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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,
6
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
7
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
8
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
9
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
10
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
12
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.
13
(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
15
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.
16
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.
17
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.
18
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.
19
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.
20
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)