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International Biodeterioration & Biodegradation 86 (2014) 317e326

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier .com/locate/ ibiod

Accelerated biodegradation of cured cement paste by Thiobacillusspecies under simulation condition

Azam Yousefi a,b, Ali Allahverdi a,*, Parisa Hejazi b

aCement Research Center, Iran University of Science and Technology, Tehran 1684613114, IranbBiotechnology Research Laboratory, School of Chemical Engineering, Iran University of Science and Technology, Tehran 1684613114, Iran

a r t i c l e i n f o

Article history:Received 24 July 2013Received in revised form22 September 2013Accepted 6 October 2013Available online 12 November 2013

Keywords:BiodegradationCement pasteBiogenic sulfuric acidThiobacillus thioparusAcidithiobacillus thiooxidans

* Corresponding author. Tel.: þ98 21 77240496; faxE-mail addresses: ali_averdi@yahoo.com, ali.allahv

0964-8305/$ e see front matter � 2013 Elsevier Ltd.http://dx.doi.org/10.1016/j.ibiod.2013.10.008

a b s t r a c t

Biodegradation is one of the most important types of cement deterioration. Complex microbial pop-ulations take part in the biodegradation process of cement-based materials. Studies in this field showthat the sulfur-oxidizing bacteria, including Acidithiobacillus thiooxidans, due to sulfuric acid formation,play a key role in this process. In this study, with the accelerated leaching process of calcium hydroxide ofcement paste, cured under running tap water and exposed to sterile biogenic sulfuric acid for 6 days, thesurface pH of the cement was reduced to a more favorable level for bacterial growth. In this case, thegrowth of Thiobacillus proceeded in the presence of cured cement paste specimens. After 90 days ofexposure to a semi-continuous culture of A. thiooxidanswith its pH less than 2 and continuous removal ofdamaged layers the compressive strength, length and mass of the samples dropped by 96%, 11% and 43%,in the order given. The mechanism of degradation and the structure of degraded specimens wereanalyzed by test laboratory techniques such as, XRD, SEM and EDAX analyses.

� 2013 Elsevier Ltd. All rights reserved.

1. Introduction

Some microorganisms can grow on cement surfaces thoughtheir thin biofilms are invisible. They are not responsible for coloredspots usually observed on the surface of concrete structures, butthey have the ability to destroy them (Escadeillas et al., 2007). Sometypes of autotrophic and heterotrophic microorganisms, notablygenus Thiobacillus, as a group of bacteria with serious destructionability, were isolated and identified from the degraded concretestructures (Nica et al., 2000; Okabe et al., 2007). Most researchreports have indicated that Acidithiobacillus thiooxidans plays a keyrole in the biodegradation process of cement (Milde et al., 1983;Sand and Bock, 1984; Diercks et al., 1991; Haile and Nakhla, 2009;Wei et al., 2010). These bacteria are available in water, air and soil(Waksman, 1922; Vidyalakshmi et al., 2009) and still the best andmost suitable growth environment is found to be in sewage pipe-lines (Roberts et al., 2002), so any cement structure located nearbyundergoes biodeterioration. These bacteria have the ability tooxidize organic and inorganic sulfur compounds in the presence ofoxygen, carbon dioxide and moisture (Waksman, 1922; Wei et al.,2010). The final product of sulfur-oxidizing bacterial activity is

: þ98 21 77240495.erdi@iust.ac.ir (A. Allahverdi).

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sulfuric acid (Diercks et al., 1991; Roberts et al., 2002). A whitegypsum layer is found by reaction of sulfuric acid and cementportlandite [Ca(OH)2]. The reaction of gypsum and aluminate phaseof the cement produces Ettringite (3CaO$Al2O3$3CaSO4$32 H2O),with expandability and low adhesion properties (Saricimen et al.,2003; Connell et al., 2010). With sulfuric acid formation by sulfur-oxidizing bacteria, the strong structure of cement is converted toa loose structure of gypsum and Ettringite at medium temperature(>15 �C) and Thaumasite (CaSiO3$CaCO3$CaSO4$15H2O) at lowtemperature (<15 �C) (Cwalina, 2008).

First, Thiobacillus does not exhibit any sign of growth on con-crete structures at initial pH of around 12e13 (Roberts et al., 2002),though from the beginning, some algae and fungi grow on cementsurfaces as a suitable substrate (Diercks et al., 1991; Jayakumar andSaravanane, 2010; Wiktor et al., 2010). At longer time, thecarbonation and leaching processes of cement portlandite occur byclimatic changes such as snow, rain and other running water andthe pH of cement surface are reduced to about 9 (Shook and Bell,1998). Thiobacillus thioparus is the first acting species of Thio-bacillus bacteria that has the ability to grow on the cement surfaceat pH 10. By production of polythionic acid, elemental sulfur andsulfuric acid, the pH of cement surface is dropped again andtherefore other Thiobacillus strains start to grow. At pH 4.5, thevarious strains of A. thiooxidans find the ability to grow on thesurface of cement (Roberts et al., 2002). By activity of these bacteria

Table 1Components of liquid culture media for T. thioparus PTCC 1668 (M1) andA. thiooxidans 1717 PTCC (M2).

Mineral salt (g)/1 l distilled water M1 M2

NH4Cl 0.40 2.43MgCl2$6H2O 0.20 0.41Na2CO3 0.40 0.00K2HPO4 2.00 0.00KH2PO4 2.00 3.00Na2S2O3$5H2O 5.00 5.00

A. Yousefi et al. / International Biodeterioration & Biodegradation 86 (2014) 317e326318

under suitable conditions, pH of the environment is dropped to lessthan 1. These bacteria have been found abundant in the 1e5 mmlayer of concrete structures, but in deeper layers, their numbers arereduced logarithmically due to lower diffusion of oxygen and car-bon dioxide gases of the air from the surface cement. So, sulfuricacid is produced on the concrete surface and penetrates into thelower layers and reacts with cement inner compounds (Nica et al.,2000; Yamanaka et al., 2002; Wiktor et al., 2010).

There are good review articles (Diercks et al., 1991; Montenyet al., 2000; Gaylarde et al., 2003; Cwalina, 2008; Connell et al.,2010) in relation to cement biodegradation. Considerable research(Magniont et al., 2011) works have been conducted on thebiodegradation of concrete-based structures by synthetic organicacids (acetic and lactic acids) and comparisons are made withbiogenic acid produced by Escherichia coli and silage effluent(acetic, propionic, butyric and isobutyric acids) (Bertron et al.,2005a,b, 2007). Biodegradation mechanism of cement structure isdifferent in domestic and industrial sewage systems from that ofagricultural silos, etc.; because of different microorganisms pro-ducing different acids. In agricultural and agro-food effluents, it isshown that some ionic salts and metals of cement are consumed bybacteria. Also it is particularly observed that bacteria cause moreintense deterioration of cement compared to a medium withoutbacteria or in a synthetic acid solution (Magniont et al., 2011). Inother studies chemical sulfuric acid (Chandra and Berntsson, 1983;Knight et al., 2002; Saricimen et al., 2003; Hewayde et al., 2007;Herisson et al., 2013) or sodium and magnesium sulfates(Monteny et al., 2000) are found to simulate the biodegradationprocess of cements, and it is shown that cement degradation withchemical sulfuric acid and sulfate salts take different course frombiogenic sulfuric acid bacterially produced, even though the so-dium and magnesium cations play important roles in degradationof cement. Some research works have focused their studies onpresenting a model for biodegradation (Kaempfer and Berndt,1999; Vollertsen et al., 2008; De Windt and Devillers, 2010) pro-cess by in situ tests (Monteny et al., 2000; Okabe et al., 2007; Alumet al., 2008; Herisson et al., 2013) and by a mixture of severalbacteria (Sand and Bock, 1984; Wei et al., 2010; Herisson et al.,2013), or just in presence of one bacteria such as A. thiooxidans(Hormann et al., 1997; Vincke et al., 1999; Knight et al., 2002; Haileet al., 2008) and finally others have dealt with biodegradation ofconcrete structures using algae (Ismail et al., 1993; Bertron et al.,2007; Escadeillas et al., 2007, 2009; Alum et al., 2008; Jayakumarand Saravanane, 2010; Wiktor et al., 2010) and fungi (Gaylardeet al., 2003; De Windt and Devillers, 2010). In these studies, a va-riety of media are used including sulfur powder and thiosulfatewith calcium, iron, aluminum and magnesium ions with the con-crete, mortar and cement paste specimens in different shapes andsizes under variable temperature and humidity conditions. Scat-tered research data with various microorganisms and methodshave produced complications to reach a unified conclusion forcomparing the observations and data by various experiments oncement biodegradation. So, in-depth understanding of variousinteractive processes in structural biodegradation of cements andtheir constant changes are necessary.

In the present work, the growth of two species of Thiobacillus isstudied under normal laboratory conditions in liquid media, freefrom ions in common with cements such as calcium, iron andsulfates, inwhich bacteria become fully colonized. In retrospect, thebehavior of Thiobacillus has been assessed in the presence of acement paste. The reduction in the pH of liquidmedium to less than2 leads to degradation in cement chemical structures. The studywas further continued to evaluate the biodegradation of the cementin curing and degradation stages by A. thiooxidans in simulatedlaboratory conditions.

2. Materials and methods

2.1. Materials

2.1.1. Microorganisms and cultivation mediaTwo oxidizing-sulfur bacteria T. thioparus PTCC 1668 and

A. thiooxidans PTCC 1717 were purchased from Persian Type CultureCollection, Iranian Research Organization for Science and Tech-nology (IROST). The optimum pH for growth of the former was 7,while it was 4.5 for the latter microorganism at 30 �C. These mi-croorganisms were kept in refrigerator at 4 �C in a liquid mediumcontaining thiosulfate and they were re-cultivated once every 2e3weeks. Liquid culture media for T. thioparus PTCC 1668 andA. thiooxidans PTCC 1717 (designated as M1 and M2 in Table 1)contained mineral salts (Merck Co., Germany) free of calcium, iron,aluminum, and sulfate ions. The optimum culture media for bac-terial growth and their corresponding data are not published yet.The culture media were sterilized at 121 �C under 1.2 bar pressurefor 20 min. The optimized volume of the inoculum in their culturemedia was 1 v/v(%) towards the end of the logarithmic phase ofbacterial growth.

Symbols for various tests are presented in Table 2.

2.1.2. Cement paste specimen preparationPortland cement was of Type II with chemical composition as

presented in Table 3 and physical properties of Blaine Fineness of302 m2/kg and density of 3120 kg/m3.

Cement paste was prepared by tap water of water/cement ratioof 0.35 and molded in 2 � 2 � 2 cm3 cube blocks. The specimenswere stored in an environment with high humidity 95% and roomtemperature for 1 day and molded and treated under curingcondition.

2.2. Methods

2.2.1. Simulation experimentsAfter removing the specimens from the molds, in order to

simulate ion leaching and pH reduction processes, the specimenswere exposed to running tap water for 27 days for hydration ofcement phases and leaching out the portlandite from their surfaceat room temperature. Then the specimens were exposed to sterilebiogenic sulfuric acid with pH less than 2 and at room temperaturefor 6 days. This acid was obtained from the growth of A. thiooxidansPTCC 1717 in M2 medium after two days at shaker incubator con-ditions (30 �C, 150 rpm). The culture growth resulted in a pH thatwas in agreement with a report given for a 20-year old sewercondition (Kaempfer and Berndt, 1999). In all the tests, the volumeratio of biogenic sulfuric acid to cement paste specimens surfaceswas equal to 5.

After simulation process and reduction of pH of the cementpaste to its required level, the biodegradation of specimens wasstudied through different pathways of slow (1) and accelerated (2)

Table 2Symbols designated for various culture media and their bacterial growth tests.

Symbol Test

M1 Culture medium of T. thioparus PTCC 1668M2 Culture medium of A. thiooxidans PTCC 1717TTP Growth test for T. thioparus in M1ATO Growth test for A. thiooxidans in M2CM1 Leaching test of cement paste specimen in M1CM2 Leaching test of cement paste specimen in M2CTTP Cement paste specimen exposed to T. thioparus in M1CATO Cement paste specimen exposed to A. thiooxidans in M2

T= 30 oC

100 rpm

V/S = 5

ATO CM2 CATO

Fig. 1. Schematic pathway of slow test (ATO: microorganism growth control, CM2:leaching process control and CATO: main test of bacterial growth on cement paste).

A. Yousefi et al. / International Biodeterioration & Biodegradation 86 (2014) 317e326 319

degradations. The surface microorganisms of cement paste speci-mens in all the tests were inactivated by immersion in 70e75%ethanol bath for 1e2 days and exposed to UV irradiation of 20W for1 h and were washed completely with sterilized distilled water.

Pathway (1): By dropping the surface pH to 7, at sterile condi-tions, the specimens were exposed to 1% inoculum of T. thioparusPTCC 1668 (CTTP test) culture growth followed by A. thiooxidansPTCC 1717 (CATO test). Each test was comprised of 2 control sam-ples; one for bacterial growth (ATO and TTP tests) and the other forleaching process (CM1 and CM2 tests) according to Table 2.

Fig. 1 shows a simplified schematic pathway (1) forA. thiooxidans PTCC 1717. One drop of reagent of bromophenol blue(0.01% w/v in distilled water) was used as an internal indicator forculture medium which turned purple at pH > 3.94 and cream atpH < 3.94 and with a further decrease in pH it appeared yellow.Changes in the color of culture medium were indications of thequality of the bacterial growth. A similar procedure was used forT. thioparus PTCC 1668 in M1 culture mediumwithout an indicator.Although this pathway took a long time to evaluate the biodegra-dation of cement in laboratory, but it was indispensable in studyingthe behavior of bacteria on cement paste. The tests were repeatedtwice.

Pathway (2): This pathway was an accelerated biodegradationmethod on cement paste.

Due to slow growth of A. thiooxidans and in order to exhibitaccelerated biodegradation of cement specimens, a semi-continuous culture of these bacteria with pH < 2 was used at30 �C. As in Fig. 2, the new specimens were exposed to biogenicsulfuric acid produced by A. thiooxidans PTCC 1717, with the pH ofabout 2, of 5/1 acid volume/cement surface. The change in the colorof the culture medium with respect to increases in pH was attrib-uted to the predominant leaching process over bacterial growth,which was an indication of lower biodegradation rate. So by in-creases in pH of the culture medium and its sudden color changesfrom yellow to purple, the microbial culture was immediatelyreplaced by a fresh one. Also, simulation was made by brushing thedegraded layers of cement paste every 10 days based on actual fieldcircumstances in which the sewage flow removes the cementdegraded layers. The whole trial lasted 90 days by removal of 9

Table 3Chemical composition of Type 2 Portland cement.

Chemical composition Concentration (% w/w)

CaO 63.26SiO2 22.50Al2O3 4.15Fe2O3 3.44MgO 3.25SO3 1.80K2O 0.65Na2O 0.20Free-CaO 0.72LOI 0.61

specimens every 10 days to measure the specimens’ compressivestrength, mass and dimensional status.

2.2.2. Measurement of degradation intensity of cement pasteThe properties of cement pastes such as compressive strength

(MPa), mass (g) and length (mm) were studied after 28-day curingand at different exposure times to culture liquid containingA. thiooxidans. The percentage of changes in properties of thespecimen in a 90-day period was determined using Eq. (1).

DXð%Þ ¼�X0 � XX0

�� 100 (1)

whereX0: Initial properties of cement paste at the end of curing

process.X: Measured properties of cement paste, after exposure to

A. thiooxidans culture, every 10 days up to 90 days.DX (%): Reduction percentage in properties in cement paste after

exposure to A. thiooxidans culture, every 10 days up to 90 days.

2.2.2.1. Measuring the compressive strength of the specimens.The surfaces of the cement paste specimens were cleaned afterreaching the desired age. For accuratemeasurement of the force thevertical load was applied on one side of the specimen which hadbeen in contact with the wall of the mold. The amount of appliedforce was measured by a hydraulic press machine until a crackappeared on the specimen. By dividing this force to the specimensurface, its compressive strength was obtained. The recordedcompressive strength was an average of 3 different specimens.

2.2.2.2. Measuring the mass of the specimens. The mass of eachcement paste specimen was taken by a digital scale with an accu-racy�0.01 g, after having been kept in an oven of 60 �C for 1 day forevaporation of surface water. The specimens were cooled in adesiccator for 1 h and their surfaces were brushed to remove thedegraded layers. The recordedmass was an average of 3 specimens.The specimens were placed in the oven after being exposed tobacterial activity and kept in a 100% power ultrasonic bath for15 min, at 30 �C and 59 kHz frequency to remove the white looselayer from their surfaces.

2.2.2.3. Measuring the length of specimens. The measured length ofeach cement paste specimen, from a specified point with a preci-sion caliper �0.01 mm, was an average of 3 specimens, under thecondition described in Section 2.2.2.2.

2.2.3. pH measurementThe pH of the bacterial suspensions and solutions containing

cement paste specimens was determined by a Percisa pH-meter,Switzerland. To measure the surface pH of the cement pastespecimens, each one was placed in 100 ml of distilled water for 1 hto achieve a stable pH for the solution. This test was repeated on 3identical specimens to obtain an average pH value.

Fig. 2. Schematic pathway of accelerated biodegradation test of cement paste specimens.

A. Yousefi et al. / International Biodeterioration & Biodegradation 86 (2014) 317e326320

2.2.4. Measuring the sulfate concentrationTomeasure the concentration of sulfate ions produced by sulfur-

oxidizing bacteria, a method based on barium chloride solution andturbidimetry measurements (Kolmert et al., 2000) were used. Inthis method, barium sulfate precipitate was formed and the cultureturbidity was measured at wavelength of 420 nm by a MetertechUVeVis spectrophotometer (SP8001 model, Taiwan).

2.2.5. Cell optical densityMicrobial culture turbiditymeasurement, as an indirect method,

was employed to measure bacterial cell concentration. UVeVisspectrophotometer was used at 600 nm to measure the opticaldensity of cells (OD600) which was correlated with the rate ofbacterial growth.

2.2.6. Changes in mineral phase compositionTo study the changes in mineral phase composition of degraded

layers of the cement paste specimens, a Bruker X-ray diffractometer(XRD, Cuka, l ¼ 0.154 nm) was used.

2.2.7. Microscopic structural changesThe microstructure of the biodegraded cement paste was inves-

tigatedusing aTESCANscanningelectronmicroscopy (modelVEEA11,Czech), and energydispersive analysis bya TNCAEDAXX-ray (Oxford,England). The linear elemental analyses of biodegraded specimenswere carried out by EDAX to identify the changes made in theirelemental compositions. The sampleswere placed in an ovenof 60 �Cfor 3e4 days. After cooling, they were gold coated in a deep vacuum.

3. Results and discussion

3.1. Curing process and pH reduction in cement paste

A 27-day curing process of the specimens resulted in hydrationof different phases of cement and reduction of their pH which were

Fig. 3. Variations in pH and sulfate ion concentration in various suspensions versus ti

accomplished by running tap water. In this process, the pH of thespecimens at around 12 dropped to about 10 at the rate ofabout �0.089 pH/day. To speed up the cement biodegradation, thesurface pH of the specimens was reduced to 7.29 at a rate ofabout �0.45 pH/day by their exposure to sterile biogenic sulfuricacid at pH of approximately 2 for 6 days.

3.2. Thiobacillus growth on cement paste through pathway (1)

At first, the cured cement paste specimens were exposed toT. thioparus culture for 13 days. By further decrease in pH, theywereexposed to A. thiooxidans growth. In each experiment, two groupsof control samples including biologically controlled tests (TTP andATO) and leaching controlled tests (CM1 and CM2) were used.

The sulfate and hydrogen ions were produced due to oxidationof sodium thiosulfate in presence of oxygen and sufficient moisturein TTP test (a microbial control) by T. thioparus as in Eq. (2). In thisprocess, the pH of solution was reduced to 4.5, from its initial valueof 7, after 6 days shown in Fig. 3. In CM1 control test of portlanditeleaching the pH increased from 7 to about 8. In CTTP procedure, asthe actual test, comprised of T. thioparus bacteria and cement paste,the overall trend in pH variation was similar to microbial controltest, but once the microorganismwas in its lag phase (initial 4-day)there was a slight increase in pH which occurred due to theleaching process. At bacterial growth phase, entering the loga-rithmic phase of 4e6 days, the pH was reduced to 5. Beyond 6 days,at stationary phase of bacterial growth the leaching of calciumhydroxide was the dominant process and, therefore, the pH of themedium followed an increasing trend.

Na2S2O3 þ 2O2 þ H2O/Na2SO4 þ H2SO4 (2)

The increasing trend in sulfate ion concentration is shown inFig. 3. It is evident that sulfur-oxidizing bacteria have consumedthiosulfate ions in the culture medium as a source of energy, andconverted them into sulfate ions (TTP test). In control test of

me (TTP: microbial control, CM1: leaching process control and CTTP: actual test).

A. Yousefi et al. / International Biodeterioration & Biodegradation 86 (2014) 317e326 321

leaching process (CM1), the sulfate ion concentration is negligibledue to low concentration of sulfate ions in initial cement paste, andso a slight increase is observed in sulfate concentration. In CTTPtest, the production of sulfate is clearly evident due to leachingprocess of cement and bacterial growth. In contrast the productionof sulfate ions in this test is higher (w70mM) thanwhat it had beenexpected (40e50mM), and this could be due to the consumption ofcement’s other components by T. thioparus and further formation ofsulfate ions from the cement paste. Formation of calcium, iron andaluminum ions of cement, exposed to sulfuric acid produced byA. thiooxidans, is less than the same test with chemical sulfuric acid(Knight et al., 2002), due to differences in chemical and biologicalsulfuric acid attacking mechanisms on cement paste, and/or it maybe linked to consumption of calcium, iron and aluminum ions byA. thiooxidans in cement.

With further reduction of pH to w5, A. thiooxidans was able togrow and colonize in cement. Fig. 4 shows variations in pH of M2culture medium versus time in solutions with and withoutA. thiooxidans and the cement paste specimen. In CM2 control test,with no bacteria, due to leaching process of portlandite withincement specimen, the pH of solution increased over time and itsamount increased from 4.52 to w6.5. In ATO test, as the microbialcontrol, due to oxidation of thiosulfate ions to hydrogen and sulfateions, the pH was lowered from 4.52 to 1.85 after 5 days. In CATOtest, there were two opposing actions of bacterial growth (reduc-tion in pH) and leaching process of portlandite (increase in pH).Once the bacteria were in their lag phase, a slight increase occurredin pH due to portlandite leaching process and when at their loga-rithmic phase (day 2e5) the pH decreased to w2.5. After 5 days,when the bacteria entered their stationary phase, the leachingprocess of portlandite overcame this phase, so the pH of the solu-tion increased again.

The maximum turbidity of bacterial growth was found in ATOtest with its OD600 ¼ 0.25. Cell turbidity of the suspension ofleaching process control (CM2) was negligible. The cell turbidity ofCATO test (bacterial growth on cement paste specimen) was verylow as well, whereas due to the presence and growth of bacteria,the OD600 was expected to be at least 0.2. This could be due toadherence of bacteria cells on the surface of the cement. After 8days, the specimen surface, exposed to A. thiooxidans growth,appeared as a soft, white and thin porous layer, but the specimenthat had been placed in solution M2 looked unchanged. In fact, thislayer seemed to have provided a suitable substrate for bacteria toadhere strongly to cement surface and therefore, resulting in lowerturbidity of solution.

After these biodegradation experiments, the specimens, placedin M1 and M2 media, were examined for their final compressivestrength, length and mass values. The results of the measurements

Fig. 4. Variations in pH of M2 culture medium versus time in solutions with andwithout A. thiooxidans and a cement paste specimen (ATO: microbial control, CM2:leaching process control and CATO: actual test).

indicated that the properties of specimens were kept intact.Therefore, M1 and M2 media were suitable for the growth of genusThiobacillus on cement pastes. Under normal conditions, however,the cement pastes in neutral states were expected to show highercompressive strength due to their hydration process, but due tonatural leaching of portlandite from inside the cement into theculture media within such short testing periods of 8 and 13 daysthere were no detectable changes observed in the general proper-ties of the specimens.

3.3. Accelerated biodegradation of cured cement pastes bypathway (2)

The results of the previous section showed that genus Thio-bacillus grewwell on a cured cement paste resulting in formation ofbiogenic sulfuric acid. This acid showed complex damaging effectson cement structure. The rate of sulfuric acid production bypathway (1) was very slow and the assessment procedure of thechanges in cement structure took much longer. Therefore, to speedup the rate of cement biodegradation and the subsequent assess-ments the simulated pathway (2) of Section 2.2.1 was adopted.

The results of the accelerated biodegradation of cement pastesby A. thiooxidans PTCC 1717 culture with pH less than 2 are shownin Fig. 5. According to Fig. 5a, the degradation depth of the speci-mens during 90 days is measured 2.5 mm, which is equivalent to11% reduction in the specimens length. By its extrapolation to oneyear, the annual rate of cement paste biodegradation is found to be10 mm/y. However, the real annual rate of biodegradation of theconcrete is reported as 2e4.7 mm/y (Roberts et al., 2002). Also thesevere degradation of Hamburg concrete sewer pipes is reported6 cm after 6 years, although the pipeline had been coated by epoxyresin with 200e300 mm thickness (Sand and Bock, 1984). Thedegradation depth of the pipeline contained 50% gypsum and thesurface pH value of the concrete was reported between 1 and 2. Inthe same sewage system, degradation took place on concrete whilein our current investigation the degradation has been evaluated oncement paste and this latter location has displayed a considerableeffect on the degradation rate. As far as we are aware, although noresearch is carried out under the exact similar condition as others,but it seems that our final results and deductions might be com-parable with the data reported by Hamburg sewage system. Inaccelerated methods, the real degradation occurs faster, so theevaluation processes and their reproducibility are easier, thoughthe natural degradation rate can be different from an accelerateddegradation rate, therefore, a fixed annual rate cannot be reportedfor biodegradation of cement and concrete, because the degrada-tion rate depends on different parameters such as the quantity ofsulfur compounds, moisture, turbulency and sewage flow rate,length of pipeline and temperature, etc.

The temperature of 30 �C, compared to an ambient condition,was an optimum at which bacterial growth reached its maximumand it accelerated the rate of cement biodegradation. It is evident inFig. 5b the cultivation of A. thiooxidans PTCC 1717 at a pH below 2with acid volume to cement surface ratio of 5 to 1, its suddenchange to a pH of w4 and subsequent replacement with the initialcultivation, and cleaning of loose porous layers in an ultrasonicbath, all contributed in a relatively high drop in mass of the cementpaste specimen. The mass reduction of 43% after 90 days is clearlymuch higher compared to other researchers’ reports listed inTable 4. It should be noted that these researchers employed con-crete and mortars which certainly influenced the degradation rate,due to aggregates which must have shown high resistance to bac-terial attack. Cleaning in an ultrasonic bath (Hormann et al., 1997)and drying in an oven of 60 �C for 1 day before weighing thespecimens accounted for high mass loss of the present study,

Fig. 5. Changes in testing parameters: (a) length (b) mass and (c) compressive strength of a cement paste specimen during a 90-day test period, and (d) reduction in compressivestrength versus mass.

A. Yousefi et al. / International Biodeterioration & Biodegradation 86 (2014) 317e326322

compared to other studies which just use an ordinary brush. It maybe deduced that there are certainly no fixed rates observed forannual mass and dimensional reductions of cement as they varyaccording to the factors mentioned above. In fact, the annual massreduction rate is a strong evidence for biodegradation of cementstructures and it is a justification for the protective measures whichshould be taken against degradation of cement-based materials.

Fig. 5c shows the changes in compressive strength of cementpaste specimens versus time. The further reduction of compressivestrength (96%) of cement paste compared to their mass and lengthreductions can be due to dimensional deformation of specimensfrom their initial cubic shape. This deformation in the shape ofspecimens prevents a uniform force to be applied on their surfacesduring compressive strength measurements. Also cement chemicalcomponents involved in chemical reactions in acidic solution arevery intense, forming expansion products in surface areas of thespecimens, causing internal tensions and weakening mechanicalstrength. After one month passed from the beginning of the testperiod, it occasionally happened that specimens were broken un-expectedly when attempts were made to brush off the thin porouslayers for final preparation of their mass and length measurements.

Fig. 5d indicates an approximate linear relationship in reductionof mass and compressive strength of cement specimens during

Table 4The reduction in mass and thickness of cement-based specimens reported in literature.

Specimen type Thicknessreduction

Mass reduction (%) Experimenttime (day)

Bacte

Concrete 0.75e0.8 mm 9e11 51 T. ne3.8 mm 13.5 360 T. thie 5.8 270 Thiob50% 35 365 Sewe

Mortar e 18e31 150 T. thiCement paste e 5.7 90 Thiom

T. fer11% 43 90 A.thi

90-day test period. The slope of the fitted line for compressivestrength and mass reductions is found to be 8.3 MPa/g.

The above tests were completed by visual observations ofspecimens. In the first and second 10-day periods, specimensshowed a normal appearance without any change in color anddimension, though there were reductions noticed in length by1.03% (Fig. 5a), mass by 2.74% (Fig. 5b) and compressive strength by26.7% (Fig. 5c). Reduction of length by 1.03% was not detectable bynaked eye after 20 days. The mass reduction of 2.74% in the sameinterval was due to leaching processes of cement components,especially the portlandite into the acidic bacterial solution.Although the reduced mass and length of the specimens were notcorrelated with any change in their appearance to be detected bynaked eye, but these reductions sufficiently agreed with thereduction of compressive strength by 26.7%. After 30 days from thestart of experiment of accelerated biodegradation process, theformation of a white, soft and low density layer with low adhesionwas observed on the surfaces of specimens. The layers were easilyseparated from the surface of the specimens, as soon as they wereremoved from bacterial acidic solution and exposed to ambient airor placed into the oven of 60 �C with severe cracks developed onthe white layers which dropped from the surface instantly. Inaddition, when in another test the dried specimen and a control

ria Ref.

apolitanusT. Thiooxidans T. ntermedius/T. novellus (Vincke et al., 1999)ooxidans (Ismail et al., 1993)acillus neapolitarius, T. intermedius and T. thiooxidans (Sand et al., 1984)r atmosphere (Okabe et al., 2007)ooxidans (Hormann et al., 1997)onas perometablis T. thiooxidans A. thiooxidans and

rooxidans T. intermedia and T. peromotabolis ProbeWei et al., 2010)

ooxidans Current research

Fig. 7. X-ray diffraction patterns of the surface layer, the deteriorating layer and theintact part of cement paste specimen (G, gypsum; P, portlandit; C, calcite; CSH, calciumsilicate hydrate).

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specimen (not having been in biogenic sulfuric acid solution) wereput into the water, plenty of bubbles were rapidly developed fromthe degraded specimen up to about 1 h. These bubbles from thedegraded specimens were attributed to the leaching process of theinternal chemical components of the cement and formation ofporous and tiny ducts within it. This phenomenon and separationof layers persisted till the end of the accelerated biodegradationperiod. In images of Fig. 6, there are changes in the appearance ofthe specimens representing the degraded paste after 60 daysexposure to cultivation of bacterial growth at pH below 2,compared to a pure cement paste. The changes in the appearance ofthe specimens during the sixth to ninth testing periods are clearlyvisible to the naked eye.

By visual assessment of the cross-section of degraded speci-mens, 3 distinct regions were noticed as the followings: first, acompletely degraded white thin layer on the surface, lacking anycementitious property; the second region, consisting of a moder-ately thin layer with cement property and distinct bright colorrelative to other intact parts of the specimen and the third regionwas the remaining part that seemed completely safe. The samplingand XRD analysis were performed on each region to study thephase constituents of degraded sections (surface white layer) andthe deteriorating layer (thin layer under the white region). Theresults of this analysis are shown in Fig. 7. By comparing the XRDdata with standard patterns (33-306, 05-0586, 04-0733, 33-0311and 41-1451) it appeared that the white loose layer formed on thecement paste was mainly consisted of gypsum (33-0311).

The comparison of XRD patterns of the deteriorating layer withstandard patterns (04-0733 and 05-0586), it was revealed that thedominant phases were indicated as calcite and portlandite and thesecondary phases consisted of calcium silicate hydrate and gypsum.The specific Ettringite phase peaks were not observed at 2q ¼ 9.17�

and 15.82�. The reason could be due to unstable Ettringite phase atpH under 10.6 (Allahverdi and �Skvara, 2000b) which because of itsclose contact with biogenic sulfuric acid solution at pH below 2, itdecomposed to gypsum and aluminum hydroxide. Also Thaumasitephase could not be produced at 30 �C test temperature which washigher than Thaumasite formation temperature (15 �C).

The XRD pattern of the intact part of the samples showed thatportlandite and calcium silicate hydrate were dominant phasescompared to the secondary phase of calcite. Fig. 7 shows thefundamental changes in mineral phase constituents of biodegradedcement paste specimen with biogenic sulfuric acid.

The presence of calcite phase in the deep regions of cement pasteafter 60 days of exposure to biogenic sulfuric acid was probably dueto either carbonation by atmospheric carbon dioxide during samplepreparation or carbonation during exposure to biogenic sulfuric acid.In fact, one can conclude that pathways must have been created fordiffusion of atmospheric carbon dioxide which reacted with por-tlandite present in deep parts of cement paste.

Fig. 6. The changes in the appearance of specimen by exposure to cultivation of A. th

According to Fig. 7, the XRD patterns of the “deteriorating layer”are comparably different from the intact part of specimen. In thislayer, there is the reduction in the amount of portlandite phase andthe high presence of calcite phase. These changes may be due to thefact that in this region the atmospheric carbon dioxide has easilyreacted with portlandite which has led to increased calcite phase.The presence of a small percentage of gypsum in this layer may alsobe attributed to the diffusion of a biogenic sulfate and its reactionwith portlandite. It is noted that at cleaning stage some specimenswere broken before weighing. This could be due to internal ten-sions created by gypsum crystals sediment in the surface regions ofthe specimen leading to micro-cracks formation.

iooxidans with pH below 2 after 60 days (control (left) and biodegraded (right).

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In the white surface layer which was completely degraded, nosign of portlandite, calcite and calcium silicate hydrate phases wereobserved. At low pH, the physical and chemical balance of thecement matrix was disrupted and its hydrated compounds startedto decompose. The first compound that began to dissolve and leachout included the portlandite followed by decalcification of calciumsilicate, calcium aluminates and calcium aluminum ferrite hydratesproducing the amorphous hydrogels. Following the sulfuric acidattack onto the cement paste, the final products consisted of cal-cium sulfate and the hydrogels of silica and aluminum and ferricoxide (Allahverdi et al., 2000a,b, 2005; Lajili et al., 2008).

In the degradation process due to biogenic sulfuric acid attack,the diffusion of the acid ions into the specimen surface may facil-itate reactions with the leaching calcium and the growing gypsum

Fig. 8. SEM images of cement paste specimen: (a) with 70 magnification and (b) 150, (c)deteriorating layer and (f) its intact part after 60 days of exposure to biogenic sulfuric acid

crystals. As the degradation process proceeds, the gypsum layerthickens further and it finally imposes a protective effect andcontrols the rate of degradation process by diffusion phenomenon.However, due to elimination of the gypsum layer in every 10-dayinterval, a fresh surface of the cement paste came into contact withthe solution of biogenic sulfuric acid, so in total, the degradationrates were controlled by the diffusion phenomenon combined withthe surface degradation processes.

Fig. 8 shows SEM images of cement paste specimen after 60 daysof exposure to biogenic sulfuric acid with different magnifications.

While preparing the samples for SEM and EDAX analyses, themajor part of the surface degraded layer that contained gypsumwas separated from the surface while drying the samples for 4 daysat 60 �C. Despite of the gypsum presence which was confirmed by

its deteriorating layer with 1000 magnification and (d) 2000, (e) the boundary of its.

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XRD in the degraded surface layer, these crystals could not be easilytraced by SEM analysis on the remaining surface layer of thespecimens. The layer boundary line of the deteriorating and theintact deep layers of the cement paste is clearly observed in Fig. 8b.

The present images in Fig. 8c and d show the deteriorating layerwith 1000 and 2000 magnifications. With regard to these images,the vertical border between the deteriorating layer of at leastw100 mm thickness and the intact area is clearly visible. Fig. 8dshows the deteriorating layer with higher magnification (2000)having a structure covered with small and shattered pieces ofparticles. Due to consumption of portlandite in the deterioratinglayer, there is no sign of its crystals present, though some scatteredmicroscopic cracks are evident for the progressive degradation ofthe cement paste.

The images in Fig. 8e and f show the deteriorating layer and theintact areas of cement paste clearly. The image on Fig. 8e showsportions of the extended vertical cracks in the boundary of thedeteriorating layer and the intact areas. The image on Fig. 8f showsthe intact part of cement paste after 60 days of exposure to biogenicsulfuric acid. The amorphous matrix of calcium silicate hydrates isclearly visible inside the circle.

Fig. 9 indicates the EDAX linear elemental analysis of the cross-section of cement paste specimen after 60 days of exposure tobiogenic sulfuric acid. The high sulfur content close to the surface isa strong evidence for the presence of gypsum, as a residual

Fig. 9. EDAX Elemental analysis from the cross-section of the cement paste specimenafter 60 days exposure to biogenic sulfuric acid.

degraded layer, compared to other areas. So in the outermost layer,the amount of silicon is low due to the formation and precipitationof gypsum and because of its insolubility, silica gel, contrary toportlandite, it cannot be leached out into the solution. But, ac-cording to the results of element analysis of the deteriorating layer,as the next layer, the amount of silicon is high while the amounts ofiron and aluminum are low. The increase in the amount of silicon isdue to the leaching portlandite and the reductions in the amountsof leaching iron and aluminum can be due to the acidic pH of thisregion which transfers iron and aluminum ions or other com-pounds into the acid solution. In the third region, the furthest awaylayer from biogenic sulfuric acid attacks, the amounts of calcium,iron, aluminum and silicon are comparatively constant. In fact,diffusion of the constituents of cement paste into the solution isdependent on the pH of different regions. Therefore, calcium canleach out from all parts of the specimen, because at pH below 12.6,the portlandite begins to dissolve, while aluminum and iron of thecement can enter into solution from the deteriorating layer and itssilica content is insoluble in all acidic conditions of the presentmedia. It may be theoretically stated that the pH increases fromw2of the surface layer to above 12 of deep intact region, although it isnot possible to measure the pH of different parts of specimen.

4. Conclusions

Biodegradation of cement is a slow process and its study needsto be accelerated by simulation. In this research, by simulation ofsewage system conditions, i.e. using running tap water and semi-continuous culture of A. thiooxidans PTCC 1717 bacteria, thebiodegradation of the cement paste has been investigated. Theresults show that:

� T. thioparus PTCC 1668 and A. thiooxidans PTCC 1717 bacteriahave well grown in the presence of cured cement pastespecimens.

� Cement biodegradation is due to sulfuric acid production andconsumption of cement components by bacteria.

� Using a semi-continuous culture of A. Thiooxidans inducesintensive biodegradation of the cured cement paste specimens.

� By bacterial attack under simulated conditions during 90 days,the compressive strength, length and mass of the cured cementpaste specimens are reduced by 96%, 11% and 43% in the ordergiven.

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