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Article

Obtaining Phlebiopsis gigantea Oidia Using Liquid- and Solid-Surface Cultivation Processes

by
Oskars Grigs
1,*,
Ilvars Sutris
1,
Karlis Roberts Gasuns
1,
Evelina Vahere-Abrazune
1,
Kristine Kenigsvalde
2,
Darta Klavina
2 and
Talis Gaitnieks
2
1
Laboratory of Bioengineering, Latvian State Institute of Wood Chemistry, LV-2169 Salaspils, Latvia
2
Latvian State Forest Research Institute “Silava”, LV-2169 Salaspils, Latvia
*
Author to whom correspondence should be addressed.
Appl. Sci. 2026, 16(6), 2897; https://doi.org/10.3390/app16062897
Submission received: 23 December 2025 / Revised: 9 March 2026 / Accepted: 13 March 2026 / Published: 17 March 2026
(This article belongs to the Section Applied Biosciences and Bioengineering)
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This work supports the development of more effective fungal-based plant protection products by identifying cultivation conditions that maximize spore production of Phlebiopsis gigantea isolate PG 182. By evaluating both liquid-surface and solid-state fermentation parameters, the study demonstrates how medium composition, substrate enrichment, and cultivation time influence spore yield. These insights can guide scalable production of high-quality fungal inoculum for forestry and agricultural applications.

Abstract

Fungal spores are the main active ingredients in fungal preparations. In this study, we evaluated vegetative spore (oidia) production of the Latvian isolate of Phlebiopsis gigantea PG 182 using liquid-surface (LSF) and solid-state (SSF) fermentation processes in the 450 mL and 700 mL jars, respectively. The effects of medium depth (0.5 or 0.7 cm), malt extract (ME) syrup concentration (25, 50, and 75 g/L) and duration time of cultivation (7, 14, 21 and 28 days) on oidia production and partly on mycelium biomass yield were evaluated in the LSF experiments. The highest spore yields (0.88 ± 0.22) × 107 and (1.10 ± 0.31) × 107 (95% CI) (oidia/g liquid medium) were achieved on day 28 in the LSF process using a medium depth of 0.5 cm and ME concentrations of 25 and 50 g/L, respectively. While in the SSF process, pine sawdust enrichment with wheat bran (0, 5, 10, 15, and 25%) and cultivation time (14, 21 and 28 days) were evaluated under conditions of 8 cm substrate depth. The most promising result was obtained on day 28 with 10% bran supplementation, reaching (24.73 ± 5.09) × 107 (95% CI) (oidia/g solid medium), which is 1.45 and 3.17 times more than after 21 and 14 days of cultivation, respectively. Our findings indicate that SSF with a 10% wheat bran additive produces superior spore yields for P. gigantea isolate PG 182, exceeding benchmarks set by comparable research. Potential for further improvement remains by optimizing the wheat bran (WB)-to-substrate ratio and refining the thermal processing of the solid substrate.

1. Introduction

Phlebiopsis gigantea (Fr.) Jülich is a white rot fungus widely used in Europe for the biological control of Heterobasidion spp., one of the main fungal pathogens reducing the economic value of conifer wood [1,2,3]. Preventive stump treatment includes the application of chemical control agents such as urea and borates, or biological control agents (BCA) containing the saprotrophic fungus P. gigantea [3], to inhibit spore germination on freshly cut stumps. Biological treatment with P. gigantea is based on rapid stump colonization and competitive exclusion of the pathogen and is increasingly favored due to its biological mode of action and compatibility with sustainable forest management practices (e.g., Rotstop® preparation use). P. gigantea represents a well-established and robust, well-characterized biocontrol organism, particularly in terms of cultivation feasibility and historical efficacy against Heterobasidion spp. and is therefore commonly used as a reference biocontrol organism [4]. Different P. gigantea strains display considerable variability in functional traits, such as mycelial growth rate, when colonizing different conifer species [5]. To provide the high efficiency of the BCA and to reduce the long-term ecological impact of the single P. gigantea isolate contained in the preparation Rotstop on the diversity of the local fungi population, it might be advisable to use P. gigantea isolates of local origin and to change the P. gigantea isolate every few years [6,7,8].
Filamentous fungi produce two types of propagules: mycelium and spores. P. gigantea produces asexual spores (oidia), which arise from segmentation of hyphae [9] and are typically produced under free availability of gaseous oxygen. The spores display distinct morphological, functional, and biochemical characteristics that enable them to persist as viable for longer periods under environmental and processing conditions [10,11]. Therefore, spores are considered the preferred propagule type for P. gigantea BCA formulations [12,13].
Solid-state fermentation (SSF) and submerged (liquid) fermentation (SmF) are established bioprocessing strategies for the controlled cultivation of fungi, enabling the production of mycelial biomass and/or spores. However, mycelium produced by SmF retains the capacity for spore production afterwards under wet and air exposure conditions. The composition of the liquid medium could also activate or inhibit growth and sporulation when environmental conditions are conducive to these fungal life-cycle stages [14]. A mixed SSF–SmF process in which a surface (liquid–air interface) exists is referred to as liquid-surface fermentation (LSF). Under these conditions, gaseous oxygen is freely available, while the submerged part contains nutrients and dissolved oxygen, forming favorable conditions for mycelium formation. In such a process, spore formation occurs in areas where the mycelium comes into contact with air.
SSF occurs in the absence or near absence of free water, typically utilizing a natural substrate as a carbon and energy source (wheat bran, rye, rice, seeds, roots, wood pieces, sawdust, etc.) and is the primary method used to produce fungal spores [15]. The principal advantages of SSF include the fungal capacity for sporulation and the utilization of potentially low-cost substrates. However, SSF does have some disadvantages, including difficulties in scale-up (due to challenges in maintaining substrate uniformity), limited process control (due to the heterogeneous nature of the solid substrate), and limited product recovery (requiring more complex downstream processing due to the presence of solids in the fermentation mixture) [15,16]. Typically, SSF processes are performed in tray, packed-bed, or rotating drum bioreactor systems [17]. Virtanen et al. [13,18] reported P. gigantea SSF cultivation in plastic bags, trays, a novel packed bed bioreactor, and a ~100 L volume bioreactor and found P. gigantea spore yields of 3.6–5.4 × 106 CFU/g. In another study, small-scale P. gigantea spore production was investigated on straw, rye bran, and coniferous sawdust, where cultivation for 28 days resulted in the highest yield on coniferous sawdust, reaching (7.9 ± 4.3) × 106 oidia/g [19].
In contrast, SmF occurs in the presence of excess water [20]. Depending on the SmF cultivation conditions, mycelium may appear as a suspension (suspension of the short mycelium fragments), clumps (localized growth of the mycelium in larger aggregates), or pellets (closely packed and uniformly dispersed form of mycelium). Typically, fungus SmF processes are performed in stirred-tank, bubble column, and concentric-tube airlift bioreactor systems [21]. Mycelium and spores of different fungi, e.g., Penicillum, Beauveria bassiana, and Trichoderma, have been cultivated in SmF [14,22,23,24]. Scarce information is available in the literature about P. gigantea SmF processes. Kuznetsov and Ruchi [25] demonstrated P. gigantea production using alcohol stillage waste products as the main medium constituents and found that the highest fungal biomass (7.9–9.8 g/L) and spore yields (1.5 × 106 CFU/mL) were achieved in a medium consisting of alcohol stillage supplemented with 5% sawdust.
The aim of our study was to evaluate spore (oidia) production of the Latvian Phlebiopsis gigantea isolate PG 182 in LSF and SSF processes. The specific aims were to assess the effect of: (i) ME type (microbiological grade or food grade syrup) and concentration in the LSF process; (ii) the proportion of wheat bran supplementation (w/w %) to pine wood shavings in SSF; and (iii) the cultivation duration over a period of 7–28 days.

2. Materials and Methods

2.1. Inoculum

For both LSF and SSF modes, the inoculum was prepared in 1 L Erlenmeyer-type flasks, containing 250 mL of microbiological grade ME (Carl Roth GmbH + Co. KG, Karlsruhe, Germany) broth at a concentration of 15 g/L. The medium was sterilized at 121 °C for 20 min. Each flask was inoculated with a mycelial disk (⌀ = 14.5 mm) taken from a P. gigantea isolate PG 182 (isolate was collected from Scots pine wood under fruit bodies of P. gigantea located in eastern Latvia) culture grown on Hagem agar medium. The inoculum flasks were incubated at room temperature (approximately 21 °C to 25 °C) under a natural light cycle (indirect sunlight during the day; irregular exposure to artificial light during dark periods). After 14 days of cultivation, the inoculum was agitated using a magnetic stirrer MSH-300 (Biosan, Riga, Latvia) set at 1250 rpm, employing a magnetic stir bar (⌀ = 8 mm; length = 25 mm) for 60 min. After agitation, the culture was left to settle for ~5 min and then decanted. Larger biomass fragments remaining in the flask were discarded, and the decanted fraction was used as the inoculum. The optical density of the inoculum (OD630, absorbance mode) was 0.10–0.12, as determined using a BK-V1000 spectrophotometer (BIOBASE, Jinan, China).
For the LSF process, the jars containing liquid media were inoculated with the fine fraction of the decanted inoculum in an amount of 1% (v/v). Following inoculation, the jars were gently hand-agitated for approximately 10 s to ensure even distribution.
For the SSF process, the jars containing solid substrate were inoculated with the decanted fraction of the inoculum at a ratio of 1 mL to 1 g of substrate. Following inoculation, the jars were vigorously hand-shaken for approximately 10 s.

2.2. LSF Process

LSF was used to examine the influence of liquid medium composition and cultivation duration on the growth and oidia production of Phlebiopsis gigantea isolate PG182 under static, controlled conditions. Cultivation was performed without agitation to promote the formation of a surface-associated mycelial layer and to avoid mechanical disturbance that could affect mycelial integrity and sporulation. Several liquid media, including ME-based and defined formulations differing in carbon source concentration and inorganic nutrient supplementation, were evaluated. The LSF system was implemented in small-volume glass jars (containing shallow liquid medium layers). Sufficient oxygen availability at the air–liquid interface was maintained by using non-hermetically sealed lids that allowed passive gas exchange during incubation.

2.2.1. Medium

The evaluated media consisted of food-grade ME syrup (ILGEZEEM, Riga, Latvia) at 25, 50, and 75 g L−1 and microbiological-grade ME at 15 g L−1 (media pH 5.1 at inoculation). The food-grade ME syrup used in the LSF experiments consisted of approximately 75% dry matter and represented a carbohydrate-rich medium (70.7 g per 100 g syrup), of which 35.6 g were sugars, with a smaller protein fraction (3.3 g per 100 g). It also contained trace amounts of B-group vitamins, including thiamine (0.2 mg), niacin (3.1 mg), vitamin B6 (0.4 mg), and vitamin B12 (0.4 mg).

2.2.2. Cultivation (LSF)

LSF was used to examine the influence of liquid medium composition and cultivation duration on the growth and oidia production of Phlebiopsis gigantea isolate PG 182 under static, controlled conditions. Cultivation was performed without agitation to promote the formation of a surface-associated mycelial layer and to avoid mechanical disturbance that could affect mycelial integrity and sporulation. Several liquid media, including ME-based and defined formulations differing in carbon source concentration and inorganic nutrient supplementation, were evaluated. The LSF system was implemented in small-volume glass jars (d = 8.5 cm, h = 10.5 cm, working volume of 480 mL) containing shallow liquid layers of 0.5 or 2.0 cm. Sufficient oxygen availability at the air–liquid interface was maintained by using non-hermetically sealed lids that allowed passive gas exchange during incubation.
The inoculated jars were then incubated in a static incubator ES-20 Orbital Shaker-Incubator (Biosan, Riga, Latvia) at 28 °C (Figure 1A). Two medium compositions were tested per experimental run, with nine inoculated jars prepared and analyzed for each composition. Samples were collected in groups of three replicates and analyzed on days 7, 14, 21, and 28 of the experiment.

2.3. SSF Process

2.3.1. Solid Substrate

A bulk solid substrate consisted of pine wood shavings, and depending on the experiment, it was enriched with wheat bran additives. Initial pine wood shavings consisted of <2 mm (25%), 2–7 mm (48%), 7–13 mm (24%) and >13 mm (3%) fractions, with the bulk density of 0.0528 g/mL. The bulk substrate was sieved, with the finer wood shavings consisting of fractions <2 mm discarded and the larger fractions recombined, leading to a substrate consisting of fractions 2–7 mm (64%), 7–13 mm (32%) and >13 mm (4%) to be used for cultivation, with the resulting density of 0.0412 g/mL. The moisture content of the wood shavings was 6% before the sieving, dropping to 1% after recombining the larger fractions. Based on preliminary experiments, a substrate moisture content of 60% was selected for cultivation experiments. The target moisture level was obtained by adding a combination of sterile distilled water and liquid inoculum to the substrate.
The bulk density of the wood shavings was measured following the procedure described by Rezaei et al. (2016) [26]. For moisture content determination, an aliquot of wood shavings was weighed into beakers using an electronic precision balance EMS 300-1 (KERN & SOHN GmbH, Balingen–Frommern, Germany) and placed in a drying oven at a constant temperature (105 °C) until the sample mass stabilized. The moisture content (on a wet basis) was calculated from the difference between the initial and final (dry) masses. To determine the saturation moisture content, wood shavings were fully submerged in water for 24 h. After soaking, excess surface water was removed by shaking the samples on a sieve. A representative aliquot was transferred to pre-weighed beakers and dried to constant weight. The moisture content at full saturation was calculated from the difference between the soaked and dried masses, representing the maximum moisture level the wood shavings could retain without further absorption.
The jars containing the selected substrate were sterilized by autoclaving at 121 °C for 20 min.

2.3.2. Cultivation (SSF)

Solid-state fermentation was conducted in a custom water-bath incubator designed to maintain a stable temperature and reduce substrate moisture loss (see Figure 1B). Fermentation jars were partially submerged in temperature-controlled water regulated by a circulating water-bath thermostat TW-2.02 (ELMI, Riga, Latvia), while humidified air was supplied at 0.5 L min−1 using an air compressor Dynair DA5001/4C (Jiangsu Dynamic Medical Technology Co., Ltd., Kunshan, China). In total, the incubator could simultaneously fit 18 of the selected 700 mL jars (d = 9.5 cm, h = 11 cm). The jars were sealed with loosely fitting metal lids. The jars were filled with 20 g (with a medium depth of 9 cm) of a solid substrate of selected composition. For cultivation purposes, the substrate moisture content was adjusted to 45% using distilled water, reaching a total of 60% when inoculum was added. In experiments where the enrichment of the substrate with wheat bran was investigated, an aliquoted amount of the wheat bran was added to the sawdust (at concentrations of 5%, 10%, 15% and 25%). For each tested substrate composition, nine replicates were prepared and analyzed. The inoculated jars were incubated statically at 28 °C, with two substrate compositions tested in each experimental run. Samples were collected in groups of three replicates and analyzed on days 14, 21, and 28 of the experiment.

2.4. Oidia Count Analysis

Oidia concentration was determined using a hemocytometer, with appropriate dilution applied when necessary, using a light microscope DM750 (Leica Microsystems, Wetzlar, Germany). For each liquid-culture jar sample, oidia concentrations were determined from three independent hemocytometer Neubauer improved 0.1 mm (Assistent, Sondheim vor der Rhön, Germany) counts, each based on enumeration of oidia in the four large corner squares of the hemocytometer chamber, whereas for solid-state jar samples, one such hemocytometer count was performed per sample.

2.4.1. LSF

For oidia quantification, the total volume in each culture-containing jar was adjusted to 100 mL with sterile 0.9% NaCl solution containing 1% Tween 80. The sample was agitated using a magnetic stirrer at 1250 rpm for a total duration of 45 min.

2.4.2. SSF

For oidia quantification, an aliquoted amount of substrate was separated from the main mass, and the spores were washed off using a 1% (v/v) Tween 80 solution. Approximately 5 g of the substrate was added to an Erlenmeyer-type flask, and 100 mL of sterile 0.9% (w/v) NaCl solution containing 1% Tween 80 was added. The oidia containing substrate was then washed by placing the flask in an orbital shaker-incubator set to 150 rpm for 30 min. After 30 min of agitation, the sample was taken out of the mixer, and the oidia concentration in the solution was determined using a hemocytometer under a light microscope.

2.5. Oidia Viability Analysis

Viability of the P. gigantea oidia was assessed by spreading the suspension onto Hagem agar medium immediately after agitation of the cultivated samples for oidia enumeration, followed by incubation at room temperature (approximately 21–25 °C) for 24 h. Viable oidia were counted using a light microscope. For each sample, five randomly selected fields of view on the agar surface were examined using 200× total magnification, with oidia assessed within a single fixed field of view per observation, without lateral movement of the stage. Oidia viability was subsequently calculated based on these observations.

2.6. Statistical Analysis and Mathematical Modelling

P. gigantea oidia production was analyzed in three to nine replicates of each tested medium/substrate in LSF and SSF. Analysis of variance (one-way and two-way ANOVA) and Tukey tests at a 95% confidence level were performed in RStudio [27] using R programming (R 4.5.2) [28] to compare the mean values of the experimental data and to evaluate the effects of wheat bran additive, ME syrup concentration, cultivation duration, and their interactions on P. gigantea oidia yields.
Programming in MATLAB (R2019a, MathWorks, Natick, MA, USA) using .m code was employed to model the investigated mycelium growth and spore production processes. The MATLAB Curve Fitting Toolbox was utilized to fit mathematical models to the experimental data and to estimate kinetic parameters.

3. Results

Phlebiopsis gigantea Latvian isolate PG 182 SSF and LSF processes were investigated.

3.1. Liquid-Surface Fermentation (LSF)

The aim of this process is to evaluate spore production using mycelium grown in a thin-layer liquid medium.

3.1.1. Mycelium and Spore Biomass

The aim of the subsequent experimental series was to ensure higher oidia yields by changing different parameters of the LSF process. The initial concept was based on the idea that the cultivated mycelium could be separated from the liquid medium and exposed to air, thereby allowing oidia to form on its surface. In a preliminary experiment, P. gigantea biomass (mainly mycelium) production was evaluated using different ME types—microbiological grade ME (15 g/L) and ME syrup (25 g/L)—with a medium depth of 2.0 cm (Figure 2).
It was found that up to day 14, a similar biomass growth rate was observed for both analyzed media; by day 21, the growth was higher in the ME syrup medium (Figure 3A). Total yield of P. gigantea biomass in ME syrup medium was 0.24 ± 0.03 g (dry weight)/g (medium), representing a 40% higher increase compared to biomass in microbiological grade ME medium. Logistic growth model fitted for biomass dry weight (W) (model parameters for microbiologic grade ME: max weight (Wmax) 0.182 g, initial weight (W0) 0.037 g, and growth rate (µ) 0.19 1/d; model parameters for ME syrup: Wmax = 0.301 g, W0 = 0.033 g, µ = 0.16 1/d).
Considering both the higher yield and the fact that ME syrup is a more industrially suitable raw material due to its lower cost, it was selected for further studies.
By reducing the medium depth from 2.0 to 0.5 cm, a similar result of 0.20 ± 0.01 g (dry weight)/g (medium) was obtained within 21 days using the ME syrup medium (25 g/L). Formation of spores started earlier at a lower medium depth because the mycelium was more exposed to the air. Model parameters for ME syrup medium depth 0.5 cm case: Wmax = 0.205 g, W0 = 0.048 g, µ = 0.22 1/d (Figure 3A). Given that biomass production reached its stationary phase (plateau), the yield was estimated at 0.32 g of mycelium per gram of ME syrup, based on a final biomass of 0.2 g in 0.025 L of medium at a 25 g/L concentration. The relationship between the maximum biomass growth rate and the concentration of ME syrup is illustrated in Figure 3B.
These observations provided the basis for revising the initially considered strategy, suggesting that mycelium obtained via the LSF process can be cultivated in a sufficiently thin liquid medium layer. As the medium gradually evaporates during cultivation, the already formed mycelium becomes drier, thereby promoting spore formation on its surface. Preliminary experiments indicated a substantial increase in spore concentration between days 14 and 21 of the process, suggesting that a longer cultivation period is required to evaluate the potential increase in oidia production. It was also determined that a liquid medium depth of 0.5 cm is the most suitable, as by day 28 the mycelium is not yet completely dried, while the depth is sufficiently low to allow timely spore formation at the liquid–air interface. As illustrated by the subsequent results, the most rapid increase in spore production primarily occurs during the third week of cultivation. By the fourth week, this trend levels off, suggesting an optimal cultivation period of up to four weeks.

3.1.2. Oidia Formation

A series of experiments was conducted to evaluate the effects of ME syrup concentrations (25, 50, and 75 g/L) on the dynamics of oidia formation over time intervals of 14, 21, and 28 days. The highest production of P. gigantea oidia was obtained after cultivation for 28 days in all analyzed variants of medium (Figure 4).
ANOVA was conducted to determine the effect of medium concentration/composition and cultivation duration on P. gigantea oidia yields. P. gigantea oidia production was significantly affected by medium concentration/composition (F2.36 = 7.41, p = 0.002) and cultivation duration (F2.36 = 61.28, p < 0.001); interaction of these two factors did not have a significant effect (p = 0.175).
P. gigantea oidia yield was the highest in ME syrup medium on day 28 at a concentration of 50 g/L—(1.10 ± 0.31) × 107 (95% CI) (oidia/g liquid medium). During the early cultivation phase (within the first 14 days), differences in ME concentration do not yet affect spore concentration. At this stage, the fungus is still adapting and establishing growth, and the average spore count across all groups remains statistically similar (p > 0.05, result of a one-way ANOVA for the separate time points). On day 21, significant differences in results were observed between concentrations (p = 0.0001). According to Tukey’s post-hoc test, significant differences were identified for 50 g/L vs. 75 g/L (p = 0.0001) and 25 g/L vs. 75 g/L (p < 0.005). The 75 g/L concentration likely exerts an inhibitory (or retarding) effect; potentially, high osmotic pressure or substrate excess delays the transition to the sporulation phase. However, by the end of cultivation, the differences had leveled out. Regardless of the initial ME concentration (25, 50, or 75 g/L), the fungus reached its maximum spore production capacity—approximately 107 oidia/g in all variants (p > 0.05).
The course of P. gigantea growth was assessed also visually (Figure 5).
P. gigantea oidia viability levels were in a similar range of 90–95% regardless of their sampling time after 14, 21, and 28 days.
Logistic growth model was fitted for oidia concentration (CO) (Figure 6A) (model parameters for ME syrup 25 g/L: oidia max concentration (CO,max) 0.91 × 107 oidia/g of liquid substrate, oidia initial concentration (CO,0) 1.65 × 103 oidia/g, growth rate (µ) 0.42 1/d; model parameters for ME syrup 50 g/L: CO,max = 1.14 × 107 oidia/g, C O,0 = 5.49 × 103 oidia/g, µ = 0.40 1/d; and model parameters for ME syrup 75 g/L: CO,max = 1000 × 107 oidia/g, C O,0 = 5.79 × 103 oidia/g, µ = 0.17 1/d).
It should be noted that the CO,max identified for the 75 g/L variant is likely unsuitable for modeling spore accumulation beyond day 28. Aside from the nutritional impact of the ME concentration, the gas-liquid interface (medium-air boundary layer) acts as a critical limiting factor. Given that all three concentrations yielded similar results by day 28, it is highly probable that the system has reached its maximum spore production capacity. The relationship between the oidia concentration and the ME syrup concentration is illustrated in Figure 6B. This mathematical relationship is described by a polynomial fit.

3.2. Solid-State Cultivation (SSF)

The effect of enriching the pine wood shavings substrate with varying amounts of wheat bran (5, 10, 15, and 25%) on P. gigantea oidia production was evaluated. The selected thermal treatment regime for the sterilization of substrate was not enough, and cases of non-sterility were common in processes with 15% and 25% bran supplementation. The data is not included.
Even a small wheat bran supplementation of 5% promoted a substantial increase in oidia production (Figure 7). ANOVA was conducted to determine the effect of substrate composition and cultivation duration on P. gigantea oidia yields. P. gigantea oidia production was significantly affected by supplementation of wheat bran (F2.63 = 26.04, p < 0.001) and duration of cultivation (F2.63 = 4.18, p = 0.019) and interaction of these two parameters (F2.63 = 3.05, p = 0.006). The effect of wheat bran supplementation was time-dependent; this was most evident in the 10% WB treatment (p < 0.05), particularly during the interval between day 14 and day 28 (p = 0.0011).
A visual insight into P. gigantea growth on pine wood shavings is provided in Figure 8.
Wheat bran supplementation at 5% and 10% yielded similar results, with a tendency for oidia numbers to increase from day 14 to day 28, and the obtained values were close within the error margins. The highest result, (24.73 ± 5.09) × 107 (95% CI) (oidia/g solid medium), was achieved with 10% bran supplementation. This is 1.45 and 3.17 times more than after 21 and 14 days of cultivation, respectively.
A linear relationship was established between spore concentration and time (t) across the series with varying wheat bran (WB) additive concentrations (Figure 9A). The oidia formation rate as a function of wheat bran (WB) concentration was modeled using a mechanistic Monod equation (Figure 9B). This model describes a system where the resource (WB additive) is the limiting factor at low concentrations (base growth rate µ0,base = 0.067 1/d at WB = 0%) but reaches saturation at higher levels (saturation constant KS = 6.09%, max growth rate µ0,max = 1.48 1/d).
P. gigantea oidia showed high viability levels of approximately 90–95% at day 28, similar to those observed in the LSF process.

4. Discussion

The aim of this study was to evaluate the yields of P. gigantea oidia in LSF and SSF processes. Our research showed great variation in P. gigantea oidia production. Also, other research has shown P. gigantea spore production from 0.7 to 271 million oidia per Petri dish with ME agar medium [19,29,30,31] and analyzed the same P. gigantea isolate PG 182 and obtained 1.5 million oidia in 1 mL suspension (ca. 0.75 million spores/Petri dish) after cultivation for 12 days on ME agar at 20 °C. Only an approximate comparison of oidia production in our research could be made with results from previously mentioned other research because the composition of media, temperature, moisture content, and other factors affect the growth of P. gigantea mycelium and oidia production; also, the correct stage of fungal development must be established and the precise physiological conditions to achieve the maximal growth of mycelium and spore production [32,33]. Virtanen et al. [13] observed that P. gigantea reached its late exponential phase in liquid medium after incubation for 5 days at 28 °C. In our experiment, we used an inoculum incubated for 14 days at 21–25 °C. Beyond the specific experimental findings, a limitation of this study was the use of a single fungal isolate, which restricts the generalization of our findings. The host tree species of origin of P. gigantea has an effect on the growth of the fungus [5].
P. gigantea oidia production in pine sawdust did not change depending on the time of cultivation; it increased only if sawdust was supplemented by wheat bran. Wheat bran contains thiamine, niacin, potassium, iron, magnesium, phosphorus, and other chemical elements that are needed by most fungi for vigorous growth [34,35]. The highest yield of P. gigantea oidia (24.73 ± 5.09) × 107 (95% CI) (oidia/g solid medium) was obtained in the SSF process using pine wood shavings enriched with 10% wheat bran after incubation for 28 days. Pine wood shavings with wheat bran supplementation above 10% in SSF may potentially yield even higher results of P. gigantea oidia production, but unfortunately, using the selected thermal treatment regime (autoclaving at 121 °C for 20 min) for substrate sterilization, cases of non-sterility were common in processes with 15% and 25% bran supplementation.
In our research, P. gigantea oidia yields after 14 days of incubation in pine wood shavings without wheat bran were four times higher than in the research of Virtanen et al. [13,18], where P. gigantea was incubated for 10 days at a lower temperature (2 days at 28 °C and 8 days at 22 °C). The most comparable experiment to our research in terms of experimental design was published by Burņeviča et al. [19], where P. gigantea was cultivated in mixed pine and spruce sawdust for 21 days. Our obtained yield of (2.03 ± 0.36) × 107 (95% CI) oidia/g on pine wood shavings without wheat bran supplementation after 21 days was approximately two times higher. This difference may be attributed to substrate morphology, as in our study, pine wood shavings provided a larger free surface area and most likely better aeration conditions than the denser pine and spruce sawdust substrate used by Burņeviča et al. [19]. Other research has shown that sawdust of different tree species has an effect on the growth of P. gigantea; the best substrates for P. gigantea cultivation were sawdust of European larch and Scots pine [36] and a mixture of linden and Scots pine sawdust [37]. The research of Fedorov et al. (1981) [38] has shown that the size of the sawdust fraction has a significant effect on the growth of P. gigantea. In further research, it would be valuable to assess how the size of the substrate fraction affects the growth of P. gigantea.
The mycelial and spore biomass yield in the LSF process—0.2 g in 0.025 L of medium at a 25 g/L concentration on day 21—is approximately threefold higher than the yield obtained using the basal medium reported by Prasher et al. [33]. In their study, the growth peak for P. gigantea biomass was reached around day 12. These findings demonstrate that ME syrup serves as a cost-effective and efficient medium for P. gigantea biomass production, addressing an important obstacle to technology scale-up.
The main conclusion of the LSF process was that the highest production of P. gigantea oidia was observed in ME syrup medium (50 g/L) after incubation for 28 days. Also, in other research where fungi from Ganoderma species were analyzed, more rapid growth was found on ME with lower concentrations compared to higher ME concentrations [19,39]. The food-grade ME syrup used in the LSF experiments contains readily available and more complex nutritional sources as well as micronutrients that are commonly associated with fungal growth media. The defined nutrient profile provides a reference for interpreting the growth conditions used in the LSF process. Our observations indicate that P. gigantea mycelium can be successfully cultivated via LSF in a thin layer (ca. 0.5 cm), provided that the medium surface is directly exposed to the atmosphere at an incubation temperature of 28 °C. By selecting a liquid medium depth that supports mycelial growth and subsequent spore formation on the partially dried mycelium, both processes could be done continuously within a single cultivation vessel. Although LSF yielded a substantially lower output per unit of substrate—approximately 20-fold lower at (1.1 ± 0.04) × 107 oidia/g of liquid medium—compared to SSF, this process offers advantages for scaling. Specifically, LSF allows for easier handling and sterilization of the culture medium. Furthermore, the final product can be more readily obtained in powder or liquid form, as spores do not require separation from wood shavings. However, the technical hurdles associated with preparing and operating numerous tray-type vessels in a sterile environment must be addressed. It should be noted that this type of LSF represents one of the least studied approaches for spore production in filamentous fungi such as P. gigantea. Our findings in this regard may support and advance further developments in the spore production of filamentous fungi using this type of process.

Author Contributions

Conceptualization, O.G.; methodology, O.G., K.K. and K.R.G.; validation, O.G.; formal analysis, O.G., K.K., D.K. and T.G.; investigation, O.G., I.S., K.R.G. and E.V.-A.; resources, O.G., K.K. and D.K.; data curation, O.G., I.S. and K.R.G.; writing—original draft preparation, O.G. and I.S.; writing—review and editing, O.G., I.S. and K.K.; visualization, O.G. and I.S.; supervision, O.G., D.K. and T.G.; funding acquisition, O.G. and T.G. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Latvian Council of Science State Research Program: Innovation in Forest Management and Value Chain for Latvia\Growth: New Forest Services, Products and Technologies\(Forest4LV), project No. VPP-ZM-VRIIILA-2024/2-0002.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

The raw data supporting the conclusions of this article will be made available by the authors on request.

Acknowledgments

We thank Elina Didrihsone for her administrative support to the project.

Conflicts of Interest

The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results.

Abbreviations

The following abbreviations are used in this manuscript:
MEMalt Extract
LSFLiquid-Surface Fermentation
SSFSolid-State Fermentation
SmFSubmerged Fermentation
nNumber of independent measurements
WBWheat Bran

References

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Figure 1. Implementation of the LSF and SSF processes in an air-heated incubator (A) and a water-bath thermostat (B), respectively.
Figure 1. Implementation of the LSF and SSF processes in an air-heated incubator (A) and a water-bath thermostat (B), respectively.
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Figure 2. Growth of P. gigantea PG 182 biomass in liquid medium (malt extract syrup) with a medium depth of 2 cm. (A) day 1, (B) day 7, (C) day 14 and (D) day 21.
Figure 2. Growth of P. gigantea PG 182 biomass in liquid medium (malt extract syrup) with a medium depth of 2 cm. (A) day 1, (B) day 7, (C) day 14 and (D) day 21.
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Figure 3. Growth dynamics and kinetic modeling of P. gigantea PG 182 mycelial biomass during liquid-surface fermentation (LSF). (A) Mycelium dry weight measurements and growth models for ME syrup at medium depths of 2.0 cm (brown) (day 7: n = 3; day 14: n = 3; day 21: n = 3) and 0.5 cm (yellow) (day 7: n = 3; day 14: n = 2; day 21: n = 3), compared to microbiological ME at a depth of 2.0 cm (blue) (day 7: n = 3; day 14: n = 3; day 21: n = 3). (B) Model of the maximum specific growth rate (µmax,ME) of mycelium across various initial ME syrup concentrations.
Figure 3. Growth dynamics and kinetic modeling of P. gigantea PG 182 mycelial biomass during liquid-surface fermentation (LSF). (A) Mycelium dry weight measurements and growth models for ME syrup at medium depths of 2.0 cm (brown) (day 7: n = 3; day 14: n = 3; day 21: n = 3) and 0.5 cm (yellow) (day 7: n = 3; day 14: n = 2; day 21: n = 3), compared to microbiological ME at a depth of 2.0 cm (blue) (day 7: n = 3; day 14: n = 3; day 21: n = 3). (B) Model of the maximum specific growth rate (µmax,ME) of mycelium across various initial ME syrup concentrations.
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Figure 4. The effect of ME syrup concentration on the dynamics of P. gigantea isolate PG 182 oidia formation. Data represent mean values ± 95% confidence intervals. Individual data points are overlaid to illustrate replicate distribution: for malt ME syrup concentrations of 25 g/L (light brown) (day 14: n = 3; day 21: n = 3; day 28: n = 3), 50 g/L (light green) (day 14: n = 6; day 21: n = 6; day 28: n = 6), and 75 g/L (light purple) (day 14: n = 3; day 21: n = 3; day 28: n = 3).
Figure 4. The effect of ME syrup concentration on the dynamics of P. gigantea isolate PG 182 oidia formation. Data represent mean values ± 95% confidence intervals. Individual data points are overlaid to illustrate replicate distribution: for malt ME syrup concentrations of 25 g/L (light brown) (day 14: n = 3; day 21: n = 3; day 28: n = 3), 50 g/L (light green) (day 14: n = 6; day 21: n = 6; day 28: n = 6), and 75 g/L (light purple) (day 14: n = 3; day 21: n = 3; day 28: n = 3).
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Figure 5. Formation of P. gigantea mycelium and oidia on the surface of a liquid medium (ME syrup 50 g/L, medium depth of 0.5 cm) on Day 14 (A), Day 21 (B), and Day 28 (C).
Figure 5. Formation of P. gigantea mycelium and oidia on the surface of a liquid medium (ME syrup 50 g/L, medium depth of 0.5 cm) on Day 14 (A), Day 21 (B), and Day 28 (C).
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Figure 6. Dynamics and kinetic modeling of P. gigantea (PG 182) oidia formation during liquid-surface fermentation (LSF). (A) Oidia concentration measurements and formation models for ME syrup concentrations of 25 g/L (light brown) (day 14: n = 3; day 21: n = 3; day 28: n = 3), 50 g/L (light green) (day 14: n = 6; day 21: n = 6; day 28: n = 6), and 75 g/L (light purple) (day 14: n = 3; day 21: n = 3; day 28: n = 3). (B) Kinetic model of the specific oidia formation rate (µO) as a function of various initial ME syrup concentrations.
Figure 6. Dynamics and kinetic modeling of P. gigantea (PG 182) oidia formation during liquid-surface fermentation (LSF). (A) Oidia concentration measurements and formation models for ME syrup concentrations of 25 g/L (light brown) (day 14: n = 3; day 21: n = 3; day 28: n = 3), 50 g/L (light green) (day 14: n = 6; day 21: n = 6; day 28: n = 6), and 75 g/L (light purple) (day 14: n = 3; day 21: n = 3; day 28: n = 3). (B) Kinetic model of the specific oidia formation rate (µO) as a function of various initial ME syrup concentrations.
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Figure 7. The effect of wheat bran supplementation on the production of P. gigantea isolate PG 182 oidia on pine wood shavings as a substrate. Data represent mean values ± 95% confidence intervals. Individual data points are overlaid to illustrate replicate distribution: pine wood shavings without wheat bran (0% WB, brown) (day 14: n = 9; day 21: n = 9; day 28: n = 6), and with WB additions of 5% (green) (day 14: n = 6; day 21: n = 6; day 28: n = 6) and 10% (purple) (day 14: n = 3; day 21: n = 6; day 28: n = 6).
Figure 7. The effect of wheat bran supplementation on the production of P. gigantea isolate PG 182 oidia on pine wood shavings as a substrate. Data represent mean values ± 95% confidence intervals. Individual data points are overlaid to illustrate replicate distribution: pine wood shavings without wheat bran (0% WB, brown) (day 14: n = 9; day 21: n = 9; day 28: n = 6), and with WB additions of 5% (green) (day 14: n = 6; day 21: n = 6; day 28: n = 6) and 10% (purple) (day 14: n = 3; day 21: n = 6; day 28: n = 6).
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Figure 8. Pine wood shavings after 28 days of cultivation: without the addition of wheat bran (A) and with a 10% supplementation of wheat bran (B). (C) microscopy image showing a colonized pine wood shaving and suspended oidia.
Figure 8. Pine wood shavings after 28 days of cultivation: without the addition of wheat bran (A) and with a 10% supplementation of wheat bran (B). (C) microscopy image showing a colonized pine wood shaving and suspended oidia.
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Figure 9. Dynamics and kinetic modeling of P. gigantea PG 182 oidia formation during solid-state fermentation (SSF). (A) Oidia concentration measurements and formation models for pine wood shavings without wheat bran (0% WB, brown) (day 14: n = 9; day 21: n = 9; day 28: n = 6), and with WB additions of 5% (green) (day 14: n = 6; day 21: n = 6; day 28: n = 6) and 10% (purple) (day 14: n = 3; day 21: n = 6; day 28: n = 6). (B) Kinetic model of the specific oidia formation rate (µO) as a function of various initial WB additive amounts.
Figure 9. Dynamics and kinetic modeling of P. gigantea PG 182 oidia formation during solid-state fermentation (SSF). (A) Oidia concentration measurements and formation models for pine wood shavings without wheat bran (0% WB, brown) (day 14: n = 9; day 21: n = 9; day 28: n = 6), and with WB additions of 5% (green) (day 14: n = 6; day 21: n = 6; day 28: n = 6) and 10% (purple) (day 14: n = 3; day 21: n = 6; day 28: n = 6). (B) Kinetic model of the specific oidia formation rate (µO) as a function of various initial WB additive amounts.
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MDPI and ACS Style

Grigs, O.; Sutris, I.; Gasuns, K.R.; Vahere-Abrazune, E.; Kenigsvalde, K.; Klavina, D.; Gaitnieks, T. Obtaining Phlebiopsis gigantea Oidia Using Liquid- and Solid-Surface Cultivation Processes. Appl. Sci. 2026, 16, 2897. https://doi.org/10.3390/app16062897

AMA Style

Grigs O, Sutris I, Gasuns KR, Vahere-Abrazune E, Kenigsvalde K, Klavina D, Gaitnieks T. Obtaining Phlebiopsis gigantea Oidia Using Liquid- and Solid-Surface Cultivation Processes. Applied Sciences. 2026; 16(6):2897. https://doi.org/10.3390/app16062897

Chicago/Turabian Style

Grigs, Oskars, Ilvars Sutris, Karlis Roberts Gasuns, Evelina Vahere-Abrazune, Kristine Kenigsvalde, Darta Klavina, and Talis Gaitnieks. 2026. "Obtaining Phlebiopsis gigantea Oidia Using Liquid- and Solid-Surface Cultivation Processes" Applied Sciences 16, no. 6: 2897. https://doi.org/10.3390/app16062897

APA Style

Grigs, O., Sutris, I., Gasuns, K. R., Vahere-Abrazune, E., Kenigsvalde, K., Klavina, D., & Gaitnieks, T. (2026). Obtaining Phlebiopsis gigantea Oidia Using Liquid- and Solid-Surface Cultivation Processes. Applied Sciences, 16(6), 2897. https://doi.org/10.3390/app16062897

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