Environmental pollution with man-made contaminants poses potential risks for humans, animals and plants due to their persistence and toxicity. Contaminants are very different in their chemical structure, show different abundance and stability in the environment and differ in the accessibility for microorganisms. In addition, they can strongly adsorb to soil particles and remain immobilized (Boonchan et al., 2000; Pinto and Moore, 2000). Contaminants have a large extent: from crude oil ingredients as benzene, toluene, ethylbenzene and xylene (BTEX), to polycyclic aromatic hydrocarbons (PAHs) from coal and tar residues, hexadecane from diesel to chemical liquid waste as polychlorinated biphenyls (PCB). The removal of these toxic compounds is a challenge and can be very cost intensive. New technologies and cleanup methods together with a better understanding of used methods, to improve degradation time and rate, are needed.
Biofilter systems are a common gas or wastewater treatment technologies that use biological mechanisms to degrade contaminants. They are cost-effective compared to physicochemical processes, especially in case of high gas flow rates with low concentrations of volatile organic compounds (Maestre et al., 2007). Mostly bacteria are used in these systems, but fungal strains can also be good candidates. Fungi have the advantage of being able to withstand harsh conditions like low water availability, varying pH values or changes in temperature and are able to degrade great variety of contaminants (Cox, 1995). Additionally, bacterial gas biofilters have poor degrading abilities concerning hydrophobic contaminants, as they are poorly absorbed due to their low water solubility (van Groenestijn et al., 2001). One technical option to overcome this problem is fungal strains grown on inert packaging material (van Groenestijn et al., 2001). Examples of successful usage are
Another option to use fungal strains is residual contaminations in soils, which often remain untreated after the excavation of highly contaminated hot spots due to the high costs. In these situations,
The group of white-rot basidiomycete fungi and their molecular degradation tools, that is, extracellular ligninolytic enzymes and accessory enzyme, are normally used for lignin degradation and has been found to be useful for contaminant degradation. However, white-rot fungi typically grow in compact wood and favor acidic condition (Marco-Urrea et al., 2015). Therefore, known non-ligninolytic fungal strains with the possibility to live on contaminants are often better candidates for bioaugmentation, as they are able to live in extreme conditions such as high or changing pH or desiccation.
To identify key players of fungal degradation in contaminated soil, for the first time we set up a microtiter plate screening method based on the screening method described by Blasi et al. (2016) and developed a quick and high throughput screening method using the contaminated soil as inoculant. Twelve soil samples were used to obtain a broad range of fungal strains and investigate differences between the sites and varying PAH contamination that acted as selective pressure for the microbiota. We were able to isolate 93 different fungal strains from the first screening where contaminated soil was used as inoculant and growth in the presence of toluene, PCB 126 and hexadecane was tested. These contaminants were chosen as they offer a broad range of different chemical structures and not many microbes are known to be able to degrade PCB. Pure fungal strains were cultivated and growth in the presence of the three contaminants was confirmed repeating the microtiter plate screening method. The best growing 11 strains were identified by ITS and partial 18S rDNA sequencing data. These strains are discussed for their potential application in the field.
Soil samples from different contaminated sites were provided by ESW Consulting Wruss ZT GmbH. The samples were dried with Na2SO4 (Merck Millipore) and 30 g of homogenized soil sample was extracted on a Soxhlet apparatus for at least 6 hours using 150 mL of cyclohexane (LGC standards GmbH) as a solvent, containing six deuterated internal standards (
Unsieved soil of each sample (approximately 0.15 g) was dissolved in 1 mL of 0.9% sterile NaCl solution and vortexed to gain a homogenized liquid inoculant. 20 μl of each inoculant were used for the microtiter plate screening method using a 96 wells plate. Three different contaminants were used to identify new fungal species being able to grow in the presence of PCB 126 (10 ng μl-1 PCP 126 in isooctane, Dr. Ehrenstorfer GmbH), hexadecane (99% analytical grade, Alfa Aesar) or toluene (Merck KGaA, Darmstadt, DE). Per contaminant 150 μl of media, 50 μl of contaminant solution and 20 μl of the inoculum inoculant were used for hexadecane and PCB 126. Plates were kept at room temperature on a shaker. Toluene (5%) was diluted with dibutyl phthalate in a beaker in a glass vacuum desiccator and plates were stored next to it to expose them to the volatile phase. Toluene plates were filled with 200 μl of medium plus 20 μl of inoculant. All tests were performed in triplicates, a negative control (medium without inoculant) was included on each plate. In addition, a positive control was carried out for each soil: 200 μl of a glucose medium and 20 μl of inoculant to investigate the number of fungal colonies deriving from the contaminated soil samples with glucose as carbon source. Growth of fungal species was measured through changes in the optical density (OD) at 700 nm with a microtiter plate reader Infinite M 1000 (Tecan, CH, set at 24°C, 70.8 rpm, wavelength: 700 nm, bandwidth: 5 nm) and OD values were corrected with values of the negative control. After 10 days, fungal growth was observed in all wells, the screening method was stopped and the liquid of the microtiter plate wells was plated on 2% malt extract agar (MEA: 2% malt extract, 2% D-glucose, 0.1% bacto peptone, 2% agar) and Rose-Bengal Chloramphenicol agar (Merck) from the wells with the contaminant but also from the positive control to isolate fungal strains growing on glucose and PAHs. Fungal strains were isolated and pure cultures were cultivated.
In order to test the ability of each isolated strain to live in the presence of the contaminants and to identify the best growing strains, the microtiter plate screening was repeated using pure cultures as inoculant rather than PAH contaminated soil. For this experiment, 1 cm2 of fungal biomass was diluted in 1 mL of 0.9% of sterile NaCl containing glass beads (Carl Roth, DE) and was homogenized on a Ribolyzer (FastPrep-24 Instrument, MP Biomedicals, CA) for 5 sec at 4 m s-1. The screening protocol for PAH contaminated soils was also used to grow strains in triplicates at room temperature in the presence of hexadecane, PCB 126 or toluene. Additionally, positive and negative controls, as previously described, were set up and the plates were incubated for 27 days.
DNA extraction of the eleven best growing strains was done according to the protocol described in Sert and Sterflinger (Sert and Sterflinger, 2010). In this procedure, the fungal cells are disrupted through glass beads using a Ribolyzer for 20 sec at 4 m s-1 twice, in-between samples were cooled on ice. DNA is recovered through Ethanol precipitation. The extracted DNA was tested on its purity on a Nanodrop 1000 spectrophotometer (Thermo Fisher) and bands were detected on an electrophoresis gel 1.5 % (w/v). PCR of the ITS or 18S rRNA sequences was done using the primer pair ITS1 / ITS4, further NL1 / NL4 and NS5 / NS8 at a concentration of 0.5 μM. For the PCR the protocol of the Phusion Polymerase (Thermo Fisher) was used, including 2 U μl-1 Phusion polymerase, Phusion HF buffer (Thermo Fisher) and 10 mM dNTPs. The Thermocycler (Biorad) was set up at 98°C (30 sec) for initial denaturation followed by 34 cycles at 98°C/20 sec, 60°C/30 sec and 72°C/105 sec and ended by the final elongation 72°C/120 sec. After checking the obtained PCR products on an electrophoresis gel 1.5% (w/v), the products were purified using the QIAquick PCR Purification Kit (Qiagen). Sequencing of the products was performed by Sanger Sequencing (Microsynth Austria). Sequencing results were compared with the online databases provided by the National Center for Biotechnology Information using the BLAST search program (Altschul et al., 1997) and sequences were deposited in the NCBI nucleotide database under the accession numbers listed in Table 1. Additionally, we did morphological and taxonomic analysis according to Domsch et al. (2008).
Phylogenetic classification of the ITS/18S rDNA coding sequences of the fungal isolates Tabelle 1. Phylogenetische Klassifikation der ITS/18S rDNA kodierenden Sequenzen der PilzisolateNo Primer Pair Closest identified phylogenetic relatives [EMBL accession numbers] Query cover Ident ACBR strain No accession No NL1/NL4 99% 99% MA6020 KY454753 O NS5/NS8 99% 100% MA6025 KY454758 Y NL1/NL4 100% 99% MA6015 KY454754 BL 3 NL1/NL4 96% 99% MA6021 KY454755 BL 4 NL1/NL4 99% 99% MA6017 KY454756 V ITS1/ITS4 100% 99% MA6016 KY454760 Z ITS1/ITS4 97% 98% MA6019 KY454761 H ITS1/ITS4 100% 99% MA6018 KY454762 NL1/NL4 99% 99% KY454757 U [EU552153.1] MA6024 NS5/NS8 99% 99% KY454759 X1 ITS1/ITS4 99% 100% MA6022 KY454763 G ITS1/ITS4 99% 99% MA6023 KY454764
The total concentration of 16 US EPA PAH ranged from 20.53 mg kg-1 DW (soil sample (S) 10) to 867.77 mg kg-1 DW (S 6). S 1—6 contained a lower proportion of high molecular weight PAHs (HMW, ≥ 4 aromatic rings, Figure 3) compared to S 7—12. According to literature (Lladó et al., 2013), samples with a high proportion of HMW PAHs are most likely aged contaminations due to the quicker dissipation of low molecular weight (LMW) PAHs in the field.
We were able to successfully adapt the microtiter plate screening method described by Blasi et al. (2016) to isolate 93 fungal colonies using the contaminated soil as inoculant. The soil contaminated with PAHs, acted as selective pressure for the microbiota to be able to isolate the fungal strains adapted to the contaminated environment. Two different microtiter plate screenings were performed:
with soil as inoculant followed by isolation of fungal colonies (3.2.1) with pure strains after isolation and cultivation from the soil (3.2.2)
As described above, soil samples varied in PAH content (Figure 3 and Table 2). This had a direct effect on the number of colonies isolated from the soil (Figure 1a, b). In the first plate screening, mixed cultures were obtained and resulted in 93 pure cultures. The highest amount of PAH was measured in S6. Nevertheless we could isolate two fungal colonies, one of them resulting in a successful candidate of the screening. In 6 out of 12 samples, a link between the proportion of HMW to total PAH concentration and the total number of isolated fungal communities was indicated by a strong linear regression (Figure 1a). The proportion of HMW / total PAH can be linked to the age of the contamination (Lladó et al., 2013). Accordingly, microorganisms in soil samples with lower proportion of HMW PAHs tend to be less adapted to contaminant degradation compared to microorganisms that could adapt to the contamination over a longer period of time in soil samples with a higher proportion of HMW PAHs. The 6 samples excluded from the regression analysis (S 7-12) were samples of very high HMW proportion, indicating high age. In these samples the microbial composition may have further changed because most degradable contaminants were already degraded. No correlation between individual PAHs and the ability to survive one of the three target contaminants could be found.
GC Results: Measured US EPA PAH concentrations and standard deviations of the 12 soil samples (S1-12). Values are presented as mg/kg dry weight and standard deviations were calculated (mg/kg dryweight±standard deviation) Tabelle 2. GC-Ergebnisse: Gemessene US EPA PAK-Konzentrationen und Standardabweichungen der 12 Bodenproben (S1-12) sind in der Tabelle dargestellt. Die Werte sind also mg/kg Trockengeweicht dargestellt und die Standardabweichung wurde berechnet (mg/kg Trockengewicht±Standardabbweichung)sample S1 S2 S3 S4 S5 S6 S7 S8 S9 S10 S11 S12 Naphtalene 5.8±1.2 4.8±1.0 12.8±2.6 9.4±1.9 6.8±1.4 31.5±6.3 0.1±0.02 <0.3(0.0) <0.3 (0.0) 0.1±0.01 <0.3(0.0) <0.3(0.0) Acenaphthylene 5.8±1.2 1.8±0.4 <2.7(0.00) 0.9±0.2 0.9±0.2 5.2±1.0 0.1±0.02 <0.3(0.0) 0.4±0.1 0.2±0.03 0.7±0.1 0.3±0.1 Acenatphene 6.9±1.4 4.3±0.9 11.2±2.2 8.5±1.7 6.8±1.4 24.9±5.0 0.6±0.1 0.4±0.1 0.4±0.1 0.1±0.02 <0.3 (0.0) 0.3±0.1 Flurene 10.1±2.0 6.3±1.3 14.8±3.0 10.3±2.1 8.3±1.7 34.3±6.9 0.5±0.1 0.4±0.1 0.4±0.1 0.1±0.02 0.5±0.1 0.6±0.1 Phenanthrene 55.20±11.0 28.3±5.7 70.2±14.0 48.3±9.7 36.5±7.3 154.0±31.0 5.9±1.2 4.4±0.9 4.1±0.8 1.0±0.2 6.4±1.3 5.7±1.2 Anthracene 22.5±4.5 11.4±2.3 22.2±4.4 18.1±3.6 14.4±2.9 67.7±13.5 1.4±0.3 1.2±0.2 1.6±20.3 0.4±0.01 2.1±0.4 1.4±0.3 Fluoranthene 78.0±15.6 29.1±5.8 48.2±9.6 39.0±7.8 30.8±6.2 141.0±28.0 7.5±1.5 12.6±2.5 10.0±2.0 2.9±0.6 17.5±3.5 9.8±2.0 Pyrene 77.8±15.6 30.4±6.1 47.9±9.6 41.6±8.3 32.2±6.4 108.0±22.0 7.5±1.5 24.4±4.9 10.0±2.0 2.3±0.5 14.5±2.9 7.7±2.0 Benz[a]anthracene 36.5±7.3 13.5±2.7 19.9±4.0 16.6±3.3 13.0±2.6 64.3±12.9 4.7±1.0 23.5±4.7 9.8±2.0 1.9±0.4 9.8±2.0 5.9±1.2 Chrysene 37.4±7.5 13.3±2.7 20.7±4.1 15.8±3.2 12.5±2.5 68.6±13.7 5.9±1.2 27.3±5.5 11.0±2.2 2.2±0.4 10.2±2.0 7.1±1.4 Benzo(b) fluoranthene 33.0±6.6 10.4±2.1 14.4±2.9 10.9±2.2 9.0±1.8 36.1±7.2 6.5±1.3 28.1±5.6 11.9±2.4 1.9±0.4 9.1±1.8 7.1±1.4 Benzo(k) fluoranthene 26.6±5.3 9.5±1.9 12.7±2.5 10.6±2.1 8.5±1.7 45.3±9.1 4.2±0.8 33.0±6.6 10.4±2.1 1.7±0.3 8.0±1.6 5.0±1.0 Benzo(a)pyrene 30.6±6.1 10.9±2.2 14.3±2.9 11.9±2.4 9.76±2.0 44.7±8.9 5.0±1.0 27.1±5.4 9.8±2.0 1.8±0.4 8.5±1.7 5.1±1.0 Indeno(1,2,3-cd)pyrene 21.3±4.3 7.0±1.4 8.5±1.7 8.3±1.7 6.0±1.2 20.1±4.0 3.1±0.6 19.6±3.9 8.2±1.6 1.7±0.3 5.2±1.0 2.8±0.6 Dibenzo(a,h) anthracene 5.9±1.2 1.7±0.4 <2.7(0.0) 1.6±0.3 1.3±0.3 4.8±1.0 1.1±0.2 5.5±1.1 2.0±0.4 0.5±0.1 1.2±0.2 0.7±0.1 Benzo(g,hi,i) perylene 19.6+3.9 6.5±1.3 7.8±1.6 6.4±1.3 5.2±1.1 17.2±3.4 3.4±0.7 22.7±4.5 7.2±1.5 1.8±0.4 4.2±0.8 2.7±0.5 sum 472.9±94.6 189.4±38.0 325.6±65.1 258.1±51.7 202.0±40.4 867.8±173.9 57.4±11.5 230.2±46.0 97.2±39.5 20.6±4.0 97.6±19.4 62.2±12.9
After the cultivation of pure cultures, the microtiter plate screening was repeated. Results are represented in Table 3. All values were corrected with the values of the negative controls.
Results of the second microtiter plate screening: Number of colonies of having the same growth pattern of all 93 fungal isolates on toluene, hexadecane and PCB 126. + = good growth, ~ = very slow growth and - = no growth. Hex = hexadecane, Tol = toluene, PCB = PCB 126 Tabelle 3. Ergebnisse des zweiten Mikrotiterplatten-Screenings: Anzahl der Kolonien mit demselben Wachstumsmuster innerhalb der 93 isolierten Kolonien in Gegenwart von Toluol, Hexadekan und PCB 126. + = gutes Wachstum, ~ = sehr langsames Wachstum und - = kein Wachstum. Hex = Hexadekan Tol = Toluol, PCB = PCB 126colony numbers Hex Tol PCB 21 + + - 14 + + ~ 9 + + + 8 + - - 6 + ~ ~ 5 + - ~ 4 - - - 3 ~ + - 3 + ~ - 2 - + - 2 ~ - - 2 ~ - ~ 2 ~ ~ ~ 2 ~ + ~ 2 + ~ + 2 ~ ~ + 1 - ~ - 1 - - ~ 1 - ~ ~ 1 ~ ~ - 1 - ~ + 1 ~ ~ ~
For the 93 isolated fungal colonies, survival was highest in the presence of hexadecane (76%) and toluene (56%), likely due to a lower toxicity compared to the chlorinated PCB (LD50 Rat oral: Toluene 2.6 to 7.5 g/kg, PCB 4250 mg/kg, Hexadecane not lethal; cf., U.S. National Library of Medicine). 20% of the colonies showed slow or no growth in the pure strain screening, which can be explained by mutualism or by an additional carbon source such as dissolved organic carbon from the soil which was missing in the second screening using pure cultures.
Interactions and mutualism of different strains in the microbiota might play an important role for degradation as a study by Boonchan et al. (2000) showed.
After eliminating similar strains based on their macroscopic and microscopic appearance, we chose 11 fungal strains (Table 4) that showed a high increase in biomass (Absorbance ≥ 0.5 for hexadecane or toluene, increasing absorbance with PCB) and more than one microtiter plate setup (Figure 2a, b, c). These strains were chosen for identification through Sanger sequencing of the ITS or 18S rDNA. We used different primer pairs to get the optimal coverage and identity using NCBI BLAST (Table 1). For most of the strains, growth in the presence of PCB 126 was poorly visible. Only strains BL3 and U, identified as
Growth pattern of the chosen fungal isolates for sequencing: + = good growth, ~ = very slow growth and - = no growth. Hex = hexadecane, Tol = toluene, PCB = PCB 126 Tabelle 4. Wachstumsmuster der zum Sequenzieren ausgewählten Pilzisolate: + = gutes Wachstum, ~ = sehr langsames Wachstum und - = kein Wachstum. Hex = Hexadekan Tol = Toluol, PCB = PCB 126No Hex Tol PCB α 14 + + ~ O + + - U + + + V + + - X 1 + + - Y + + - Z + + ~ BL 3 + + + BL 4 ~ + ~ G + - ~ H + - ~
The two fungal strains only increased their biomass at the end of the test, and therefore, might need longer growth time. For 9 strains, an increase of OD in the presence of toluene was visible.
Nine out of 11 strains (as given in Table 1) have previously been reported as isolated from the contaminated sites. Especially good results in the microtiter plate screening showed
The isolation of
Another good degrader for hexadecane might be
In our study, we present the successful usage of a microtiter plate screening method for isolating the fungal strains from PAH contaminated soil. Microbiota originating from the contaminated soil is reported by Garon et al. (2004) to degrade contaminants better than the collection of originating reference strains. Our study combines the advantage of the quick, high throughput screening method and the selective pressure of contaminated soil. The method was proven successful not only of being able to isolate 93 cultivable colonies but also 9 strains could be identified that are connected to the degradation of organic contaminants. The 11 identified strains could potentially be applied to bioremediation or biofilter systems. The strains are isolated from specific sites in Austria and should be well adapted for similar contaminated sites.
In 6 out of 12 samples, a link between the proportion of HMW to total PAH concentration and the total number of isolated fungal communities could be observed. Additionally, 20% of the strains were not able to grow the presence of the contaminants as a pure culture, which might be explained by mutualism with other fungal strains, other members of the soil microbiota, lack of substrate from the soil inoculant or difference in toxic pressure from the contaminant substrate.
The identified 11 strains can be used as a starting point for detailed research on carbon utilization and degradation performance. Especially, the use of the contaminants as sole carbon source without producing toxic cometabolites or additional carbon sources such as dibutyl phthalate from the microtiter plate material needs to be confirmed. Future work will have to address the toxicity of intermediate products, fate, degradation pathways, enzymes used and up and downregulated genes. While for ligninolytic fungi, a lot of studies concerning the enzymes involved in PAH degradation are available; for non-ligninolytic fungi, this information is lacking (Marco-Urrea et al., 2015). The correlation of 7 strains with the suggestion of Prenafeta et al. (2006), proposing a link between the capabilities of fungal strains to degrade contaminants and being neurotropic agents for warm-blooded vertebrates, has to be further investigated and might also help to understand fungal pathogenicity. Therefore it is of utmost importance to understand fungal degradation mechanisms and strains contributing to bioaugmentation to reduce the risk of spreading pathogenic microbes. More studies are needed to enable an effective usage of non-ligninolytic fungi, understand their degradation mechanisms, interaction with other microorganisms in degradation application and anticipate new effective technologies.