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THE USE OF MICROORGANISMS FOR THE REMOVAL OF NITRATES AND ORGANIC SUBSTANCES ON ARTISTIC STONEWORKS

RANALLl,G.

DISTAAM, Universitlll degli Studi del Molise. 86100 Campobasso (Italy).

CHIAVARINl,M., GUIDETil,V., MARSALA,F.

SYREMONT, Unitlll di Novara. 28100 Novara (Italy).

MATIEINl,M.,

Opificio Pietre Dure, Fortezza da Basso. 55100 - Firenze (Italy).

ZANARDINl,E. AND SORLINl,C.

DISTAM, Universitlll degli Studi di Milano. 20133 Milano (Italy).

1. INTRODUCTION

It is common knowledge that the chemical and microbial pollutants of the atmosphere contribute to accelerate the degradation of artistic stoneworks [1]. This particular phenomenon is expecially true for artworks exposed in the open air that are influenced directly by the environment: humidity, wind, rainfall, thermal excursions, pollutant gasses and other pollutans.

The increment of pollutants especially in the atmosphere (nitrogen, sulphur and carbon oxides etc.) due to the increase of industrial activity, urbanisation and transport in the last decades have caused a significant acceleration of the degradation processes affecting artworks, which had otherwise been well preserved over the last centuries. The research was aimed at developing an advanced system of biological removal of nitrates and organic substances present on artistic stoneworks (in situ) based on the use of microbial cultures carefully selected and grown on a microbial support.

2. MATERIALS AND METHODS

2.1.Screening of microorganisms having a high denitrifying activity The denitrifying activity of pure cultures of Pseudornonas aeruqinosa (RZ94) and Pseudomonas

stutzeri (GB94), obtained from DISTAM, Milan, was tested. Mixed cultures obtained from samples of powdered plaster from the facade of buildings where denitrifying bacteria had been previously found

were also tested. Media used for preliminary batch tests were n° 207 (ATCC, Rockerville, USA) and medium containing known quantities of nitrates, according to Pochon [2] and Normal B [3]. Incubation at 28 °C for 48-72

h, under anaerobic conditions. Denitrifying activities of pure and mixed cultures were evaluated at time zero and after 2, 4, 8, 10, 15 days, by determining residual nitrates content on the fermentative substrates, using a Beckman mod.

24 spectrometer (absorbance at 515 nm).

2.2.Use of microbial support Recent laboratory tests [4] have demonstrated that sepiolite (Tulsa, Spain) resulted to be the best

inert for the developing of microbial biofilms with a high active biomass per cm3

. This material was therefore selected as microbial support. Before direct use, the support underwent a pretreating process to eliminate or reduce interferences and to release undesirable ions and contaminant traces. The main characteristics of the sepiolite selected as microbial support are reported in Table 1.

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Table 1 - The main characteristics of the sepiolite selected as microbial support.

Particle size (meshI 30/60 Si0.2.1%_l 30.5 AJ2.0-3. 5.4 M_g_O 4.3 F~0--3. 2.2 Cao 26.3 N~O 1.0 ~o 1.2

.£.H 8.2 Humidi!Y_ (o;.;) 15.0 S..e_ecific _g_ravi!Y]g_tcm J. 2.5 Surface area ]m--.:z91 330.0 Water carrying caQ_acfil0/tl 80/90

2.3.Sepiolite colonisation

Aliquots of sepiolite were put in flasks and were submitted to two sterilisation processes at 121 °C for

20 minutes, they were then added with a sterile cultural broth (Figure 1) and inoculated with a 1-5%

suspension of selected denitrifying bacteria at a concentration of 3x1 o5 cell/ml. Flasks were

incubated under aerobic conditions for 10-14 days at 18 °C. Microbial counts, microscopic

observation, scanning electron microscopy (Figure 2) as well as determinations of microbial activity confirmed that colonisation had occurred.

2.4. Preparation of stone samples artificially enriched with nitrates Two different stone samples (brick and Lecce stone measuring 5x5x2 and 3x3x3 cm, respectively)

were artificially enriched with nitrates by immersion in a KN03 saturated solution for 48 hours.

Nitrates resulted to be reasonably well distributed over the surface of stone samples. Considering the

different porosities of the stones, the reproducibility of the method was acceptable (nitrate dispersion

was only 10-15%). The enriched samples were then used to assess the denitrifying efficiency of the

selected bacteria (P. stutzeri and P. aeruginosa) adhering to colonised sepiolite.

2.5. Tests to assess the removal of nitrates on artificially enriched stone and real samples.

Determination of nitrate content by ion chromatography (Dionex, DX 100 mod., USA; column, lonPac

AS4A; eluent, 1.8 mM Na2C03 and 1. 7 mM NaHC03, flow rate 2 mUmin; injection volume 1 O µL) in stone samples was tested; sampling of the stone was carried out according to Normal method 28/88 [5].

Artificially enriched samples were put in contact, for different periods of time, with colonised sepiolite

samples. Sepiolite samples were previously washed twice with a nitrate-free medium in order to

eliminate exhausted cultural broth. Controls (blanks) were prepared using a non-inoculated sepiolite

sample for each stone sample. Tests were carried out in duplicate. The efficiency of nitrate removal

was subsequently assessed by means of ion chromatographic analyses using standard curves.

A preliminary test was conducted in an anaerobic glove cabinet (Forma Scientific, Inc., 1029 mod.,

USA) (N2:H2:C02 85:10:5), on a real sample represented of a fragment from a statue in Vicenza

stone having an initial nitrate content of 1100 ppm. The fragment was put in contact for 30 hours with sepiolite inoculated with P. stutzeri and with non-inoculated sepiolite.

2.6. Screening of microorganisms having a high biodegradative activ ity.

Pseudomonas aeruginosa and Pseudomonas stutzeri, as well as Pseudomonas, P. putida, P. cepacia, P. fluorescens, P. testosteroni and P. acidovorans were tested to evaluate their capability of

utilising different organic substances, supplied separately, as sole carbon source. The organic

substances supplied were selected in v irtue of their common use in the making of works of art or in

restoration processes. They consisted of casein, linseed oil, egg yolks, walnut oil , beeswax and shellac.

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Biodegrability tests were conducted using flasks containing a "DR" mineral medium added separately with 1 % of organic substances. The flasks were then inoculated with the microbial cultures at a concentration of 1-5% final volume. They were incubated at 28 °C in a thermostat chamber and were submitted to agitation if in aerobiosis or left stationary if in anaerobiosis. Removal efficiency was assessed at time zero, at intervals varying from 12 hours to 4 days by photometrical measurements, determination of ATP content (Bioluminometer Lumac, mod. P 1500, The Netherlands) and microscopic observation (Axioplan Zeiss microscope).

2. 7. Preparation of stone samples artificially enriched with organic substances.

The biodegradative efficiency of the bacterial species selected in batch tests (P. stutzeri and P. aeruginosa) adhering to sepiolite was assessed by applying known quantities of colonised sepiolite on

stone samples (marble 3x3x3 cm, Lecce stone 5x5x2 cm) previously streaked with beeswax (5% w/w solution in chloroform) and casein (10% w/w solution in water at 50-60 °C). The amount of organic substances streaked was about 20-30 gtm2.

2.8. Tests to assess biodegradability on artificially enriched stone samples Tests were conducted as described in Paragraph 2.5. Beeswax biodegradability was assessed by

contact angle measurements (Lorentzen and Wettre apparatus, Sweden; according to Normal 33/89) [6] and FT-IR analyses (Nicolet, 740 mod., USA; sampling the stone according to Normal 22188 [5]), and casein by measuring nitrogen content before and after treatment (CHL-S-0, C.E. Instruments,

Italy).

3. RESULTS AND DISCUSSION 3.1 Nitrate removal efficiency

3.1.1 Batch tests The results obtained are reported in Figure 3. No significant nitrate removal was achieved with mixed cultures. Nitrate content in cultural broth after 4 days of incubation was nearly the same that at time zero. This is in agreement with th ATP content that indicated that very little microbial activity had taken place. Nitrate removal was better achieved with pure cultures of P. stutzeri. A 66% reduction was recorded after two days of incubation reaching 99% after four days. Nitrate was completely

removed by pure cultures of P. aeruginosa after only 2 days of incubation.

3.1.2 Tests on stone samples artificially enriched with nitrates The results, expressed as% of removal efficiency, are reported in Table 2. A partial nitrate removal was achieved with non-inoculated sepiolite (blanks). This could be due to the fact that nitrates are highly soluble. The removal efficiency was, however. greater with colonised sepiolite and it reached

more than 90% after only 24 hours.

3.1.3 Tests on real samples On Vicenza Stone real sample, a 20% nitrate removal was achieved with non-inoculated sepiolite

and a 88% removal with inoculated sepiolite. Unexpectedly, denitrifying bacteria were also efficient in removing sulphates present in the fragment up to 45%, while no removal was achieved with non-

inoculated sepiolite.

3.2 Biodegradability of organic substances

3.2.1 - Batch tests The results obtained for ATP content, as indicator of microbial acivity, are reported in Figure 4. Pure cultures of Pseudomonas stutzeri and P. aeruginosa showed a good capability of utilising organic substances. Growth was particularly significant with linseed oil, walnut oil, egg yolks and beeswax.

3.2.2 Tests on artificially enriched stone samples The results of samples enriched with beeswax and casein are reported in Table 3. The removal of both organic substances on marble by the two bacterial species was very good. The partial removal

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achieved by non-inoculated sepiolite (blanks) could be attributable to the low porosity of this materail

(around 1 %). Casein was better removed on Lecce stone than beeswax. The best results were obtained with P.aeruginosa that almost completely removed casein. No significant difference was registered for beeswax removal on Lecce Stone, probably due to the high porosity of this material

(around 40%). In conclusion, the results obtained on stone samples artificially enriched with nitrates and organic

substances were quite satisfactory and they confirm the validity of the proposed method.

ACKNOWLEDGMENTS This work was developed for a National Program Research on Chemical, 2nd phase, supported by "Ministero dell'Universita e Ricerca Scientifica e Tecnologica" and assigned to Syremont S.p.A. •

Milano (Italy).

REFERENCES (!] C. Sorlini., D. Falappi. , G. Ranall i. Biodeterioration preliminary tests on Serena stone treated with

resin. Ann. Microbiol. 1991 , 41 :71-79. (2] J. Pochon , P. Tardieux Techniques d'analyses microbiologie du soil. Ed. La Tourelle. St.Maude (Seine),

1962. [3] Commissione Normal 9/88, Raccomandazioni Normal B, Microflora autotrofa ed eterotrofa: tecniche di

isolamento in coltura, CNR-ICR, Rome, 1988. (4] G. Ranalli , P. Balsari , S. Merlo, C. Sorlini. Preliminary research on support matrices to utilize in anaerobic expanded-bed digester. 5th Int. Symposium on Anaerobic Digestion, Bologna (Italy), May 22-26, 1988, pp.331-

334. (5] Commissione Normal 28/88, Raccomandazioni Normal C, Composizione chimica dei materiali lapidei, CNR-

ICR, Rome, 1988. [6] Commissione Normal 33/89, Raccomandazioni Normal F, Misura dell'angolo di contatto, CNR-ICR, Rome,

1989.

Table 2 - Test on biological removal of nitrates

Matrices Test Time (h) Removal efficienc:y1%)

blank 51.0 Brick P.aerl!.IJ!.n. 7 83.0

P.stutzeri 85.7 blanc 59.1

Brick P.aerL!E}n. 12 86.9 P.stutzeri 87.1 blank 66.7

Brick P.aerl!.IJ!.n. 24 91 .7 P.stutzeri 93.7 blank 67.8

Brick P.aen.!SJ!.n. 48 93.3 P.stutzeri 93.0 blank 67.7

Brick P. aerL!E}n. 72 94.8 P.stutzeri 94.1 blank 53.8

Lecce Stone P.aerL!E}n. 12 87.7 P.stutzeri 88.8 blank 64.8

Lecce Stone P.aerL!E}n. 24 94.0 P.stutzeri 93.4 blank 70.4

Lecce Stone P. aer'!f}jn. 30 99.8 P. stutzeri 100.0

(Initial nitrates content: 3.0 % and 6.0% on brick and Lecce Stone, respectively) .

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Table 3 - Biological efficiency removal organic matters

(caseine and beeswax).

Organic matters Matrices Test

Caseine Lecce Stone Blank

Ca seine Lecce Stone P. aeruginosa

Ca seine Lecce Stone P. stutzeri

Beeswax Lecce Stone Blank

Beeswax Lecce Stone P. aeruginosa

Beeswax Lecce Stone P. stutzeri

Caseine Marble Blank

Caseine Marble P. aeruginosa

Caseine Marble P. stutzeri

Beeswax Marble Blank

Beeswax Marble P. aeruginosa

Beeswax Marble P. stutzeri

Analyses results

N: 7.7%

N: 1 %

N: 4.5%

FT-IR: -

an_g_, cont. 107°

FT-IR:+

an_g_, cont. 99.8°

FT-IR: +

ang. cont. 98. 7°

N: 2.2%

N: 0%

N: 0%

FT-IR: +

ang. cont.89.4°

FT-IR: ++

ang, cont.n .d.

FT-IR: +++

an_g_, cont. n.d.

Initial concentration of elementary N: 8.1 % in Lecce Stone, 5.6% on Marble;

Initial contact angle: 108.3° on Marble and 110.2° on Lecce Stone.

Qualitative results of FT-IR:

(-)

(+)

(++)

(+++)

= no reduction of organic substances peaks

= low reduction of organic substances peaks = high reduction of organic substances peaks = quite total disappearance of organic substances peaks

n.d. = not detectable

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Figure 1 - Flasks with sepiolite and sterile culturale broth

Figur~ 2 - Sepiolite colonised by Pseudomonas Stutzeri (SEM - 15000x)