Characteristics of Mould Growth in Pine and Spruce Sapwood and Heartwood under Fluctuating Humidity

Abstract

The importance of maintaining a healthy indoor climate has recently increased, as has the durability of building structures, and for this issue, we need to predict mould growth. To prepare this model under real conditions is challenging, and this work aimed to contribute data to this model. This article presents the findings of a laboratory study investigating the effects of fluctuations in the relative humidity and temperature conditions on mould growth on pine and spruce. The study compared the results to a previous steady-state experiment, demonstrating that fluctuations in relative humidity and temperature prolonged the onset of mould growth. The mould growth observed depended on the type of wood with pine or spruce wood exhibiting different growth patterns compared to heartwood or sapwood. In sapwood, mould growth was found to be almost independent of the direction of the fiber. The first microscopic indications of mould growth on pine sapwood were observed around day 76, with the first macroscopic indications observed around day 90. On the contrary, spruce sapwood demonstrated a limit for mould growth. The mould growth was only visible under the microscope with the first indications observed between the 72nd and 80th day. Furthermore, heartwood was found to be unsuitable for mould growth under fluctuating conditions.

1. Introduction

Mould is a small organism that occurs on many types of surfaces and is widely disseminated through spores. The mould is important in the biochemical cycle of elements, the movement and transformation of chemical elements, and compounds between living organisms, and it enables the decomposition of materials and therefore is necessary in life. For humans, they can cause several health problems, especially immunodeficiency ones [1,2,3,4,5]. The most dangerous mould for human health is species such as Aspergillus spp. or Stachybotris spp. [6]. In buildings, they can lead to the deterioration of interior and exterior finishes as well as other structural components. This deterioration can have adverse effects on both the physical condition of the building and human health, resulting in economic and social costs. Additionally, the presence of mould is indicative of high humidity, which can accelerate the degradation of structures and lead to an uncomfortably humid environment for occupants.

Because of the above, knowledge of the probability of occurrence of mould in a building is very important. The mould requires for its life available nutrients and humidity as well as a sufficient pH [7,8]. The amount of humidity required for the germination and subsequent development of mycelium and the dissemination of mould vary depending on the species [9]. The amount of humidity on finishing materials is dependent on the ratio of environmental humidity and temperature. Generally, the building materials in conditions below 80% relative humidity (RH) do not support mould growth [10].

In order to assess the parameters that contribute to mould growth on wood, it is necessary to consider the effect of temperature and RH. To accurately simulate the conditions that occur in real building conditions where mould growth occurs, it is necessary to employ a dynamic prediction model of mould growth that includes changing conditions over time. Furthermore, it is crucial to consider the specific species of wood, the portion of the tree from which the samples were obtained, and the direction of the cut [11,12,13,14]. Furthermore, the species of wood, the part of the tree, and the method of cutting the sample are important information [15,16].

The more important factor for mould germination is the moisture of the substrate rather than the environment RH [17]. In general, a higher humidity is required for germination and then for subsequent growth [18]. The minimum humidity requirements to growth are dependent on the species, which are divided into three groups. Hydrophilic fungi require relative humidity above 90%, moderately xerophilic fungi begin to grow at RH 75–79% and slightly xerophilic fungi need RH in the range of 80–89% [19,20,21]. For example, Aspergillus spp., Penicillium spp. and Alternaria spp. begin germination around 85% RH [22,23,24,25]. These moisture ranges for mould growth were calculated from the experiments based on laboratory experiments on nutrient agar, where the nutrient condition is optimal [25,26,27]. For building materials, where nutrient availability is not optimal but rather limited, the requirement for available moisture is probably slightly higher than mentioned above [28,29]. Moisture requirements are also related to temperature; at lower temperatures, the mould requires more available humidity to germinate and grow [14,30]. The growth of mould is also dependent on temperature even when other conditions remain constant [14,31]. A reduction in temperature from 20 to 5 °C has been observed to result in a fourfold reduction in the rate of mould growth. According to Ref. [14], the other requirements for mould grow are nutrients such as sources of carbon, nitrogen, etc. Especially when glucose is available as a carbon source, germination is faster [32].

The majority of published results pertaining to mould growth are focused on pine wood [11,15,33,34]. Under constant conditions (approximately 90% relative humidity and 22 °C, on pine sapwood), initial mould growth is observed around day 7, while macroscopic mould growth is visible between days 10 and 22, which depends on surface preparation [8,33]. The length of time required for germination is influenced by environmental conditions and the duration of unfavorable conditions. In cases where the temperature and RH fluctuate, prolonging the duration of unfavorable conditions has been associated with an increase in the time required for germination to occur. A weekly RH cycle (90/60% RH, 22 °C) is only suitable for the germination and formation of a limited number of hyphae. A 12 h cycle of fluctuating relative humidity (90/60% RH, 22 °C) ultimately results in a change in germination time over a six-week period. Following germination, however, the fungal growth curve remained identical to that observed under constant conditions. A weekly temperature cycle (90% RH, 5/22 °C) resulted in a prolongation of the germination period (approximately 49 days) and a slowing down of fungal regrowth [31].

This article examines the impact of fluctuating conditions on the growth of mould on wood. The conditions under which mould growth is most likely to occur were selected for examination rather than those that are commonly found in buildings or structures. In alignment with the findings of the literature review, this study assesses mould growth from a multifaceted perspective, extending beyond the conventional parameters of temperature and humidity fluctuations. It considers the influence of wood species and wood type (pine and spruce, heartwood and sapwood) as well as the direction of the cut used to obtain samples (tangential, radial, and transverse). The samples with different wood fiber directions were chosen to see if, even under fluctuation conditions, mould growth would be different for each direction.

2. Materials and Methods

This paper presents the results of a laboratory experiment on pine and spruce samples from sapwood and heartwood, which were prepared using a single beam with respect to fiber directions. The samples were exposed to fluctuating humidity and temperature in 18/6 h cycles. The temperature varied between 25 and 15 °C, while the humidity varied between 98% and 73% with the higher condition present for 18 h. The experimental condition was monitored by the data logger. Mould growth was observed weekly using a 40× and 100× magnification microscope and the naked eye (1×).

Additionally, the live phase of the mould was also recorded, including germination, hyphae, sporulation, and decay. The more important stage was recorded by images. This research is notable for its use of samples with respect to the direction of wood fibers. The assessment of mould growth was conducted based on evaluations of mould coverage of samples and the life phase of mould.

The primary findings are presented in the form of a time-dependent mould index. The mould index is a dimensionless quantity used to model mould growth. For a detailed description of this index, please refer to the section on Evaluation, specifically Section 2.3. The characteristics of specific MI and typical figures are presented for enhanced comprehension; see tables in Section 2.3 and figures in Section 3.

The experiment was conducted in several steps; see Figure 1. The initial step of the experiment, see Section 2.1 (Preparation), involved sample preparation, which included their sterilization and humidity conditioning. Additionally, a spore suspension was prepared for inoculation. The second stage, see Section 2.2 (Main experiment), involved conducting the experiment within the experimental chamber described in [35]. The condition is described in Section 2.2. The last step, see Section 2.3, was the assessment of mould growth, which was based on the intermediate evaluations of the individual samples, notes, and photographic records.

2.1. Preparation

2.1.1. Materials and Samples

The wood samples were prepared from spruce (Picea abies (L.) H. Karst.) and pine (Pinus sylvestris L.). The origin boards for preparing samples were 4 m long boards, which were obtained from a commercial lumberyard in 2017. The boards were free of any defects, including warping, blue stain mould, knots, or resin in pockets. The wood was dried at a temperature below 60 °C. The dry bulk density was 387–454 kg/m³ for spruce and 478–565 kg/m³ for pine. The variation in dry bulk density for the wood samples is expected to be lower due to the similar position on the trunk. The boards were stored under controlled environmental conditions (temperature 20–25 °C, relative humidity 50–60%) before preparing the samples.

The samples were prepared from sapwood and heartwood portions and oriented according to the anatomical direction of wood fibers. Samples were prepared in 12 modifications, regarding anatomical species and fiber direction; see

Table 1. The nomenclature and amount of samples.

Tree Species	Direction of Wood Fiber	Nomenclature	Amount
Pine—Sapwood	Tangential surface	${\mathrm{P}}_{S}T$	6
	Radial surface	${\mathrm{P}}_{S}R$	6
	Transversal surface	${\mathrm{P}}_{S}X$	6
Pine—Heartwood	Tangential surface	${\mathrm{P}}_{H}T$	6
	Radial surface	${\mathrm{P}}_{H}R$	6
	Transversal surface	${\mathrm{P}}_{H}X$	6
Spruce—Sapwood	Tangential surface	${\mathrm{S}}_{S}T$	6
	Radial surface	${\mathrm{S}}_{S}R$	6
	Transversal surface	${\mathrm{S}}_{S}X$	6
Spruce—Heartwood	Tangential surface	${\mathrm{S}}_{H}T$	6
	Radial surface	${\mathrm{S}}_{H}R$	6
	Transversal surface	${\mathrm{S}}_{H}X$	6

and illustrative images in Figure 2. A particular type of samples was prepared in six repetitions.

The dimensions of the samples were 20 mm × 20 mm × 3 mm. Inoculation and mould growth observations were conducted on the main top surface of each specimen, specifically the 20 mm × 20 mm square.

The surface of the samples was treated with a planer (Makita 2012NB) in order to achieve uniform surface quality with the exception of the transverse surface. The tangential surfaces of the specimens were composed exclusively of early wood. The samples were subjected to UV radiation for a period of 30 min on each side in order to be sterilized. Prior to the commencement of the main experiment, the samples were conditioned to achieve an equilibrium moisture content with an RH of 86% at 22 °C of the environmental conditions; see the Figure 3. The mentioned RH was established using saturated salt solutions (Na₂SO₄ × 10 H₂O). This preconditioning was conducted for a period of seven days.

2.1.2. Preparation of the Inoculate

The experimental inoculum was prepared from mould species commonly found in the Czech Republic and known to occur in the wood built into the construction [12,36,37]. The selected species were obtained from the Czech Collection of Microorganisms at the Faculty of Science, Masaryk University, Aspergillus niger van Tieghem (CCM 8189), Penicillium cf. brevicompactum Dierckx (CCM 8288) and Alternaria alternata (Fries) von Keissler (CCM F-397); their accession numbers of species are in the brackets.

Before inoculation, the moulds were reinoculated and the spores were collected as described in [1]. Each spore suspension was adjusted to a concentration of 1 × 10⁶ CFU/mL (colony formation units per milliliter), and the experimental spore suspension was prepared in a 1:1:0.25 ratio of 1:1:0.25 with the lowest level of Aspergillus sp. present.

2.2. Main Experiment

The samples were placed in an aluminum tray to facilitate handling during microscope evaluation and to limit the effect of moisture and heat to only one side of the samples (1D transport), as illustrated in Figure 4. Bitumen sealant was applied to the sides of the samples to prevent heat and moisture transport through other locations. This methodology for sample deposition was employed due to the continuity of these experiments with those where dynamic moisture transport was measured [35].

Each tray always contained samples from one species of wood and samples with all directions (T, R, X). A total of six trays were prepared for each kind of wood; see Table 1. The trays were placed in the special box in rows from the top: pine sapwood, pine heartwood, spruce sapwood, and spruce heartwood. The special box (see Figure 5) was used to create an environment with the desired relative humidity, as described in detail in Richter et al. (2023) [35]. Fans and settling chambers were used to maintain a constant laminar flow of treated air over the samples. The velocity of the airflow was measured at a height of 1.0 cm above the exposed surface of the sample and was found to be 0.3 m/s. The temperature and RH were monitored using COMET dataloggers, which exhibited an accuracy of ±0.3 °C and ±2.5% RH, respectively.

The experimental conditions were set up as follows: 98% RH at 25 °C for 16 h and 73% RH at 15 °C for 8 h; see Figure 3. To maintain lower relative humidity, a saturated sodium chloride (NaCl) solution was employed, while distilled water was utilized to maintain higher relative humidity. The main experiment was conducted over a period of five months; the conditions are shown in the graph in Figure 3.

2.3. Evaluation and Base of Assessments

Mould growth was observed using microscopic (BA 410E Epi microscope, Motic, Barcelona, Spain) and macroscopic methods. Microscopic images were taken with a Promicam Pro 3-3CP camera attached to the microscope, and macroscopic images were taken by a photographic camera. The observation had 40–100× magnification. Observations were made every three days at the beginning of the experiment. During the middle phase of the experiment, the interval was extended to seven days, and at the end of the experiment, it was extended to 14 days.

The experiment was carried out over five months. Both quantitative and qualitative indicators describing mould growth were recorded and used to determine the mould index (MI); see

Table 2. The definition of mould index and subsequent mould subindex, MI—mould index and SMI—mould subindex.

MI	SMI	Magnification of Observation		Description
[-]	[-]	40×	1×
0	0	0	0	No growth, only spores
	0.5	0.1–1		Germination of spores
1	1	1–5	1–5	Initial growth, first visible hyphae
	1.5	5–10		Initial growth hyphae clearly visible
2	-	10–30		Development of mycelium with first aerial hyphae, moulds are almost exclusively visible under a microscope
3	3	30–50	5–25	Development of mycelium with sporangium, Moulds start visible macroscopically
	3.5	50–70	25–50	Development of mycelium with sporangium, Moulds are visible macroscopically
4	-	70–90	50–70	Decay of mould or huge sporulation
		90–100	70–100

and

Table 3. Comparison of MI (mould growth rating scale) by several authors, Ryp—the mould index is used in this articles at [8], VTT—mould index defined at [14], Joh—mould index defined at [19], mould coverage of the sample was divided according to the method of observation with the following divisions: 1×—macroscopically by the naked eye and 40×—microscopically with a magnification of 40×, MI—mould index, SMI—mould subindex.

Magnification		MI by VTT	MI by Joh and Ryp	SMI by Ryp
40×	1×
0	0	0	0	0
0.1–1		1		0.5
1–5	1–5		1	1
5–10		2		1.5
10–30			2	-
30–50	5–25	3	3	3
50–70	25–50	4		3.5
70–90	50–70	5	4	4
90–100	70–100	6

.

The scale of the MI was developed based on the work of Johansson [3] and subsequently augmented by the addition of the mould subindex (MSI), which provides a more accurate description. The MI and MIS are determined by the extent of visible coverage under 40× and 1× magnification as well as the phase of mould growth.

The microscopic coverage of the surface was estimated through microscopic observations at 40× magnification, while the phase of mould growth was determined through observations at 100× magnification. Both approaches were utilized to ascertain the MI, or if it was possible, the MIS was determined. Macroscopic coverage, beginning at MI = 3, was gauged using photographic documentation at 40× and 1× magnifications. Additionally, the presence and condition (visibility) of hyphae and sporangia were documented.

The decision between MI 1 and 1.5 or between 3 and 3.5 is based on the rate of coverage. In general, MI Mould index 0 indicates no growth or germination, while MI 0.5 (germination) was not observed on wood samples. The MI 2 indicates microscopic visible mould growth, whilst the MI 3 indicates macroscopic visible mould growth. Indexes 3 and 4 include macroscopic mould growth.

3. Results

Mould growth had evaluated as a time-dependent MI. The attainment of a specific MI at a given time was initially evaluated separately for all samples and subsequently subdivided into sample types and an additional subgroup (see P_SRA). Subsequently, for each group, the date of attainment of a given MI was calculated as the average of all samples in that group, which was rounded to the nearest day. The samples were divided into three main groups. The first group consisted of heartwood samples regardless of tree species. The second group consisted of pine sapwood samples, and the third group included spruce sapwood.

3.1. Pine and Spruce Heartwood

The results showed that the heartwood is similar to other woods and the direction of the grain at the top site does not affect mould growth.

A total of 36 heartwood samples were analyzed without regard for the botanical species or the way the samples were cut. In most cases, mould growth was not observed on the heartwood samples. The mould growth observed on spruce heartwood samples was limited to two samples with the maximum MI reaching 1.

3.2. Pine—Sapwood

Mould growth was observed in all samples made of pine sapwood. When mould growth was evaluated in terms of wood fiber direction, a single group of three samples exhibited significantly different mould growth and was designated as P_SRa with the index “a” added to the designation.

The initial manifestation of growth in P_SRa was observed on the 27th day, yet in general, the growth rate was notably slower than that observed in other samples from pine sapwood. Upon completion of the experiment, the mould coverage reached a maximum of MI = 4.

The maximum value of mould growth for the remaining samples was recorded as MI = 3.5. The value of MI = 3 was recorded on the days indicated in parentheses. The specific days on which this value was recorded are as follows: P_SX (101), P_SR (110) and P_ST (120). The highest mould index (MI) of 4 was observed exclusively in the P_SX samples. For further details regarding the relevant dates, please refer to

Table 4. The time dependence of achieving a given MI for pine sapwood. The MI was calculated as the average of all results in a given group with the expression of mould growth taken into account.

Samples MI [-]/Time [day]	PST	PSX	PSR	PSRa
1	76.25	77.75	76	27
1.5	-	-	79	45.7
2	81.3	79.5	82	69
3	90.8	92.3	89.7	79
3.5	120	101.5	110	101
4	-	103.3	-	143

. Illustrative images can be found in Figure 6, Figure 7 and Figure 8.

3.3. Spruce—Sapwood

Mould was observed only in the S_ST and S_SR samples of the spruce sapwood samples, as shown in

Table 5. The time dependence of achieving a given MI for spruce sapwood. MI was calculated as the average of all results in a given group with the expression of mould growth taken into account.

Samples MI [-]/ Time [day]	SST	SSX	SSR
1	70	-	72
1.5	91	-	98
2	110	-	120
3	-	-	-
3.5	-	-	-
4	-	-	-

and Figure 9. It is worth noting that the growth was only observed microscopically. The growth was slower in the SR samples. No growth was observed in the SX direction samples until the end of the experiment.

4. Discussion

This study confirms that mould growth on the surface of heartwood has occurred in a negligible number of instances under fluctuating conditions (98/75% RH, 25/15 °C in cycles of 16/8 h). The frequency of mould growth on heartwood was found to be only on 2 samples out of 36 regardless of the wood species from which the samples were derived, whether pine or spruce. These results are probably because of the low amount of nutrient for mould growth in the heartwood part of the tree [11,19,21,38], which consists of mainly saccharides and is a source of basic elements such as nitrogen and phosphorous.

The mould growth on pine sapwood showed that the samples had different properties, because the mould growed on the four samples differently than it did on the 12 other samples. The three samples were P_ST and one P_SX, which were more problematic samples for preparation with respect both to the direction of the fibers and the differentiation of sapwood and heartwood. The groups are labeled as P_SRa and showed similar curve as [15] for pine sapwood at a constant RH and fluctuation temperature (RH 90%, 22 °C/5 °C in weekly cycles). The difference was that the maximal MI was 4. The mould growth curve under fluctuation conditions (RH 97% at 25 °C for 16 h/ RH 73% at 15 °C for 8 h) for other pine sapwood samples (P_ST, P_SX, P_SR) have shifted over the 50 days for the germination (MI = 1) compared to the samples P_SRa. Generally, pine sapwood samples have a shift in germination and regrow a little slower than the same samples made from the same trunk and placed in constant conditions (RH 95% at 22 °C, RH 87% at 22 °C) [8].

Given the above, it seems that the mould growth under fluctuating conditions depends on the duration of uncomfortable conditions for growth and how far the conditions are from optimal. When the conditions are not far from optimal, the rate of growth depends on the availability of nutrients. Mould growth is more affected by uncomfortable RH than by uncomfortable temperature. The temperature around 15 °C and 73% RH for 8 h (uncomfortable condition) makes germination slower by seven weeks but does not affect the rate of regrowth.

The spruce sapwood showed more effects from fluctuating conditions compared to samples made from the same trunk at constant conditions (87% RH and 95% RH at 23 °C). For pine sapwood, the direction of fibers did not show any difference. The germination had a shift around 3 months, and the maximal MI was 2. It confirms that nutrients composed of spruce are not suitable for mould growth under fluctuating conditions, and the composition of wood is an important feature for the rate of mould growth.

The hypothesis that different growth rates would be observed under fluctuating conditions depending on the direction of wood fiber was not substantiated by the findings. It is probable that this was due to the small height of the samples and the fact that they were sealed on all sides except the top. It can be assumed that equilibrium moisture content was reached in a similar time frame.

5. Conclusions

Mould growth in wood under fluctuating conditions (98–73% RH, 18/6 h) depends on the type of wood, such as botanical species (pine or spruce) or the type of wood (heartwood or sapwood). In pine sapwood, mould growth is almost independent of the direction of the fiber. The first microscopic signs of mould growth in pine were observed around day 76, and the first macroscopic signs were observed around day 90 from the beginning of the experiment. The spruce sapwood showed nutrient limits for mould growth. Mould growth was only visible microscopically, and the first signs were seen between the 72nd and 80th day. The heartwood was not suitable for mould growth under fluctuating conditions. The evaluation of mould growth under fluctuating conditions depends on the duration of uncomfortable conditions and the availability of nutrients.