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Bioremediation of Some Deterioration Products fromSandstone of Archeological Karnak Temple UsingStimulated Irradiated Alkalo-Thermophilic PurifiedMicrobial EnzymesNeveen S. I. Geweely a & Hala A. M. Afifi ba Botany Department, Faculty of Science , Cairo University , Giza, 12613, Egyptb Conservation Department, Faculty of Archaeology , Cairo University , Giza, 12613, EgyptPublished online: 07 Jan 2011.

To cite this article: Neveen S. I. Geweely & Hala A. M. Afifi (2011) Bioremediation of Some Deterioration Products fromSandstone of Archeological Karnak Temple Using Stimulated Irradiated Alkalo-Thermophilic Purified Microbial Enzymes,Geomicrobiology Journal, 28:1, 56-67, DOI: 10.1080/01490451.2010.498296

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Geomicrobiology Journal, 28:56–67, 2011Copyright © Taylor & Francis Group, LLCISSN: 0149-0451 print / 1521-0529 onlineDOI: 10.1080/01490451.2010.498296

Bioremediation of Some Deterioration Products fromSandstone of Archeological Karnak Temple Using StimulatedIrradiated Alkalo-Thermophilic Purified Microbial Enzymes

Neveen S. I. Geweely1 and Hala A. M. Afifi2

1Botany Department, Faculty of Science, Cairo University, Giza, 12613, Egypt2Conservation Department, Faculty of Archaeology, Cairo University, Giza, 12613, Egypt

The archeological temples of Karnak in Luxor city are the mostimportant records of the history and civilization of Egypt thatbelonged to the Middle Kingdom to the reign of the Ptolemies.Many parts in Karnak temple are suffering from different typesof biological deterioration products (visible fungal colonies, blooddrops, and celluloytic wild bees’ nests). The deteriorated samplesof archeological sandstone of Karnak temple were analyzed byX-ray diffraction (XRD) followed by energy dispersed X-rayanalyses (EDX), Fourier Transform Infrared Spectroscopy (FTIR)investigations, scanning electron microscope (SEM) and polarizingmicroscope (PLM). The major component was quartz. In vitroantagonistic activity of Trichoderma reesei against deteriorativeisolated fungal species on Karnak Temple stone was carried out.Trichoderma reesei and Fusarium oxysporum, a cellulolytic andfibrinolytic microorganism, were subjected to mutagenesis usingthree types of radiations (UV, gamma and laser radiation). Stimula-tion of cellulytic and fibrinolytic microbial enzymes were obtainedafter 5- and 7.5-min exposure times to laser irradiation in absenceof the photosensitizer, respectively. Cellulolytic and fibrinolyticenzymes recovered from non-irradiated and irradiated microbialcells were purified to homogeneity by salting out with ammoniumsulphate, dialysis and chromatography through (Sephadex G-200,Sephadex G-100 and diethylaminoethyl cellulose columns) and testfor purity by simple polyacrylamide gel electrophoresis techniquewas carried out. The enzymes recovered from irradiated hyper-producing mutant microbes was found more efficient accompaniedwith low molecular weights compared with the non-irradiated pu-rified enzymes. Characterization of the irradiated purified efficientenzymes revealed that the enzymes were alkalo-thermophilic.

Keywords temples of Karnak, Bioremediation, T. reesei, F. oxys-porum, wild bees’ nests, bat drops, radiation, enzymepurification

Received 11 January 2010; accepted 19 May 2010.Address correspondence to Neveen S. I. Geweely, Botany Depart-

ment, Faculty of Science, Cairo University, Giza, 12613, Egypt. E-mail:ngeweely@yahoo.com

INTRODUCTIONArcheological stone represent an important part of our

world’s cultural heritage. The history of human has been ac-companied by use of natural stones specially sandstone. Theprocesses leading to the deterioration of stone buildings havebeen the subject of numerous publications (James 1980). Thedeterioration of stones in monuments is a combined processcaused by physical, chemical and biological factors. The rela-tive importance of each factor varies according to the environ-mental conditions, the stone type, its preservation state and itslocation on the monument (Papida et al. 2000). Deterioration ofstones begins from the moment it was quarried due to naturalweathering processes (Webster and May 2006). There are otherphenomena as crystallization of soluble salts, pollution, biolog-ical colonization, soot, bat blood drops, and wild bees’ nests(Sleater 1973).

Bats can damage stone substrata by trampling and blooddropping. These drops can induces discoloration of painted sur-faces and serve as nutritive substrata for bacteria and fungi(Winkler 1975). Wild bees’ nest removal is considered a dif-ficult problem in cleaning processes due to its solidity. Theaction of the bee insect is boring to build his cellulosic nestinto stones by removing clay ground materials with some otherorganic materials (Lioyd 1976). Attempts to restore archeo-logical stone surfaces by removal of deterioration productsusing mechanical and chemical cleaning that lead to severaltypes of damage were discussed. (Maxwell 1992; Webster1992).

The term “bioremediation” covers a range of processes thatuse microorganisms, or their enzymes, to return deterioratedarcheological objects to their original condition (Atlas 1995).Microorganisms have been used as biocides and are sometimesmentioned for cleaning objects of historical and cultural sig-nificance (Young and Wainwright 1995). The possibility ofusing biological agents such as antagonistic species from thegenus Trichoderma are reported in the literature, and a biofungi-cide formulated with T. harzianum, named Trichodex was used(Samuels 1996). These Trichoderma species are well studied

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and have shown efficiency in biocontrol of different deteriorat-ing fungal species (Melo 1996).

Microbial enzymes have been used for cleaning archeolog-ical objects by removal of crusts, but reports have indicateddisadvantages in the practical difficulty of maintaining a goodenzymatic activity, as a function of temperature and pH (Sorlini1984). So, every effort must be directed to stimulate micro-bial enzyme activities with wide ranges of temperature and pH.These enzymes could be very useful as preventative, takinginto account the difficulty for liquid biocides to deeply pene-trate inside the archeological object. Microbial cellulolytic andfibrinolytic enzymes were used for hydrolyses of deteriorationproducts of cellulolytic substances and blood drops, respectively(Swenson et al. 2000; Kasab 2007).

Nowadays, increasing the efficiency of microbial enzymesthat are used in the bioremediation process by low intensityradiation has gained great interest. The importance of under-standing the effects of electromagnetic radiation on biologicalenzymes has spurred a great research effort, not only becauseof its basic biological interest, but also for technological appli-cations (Trombert et al. 2007). A number of studies have beencarried out to investigate the use of different types of radiation toincrease the enzymatic activity of microorganisms (Vladimirovet al. 2004). Several types of low-intensity laser radiation, in-cluding helium neon laser (He–Ne), were tried against differ-ent microorganisms to stimulate the microbial enzymes (Karu2003).

Improvement of cellulase production by mutation using UV-irradiation was found by Fang et al. (2009). Effect of low in-tensity electromagnetic radiation on fibrinolytic properties wasstudied by Lohinov et al. (2001). Shi et al. (2008) stated that af-ter mutagenesis treatments by UV and gamma-radiation, a highfibrinolytic enzyme producing a fungal strain was obtained.

The aim of the present work was to remove the biologicaldeterioration products (fungal colonies, blood drops and bees’nests) that represent a big problem in cleaning processes. Thus,we must study the chemical composition and deterioration prod-ucts of sandstones from archeological Karnak temple in LuxorCity, Egypt. Isolation of deteriorating fungal species on sand-stones of Karnak temple with studying the antagonistic activityof T. ressei against them was recorded. Bioremediation of de-terioration products was carried out by comparative stimulativeeffect of UV, gamma and laser radiation on the production ofcellulolytic and fibrinolytic enzymes from T. reesei and F. oxys-porum, respectively. The research will also include comparativestudy on purification, molecular weights and characterization ofboth non-irradiated and irradiated cellulolytic and fibrinolyticenzymes to improve their bioremediation efficiency.

MATERIALS AND METHODS

Sources of IsolationThe archeological temples of Karnak in Luxor city are the

most important records of the history and civilization of Egypt

FIG. 1. Map of Luxor city, Egypt.

that belonged to the Middle Kingdom to the reign of thePtolemies (Figures 1 and 2). The color of stone surface waschanged by bats’ blood drops with deteriorated stone surfaceby wild bees’ nests and deteriorated fungal colonies. Parts ofdeteriorated sandstone at Karnak temple and the deteriorationproducts of wild bee nests samples were showed in Figures 3and 4.

Investigations and Analyses of Deteriorated ArcheologicalKarnak Temple Stone and Deterioration Products

X-ray diffraction (XRD) and Energy Dispersive X-ray Anal-ysis (EDX). The samples collected from the deterioratedsandstone and a wild bees’ nest from the Karnak temple inLuxor City, Egypt were analyzed by X-ray diffraction analysis.The samples were ground, pressed into the specimen holder,and mounted in a Phillips X-ray diffraction equipment modelPW/1840 with Ni filter, Cu radiation 1.54056 A◦ at 40 KV,25mA, 0.05 /sec. An energy dispersive X-ray analysis wasused to study the elements qualitatively and quantitatively usingan EDX unit Model Phillips XL30 with accelerating Voltage25KV.

Fourier Transmission Infrared Spectroscopy (FTIR). In-frared spectroscopy is one of the most widely used techniquesin the field of art conservation. Its versatility and ability for pro-viding structural information of both inorganic and organic ma-terials was useful (Derrick et al. 1999). Transmission IR spectrawere recorded using a Thermo Nicolet FTIR spectrophotometerto assure that red spots in the temple walls were due to batblood drops. The powdered sample was examined between4000–400 cm−1.

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58 N. S. I. GEWEELY AND H. A. M. AFIFI

FIG. 2. Plan of Karnak temple, Luxor city, Egypt.

Scanning Electron Microscope (SEM). The alteration ofsurface morphology and the fungal deteriorated sandstone sam-ple were carried out by using SEM Model Phillips XL30 withaccelerating Voltage 25KV (Goldstein et al. 1992).

Polarizing microscope (PM). Sample from deterioratedsandstone of the temple have been examined in thin sectionby using transmitting polarizing microscope to get details onthe texture components due to fungal growth.

FIG. 3. Deteriorated parts of carved sandstone of archeological Karnaktemple.

FIG. 4. The deterioration products of wild bee nests on the Karnak temple(scale bar 20 µm).

Isolation and Identification of Fungal Species onArcheological Karnak Temple

Many parts in Karnak temple are suffering from visiblecolonies of microscopic fungi, blood drops and cellulolytic wildbees’ nests. Swabbing with sterile cotton swabs and scalpel frommarkedly damaged surfaces of the sandstone of karnak templewith visible colonies of fungi was carried out from the two sites.The first isolation site was collected from the blood drops’ site,while the second isolation site were performed from the site ofcellulolytic wild bees’ nests. In the laboratory, swab sampleswere shaken mechanically for 10 min in 10 mL sterile distilledwater and 1 mL aliquots of the resulting suspensions used toprepare spread plates on Czapeck’s Dox agar. Plates were in-cubated in the dark at 27◦C for 7 days, and the microscopicfungi were identified using the diagnostic keys (Gilman l957;Barnett and Hunter 1972; Samson and Reenen-Koekstra 1988;Moubasher l993; Kern and Blevins 1997).

Test OrganismsIn vitro antagonistic activity of T. reesei against deteriorative

isolated fungal species on Karnak Temples was carried out. Theeffect of non-volatile and volatile metabolites from Trichodermaspecies was tested against the isolated fungal species (Lund-berg and Unestan 1980; Dennis and Webster 1971a; Dennis andWebster 1971b), respectively. Growth rates, in both assays, wererecorded daily by measuring colony diameter according to Lillyand Barnett (1951). The inhibition percent was obtained usingthe formula: I% = [ (C2 – C1)/C2) ] × 100 (Edington et al.1971), where C1 represents growth of isolated fungi and C2represents growth of control.

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Radiation Sources and Cultivation of MicroorganismsThe removal of blood drops and cellulolytic wild bees’ nests

on Karnak Temples were carried out by irradiated purifiedcellulolytic and fibrinolytic enzymes of T. reesei and F. oxyspo-rum, respectively. The source of UV radiation was Philips TUV-30-W-245 nm Lamp, type No. 57413-P/40 at a distance of20 cm at exposure time (0, 5, 10, 20, 40 and 60 min). Gammairradiation was Cobalt-60 gamma cell 3500, which was locatedat the Middle Eastern Regional Radioisotopes Center for Arabcountries (Dokki, Cairo) using exposure times of 0, 5, 10,20, 40 and 60 min. Laser source was located at the NationalInstitute of Laser Enhanced Science (NILES), Cairo University.The laser used was a He–Ne gas laser (NEC, Japan), which hasa power output of 7.3 mW; it emitted light with a wavelength of632.8 nm in a beam of diameter 1.3 mm. Three photosensitizers(0.5 mg/ml, incubated with the test fungus for 5 min beforeirradiation) were used, which were crystal violet (CV, Sigma),toluidine blue O (TBO, Sigma) and hematoporphyrin (HP,Porphyin products-Logan, Utah). The exposure time was (0, 3,5, 7.5, 10 and 20 min.). A photosensitizer-free sample was usedfor comparison.

Assay of Cellulolytic and Fibrinolytic EnzymesThe irradiated (UV, gamma and laser) and non-irradiated

spores of cellulolytic T. reesei were spread into Czapek–Dox’smedia using sucrose as substrate. On the other hand, the basalmedium was a modified Czapek medium using casein as asubstrate, consisting of the following ingredients (g/l): casein,5; sucrose, 30; K2HPO4, 1.0; MgSO4 7H2O, 0.5; KCl, 0.5;FeSO4 7H2O, 0.01 was used for isolation of fibrinolytic organ-ism (F. oxysporum) (Ali and Ibrahim 2008). All flasks wereincubated in the dark at 50◦C. The optimum pH of growth ofthe test fungi is pH 8 for 7 days. The activities of irradiated andnon-irradiated cellulolytic and fibrinolytic enzymes were de-termined in the culture filtrate (Rodionova et al. 1966; Egorovet al. 1982). All assays were conducted in triplicate.

Comparative Purification Scheme, Molecular Weights andCharacterization of Cellulolytic and Fibrinolytic Enzymes

A crude extract of cellulolytic and fibrinolytic enzymes wereprepared by precipitation with ammonium sulfate followed bydialyses. The dialyzed fractions of cellulolytic and fibrinolyticenzymes were chromatographed on Sephadex G-200, G-100and DEAE-Sephadex columns, respectively. The protein con-tent and the activity of cellulolytic and fibrinolytic enzymeswere determined for each fraction (Segel 1968). Estimation ofthe molecular weight of the purified enzymes were recorded us-ing sodium dodecyl-sulfate polyacrylamide gel electrophoresis(SDS-PAGE) (Laemmli 1970). Characterization of the purifiedenzymes was recorded by the effect of different reaction tem-perature (30, 45, 50, 70, 80 and 90◦C) on the enzyme activitiesfor 5 min. The effect of different pH values ranging from 3 to10 on the activity of the purified enzymes was tested.

FIG. 5. XRD pattern of sandstone sample showing quartz as the main com-ponent of Karnak temple stone.

RESULTS AND DISCUSSION

Investigations and Analyses of Karnak Temple Stone andDeterioration Products

Archeological sandstone sample from the carved wall in thetemple of Karnak was taken from deteriorated parts and studied.X-ray diffraction was used to investigate the nature of this sand-stone. The result in Figure 5 revealed that quartz SiO2 is the maincomponent of the temple sandstone, with different amounts ofplagioclase (NaAlSi3O8, CaAl2Si2O8) and severe action of saltefflorescence and the salts are crystallized as halite (NaCl). Thedata in EDX pattern (Figure 6) assured the same data in XRDanalysis, which indicated the presence of Si as quartz SiO2 as themain component of archeological sandstone which may stim-ulate the growth of deteriorated fungal species. The obtainedresults agree with that reported by David and Bruceþ (1992)who stated that fungi grow best on quartz.

Analyses of the deterioration products on the Karnak tem-ple stone assured the presence of cellulose material as the maincomponent of wild bee nest (Figure 7), which determined theenzymes that could be used to clean it. FTIR pattern of dete-riorated blood stained archeological sandstone was recorded in

FIG. 6. EDX pattern of sandstone sample showing presence of Si confirmingthat quartz is the main component of Karnak temple stone.

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FIG. 7. XRD pattern identifying cellulose as the main component of thedeterioration products of wild bees’ nests on Karnak temple stone sample.

Figure 8, where it contained mainly blood proteinaceous materi-als. Proteins were characterized by the presence of amaide I andamaide II bands at 1644.02 as shown in peak No. 9. The bands(1100–1000 cm−1) referred the presence of the silicon dioxide(quartz), which was observed in the peak No. 11 at 1084.76cm−1.

FIG. 8. FTIR pattern of the deterioration products of sandstone of bat dropsidentifying the blood drops on Karnak temple stone.

FIG. 9. SEM photomicrograph showing the different sizes of quartz grains asthe main component of Karnak temple stone sample (scale bar 20 µm).

FIG. 10. SEM photomicrograph showing fungal hyphal growing and sporegermination between the sand stone grains of Karnak temple (scale bar 50 µm).

The scanning electron microscope showed that the main min-eral constituents of Karnak temple are quartz grains as maincomponent of archeological sandstone (Figure 9). The data inFigure 10 reveal the presence of some fungal hyphal growing’sbetween the sandstone grains and the spore germination of somedeteriorating fungal species. The chemical reactions with stoneminerals may be due to metabolic by-products of fungi, whichhave negative effects on monumental stone materials (Orial et al.1993). The disintegration in the quartz grains of the tested arche-ological sandstone due to fungal hyphal growth was recordedby using Polarizing microscope (PM) (Figure 11).

Investigation of the tested archeological sandstone samplesof Karnak temple reveal that the biological deteriorated productsare blood drops and cellulolytic wild bees’ nests, so fibrinolyticand cellulytic enzymes were radio-stimulated and purified forenhancing bioremediation process.

FIG. 11. Photomicrograph showing disintegration in quartz grains due tofungal hyphal growth (scale bar 20 µm).

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TABLE 1Fungal diversity from the two-isolation sites (blood drops siteand cellulolytic wild bee’s nests) on the archeological Karnak

temple stone.

Count of species

Isolation sites Blood drops Cellulolytic wildFungal species site bee’s nests

Alternaria alternata 8 4Aspergillus flavus 6 24A. niger — 25A. ochraceous — 17A. parasiticus — 15Cladosporium herbarum 5 —Curvularia eragrostidis 7 2Total count 26 87Number of species 4 6

Frequent Occurrence and Antagonistic Activity of T.reesei Against the Deteriorated Fungi Isolated fromArcheological Karnak Temple

Seven deteriorating fungal species (Alternaria alternata, As-pergillus flavus, A. niger, A. ochraceous, A. parasiticus, Cla-dosporium herbarum, and Curvularia eragrostidis) accounting113 colonies were isolated from the two isolation sites (cellu-lolytic wild bees nests and blood drops) on the archeologicalsand stone of Karnak temple in Luxor city (Tables 1 and 2).Except C. herbarum, the bulk of all isolated fungal species (sixfungal species) were recovered from the cellulolytic wild bees’nests site, on the other hand, only four fungal species (A. alter-nata, A. flavus, C. herbarum and C. eragrostidis) were isolatedfrom blood drops site on the archeological sand stone of Karnaktemple. The disappearance of some tested fungal species in onesite and appear in other may be due to the presence or absenceof the homologous enzyme system.

Our findings agree with Kumar and Kumar (1999), whostated that Alternaria sp., Aspergillus flavus, Aspergillus niger,Cladosporium and Curvularia verrugulosa cause deteriorationof archeological sandstone. Mechanisms of the microbial degra-dation of minerals in sandstone monuments was detected byEckhardt (1985). Petersen et al. (1988) recorded the distribu-tion and the effects of fungi on sandstone. The tested genusAspergillus contributed the broadest spectra where A. flavusand A. niger showed the highest occurrence, followed by amoderate occurrence of A. ochraceous, A. parasiticus and Al-ternaria alternata. The presence of blood drops and cellulosicwild bees’ nests on the tested Karnak temple may enhance thedominance of the isolated fungal species. Corey et al. (1997)isolated Alternaria, Aspergillus, Cladosporium and Curvulariaspecies on blood samples and also the most frequent airbornefungi were collected on mixed cellulose and protein sampleswere Alternaria, Aspergillus, Cladosporium and Penicilliumspecies (Green et al. 2005). On the other hand, two tested fungalspecies (C. eragrostidis and C. herbarum) were isolated in lowoccurrence.

Non-volatile substances were highly significantly respon-sible for most of the antagonistic activity of T. reesei (70–85%) against the isolated deteriorating fungal species comparedwith the inhibitory effect of the volatile substances (5–15%).The most distinguished inhibitory effect was observed with A.flavus (85%) (Table 2). These findings were additionally sup-ported by the study of Sarath et al. (1989) who found that thevolatile antifungals of Trichoderma such as 6-pentyl-2-pyronewere much less effective against fungi. Also Doi and Mori(1994), demonstrated that volatiles from Trichoderma specieswere able to arrest the growth of different fungal species. Veyet al. (2001) stated that Trichoderma strains produce volatileand non-volatile toxic metabolites, such as harzianic acid,alamethicins, and tricholin, that hinder the growth of othermicroorganisms.

These compounds may be responsible for the inhibition ofthe deteriorating isolated fungal species (Gupta et al. 1995). The

TABLE 2Survey of species, frequency of occurrence and antagonistic activity of T . reesei against the tested fungal species isolated from

archeological Karnak temple. High (H) 20-30, Moderate (M) 10-20, Low (L) 0-10. LSD = 11.2

Antagonistic activity of T. reesei (Inhibition %)

Fungal species Count of species Frequency of occurrence Non-volatile substances volatile substances

Alternaria alternata 12 M 70 8Aspergillus flavus 30 H 85 10A. niger 25 H 83 7A. ochraceous 17 M 80 12A. parasiticus 15 M 80 15Cladosporium herbarum 5 L 75 5Curvularia eragrostidis 9 L 75 6Total count 113Number of species 7

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62 N. S. I. GEWEELY AND H. A. M. AFIFI

antagonistic activity of T. reesei has been shown to be efficientagainst Cladosporium and Alternaria (Barbosa et al. 2001). Dif-ferent mechanisms have been suggested that, the biocontrol ac-tivity of Trichoderma was referred to competition for space andnutrients, secretion of enzymes, production of inhibitory com-pounds, high reproductive capacity and strong aggressivenessagainst other microbes, where Trichoderma isolates displayedvarious extracellular enzymatic activities including cellulolyticenzymes to fight off other fungal competitors, so Trichodermaenzymes can be exploited as potential antimicrobial candidatesagainst other microbes (Gachomo and Kotchoni 2008).

Comparative Stimulative Effect by the Low IntensityRadiation on Cellulolytic and Fibrinolytic Enzymes

In the absence of photosensitizers, the laser irradiation causeda gradual significant increase in cellulolytic and fibrinolyticenzymes activities reaching the maximum values (50.3 and44.5 U/ml) after 5 and 7.5 min, respectively compared with thestimulative effect of UV and gamma rays (40.0, 31.5 and 39.4,35.0 U/ml) after 40 and 10 min. exposure time, respectively(Table 3). The observed results suggest that the radiation maystimulate the gene responsible for enzyme production. Kiharaet al. (2004) showed that genes encoding the enzymes synthe-sis are specifically enhanced by radiation in a dose-dependentmanner.

Our results substantiate with those formerly reported byVladimirov et al. (2004) who stated that the laser radiation canpenetrate the fungal cells where it accelerates their division andprotein synthesis. Radiation with the He–Ne laser for 10 minmay increase the mitotic index of the cells on the third and fourthday after irradiation (Gamaeva et al. 1983). Wang et al. (2007)found that after mutagenesis treatments by UV, and γ -radiation,a high fibrinolytic enzyme produced.Kemar et al. (2003) foundpoint mutations due to ultraviolet radiation, which apparently

caused the mutant to evolve with enhanced enzyme activity thatdegraded the precursor and accumulated enzymes. Santiago etal. (2006) stated that mutant strains from A. niger were producedby UV radiation to increase their cellulolytic activity production.

Using photosensitizers with laser irradiation on the testedfungal species caused a significant inhibition in both enzymeactivities, where the data indicate that HP was the most effec-tive photosensitizing agent in reducing enzyme activities. Thismay be due to the high light absorbance of HP at the wave-length emitted by the light source as compared with TBO or CV(Millson et al. 1996). Our finding agreed with that reported byBhatti et al. (1998), who suggested that during the lethal photo-sensitization, singlet oxygen, which is the main reactive speciesproduced by the above photoexcited photosensitizers, is knownto efficiently react with highly susceptible amino acid residuessuch as cysteine, histidine, methionine, tryptophan and tyrosinegenerating protein oxidation products including hydroperoxidesthat cause growth inhibition. The tested higher laser doses inhibitthe growth of microorganisms, particularly when the radiationis applied in the presence of a photosensitizer. Blank and Corri-gan (1995) stated that the lowest dose of radiation enhanced theproduction of enzymes, while the higher doses were inhibitoryto growth.

Comparative Purification Schemes, Molecular Weightsand Characterization of Cellulolytic and FibrinolyticEnzymes

It is clear from the previous experiment that a 5- and 7.5-minexposure time of non-photosensitizer laser irradiated T. reeseiand F. oxysporum gave the highest significant values of thecellulolytic and fibrinolytic enzymes, respectively. The highestprecipitation of both enzymes was obtained by 80% ammoniumsulfate. Dialyses of the precipitated products raised the specificactivities to 1.9, 1.8 and 2.2, 1.9 folds over the crude extract

TABLE 3Comparative stimulative effect by low intensity UV, gamma and laser radiation on T. reesei cellulolytic (C) and F. oxysporum

fibrinolytic (F) enzymes at different exposure times

Laser radiation

PhotosensitizerUV Gamma No Crystal Hematoporhpyrin Toluidine

radiation radiation photosensitizer (CV) (HP) (TBO)Exposure Exposuretime (min) C F C F time (min) C F C F C F C F

0 (Control) 30.0 28.5 30.0 28.5 0 (Control) 30.0 28.5 28.2 25.0 22.5 20.1 25.1 23.05 32.1 29.5 33.1 30.6 3 42.7 35.8 25.5 23.2 17.5 15.7 22.3 20.510 33.5 30.0 39.4 35.0 5 50.3 40.2 22.2 18.5 11.2 10.2 18.5 17.020 35.0 31.0 25.6 31.2 7.5 38.0 44.5 15.0 11.8 9.1 7.4 14.1 12.140 40.0 31.5 18.0 25.0 10 20.1 25.0 8.6 9.3 7.5 5.2 10.5 8.660 22.0 18 8.0 12.0 20 11.5 9.2 3.2 4.5 2.7 3.1 5.4 3.8LSD at 0.05 2.0 0.62 3.4 4.0 5.1 3.3 2.4 1.6 3.0 1.7 3.9 2.5

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TABLE 4Comparative purification scheme of non-irradiated cellulolytic (NIC), laser irradiated cellulolytic (IC), non-irradiated fibrinolytic

(NIF) and laser irradiated fibrinolytic (IF) enzymes.

Specific activityTotal protein (mg) Total activity (U) (U/mg protein) Purification fold Recovery (%)

Purification steps NIC IC NIF IF NIC IC NIF IF NIC IC NIF IF NIC IC NIF IF NIC IC NIF IF

Crude extract 3.58 8.1 38.5 40.5 35.7 82 251.1 290.0 9.9 10.1 6.5 7.1 1.0 1.0 1.0 1.0 100 100 100 100(NH4)2SO4 2.31 4.0 22.7 24.0 30.2 77.0 228.5 272.0 13.0 19.2 10.0 11.3 1.3 1.9 1.5 1.6 84.5 93.9 90.9 93.7Dialyses 1.4 3.2 10.1 15.5 28.8 73.0 119.5 211.6 19.8 22.8 11.8 13.6 1.9 2.2 1.8 1.9 80.6 82.4 47.5 72.9Sephadex G-200 0.7 1.8 1.3 3.1 25.0 68.4 60.9 170.4 35.2 38.0 46.1 54.9 3.5 3.7 7.0 7.7 70.0 77.2 24.2 58.7Sephadex G-100 0.4 1.0 0.7 2.0 23.3 60.0 40.3 130.3 54.2 60.0 53.0 65.1 5.4 5.9 8.1 9.1 65.3 67.7 16.0 44.9DEAE-Sephadex 0.3 0.6 0.3 1.2 21.5 53.3 26.5 101.0 71.6 88.8 71.6 84.1 7.1 8.7 10.9 11.8 60.2 65.5 10.5 34.8

FIG. 12. Typical elution profile for the behavior of cellulolytic enzyme recovered from non-laser irradiated (A, C) on Sephadex G-100 and DEAE-Sephadex,respectively and laser irradiated (B, D) T. reesei on Sephadex G-100 and DEAE-Sephadex, respectively.

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64 N. S. I. GEWEELY AND H. A. M. AFIFI

FIG. 13. Typical elution profile for the behavior of fibrinolytic enzyme recovered from non-laser irradiated (A, C) on Sephadex G-100 and DEAE-Sephadex,respectively and laser irradiated (B, D) F. oxysporum on Sephadex G-100 and DEAE-Sephadex, respectively.

FIG. 14. Estimation of the molecular weights of cellulolytic and fibrinolytic enzymes recovered from the non-irradiated and laser irradiated T. reesei and F.oxysporum, respectively by SDS-PAGE profile. Lane 1: standard marker proteins (Maltose-binding proteins: 42.7 kDa, Carbonic anhydrase: 31 kDa, trypsinogen:24 kDa and Lysozyme: 14.4 kDa).

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MICROBIAL ENZYMES AND BIOREMEDIATION IN EGYPTIAN ARCHEOLOGY 65

FIG. 15. Effect of temperature and pH of the reaction mixture on the activity of the purified laser irradiated and non-irradiated T. reesei cellulolytic and F.oxysporum fibrinolytic enzymes, LSD = 13.2, 10.1, respectively.

of non-irradiated and irradiated cellulolytic and fibrinolytic en-zymes, respectively. This increase in activity is presumably dueto the removal of low molecular weight inhibitors, releasedfrom the growth substrates. The elution profile shows that ir-radiated cellulolytic and fibrinolytic enzymes were eluted fromSephadex G-200, followed by Sephadex G-100 column with aspecific activity of 60.0 and 65.1 U/mg, which represents a 5.9-and 9.1-fold purification over the crude extract with the recoveryof 67.7 and 44.9%, respectively (Figures 12 and 13, A, B, Table4). Singh et al. (1995) stated that the radiation mutagenesis ofFusarium oxysporum enhances the activity of enzymes by morethan 3-fold.

The lyophilized fraction recovered from Sephadex G-100column was applied to DEAE-Sephadex column. From theelution profile in Figures 12 and 13 (C, D) and Table 4, itcan be seen that seven active fractions from 21 to 27 onDEAE-Sephadex column were pooled from irradiated T.reesei cellulase and the maximum activity was established infraction 23. The specific activity raised to 88.8 U/mg, whichrepresents a 8.7-fold purification over the crude extract with65.5% recovery compared with the non-irradiated control.Irradiated F. oxysporum fibrinolytic enzyme was eluted from theDEAE-Sephadex column in one peak of activity accompaniedwith one peak of protein in the fractions (10–14). The specificactivity in this peak was raised to 84.1 U/mg, which representan 11.8-fold increase over the crude enzyme, and the recoverywas 34.8% compared with the lower activities in non-irradiatedcontrol. This indicates that the laser radiation may inducemutation of the test fungus, leading to an enhancement in thecharacters and activity of the produced enzyme.

Our finding agrees with that recorded by Kapelev (1989),who found that the exposure to 2 mW of He–Ne laser radiation at632.8 nm caused a 22–29% increase in enzyme activity. Gadgilet al. (1995) found enhanced cellulase production of 1.5 to1.75-fold by a mutant of Trichoderma reesei by using radiation.Gherbawy (1999) recorded that irradiation caused the enzymatichydrolysis to increase by more than 3-fold.

The estimated molecular weights of cellulolytic and fibri-nolytic enzymes derived from non-irradiated and irradiated T.

reesei and F. oxysporum were (31, 27 kDa) and (37.7, 14.4 kDa),respectively as shown in Figure 14. The low molecular weightof the enzyme recovered from the irradiated fungus may be suit-able for high molar activities toward the substrate or might beexplained by the higher specific adsorption of enzyme on thesubstrate (Du et al. 1997).

The optimum pH and temperature for the production of thetested laser-irradiated cellulolytic and fibrinolytic enzymes were(8, 9 and 50◦C, 45◦C), respectively (Figure 15). The optimumactivity of the purified fibrinolytic enzyme was reached at 40◦Cand pH 8.2 (Ali and Ibrahim 2008). Peng et al. (2003) foundthat the optimal pH and temperature were 9.0 and 48◦C forthe fibrinolytic enzyme. Sun et al. (2006) obtained best fibri-nolytic enzymes at pH (7.5–8.3). Ali et al. (1991) reported thatmaximum yield of cellulase was at pH 6.0 and 40◦C. Maxi-mum cellulase was obtained at pH 7, 40◦C (Immanuel et al.2007). It is well known that pH affects the availability of certainmetabolic ions and permeability of fungal cell membranes andalso temperature is a cardinal factor affecting the amount andrate of growth of an organism (Garg et al. 1985).

Increasing temperature has the general effect of increasingenzyme activity, but the enzyme begins to suffer thermal inac-tivation at higher temperatures (Moore-Landecker et al. 1990).Fibrinolytic enzyme from Fusarium sp. was purified with asingle protein molecular weight of 28 kDa, the optimum tem-perature and pH value were 45◦C and 8.5, respectively (Wu etal. 2009).

CONCLUSIONThe obtained results showed that the main component of

sandstone, which was used in construction of archeologicalKarnak temple in Egypt, is composed of quartz grains, whichsimulate material for deteriorated fungal growth. Serious bio-logical deterioration products (fungal colonization, wild bees’nests and blood drops) are present in the sandstone’s sur-faces. Mechanical and chemical cleaning processes producesome deterioration aspects, such as erosion and surface loss, sonew methods of bioremediation are necessary to remove thesedeterioration products without damaging the surface.

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66 N. S. I. GEWEELY AND H. A. M. AFIFI

The purified alkalo-thermophilic cellulolytic and fibrinolyticenzymes recovered from the low-intensity, laser-irradiated cellsof T. reesei and F. oxysporum were preferable enzymes for thebioremediation process of archeological Karnak temple stone.The most advantageous working conditions for the applicationof radiostimulated purified enzymes in preservation of arche-ological objects are high temperature and alkaline conditions(more tolerant to weather fluctuation). This study found severaluseful remedies for the deteriorating factors using biologicalsystems: the antifungal activity of Trichoderma (to remediatethe visible fungal colonies) and irradiated cellulolytic and fibri-nolytic enzymes (to remediate cellulolytic wild bees’ nests andblood drops). Further production, purification and application ofradiostimulated enzymes for conservation of archeological ob-jects were preferable instead of using chemicals and mechanicalcleaning to avoid the unfavorable side effect on the propertiesof archeological stone, public health and environment.

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