International Biodeterioration & Biodegradation 169 (2022) 105374

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International Biodeterioration & Biodegradation

journal homepage: www.elsevier.com/locate/ibiod

Metabolomic analysis of photosynthetic biofilms on building façades in

temperate climate zones
Michał Komar a, *, Paulina Nowicka-Krawczyk b, Tomasz Ruman c, Joanna Nizioł c, Piotr Konca d,
Beata Gutarowska a
a
Department of Environmental Biotechnology, Faculty of Biotechnology and Food Sciences, Lodz University of Technology, Wólczańska 171/173, Łódź, 90-924, Poland
b
Department of Algology and Mycology, Faculty of Biology and Environmental Protection, University of Łódź, Banacha12/16, Łódź, 90-237, Poland
c
Faculty of Chemistry, Rzeszów Univeristy of Technology, al. Powstańców Warszawy 6, Rzeszów, 35-959, Poland
d
Department of Building Physics and Building Materials, Lodz University of Technology, Al.Politechniki 6, Łódź, 90-924, Poland

A R T I C L E I N F O A B S T R A C T

Keywords: The aim of the study was to incorporate a multidimensional approach to the biodeteriorative influence of aer­
Algae ophytic algal biofilms colonising building materials, such as bricks and plasters in temperature climate zones.
Biofilms Stichococcus sp., Klebsormidium sp., Chlorococcum infusionum, Chlorella vulgaris and Pseudochlorella signiensis were
Biodeterioration
detected in green biofilms covering internal and external walls of buildings in Poland. Their growth led to
Brick
Plaster
changes in the material’s colour in the range of ΔE = 10.82–37.67, as well high water retention and absorptivity.
Laser desorption/ionisation time-of-flight mass The moisture content of analysed materials ranged from 9.00 to 14.59%. Further, the direct influence of algal
spectrometry growth on the mechanical properties of the analysed materials was not found. For metabolome analysis laser
desorption/ionisation time-of-flight mass spectrometry with silver-109 and gold nanoparticles was used. De­
rivatives of sulfuric compounds and organic acids were detected but this was not the case for metabolites with
well-documented biodeteriorative potential. 109AgNPET LDI MS allowed for the detection of 43 different
metabolic pathways, using AuNPET LDI MS 25 pathways. In total, 110 metabolites were found. Although the
spectrum of metabolites detected using 109AgNPET LDI MS was broader, AuNPET LDI MS allowed for the
detection of pathways that were not found by 109AgNPET LDI MS. The combination of both methods allowed for
the widest range of results.

1. Introduction microorganisms are aerophytic algae (= terrestrial or airborne algae)

The process of material deterioration occurring in response to the life
processes of organisms, i.e. biodeterioration, has been the subject of
many previous studies (Hueck, 1965). Of all the factors influencing
biodeterioration, heterotrophic microorganisms tend to pose the great­
est problem due to their ability to decompose colonised substrates
chemically (Sterflinger and Piñar, 2013). In the terrestrial environment,
especially in the tropical climate zone, characterised by relatively high
temperatures and humidity, cyanobacteria, a form of photosynthetic
bacteria, are also considered to play a significant role in biodeteriora­
tion, as they can form microfractures in substrates by transforming
mineral compounds and growing inside the gaps (Crispim and Gaylarde,
2005). These, however, are not the only photosynthesising microor­
ganisms (phototrophs) that colonise various types of materials in
terrestrial ecosystems; in temperate climate zones, the dominant
(Samad and Adhikary, 2008). These microscopic, eukaryotic photo­
synthetic microorganisms have adapted to living in a terrestrial envi­
ronment and being spread by wind. Moreover, their dependence on
water is limited only to the presence of water vapour in the air. They
possess a specific metabolism that allows them to survive and reproduce
successfully in conditions deficient of dissolved mineral compounds,
with high intensity of light and rapid desiccation (Sharma, 2015). With
regard to the ecological processes occurring in ecosystems, aerophytic
algae are pioneering organisms, inhabiting places yet inaccessible to
heterotrophs; their life processes, and above all the death of their cells,
transform habitats, providing organic matter essential for the develop­
ment of bacteria and fungi; thus, their presence represents an essential
link in the succession of heterotrophic microflora (May et al., 2011;
Tomaselli et al., 2000). Such ecology characterises some species of green
algae (Chlorophyta), streptophytes (Streptophyta) and ochrophytes

* Corresponding author.
E-mail address: michal.komar@dokt.p.lodz.pl (M. Komar).

https://doi.org/10.1016/j.ibiod.2022.105374
Received 17 June 2021; Received in revised form 27 December 2021; Accepted 12 January 2022
Available online 29 January 2022
0964-8305/© 2022 Elsevier Ltd. All rights reserved.
M. Komar et al.
(Ochrophyta), among which few diatoms (Bacillariophyceae) and xan­
thophycean algae (Xanthophyceae) inhabit the terrestrial environment
(Nowicka-Krawczyk et al., 2014).
The cells of algae are carried by the wind to the surfaces of various
types of substrates. Should the environmental conditions be suitable,
successful colonisation by an alga is followed by a process of intensive
cell division, leading to the formation of a photosynthetic biofilm. The
first symptoms of colonisation visible to “the naked eye” can be seen
after only three weeks, while the time necessary for the formation of a
“green biofilm” depends on the species and the intensity of changes in
environmental conditions: the process has been estimated to last be­
tween 40 and 60 days (Leadbeater and Callow, 1992). In a temperate
climate, terrestrial photosynthetic biofilms are widespread. They occur
on natural substrates, such as tree bark, wood and rock formations, and
man-made ones such as brick, plaster, concrete and plastic (Samad and
Adhikary, 2008). Following colonisation, biofilms of terrestrial algae
negatively alter the appearance of buildings and historic objects (Stupar
et al., 2012). In addition, although it has been proposed that aerophytic
algae may exert a destructive influence on the technical state of building
materials (Samad and Adhikary, 2008; Grbić et al., 2010), this has not
yet been confirmed by comprehensive studies, and it is still widely
believed that their negative impact on materials is purely aesthetic
(Ortega-Calvo et al., 1995; Gulotta et al., 2018), with some reporting on
their ability to change the colour of the substrate (Cutler et al., 2013).
The foundation to understanding all phenomena, especially those
with negative effects on humans, is the correct diagnosis of their cause.
Remedial action against biodeterioration is typically undertaken when
its symptoms are noticeable to the naked eye. The visual signs of the
negative impact of microorganisms on the structural integrity of sub­
strates, however, tend to include the presence of various types of
microfractures on the surface, or cracks inside the substrates resulting
from the ingrowth of microorganisms (Brehm et al., 2005; Gorbushina,
2007; Hauer, 2010). These phenomena lessen the mechanical durability
of materials; therefore, this type of microbial activity plays a significant
role in biodeterioration, because it not only causes the mechanical
erosion of materials, but also accelerates water erosion by increasing the
water content in the interior.
Heterotrophic microorganisms produce various types of organic and
inorganic compounds, some of which may have a corrosive effect on
building materials, and release them into substrates. As this type of
activity poses a great threat to the technical state of materials, it is
regarded as a major factor affecting biodeterioration (Gaylarde et al.,
2003). Microbial metabolism, which can affect and alter the chemical
composition of building materials can be evaluated using metabolomics
analyses, and this methodology has already been used to assess the
deteriorative activity of microorganisms growing on historic buildings
(Gutarowska et al., 2015).
This study presents an investigation in the biodeterioration abilities
of aerophytic algae forming green biofilms in temperate climate zones
on brick and plaster, which are the most commonly used façade mate­
rials. The scope of research included the qualitative and quantitative
analysis of algae in biofilms, the assessment of colour and durability of
substrates due to green biofilm development, and the investigation of
metabolites on building materials colonised by photosynthetic biofilms.
2. Materials and methods
2.1. Sampling
For the purpose of this study, bricks and plaster samples were chosen
as the typical materials in Central European architecture. Fragments of
building materials covered in algal biofilm were selected. As a reference,
samples (control) of plasters and bricks without biofilm coverage were
used.
For the purposes of algological identification, biofilm covering the
walls of investigated sites was collected by scraping with a soft sterile
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International Biodeterioration & Biodegradation 169 (2022) 105374
brush, fixed with liquid Bold’s Basal Medium, prepared from scratch in
accordance with (Andersen, 2005). Material was gathered from 12 cm2
of surface area and transferred to a BBM agar slant (1,5% Agar, Difco®).
For colour change measurements, elemental composition tests, pH
measurements surface fragments of bricks and plasters covered in bio­
film and without biofilm coverage (control) were acquired using a sterile
scalpel and chisel, stored in sterile plastic containers, and transferred to
the laboratory. In order to measure the moisture and compressive
strength of the material, full-sized bricks were taken from sampling sites.
Metabolome analysis profiling was performed on flat surface fragments
of bricks and plasters collected with the sterile scalpel and placed in
sterile plastic containers.
Two nineteenth-century buildings were selected as sampling sites.
Sampling site 1 entailed the exterior walls of a deteriorating building
located in Lodz, Central Poland (51o 49′ 14,118′′ N, 19o 24′ 20,959′′ E).
Sampling site 2 involved the interior walls of a building located in
Gdynia, Northern Poland (54o 30′ 47.011′′ N, 18o 32’ 48.973”). Visual
evaluation of the research sites revealed that in sampling site 1, the
southern and eastern walls were visibly covered in characteristic light-
green patches, while in sampling site 2 dark green biofilm was present
on the southern and western interior walls (Fig. 1A–D); in both cases
indicating algal colonisation. The environmental conditions, as well as a
description of the sampling sites are presented in Table S1. Air tem­
perature and air humidity were measured using Testo 606-2 moisture
meter (Testo, Pruszków, Poland). For light intensity measurement
Albatronic AB-8809A (Mera, Warsaw, Poland) was used. The measure­
ments of environmental parameters and material sampling were per­
formed during a single session in late summer.
2.2. Algological analysis
The number of cells was determined using the droplet method on
microscope slides (Starmach, 1989). Total cell count in biofilm per 1 cm2
of collected material was calculated using formula (1):
n x Pp
Lx = (1)
Pi x V
where: Lx – cell count per 1 cm2 of tested material, n – number of cells
counted, Pp – surface area of coverslip (400 mm2), Pi – surface area of
preparation viewed under microscope (mm2), V – surface area of biofilm
sample applied to a microscope slide (cm2).
The biomass of algae in biofilm samples was estimated on the basis of
the ratio of the average cell volume of the species (SOT [μm3]), and the
number of cells (Lx) in 1 cm2 of the biofilm according to equation (2):
Bi = SOT × Lx (2)
where: Bi – Biomass of algae in collected biofilm samples (μm3/cm2),
SOT – average cell volume of the species (μm3), Lx – number of cells in 1
cm2 of the tested material (cm2).
Remaining biofilm was transferred to plates with solid BBM. Samples
were incubated on shelves in room conditions for 21 days under artifi­
cial light from fluorescent tubes (Osram FLUORA T8 L 36W/77) of 2800
Lux in a 16 h/8 h day/night period, with a temperature of 22 ◦ C ± 0.2 ◦ C
and air humidity of 50% ± 5%. After 21 days of incubation, biofilm
cultures were observed under a Nikon Eclipse 50i light microscope
equipped with an Opta-Tech digital camera. The identification of algal
taxonomical units was carried out based on microscopic observations
and the morphological characteristics of algal cells described previously
in (Ettl and Gärtner, 1988; Hallmann et al., 2010, 2016). Microimages of
algae present in biofilm samples are shown in Fig. 1E and F.
2.3. Colour change evaluation
The colour of samples was measured in a CIE L*a*b colour model
using a Chroma meter Minolta CR 400 colorimeter with Spectra Magic
M. Komar et al.
NX 1.3 software (Konica Minolta, Japan) software. The numerical value
of colour change (ΔE) was calculated using formula (3):
√̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅̅
ΔE = (ΔL)2 + (Δa)2 + (Δb)2 (3)
where: ΔE – numerical value of colour change between tested and
reference sample, ΔL – difference in lightness (white - black) between
tested and reference sample, Δa -difference in (green – red) colour be­
tween tested and reference sample, Δb - difference in (blue – yellow)
colour between tested and reference sample.
2.4. Material tests
Samples were tested for compressive strength (S) in accordance with
the (“PN EN 772 1 +A1:2015 10 (Methods of testing masonry elements -
Part 1: Determination of compressive strength),” 2015) and for material
moisture (M).
The material moisture (M) of samples was measured using gravi­
metric method with RadWag 110NH moisture analyzer and calculated
using formula (4):
Mw − Md
M= × 100% (4)
Md
where: M – mass moisture, Mw – wet mass of sample, Md – dry mass of
sample.
The moisture results were juxtaposed to a building wall moisture
scale adapted from (Adamowski, 2005).
Elemental composition (C, N, H, P, S) was determined using a Flash
Elemental Analyzer (Thermo Finnigan, Italy), following the
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International Biodeterioration & Biodegradation 169 (2022) 105374
Fig. 1. Images of studied sites and biofilms;
(A) sampling site 1, exterior walls of build­
ing located in Lodz, Poland, (B) wall frag­
ment covered with algal biofilm; (C)
sampling site 2, south-west interior walls of
building located in Gdynia, Poland, (D) wall
fragment covered with algal biofilm; (E)
microscopic image of biofilm from sampling
site 1 (visible Chlorococcum infusionum cells),
(F) microscopic image of biofilm from sam­
pling site 2 (visible 1. Chlorella vulgaris; 2.
Pseudochlorella signiensis; 3. Stichococcus sp.
cells).
manufacturer’s procedures. The total carbon was divided by the total
nitrogen to obtain the C/N ratio. This method was also described pre­
viously in (Gutarowska et al., 2018).
The pH measurements were conducted on the surface fragments of
bricks and plasters covered in biofilm. As a reference (control) samples
fragments of same materials without biofilm coverage were used.
Methodology was based on (Wang et al., 2021) with minor modification.
Samples were mechanically crushed into fragments of diameter larger
than 500 μm and then weighted on Radwag Ps 8100.R2 precision bal­
ance. The samples were suspended in a 1: 1 ratio in deionized water.
Suspensions were stirred for at least 5 min. Afterwards the pH of the
suspension was measured using (Mettler Toledo FiveEasy Plus pH meter.
Samples were than filtered through SJM_110 Euro 3w 110 mm filters
(SLINAP, Poland) and the pH measurement was repeated. Results were
presented as statistical mean ± standard deviation of both
measurements.
2.5. Metabolome analysis
For the purposes of metabolome analysis, flat surface fragments of
bricks and plasters were collected with a sterile scalpel and placed in
sterile plastic containers. Analysis was performed by high-resolution
laser desorption/ionisation time-of-flight mass spectrometry using
AuNPET and 109AgNPET plates. A hundred mg of sample and 1 mL of 2-
propyl alcohol were homogenised in 2 mL vials containing three 3-mm
stainless steel balls using a bead homogeniser (4000 rpm, 60 s). The
resulting liquid material was directly placed on AuNPET or 109AgNPET
plates. Preparation of the AuNPET target plate was carried out according
to the methodology described previously in (Sekuła et al., 2015). Target
M. Komar et al. International Biodeterioration & Biodegradation 169 (2022) 105374

plates covered with silver-109 nanoparticles (109AgNPET) were pre­ Table 2

pared according to (Nizioł et al., 2013). Experiments were conducted Results of elemental composition measurements on tested surfaces.
with a Bruker Autoflex Speed time-of-flight mass spectrometer with a Material Sample Percentage share of elemental composition [%]
SmartBeam II laser (352 nm) in the m/z 80–2000 range in reflectron
C N P H S
mode. Laser impulse energy was approximately 100–190 μJ with 1000
Hz repetition rate. First reflector voltage was 21 kV and the second was Silicate brick Biofilm 0.56 0.09 <0.01 0.19 <0.01
Control 0.09
held at 9.55 kV. Spectra were calibrated with silver 109Ag + -109Ag10+
<0.01 <0.01 <0.01 <0.01
Red brick Biofilm 0.89 0.04 <0.01 0.19 <0.01
(109AgNPET) and gold Au+-Au7+ (AuNPET) ions. For the purposes of Control 0.11 <0.01 <0.01 <0.01 <0.01
data analysis, the Flex Analysis 4.0 software was used. Identification of Plaster Biofilm 0.93 0.11 <0.01 0.26 0.02
potential metabolites was conducted with lab-made software based on Control 0.18 0.02 <0.01 <0.01 <0.01
metabolomic databases (Lipidmaps, MetaCyc, FOODB, HMDB, KEGG,
Peptides). Results were additionally analysed using the PATHOS
between tested and control samples were visible in carbon, nitrogen and
metabolomic tool. The acquired metabolomic profiles were subjected to
hydrogen compositions. Highest difference (0.78%) was observed for
statistical analysis using MetaboAnalyst 3.0 and the XCMS Online soft­
the carbon composition in case of red brick samples. Differences in the
ware, allowing for the assessment of metabolite variability in the tested
composition of phosphorous and sulfur were negligible. The composi­
samples.
tion of phosphrous in all samples and all tested materials was lower than
0.01%.
3. Results Results of pH measurements of tested materials with and without
green biofilm coverage were presented in Table 3. The highest difference
3.1. Algological analysis in pH value (ΔpH = 0.42) was observed for plaster samples, and lowest
(ΔpH = 0.22) for silicate brick. All obtained values were statistically
Five taxonomical units of Chlorococcum infusionum, Chlorella vulgaris, significant. Highest standard deviation equal to 0.56 was noted for sil­
Pseudochlorella signiensis, Klebsormidium sp. and Stichococcus sp. were icate brick control samples.
identified in biofilm covering bricks and plasters in the analysed
research sites. In biofilm collected from sampling site 1 (samples 1–4)
only Chlorococcum infusionum cells were identified. Sampling site 2 3.3. Metabolome analysis
(samples 10–13) was more diverse taxonomically, with four tax­
onomical units detected in the tested biofilm. Total cell count in 1 cm2 of Metabolomics using 109AgNPET and AuNPET LDI MS revealed 59
biofilm acquired from sampling site 1 ranged from Lx = 7.48 × 102 to Lx different metabolic pathways occurring in biofilm covering the tested
= 8.52 × 102 (Table S2). Samples 10–13 were characterised by signifi­ building materials (Table 4). The 109AgNPET LDI method revealed 43
cantly higher total cell count in 1 cm2 of biofilm, ranging from Lx = 2.11 different metabolic pathways, while AuNPET LDI MS allowed for the
× 106, detected in sample 10, to Lx = 4.20 × 106 in sample 13. The most detection of 25 metabolic pathways. In total 110 metabolites were
dominant taxa of algae found in samples 10–13 were Stichococcus sp. and present in the analysed samples (Table S3 and Table S4). Differences in
Chlorella vulgaris. The studied biofilms significantly differed in terms of the metabolic profiles acquired with both methods were visualised with
taxonomic composition. Principal Component Analysis (Fig. 2) and Heatmaps (Figs. S1–S2).
Performed statistical analysis revealed high variation in the meta­
bolic profiles obtained in studied samples. Differences occurred both
3.2. Characteristics of building materials among the analysed samples, as well as between samples and their
corresponding controls. For the 109AgNPET LDI MS method, total vari­
All samples showed a numerical value of ΔE in the range of ance of 24.8% was noted (20.2% – PC1, 4.6% – PC2). The largest vari­
10.82–37.67, with 88% within the range of ΔE = 10–20, and 13% within ance was noted between samples 1 (southern, exterior wall of sampling
the range ΔE = 20–40 (Table 1). The results acquired corresponded with site 1) and 12 (southern, interior wall of sampling site 2). For the
visual evaluation during sampling (see Fig. 1A–D). AuNPET LDI MS method, total variance of PCA analysis equalled to
The material characteristics of analysed substrates are shown in 13.8% (8.7% – PC1, 5.1% – PC3), with the largest variation in the
Table 1. The material moisture (M) of the studied walls ranged from metabolome characteristics also being between samples 1 (southern,
9.00% to 14.78%, where 69% of analysed samples scored within 8–12%, exterior wall of sampling site 1) and 12 (southern, interior wall of
and 31% within 12–15%. The compressive strength (S) of tested bricks sampling site 2).
ranged from S = 5.6 N/mm2 to 12.1 N/mm2 placing all tested bricks
within the range of compressive strength of full bricks available 4. Discussion
commercially (S = 5–20 N/mm2 depending on brick grade). Twenty-
seven percent of tested samples were within the lower range of brick 4.1. Algological analysis
grade compressive strength, while 73% showed compressive strength
corresponding to mid and high grades of full bricks (S = 10–15 N/mm2). All identified algae taxa occurring in the biofilm collected from both
Elemental composition results were shown in Table 2. Differences sampling sites belong to green algae. The presence of algae of the genus
Chlorella, Klebsormidium and Chlorococcum infusionum on brick-type
Table 1
Material characteristics of the analysed substrates. Table 3
Statistical Focus Material moisture Compressive Colour change Results of pH measurements of tested materials with and without green biofilm
M [%] strength S [N/mm2] [ΔE] coverage.
Mean 11.55 9.97 18.44 Material type Sample type Mean pH value Standard deviation (SD)
SD 2.23 2.33 8.22
Silicate brick Control 7.82 ±0.56
Median 11.50 10.80 16.80
Biofilm 7,60 ±0.54
Min 9.00 5.60 10.82
Red brick Control 7.81 ±0.13
Max 14.78 12.10 37.67
Biofilm 7.51 ±0.39
Distribution 8–12 12–15 5–10 10–15 10–20 20–40 Plaster Control 8.08 ±0.24
% sample share 69% 31% 27% 73% 88% 12% Biofilm 7.66 ±0.32

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M. Komar et al. International Biodeterioration & Biodegradation 169 (2022) 105374

Table 4
Metabolic pathways detected in building material samples by109AgNPET and AuNPET LDI MS.
No Metabolic pathway Detected by109AgNPET in building materials samples Detected by AuNPET in building materials samples

1 4 5 11 12 1 4 5 11 12

1 1,4-Dichlorobenzene degradation x
2 2,4-Dichlorobenzoate degradation x
3 Acridone alkaloid biosynthesis x x
4 alpha-Linolenic acid metabolism x
5 Amino sugar and nucleotide sugar metabolism x
6 Aminoacyl-tRNA biosynthesis x
7 Anthocyanin biosynthesis x
8 Arachidonic acid metabolism x
9 Ascorbate and aldarate metabolism x
10 beta-Alanine metabolism x
11 Betalain biosynthesis x
12 Biosynthesis of 12-, 14- and 16-membered macrolides x
13 Biosynthesis of ansamycins x
14 Biphenyl degradation x
15 Bisphenol A degradation x
16 Butanoate metabolism x
17 C5-Branched dibasic acid metabolism x x
18 Caffeine metabolism x
19 Carotenoid biosynthesis x
20 Cysteine and methionine metabolism x x
21 Diterpenoid biosynthesis x
22 Drug metabolism - cytochrome P450 x
23 Flavone and flavonol biosynthesis x
24 Folate biosynthesis x
25 Fructose and mannose metabolism x
26 gamma-Hexachlorocyclohexane degradation x
27 Geraniol degradation x
28 Glucosinolate biosynthesis x x
29 Glycine, serine and threonine metabolism x
30 Histidine metabolism x x
31 Insect hormone biosynthesis x x
32 Isoflavonoid biosynthesis x x
33 Isoquinoline alkaloid biosynthesis x
34 Limonene and pinene degradation x
35 Lysine biosynthesis x
36 Lysine degradation x x x
37 Metabolism of xenobiotics by cytochrome P450 x
38 Methane metabolism x x
39 Monoterpenoid biosynthesis x
40 Naphthalene and anthracene degradation x x x
41 Nicotinate and nicotinamide metabolism x x
42 Penicillin and cephalosporin biosynthesis x
43 Phenylalanine, tyrosine and tryptophan biosynthesis x
44 Polyketide sugar unit biosynthesis x
45 Porphyrin and chlorophyll metabolism x
46 Primary bile acid biosynthesis x x x
47 Purine metabolism x x
48 Secondary bile acid biosynthesis x
49 Sesquiterpenoid biosynthesis x
50 Steroid biosynthesis x x x x
51 Steroid degradation x x
52 Steroid hormone biosynthesis x x
53 Sulfur metabolism x
54 Taurine and hypotaurine metabolism x
55 Tetrachloroethene degradation x
56 Trinitrotoluene degradation x
57 Tropane, piperidine and pyridine alkaloid biosynthesis x
58 Tryptophan metabolism x x
59 Ubiquinone and other terpenoid-quinone biosynthesis x

materials was also confirmed in (Nowicka-Krawczyk et al., 2014). The
occurrence of Stichococcus sp. algae on plasters has been described in
(Häubner et al., 2006) and (Nakajima et al., 2020). These taxa are,
therefore, commonly found in biofilms that cover bricks and plasters.
Significantly less scientific data can be found on the presence of Pseu­
dochlorella signiensis (=Pabia signiensis) on the tested materials. Its
presence has been so far described on stone (Hallmann et al., 2013) and
plastic (Hallmann et al., 2016) surfaces. In this study, however, this
species was detected in only one sample (sample no. 13) and was not
identified in the remaining biofilm samples. The number of Pseudo­
chlorella signiensis cells detected in sample 13 (7.35 x105) was significant
(higher than the number of Klebsormidium sp.) and may indicate that this
species may also occur on substrates such as plaster in temperate climate
zones. Environmental conditions may influence development and
taxonomic composition (Nowicka-Krawczyk et al., 2014; Piotrowska
et al., 2014) and were the most probable reason behind differences in the
analysed samples. Lack of diversity and the lower abundance of the taxa
detected in the biofilm covering the southern and eastern exterior walls
of sampling site 1 could be correlated with higher exposure to weather
conditions, lower air humidity than in sampling site 2 and possibly older
age. While exposure to rainfall may promote biofilm development and
affect its taxonomic diversity, exterior walls are also more susceptible to

5
M. Komar et al.
Fig. 2. PCA statistical analysis of metabolomic profiles detected by 109AgNPET LDI MS method (A) and AuNPET LDI MS method (B); 1, 4, 5, 11, 12 – analysed
material in samples covered with biofilm; 1 C, 4 C, 5 C, 11 C, 12 C - corresponding reference (control) samples.
drying, especially in summer.
The role of phototrophs such as algae on building materials is usually
associated with initial colonisation as they allow for inorganic carbon
present in the atmosphere to be incorporated into biomass. This enables
the carbon cycle to occur, leading to the development of microbial
community and simultaneously modifies both the surface properties. In
turn growth of organisms of documented biodeteriorative potential is
enabled (May, 2010; Borderie et al., 2015; Zhang et al., 2019). The role
of other autotrophic organisms in the cycles of elements such as carbon
and nitrogen was described in other studies (Sepehri and Sarrafzadeh,
2018).
4.2. Characteristics of building materials
The colour change assay showed a significant influence of algal
biofilm on the colour change of the analysed substrates. The detected
values were numerically significant. Since all results were greater than
2, an inexperienced observer should be able to notice visually differ­
ences in colour between colonised and non-colonised material. As the
determined ΔE value was greater than 5, the difference in colour should
be explicit (Mokrzycki and Tatol, 2011). The tests performed clearly
show the significant influence of algal biofilms on the discolouration of
building material surfaces, confirming their influence on the aesthetic
biodeterioration of building materials and façades.
The comparison of the obtained moisture results with the building
wall moisture scale adapted from (Adamowski, 2005) showed that the
analysed building walls can be classified as very damp walls (M =
8–12%) or wet walls (M > 12). Higher moisture values were mostly
observed in samples collected from the interior walls of sampling site 2,
which were protected from external environmental conditions, and at
the same time they were overgrown by a higher biomass of algae. The
high material moisture of analysed material samples suggests the in­
fluence of phototrophic biofilms on water absorption and retention.
Water absorbed on the surface of inorganic materials is critical for
the development of microbial community and succession of organisms,
including those of proven, high deteriorative potential. Moreover, with
the presence of stagnant water the concentration of salts on the surface
of inorganic materials may change, leading to the biogeochemical
biodeterioration process (Gu, 2018). Additionally, biophysical mecha­
nisms of deterioration may also occur as a result of biofilm hygrothermic
expansion, especially during freeze/thaw cycles and changes in thermal
conductivity (Gaylarde et al., 2003; Peraza Zurita et al., 2005; May,
2010). These algal properties were also noted briefly in (Rajkowska
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International Biodeterioration & Biodegradation 169 (2022) 105374
et al., 2014).
Elemental composition tests have shown differences between surface
fragments of bricks and plasters covered in biofilm and without biofilm
coverage. These are especially visible in case of carbon (C). and might be
correlated with the ability of photoautotrophs to fix atmospheric carbon
dioxide (Gaylarde et al., 2003) which in turn is transferred to organic
carbon in form of dead organic matter. Increasing the bioavailability of
elements for heterotrophs is primary indirect mechanism of biodeteri­
oration associated with algal activity (Borderie et al., 2015). On the
other hand, it should be noted that collected biofilms were non-axenic
which was also indicated by present metabolomic studies, suggesting
presence of non-photoautotrophic metabolites and organisms such as
bacteria on tested surfaces. Higher percentage share of hydrogen (H)
was also observed but differences in other elements were negligible.
Material surface pH measurements have shown a slight decrease in
the pH values of colonized material surface fragments. This trend can be
observed regardless of the type of tested material and suggests that
biofilms covering building materials may exert influence on the acidity
of colonized surfaces. It cannot be unambiguously assigned as the
acidifying activity of photoautotrophic biofilms and require further tests
conducted in model environment with algal monocultures.
Considering all tested samples placed within compressive strength
values of commercially available bricks any further direct influence of
algal biofilms on building materials was not found, as the correlation
between their growth and compressive strength reduction was not
determined.
4.3. Metabolome analysis
Results of metabolomic studies strongly suggest that laser desorp­
tion/ionisation time-of-flight mass spectrometry methods using gold
and 109-silver nanoparticles can be successfully incorporated in
metabolome studies of phototrophic biofilms covering building mate­
rials. The use of the 109AgNPET method allowed to obtain more results,
including a greater number of both metabolic pathways and metabolites
detected. AuNPET LDI MS, however, enabled the identification of
pathways and metabolites not detected with the 109AgNPET method (e.
g. biphenol degradation, bisphenol A degradation, folate biosynthesis,
tryptophan metabolism). As such, the combination of both methods al­
lows for the widest possible range of results.
Apart from: 1,4-dichlorobenzene degradation, acridone alkaloid
biosynthesis, biosynthesis of 12-, 14- and 16-membered macrolides,
bisphenol A degradation, diterpenoid biosynthesis, flavone and flavonol
M. Komar et al.
biosynthesis, glucosinolate biosynthesis, isoflavonoid biosynthesis, sec­
ondary bile acid biosynthesis and steroid degradation pathways, all 48
remaining metabolic pathways (Table 4) have been at least partially
present in algal metabolism, according to the KEGG database (Kanehisa
and Goto, 2000). Metabolic pathways, such as flavone and flavonol
biosynthesis, and isoflavonoid biosynthesis are usually associated with
the metabolic activity of plants (Kanehisa and Goto, 2000; Falcone
Ferreyra et al., 2012). These were identified only in samples (sample 1
and 4) acquired from the exterior walls of sampling site 1. In their close
vicinity, lush vegetation was present which probably resulted in the
presence of phytocompounds in these biofilms. These pathways were
not detected in any sample acquired from external walls of sampling site
2. Similarly, the glucosinolate biosynthesis pathway, assigned mostly to
the Brassicaceae family of plants (Kanehisa and Goto, 2000; Chhajed
et al., 2020), was only detected in samples 4 and 5.
Secondary bile acid biosynthesis is a metabolic pathway attributed to
the activity of gut microbiota (Kanehisa and Goto, 2000; Jia et al., 2019)
and was detected in sample 1 (silicate brick, southern wall, 1,2 m
acquisition height). Although algal cells and their metabolites were
detected in all analysed samples, usually biofilms are not homogenic and
often contain cells of other organisms. Identification of secondary bile
acid biosynthesis might indicate that some bacterial cells were also
present on the analysed materials. The presence of the secondary bile
acid biosynthesis pathway might also indicate the activity of animals
(mammals) in the vicinity of sample site 1.
A steroid degradation pathway was detected in samples 4 and 11.
According to (Chiang et al., 2020) some bacteria and microalgae can
transform metabolites, such as steroids. Their complete mineralisation,
however, is carried out only by bacterial cells. Compounds such as ste­
roids are abundantly common in various environments. In the identified
pathway, cholesterol was detected. This compound was also found in
other metabolic pathways, such as histidine metabolism.
Metabolomic studies allowed for the detection of primary meta­
bolism pathways (e.g. methane metabolism, porphyrin and chlorophyll
metabolism, histidine metabolism, steroid biosynthesis, anthocyanin
biosynthesis, lysine biosynthesis and degradation, carotenoids biosyn­
thesis etc.) and primary metabolites (inter alia, steroids, homocysteine,
glycine, diprotic acids, anthocyanins). The presence of primary meta­
bolic pathways and metabolites (such as lysine degradation, histidine
metabolism, steroid biosynthesis etc.) indicates ongoing, active cellular
processes, while the occurrence of steroids, strigalactones and phytos­
terols suggests the presence of phototrophic cells. In secondary meta­
bolism pathways, isoflavonoid biosynthesis, penicillin and
cephalosporin biosynthesis, tetrachloroethene degradation, metabolism
of xenobiotics by cytochrome P450, steroid degradation etc. were also
detected.
The detected metabolites also suggest the presence of other organ­
isms than algae in the studied biofilms. Metabolites, such as O-phospho-
L-homoserine, L-2-aminoadipate, L-threonine O-3-phosphate, 8,8a-
deoxyoleandolide, 4-methyl-L-glutamate and L-rhamnulose 1-phos­
phate, are usually attributed to the metabolic activity of bacteria
(Hastings et al., 2015; Kim et al., 2020). Conversely, compounds such as
(+)-7-isomethyljasmonate, methyl jasmonate, betanin, gomphrenin-I,
acacetin, geraniol, nerol, desulphoglucotropeolin, 3-methylthiopropyl-­
desulphoglucosinolate, (− )-maackiain, (+)-maackiain, 2′ -hydrox­
yformononetin, biochanin, calycosin, prunetin, rotenone,
dihydrocarveol, (+)-isomenthone, (− )-linalool, (− )-menthone,
(− )-alpha-terpineol, (− )-endo-fenchol, 1,8-cineole, sabinene hydrate,
1-methylnaphthalene, lupeol, 24-ethylidene lophenol, cycloartenol and
cycloeucalenol, are associated mostly with the activity of plants.
Most questionable is the presence of metabolites of mostly animal
origin, such as 11(R)-HPETE, leukotriene B4, 3-(methylthio) propionic
acid, bromobenzene and cinnavalininate, especially detected in the
samples collected from sampling site 2 (e.g. 7-dehydrodesmosterol).
These (apart from cinnavalininate) belong to the steroid degradation
pathway. The ability of algae and bacteria to transport metabolites of
7
International Biodeterioration & Biodegradation 169 (2022) 105374
this pathway, and their ubiquitous occurrence in various environments
has been described above, while the presence of metabolites of animal
origin in samples taken from sampling site 1 can be explained by the
presence of animals in the vicinity of the walls tested.
It should be noted that these compounds act as intermediaries in
metabolic pathways, including those found in green algae, and are
further transformed into other compounds. Thus, their identification
may not unequivocally point to the presence of other organisms than
those identified, but merely suggest it. Metabolites with a strongly
documented biodeteriorative effect on building materials have not been
detected. Compounds present in the studied samples, such as salts of
benzoic acid and naphthalene, may indicate the ongoing processes of
degradation of organic substances (Szulc et al., 2017). Identified sulfur
compounds, i.e. sulphoacetaldehyde, sulphosalicylic acid, sulpho­
benzoic acid as well as tetrachloroethene (being an organic solvent) may
exhibit deteriorative influence. The presence of fatty acids, their de­
rivatives (e.g. hepoxylins and laukotrienes) and organic acids, such as
propionic acids, was also detected but the direct influence of these
compounds on the destruction of inorganic substrates has not been
proven.
Most biodeteriorative influence on inorganic materials (including
building materials) is associated with biogeochemical mechanisms such
as production and excretion of oragnic acids, sulfur cycle and oxidation,
and biochemical transformations of nitrogen. These were not confirmed
by presented studies (lack of biodeteriorative metabolites and no change
in material compressive strength) and are usually associated with the
activity of other microorganisms such as fungi and bacteria (Gu, 2018;
Zhang et al., 2019). Acquired results were reinforced by statistical
analysis indicating the high metabolic activity of biofilm covering the
analysed materials and the high variation of the obtained metabolomic
profiles.
AgNPET and AuNPET LDI MS methods can be therefore, successfully
used in metabolome analysis of microbial communities covering sur­
faces of building materials. Additionally, these can be applied directly
on environmental samples and does not require culturing simulta­
neously allowing to assess metabolic processes occurring in the whole
biofilm system, where different organisms coexist.
5. Conclusions
Bricks and plasters used in temperate climate zones can be colonized
by Chlorococcum infusionum, Chlorella vulgaris, Pseudochlorella signiensis,
Klebsormidium sp. and Stichococcus sp., leading to aesthetic biodeterio­
ration and strong discolouration, easily observed even by inexperienced
observers. Influence on water absorption and retention was confirmed
but no other direct biodeterioration mechanism was detected. Research
revealed that phototrophic biofilms covering building materials by
conducting various metabolic processes participate in total metabolome
profile of building materials and that 109AgNPET and AuNPET LDI MS
techniques complement each other giving satisfying results in metab­
olomic studies.
Declaration of competing interest
The authors declare that they have no known competing financial
interests or personal relationships that could have appeared to influence
the work reported in this paper.
Acknowledgments
The authors would like to acknowledge Associate Professors Joanna
Żelazna-Wieczorek and Dorota Żyżelewicz for the indispensable support
they provided in conducting the present research.
This work has been completed while the first author was the Doctoral
Candidate in the Interdisciplinary Doctoral School at the Lodz Univer­
sity of Technology Poland.
M. Komar et al.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://doi.
org/10.1016/j.ibiod.2022.105374.
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