Structural Interactions among Epilithic Cyanobacteria andHeterotrophic Microorganisms in Roman Hypogea

P. Albertano,1 C. Urzı2

1 Department of Biology, University of Rome “Tor Vergata,” via della Ricerca scientifica, 00133 Roma, Italy2 Institute of Microbiology, Faculty of Science, University of Messina, 98166 Villaggio S. Agata Messina, Italy

Received: 17 February 1999; Accepted: 19 May 1999

A B S T R A C T

Phototrophic microbial communities present in the Roman Catacombs were characterized and

different species of terrestrial epilithic cyanobacteria were found to occur as dominant organisms.

Eucapsis, Leptolyngbya, Scytonema, and Fischerella were the most frequently encountered cyanobac-

terial taxa, while a few species of green algae and the diatom Diadesmis gallica occurred in minor

amounts. Streptomyces strains, a few genera of eubacteria, and to a lesser extent fungi were always

present in the same microhabitats and contributed to the deterioration of stone surfaces. The

combined use of light and electron microscopy evidenced the structural relationships among rod-

shaped or filamentous bacteria and cyanobacterial cells, as well as the presence of polysaccharide

capsules and sheaths, and of mineral precipitates on S. julianum filaments. The significance of the

intimate association among the microorganisms was discussed in relation to the damage caused by

the growth of biological patinas on stone surfaces.

Introduction

Different species of terrestrial epilithic cyanobacteria occur

as dominant phototrophic microorganisms in Roman hypo-

gea [3, 5]. These archaeological sites are characterized by

high relative humidity (<90%), constant temperature

throughout the year, and extremely low photon fluxes. Ir-

radiance is usually provided by the lighting systems that are

used in the Catacombs and the Necropolis to allow the il-

lumination of frescoes, stuccoes and marbles of historic and

artistic value during visitors’ hours. Few species of eukary-

otic phototrophs are able to withstand the low irradiance

available in these sites, and only terrestrial species of sciaphi-

lous coccal and filamentous green algae, diatoms, and

mosses have been reported [8, 10, 11, 27].

However, bacteria and, to a lesser extent, fungi are always

present in the same microhabitats colonized by cyanobacte-

ria and microalgae [2, 50], and populations of heterotrophic

bacteria and actinobacteria have been found in other ar-

chaeological and natural hypogea [42, 46, 49]. Nevertheless,

heterotrophs can sometimes develop even in the absence of

phototrophic microbiota probably due to the organic matter

present in mortars and frescoes and to the stable microcli-

matic conditions (humidity and temperature) that charac-

terize the hypogea. In such circumstances, actinobacteriaCorrespondence to: P. Albertano; Fax: +39 06 202 3500; E-mail:

albertano@uniroma2.it

MICROBIALECOLOGY

Microb Ecol (1999) 38:244–252

DOI: 10.1007/s002489900170

© 1999 Springer-Verlag New York Inc.

produce filamentous structures that penetrate into the soft

substratum and produce propagules and spores for repro-

duction. Extensive growth of Streptomyces strains, for in-

stance, can occur as a white veil on the surface and is easily

perceptible as a characteristic smell of soil [2].

However, once the hypogean environments start to be

illuminated by any natural or artificial light source, eukary-

otic algae and cyanobacteria become predominant, them-

selves causing aesthetic, physical, and chemical damages [8,

12]. At that moment, a synergic biodeteriorative effect on

stone surfaces is possibly achieved by the concomitant

growth of phototrophic and heterotrophic populations, as

has been shown for other microbial communities in con-

fined environments [28]. Bacteria and fungi, in fact, are able

to use the organic matter produced by phototrophs to re-

lease acidic organic compounds and solubilize the minerals

of the substratum [23, 31, 32, 45].

In recent years, studies on the phototrophic microbial

communities present in the Roman Catacombs have been

undertaken in order to characterize the adaptive structural

features and the ecophysiology of the cyanobacterial species

involved [3, 7, 9, 18], but little attention has been paid to the

heterotrophic component. The first results on the taxa char-

acterizing heterotrophic microflora of the Roman Cata-

combs are, therefore, reported in this study, in which special

interest has been devoted to the understanding of organism

relationships within the polymicrobial population. Natural

samples and cultures were studied using light and electron

microscopy methods and focusing on the interactions either

among phototrophs and heterotrophs or between mineral

substratum and microorganisms.

MethodsPhysicochemical Parameters

Climatic parameters were recorded at each site. Relative humidity

(RH%) and temperature (T °C) were measured using a Hanna

Instruments Thermo-Hygrometer model H18564. Light measure-

ments were done with a LI-Cor Quantum/Radiometer LI-185B

equipped with a LI-190SB Quantum, LI-200SB pyranometer, and

LI-210SB photometric sensors for photosynthetic photon flux flu-

ence rate (µmol m−2s−1), total irradiance (W m−2), and illumi-

nance (lux), respectively.

Sampling and Cultural Analyses

Samples were collected on January 1997 among blue-green to dark

biological patinas inside the Catacombs of St. Callistus and Priscilla

in Rome (Italy) using sterile materials, and divided in aliquots. Part

was used for in situ pH measurements by placing sample fragments

on the flat surface of a portable Sensotron pH meter and for the

direct inoculation in agarized BG11 [44], BBM [20], and Zehnder

[48] culture media for the growth of phototrophs. The other ali-

quots were subsequently processed for cultural and microscopical

analyses. For qualitative determination of chemorganotrophic bac-

teria and fungi, fragments were suspended in 0.9% NaCl supple-

mented with 0.001% Tween 80 and further decimal solutions were

prepared. Then, 100 µl of each suspension/dilution were inoculated

on the agar surfaces of BRII and DRBC nutrient media for the

growth and isolation of chemorganotrophic bacteria [19] and fungi

[51], respectively.

Determination of cyanobacterial taxa was carried out according

to Komarek and Anagnostidis [29, 30] and Anagnostidis and Ko-

marek [13, 14]. Bacteria were identified at genus level following the

procedures of classical manuals [47, 52]. Fungi were classified fol-

lowing the keys of Barnett and Hunter [16] and Ellis [24, 25].

For both bacteria and fungi, a general screening for the ability

to solubilize and precipitate CaCO3 was performed, using CaCO3

glucose agar (glucose 1%, CaCO3 0.5%, agar 1,5%) and B4 me-

dium, respectively [17].

Microscopical Analyses

The development of mixed cultures of photo- and heterotrophs

was followed using a Zeiss Televal 31 inverted microscope, and

features of individual species were observed with a Zeiss Axioskop

equipped with Differential Interferential Contrast (DIC).

Light microscopy was performed either with acridine orange or

without staining using an Aristoplan Leica microscope equipped

with a 100 W mercury lamp and a set of green and blue filters for

epifluorescence. Environmental scanning electron microscopy

(ESEM) observations were carried out on untreated fresh samples

using a Philips Electroscan ESEM (Eindhoven, The Netherlands) at

an operating voltage of 15 kV, in 5.7 torr water vapor. For con-

ventional scanning electron microscopy (SEM), samples were fixed

in 2.5% (w/v) glutaraldehyde in 0.2 M phosphate buffer at pH 7.0,

postfixed in 1% OsO4, dehydrated in ethanol series, critical point

dried, gold sputtered, and observed using a Zeiss DSM 950 scan-

ning electron microscope at 10 kV. For transmission electron mi-

croscopy (TEM), samples were fixed and dehydrated as reported

above, embedded in an 812 Resin Kit (Multilab, England), thin

sectioned, and observed with a Zeiss CEM 902 transmission elec-

tron microscope at 80 kV.

Results

The climatic conditions and pH values recorded at each

sampling site are shown in Table 1. No significant dif-

ferences were evidenced between St. Callistus and Pris-

cilla Catacombs and within each sampling site. Relative hu-

midity was always more than 90%, temperature ranged be-

tween 15.6 and 18°C; either light irradiance (0.5–7.5

Microbial Communities in Roman Hypogea 245

W ? m−2) or photon flux available for photosynthesis (0.2–

2.5 µmol ? m−2?s−1) were extremely low. Values of pH mea-

sured on sample fragments were all slightly above or below

neutral, with the exception of sample CSC 13 (pH = 5.63)

and CP 7 (pH = 5.99).

Data obtained by microscopic and culture analyses on the

composition of microbial communities living at each sam-

pling site are summarized in Table 2 and shown in Figs.

1–12. No relationship between the presence of dominant

taxa of cyanobacteria, bacteria and fungi and the intrinsic

properties of the substratums was observed.

Direct light microscopy, epifluorescence, and ESEM ob-

servations of fresh samples revealed a number of rod-shaped

and filamentous bacteria closely associated to phototrophs

(Figs. 1–6). The chroococcal Eucapsis (Fig. 11), Oscillatori-

alean Leptolyngbya spp. (Figs. 1, 9, 10), and the diazotrophic

Scytonema julianum (Figs. 5, 6), S. ocellatum, and Fischerella

maior (Fig. 10) were the most abundant cyanobacterial gen-

era, while a few species of green algae, the diatom Diades-

Table 2. List of phototrophic and chemoorganotrophic organisms colonizing different substrata in St. Callistus’ and Priscilla’s Catacombs

in Romea

Sample Substrate PhototrophsChemoorganotrophic

bacteria Fungi

CSC 5 Tufa C: Eucapsis, Leptolyngbya,Plectonema

E: Diadesmis, mosses,Pseudopleuroccoccus

Bacillus, Micrococcus Aspergillus, Cladosporiumsphaerospermum,Sporotrichum

CSC 6b Gypsum C. Chroococcales, LeptolyngbyaE: Pseudopleurococcus

Streptomyces, Bacillus A. flavus, C. sphaerospermum

CSC 7 Brick C: Eucapsis, Fischerella,Leptolyngbya, Scytonema

E: Coccal greens, Diadesmis

Actinobacteria, Bacillus,Streptomyces

Aspergillus sp., A. flavus, C.sphaerospermum, Penicillium

CSC 9c Plaster C: Eucapsis Fischerella,Leptolyngbya, Scytonema

E: Diadesmis, mosses

Streptomyces Cladosporium. Sporotrichum

CSC 12b Brick C: Fischerella, LeptolyngbyaE: Coccal greens

Streptomyces Aspergillus, C. sphaerospermum

CSC 13 Plaster C: Fischerella, Leptolyngbya Streptomyces Aspergillus, C. sphaerospermum,Sporotrichum

CP 5b Frescoes E: Mosses Bacillus, Micrococcus,Streptomyces

C. sphaerospermum

CP 6 Plaster C: Leptolyngbya, Scytonema,E: Diadesmis, mosses

Streptomyces A. flavus

CP 7 Plaster E: Mosses Bacillus A. flavus, TrichosporonCP 8a Tufa C: Eucapsis, Leptolyngbya,

ScytonemaE: Coccal greens, Diadesmis,

mosses

Actinobacteria, Bacillus,Streptomyces

Doratomyces (occ.), Penicillium

CP8b Brick C: Eucapsis, Leptolyngbya Streptomyces PenicilliumCP 9 Tufa C: Leptolyngbya, Scytonema,

E: Diadesmis, mossesStreptomyces A. flavus, A. niger, Glycomax

murorum, Paecylomyces

a Dominant genera are evidenced in bold. Identification of phototrophic taxa was based on observation either of natural samples or mixed and monospecificcultures (C = cyanobacteria, E = eukaryotes.).

Table 1. Physicochemical data of sampling sites in St. Callistus’

(CSC) and Priscilla’s Catacombs (CP) in Rome (Italy)

SiteRH(%)

T(°C)

µmolm−2s−1 W m2 lux pH

CSC5 95.0 16.9 1.7 7.5 240 6.29CSC6b 96.5 16.8 0.9 1.9 50 6.67CSC7 90.6 17.1 0.8 1.6 70 6.28CSC9c 94.0 17.2 0.6 1.4 35 8.15a

CSC12b 97.9 16.8 2.5 5.0 140 6.85CSC13 99.9 18.0 — — — 5.63CP5 99.9 16.9 0.5 1.4 27 7.59CP6 99.9 15.6 0.2 0.6 12 7.57CP7 91.6 16.2 0.4 1.3 28 5.99CP8a 99.9 16.5 0.6 1.4 31 7.34CP8b 99.9 16.6 0.2 0.5 9 7.28CP9 99.9 17.8 0.5 1.3 24 7.58

a Laboratory measurement with addtion of 10 µl H2O

246 P. Albertano, C. Urzı

mis gallica (Fig. 12) and moss protonemata (Fig. 9) occurred

in minor amounts. Most of the prokaryotic and eukaryotic

phototrophs showed a wide distribution over the range of

irradiances recorded. A weak trend in the distribution of

diazotrophic cyanobacteria could be evidenced that might

indicate a preference of F. maior for irradiances higher than

0.6 µmol m−2s−1, and below 0.8 µmol m−2s−1 of Scytonema

species.

Cultural analyses for heterotrophic microorganisms have

shown that Streptomyces strains (Fig. 2) were present in most

of the samples as major colonizers. Other bacteria, such as

Bacillus and Micrococcus species, and sometimes fungi oc-

curred as accompanying microflora, especially in locations

close to the entry of the Catacombs. In few cases, and only

in St. Callistus’s Catacomb, a white filamentous fungal strain

belonging to Sporotrichum sp. was isolated as main coloniz-

ers.

When the type of substratum, the phototrophic species

found at the different sampling sites, or the climate of the

various microhabitats were considered, no apparent specific

relationship of the accompanying heterotrophic microbiota

could be detected on the basis of cell morphology in fresh

samples and identification procedures in cultures.

Accordingly, the lack of significant differences in the spe-

cies composition of the communities under investigation did

not allow a conclusion as to the differences detected in pH

values. In addition to the possible excretion of acidic com-

pounds, fluctuation in H+ concentration during light/dark

cycles should be expected in each microhabitat because of

the changes in CO2 emission and consumption by microbial

respiration and photosynthesis. Further use of pH micro-

electrodes is, therefore, needed to better estimate variation in

this parameter.

Figs. 1, 2. Epifluorescence images of fresh samples after staining

with acridine orange. (1) Sample CSC7 from St. Callistus’ Cata-

comb in which microorganisms appeared intimately associated and

bacteria surround a cyanobacterial filament (arrow). (2) Hyphae

and spores of one strain of Streptomyces sp.

Figs. 3, 4. ESEM images of hydrated samples from Priscilla’s

Catacomb CP6. Branching filamentous cyanobacteria (arrow) ap-

peared surrounded by “clouds” of thin bacterial filaments. Bars =

10 µm.

Microbial Communities in Roman Hypogea 247

Electron microscopy of natural samples evidenced the

ultrastructural relationships between microorganisms and

substratum (Figs. 6–10). A common feature among photo-

trophic prokaryotes was the production of polysaccharide

capsules and sheaths. What is noteworthy is that one of the

Scytonema species, S. julianum, always showed heavy pre-

cipitation of crystals along the sheath enclosing the tri-

chomes (Figs. 5, 6). Specific tests carried out in laboratory

conditions showed that also almost all the Streptomyces iso-

lates were able to precipitate calcium carbonate crystals

around the colonies (Fig. 13). Newly formed crystals were

usually single, did not form a layer, and tended to stick

around the filaments or on the top of colonies (Fig. 14).

However, the shape of these crystals was different from those

present on the sheath of S. julianum. Neither Streptomyces

nor fungi were able to solubilize calcium carbonate, with the

exception of two fungal strains, Doratomyces sp. and Paecy-

lomyces sp., found only occasionally in Priscilla’s Catacomb.

Discussion

These results report for the first time on the structural re-

lationship among phototrophic and heterotrophic microor-

ganisms in hypogean environments, and more generally in

archaeological sites. The combined use of light and electron

microscopy and culture methods has proven the recurrent

intimate association between actinobacteria, mainly Strepto-

myces species, and filamentous cyanobacteria in Roman

Catacombs.

Numerous studies have dealt with the intimate relation-

Figs. 5, 6. ESEM images of hydrated samples of S. julianum from

Priscilla’s Catacomb CP6. (5) Filaments showing the precipitation

of crystals on the sheaths (arrow). (6) One filamentous bacterium

(arrow) appears tightly adhering to the cyanobacterium. Bars =

10 µm.

Figs. 7, 8. SEM images of samples from St. Callistus’ Catacomb

CSC7. (7) Thin cyanobacterial filaments interspersed among min-

eral particles detached from the stone surface. (8) The appearance

of the mucilagineous matrix (m) in which coccal (on the left) and

filamentous microorganisms (on the right) are embedded. Bars =

10 µm.

248 P. Albertano, C. Urzı

ships between phototrophic and heterotrophic microorgan-

isms because of their frequent occurrence in natural envi-

ronments and laboratory conditions. However, most of the

interactions between cyanobacteria and other prokaryotes

have been investigated in aquatic environments [38, 39, 40].

One of the pioneer papers on this topic was published by

Engelmann [26], who first evidenced that aerotactic bacteria

were attracted by the oxygen released photosynthetically by

chloroplasts. Later on, first Paerl [37] and then Lupton and

Marshall [33] reported on the preferred localization of bac-

teria on cyanobacterial heterocysts. In both aquatic and ter-

restrial cyanobacterial-dominated mats and biofilms, the

consortium with bacteria offers a successful strategy to over-

come the prokaryotic structural/functional limitations pro-

viding opportunities to fulfill specific metabolic or growth

needs through extracellular means as opposed to cytoplas-

mic compartmentalization [39]. Metabolic activities such as

photosynthetic oxygen evolution and respiratory consump-

tion; production of organic carbon compounds; bacterial

excretion of growth factors and vitamins; nitrogen transfor-

mation steps such as N2-fixation, nitrification, denitrifica-

Figs. 9, 10. TEM images of samples from St. Callistus’ Catacomb.

(9) Thin Leptolyngbya sp. filaments (arrow) were visible on the

outermost layer of the microbial community section closely adher-

ing to a mineral particle (p). The inner layers were occupied by

moss protonemata cells (M), bacteria, and mucilage (m). (10)

Cross-section of one Scytonema julianum filament (S) showing

crystal precipitates on the sheath surface (arrow), and the longitu-

dinal section of one Leptolyngbya sp. (L) with bacterial cells within

the common exopolymeric matrix. Bars = 1 µm.

Figs. 11, 12. TEM images of samples from Priscilla’s Catacomb.

(11) Chroococcal cyanobacteria (C) appeared surrounded by thick

layered capsules sticking to other fibrillar material of bacterial ori-

gin (b). (12) Empty frustules of the small diatom Diadesmis gallica

appeared heavily colonized by bacteria (arrow). Bars = 1 µm.

Microbial Communities in Roman Hypogea 249

tion, and ammonification; and mobilisation of mineral con-

stituents can create microenvironments suitable for both

auto- and heterotrophic microorganisms [38]. In hypogea,

the physical contact among microorganisms was favored by

the presence of the polysaccharide fibrils forming capsules

and sheaths in both cyanobacteria and epiphytic bacteria. In

these peculiar habitats, as in almost all terrestrial environ-

ments, in contrast to aquatic systems, gram-positive bacteria

appear to predominate as cyanobacterial epiphytes [38].

However, no direct contact between them was observed.

This fact confirms the exceptional finding of a direct cell-

wall association that has previously been reported between

Leptolyngbya sp. and one filamentous bacteria, possibly

Streptomyces sp., from a similar hypogean environment [12].

In addition, extracellular polymeric substances produced by

cyanobacteria can mediate metabolite exchange and serve as

an excellent growth substratum for a variety of heterotrophic

bacteria [39].

The growth of mixed communities in form of blue-green,

brown, gray or white biological patinas on stone surfaces is

thus the consequence of the favorable climatic conditions. In

Roman hypogea, such as the Roman Catacombs studied in

our research, the whole habitat fits well for both photo-

trophic and heterotrophic populations [2]. The cyanobacte-

rial and microalgal species found possess adaptive characters

that allow them to thrive on the extreme low photon flux

densities and the spectral quality provided by the artificial

lighting systems present in such environment [3, 4, 5]. It is

worthy of mention that most of the taxa of phototrophs

found in these Roman Catacombs have not yet been re-

ported for similar environments or other archaeological and

historical sites [34–36, 41]. In particular, Eucapsis sp., one

red Leptolyngbya sp., Scytonema ocellatum and Fischerella

maior (Albertano, unpublished), and Diadesmis gallica seem

to be characteristic components of the microbial community

of hypogea in Rome [3, 5, 11].

Finally, mineral precipitation in the form of crystals has

been observed in some cyanobacterial species, e.g., S. julia-

num, and most of the Streptomyces isolates. Scytonema ju-

lianum is a typical inhabitant of natural and archaeological

environments characterized by high humidity and low irra-

diance where erect calcified filaments usually develop on

limestone surfaces [1, 15, 43]. The precipitation of calcium

carbonate on the surface of the polysaccharide sheath of

cyanobacteria in the form of calcite crystals is known to be

promoted by live bacteria [21]. In our investigation, Strep-

tomyces was always found in association to S. julianum, and

could, therefore, be a candidate for such biologically induced

calcification. Undoubtedly, carbonate precipitation and/or

solubilization are major factors in the biotransformation of

calcareous substrata.

Conclusions

The epilithic microbial community established in Roman

Catacombs can be associated to different biodeteriorative

effects [22]. Aesthetic damage is caused by the development

of colored biological patinas. A marked softening of the

substratum and the progressive deepening of the biological

growth in the layers beneath the surface is due to the mo-

bilization of elements and to enhanced water retention by

polysaccharide sheaths. A white crust formation is associated

to the surface deposition of newly formed crystals and results

in a stromatolithic layering on the stone surface.

Figs. 13, 14. Precipitation of crystals by the Streptomyces strain BC

598 as seen with the dissecting microscope on medium B4. (13)

Note the front of precipitation between two colonies (arrow). (14)

Big crystal (c) precipitation occurs on the top of colonies and close

surroundings.

250 P. Albertano, C. Urzı

Acknowledgments

This work was supported by the financial contribution of

the National Research Council of Italy, C.N.R.—Progetto

Finalizzato Beni Culturali, grants n. 96.01066.PF36,

97.00578.PF36 and n. 97.00720.PF36, and EC contract n.

ENV4-CT98-0707. We gratefully thank Professor F. Bisconti

and Dr. R. Giuliani of the “Pontificia Commissione di Ar-

cheologia Sacra” in Rome for the fruitful collaboration and

the permission to sample in the Catacombs. Palma Mattioli,

University of Rome “Tor Vergata,” for her skilful support in

image digitalizing, and William Fenton, University of Mes-

sina, for his help to improve the English text, are gratefully

acknowledged.

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