Quantification of heavy metals of soil samples
Little is known about the profiles of microbial community composition and structure in these metal contaminated soils. Since a small number of eukaryotes have been investigated with respect to heavy metal resistance so far, the present work presents a solid investigation on the microbial communities from soils contaminated by heavy metal. As part of this investigation, it was carried out the quantification of the metals contained in the samples analyzed, in order to robustly relate the presence of gene sequences and heavy metal contamination. The results are disposed in Table 1.
The genus Aspergillus and Neosartorya have already been found in heavy-metal polluted environments in previous researches. In 2016, Khamesy and co-workers identified two isolates from Aspergillus genus that were able to tolerate the presence of high concentrations of lead and cadmium, between 0 to 2500 mg L-1 in waste deposits. According to Qayyum and co-workers (2016), three strains related to genera Aspergillus and one related to genera Neosartarya were found to be heavy metal resistants. These strains were isolated from sites under a slag heap at the Smelting Industry located in Weifang city, Shandong Province, China. In this work, the authors showed that the fungal isolates tested were able to grow in Sabouraud Dextrose Agar medium containing 50 and 100 mg.L-1 of lead and chromium.
According to Vale et al. (2011), an Aspergillus niger strain was able to develop in culture medium supplied with growing concentrations of Zn and Cr until 100 mg. L-1. Authors found that ion Cr showed higher toxicity than ion Zn, inhibiting the capacity of the fungus to develop during the germinative phase. Zinc ions showed to be beneficial to the biological membranes and required for the growth of microorganisms, however at high concentrations may be inhibitory or toxic to growth.
The RG and RM samples presented the highest abundance of OTUs given the large amount of organic carbon found in these samples (Table 1). Similarly, a high concentration of nitrogen could be observed in the RM sample. These soil compositions may have influenced the microbial community bioadsorption among the studied samples.
In silico data analysis and assembly of sequences
After quality control performed by FastQC software, the metagenomes sequenced by Illumina MiSeq plataform yielded approximately 674 thousand high-quality short DNA sequences. Annotation was performed using MG-RAST for taxonomic affiliation and putative protein-coding sequences related to heavy metal metabolism were searched against the databases including COG, KEGG Orthology and NOG. Principal properties of metagenomes, analysis statistics and the total of significant BLAST OTUs obtained are showed in Table 2.
Taxonomic classification and heavy metal functional affiliation of fungi sequences
High fungal abundance was found in all samples, totalizing 2.583 OTUs (10.5%) from Ascomycota and 273 OTUs (1.1%) from Basidiomycota phylum. However, low sequences from Mortierellomycotina (RM – 4 OTUs; RG – 8 OTUs) and from Entomophthoromycotina (RG - 3 OTUs; RM – 1 OTU) subphyla were found. Among the four samples, the greater fungal abundance was revealed in RG site, i.e., the less contaminated sampled spot, with 964 and 116 sequences from Ascomycota and Basidiomycota, respectively. Within RA sample, 759 sequences related to Ascomycota and 56 assigned to Basidiomycota phylum were found, representing the second most abundant site sampled. The RM sample, highly contaminated with heavy metals, presented 716 and 65 Ascomycota and Basidiomycota related sequences, respectively. From RS sample, 144 and 36 OTUs from Ascomycota and Basidiomycota were recovered, respectively (Figure 2 and Table 3).
The great fungal diversity was verified by the presence of representatives of 12 orders including Capnodiales, Pleosporales, Eurotiales, Onygenales, Helotiales, Pezizales, Saccharomycetales, Schizosaccharomycetales, Hypocreales, Magnaporthales, Phyllachorales and one unclassified taxonomic group from the phylum Ascomycota, suggesting a new sequence yet to be classified. On the other hand, five orders belonging to the phylum Basidiomycota were found: Agaricales, Polyporales, Malasseziales, Tremellales and Ustilaginales. One (Mortierellales) and two (Entomophthorales and Mucorales) orders were found from Mortierellomycotina and Entomophthoromycotina subphyla, respectively. Blastocladiales order was found from Bastocladiomycota phylum and Chytridiomycota phylum was represented by Cladochytriales, Chytridiales, Spizellomycetales and Monoblepharidales orders (Figure 2 and Table 3).
The majority of Ascomycota OTUs in all samples were related to the classes Eurotiomycetes (910 OTUs) and Sordariomycetes (1.030 OTUs) (Figure 3). From Eurotiomycetes class, the order Eurotiales was the most diverse, containing 238, 75, 282, 98 and 41 sequences from genera Aspergillus, Emericella, Neosartorya, Penicillium and Talaromyces, respectively (Table 3). The greatest diversity was found in GR sample (control), showing that the absence of heavy metals preserves the characteristics of the environment, allowing the development of different classes of these microorganisms, thus increasing fungal diversity. The sample RS, highly contaminated with heavy metals, presented the lowest microbial diversity belonging to these two classes, revealing that the presence of such substances have undermined the survival and/or the development of some classes which were not able to adapt to the contaminated environment. Presence of species belonging to Eurotiales order, such as Penicillium and Aspergillus, in sites contaminated with heavy metals, occurs due to the ability of individuals of these species to remove heavy metals from the environment, such as chromium and nickel (Iram et al. 2012)
From Sordariomycetes class, the orders Sordariales and Hypocleales were the most abundant, mainly within RA (higher Sordariales sequences OTUs) and RG samples (higher Hypocreales sequences OTUs) (Figure 4 and Table 3). In total, 334 sequences related to Hypocreales and 552 related to Sordoriales orders were found. From Sordariales order, sequences OTUs from genera Chaetomium (184 OTUs), Neurospora (222 OTUs), Bombardia (2 OTUs) and Podospora (144 OTUs) were found. Concerning to Hypocreales order, sequences OTUs belonged to the genera Gibberella (238 OTUs), Nectria (92 OTUs), Cordyceps (1 OTU) and Hypocrea (3 OTUs) (Table 3). At class level, Eurotiomycetes and Sordariomycetes were the most abundant taxonomic groups within Ascomycota phylum (Figure 2). Sequences retrieved from sample RA belonged to genera Chaetomium (115 OTUs), Bombardia (2 OTUs), Neurospora (110 OTUs), and Podospora (77 OTUs), same genera recovered (except Bombardia genus) from RG sample sequences which, however, were less abundant, with 27 sequences OTUs from Chaetomium, 63 from Neurospora and 32 from Podospora. In these two locations, the higher frequencies of sequences OTUs related to those genera are possibly due to the lower concentration of heavy metals, which allowed these microbial communities to thrive. Considering that it was possible to observe a reduction in the number of sequences related to the same genera found in the samples contaminated with greater concentration of heavy metals (RS and RM), it is possible to infer that the presence of these contaminants interfered in the homeostasis of the environments, culminating in the reduction of these microbial communities.
A considerable number of OTUs related to Saccharomycetes class was observed in all samples sites, reaching 54, 43, 57 and 52 OTUs from RM, RS, RG and RA, respectively (Table 3). Yeasts have robust strategies of resistance to heavy metals including mechanisms that lead to the detoxification of the cytoplasm, exerted by transporting the metals to the outside of the cell by specific carriers, or by accumulation of metals, normally complexed with thiolated peptides, in organelles such as vacuoles (Fidalgo 2011; Tsai et al. 2009). In addition, the composition of sampling site, composed by 30.16 g.Kg -1 of total organic carbon (data not shown), provided a large amount of nutrients for the development of Saccharomycetes and other filamentous fungi.On the other hand, very low unclassified OTUs related to Ascomycota were found in RG sample (control) (Figure 3), showing that the contamination by heavy metals can affect the composition of any microbial community.
The higher Basidiomycota abundance found in this work was related to the Agaricomycetes class derived from samples RA, RG, RM and RS, totalizing 26, 53, 37 and 18 OTUs, respectively (Figure 5). Again, the majority of sequences OTUs from these groups were recovered from RG sample (control), the less heavy metal contaminated site (data shown on Table 1). The orders Agaricales and Tremellales were the most abundant taxonomic groups, with 122 and 67 OTUs respectively. Agaricales order was represented by sequences of Coprinopsis (38 OTUs), Moniliophthora(14 OTUs), Schizophyllum (26 OTUs) and Laccaria (44 OTUs) genera. Concerning Tremellales, only sequences from Filobasidiella genus were found (65 OTUs). Sequences related to Blastocladiomycota and Chytridiomycota phyla were found from samples RM, RG and RA. Chytridiomycota was represented by sequences from genera Cladochytrium (2 OTUs), Olpidium(1 OTU), Spizellomyces (4 OTUs) and Harpochytrium (2 OTUs), while Blastocladiomycota was represented by sequences from Allomyces (2 OTUs) and Blastocladiella (1 OTUs) genera (Table 3). These two last phyla were not represented by any sequence of RS sample, problaby due to the great contamination of all heavy metals, mainly Cr, Pb and Zn. However, as the other contaminated sites presented sequences from Chytridiomycota and Blastocladiomycota phyla, it is possible to affirm that these taxonomic groups are capable of thrive under certain concentrations of heavy metals, but can not tolerate high concentrations of lead specifically.
The fungi classified in the phyla Blastocladiomycota and Chytridiomycota present as similarity the production of spores with a single smooth flagellum. Some representatives of these phyla are cosmopolitan found in different ecosystems and terrestrial ecosystems (Jerônimo et al. 2015). The knowledge about tolerance to heavy metals by genus from Blastocladiomycota and Chytridiomycota phyla is very poor. In the work made by Jia and co-workers (2018) representantives from Chytridiomycota phyla were not affected by the presence of heavy metals Cd (5.27 mg.kg−1) and Cu (553.53 mg.kg−1). This could be related to the great amount of extracellular polymeric substances (EPS) that same fungi may produce by their metabolism. Georg and Gomes (2007), performed a study were the aquatic fungus Blastocladiella emersonii was submited to a global transcriptional response by using cDNA libraries. The author found genes related to cellular transport function during exposure to 50 µM Cd.
The sequences from Entomophthorales and Mucorales orders from Entomophthoromycotina subphylum belonged to species from genera Basidiobolus (RM – 1 OTU) and Rhizopus (RG – 3 OTUs). Subphylum Mortierellomycotina was represented by sequences of Mortierella genus, being 9 OTUs from RG site and 4 OTUs from RM site (Table 3).
Filamentous fungi are known to be resistant to heavy metals due to their ability to detoxify these compounds by several mechanisms such as i) valence transformation; ii) active uptake; iii) impermeability and sequestration; iv) extra and intracellular precipitation; v) biosorption to cell wall; vi) transformation of metals; vii) defense mechanisms including immobilization of heavy metals using extracellular and intracellular chelating compounds and viii) presence of transporter systems for the uptake of essential metals in the cell membrane (Islam et al. 2008; Qayyum et al. 2016). In the Agaricomycete Paxillus involutus, the uptake of Cd is made by the slow carrier-mediated transport into the cell after a rapid binding to the cell wall. The uptake partially depends on the membrane potential and it is linked to the transport of calcium (Baldrian 2003). Only one OTU from metagenomic sample RA related to environmental processes by signal transduction from calcium signaling pathway was found. Ahmad et al. (2005) evaluated an Aspergillus sp. strain for the biosorption potential of Cr and Cd and observed that bioadsorption of these metals ranged from 6.20 - 9.5 mg.g-1 for Cr and 2.3 - 8.21 mg.g-1 for Cd. According to the authors, results revealed that fungi from metal polluted sites showed higher metal tolerance and bioadsorption capacities. In white-rot fungi, the heavy metals are intracellularly chelated by peptidic low molecular weight compounds phytochelatins or metallothioneins. In the environment, heavy metals can interact with extracellular enzymes of fungi and must be taken up by the fungus to promote a physiological response Baldrian (2003). According to Cui et al. (2017), the Mortierella sp. species showed not only the potential to effectively reduce the heavy metal activities of Zn, Pb and Mn up to 74, 85 and 79%, respectively from mine tailings, but also significantly shorten the remediation period of the environment with a consequent improvement in the site living conditions.
According to Colwell and Coddington (1994), species accumulation curves plot the cumulative number of species recorded as a function of sampling and effort, showing the rate at which new species are found. Thus, statistical analyzes from the data set with complete coverage is expected to result in a plateau shaped curve (Oliveira et al. 2011). In this work, the fungal communities from all analyzed samples exhibited similar relative species richness in accordance with the rarefaction curves, and the profile of these curves suggests that the sampling was able to cover the fungal diversity of these samples (Figure 6).
Alpha diversity (α) is the total number of species in a habitat (Nogueira et al. 2008; Magurran 2004). In our work, a great species richness (alpha diversity) was found in all samples. The diversity found in RG sample totalized 68 distinct species (55 from Ascomycota, 8 from Basidiomycota, 2 from Chytridiomycota, 1 from Blastocladiomycota, 1 from Entomophthoromycota and 1 from Mortierellomycota), followed by RM sample, which recovered 68 species (52, 8, 4, 1, 1 and 2 from Ascomycota, Basidiomycota, Chytridiomycota, Blastocladiomycota, Entomophthoromycota and Mortierellomycota, respectively). Samples RA and RS recovered, respectively, 50 and 40 distinct species of Ascomycota, and 8 Basidiomycota species each. Only RA sample was able to recovered 3 species from Chytridiomycota (Table 4).
In general, the RS sample showed a considerable reduction (about 90%) in the amount of genera representative of the Ascomycota phylum (n = 57), mainly due to the presence of lead, chromium and zinc, when compared to the control sample (n = 559), followed by RA (n = 515) and RM (n = 489) with a reduction of 7.8% and 12.5%, respectively. In the same way, the genera belonging to the phylum Basidiomycota also suffered a reduction in numbers, totaling RS (n = 25; 66.2%), RA (n = 40; 46%) and RM (n = 48; 35%) compared to the control (n = 74) (Table 3).
Despite the higher contamination with Cd, Cu, Ni and Zn metals in the RM sample in comparison to RS sample, RS site presented lower α-diversity (48 distinct species), and, comparing the contaminations of all samples, the possible responsible for the lower diversity was the higher concentration of lead in this area (Table 1), as the presence of other heavy metals such as Zn and Cr did not interfere in the diversity of the other samples and lead is considered a non-essential and environmental micro-contaminant, being more harmful to the fungal communities (Rasool and Irum 2014; Woldeamanuale 2017). The total alpha-diversity of, RM and RG samples had the same number of species recovered, while RA sample had only about 10% of the loss of alpha diversity (Table 4). These results may be explained by the difference in the number of OTUs related to some genera found in the four samples studied (Table 3). The genera that emphasize this find are Aspergillus and Chaetomium. In the RG sample, 87 sequences OTUs of Aspergillus and 27 of Chaetomium were found. Meanwhile, in the RM sample, were recovered about 25% and 51% more sequences OTUs from Aspergillus and Chaetomium, respectively. These two genera may be acting as the main groups involved with the expression of genes related to heavy metal tolerance, since the other genera showed less number of OTUs recovered. In the same way, RA sample (moderate contamination of all heavy metals), was able to recovered about 3.5 times more OTUs of Chaetomium, 1.4 times more OTUs of Podospora andabout 74% more OTUs of Neurospora, compared to RG sample. The number of sequences assigned to genus Neosartorya in RG and RM samples was practically the same (106 and 104, respectively), showing that the concentration of metals found within RM sample did not affect the prevalence of this group of organisms. From these results, it is possible to affirm that the genera Neurospora, Aspergillus, Podospora, Chaetomium and Neosartorya are tolerant to high levels of heavy metals.
The genera Neurospora and Aspergillus have been reported as heavy metal tolerant fungi (Iram et al. 2009; Joshi 2014; Oladipo et al. 2016; Qayyum et al. 2016). According to Haruma et al. (2018), the root-endophytic Chaetomium cupreum, which produced siderophores, enhanced aluminium stress tolerance in Miscanthus sinensis (a plant species) by accumulating aluminium in mycelia around the roots. Xu et al. (2015), performed a work were the diversity of dark septate endophytes and their cadmium tolerance were investigated from mine tailing soils that contained excessive Pb, Zn, and Cd. In this work, strains of the genus Podospora were isolated from this environment. The Neosartorya genus were already found in heavy metal sediments, in a study conducted by Abdel-Azeen and coworkers (2015), in which this genus was isolated from Lake Manzala sediments, in Egypt, contaminated by industrial activity. Urík and coworkers (2010) verified the thallium (TI) metal bioabsorbability and bioremediation ability of this genus and, in 2016, Onn et al. isolated endophytic fungi from mangrove plants and soil and verified that an isolate of Neosartorya sp. showed maximum Cu and Zn biosorption ability.
According to Kacprzak and Malina (2005) the genus Mortierellawas most frequently present in soil with high contents of metals including zinc and iron, although, it did not occur in non-contaminated soil samples. On the other hand, the genus Basidiobolus probably has not yet been described as being a genus of fungus resistant to heavy metals.
Concerning Entomophthoromycotina and Mortierellomycotina subphyla, similar results were found as the number of sequences assigned to these taxonomic groups were higher in the RG sample (11 OTUs) in comparison to the RM sample, which showed 4 representatives from the Mortierellales order (Mortierella genus – data not shown) and 1 sequence OTU assigned to Entomophthorales orders (Basidiobolus genus – data not shown). Interestingly, Entomophthorales order was only found in a contaminanted site (RM), and as there is no information available in the literature associating these organisms to heavy metal contamination, this finding shows the promising capability of this group for future studies. Concerning the Mucorales, sequences assigned to this group were only found in the less contaminated site (RG), showing the sensitivity of these organisms to heavy metal contamination. No sequences of Entomophthorales and Mucorales were found in the RS sample, the site with the highest lead contamination. Regarding Blastocladiomycota and Chytridiomycota subphyla, no sequences assingned to these two groups were found within RS sample, showing that lead contamination may have been the most striking for the survival of these organisms in the environment. However, members of Chytridiomycota subphylum, i.e.Cladochytridium, Olpidium and Harpochytrium, were only found in RM sample, considered highly contaminated. There is no available information about Cladochytrium and Harpochytrium heavy metal resistance in the literature. Olpidium zoospores were killed under the presence of high concentration of copper and zinc in a study conducted by Tomlinson and Faithfull (1979), however the effects of heavy metals in other zoosporic fungi remains poorly studied (Gleason et al. 2010).
Fungal taxonomic affiliation of heavy metal metabolism and tolerance and biodegradation
With respect to tolerance, functional metabolism and biogradation of heavy metals, analyzes of the Ascomycota sequences of the four samples utilizing the KO (KEGG Orthology) database revealed a high number of sequences OTUs related to genes responsible for the metabolic pathways of the target compounds, with a total of 635 OTUs, being 161, 264, 157 and 53 sequences from RA, RG, RM and RS, respectively. The sequences were shown to be associated mainly to Metabolism (307 OTUs) and Genetic Information Processing (174 OTUs), followed by Cellular Processes (67 OTUs), Human Diseases (68 OTUs), Environmental Information Processing (23 OTUs) and Organismal Systems (6 OTUs) gene (Figure 7A). In the Basidiomycota sequences analyzes, positive OTUs related to Metabolism (17 OTUs) and Genetic Information Processing (12 OTUs) functions were found within all samples (Figure 6B). Functional analyzes performed by COG database revealed one OTU related to predicted divalent heavy-metal cations transporter and one OTU related to copper chaperone in RA sample. The copper chapenone is a metalloprotein responsible for the delivery of Cu to Superoxide dismutase that constitutes a very important antioxidant defense against oxidative stress (Younus 2018). According to Zhang et al. (2016) heavy metals such as lead- and cadmium- caused chemical stress and reactive oxygen species formation in Phanerochaete chrysosporium, a species from the Basidiomycota phylum. The authors reported the lead- and cadmium - induced oxidative stress on the activities of catalase enzyme. In the same way, in your study, were found same OTUs related to predicted Zn-dependent proteases (2 OTUs), predicted Zn-dependent hydrolases of the beta-lactamase fold (3 OTUs), predicted metal-dependent protease of the PAD1/JAB1 superfamily (2 OTUs) and cytosine deaminase and related metal-dependent hydrolases (2 OTUs), data suggesting the use of heavy metals for cellular activity. The results, especially from COG database, showed the recovery of a large amount of potential unknown functional genes sequences. In addition, a large number of genes sequences associated with basal metabolism were retrieved from all four samples, such as amino acid transport and metabolism, suggesting the presence of a powerful metabolic energy in the environment highly contaminated with heavy metal, which allows the maintenance of the basic microbial activities.
Sequences associated with DNA repair genes and affiliated to Ascomycota phylum were retrieved within three samples, including two contaminated with heavy metals (RA and RS), totalizing 12 OTUs (Table 5). In the RM sample, the most contaminated with metals, no DNA repair genes were found, contrary to what was expected, since the presence of heavy metals such as chromium and cadmium may activate stress-responsive pathways in yeasts and filamentous fungi (Pócsi 2011; Viti et al. 2014). However, the sample presented the higher number of genes related to heavy metals biosorption processes, which shows that the biosorption is the main tolerance activity executed by the fungal community inhabiting this site. The RA site, moderately contaminated with heavy metals, presented both DNA repair genes and genes related to heavy metals biosorption processes, which indicates that fungal communities inhabiting this site have developed different adaptation mechanisms to confront the contamination. No sequences related to genes associated to repair of DNA and membrane transport processes assigned to Basidiomycota, Mortierellomycotina and Bastocladiomycota phyla and Entomophthoromycotina and Mortierellomycotina subphyla were found. Thus, must probably, the fungal sequences assingned to these taxonomic groups may not have the capacity to maintain microbial cellular life and/or to biosorb the heavy metals present in these samples. On the other hand, a large number of fungal genera were found (RM = 53 OTUs, RS = 25 OTUs and RA = 43), suggesting the ability to tolerate the contamination heavy metal in each sample site.
The mechanism involved in the bioadsorption process can range from ion exchange to membrane diffusion that can be influenced by biomass (carbon) and solution chemistry (nitrogen source). Factors such as cell age, environmental and nutritional conditions interfere with the preference of living biomass in the bonds with metallic ions. To overcome the toxic effect of metals at high concentrations, dead biomass may be preferred in these bioadsorption processes (Ahmad et al. 2005). The biosorption capacity of several metals such as Zn, Ni, Cd, Cu, Co by filamentous fungi biomass has been reported. In addition, metal concentration as well as factors such as temperature and pH are known to influence the biosorption process (Ahmad et al. 2005).
Cr (VI) biosorption decreases as the pH of the solution increases. In this study, an experiment performed on membrane reactors, the authors showed that a large COD removal can be achieved at a pH of 7 to 8 (Chen 2012). In our work, all samples had pH values above 8.0 (except the control) as well. , the microbial community could be adsorbing metals due to a relatively high pH range.
Another important point of the bioremediation of metals is regarding the presence of a high percentage of cell-wall material within fungal biomass, which increases the variety of functional groups involved in metal binding (Dhankhar and Hooda 2011). In this sense, it is possible to establish a relationship between the enzymes or proteins involved in membrane transport processes and the biosorption of heavy metals by filamentous fungi. In our work, were found in RM and/or RA samples, proteins sequences involved in signal transduction including two-component system (11 OTUs) and calcium signaling pathway (1 OTU) as well as proteins involved in membrane transport such a ABC transport (3 OTUs), endorsing the role of these proteins in the adaptation of filamentous fungi in contaminated environments. The properties of environments, especially those heavily contaminated with heavy metals from manufacturing industries of aluminum, leather dyeing and those that utilize toxic, corrosive and flammable products, may influence and modify the microbial community composition and functionality. By using metagenomic approach, it was demonstrated that the diversity and dynamics of metabolic activities in the microbial communities from banks of stream, conferring these communities the role of recyclers of circulating compounds and making the filamentous fungi a key point in the biosorption of heavy metals, resulting in the reduction of these compounds in the environment. The environment impacted with heavy metals is a drastically modified habitat, and the microbial communities must adapt to the new toxic conditions. Filamentous fungi has the ability to adjust throught the expression of genes involved to heavy metal bioremediation process, aiming to survive and proliferate in extremely toxics environments surrounding manufacturing industries that disposal toxic products including heavy metals. The results obtained from the metagenomic approach from this work provide valuable and robust information for subsequent studies focused on the development and improvement of selective culture media for the isolation of fungal strains adapted to bioremediation processes from environments contaminated with heavy metals.