Bioaccumulation of toxic metals by fungi of the genus Aspergillus isolated from the contaminated area of Ostramo Lagoons

The study compares the ability to bioaccumulate toxic metal ions using microscopic filamentous fungi of the genus Aspergillus isolated from the anthropogenically contaminated site of the Ostramo Lagoons (Ostrava, Czech Republic). The experiment comprised six species of indigenous fungal isolates: A. niger, A. candidus, A. iizukae, A. westerdijkiae, A. ochraceus and A. clavatus. Nutrient liquid media enriched with Cu(II), Zn(II), Ni(II) and Cr(III) were individually inoculated with spores of these fungi. After thirty days of incubation, the content of metal ions in the dried fungal biomass and medium was measured by the AAS. It was found that the average bioaccumulation capacity of selected toxic metal within the tested strains decreases in the following order: A. ochraceus > A. candidus > A. clavatus > A. westerdijkiae > A. iizukae > A. niger. The highest bioaccumulation efficiency was achieved by the A. ochraceus strain which accumulated Cu(II) with an efficiency of 57.42 %, Zn(II) with 56.88 %, Cr(III) with 37.73 %. When comparing the ability of bioaccumulation of the toxic metals, the following was found: Zn(II) > Cu(II) > Cr(III) > Ni(II). Understanding of bioaccumulation processes that take place in fungal cells at the molecular level may lead to better strategies for the application of these interesting microorganisms in bioremediation processes.


Introduction
Microorganisms, including microscopic filamentous fungi, have adapted to the presence of toxic metals through various resistance mechanisms. Tolerance/resistance of microscopic filamentous fungi to toxic metals is conditioned by their environment [1,2]. Microorganisms that show a high level of tolerance to toxic metal ions were isolated from environments with a high incidence of these metals [3, 4, 5], respectively toxic metals in combination with other toxic chemicals, both organic and inorganic [6,7]. It has previously been confirmed that toxic metals can accumulate in the living biomass of resistant fungi strains growing on organic waste material [8,9]. The Ostrava lagoons Ostramo represented an old ecological burden of extraordinary scale with high concentrations of hydrocarbons and toxic metals [10] and thus represent a unique environment suitable for the adaptation of microorganisms resistant to these substances. This study compares the ability of six different Aspergillus species isolated from the Ostramo Lagoons to bioaccumulate Cu(II), Zn(II), Ni(II), Cr(III) toxic metal ions.
Contamination of the environment with toxic metals poses a serious environmental risk, especially due to the bioaccumulation of metals in living organisms and their subsequent distribution through the food chain, which leads to serious ecological and health risks [11]. The ability of living organisms to resist 2 the toxic effects of metals and subsequently accumulate these metals in living cells can also be used in bioremediation processes. The method using the properties of cells of living organisms, including their metabolism, is called bioaccumulation. Bioaccumulation of toxic metals is a complex process involving the active transport of metals through cell membranes into the cell and using physical, chemical, and biological mechanisms [12]. It is a combination of surface reactions, intra-and extracellular clotting, and complexation reactions [13]. Bioaccumulation depends on the internal structural and biochemical properties of cells, on genetic and physiological adaptation of the organism, environmental modification of the metal, its availability, and toxicity which is mainly influenced by the oxidation state of the metal [14]. Currently, the application of bioaccumulation processes is one of the adaptation mechanisms for the removal of pollutants from contaminated environments, including wastewater and semi-liquid sludge [15].

Interactions of microscopic fungi with toxic metals
Fungal metabolism can very significantly affect the mobility and toxicity of metals and metalloids. There are various ways in which metals can be transformed into a form that is suitable for interactions with cell mass [5]. One of these methods is solubilization, which involves, for example, the formation of complex with organic acids, other metabolites or siderophores [16]. In contrast, metal immobilization results from sorption to cellular exopolymers (cell walls), cell transport, and intra-and extracellular sequestration or precipitation [17]. Dissolution of metal compounds and immobilization of metals are key elements of the biogeochemical cycles of toxic metals. Metals are directly and indirectly involved in all stages of microbial growth, physiology, and morphogenesis [18].
Resistance/tolerance of organisms to toxic metals is defined as the ability to resist metal toxicity using one or more resistance mechanisms that are activated in direct interaction with a particular metal [19,20]. Some metals, such as cobalt, copper and nickel, serve as micronutrients and are used during redox processes to stabilize the molecule through electrostatic interactions or as components of certain enzymes, such as to regulate osmotic pressure in a cell. However, many metals do not play a significant role, have no nutritional value and are potentially toxic to microorganisms. All metals interact with cellular structures through covalent and ionic bonds. Essential and non-essential metal ions interact with fungal cells and are accumulated by physico-chemical mechanisms and transport systems of varying specificity [21]. In high concentrations, essential and non-essential metals can damage cell membranes, alter the specificity of enzymes, disrupt cellular functions and damage DNA. In these cases, different mechanisms of resistance of microorganisms to toxic metals may apply, which may vary depending on the microorganism, the metal, and the environment (pH, metal ion concentration, etc.) [22]. The key to understanding these mechanisms is, among other things, their genetic nature. These complex processes taking place at the molecular level in the cells of microorganisms may lead to better strategies for their detoxification from the environment.

Origin of microscopic filamentous fungi of the genus Aspergillus
The landfill, also known as OSTRAMO lagoons (Ostrava, Czech Republic), was established at the end of the 19th century by depositing waste from refinery production. Since 1965, waste from the regeneration of used lubricating oils has also been deposited here, later also other toxic waste, which was often not specified. The landfill consisted of several lagoons originally designated R1, R2 and R3, which were separated by dikes with earth mounds 5 m above the surrounding terrain; lagoon R0 was located in the earth pit of the former brickyard, the existence of which was confirmed only in 1999 [23]. The Ostrava lagoons are classified as very serious and old ecological burdens of extraordinary extent. The actual area of the lagoons was burdened by pollution of the rock environment mainly by organic compounds. The main organic contaminants were polycyclic aromatic hydrocarbons, polychlorinated 3 biphenyls, and phenols. In addition to organic pollution, there were several toxic metals -As, Cd, Cu, Hg, Ni, and Pb, the pH of the lagoons was acidic to neutral [23]. Microscopic fungi were isolated from lagoons during their microbiological survey in the period 2012-2015. Isolates were identified at the workplace of the Slovak Academy of Sciences in Bratislava and their sequences were stored in the GenBank database: MK243706 -MK243708, MG639905 -MG639907.

Sampling
Samples of semi-liquid sludge were taken from the R2 lagoon area at a depth of 0.1 m, then individual strains of microscopic filamentous fungi were isolated and cultured according to a standard procedure using Sabourad Dextrose Agar (HiMedia Laboratories, India). Identification and taxonomic classification of individual species was performed by sequence analysis of the Internal Transcribed Spacer of Ribosomal DNA.

Microorganisms
The following Aspergillus isolates from the contaminated site of the Ostramo lagoons were used in the study: A. niger, A. candidus, A. iizukae, A. westerdijkiae, A. ochraceus, A. clavatus. For a comparative study of the bioaccumulation potential of these species, 2 ml of a spore suspension was used. This suspension was aseptically transferred to a mixture of nutrient medium and metal solution.

Chemicals and media
The spore suspension was transferred to a nutrient medium containing the appropriate metal ions. First, the amount of the respective salt was determined so that the final concentration of metal ions in the medium was 1000 mg L−1. The following chemicals were used as a source of metal cations (Table 1). Sabouraud Dextrose Broth (Sabouraud liquid medium, HiMedia Laboratories, India) was prepared according to the manufacturer's instructions and used to cultivate the microscopic filamentous fungi. Demineralized water was used to prepare the medium and the resulting pH of the nutrient medium was 5.6 ± 0.2. The medium was autoclaved (15-lbs, 121°C). The appropriate amount of metal salt was quantitatively transferred to 1 L of sterile medium. This was followed by further dilution of the solution with sterile metal-free nutrient medium in a ratio of 1:10 so that the final metal concentration in the solution was 100 mg·L -1 . The experiments were then performed in 1 L Erlenmeyer flasks.  was calculated by the ratio of the amount of metal accumulated by the microfungal biomass after 30 days to the original amount of metal in the nutrient medium before culturing the microscopic fungi.

Results
There were 6 × 0.5 L of nutrient media for each metal ion -Cu(II), Zn(II), Ni(II) and Cr(III) at a mass concentration of 100 mg·L-1. After 30 days of incubation, the average weight of the harvested and dried biomass of Aspergillus species depending on the type of toxic metal cation in the medium as follows: Cr(III) > Cu(II) > Zn(II) > Ni(II) ( Table 2).  . This result showed the ability of the biomass of microscopic fungi of the genus Aspergillus to accumulate toxic metal ions (Table 3). with the smallest amount of grown biomass after 30 days of cultivation, thus accumulating up to 92.52% of the initial amount of Zn(II) in the medium. The remaining strains accumulated on average per 1 g of dry microfungal biomass toxic metal ions in the following order: Zn(II) > Cu(II) > Ni(II) > Cr(III). The lowest amount of toxic metal accumulated in 1 g of dry microfungal biomass was found in A. niger, the best result was obtained in A. candidus for Zn(II), Cu(II) and Ni(II). The bioaccumulation capacity of this fungi for the indicated toxic metal ions in 1 g of dry microfungal biomass is as follows: A. candidus > A. ochraceus> A. clavatus > A. westerdijkiae > A. iizukae > A. niger. We have also calculated the bioaccumulation efficiency of the data obtained. The total amount of bioaccumulated toxic metal in the dried biomass, expressed as a percentage, is shown in Table 4. Bioaccumulation efficiency was calculated for each strain examined and for each toxic metal applied. The value was calculated according to the following formula: where η is the bioaccumulation efficiency [%], macc is the amount of metal accumulated in the total amount of dried microfungal biomass and min is the amount of metal in the medium at the beginning of the incubation, in this case the metal concentration is 100 mg L −1 in 0.5 L of medium (min = 50 mg).
The results of bioaccumulation efficiency show that A. ochraceus best accumulated ions of the toxic metals, which achieved an efficiency of 57.42 % during Cu(II) bioaccumulation, 56.88 % during Zn(II) bioaccumulation and 37.73 % during Cr(III) bioaccumulation. This strain is characterized by a high ability to resist the toxic effects of these metals, which are classified as transition metals. At the same time, it is able to bioaccumulate these metals into mycelial cells with relatively high efficiency and thus use the potential of its metabolism. The maximum of bioaccumulation potential was found in A. candidus for Zn(II), the efficiency of bioaccumulation in this case was up to 92.52 %. Thus, this strain showed a high degree of adaptation to the presence of Zn(II) ions in the environment, although a significant inhibitory effect of these ions on mycelial growth was demonstrated. Interesting results were also obtained for the A. clavatus strain, where the efficiency of copper bioaccumulation was 33.35 % and the efficiency of zinc bioaccumulation was 30.84 %. A. westerdijkiae accumulated Zn(II) the best with an efficiency of 47.66 %. Ni(II) the best accumulated the A. iizukae, which, however, was not very successful in the bioaccumulation of other metals. It remains interesting that Ni(II) accumulated the best, which was accumulated overall by all Aspergillus species with the lowest efficiency. The average bioaccumulation capacity of selected toxic metals within the tested strains decreases in the following order: A. ochraceus > A. candidus > A. clavatus > A. westerdijkiae > A. iizukae > A. niger. A. ochraceus accumulated metals with the highest efficiency, A. candidus, which showed the highest content of metal ions in 1 g of dry biomass, was placed behind it.