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CHAPTER 2

LITERATURE REVIEW

2.1 GENERAL

In this chapter, comprehensive review of literature highlighting biological influences

on construction materials; biodeterioration of various live organisms like bacteria,

lichens, mosses, fungi and algae; interaction of weak acid with cement / cementitious

materials, are presented. Finally, critical observations based on the above review

highlighting the ample scope for research in the area of biodeterioration has been

highlighted.

2.2 BIOLOGICAL INFLUENCES ON CONSTRUCTION MATERIALS: AN

OVERVIEW

2.2.1 Durability

Traditionally, a variety of materials (both natural and man made) have been

extensively used in construction activities, all over the world. Of them, concrete has

been used very extensively due to its versatility, among other reasons (Mehta and

Monterio, 1999). In recent years, the emphasis is on understanding the strength and

durability of construction materials, and concrete is no exception to the above

approach.

Durability is defined as the service life of a material under given environmental

conditions. Durability of a variety of construction materials and mechanism / (s) of

their deterioration have been studied and reported. ACI Committee 201 has adopted a

separate definition for durability of concrete (Mehta and Monteria, 1999) and Mehta

and Gerwick, (1982), have grouped the causes of concrete deterioration. In general, all

factors that might cause a material / structural system to perform unacceptably at any

point during its life time have to be considered for service-life prediction and a

comprehensive life-cycle cost analysis. From the above perspective, apart from

extreme events (like earthquake, cyclones etc.), the environmental factors have made

the broadest impact on the long-term performance and hence, the largest potential

economic consequences. In that context, in recent times, the focus of study world over,

is on biodeterioration.

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2.2.2 Biodeterioration

Deterioration is defined as a loss of structural capacity with time as a result of the

action of the external agents or material leaching (Sanchez-Silva et al. 2008).

Biodeterioration in its widely accepted form of definition is: “any undesirable change

in the properties of a material caused by the vital activities of organisms” (Hueck,

1968). Another definition is: “the process by which biological agents (i.e. live

organisms) are the cause of the (structural) lowering in quality or value” (Rose, 1981).

There is a distinction between ‘biodegradation’ and ‘biodeterioration’ (Allsopp et al.

2006). Whereas ‘biodegradation’ is concerned with the use of microorganisms to

modify materials with a positive or useful purpose, ‘biodeterioration’ refers to the

‘negative impact of live-organisms activity’. While the biological process/(es) are the

primary cause of deterioration in biodeterioration, the conventional physical and

chemical processes are the primary causes associated with durability and hence in the

deterioration studies of construction materials.

2.2.3 Classification of Biodeterioration

Biodeterioration can be broadly classified into three categories namely: (i) biophysical

(ii) biochemical and (iii) aesthetic (Allsopp and Seal, 2006 and Gaylarde et al., 2003)

Depending on the biodeteriogens, the nature of material and environmental conditions,

the above processes may occur separately or simultaneously.

Biophysical or biomechanical deterioration refers to actions that directly affect the

component’s material and mechanical properties. This often is related to the process

by growth or by movement, but, do not use the material as a food source (e.g. root

damage, gnawing by rodents).

Biochemical deterioration can be further divided into: (i) assimilatory and

(ii) dissimilatory. Assimilatory process occurs when the organisms use the component

as a source of food (i.e. carbon and / or energy source), thus modifying the properties

of the material (e.g. degradation of fuels, metals). However, in a dissimilatory process,

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the live-organisms excrete waste products or other substances (e.g. H2

Biodeterioration is usually concerned with the consequences of relatively small

organisms (i.e. microorganisms and fungi). The development of specific biological

species on a particular construction material is determined by the nature and properties

of the material (mineral constituents, pH, relative percentage of various minerals,

salinity, moisture content and texture). It also depends on certain environmental

factors (i.e. temperature, relative humidity - RH, light conditions, oxygen, nitrogen,

atmospheric pollution levels, wind and rainfall). In addition to light, microbial

invasion also requires the existence of nutrients. Apart from light, the two major

classes of nutrients are those that provide a source of energy and nitrogen. They are

S; FeS) that

react chemically with the component, thus adversely affecting the material.

Aesthetic or fouling or soiling biodeterioration is caused by the presence of organisms,

their dead bodies, excreta or metabolic products forming a microbial layer on the

surface of the component known as ‘biofilm’. This type is primarily associated with

the presence of microorganisms causing unacceptable appearance. Even though the

performance of the materials is not affected initially, with passage of time, the fouling

may exceed the purely aesthetic consideration and may cause physicochemical damage

to the component / material.

A glossary of terms associated with biodeterioration and the various methods /

techniques of assessment of biodeterioration are given in Appendix A1.

2.2.4 Live organisms associated with biodeterioration

The most common live-organisms associated with biodeterioration of construction

materials may be grouped as: (1) Marine borers (e.g., gribble and shipworms);

(2) Insects (e.g., termites and wood-boring beetles); (3) Fungi (soft rots, white and

brown rots), primary and secondary molds, strainers algae, and lichens; and

(4) Microorganisms (e.g., bacteria).

2.2.5 Ecological aspects of biodeterioration

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provided by the enzymatic breakdown of compounds in the materials and by the

environment. In general and in very simple terms, the response of living organisms to

a potentially colonizable surface depends on the ecological and physiological

requirements of the biological species involved (Caneva and Salavadori, 1988).

2.2.6 Nutritional requirements of living organisms

All living organisms can be broadly classified as: (i) autotrophs (ii) heterotrophs,

based on their nutritional requirements. For all autotrophic organisms, inorganic

surface constituents represent potential nutritive substances and are important factors

that condition their growth. On the other hand, heterotrophic organisms grow only

when organic matter is present on the surface. Most organisms, except, xerophilous

species, prefer surface with a high moisture content. Classifications of organisms

based on their nutritional requirements are summarized in Table 2.1.

2.2.7 Characterization of biodeteriogens

An understanding of morphological and physiological characteristics of biological

agencies is required to identify accurately the biological species that have established

themselves on the surface of and sometimes within, the construction material. It is not

only essential to establish the exact characterization (both qualitatively and

quantitatively) of the organisms active on the construction material, but, it is also

important to assess the cause-effect of biodeterioration action of a specific identified

biological agent. The above aspect is critical from the point of view of appropriate

choice of preventive and remedial methods / measures.

While it is easy to identify using visual observations in the field and in the laboratory

through microscopic diagnostic methods the organisms such as: lichens, mosses and

liverworts, organisms such as: bacteria, fungi and algae are not easily identifiable

through direct visual examination. Hence this study involves the isolation and

characterization of the active microbial agents in a field sample, re-creation of

geomicrobial process under laboratory conditions using an enriched / pure culture

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from the field sample, and characterization of the reaction mechanisms of active

microbial agents (Ehrlich, 1981). Microbes, especially bacteria, algae and fungi from

field samples may be cultured aerobically on a variety of media (with / without prior

environment) or anaerobically cultivated in liquid and solid media (e.g. agar-shake

cultures).

Different organisms may be isolated from field samples using standard

microbiological techniques such as: staining (Curri and Paleni, 1981) and enzymatic

testing (Curri and Palein, 1976, Warscheid, 1990). Isolated colonies of

microorganisms grown in the culture can then be investigated by optical and electron

microscopy.

2.2.8 Assessment of biodeterioration

An appropriate assessment of biodeterioration and weathering of a material requires a

combination of micrological, surface analysis and material characterization techniques.

Evaluation of biodeterioration typically involves: (i) identification of the major types

of organisms; (ii) microscopic observation of the interface biofilm / material and of the

material itself after removal of the biofilm; (iii) elemental and mineral analysis of the

damaged material and (iv) a final interpretation consisting of the correlation between

the morphological and metabolic properties of the identified organisms, the

morphology of the decay and the chemistry of the altered material.

(A) Analytical methods

Several methods for material characterization and surface analysis such as:

(i) scanning electron microscopy (SEM); (ii) energy dispersion X-ray analysis (EDX);

(iii) environmental scanning electron microscopy (ESEM); (iv) petrographic analysis;

(v) Mossbauer spectroscopy (MS); (vi) conventional X-ray diffraction (XRD);

(vii) grazing incidence diffraction (GID); (viii) Raman spectroscopy (RS); (ix) Other

spectroscopic techniques like X-ray photoelectron spectroscopy (XPS), reflection

electron energy loss spectroscopy (REELS) and advanced combined applications of

synchrotron based µ-X-ray (SR- µXRD / µXRF) have been adopted so far (Herrera

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and Videla,2009). Apart from the above methods, DNA based molecular biology

techniques can be advantageously used to identify components of microbial biofilms

(Herrera and Videla, 2009). Herrera and Videla (2009) have cited different examples

on the use of the above methods in the field of preservative cultural property. A brief

outline of the above (analytical) methods are given in Appendix -A2.

(B) Phycochemical Studies

Phycochemical is a new term reprorted to be first used by Shameel (1990). It is

actually the study of natural products and chemical constituents from a biological

viewpoint, which is very extensively used in algal studies. It was primarily directed to

the investigation and distribution of ‘secondary metabolites’ in different parts of algae

under different seasons and variety of habitat conditions (Shameel, 1991). Since

1990s, effort was directed by many phycologists all over the world, for the study of

different types of natural products occurring within algae. Typically, a detailed

phycochemical is made to analyze their saturated and unsaturated acids, sterols,

terpenoids and other chemical constituents. Rehman (1994) has summarized the

phycochemical studies on various species of algae, especially found on the coast of

Karachi, Pakistan, and has reported that unsaturated acids were found in large

proportion than saturated ones. Further, their compositions varied from species to

species. A brief outline of the standard testing methods for isolation of fatty acids is

given in Appendix- B4.

2.2.9 Biofouling

Biofouling, especially, marine biofouling is caused by the adhesion of barnacles,

macroalgae and microbial slimes and it is a dynamic process.

(A) Micro and Macro - fouling

When a clean surface is immersed in water, it absorbs within minutes, a molecular

‘conditioning film’ consisting of dissolved organic material. Bacteria colonize within

hours, as may unicellular algae and cyanobacteria (blue-green algae). These early

small colonizers form a biofilm: an assemblage of attached cells, sometimes referred

to as ‘microfouling’ or ‘slime’. Diatoms (unicellular algae) predominate in biofilms

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and adhere to certain types of antifouling coating, by secreting extracellular polymeric

substances (EPS).

A macrofouling community (consisting of either ‘soft fouling’ or ‘hard fouling’) may

develop and overgrow the microfouling. Soft fouling comprises algae and

invertebrates, such as, soft corals, sponges, anemones etc., while hard fouling

comprises invertebrates such as: barnacles, mussels and tubeworms. However, the

specific organisms that develop in a fouling community depend on: the substratum,

geographical location, the season and factors such as: competition and predation.

(B) Major Macrofouling Alga

The dispersal stage of organisms is the most important stage in biofouling and its

control. Larvae of invertebrates and spores of algae need to quickly find and bind to a

surface in order to complete their life history. This adhesion takes place within

seconds, under water, to a wide range of substrates, over a wide range of temperatures

and salinities, and in conditions of turbulence. This phase of initial, or first-contact,

adhesion to a substrate is shown by diverse single and multi-cellular fouling organisms

and has been, termed as ‘the first kiss’.

(C) Colonization of ‘Enteromorpha’

The green algae Enteromorpha – the slippery grass like plant that covers rocks in the

intertidal zone, is the major macrofouling alga. It colonizes on new surfaces through

the production of vast quantities of microscopic motile spores [Fig. 2.1; Callow and

Callow, 2002]. Swimming spores attach rapidly once they have ‘detected’ a suitable

surface for settlement, resulting in firm attachment to the substratum (Fig. 2.2). Spore

germination often occurs within a few hours, giving rise to germlings. These are

attached to the substratum by adhesive that is secreted by the rhizoids. This is followed

by an irreversible commitment to adhesion involving withdrawal of flagella and

secretion of a powerful adhesive (Fig. 2.3). A number of ‘cues’ (like negative

phototaxis and chemical cues) are involved in attracting spores to a particular surface,

on which to settle and attach and that settlement is strongly influenced by a number of

surface properties including wettability and microtopography.

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Once a suitable area for settlement is located, the spore then secretes a glycoprotein

adhesive by exocytosis of the content of membrane bound cytoplasmic ‘adhesive’

vesicles, formed from the Golgi apparatus [Fig. 2.4; Callow and Callow, 2002)].

Recently, a relatively new technique, known as atomic force microscopy (AFM), to

examine some of the properties of spore glue has been reported (Callow and Callow,

2002).

(D) Atomic Force Microscopy (AFM)

It is a useful investigative tool that provides 3-D images of surface topography of

biological specimens in ambient liquid or gas environments. The advantage with this is

that the samples do not need to be fixed, dehydrated, coated or frozen. Further, the

instrument neither uses optical or electronic lens. Instead it relies upon sensitive laser

detection of deflections to a small cantilever mounter tip, which occur in response to

intermolecular forces. The tip is raster - scanned across a surface.

AFM can also used to measure visco-elastic properties of materials such as: adhesive

strength, hardness and elasticity. AFM has been used to make measurement on

Enteromorpha adhesive pad in its hydrate state and the freshly released adhesive was

found to have ‘adhesion strength’ of approximately 500m N/m (indicating a very

sticky material) and its compressibility is similar to a 20% solution of gelatin. It was

also reported that within minutes of release the adhesive undergoes a progressive

‘curing’ process, presumably by cross-linking, becoming less sticky and more

compressible and assuming a consistency similar to natural rubber (Callow and

Callow, 2002).

2.2.10 Biodeterioration Mechanisms

(A) Bioreceptivity

A number of factors such as: chemical nature, physical structure and geological origin

(for construction material like stones) and environmental factors such as: water

availability, pH, exposure climatic conditions, nutrient sources and petrologic

parameters such as: mineral composition type of cement as well as porosity and

permeability of the rock material, in general, influence biodeterioration and

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bioreceptivity of construction materials (Warscheid and Braams, 2000). Large pore

sandstones promote microbial contamination only temporarily, whereas, small pore

stones offer more suitable conditions for bioreceptivity. The presence of significant

amounts of carbonate compounds (e.g. > 3% w/v CaCO3) in calcareous sandstones,

concrete or lime mortars, results in the buffering of biogenic metabolic products

producing a constant suitable ph-milieu, for the growth of bacteria. Dense calcareous

matrix a superficial microbial contamination and subject to lichens and fungal attack,

but, the degree of attack depend on the pore size distribution as well as on the

alkalinity of the artificial stones. Organic adhesives, which are present in ancient brick

and mortar, increase the susceptibility of the mineral substrate to microbial attack

(Warscheid and Braams, 2000).

(B) Microbial Induced Deterioration (MID): General Mechanistic Overview

The term ‘microorganism’ covers a wide variety of life forms. Bacteria, cyanobacteria,

algae, lichens and fungi, together with protozoa, are classified as microorganisms. Due

to their diversity, they are able to degrade nearly all naturally occurring compounds

(Sand, 1997). Pure cultures do not exist under natural conditions. Mixed culture, called

‘biocoenoses’ are active by usually, creating favorable growth conditions for the

microorganisms. Moreover one microorganism may exert multiple detrimental effects,

and the substratum (i.e. base material) that are attacked may also include a (large)

variety of different compounds, causing complexity in the analysis. The various

categories under which biodeterioration can be grouped are summarized and given in

Table 2.2. A brief description of the microbial action, summarized under nine main

categories are given below (Sand, 1997):

(1) Physical presence of microbial cells

Sometimes, the physical presence of microbial cells is sufficient to cause damage to

equipment. Sediment microbial cells with dimensions of about 0.3 -2 μm diameter and

fungal cells of about 5 μm and above will cause damage to the electronic chips. Hence

clean air technology has to be used to keep such failure to a minimum.

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(2) Inorganic acids (including CO2)

Two Inorganic acids (nitrite and sulfuric acid) are produced by microorganisms.

Sulfuric acid is generated mainly by bacteria belonging to the genus ‘Thiobacillus’ and

nitric acid by nitrifying bacteria of the genera Nitrisimonas and Nitrobacter.

Thiobacillus are acidophilic (i.e. acid-tolerant) and are able to fix CO2 and their

sources of energy are reduced inorganic sulfur compounds. Their growth is often

inhibited by organic compounds. Nitrifying bacteria are not as acid-resistance as

Thiobacilli and their source of energy are: ammonium compounds, urea, nitric and

possibly NO. however, both cause considerable damage to mineral materials, like,

concrete, natural stone, glass etc., Further, cementitious- bound materials like concrete

and other materials such as limestone, marble etc, are also susceptible for

carbonization and weak-acid attack due to metabolic activity of organisms.

(3) Organic acids

Generally, most micro-organisms excrete organic acids while metabolizing organic

and inorganic compounds. Organic acids react with (substratum) materials by two

mechanism: (i) by the action of protons and (ii) by chelation of metal ions.

Eventhough the acidic effect of organic acids may be comparable to that of mineral

acids, a distinction needs to be made between the strong and weak organic acids (For

example: acetic, gluconic, oxalic, oxalacetic, succinie, malic, glyoxylic acid etc.,).

Besides ‘simple’ organic acids, molecules, such as: amino acids or polysaccharides

with ionic groups may be excreted into the medium during metabolism.

(4) Organic Solvents

Many microorganisms are capable of metabolizing organic compounds via

fermentation as organic solvents (e.g. organic acids like: acetic, formic or butyric acid;

alcohols like: ethanol, propanol, butanol etc and ketones). These solvents may react

with materials of natural and / or synthetic origin, causing swelling, total / partial

dissolution and finally deterioration.

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(5) Salt stress

Anions – the final product of microbial metabolism react with cationic compounds of

ceramic materials to salts, which are highly water soluble and thus are hydrated. Their

presence results in increased water content of porous mineral materials and on drying

resulting in salt crystals, which require increased volume and causes a ‘blasting’

deterioration of porous materials. In this form of attack, physical attack and

microbiological attack cannot be distinguished and hence not quantified.

(6) Noxious compounds- hydrogen sulfide (H2S) and nitrogen oxides

In sewage, due to aerobic degradation H2S is released (H2S – weak acid, from the

chemical point of view), reacts with cations to sulfidic compounds, resulting in severe

metallic corrosion. In the case of mineral materials (say like concrete), H2S is the

nutrient source for aerobic Thiobacilli producing sulfuric acid and resulting in

deterioration of concrete. The action of nitrogen oxides (occurring in atmosphere, soil,

water) on mineral materials may be described as an enhancement of an acid attack and

/ or as the generation of acidic reactants from gaseous atmospheric pollutants.

(7) Biofouling and biofilm

Microorganisms growing on and / or in mineral materials excrete exopolymers, which

contain ionic groups, and hence function like ion-exchangers. Biofilm and

exopolymers result in clogging of pores of materials, thus reducing evaporation of

water and reduced penetration of protecting agents, cruise velocity / increased fuel

consumption and biocorrosion due to enhanced activity of SRB on material iron.

Microorganisms living on insoluble compounds often excrete exoenzymes to degrade

these into small fragments. Examples of the above are biodeterioration of wood,

whereby it is degraded to cellubiose and finally glucose, which is used as thee

substrate. In the case of purely mineral materials, exoenzymes are not important.

However, certain, exoenzymes substances, such as; resins, waxes, carbohydrates or

(8) Exoenzymes

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other compounds are added to inorganic materials to achieve improved virtues, and

render them susceptible to attack by exoenzymes.

(9) Chelating agents, emulsifying compounds

Besides organic acids, exopolymers of microorganisms, containing anionic groups

such as: amino acids, peptides or sugar acids, may act as complexing agents. Further,

emulsifying compounds, such as, phospholipids (excreted by microorganisms), are

known to be involved in the biological degradation of insoluble compounds, such as

pyrite, sulfur etc., The presence of emulsifying agent, increases the biogradability, by

increasing the hydrophilicity of substances, which were formerly hydrophobic.

2.3 REVIEW OF WORKS OF EARLIER INVESTIGATORS

2.3.1 Biodeterioration by Bacteria

Bacteria are a group of prokaryotic unicellular or colonial organisms of various shapes

(spherical, rodlike, or spiral). They may be motile or immotile. They include

autotrophic and heterotrophic species. Owing to their simple ecological and nutritional

needs, they develop easily on outdoor objects, especially where the surface exhibits

high water content (Kumar and Kumar, 1999).

Midle et al. (1983) identified the presence of thiobacilli on the corroded concrete

walls of the Hamburg sewer system. They estimated thiobacilli from the samples

collected from six sites of Hamburg sewer systems which were showing different

degree of concrete corrosion. There was a marked enrichment of thiobacilli on the

sewer pipe surface above the sewage level in comparison to the liquid phase. The

highest number [108 thiobacilli (mg protein)-1] was found at the site of the greatest

corrosion. Ten isolates of the genus Thiobacillus were characterized and identified and

it was found that facultative chemolithotrophic bacteria predominated at sites of early

corrosion, whereas T. thiooxidans was most abundant in severely corroded areas.

Further they suggested that the cell number of T. thiooxidans could be greatly

decreased by aerating the sewage with pure oxygen.

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Sand and Bock (1984) investigated the biogenic sulfuric acid corrosion of sewer

network of Hamburg sewer. A field study indicated thiobacilli of the species

Thibacillus neapoplitanus, T. intermedius, T. novellus and T. thio-oxidans. A positive

correlation between the cell numbers of T. thio-oxidans and the grade of corrosion was

noted. As sources of sulfur the volatile compounds hydrogen sulfide, sulfite,

methylmercaptane, dimethylsulfide and dithiabutane are possible. Biogenic concrete

corrosion was simulated in a strictly controlled H2

Sand and Bock (1991) explained the biodeterioration of mineral materials by

microorganism with respect to concrete and natural stone. Microorganisms such as

chemolithotrophic and chemoorganotropic bacteria, cyanobacteria, algae, fungi and

lichens contribute substantially to the deterioration of mineral materials such as

natural stone, concrete, ceramics, and glass. In their study three simulation apparatuses

were constructed; each allowed the incubation of test materials under

microbiologically optimized conditions and biodeterioration involving biogenic

sulfuric acid corrosion, which under natural conditions needs eight times as long, was

detectable within a few months. In the case of biogenic sulfuric acid corrosion,

S breeding chamber and differences

among the various concrete types studied were reproducibly demonstrated.

Sand et al. (1984) have explained the role of sulfur oxidizing bacteria in the

degradation of concrete. They devised a specially designed chamber containing

concrete test blocks where the temperature, humidity, hydrogen sulfide, and exposure

to aerosols of different Thiobacilli, can be controlled. From the above simulation

studies, it has been shown that the rate of concrete degradation in the test chamber is

accelerated so that degradation that required at least 4 years in sewer systems was

reproducibly seen in 9 months. With the above system the rates of degradation were

shown to correspond most closely to the cell numbers of T.thiooxidans,

T.neapolitanus, T.intermedius, and T.novellus found on the concrete. Thiobacilli

contain a relatively unusual polar ornithine lipid that can be used to chemically

monitor the biomass and activity of these organisms that facilitate concrete

degradation.

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simulation experiments demonstrated differences in resistance of various concrete

types, which ranged from 1 to 20 % weight loss of test blocks within 1 year. They

have emphasized the importance role of biotests over physical / chemical test methods

in biodeterioration studies and the selection of appropriate materials from many

different ones.

Sand et al. (1994) emphasized the need for a biotest to understand and estimate the

biogenic sulfuric acid corrosion which happens routinely in sewer systems causing

extensive damages. A laboratory based simulation test which is an accelerated test to

monitor the above corrosion in cement based materials has been highlighted. The

result of the accelerated test has been stated to match the long - term field

observations, and hence proving that the above test could be used as a standard in such

biodeterioration studies.

Davis et al. (1998) analyzed the concrete corroded from sewer pipes. The microbial

populations in the loose outer corrosion layer (OCL) and the bound inner corrosion

layer (ICL) of concrete from a corroded sewage collection system were enumerated.

Chemical and physical studies were performed to determine the mineralogical

composition strength of the samples. The average number of acidophilic sulphur-

oxidizing microorganisms (ASOM) were found to be 14,500 and 16,000 MPN/g

(OCL) and 12,500 and 100 MPN/g (ICL) at the crown and springline. Whereas, the

average numbers of neutrophilic sulfur-oxidizing microorganisms (NSOM) were

108,000 and 114,000 MPN/g (OCL), and 5 and 300 MPN/g (ICL) at the crown and the

springline. It was found that the average compressive strength of the concrete

undergoing corrosion was reduced by 20% and the results suggest an initial ecological

succession occurred on the concrete surface and that the progressing of the corroding

front into the concrete was controlled by the penetration of acid produced by ASOM

followed by the ASOM themselves. However, it was found that NSOM did penetrate

the concrete.

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Vincke et al. (1999) have devised a new test method for biogenic sulfuric acid

corrosion of concrete, more specifically in sewer conditions, with the aim to develop

an accelerated and reproducible procedure for monitoring the resistance of different

types of concrete with regard to biogenic sulfuric acid corrosion. The experimental

procedure reflected the worst-case condition by providing besides H2S, also an

enrichment of thiobacilli and biologically produced sulfur. By simulating the cyclic

processes occurring in sewer pipes, significant differences between concrete mixtures

could be detected after 51 days. Concrete modified by a styrene-acrylic ester polymer

demonstrated a higher resistance against biogenic sulfuric acid attack.

Monteny et al. (2000) have presented an overview of the recent developments in the

test methods of biogenic sulfuric acid corrosion and have delineated the possible

differences between biogenic sulfuric acid corrosion and chemical sulfuric acid

corrosion. Based on the test results of simulation test, in-situ test and observations and

chemical test, it has been stated that high-alumina cements are seen to give the best

results concerning biogenic sulfuric acid corrosion.

Videla et al. (2000) studied the biodeterioration of Mayan archaeological site in the

Yucatan Peninsula, Mexico. Two different sites were chosen at the archaeological site

of Uxmal in the Yucatan Peninsula, Mexico. Heterotropic bacteria, cyanobacteria and

different fungi were isolated and classified taxonomically. The other archeological site

chosen for the study was the fortress of Tulum, located at the side of the Caribbean sea

and exposed to chloride of marine spray and sand erosion. Here heterotropic aerobic

and anaerobic bacteria, cyanobacteria and fungi were isolated from the four sampling

areas selected. In both archeological sites crust deposits were observed using light

microscopy, SEM and ESEM. Surface analysis were made by means of EDAX and

electron microprobe. The above mentioned analysis suggested that the biodeterioration

may be performed through a biosolubilizaion mechanism involving the production of

metabolic acids by bacteria and fungi. In Tulum, rock decay would be also markedly

affected by the aggressive marine atmosphere, as evident from the high percentage of

chloride and calcium found in the EDAX profiles.

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Papida et al. (2000) studied the enhancement of physical weathering of building

stones by microbial populations. Two limestones from Crete, Greece and a dolomite

from Mansfield, UK were subjected to combined microbial and physical weathering

simulation cycles, in an attempt to assess the contribution of each agent of decay.

Sound stone discs were exposed to different temperature and wet / dry cycling regimes

involving treatment with distilled water or solutions of sodium chloride or sodium

sulphate. Before the weathering cycles, half of the discs were inoculated with mixed

microbial populations (MMP), originally recovered from decayed building stone of

Portchester Castle, Hampshire, UK. The presence of MMP greatly accelerated the

rates of deterioration of stone of all treatments, measured by weight change and

alteration of hydraulic properties of stone. A combination of physical and biological

processes significantly enhanced the extent of decay when compared with the physical

or biological agents acting alone. Populations of heterotrophic, sulphur-utilising,

halotolerant and moderately halophilic bacterial populations remained large

throughout the experiment. Biofilms formed by populations of microorganisms were

visualised by staining and assessed by colorimetric measurement of total carbohydrate

in the stone substrate.

Saiz - Jimenez et al. (2000) investigated the occurrence of halotolerant / halophilic

bacterial communities in deteriorated monuments. They have stated that the use of

traditional microbiological methods help the isolation of a large number of

heterotrophic bacteria on deteriorated monuments, the spectrum of isolated bacteria

changed when the protocols used in studies of halophilic bacteria were applied to

mural paintings, efflorescences or mineral deposits. It is found that the enumeration of

the heterotrophic viable bacteria indicates that the higher counts were, generally,

obtained in media with 10% of salt concentration and that media with magnesium

sulphate always yielded higher counts than sodium chloride, particularly in

environments where magnesium salts were abundant.

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Lamenti et al. (2000) studied the colonisation of ornamental marble statues in the

Boboli Gardens of Florence (Italy) by photosynthetic micro-organisms. The green

microalga Coccomyxa was the first colonizer of newly restored marble surfaces,

appearing one year after the periodic cleaning and restoration of the statues. Two years

after restoration this alga gave rise to very thin green biofilms and later, the biofilms

were enriched by cyanobacterial forms, which became dominant. The secretion of

polysaccharidic substances and cell surface hydrophobicity enhancing the capacity to

adhere, favoured permanent colonisation of the cyanobacterial population.

Monteny et al. (2001) has simulated sulfuric acid corrosion of polymer-modified

concrete using chemical and microbiological tests. They used five different concrete

compositions for the test, including a reference mixture with high sulfate resistant

portland cement and four different polymer cement concretes with styrene–acrylic

ester polymer, acrylic polymer, styrene butadiene polymer and vinylcopolymer,

respectively. The concrete composition with the styrene–acrylic ester polymer showed

in both tests a higher resistance than the reference mixture while the compositions with

the acrylic polymer and the styrene butadiene polymer had a lower resistance than the

reference mixture. The concrete composition with the vinylcopolymer did not induce

the same results in both tests. The results of the chemical test indicated a slight

increase in resistance when compared to the reference mixture while the opposite was

noticed in the microbiological test.

Vincke et al. (2001) conducted a case study on the microbial communities on

corroded concrete sewer pipes. Conventional as well as molecular techniques have

been used to determine the microbial communities present on the concrete walls of

sewer pipes. The genetic fingerprint of the microbiota on corroded concrete sewer

pipes was obtained by means of denaturing gradient gel electrophoresis (DGGE) of

16S rRNA gene fragments. The DGGE profiles of the bacterial communities present

on the concrete surface changed as observed by shifts occurring at the level of the

dominance of bands from non-corroded places to the most severely corroded places.

By means of statistical tools, it was possible to distinguish two different groups,

21

corresponding to the microbial communities on corroded and non-corroded surfaces,

respectively. Characterization of the microbial communities indicated that the

sequences of typical bands showed the highest level of identity to sequences from the

bacterial strains Thiobacillus thiooxidans, Acidithiobacillus sp ., Mycobacterium sp.

and different heterotrophs Proteobacteria, Acidobacteria and Actinobacteria.

Hernandez et al. (2002) have made an in - situ assessment of active Thiobaccillus

species in corroding sewers using fluorescent RNA probes. Active populations of

selected Thiobacillus, Leptospirillum, and Acidiphilium species were compared to total

bacterial populations growing on the surfaces of corroding concrete using three

oligonucleotide probes that have been confirmed to recognize unique sequences of 16S

rRNA in the following acidophilic bacteria: Thiobacillus ferrooxidans and

Thiobacillus thiooxidans (probe: Thio820), Leptospirilium ferrooxidans (Probe:

Lept581) and members of the genus Acidiphilium (probe: Acdp821). With these

genetic probes, fluorescent in situ hybridizations (FISH) were used to identify and

enumerate selected bacteria in homogenized biofilm samples taken from the corroding

crowns of concrete sewer collection systems operating in Houston, Texas, USA. Direct

epifluorescent microscopy demonstrated the ability of FISH to identify significant

numbers of active acidophilic bacteria among concrete particles, products of concrete

corrosion (e.g. CaSO4

Vincke et al. (2002) have studied the influence of polymer addition on biogenic

sulfuric acid attack of concrete. They used simple and reproducible microbiological

simulation procedure in combination with a chemical procedure to test concrete for its

potential resistance towards biogenic sulfuric acid. It was shown particularly that the

), and other mineral debris. As judged by FISH analyses with the

species-specific probe Thio820, and a domain-level probe that recognizes all Bacteria

(Eub338), T. ferrooxidans and T. thiooxidans comprised between 12% and 42% of the

total active bacteria present in corroding concrete samples. Although both

Acidiphilium and Leptospirillum have also been postulated to have ecological

significance in acidic sulfur-oxidizing environments, neither genera was detected using

genus-specific probes.

22

penetration of H2

De Belie et al. (2004) predicted the effect of chemical and biogenic sulfuric acid on

different types of commercially produced concrete sewer pipes. New equipment and

procedures for chemical and microbiological tests, simulating biogenic sulfuric acid

corrosion in sewerage systems were adopted. Both chemical and microbiological tests

showed that the aggregate type had the largest effect on degradation. Concrete with

limestone aggregates showed a smaller degradation depth than did the concrete with

inert aggregates. The limestone aggregates locally created a buffering environment,

protecting the cement paste. This was confirmed by microscopic analysis of the eroded

S inside the concrete crevices accelerated the corrosion process. The

influence of different polymer types and silica fume additions on the resistance of the

concrete samples was determined. The addition of the styrene acrylic ester polymer

resulted in an increased resistance while the addition of the acrylic polymer or silica

fume caused less resistant concrete. For the vinylcopolymer and the styrene butadiene

polymer, no significant effect was observed on the resistance of the concrete samples.

The results of the two different test methods confirmed the difference between

corrosion due to purely chemical sulfuric acid and corrosion due to microbiologically

produced sulfuric acid.

Herrera et al. (2004) studied the biodeterioration of peridotite and other construction

materials in a church which is a part of the Columbian cultural heritage using

microbiological and surface analysis techniques. The facade of the church was built

with peridotite, an ultrabasic igneous rock containing >90% of iron and magnesium

minerals such as olivine and pyroxene. Assessment showed that the atmospheric

characteristics in the city if Medellin are only slightly aggressive, suggesting that

weathering would not be the main cause of decay of the material. The main

microorganisms isolated from the façade of the church were heterotrophic bacteria,

fungi and phototrophic microalgae and cyanobacteria. Lichens and mosses are also

found to colonizing the rock. Experimental evidence suggests that deterioration of the

peridotite is mainly is due to acidifying bacteria and other microbial contamination and

that atmospheric factors would only play a secondary role in the decay.

23

surfaces. The production method of concrete pipes influenced durability through its

effect on W/C ratio and water absorption values. In the microbiological tests,

HSR (high sulfate resisting) Portland cement concrete performed slightly better than

did the slag cement concrete. A possible explanation can be a more rapid colonisation

by microorganisms of the surface of slag cement samples. A new method for

degradation prediction was suggested based on the parameters alkalinity and water

absorption (as a measure for concrete porosity).

Kawai et al. (2005) studied concrete deterioration caused by sulfuric acid attack was

investigated considering the effects of the flow of acid solution over the surface of

concrete and thus simulating the shearing force of the fluid that erode the surface of

concrete. Further a prediction model for the deterioration of concrete due to sulfuric

acid was also attempted. Cylindrical concrete specimens and mortar prisms were

immersed in various concentrations of sulfuric acid. In certain tests the sulfuric acid

solution was circulated onto concrete specimens. In both instances, the depths of zones

eroded and neutralized by acids were measured. As well, the zones of deteriorated

concrete were analysed with an XRD and an ion chromatoanalyzer. It was found that

the rate of concrete deterioration caused by sulfuric acid attack depended on the pH

value of acid solutions and that the depth of erosion of concrete was nearly

proportional to the exposure time of flowing acid solution to which concrete was

exposed.

Crispim and Gaylarde (2005) presented a review on biodeterioration of cultural

heritage by cyanobacteria. They emphasized the importance of cyanobacteria as

deteriogens and also discussed the traditional and more modern molecular methods

used to detect these microorganisms. It has been stated that the development of

molecular techniques for the rapid identification of cyanobacteria without the need for

culture and isolation is fundamental, if the knowledge of these communities in

biofilms on the surfaces of historic buildings is to be extended.

24

De Graef et al. (2005) used X-ray microtomography (µCT) for monitoring the

biological weathering of natural building stones and concrete due to its non-destructive

character. In depth changes of porosity of concrete and stone specimens due to

bacterial weathering were assessed in this work. Also, porosity was visualised based

on 3D data with homemade software. Scanning electron microscopy (SEM) images

provided additional information and supported conclusions drawn from the X-ray μCT

data. It has been stated that resolution improvement will make the study of

petrophysical aspects of physical weathering and / or biological deterioration processes

of natural building stones and concrete a promising subject for further μCT-application

Seth and Edyvean (2006) studied the function of sulfate-reducing bacteria (SRB) in

corrosion of potable water mains. In this study, the presence of SRB in sampled water

mains, in a region that would otherwise be expected to be stable, both by direct

sampling, by using coupons in a Robbins device installed in the distribution network,

and by sampling from laboratory tanks. It has been stated that samples of pipes of

various materials show a high frequency of SRB and that cast iron coupons from the

Robbins device gave positive results for SRB after only 1 month in the distribution

system, indicating microbially induced corrosion where as, laboratory coupon tests

indicated the absence of SRB.

Crispim et al. (2006) studied the effect of cyanobacterial biofilm communities on

external building surfaces. They established cyanobacterial species using both

established and molecular techniques and have concluded that their results indicate

that cyanobacteria from external walls are an ecologically isolated group.

Lors et al. (2009) investigated the concrete biodeterioration by varying the pH of two

buffered media having their initial pH ranging between 3.5 and 8.5 during the growth

of Acidithiobacillus thiooxidans. The first media was buffered with tricyclic phosphate

whereas the second one contained phosphate ions and thus exhibited a stronger buffer

capacity. Bacterial growth was not observed in any of the two media when the initial

pH was higher than 5.5. On the other hand, for initial pH lower than 5.5, bacterial

25

growth induced pH drops in both media. It has been observed that the drop in pH was

preceded by a lag phase during which the pH remained unchanged. However, in the

medium buffered with phosphate ions, the lag periods were longer. As these media

were developed for designing a bioleaching test to evaluate concrete biodeterioration

caused by A. thiooxidans, it has been concluded that the medium containing tricyclic

phosphate appeared to be the most appropriate.

2.3.2 Biodeterioration by Lichens

Lichens are a large group of composite organisms formed by the symbiotic association

of Chlorophyta or Cyanobacteria and a fungus. Due to their resistance to desiccation

and extreme temperature and efficiency in accumulating nutrients, lichens occur in a

wide range of habitats, including those normally hostile to other life forms. Together

with Cyanobacteria they play an important role as pioneer organisms in colonizing

substrate. The types of lichens that attach themselves to the surface with devices such

as rhizoids (foliose and fruticose lichens) and hyphae (crustose lichens) have been

isolated in tropical regions. They can be epilithic (living over substrate) or endolithic

(entirely living beneath a substrate) (Kumar and Kumar, 1999).

Cooks and Otto (1990) have studied the effect of the weathering processes generated

by Lecidea aff. Sarcogynoides (Koerb.) on the substrate by means of a SEM. The

elements present in the substrate (Magaliesberg quartzite) and in the lichen thallus

were determined by X-ray fluorescence spectrometry for the purpose of comparison.

The elements present were mostly similar although a few were present in the thallus

which were not observed in the quartzite. It is possible that those elements present in

the lichen thallus which were not present in the substrate may have been extracted

from the atmosphere. The occurrence of small hollows (weathering pits) in which the

early stages of plant development occurs, and the disintegration of the rock indicate

that Lecidea aff. sarcogynoides (Koerb.) contributes to the chemical weathering

processes by chelation and mechanically by the penetration and expansion of hyphae.

A model is proposed in which a possible mechanism for these weathering processes

has been suggested.

26

Lamas et al. (1995) studied the colonization of granite churches in Galicia (northwest

Spain) by lichens, and the contribution of lichens to weathering. Lichens were sampled

from 20 rural churches, with the aims of identifying the commonest species and

investigating environmental correlates of species distribution. Species composition

was basically similar at all 20 sites, but microenvironmental factors (for example,

aspect and microsite humidity) had clear effects on the distribution of many species.

As regards weathering, the most common effect of lichens was disaggregation

associated with hyphal penetration of intergranular voids. Lichens also appear to affect

biotite, encouraging transformation to vermiculite and provoking release of Fe which

is precipitated as noncrystalline oxyhydroxides. A neoformation mineral (whewellite)

was detected in thalli of Ochrolechia parella.

Ariño et al. (1995) have studied the effect of lichen colonization on the first century

A.D. pavement of the forum at Baelo Claudia, a Roman city located in southern Spain.

Lichen colonization was found to be scarce, covering only 13% of the total surface,

whereas, the rest of the flagstones are mostly uncovered but show strong physico-

chemical weathering. The flagstones colonized by lichens do not show weathering.

The distribution of the species is influenced by environmental factors, confirming the

role of lichens as bioindicators of different habitats. The lichen / sandstone interface

shows some weathering, but nevertheless, the protective role of lichens in an

aggressive environment is noticeable.

Romao and Rattazzi (1996) has investigated the lichen colonization on the granite

rocks used as building material in Tapadao and Zambujeiro dolmens (Alentejo,

southern Portugal). The field survey undertaken in both archaeological sites has made

possible some correlations between diversity, frequency and distribution of the

species, and the climatic conditions, deterioration forms, inclination and exposure to

rainfall of the lithic pieces. Preliminary laboratory tests indicate a deep penetration of

hyphae into the substrate, resulting in remarkable ‘canalization’ and fragmentation of

all granite minerals. The consequent biogeophysical and biogeochemical damage

reveals lichens as important weathering agents.

27

Arino et al. (1997) studied the detrimental effect of lichen on ancient mortar. The

study was done in three archaeological sites of southern Spain and it showed that

mortar is a building material easily colonized by a diversity of calcicolous and rather

nitrophilous lichens. The interface between lichen and mortar showed an intense

chemical activity of the hyphae producing extensive alteration on the surface. It has

been concluded that the nature and amount of the mortar components greatly influence

the colonizing species and the patterns of alteration.

Ascaso et al. (1998) studied the biogenic weathering of calcareous litharenite stones

caused by lichen and endolithic microorganisms living in calcareous rock of the

Roman Cathedral of Jaca, Spain. Samples taken from the Cathedral Arnold were

examined with SEM equipped with a back scattered electron detector and energy

dispersive x-ray spectroscopy. The results demonstrated various types of stone

damage caused by lithobiontic microorganisms; Chlorite sheet separation by algae,

calcium carbonate nodule trapping by hyphae, and cross linear alteration and

subsequent pellicular alteration patterns in calcite grains by hyphae - were the main

bioweathering features of the calcareous substrata. Both epilithic and endolithic fungal

cells were found to be responsible for stone decay by altering calcite.

Chen et al. (2000) have comprehensively reviewed the weathering of rocks induced

by lichen colonization. It has been stated that the by the interface between lichens and

their rock substrates strongly suggests that the weathering of minerals can be

accelerated by the growth of at least some lichen species and the effects of lichens on

their mineral substrates can be attributed to both physical and chemical processes. As a

result of the weathering induced by lichens, many rock-forming minerals exhibit

extensive surface corrosion. The precipitation of poorly ordered iron oxides and

amorphous alumino-silica gels, the neoformation of crystalline metal oxalates and

secondary clay minerals have been frequently identified in a variety of rocks colonized

by lichens in nature.

28

Tomasell et al. (2000) has made a critical survey of literature data for the

biodeteriogens acting on stone monuments and combined it with the results of the

investigations performed by traditional and biomolecular (ARDRA) methods and

showed that the photosynthetic micro-organisms dwelling on stone monuments have a

rather ample biodiversity, which was also confirmed by the data on axenic

cyanobacterial strains isolated from Italian monuments. The correlation between the

literature data reporting the presence of photosynthetic micro-organisms, and the

nature of the stone substrate showed that the cyanobacteria Chroococcus,

Myxosarcina, Pleurocapsa and Scytonema, and the chlorophyta Apatococcus and

Stichococcus were associated with calcareous substrates, while Nostoc spp. were more

frequently associated with artificial substrates. They also demonstrated that Lyngbya

B2 and Apatococcus B4, isolated from monuments inoculated on stone slabs differing

in porosity and surface roughness, had a preference for calcareous lithotypes with high

values of roughness and porosity.

Carballal et al. (2001) have investigated the lichen colonization of four granite

churches situated in coastal areas in Galicia (NW Spain) were studied with the aim of

understanding relationships among lichens, salts and biodeterioration. The results

obtained from the study were compared with those from previous studies on lichen

colonization of non-coastal churches and it has been concluded that there is a group of

characteristic species on granite monuments whatever the environmental conditions

are. Besides this group of characteristic species, a large number of species were

identified on each coastal church that brought some important data to the relationship

between salts weathering and the protective action of lichens.

Williamson et al. (2002) studied the application of element mapping in SEM with

electron probe microanalysis across the lichen–rock interface, with reference to

Trapelia involuta growing on a granite substratum. The preparation of samples

containing both organic and mineral components required the development of

specialized techniques to maintain both chemical and structural integrity at the 2 μm

resolution of the X-ray element maps. X-ray element maps show the distribution of

29

entrained rock particles at the lichen–rock interface and chemical localization which is

strongly related to anatomical structure for the essential elements S, Fe, Ca, Na, K and

P. The ability to map element distribution across the lichen–rock interface has wide-

ranging potential applications in studies such as the biodeterioration of buildings and

monuments and the mobilization and uptake of toxic elements from contaminated

substrata.

De Graef et al. (2005) has used Thiobacilli as a aid for cleaning of concrete fouled by

lichens. A mixture of sulphur oxidising bacteria of the genus Thiobacillus

supplemented with an appropriate nutrient was applied to a fouled concrete surface,

either by sprinkling or by submersion to remove the fouled layer in uniformly. The

general effect of the technique was evaluated by colorimetry and microscopy. Two

sets of weathered concrete specimens, containing blast furnace slag cement or ordinary

portland cement, were investigated. The effectiveness of the technique depended on

the cement type of the concrete specimens. The effect on the OPC concrete specimens

was in some cases up to a factor 2 stronger than the result on the blast furnace slag

cement specimens. The sprinkling treatment was about 50% as effective as the

submersion treatment but was very promising in the case of in situ acidification. A

side effect was the formation of a gypsum layer on some of the specimens, resulting in

a whiter colour.

Gaylarde et al. (2006) have studied the effect of lichen colonization on limestone

monuments. Biofilms were collected from discoloured limestone samples and on

adhesive tape from historic buildings at the Mayan site of Edzna, in Campeche,

Mexico. Grey, brown, and black areas were colonised predominantly by coccoid and

colonial cyanobacteria, also detected as endoliths. The major biomass on the pink

stone surface was Trentepohlia, which caused severe local erosion (pitting) and, when

present as a more uniform biofilm, the well-known pink surface discoloration.

Watanabe et al. (2006) have studied the elemental behaviour, during the process of

weathering of glazed sekishu roof-tiles affected by Lecidea s.lat. sp. (a crustose lichen),

using optical and fluorescence microscopy, field emission scanning electron

30

microscopy (FE-SEM) and transmission electron microscopy. Sekishu roof tiles have

an opaque reddish brown glaze on their surfaces which consist of an alkali feldspar-

type X-ray amorphous glass recrystallized at 1200°C. Optical and fluorescence

microscopy revealed the presence of corrosion pits (at a depth of ~50 µm) at the

lichen-glaze interface. Elemental mapping by FE-SEM identified the concentrations of

Ti and Fe in the section of the glazed tile analysed. The behaviour of C was correlated

with those elements, suggesting the possibility of biomineralization.

Áková et al. (2008) studied the biocorrosion of concrete sewer pipes. They simulated

a biocorrosion to study the effect of simultaneous action of Acidithiobacillus

thiooxidans ( A.t. ) and sulfate-reducing bacteria on concrete samples under model

conditions. The biocorrosion effect has been proved and a further study is planed.

Duane (2006) studied the coeval biochemical and biophysical weathering processes on

Quaternary sandstone terraces south of Rabat (Temara), northwest Morocco. It has

been stated that inspite of numerous investigations on substrate-inhabiting microflora,

especially lichens, very little is known about the colonization of coastal escarpments

by lithobiontic micro-organisms, inland of a retreating coastline in Africa. The results

of a combined field observation and microscopy study focusing on the connection

between microrelief of the substrate, colonies of lithobiontic micro-organisms (in

particular the lichen Xanthoria parietina) and microstructures of putative bacterial

origin were reported. The occurrence of weathering pits in which the early stages of

the biotic development occurs, and the subsequent disintegration of the rock indicate

that lichens, mosses and fungi act synergistically by alternating chemical and

mechanical weathering. Penetration of grains by expansion and contraction of the

hyphae depletes the rock matrix and contributes to the mechanical breakdown of the

rock. A model has been proposed, firstly indicating early-stage biochemical

weathering, followed by biophysical weathering. Disintegration of the rock outcrops in

due to a complex interplay of several events, probably beginning at the nanoscale with

penetration of sites on crystal faces.

31

Gazzano et al. (2009) made an index of Lichen Potential Biodeteriogenic Activity

(LPBA) to quantify the overall lichen impact on stonework on the basis of the volume

of influence of each species, quantified both on the surface of and within the

substratum, and of other parameters related to reproduction, physico-chemical action,

and bioprotection. Ordinal scales were introduced for each parameter with reference to

experimental data and literature on the current evaluation approaches. The index was

designed in such a way that a lower knowledge of the colonization phenomena may

yield an overestimation of the lichen impact, but not an underestimation, thus assuring

a precautionary approach, which is functional to conservation programs.

Representative case studies from the north-western Italy were examined to highlight

the range of applicability of the index from small-sized stone substrata, to buildings, to

whole archaeological areas. The application of the index has shown that the

consideration of the different factors involved in the lichen biodeteriogenic activity

gives a different and more exhaustive evaluation of biodeterioration with respect to

cover only and confidently describes the lichen effect on stonework with reference to

the substratum damage. The necessity of a wide research network has been emphasied

to move towards a statistical validation of the index developed.

Nascimbene et al. (2009) has evaluated the effectiveness and life-strategies of

freshwater lichens in colonizing newly constructed stone structures in low-elevation

streams in a small nature reserve in northern Italy. Species richness, size of thalli,

morphological and ontogenetic traits of the species was related to the age of restored

habitats. Lichen colonization was surprisingly rapid, indicating the high potential of

these organisms in colonizing restored habitats. However, the species pool found in the

restored habitats was different than that found in natural sites in the same study area.

The age of newly constructed habitats influenced both species richness and thallus size

of the two most frequent Verrucaria species. Verrucaria aquatilis was a rapid

colonizer invading the substrate by several small-sized and thin thalli which soon

supported a large number of small perithecia whose development began in the earlier

phase of thallus formation. V. elaeomelaena, on the contrary, developed according to a

different strategy, establishing a thick thallus on which relatively large perithecia were

32

formed much later than in V. aquatilis. The main practical implication of the study is

reflecting to be the value of small stone structures, such as riffles and ramps, for

enhancing the establishment of pioneer freshwater lichens to rapidly colonize newly

available substrata.

Ríos et al. (2009) have investigated the deterioration effects of lichens and other

lithobionts in a temperate mesothermal climate. They examined samples of dolostone

and limestone rocks with visible signs of biodeterioration taken from the exterior wall

surfaces of four Romanesque churches in Segovia (Spain): San Lorenzo, San Martín.

Biofilms developing on the lithic substrate were analyzed by SEM and the most

common lichen species found in the samples were recorded. Fungal cultures were then

obtained from these carbonate rocks and characterized by sequencing Internal

Transcribed Spacers (ITS). Through SEM in back-scattered electron mode, fungi

(lichenized and non-lichenized) were observed as the most frequent microorganisms

occurring at sites showing signs of biodeterioration. The colonization process was

especially conditioned by the porosity characteristics of the stone used in these

buildings. While in dolostones, microorganisms mainly occupied spaces comprising

the rock's intercrystalline porosity, in bioclastic dolomitized limestones, fungal

colonization seemed to be more associated with moldic porosity. Microbial biofilms

make close contact with the substrate, and thus probably cause significant deterioration

of the underlying materials. They described the different processes of stone alteration

induced by fungal colonization and discuss the implications of these processes for the

design of treatments to prevent biodeterioration.

2.3.3 Biodeterioration by Mosses

Mosses are bryophytes, a transitional group of the kingdom Plantae. They represent a

bridge between primitive plants without tissues or organs and evolved plants with

differentiated tissues and organs. They are simple photoautotrophic organisms that

contain pigments (chlorophyll and carotenoids) and possess rudimentary rootlike

organs (rhizoids) but no vascular tissues or transport organs (phloem and xylem). They

33

frequently occur in association with algae in a variety of damp habitats from fresh

water to damp surfaces in tropical regions (Kumar and Kumar, 1999).

Altieri and Ricci (1997) studied the biodeterioration of stone substrata by bryophytes

(Musci and Hepaticae) due to biogeochemical and biogeophysical mechanisms. The

biogeochemical damage caused by epilithic moss species, found in archaeological sites

of Rome, was investigated. The cellular calcium content in specimens of Grimmia

pulvinata (Hedw.) Sm. was analysed using a sequential elution technique on moss

specimens sampled from different lithotypes and in different seasons in order to

measure the calcium concentration both in relation to the mineral composition of the

stone and to the plant physiology. The highest calcium concentration of the

extracellular exchangeable fraction was detected in the samples grown on marble and

in the waterlogged specimen sampled in spring time.

Shirzadian et al. (2008) investigated the role of mosses on biodeteriorative impacts

on bridges over Zayand-e-Rood river (Iran) as well as their control / elimination

measures. Mosses provided a suitable habitat for small organisms and a base for

proliferation and invasion of higher plants that accelerate deterioration due to

penetration of their roots. Environmental factors in biodeterioration (pH, water,

relative humidity and temperature) were determined and chemical analysis of mosses

specimen were carried out. It confirmed that the presence of mosses, algae, aquatic and

terrestrial plants in bridge causes chemical and mechanical deterioration and that loss

of heavy metal from bridge structural material reduces the material strength causing

degradation and weathering of bridge components.

2.3.4 Biodeterioration by Fungi

Fungi are a group of chemoheterotrophic organisms characterized by unicellular or

multicellular filamentous hyphae. They lack chlorophyll and, thus, the ability to

manufacture their own food by using the energy of sunlight. Hence, they cannot live

on substrate, unless some organic food is present. The waste products of algae and

34

bacteria (or the dead cells of these organisms), decaying leaves, and bird droppings

can provide such food sources (Kumar and Kumar, 1999).

Gomez-Alarcon et al. (1994) isolated the fungal stainds from decayed sandstone from

the church of Carrascosa del Campo (Spain) and tested for their capacity for excreting

organic acids when cultured in Czapek-Dox broth, as well as in the presence of algal

biomass. Strains of the geneva Penicillium and Fusarium excreted oxalic, fumaric and

succinic acids with corrosive effects on the stony materials. Moreover,

P. corylophilum was able to produce oxalic acid when cultured in the presence of the

algae M. braunii as the only source of carbon and nitrogen. SEM observations showed

that fungal spores inoculated together with algal biomass on sandstone cubes,

germinated and resumed a regular growth.

Diakumaku et al. (1995) have investigated the interaction of black fungi such as

Phoma and Alternaria with marble surfaces, the pattern of growth of these organisms

and the causes and reasons of physical and chemical damage. Special attention was

given to the penetration pattern of the organisms, as well as to their capability of

altering the colour and mineralogical composition of rock surfaces. Some attention

was given to special techniques of differentiating the pigmentations of rock surfaces

from cultural monuments.

Gómez-Alarcón et al. (1995) have studied the microbial communities and their

alteration processes of monuments at Alcala de Henares, Spain. Fungi belonging to the

genera Alternaria, Penicillium, Phoma, Trichoderma, Mucor, Ulocladium,

Dictyodesmium and Phialostele colonize the building materials of several monuments.

Bacteria of the genera Bacillus, Micrococcus and Thiobacillus were also isolated.

Microbial activity in the surface layers of the stone was determined following the

dehydrogenase activity test. Gypsum formation and weathering of some mineral

components (e.g. feldspar, mica and calcite) were shown by FT-IR and SEM-EDX, as

well as an enrichment in S due to air pollution and in P due to biological input.

35

Gutiérrez et al. (1995) have studied the extracellular polysaccharides produced by

some fungi involved in the deterioration of wood (Pleurotus species) and stone

(Ulocladium atrum). The Pleurotus glucans present the most complex structure and

the study was followed by the analysis of the low-molecular weight products and the

partially degraded polysaccharides obtained after periodate oxidation or acetolysis.

The Pleurotus species produced ligninolytic enzymes which play a role in wood

deterioration. On the other hand, Ulocladium atrum produces black pigments

(melanins) involved in stone biodarkening, which were studied by analytical pyrolysis

and chemical degradation. The occurrence of similar extracellular polysaccharides in

fungi from very different taxonomic groups, i.e. ascomycetous dematiaceous and

white-rot basidiomycetes, suggests that such polysaccharides are playing some basic

functions in hyphal growth on different substrates. In addition, they probably play

specific roles in biodeterioration of stone, including the formation of extracellular

melanin-polysaccharide stable complexes; and wood, providing a microenvironment

for the action of ligninolytic enzymes and redox intermediates.

Wollenzien et al. (1995) have isolated several filamentous and microcolonial fungi

(MCF) from stone monuments and natural rock outcrops, mainly in the Mediterranean

area. Most strains are characterized by melanin production. MCF proved to be

meristematic; some showed dimorphic yeast-like growth. Meristematic development

and melanin production is supposed to play a key role in the survival of MCF on white

marble in dry and hot environments. Meristematic swelling of cells with thick cell-

walls containing melanin and formation of endoconidia support their water

independence and desensitivization against UV-radiation. MCF are therefore supposed

to be the resident flora while filamentous fungi could only be contaminants. They are

not lichenized, since no algae could be found in association with them. Optimal

isolation techniques are discussed. Fungi are described after their in vitro morphology.

The taxonomic status of these organisms is considered.

36

Arocena et al. (2003) have studied the fragments of weathered granitic rocks from the

Kunlun Shan, Qinghai Plateau (China) to elucidate the influence of biotic crusts on the

breakdown of granitic rocks in an alpine environment. SEM (with energy dispersive

system) and X-ray diffractometry were used to describe the nature and properties of

mineral accumulations on the rock surface. Results showed that organic salts such as

calcium oxalate and calcium formate are associated with Aspicilia caesiocinera

(Nyl.ex Malbr.) Arnold, Caloplaca sp., Xanthoria elegans (Link) Th.Fr., and Lecidea

plana (Lahm) Nyl. Secondary accumulations of 2 : 1 clays minerals are found in

A. caesiocinera while oxides of manganese are associated with X. elegans. Coatings of

goethite (iron oxides) are believed to form from biological activity associated with the

presence of hyphae and rodlet structures on the flakes. Calcium oxalate crystallizes

into several morphologies such as druse, hexagonal plates, and lenticular containing

between 20 and 48 per cent calcium by weight. Calcium formate and iron oxide

(goethite) occur together in the form of red desert varnish. Observed black coatings

contain as much as 37 per cent manganese and 22 per cent iron. Clay accumulations

have plate-like morphology and contain c. 2 : 1 silicon to aluminium contents. They

argued that organic acids from the activities of biotic crusts contribute to the

breakdown of granitic rocks. Fungi accelerate the breakdown of granitic rocks through

the growth of fungal hyphae along the 001 cleavage planes in primary chloritic

minerals.

Shirakawa et al. (2003) developed a standardised accelerated laboratory test for

detecting bioreceptivity of indoor mortar to fungal growth. To determine the

predominant fungal species under field conditions, isolation was carried out using

mortar samples collected from 41 buildings in two cities of São Paulo State in the

South East of Brazil and it was found that Cladosporium is most frequently recovered

from field specimens. Based on the results of laboratory trials strain

C. sphaerospermum was chosen as a test microorganism. Four different mortars, two

laboratory-manufactured mortars composed of ordinary Portland cement, high calcium

hydrated lime and standardised sand, and two different ready-mixed building mortars

from the Brazilian market, were investigated for their susceptibility to colonisation by

37

C. sphaerospermum. Several parameters were tested to determine factors influencing

fungal bioreceptivity. The type of mortar, degree of carbonation and pH values of

mortars, as well as relative humidity of environment effected colonisation of C.

sphaerospermum. All except one mortar samples showed significant fungal growth,

however, the growth occurred only at 100% relative humidity. Interaction of C.

sphaerospermum with mortar specimens was studied using techniques of scanning and

environmental scanning electron microscopy combined with energy dispersive X-ray

analysis.

Karys and Wazny (2007) have presented an overview of the biodegradation of porous

building materials such as concrete, brick, mortar and plaster by wood dry rot fungi in

buildings. The biodegradation mechanism and the effect of dry-rot fungi on the

properties of building materials were discussed and presented.

Wiktor et al. (2009) made an accelerated laboratory test to study the biodeteriorative

effect of different fungal strains to a cementitious matrix. The test developed in this

study permits to obtain a rapid fungal development on cement specimens which is

claimed to be shorter than other test developed to date to study fungal biodeterioration.

Results are mainly related to aesthetical biodeterioration. Results show that in these

experimental conditions, fungal growth occurs since the first week of incubation.

Stereomicroscopy observations showed that microbial growth was noticed only on the

surface of specimens, while PAS staining revealed the real extent of microbial growth

on and within the matrix, as later confirmed by SEM observations. It has been stated

that test can be used with short time of incubation, to test and to compare

bioreceptivity of cement-based materials; and several months of incubation should

allow the study of mechanisms involved in biodeterioration.

Giannantonio et al. (2009) have studied the fouling of concrete surfaces by diverse

fungal genera under controlled laboratory conditions. A circulating flow-through

chamber was designed for testing the effects of different concrete compositions and

exogenously added nutrients on fungal colonization and fouling. Fungal strains

38

belonging to the genera Alternaria, Cladosporium, Epicoccum, Fusarium, Mucor,

Penicillium, Pestalotiopsis, and Trichoderma were cultured directly from visibly

fouled concrete structures and used individually and in a mix to inoculate mortar tiles

varying in cement composition, supplementary cementitious material additions, water-

to-cement ratio, and surface roughness. A strong positive relationship was observed

between tile water-to-cement ratio and the amount of biofouling. In addition, cement

containing photocatalytic titanium dioxide and exposed to artificial sunlight strongly

inhibited fungal colonization and fouling. Mortar tiles coated with form-release oil and

incubated with sterile rainwater were also capable of supporting fungal colonization.

The results obtained indicate that the fouling of concrete surfaces by fungi can be

influenced by variations in concrete composition and available nutrients.

2.3.5 Biodeterioration by Algae

(A) Definition, Classification

Algae are diverse groups of eukaryotic, unicellular or multicellular, photoautotrophic

organisms of various shapes (filamentous, ribbonlike, or platelike) that contain

pigments such as chlorophyll, carotenoids, and xanthophylls. Some algae are also able

to survive heterotrophically when necessary. The details of classification of algae

based on pigmentation into ten classes and based on location at which algae colonizes

into three classes, are briefly described in Appendix A3.

(B) Factors affecting the growth of algae

Water, light, temperature and inorganic compounds are some of the important factors

affecting the growth of algae, in general. Light is essential for photosynthesis, but the

intensity for growth varies from species to species. Temperature has an important

effect in the acceleration / retardation of growth and the reproduction of algae. The

essential elements for growth of algae are same as those necessary for the growth of

higher plants. Carbon, hydrogen, oxygen, nitrogen, phosphorous, calcium and

magnesium etc., are essential for the metabolic activities. Calcium is essential for cell

wall formation. Diatoms requires silica. Trace elements like iron, zinc, cobalt,

manganese and sulphur are required to activate enzymatic actions and for oxidation /

39

reduction reactions. Iron is essential for chlorophyll formation (Powar and

Daginawala, 2007)

Viles (1987) studied the effect of blue-green algae on limestone weathering of Aldabra

Atoll, Indian Ocean. Three different habitats can be identified on the rock surface, i.e.

epilithic, chasmolithic, and endolithic. Algae in each habitat may affect weathering in

various ways. Samples of blue-green algae and rock were taken from various

terrestrial and coastal environments on Aldabra Atoll. Samples of limestone tablets

and calcite crystals after one year in situ were also studied. Light and S.E.M.

microscopy revealed that endolithic boreholes were present on many samples,

especially those from frequently wetted sites, to a maximum depth of 800 m. An

altered zone of micrite and algal filaments was also discovered in many samples.

From morphological and petrographical evidence blue-green algal influences on

weathering on Aldabra Atoll seem to be very complex and cannot easily be related to

small scale landforms.

Guillite and Dreesen (1995) conducted laboratory chamber studies and petrographical

analysis as bioreceptivity assessment tools of building materials. Samples of selected

building materials (including natural rocks, bricks, mortars and aerated concrete) were

exposed over a 9-month period to intermittent sprinkling on steeply inclined runoff

surfaces. The sprinkling liquid consisted of nutrient-rich tap water containing a

mixture of pioneer colonising plant diaspores. A quantitative assessment of the

colonised surfaces revealed the taxonomic groups identified as cyanobacteria, green

algae, diatoms and mosses. Petrographical analysis revealed the exact mineralogical

nature of these building materials, while automated image analysis allowed the

quantification of some selected physical parameters (e.g. porosity). A major finding of

the petrographical investigation was the observation of conspicuous stratification of

the colonising plant associations and a variable penetration depth of the rhizoids and

algae. A preliminary evaluation of the biodeterioration potential of the organisms was

made. The study has demonstrated that the bioreceptivity (which refers to the aptitude

of a material to be colonized by living organisms) of building materials is highly

40

variable and that it is controlled primarily by their surface roughness, initial porosity

and mineralogical nature.

Ortega-Calvo et al. (1995) have studied the factors affecting the weathering and

colonization of monuments by phototrophic microorganisms. Phototrophic

microorganisms are common inhabitants of monuments. They reviewed different

aspects of their culture, ecology and deterioration mechanisms. Opportunistic species

of cyanobacteria and chlorophytes, present in soils and in the air, are commonly found

on the surfaces of monuments. Their growth represents a significant input of organic

matter to the stone, as estimated through chlorophyll a quantification. Monuments

provide unusual niches for the growth of algal communities, as in the case of black

sulfated crusts, or endolithic and hypogeal niches, where more specific processes and /

or communities occur.

Flores et al. (1997) investigated the growth of algae and bacteria on historic

monuments. Bacteria belonging to the genera Bacillus, Micrococcus and Thiobacillus,

yeast and microalgae of the Apatococcus genus were identified to be colonizing the

building of two monuments at Alcalá de Henares (Spain). Microbial activity in surface

layers of the stone was revealed by means of the TTC (triphenyltetrazolium chloride)

test and with SEM (scanning electron microscopy), the weathering of the substrate was

observed, as well as the presence of pollution particles and microbial structures.

Bolívar and Sánchez-Castillo (1997) studied the biomineralization processes in the

fountains of the Alhambra, Granada, Spain. The most notable form of deterioration in

the fountains is mineralization, which can cover practically the entire surface of basins

colonized by algae. They have studied two types of alterations related to the

mineralization process namely, accretion, which occurs as a result of precipitation and

mineral aggregation, and concretions related to splashing of the spout. The change in

texture and composition also results in the loss of visual characteristics on the surface

of the marble. In addition, mineralization in the walls of the endolithic cells has been

41

studied by comparing SEM images, explaining the resistance of these species to

algicides currently in use.

Dubosc et al. (2001) carried out investigations on concrete walls stained by biological

growth. Pieces of this material were removed down and observed using optical

microscopy, low-vacuum scanning electron microscopy (LVSEM) and normal SEM.

The results show that biological stains are due to two different kinds of microscopic

algae, Chlorophyceae and Cyanophyceae, whose presence depends on the amount of

moisture on the concrete wall. Accelerated laboratory tests confirm the effect of

mortar characteristics on algal development, particularly that of porosity.

Viles et al. (2001) made observations for 16 years on microfloral recolonization data

from limestone surfaces, Aldabra Atoll, Indian Ocean, for their implications of

biological weathering. A rich microflora community (or biofilm) dominated by

cyanobacteria coats most exposed rock surfaces (in both marine and terrestrial

environments) in the tropics. Such biofilms are thought to play a role in weathering,

crust formation and nutrient cycling. An initial, short-term study (1982-83) of

microfloral colonization on 0·1 × 0·1 m cleared plots on Quaternary reef limestones on

Aldabra Atoll revealed considerable variability in colonization rates, with wetter sites

and softer rocks prone to more luxuriant growths. This study reported on further

studies of colour, biomass and effects of the developing microfloral communities from

almost 100 out of the c. 300 original plots which were revisited in 1998. Many sites

still show limited colonization. Where a microfloral community has re-established,

weathering effects are notable and consistent with those found on surrounding rock.

Bellinzoni et al. (2003) have studied the distribution and amount of biological

colonisation on the “Lungotevere” walls (Rome) were analysed to assess the

ecological role of the main environmental parameters (solar irradiation, rain winds,

prevailing winds) and of the nearby tree covering. The floral data indicate a close

similarity of species in the stations examined and dominance of Chroococcus

lithophilus Ercegovic among the cyanobacteria and of Erigeron karvinskianus DC

42

among the vascular plants. In these very hard conditions the exposure and amount of

the water input do not seem to influence the flora qualitatively as much as it does

quantitatively. The low value of microflora, as found in areas characterised by the

presence of trees, can be referred to as resulting from the “umbrella” effect of the

plants. The macroflora is less influenced by such effects due to the greater capacity of

these plants to derive moisture through their roots.

Crispim et al. (2003) studied the biofilm due to algae and cyanobacteria on calcareous

historic buildings and the major microorganisms in biofilms on external surfaces of

historic buildings were found to be: algae, cyanobacteria, bacteria, and fungi, their

growth causing discoloration and degradation. They compared the phototrophs on

cement-based renderings and limestone substrates at 14 historic locations (47 sites

sampled) in Europe and Latin America and found that most biofilms contained both

cyanobacteria and algae. Single-celled and colonial cyanobacteria frequently

constituted the major phototroph biomass on limestone monuments (32 sites sampled).

Greater numbers of phototrophs, and especially of algae and of filamentous

morphotypes, were found on cement-based renderings (15 sites), probably owing to

the porosity and small pore size of the latter substrates, allowing greater entry and

retention of water. All phototrophic groups were more frequent on Latin American

than on European buildings (20 and 27 sites, respectively), with cyanobacteria and

filamentous phototrophs showing the greatest differences. The results confirm the

influence of both climate and substrate on phototroph colonization of historic

buildings.

Tripathy et al. (2004) have studied the occurrence of blue-green algae as blackish

brown crust / tuft on the exposed rock surfaces of 30 different temples and monuments

of different regions of India. A total number of 30 species attributed to 13 different

genera were encountered. Seven species of Tolypothrix, and one each of

Gloeocapsopsis, Lyngbya, Phormidium and Plectonema are the major componentes of

the crusts / tufts. There were also species belonging to Gloeothece, Myxosarcina,

Chroococcidiopsis, Plectonema, Nostoc, Calothrix, Chlorogloeopsis, Fischerella and

43

Hapalosiphon which appeared in the enrichment culture as minor component along

with the dominant species.

Zurita et. al. (2005) studied the microalgae associated with deteriorated stonework of

the fountain of Bibatauin in Granada, Spain. Colonization by microalgae and its effects

on the fountain were investigated. The microorganisms from representative sampling

areas were identified by optical microscopy, and the biogenic carbonate crusts they

formed analysed by X-ray diffraction and field emission scanning electronic

microscopy. It has been stated that the most representative genera found were

Cosmarium, Phormidium and Symploca, and the main mineral was calcite.

Schumann et al. (2005) have quantified the green microalgae colonizing building

facades, using chlorophyll extraction methods. The shortcoming in the assessments so

far used, such as, lack of inter-calibration, colour modifications due to of co-occurring

of fungi or background properties, have been highlighted. By using chlorophyll a as a

specific biomarker of aeroterrestrial microalgae, an extraction method was therefore

developed to quantify biomass. Two green microalgae, Stichococcus sp. and Chlorella

sp., isolated from facades of buildings and established as monocultures were used in

this study. Dimethyl formamide (DMF), the best extraction solvent was three times.

Using this biomarker assay, up to 313 mg chlorophyll a m−2

Miller et al. (2008) investigated the reproducibility of photosynthetic-based

colonization of stone monument under laboratory conditions. For this study, a natural

green biofilm from a limestone monument was cultivated, inoculated on stone probes

of the same lithotype and incubated in a laboratory chamber. This incubation system,

which exposes stone samples to intermittently sprinkling water, allowed the

development of photosynthetic biofilms similar to those occurring on stone

monuments. Denaturing gradient gel electrophoresis (DGGE) analysis was used to

evaluate the major microbial components of the laboratory biofilms. Cyanobacteria,

was obtained from

building facades, equivalent to c. 100 g algal fresh weight, which represents a high

organic load.

44

green microalgae, bacteria and fungi were identified by DNA-based molecular

analysis targeting the 16S and 18S ribosomal RNA genes. The natural green biofilm

was mainly composed by the Chlorophyta Chlorella, Stichococcus, and Trebouxia, and

by Cyanobacteria belonging to the genera Leptolyngbya and Pleurocapsa. A number of

bacteria belonging to Alphaproteobacteria, Bacteroidetes and Verrucomicrobia were

identified, as well as fungi from the Ascomycota. The laboratory colonization

experiment on stone probes showed a colonization pattern similar to that occurring on

stone monuments. The methodology described in this study allowed reproducing a

colonization equivalent to the natural biodeteriorating process.

Macedo et al. (2009) reviewed comprehensively the literature on chlorophyta that

cause deterioration of stone cultural heritage (outdoor monuments and stone works of

art) in European countries of the Mediterranean Basin. Some 45 case studies from 32

scientific papers published between 1976 and 2009 were analysed. Six lithotypes were

considered: marble, limestone, travertine, dolomite, sandstone and granite. A wide

range of stone monuments in the Mediterranean Basin support considerable

colonization of cyanobacteria and chlorophyta, showing notable biodiversity. About

172 taxa have been described by different authors, including 37 genera of

cyanobacteria and 48 genera of chlorophyta. The most widespread and commonly

reported taxa on the stone cultural heritage in the Mediterranean Basin are, among

cyanobacteria, Gloeocapsa, Phormidium and Chroococcus and, among chlorophyta,

Chlorella, Stichococcus and Chlorococcum. The results suggest that Cyanobacteria

and green algae play an important role in the deterioration of monuments and other

stone works of art, being responsible for aesthetic, biogeophysical and biogeochemical

damage.

Grbic et al. (2009) investigated the biofilm of cyanobacteria, algae and fungi on

sandstone substrata of Eiffel´s Lock in Becej (Serbia), which contained a complex

consortia of algae, cyanobacteria and fungi. Filamentous cyanobacteria (Nostoc sp,

Leptolyngbia sp., Stigonema ocellatum) and green algae (Desmococcus olivaceus and

Haemaotococcus pluvialis) formed dense mucous layer with characteristic coloration

45

of substrata. Melanized fungal structures (hyphae, chlamydospores and conidia) were

intertwined with cyanobacteria and algae formed biofilm. Dominant fungal genera

were Alternaria, Aureobasidium, Bipolaris, Cladosporium, Drechslera,Epicoccum,

belongs to Dematiaceous hyphomycetes. It has been stated that the biofilm

constituents’ interaction results in the bioweathering of the sandstone substrata through

mechanical penetration, acid corrosion and production of secondary mycogenic

biominerals.

Javaherdashti et al. (2009) have made a mechanistic review on the impact of algae

on accelerating the biodeterioration / biocorrosion of reinforced concrete, the

complexities involved in both microbiologically influenced corrosion and deterioration

of reinforced concrete structures by algae. It is prudent to consider two processes

namely: (i) microbiologically influenced corrosion (MIC) of the steel reinforcement

and (ii) microbiologically influenced deterioration (MID) of the concrete. Further, they

have stated that five possible corrosion / deterioration mechanisms may be expected.

It has been highlighted that portlandite and hydrated products in OPC when dissolved

by the excreting organic acids of algae, they form the corresponding calcium salts of

the attacking acid, it is the solubility of the above calcium salt that primarily controls

the rate of deterioration in hydrated OPC concrete, and not the strength of the acid

(Allahverdi, A., Škvára, F., 2000).

2.3.6 Overview of Earlier Investigations

Table 2.3 summarizes the investigations that have been carried out on the

biodeterioration of various construction materials due to various live organisms.

Further, the various sophisticated analytical techniques that have been adopted for the

above studies have also been summarized in Table 2.4. It can been seen very clearly

that the order of increasing importance given, with respect to the type of live

organisms, ‘decreases in the order of: bacteria, lichens, algae, fungi and mosses’. The

reported studies due to algae were mostly related to stones, barring a few exceptions.

Of the various analytical techniques used in biodeterioration studies, SEM & EDAX

46

and XRD have been used by almost all investigators and for all types of substrate of

construction materials.

2.4 INTERACTION OF WEAK ACIDS WITH CEMENT / CEMTITIOUS

MATERIALS

2.4.1 Definition, Types and Sources of Weak Acids

An ‘acid dissociation constant’ (Ka) (also known as: acidity constant or acid-

ionization constant) is a quantitative measure of the ‘strength of an acid’ in solution. It

is the ‘equilibrium constant’ for a chemical reaction known as ‘dissociation’ in the

case of acid-base reactions. Due to many orders of magnitude spanned by Ka values, a

‘logarithmic’ measure of Ka, namely (pKa) is commonly used. It is equal to

[-log10

Four compounds are usually regarded as the major constituents of cement:

(i) tricalcium silicate (3CaO. SiO

(Ka)] and may also be referred to as an acid dissociation constant.

The larger value of (pKa), the smaller is the extent of dissociation. A ‘weak acid’ has

(pKa) value in the (approximate) range of (-)2 to (+)12 in water. Acids with a (pKa)

value, less than (-)2 are said to ‘strong acid’, which are almost completely dissociated

in aqueous solution, to the extent that the concentration of the ‘undissociated acid’

becomes undetectable.

Generally inorganic acids are considered as ‘strong acid’, whereas, organic acids are

considered as ‘weak acid’.

2.4.2 Mechanism of Acid Attack on Concrete

(A) Chemical Composition and Hydration of Portland Cement

‘Cement’ is a finely pulverized material which by itself is not a binder, but, develops

the binding property as a result of hydration (i.e. from chemical reactions between the

cement minerals and water). A ‘hydraulic cement’ is one whose hydration products are

‘stable in an aqueous environment’ for making concrete.

2 - C3S); (ii) dicalcium silicate (2CaO. SiO2 -C2S);

(iii) tricalcium aluminate (3CaO. Al2O3 – C3A) (iv) tetracalcium aluminoferrite

(4CaO.Al2O3. Fe2O3 - C4AF). Generally, the oxide composition of cement of OPC is:

47

(i) calcium oxide (CaO) ≈ 60 -67%; (ii) silicondioxide (SiO2) ≈ 17 -25% and (iii)

aluminum oxide (Al2O3

By virtue of OPC concrete being highly alkaline, is not resistant to attack by ‘acids’ or

compounds which may convert to acids. In general, chemical attack of concrete, say,

acid attack occurs by way of decomposition of the products of hydration and formation

of new compounds, which, if soluble, may be leached out, and if not soluble, may be

disruptive in-situ (Neville, 2004). The attacking compounds must be in solution.

Ca(OH)

) ≈ 3 -8% apart from other minor oxides of iron, magnesium

etc.,

Concrete is a composite material consists essentially of a binder (say ‘cement’), within

which are embedded particles or fragments of aggregates. In the case of OPC concrete

or plain concrete or simply concrete, OPC is the binder used along with aggregates and

water to produce concrete. In the presence of water, silicates and aluminates (as stated

above) form products of hydration which over time form a hard mass and are stable in

aqueous environments.

Calcium silicate hydrates (C-S-H) which makes upto 50-60% of volume of solids in a

completely hydrated Portland cement paste, is the one primarily responsible for the

strength of the material. Calcium hydroxide crystals (also called portlandite) constitute

only 20-25% of solids volume in the hydrated paste and therefore play only a minor

role in the strength - property relationship.

(B) Acid Attack on Concrete- An Overview

2 is the most vulnerable cement hydrate, but C-S-H can also be attacked.

Further, calcareous aggregates are also vulnerable. Neville., (2004) has listed some

substances which cause severe chemical attack of concrete like various inorganic acids

(carbonic, hydrochloric, hydrofluoric, nitric, phosphoric and sulfuric); organic acids

(acetic, citric, formic, humic, lactic and tannic) and other substances (aluminum

chloride and animal fats, vegetable oils and sulfates). The products of reaction between

calcium hydroxide and oxalic, tartaric, tannic, humic, hydrofluoric or phosphoric acid

belong to the category of insoluble, non-expansive, calcium salts. Lactic and acetic

acid combine with free lime Ca(OH)2 in the alkaline concrete (pH about 13) to

produce highly soluble calcium salts. If the salts are leached, the pH in the concrete

48

pores decreases and the binding agents of the cement paste are left in an unstable

condition. The unstable material is then easily removed by mechanical impact from

animals or cleaning (Mehta and Monteiro, 1999).

The consumption of alkali materials in pore water, especially Ca(OH)2 and its rapid

depletion from the cement matrix is called ‘decalcification’. The dissolution of

calcium hydroxide crystals and the extensively decalcified CSH gels results in

increased porosity and enlarged threshold capillary pores in the leached layers. This

causes self-accelerating leaching and matrix deterioration. These changes would cause

progressive microstructural breakdown and loss of mechanical strength, which

eventually leads to complete disintegration of concrete (Asavapisit, 2002).

VFA (Volatile fatty acids) such as acetic, propionic, butyric, isobutyric and valeric

acids are found in liquid manure, apart from mineral compounds in various quantities.

Because of VFAs, liquid manure constitutes a chemically aggressive environment

towards concrete, whereby, the organic acids react with several hydrates of cement

paste (portlandite, C-S-H and hydrated aluminates) to produce calcium and aluminum

salts, whose stability in water varies from high to very high. In an immersed situation,

those actions on concrete lead to the hydrates lixiviation, increase in (paste) porosity

and decrease in mechanical resistance, resulting in reinforcement corrosion (Berton,

2004)

In the case of cementitious material like fly ash, the hydration products are essentially

the same as those of OPC under normal conditions. But, a cement fly ash paste

contains more C-S-H gel, with a lower Ca/Si ratio and less Ca(O)2

De Belie et al. (1997) have investigated the attack by lactic and acetic acid, which

were formed in spilled and soured meal-water mixtures on concrete floors in pig

houses. Accelerated degradation tests were performed. It was found that the addition

of about 10% low-calcium fly ash (by weight of cement) to OPC reduced the

than OPC alone,

due to pozzolanic reaction. Thus, fly ash helps to reduce the vulnerability of acid

attack, apart from good concrete quality due to homogenous paste (De Belie, 1997).

2.4.3 Review of Earlier Works

49

degradation significantly. Additions of flyash to OPC and to sulphate resisting

Portland cement have shown almost equal performance to the attack of the meal acids.

De Belie et al. (1996) studied the influence of four types of cement on the resistance

of concrete to feed acids, namely lactic acid and acetic acid. It has been concluded that

the cement type appears to have an important influence on the corrosion of concrete by

feed acid and that the four types of cement with decreasing change in volume in terms

of percentage and mass loss per unit area are: (i) portland cement without C3A, (ii)

OPC; (iii) cement containing fly ash; (iv) blast furnace slag cement. Further, it is

concluded that the percentage of slag cement and the cement content of the pozzolanic

cement have no significant influence.

Asavapisit et al. (2002) studied the durability or cement-based solidified wastes

against different acid attacks including that of acetic acid. It was concluded that the

resistance of the cement-based solidified waste matrices against acid attack was in the

following order: sulfuric > acetic > nitric acid.

Berton et al. (2004) analyzed the mechanisms of organic acid attack on cementitious

materials and identifying the cement composition parameters influencing the durability

of concrete. The study concentrated on three types of cement (i) OPC; (ii) low – C3

(i) The advent and use of sophisticated instrumentation, has greatly influenced the

field observation and laboratory quantification and measurements, with regard

to biodeterioration, and thus has helped in better understanding of the role of

living organisms in influencing deterioration of a variety of construction

materials;

A

OPC and (iii) slag cement. Several organic acids simulating liquid manure used in

agriculture was considered. The results show the altered zone, and a modification of

the microstructure manifesting itself by progressive dissolution of all the crystallized

phases. Thus, the attached zone has exhibited poor mechanical resistance.

2.5 CONCLUDING REMARKS

Based on the extensive literature review carried out, following critical observations are

made:

50

(ii) SEM & EDAX and XRD have been used in almost all biodeterioration studies

and for all substrata of construction materials;

(iii) Extensive studies have been carried out and reported on the biodeterioration of

bacteria on a variety of substratum / materials. However, studies on algae, more

so, on marine algae and their biological influence on materials are rather scarce.

(iv) Biological influences on concrete, in general has been very scarcely

investigated, and more so due to algae.

(v) Mechanisms of microbially induced deterioration for all combinations of live

organisms and construction materials (natural and man made) has not been

investigated in detailed and established fully. However, such studies are needed

for evolving preventive / remedial measures for bioremediation.

Hence, there is ample scope for carrying out systematic and scientific investigations in

the area of biodeterioration especially due to marine algae on concrete. The above has

resulted in setting out objectives of the present study as given in Chapter-1.

51

Table 2.1 Classifications of organisms based on their nutritional requirements (Kumar and Kumar, 1999)

Nutritional Category

Energy Source Carbon Source

Electron Donors

Electron Acceptors

Groups of Organisms

Photoautotrophs or photolithotrophs

Sunlight (photosynthetic organisms )

CO2 Water Oxygen Organics

Aerobic organisms: Cyanobacteria, Algae (Bacillariophyta or Diatoms), Algae (Chlorophyta) Lichens, Mosses and liverworts Higher plants

Chemoautotrophs or chemolithotrophs

Redox reaction s (chemosynthetic organisms )

CO2 H2 , Fe2+ NH4+, NO2-, S, S2O3

Oxygen

2-

Aerobic organisms: Hydrogen bacteria, Iron bacteria Nitrifying bacteria, Sulfur-oxidizing bacteria

Photoheterotrophs or photoorganotrophs

Sunlight (photosynthetic organisms )

Organics Organics, H2S, H

Oxygen 2

Aerobic organisms: Photosynthetic bacteria, Some algae

Organics Anaerobic organisms: Green and purple sulfur bacteria Purple nonsulfur bacteria

Chemoheterotrophs or chemoorganotrophs

Redox reaction s (chemosynthetic organisms )

Organics Organics

S, S 2O3 2-

H2

Oxygen

S

Aerobic organisms: Actinomycetes, Animals, Fungi, Respiratory bacteria

Organics NO3 –

SO4

Anaerobic organisms: Fermentativ e bacteria, Denitrifying bacteria,

Sulfur-reducing bacteria 2–

52

Table 2.2 Various categories of biodeterioration (Sand, 1997)

Mineral acids Sulfuric acid (H2SO4) Nitrous acid (HNO2) Nitirc acid (HNO3)

Organic acids Carbonic acid (CO2 / H2CO3) Oxalic acid (H2C2O4) Gluconic acid (H8C6O7) Critic acid (H8C6O7) Formic acid (H2CO2) - and many more

Organic solvents Hydrogen sulfide

Ethanol, acetone, proponal, butanol H2S- sulfuric acid -metal sulfide precipitate

Nitrous oxide salts

NO, NO2 Hygroscopic – increased water content Increase of crystal volume by inclusion of water molecules – swelling attack in pores

Biofilm Clogging of pores Decrease of porosity Increase of humidity (enhance physical attack as freezing-thawing)

Enzymes Complexing / emulsifying compounds

Degradation of organic constituents Organic acids Phospholipids Lipoproteins Lipopolysaccharides

53

Table 2.3 Overview of investigations on biodeterioration of various materials Sl.No.

Name of Investigator/ (s) Year Material

Studied Review of

Biodeterioration Mechanism of

Biodeterioration Biodeterioration studies on

Bacteria Lichen Mosses Fungi Algae 1 Wolfgang Sand 1997 - 2 Warscheid and

Braams 2000 -

3 Sanchez Silva. M et al

2008 -

4 Midle et al. 1983 Concrete 5 Sand and Bock 1984 Concrete 6 Sand et al. 1984 Concrete 7 Sand and Bock 1991 Concrete 8 Sand et al. 1994 Concrete 9 Davis et al. 1998 Concrete 10 Vincke et al., 1999 Concrete 11 Monteny et al. 2000 Concrete 12 Videla et al. 2000 Stone 13 Papida et al. 2000 Stone 14 Saiz- Jimenez et al. 2000 Stone 15 Lamenti et al. 2000 Marble 16 Monteny et al. 2001 Concrete 17 Vincke et al. 2001 Copncrete 18 Hernandez et al. 2002 Concrete 19 Vincke et al. 2002 Concrete 20 Herrera et al. 2004 Stone 21 De Belie et al. 2004 Concrete 22 Kawai et al. 2005 Concrete

Contd…

54

Sl.No.

Name of Investigator/ (s) Year Material

Studied Review of

Biodeterioration Mechanism of

Biodeterioration Biodeterioration studies on

Bacteria Lichen Mosses Fungi Algae 23 Crispim and Gaylarde 2005 N.S 24 De Graef et al. 2005 Stone and

Concrete

25 Seth and Edyvean 2006 Concrete 26 Crispim et al. 2006 Stone 27 Lors et al. 2009 Concrete 28 Cooks and Otto 1990 Rock 29 Lamas et al. 1995 Granite

rocks

30 Ariño et al. 1995 Flagstone 31 Romao and Rattazzi 1996 Granite

rocks

32 Arino et al. 1997 Mortar 33 Ascaso et al. 1998 Stones 34 Chen et al. 2000 Stone

35 Tomasell et al. 2000 Stone 36 Carballal et al. 2001 Stone 37 Williamson et al. 2002 Rock 38 De Graef et al. 2005 Concrete 39 Gaylarde et al. 2006 Stone 40 2006 Watanabe et al. glazed

sekishu roof-tiles

41 Duane 2006 Sandstone

Contd…

55

Sl.No.

Name of Investigator/ (s) Year Material

Studied Review of

Biodeterioration Mechanism of

Biodeterioration Biodeterioration studies on

Bacteria Lichen Mosses Fungi Algae 42 Áková et al. 2008 Concrete 43 Gazzano et al. 2009 Stone 44 Nascimbene et al. 2009 Stone 45 Ríos et al. 2009 Dolostone

and limestone

46 Altieri and Ricci 1997 Stone 47 Shirzadian et al. 2008 Concrete 48 Gomez-Alarcon et

al. 1994 Stone

49 Diakumaku et al. 1995 Marble 50 Gómez-Alarcón et al. 1995 Stone 51 Gutiérrez et al. 1995 Wood 52 Wollenzien et al. 1995 Stone 53 Arocena et al. 2003 Granitic

rocks

54 Shirakawa et al. 2003 Mortar 55 Karys and Wazny 2007 Porous

building materials

56 Wiktor et al. 2009 Cement 57 Giannantonio et al. 2009 Concrete 58 Viles 1987 Lime stone 59 Guillite and Dreesen 1995 Concrete

Contd…

56

Sl.No.

Name of Investigator/ (s) Year Material

Studied Review of

Biodeterioration Mechanism of

Biodeterioration Biodeterioration studies on

Bacteria Lichen Mosses Fungi Algae 60 Ortega-Calvo et al. 1995 Stone 61 Flores et al. 1997 Stone 62 Bolívar and Sánchez-

Castillo 1997 N.S

63 Dubosc et al. 2001 Concrete 64 Viles et al. 2001 Lime stone 65 Bellinzoni et al. 2003 Stone 66 Crispim et al. 2003 Lime stone 67 Tripathy et al. 2004 Rock

surface

68 Zurita et. al. 2005 Stone 69 Schumann et al. 2005 Stone 70 Miller et al. 2008 Stone 71 Macedo et al. 2009 Stone 72 Grbic et al. 2009 Stone 73 Javaherdashti et al. 2009 Concrete

Note: N.S – Not Specified

57

Table 2.4 Overview of various analytical methods used by various investigators

Sl.No Analytical Method

Purpose Type of organism Reference

Bacteria Lichens Mosses Fungi Algae 1 SEM and

EDAX Morphological studies Viles (1989)

Cooks and Otto (1993) Sand (1994) Gomez-Alarcon (1994) Gómez-Alarcón et al. (1995) Arino (1997)

Bolívar and Sánchez-Castillo (1997)

Ascaso (1998) Kumar (1999) Arocena et al. (2000) Videla et al. (2000) Monteny (2000) Chen (2000) Dubosc et al. (2001) Williamson et al. (2002) Shirakawa et al. (2003) Herrera et al. (2004) Zurita et. al. (2005) Graef et al. (2005) Ríos et al. (2009) Wiktor et al. (2009)

Contd…

58

Sl.No Analytical Method

Purpose Type of organism Reference

Bacteria Lichens Mosses Fungi Algae 2 ESEM Morphological studies Videla et al. (2000) Watanabe et al. (2006)

3 LVSEM Morphological studies Dubosc et al. (2001)

4 FTIR Finger print of compounds Gómez-Alarcón et al. (1995)

5 XRD Mineralogical composition Silva (1996) Arino (1997) Kumar (1999) Herrera et al. (2004) Kawai et al. (2005) Zurite (2005)

6 TGA Change in weight of the sample while it is heated at a constant rate

Silva (1996)

59

Fig- 2.1 SEM of swimming, quadriflagellate zoospores

(Callow and Callow, 2002)

Fig- 2.2 ESEM of a settled, adhered zoospore (Callow and Callow, 2002)

60

Fig- 2.3 Representation of the stages involved in zoospore settlement and adhesion

(Callow and Callow, 2002)

Fig- 2.4 Electron micrograph of a section through the apical region of a swimming

zoospore showing the extensive adhesive vesicles with electron-opaque deposits of the primary adhesive (Callow and Callow, 2002)