIPLE O2 BIO/BASED SEL2 CRAC1 &EALING PROCESS &n concrete the cracks up to /.0 mm wide are healed autogenously. Such micro cracks are acceptable as these do not directly influence the safety and strength of the concrete. )esearch has shown that 5autogeneous2 healing happens due to hydration of non-reacted cement particles present in the concrete matri8 when comes in contact with ingress water resulting in closure of micro cracks. :owever, because of the variability of autonomous crack healing of concrete micro cracks can still occur. The inbuilt bacteria-based self-healing process was found to heal cracks completely up to /.? mm width. (icrobiologically &nduced calcite precipitation "(&C@# was a techni ue that comes under a wider category of science known as )io!i"era$i3a io". The process by which living organisms form inorganic solids. Bio-minerali7ation by *mmonification "!mmo acid degradation4 comprises of series of comple8 biochemical reactions. !mino acids released during proteolysis undergo deamination in which nitrogen containing amino "-=:0# group is removed. Thus, process of deamination which leads to the production of ammonia is termed as AammonificationA. The process of ammonification is mediated by Bacillus subtilis $C%. !mmonification usually occurs under aerobic conditions "known as o8idative deamination# with the liberation of ammonia "=: %# or ammonium ions "=: 1# when dissolved in water .The processes of ammonification is represented as follows. C:%C:"=:0#C,,: "@eptone# > B,0 ---------C C0:0 > :0C,% > =:% "ammonia# :0C,% ----------C :> > :C,%=:% > :0, --------C =:1> > ,:This process does not only produce calcium carbonate directly due to microbial metabolic process but also indirectly due to autogeneous healing. This process results in efficient bio-based crack sealing techni ue. Bacillus subtilis $C% can able to precipitate CaC, % in the high alkaline

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

environment by converting peptone into ammonium and carbonate. The ammonia degradation of peptone based nutrient locally increases the p: and promotes the microbial deposition of carbonate as calcite crystals in a calcium rich environment along with maintaining the p: of concrete. These precipitated crystals can thus seal the cracks. The ammonia is responsible for p: increase, which in turn shifts the bicarbonate e uilibrium, resulting in the formation of calcium carbonate ions. Since the cell membrane of the bacteria is negatively charged, the bacteria draw cations from the environment, including Ca0>, to deposit on their cell surface. The Ca0> ions subse uently react with the C,% that serves as a nucleation site. Ca0> > Cell E Cell- Ca0> Cell- Ca0> > C,%0- E Cell- CaC,% BACILLUS SUBTILIS %C5 6 AN AL1ALIP&ILE !lkaline environments are marginal environments for biological activity but certain microorganisms have physiological adaptations that make life at high p: possible. The surface of concrete and concrete pre waters tend towards these alkaline levels due to abundance of lime. !lkaliphile bacteria grow optimally at p: values above F. The strain Bacillus subtilis $C%, isolated from soil, incessantly precipitates dense insoluble calcium carbonate crystals metabolically by o8idative deamination of amino acids "ammonification# through nitrogen cycle. !mmonification of nutrients through nitrogen cycle produces ammonia and carbamate. Carbamate decomposes to ammonia and carbonic acid. The ammonia and carbonic acid subse uently e uilibrate in water with their deprotonated and protonated forms, resulting in an increase in the p:. +ifferent cell concentrations were derived from the bacterial growth culture
0D

ions, leading to the precipitation of CaC,% at the cell surface

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

by serial dilution method. &t was observed at cell concentration more than -/ ?cell4ml of water, strengths are reduced due to disruption of cement sand matri8 integrity. C+$ +re of Ba- eria The pure culture was isolated from the soil sample of $=T3 and is maintained constantly on nutrient agar slants. &t forms irregular dry white colonies on nutrient agar. Whenever re uired a single colony of the culture is inoculated into nutrient broth of 0? ml in -// ml conical flask and the growth condition are maintained at %GHC temperature and placed in -0? rpm orbital shaker. The medium composition re uired for growth of culture is - @eptone9 ? g4lit., =aCl9 ? g4lit., Ieast e8tract9 % g4lit. BIOMINERALI7ATION B0 BACILLUS SUBTILIS %C5 Bio!i"era$i3a io" is the process by which living organisms form inorganic solids. Bacillus subtilis $C%, a commonly known soil bacterium can bring about the precipitation of calcite. CaC,% acts as a microbial sealant, e8hibits its positive potential in selecting consolidated fractures and surface pore in concrete as well as the consolidation of sand. (&C@ was a highly suggested because the calcite precipitation bring about as a result of comple8 biological microbial activities, was environmental friendly. This techni ue can be used to improve the compressive strength and stiffness of cracked concrete specimens as already states. There are a number of species of CaC, % minerals associated with bacteria, for e8ample calcite by bacillus pasturii , vaterite formation by !cinobacter sp., aragonitic sherulites by +eleyahlophila ")ivadeneyra et al., -FFJ#, calcite by Bacillus subtilus "( K Seshagiri )ao et al., 0/-/# and magnesium calcite spherulites and dumbbells by the slime-producing bacteria, (y8ococcus 8anthus ";on7Lle7-(uMo7 et al., 0///N :olt et al.,-FF%#. ,ne of the most robust bacteria is Bacillus subtilis which is an aerobic, spore forming, rod shaped and gram positive

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

bacterium. &t uses peptone as an energy source and produces ammonia which increases the p: in the environment and generates carbonate, causing Ca0> and C,%0- to be precipitated as CaC,%. !lkaline p: is the primary re uirement for microorganisms for calcite precipitation. The more CaC,% precipitates, the better the self-healing effect will be. The concentrations of bacteria, urea and Ca0> will greatly affect the amount of precipitated CaC, %. &n bacteria incorporated mortar samples, the live cells of optimum concentration were added directly in suspension along with mi8ing water to the cement sand mi8ture to study the application of microbiologically induced calcite precipitation in cement based materials. Mi-roorga"is! +se( for Ca$-i+! Car)o"a e Pre-ipi a io" i" Co"-re e T'pe of S's e! Cr's a$ 'pe Reference !i-roorga"is! @hotosynthetic (eromictic lake Calcite "CaC,%# (+ou$las and organism 9 Beverid$e, 1,,-) Synechococcus ;O01 @hotosynthetic Ourcene Oake Calcite "CaC,%# organism 9 (+ittrich, 200.) Chlorella Sulfate reducing !no8ic hypersaline +olomite "Ca"(g# bacteria9 / lagoon C,%# &solate S)B OKformJ 3rea degradation in (Mc.'onnau$hey, =itrogen cycle synthetic Calcite "CaC,%# Bacillus subtilis 2000) medium =itrogen cycle !mmonification and ('astanier et al., Calcite "CaC,%# Bacillus cereus nitrate 1,,,) reduction #M V Seshagiri Rao *mmonification &itro$en cycle 'alcite ('a'1)) an! Ch Sasi(ala, (*mmo acid Bacillus subtilis 0') $%&%' de$radation) JNTU Hy!era"a! MEC&ANICAL AND DURABILIT0 PROPERTIES O2 BACTERIAL CONCRETE The concrete specimens treated with bacteria are e8posed to chloride, sulfate and free7e-thaw environments to study its durability aspects along with the effect of bacteria addition on

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

mechanical characteristics. The study showed that a 0?P increase in 0Q day compressive strength of cement mortar was achieved. The strength improvement is due to growth of filler material within the pores of the cementRsand matri8. The effect of various concentrations of bacteria on the strength and durability of concrete was also studied. &t was found that the bacterial cell concentration of - 8 -/
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cells per ml of mi8ing water will give ma8imum

performance in enhancement of strength and durability of concrete. (icrobial calcite precipitation was visuali7ed by Scanning 'lectron (icroscope "S'(# as shown in Sig --% and was uantified by *-)ay +iffraction "*)+# analysis in Sig 1. The S'( uni ue imaging and microanalysis capabilities established the presence of calcite inside the cracks, bacterial impressions and calcite layer on the surface of concrete. This calcite layer improves the specimen2s impermeability, thus improving its resistance to acid, chloride, sulfate and free7ethaw attacks. The precipitation of calcite crystals in the pores and cracks of the concrete will enhance the pore structure of the concrete there by reducing the porosity of the material which is confirmed by the porosity and water permeability tests conducted on the bacteria incorporated specimens. The produced bio-minerals block and seal cracks resulting in a delay of further ingress of water as well as to a decrease of inward diffusion rate of chloride and o8ygen. (oreover, as the metabolically active-bacteria consume o8ygen, the agent acts as an o8ygen diffusion barrier protecting the embedded passivated steel reinforcement against corrosion. &n this way the reinforcement will be protected for substantially increased periods, even after breakdown of the passivated layer, as a lack of o8ygen prevents further corrosion. @ore si7e and structure studied through B'T nitrogen adsorption test shows bacterial specimens have modified dense pore structure due to calcite precipitation. Chloride penetrability studies

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

done using )apid chloride penetration test "!ST( C -0/0# shows that charged passed is very low to negligible in bacterial concrete specimens. Bacterial concrete has improved microstructure and permeation properties than controlled concrete. Studies also showed that bacterial concrete has better acid resistance in aggressive environments. ELEMENTAL COMPOSITION USING C&EMICAL ANAL0SIS Sample consisting of hardened cement mortar broken pieces and fine powder, was analy7ed chemically in order to determine the elemental composition of control specimen and bacteria treated specimen. Control Specimen Constituent Ooss on &gnition Soluble Silica"Si,0# Serric ,8ide "Se0,%# !lumina"!l0,%# Calcium ,8ide"Ca,# (agnesia"(g,# Sulphuric !nhydride"S,%# &nsoluble Silica"Si,0# - Sand "i#Sodium ,8ide !lkalies "=a0,# "ii#@otassium P by weight of the sample taken /.Q% ?./1 -./J -.0? -J.00 /.G? /.GG1.?F /./? /.-/.-0 /./% /./1 /.//0 /./G APPLICATIONS The use of bacterial concrete can in theory lead to substantial savings, especially in steel reinforced concrete. &t will also mean durability issues can be tackled in a new and more economical way when designing concrete structures. Bacterial concrete is ideal for constructing Bacteria Treated Specimen P by weight of the sample taken /.QF 1.FG -./0 -./F %J.00 /.G0 /.G% ?1.?F /./? /.-0 /.-/./% /./1 /.//0 /./G

,8ide"<0,# Titanium +io8ide"Ti,0# @hosphorous @ento8ide"@0,?# (anganic ,8ide"(n0,%# Total Chlorides Sulphide Sulphur

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

underground retainers for ha7ardous waste, as no humans would have to go near it to repair any occurring cracks. Sor residential buildings, however, it does seem the traditional repairing of cracks will remain the most economically attractive solution for now. Currently, research focuses on creating the right conditions for the bacteria to produce as much calcite as possible and on optimi7ing the distribution of food for the bacteria. &n addition, we are also looking at the selfhealing ability of bacterial concrete and how this is affected by the various deterioration mechanisms involved, such as sulfate attacks or temperature fluctuations. Self-healing concrete could vastly increase the life of concrete structures, and would remove the need for repairs, reducing the lifetime cost of a structure by up to ?/ per cent. ,ver seven per cent of the world2s C,0 emissions are caused by cement production, so reducing the amount re uired by e8tending the lifetime of structures and removing the need for repairs will have a significant environmental impact. &ncluding bacteria in concrete offers a double layer of protection in preventing steel corrosion. =ot only do the bacteria work to plug cracks in the concrete, the process of doing so uses o8ygen present which would otherwise be involved in the corrosion process of the steel bars CONVENTIONAL CRAC1 REPAIR S0STEMS )epair of cracks in concrete structures usually involves applying a concrete mortar which is bonded to the damaged surface. Sometimes, the mortar needs to be keyed into the e8isting structure with metal pins to ensure that it does not fall away. )epairs can be particularly be time consuming and e8pensive 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. Sor crack repair, a variety of techni ues is available but traditional repair systems have a number of disadvantageous aspects such as different thermal e8pansion coefficient compared to concrete and also have impact on

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Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

environment and health. Therefore, bio based calcite precipitation has been proposed as an alternative and sustainable environmental friendly crack repair techni ue. CONCLUSIONS &n bacterial concrete, induction of microorganisms inside the concrete has enormous effect on the porosity within the cement matri8 paste, on the particle si7e distribution of the crystalline phases and on the presence of in-homogeneities within the hydrated paste due to mineral precipitation. Calcite mineral precipitation results in less capillary porosity in the hardened paste and hence a greater strength. This reduced capillary porosity also favours the formation of fine-te8tured hydration products with optimi7ed particle si7e distribution of the cementitious materials in order to increase the potential packing density. So bacteria incorporated concrete has increased packing density and reduced capillary porosity. The calcite crystals formed will glue together the hydrated particles which reduce the interstitial porosity between them. The incorporation of microorganisms into concrete confers enhanced durability on the concrete. &n bacterial concrete significant reductions in water permeability and chloride ingress have been observed along with its increased resistance to attack by aggressive chemicals. The compressive strength, split tensile strength and fle8ural strength of concrete, incorporated with - 8 -/? cells4ml concentration of bacteria, increases profoundly due to pore plugging by calcite mineral precipitation during microbial metabolic process of Bacillus subtilis $C%. The Bacterial concrete mi8es have shown improved stress values for the same strain levels compared to that of controlled concrete mi8es in low, medium and high strength grades resulting in the increase of elastic modulus. Bacteria treated concrete samples gave the lower water absorption, sorptivity and porosity values compared to control concrete. This means that the time taken for the water to rise by capillary

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Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

action in bacterial concrete are longer and thus proved that these concrete are less porous compared to the control concrete. The possible reason for this is calcite mineral precipitation in the pores reduced the average pore radius of concrete by blocking the large voids "pore discontinuity# in the hydrated cement paste. Since interconnected pores are significant for permeability, the water permeability and chloride ion penetration are reduced relatively in bacteria treated specimens.

Sig -9 S'( images of precipitated rhombohedra calcite crystals, rod shaped bacterial spores

Sig 09 @recipitation of calcite crystals within the crack

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Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

Sig % "a# Cell Concentration R=il "Control Specimen#

Sig % "b# Cell Concentration R -/?4ml ",ptimum#

-%

Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

Sig 19 +iffractogram of bacteria incorporated mortar and concrete specimens shows the abundant presence of Ca and precipitation was inferred to as calcite "CaC,%# crystals

RE2ERENCES -. )amachandran, S.<., )amakrishnan, K. and Bang, S.S., T)emediation of concrete using microorganismsT, *'2 Materials 0ournal FQ "-# "0//-# %-F 0. +e (uynck W, Co8 <, +e Belie = and Kerstraete W Bacterial carbonate precipitation as an alternative surface treatment for concrete. 'onstr Build Mater "0//Q# 009 QG? -QQ? %. $onkers :( Self healing concrete9 ! biological approach. &n Self healing materials R !n alternative approach to 0/ centuries of materials science "ed. S. van der Uwaag#, "0//G# pp. -F? R0/1. S!rin$er, the =etherlands 1. +e Belie, =. and +e (uynck, W., TCrack repair in concrete using bio-depositionT, in !le8ander et al. "eds.# TConcrete )epair, )ehabilitation and )etrofitting &&T, roceedin$s

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Keynote Lecture of Prof. M V Seshagiri Rao, JNTUH College of ngineering Hy!era"a!

of an 2nternational 'onference, Cape Town, =ovember, 0//Q "3aylor 4 5rancis 6rou!, Oondon, 0//F# GGG-GQ?. )ai.iwala +. B. VBacterial Concrete9 ! Self-:ealing Concrete6 3he 2'5*2 7niversity 0ournal of Structural 8n$ineerin$, Kol. &, =o. 0, pp. ?J-J%, ,ctober 0//Q J. Stocks-Sischer, S., ;alinat, $. <., W Bang, S. S. "-FFF#. (icrobiological precipitation of CaC,%. Soil Biolo$y and Biochemistry, %-"--#, -?J%--?GG. Seshagiri )ao ( K , Ch Sasikala, K Srinivasa )eddy V! Biological !pproach To 'nhance Strength !nd +urability &n Concrete Structures6, 2nternational 0ournal of *dvances in 8n$ineerin$ 4 3echnolo$y (20*83#, Sept 0/-0 Kol. 1, &ssue 0, pp. %F0-%FF

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