1. Introduction
Composting represents one of the most efficient methods for sustainable waste management. As already highlighted by other authors, this methodology depends on many factors, the most important being the nature, composition, sizing, and quality of the substrate [
1,
2], environmental parameters during the process [
2,
3], and the microbial populations present during composting [
2,
4,
5,
6,
7]. Different methodologies and technologies were applied on the basis of processed materials [
1,
2,
6,
8]. Regardless of the used technology, all composting processes reach high temperatures of about 40–55 °C degrees that are limiting and even lethal for many organisms. The high temperatures of the composting process create strong selection on the biological components: on the one hand, it eliminates many mesophilic organisms that are threatening for plants, but selects for extremophilic and extremotolerant organisms particularly adapted to high temperatures. These thermophilic and thermotolerant micro-organisms are fundamental in biochemical processes since several species have key roles for their effectiveness in lignin degradation and thermoresistant- or thermostable-enzyme production. However, potentially dangerous propagules of the same organisms can be inhaled, becoming harmful for workers who handle daily large quantities of compost, and for end users that live or work, especially in indoor environments, near potentially contaminated sources (e.g., potted plants). Therefore, knowledge of the microbiological components of compost is essential to optimize the process and to make a product of excellent quality, and free of hazards for workers, consumers, and the environment.
In the last few decades, some authors studied the microbial components of compost, mainly focusing on the bacterial components [
7,
9,
10,
11], and other studies listed potentially dangerous species for humans [
12,
13]. Thanks to these studies, commercial products with special bacterial starter cultures, endomycorrhizal fungi, and chemical activators (in particular nitrogen) were developed, and are useful both on the domestic and industrial scale, resulting in the improvement of the composting process and lowering production costs.
With the advent of new high-throughput sequencing technologies, nonculture-based approaches were applied to characterize microbial communities, giving a wider picture on them than classical culture-based approaches [
7,
9,
12,
13,
14,
15]. These methods provided excellent indications of the community, but did not allow the isolation of organisms involved in the process. For these reasons, a culture-dependent method is still essential to isolate vital strains present in the matrix. In fact, isolation of the organisms into an axenic culture allows: (i) to identify organisms, safely discriminating potentially dangerous from harmless species; (ii) verification of the effective viability of strains present in the investigated matrix; (iii) isolation and preservation of living organisms; and (iv) study and exploitation of the organisms’ biochemical characteristics.
In the last decade, studies investigated the fungal communities in compost, confirming that fungi are a significant part of the microbial community, in particular during the maturation phase [
1,
8,
14,
16,
17,
18,
19,
20,
21,
22,
23]. Moreover, some of these studies detected fungal species that are useful during stages of maturation [
19,
21,
24] and/or potentially harmful [
12,
17,
20,
25]. For these reasons, it is fundamental to conduct studies on fungal communities involved in the composting processes in order to select both useful populations to improve the process and to investigate the presence of potentially harmful species.
This work contributes to the knowledge of thermotolerant and thermophilic fungal communities in compost. We characterized the fungal community of compost samples taken during the ripening of the product at five different stages in a composting plant in northwestern Italy. After isolation of the strains in pure culture, they were identified at the species level using the latest taxonomic insights.
3. Results
The heat map in
Table 1 represents the amount of CFUs observed during the monitoring period at different incubation temperatures. The order of magnitude observed of CFUs per gram of compost rose from 1 × 10
4 CFU g
−1 to 3 × 10
5 CFU g
−1 during a maturation period of 21 days.
The isolated strains belonged to 25 genera and were identified as 45 different species. The obtained sequences were deposited in GenBank with the following accession numbers: MT316336-MT616380, MT312848-MT312857, MT420412-MT420424, and MT433447-MT433470. The heat map in
Table 2 reports the taxonomic assignment of the isolated strains and their abundance (calculated as CFU per gram of compost) observed in each site at each time, and their related GenBank accession numbers. The heat map gives an overview of the occurrence of different strains during the maturation period, highlighting the presence of some species in several steps of maturation. The most frequently occurring fungal species (isolated in all five samples) were
Scedosporium apiospermum, Thermomyces dupontii (=
Talaromyces thermophilus) and
Thermomyces lanuginosus. Strains of
Aspergillus chevalieri, A. terreus, and
Talaromyces trachyspermus were isolated in 4 of the 5 samples. They were followed by six species that were isolated in three of the five samples:
Aspergillus fumigatus, Geotrichum sp.,
Malbranchea cinnamomea,
Rasamsonia emersonii (=
Talaromyces emersonii),
Scedosporium aurantiacum, and
Scopulariopsis brevicaulis.Figure 1 reports the total generic distribution and the related distribution at each temperature, expressed as observed percentage in the samples during the maturation period. The cumulative Shannon’s index of all samples, as shown in
Table 3, ranged from 2.60 to 3.44, and evenness increased from 0.67 to 0.84 during the maturation process, highlighting equality in the level of distribution of individuals among the various species present in the mature samples. Results about the assessment of biodiversity at the different temperatures are also reported in
Table 3.
Table 4 reports the results for Jaccard specific similarity between the analyzed samples. During the maturation period, the Jaccard value was higher between adjacent samples while samples distant over time were more different.
4. Discussion
Composting is a complex process based on the biodegradation of organic matter in which fungi play a key role during the ripening phase. Our data, collected in an active plant, confirmed the presence of thermotolerant and thermophilic fungi. During the maturation period, the number of CFUs increased, biodiversity rose, while mycobiota evolved from unequal distribution among species at the beginning of maturation to fairer distribution between species at maturity. Despite this evolution, we confirmed that the mycobiota were rather stable from a qualitative point of view. However, several potentially pathogenic species emerged in all phases of the cycle, and, for this reason, the periodic monitoring of the concentrations of certain species during the production cycle is necessary. For example, in our case, the opportunistic pathogenic fungal species Aspergillus fumigatus, A. terreus, and Scedosporium spp (in particular S. apiospermum) were found in high concentrations.
Analyzing our data, we observed an increase in the number of CFUs during the maturation period up to 10 times in the final product compared to the digestate and the mixed product sampled after one day of maturation (see
Table 1). The high temperatures at the beginning of the process probably temporarily inhibited germination that restarted once the pile reached a temperature of around 45 °C.
With regard to the specific composition of the sampled populations, about 25% of the isolated strains were shared in at least 3 of the 5 samples, thus present in at least 60% of the process (
Table 2). The Jaccard index shown in
Table 3 indicates a similarity between samples ranging from 0.16 to 0.46. The Jaccard index reported in
Table 4 shows that the similarity between successive samples increased over time, and the specific composition of the sample became more stable. Furthermore,
Figure 1 shows that, in the first four samples (TQ, 1D, 7D, 14D), there was a strong predominance of genera
Thermomyces,
Scedosporium, and
Aspergillus, while the mature sample (21D) was clearly more homogeneous. This was further confirmed by the diversity indices shown in
Table 2. In fact, the biodiversity index rose during maturation from 2.60 to 3.44. More interesting is the trend of evenness that rose steadily to reach a value of 0.84 in the mature sample. This confirmed that, with the progress of the maturation process, distribution in the species within the community became increasingly homogeneous and balanced.
Results concerning the isolated fungi are consistent with previously published data on compost samples [
17,
18,
19,
21]. The main differences could be traced back to changed taxonomic classifications. For example, key species
Thermomyces dupontii was, until 2014, known under its old name,
Talaromyces thermophilus [
32]. Other important compost-associated species that underwent name changes are
Rasamsonia emersonii and
Mycothermus thermophilus, previously known as
Talaromyces emersonii and
Scytalidium thermophilum, respectively. The isolates were primarily identified using ITS sequencing. However, this locus lacks resolution at the species level for some genera detected in the compost, such as
Aspergillus,
Penicillium,
Scedosporium,
Scopulariopsis, and
Talaromyces. In order to obtain reliable species identifications, additional genes (so-called secondary barcodes) were generated to confirm the presence of key species in the composting process. Because information is often linked to a species, correct identification is important from a biotechnological and medical point of view. Accurately identified isolates in this study could have added value for metagenome studies where only partial ITS sequences are often used.
We isolated several thermophilic fungi capable of producing enzymes such as amylase, xylanase, phytase, and chitinase, among which were
Thermomyces dupontii (=
Talaromyces thermophilus),
Thermomyces lanuginosus, and
Thermoascus aurantiacus that degrade the woody components in the compost. However, opportunistic pathogenic species were also found in the samples. The most recurrent genus was
Scedosporium. In particular,
Scedosporium apiospermum was found in all samples, including the mature compost. Five other species,
S. aurantiacum,
S. brevicaulis,
S. dehoogii, S. minutisporum, and
S. prolificans, were found in decreasing numbers. Moreover, a strong presence of
Aspergillus fumigatus was detected in the mature samples. The high temperatures, therefore, shaped the mycobiota during compost maturation. Only some specialized species can survive and proliferate in these particularly hostile conditions. Our results agreed with those of López González et al. [
19]. In fact, we found some predominant species throughout the process (resident mycobiota) in contrast to others occasionally found (transient mycobiota).