TITLE: Wastewater-Agar as a selection environment: a first step towards a fungal in-situ bioaugmentation strategy
AUTHORS: Valeria Tigini1*, Federico Bevione1, Valeria Prigione1, Anna Poli1, Lucrezia
Ranieri1, Francesco Spennati2, Giulio Munz2, Giovanna Cristina Varese1
AFFILIATION: 1 Department of Life Sciences and Systems Biology, University of Turin,
viale Mattioli 25, 10125 Turin, Italy. 2 Department of Civil and Environmental Engineering,
University or Florence, via Santa Marta 3, 50139 Firenze, Italy. 1 2 3 4 5 6 7 8
Abstract
Viable and metabolically active fungi in toxic mixed liquors, treating landfill leachates and municipal wastewaters, were identified by culture depending methods. A selective culture medium consisting of wastewater and agar (WA) restrained fungi that could be randomly present (94% of the 51 taxa retrieved on WA were sample-specific), overcoming the problem of fast growing fungi or mycoparasite fungi. Moreover, WA allowed the isolation of fungi with a possible role in the degradation of pollutants typically present in the two wastewaters. Phoma medicaginis var. medicaginis, Chaetomium globosum, and Geotrichum candidum were mainly found in municipal wastewater, whereas Pseudallescheria boydii, Scedosporium apiospermum, Aspergillus pseudodeflectus, and Scopulariopsis brevicaulis were typical of landfill leachate.
Key Words: Landfill leachate, municipal wastewater, activated sludge, autochthonous fungi, ecotoxicity, bioremediation.
1 Introduction
Traditional wastewater treatments are mainly based on biological action of activated sludge, technically called secondary treatment (Orhon, 2015). However, this technique has a limited effectiveness in case of recalcitrant or toxic pollutants. In particular, the composition of municipal wastewaters and landfill leachates has a strong impact on the microbial
community of the activated sludge systems and on the growth and metabolic activity of microorganisms capable of degrading recalcitrant compounds (Ren, 2004; Schiopu and Gavrilescu, 2010; Tran et al., 2017). In such cases, fungal bioaugmentation can be a valid tool for the implementation of secondary treatments (Loffredo et al., 2016; Zhao et al., 2017). Nevertheless, this alternative strategy can often fail on account of the scarcity of knowledge 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33
on the composition and dynamics of the community that contributes to the depuration process (Herrero and Stukey, 2015).
Recently, the literature has pointed out the presence of autochthonous fungi in the activated sludge of WWTPs treating municipal wastewaters and landfill leachate (Awad and Kraume, 2011a; Evans and Seviour, 2012; Fathi et al., 2017; Korzeniewska, 2011).
Nevertheless, these studies are generally focused on the sanitary risk associated to the work environments, since several pathogens or toxigenic species have been identified. Conversely, the role of these fungi in the depuration process is quite unknown (Guest and Smith, 2002; Tigini et al., 2014). They could be underestimated since hyphal colonisation of flocks in activated sludge is not predominant during normal operational conditions (Awad and Kraume, 2011b; Liu et al., 2017). Nevertheless, even though the organisms that quantitatively
dominate in activated sludge are the main organisms responsible of the degradation of pollutants, it is not to be excluded that fungi may have relevant ecological functions. In fact, as described by 20-80 Pareto's principle, that finds application also in ecological function of species, a minor part (roughly 20%) of factors can determine the majority (80%) of the effects. Fungi could be involved both in the direct degradation of pollutants and in the formation and stabilisation of the adequate ecosystems for the development of degrading organisms (Liu et al., 2017). Thus, the autochthonous mycobiota in WWTPs could represent a source of organisms with great potential for an in-situ bioaugmentation strategy (Djelal and Amrane, 2013; Herrero and Spina et al., 2018; Stukey, 2015; Tigini et al., 2018). However, in order to identify the fungi potentially involved in the degradation of recalcitrant pollutants, a selection based on adequate culture should be performed. In fact, not only the presence or viability of fungi is relevant, but also on the capability to exploit certain substrates as source of nourishment (Mara and Horan, 2003).
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The toxicity of the influent to be treated is another aspect that must be addressed, since this is a critical factor that can negatively affect the wastewater biological treatability (Adams et al., 2015). Several methodologies for the evaluation of the toxicity towards activated sludge have been reported in the literature (Ren, 2004). However, these techniques are focused on the impact of pollutant towards prokaryotes (i.e. respirometry, ecotoxicity tests using bacteria, etc.), ignoring the effect on other organisms. A standard method for the ecotoxicity evaluation based on fungal bioassay has not been accepted world wide yet. Consequently, the toxicity of samples should be evaluated towards different organisms, in order to acquire more
information on impact on a complex ecosystem such as that of activated sludge, which includes fungi (eukaryotes).
This work represents a preliminary study aimed at evaluating the implementation of in-situ bioaugmentation of activated sludge systems with fungi to improve the removal of recalcitrant compounds from municipal wastewaters and landfill leachates. Firstly, the ecotoxicity of the samples was assessed with four bioassays exploiting eukaryotic species (two aquatic and two terrestrial), in order to acquire information about the impact of the samples on living organisms belonging to the same domain of fungi. Moreover, the
autochthonous mycobioma, coming from mixed liquors of two WWTPs treating municipal wastewaters and landfill leachates, was cultured on selective media, in order to isolate strains with pollutant biodegradation capabilities. The incubation was conducted at two temperatures to mimic seasonal fluctuations, in order to assess their influence on fungal development. The final goal was to select fungi for the in-situ bioaugmentation treatment, which could take into account a direct enrichment of the activated sludge with the fungal biomasses (cultured in loco exploiting a recirculating side stream flux), or an eventual use of the biomass to treat the effluent after secondary treatments.
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2 Materials and Methods 2.1 Mixed liquor samples
Culturable fungi were isolated from samples of mixed liquor collected in two WWTPs located in Tuscany (Italy):
M – mixed liquor collected from the aerobic reactor of the San Colombiano WWTP treating most of the municipal wastewater produced by the city of Florence (Italy) and managed by Publiacqua;
L - mixed liquor collected from the aerobic reactor of a membrane bioreactor (MBR) used as pre-treatment of landfill leachate in the Calice WWTP managed by Gida Spa in Prato (Italy).
San Colombano WWTP treats wastewaters of the municipalities of Florence, Campi Bisenzio, Calenzano, Sesto Fiorentino, Signa, Lastra a Signa and Scandicci. The plant
capacity is about 600,000 equivalent inhabitants. The influent is pretreated through coarse and fine screens, degritting and oil removal) and then partitioned among three identical biological activated sludge sections. The biological treatment is composed by predenitrification,
nitrification (34,000 m3 of volume in total) and settling. Phosphorus is removed by chemical
dosing (alum) inside the biological reactors. The influent flow rate is about 9,000 m3 h-1, the
retention time (HRT) is about 8 h and the solids retention time (SRT) is about 14 days. Calice WWTP treats about 39,000 m3 d-1 of effluents coming from sewage (domestic
and textile wastewaters coming from the district of Prato, one of the main textile area in Europe) and about 700 m3 d-1 of truck-transported liquid wastes. The four oxidation tanks
(about 15000 m3 total volume) are in carousel shape. The tanks are equipped with a joint
system for mixing and airing. In each oxidation tank there is a system of mixing/aeration. The landfill leachate, transported by trucks, is pre-treated in a MBR (predenitrification,
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nitrification and filtration unit equipped with ultrafiltration membrane). In the MBR the HRT is 5 d and the SRT is about 50 days. Subsequently, the permeate leachate is mixed with the main flux after the primary settling of the WWTP.
2.2 Chemical and ecotoxicity analyses
The pH was determined with a Hach-Lange’s probe. The ammonium was measured by means of a nitrogen analyser (TOC-L, Shimazdu). Chlorides and phosphate ions were
measured by means of an inductively coupled plasma mass spectrometry (ICP-MS) (Perkin Elmer). The Chemical Oxygen Demand (COD) and soluble Chemical Oxygen Demand (sCOD) were determined using Hach-Lange’s cuvettes. The Total Suspended Solids (TSS) were measured as the dry weight (1 hour at 105 °C) of the residue of the filtered sample. The Volatile Suspended Solids (VSS) were measured as the dry weight (30 min at 570 °C) of the residue of the filtered sample.
Four bioassays were used to evaluate the ecotoxicity of the samples. The target
organisms were: the unicellular green alga (I) Raphidocelis subcapitata (Korshikov) Nygaard, Komárek, J.Kristiansen & O.M. Skulberg (UNI EN ISO 8692:2005); the terrestrial plants (II) Cucumis sativus L. and (III) Lepidium sativum L. (UNICHIM N. 1651, 2003); and the aquatic plant (IV) Lemna minor L. (ISO SO/WD 20079). The samples were used such as (mixed liquors, i.e. liquid phase and biomass together), with the exception of the algal test, in which filtration (Whatman type 1) was needed to avoid interferences with spectrophotometric lectures. The results were expressed as inhibition percentage with respect to the average (at least three repetitions) of opportune control in which the sample was substituted with distilled water or salin solution, depending on the method.
2.3 Isolation and identification of fungi 108 109 110 111 112 113 114 115 116 117 118 119 120 121 122 123 124 125 126 127 128 129 130 131 132
Aliquots of 1 mL of each sample were placed in Petri dishes (16 cm diam.) containing 30 mL of culture medium. Three different media were used: a modified Malt Extract Agar (MEAp: agar 20 g, glucose 2 g, malt extract 2 g, peptone 0.2 g, water up to 1 L); Dichloran Rose Bengal Agar (DRBC: 31.5 g, water up to 1 L); Wastewater-Agar (WA: agar 20 g, glucose 2 g, mixed liquor supernatant after 5 min at 10,000 rpm up to 1 L). A set of three antibiotics was added to all media: streptomycin 0.015 g L-1, chloramphenicol 0.05 g L-1, and
ampicillin 0.05 g L-1. The trial was performed with 20 replicates. Ten of them were incubated
at 25 °C, and the remaining ten were incubated at 15 °C. All the plates were incubated in the dark for 30 days.
The colony forming units (CFUs) were counted and the different fungal morphotypes were isolated in pure culture. Fungi were identified through macroscopic and microscopic features, by means of selected genus monographs for species identification. Moreover, molecular identification of fungi was performed by amplification and sequencing of specific markers: actin for Cladosporium (Bensch et al., 2012); β-tubulin for Penicillium and
Aspergillus (Glass and Donaldson 1995); D1/D2 region for yeasts (Fell et al., 2000); Internal Transcribed Spacer (ITS) for other genera (White et al., 1990). The resulting sequences were compared with reference sequences in online databases provided by the CBS-KNAW
Collection (Westerdijk Fungal Biodiversity Institute, Utrecht, The Netherlands) and the NCBI National Centre for Biotechnology Information (USA). Representative strains isolated in pure culture during this work were preserved at Mycotheca Universitatis
Taurinensis (MUT, www.mut.unito.it) of the Department of Life Sciences and Systems Biology, University of Turin (Italy). Newly generated sequences (ITS, actine and β-tubulin) were deposited in GenBank with the following accession numbers MK029865-MK029867, MK034868-MK034870, and MK036877-MK036883. 133 134 135 136 137 138 139 140 141 142 143 144 145 146 147 148 149 150 151 152 153 154 155 156 157
2.4 Statistical analyses
The nonparametric Mann-Whitney test (p≤0.05) was run to assess the significance of quantitative (fungal load) differences in relation to three variables: culture medium,
incubation temperature and sample. Differences among factors (sample, incubation
temperature and culture medium) were evaluated by applying a Permutational Multivariate Analysis of Variance (PERMANOVA; P<0.05) and visualised by Non-Metric
Multidimensional Scaling (NMDS). The contribution of individual species (in percentage) to the diversity observed was assessed by SIMilarity PERcentage (SIMPER) analyses.
3 Results and discussion
3.1 Chemical and ecotoxicological analyses
The results of chemical analyses are shown in Table 1. The samples showed typical values of suspended solids in activated sludge systems, even though in the MBR (L) higher values could be expected. The ratio between organic (represented by COD and VSS) and inorganic fractions of biomass was also conventional for mixed liquors of suspended systems. The values reported for SRT were compatible with a stable and complete nitrification process, as shown by the low ammonium concentration. The phosphate concentration was relatively low. This parameter, however, could be subjected to fluctuations, depending on influent characteristics for L sample, and on the inline dosing of alum for P removal in M sample. The soluble fraction of COD (sCOD) was very low in M sample due to both efficient removal of COD and the specificity of the sewage system of the city of Florence, where buildings are equipped with septic tanks. The sCOD of M sample was higher with respect of L, consistently with origin of the influent. Eventually, supernatant colour was dark brown for P; whereas, M was almost colourless.
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Five ecotoxicological endpoints (four bioassays) were exploited for the evaluation of the impact of these samples on living organisms.
The growth inhibition of the alga R. subcapitata is shown in Figure 1. The recorded data were linearly distributed for L sample (R2=0.97), whereas M showed a lower linear
correlation between dose and effect (R2=0.86). Surprisingly, M was more toxic than L; in fact,
the first achieved 100% alga inhibition with dose over 40%, while the second did not exceed the 20% inhibition.
The results of root elongation inhibition of L. sativum and C. sativus are reported in Figure 2. Linear distribution of dose-effect data for C. sativus test was absent (R2<0.002). L.
sativum, instead, showed a linear distribution only for L sample (R2=0.85). Moreover, this
was the sole case in which the toxicity was consistent, achieving 80% inhibition at 80% L dose. On the contrary, in all the other cases the inhibition effect did not exceed 40%.
L. minor was never sensitive towards the two samples, considering both the endpoints, fronds number (0% inhibition) and biomass development (up to -15% inhibition).
R. subcapitata was the most sensitive organism, with inhibition up to 100% at 80% sample dose. Its higher sensitivity was already reported in studies on landfill leachate (Tigini et al., 2014) and municipal wastewaters (Spina et al., 2015). Moreover, it must be emphasized that the sample filtration, performed for this test only, could have removed some elements (bacteria, protozoa, pollutant aggregates, etc.) that potentially cause algal inhibition. With respect of the other three tests, therefore, the results could be and underestimation of the actual ecotoxicity. Thus, this organism can be recommended for the evaluation of ecotoxicity of this kind of samples, and for the evaluation of toxicity variation after bioremediation treatments.
Surprisingly, only M was highly toxic, exceeding 50% inhibition at 100% dose; whereas, L showed inhibition effects always below 20%. Intuitively, landfill leachates are 183 184 185 186 187 188 189 190 191 192 193 194 195 196 197 198 199 200 201 202 203 204 205 206 207
often considered more toxic than other wastewaters; this is due to several reasons, including their dark colour that can impact on the photosynthetic process in aquatic algae, causing a sort of physical toxicity (Cleuvers and Ratte, 2002). Moreover, their toxicity can be caused by high ammonium concentration, which characterises landfill leachates (Tigini et al., 2014). However, in this case, the sample was not a raw leachate, and the concentration of ammonium in the filtered mixed liquor was very low, since complete nitrification was occurring in the MBR.
Treated municipal wastewaters are characterised by a huge variety of pollutants, including contaminants of emerging concern at very low concentration, that are generally indicated to interfere with the endocrine system and have chronic toxicity effects towards aquatic animals and phytoplankton (Szymonik et al., 2017). Acute effects are generally not observed in treated wastewaters (Spina et al., 2015). However, biosorption on activated sludge could concentrate pollutants, causing acute effects. This could be the case of the tests performed with plants, where the samples were used as such, including the solid fraction of the mixed liquors (Figure 2). Nevertheless, even when filtered (algal test), M was still more toxic than L sample.
On the base of the ecotoxicity results towards eukaryotic organisms, the mixed liquor of WWTPs treating landfill leachate seems to be a good target for in-situ additional fungal bioremediation strategies. The fungal biomass could be used to treat the effluent after secondary treatment. On the other hand, fungi could be cultured in loco exploiting a
recirculating side stream flux and exploited for direct bioaugmentation of the activated sludge. Additionally, results also suggest that fungi may be a very powerful bioresource for the pre-treatment of sludge to reduce its ecotoxicity for an eventual soil application of the dewatered excess sludge. 208 209 210 211 212 213 214 215 216 217 218 219 220 221 222 223 224 225 226 227 228 229 230 231
The isolation of fungi capable to actively grow on the wastewaters, tolerating their toxicity and possibly using wastewater components for their development, is a condicio sine qua non to achieve this ambitious goal.
3.2 Suitable conditions for the isolation and identification of typical culturable mycobiota The total fungal load was high in both samples (Table 2). In M it ranged from 7,060 CFU 100 mL-1 (WA 25 °C) to 5,8170 CFU 100 mL-1 (DRBC 25 °C). In L the fungal load
ranged from 4,344 CFU 100 mL-1 (DRBC 15 °C) to 12,190 CFU 100 mL-1 (WA 25 °C). M
generally had a higher fungal load compared to L. Samples had comparable fungal load only when inoculated on WA.
The two samples (M and L) were qualitatively different, too. Among the 74 taxa (complete list in Supplementary materials), only 22 (30%) were detected in the two samples (M and L). Most of the isolated taxa were exclusive of a single sample, indeed. In particular, 19 (26%) were exclusive of M, 34 (44%) were exclusive of L. The difference in the retrieved fungal species could partially be due to the different process configurations adopted in the two WWTPs. Besides differences in the influent origin and characteristics, WWTPs are operated at very different SRT (12 d for M and about 50 d for L) and this can affect both the survival and the predation on fungi. Moreover, the M mixed liquor is selected through settling, whereas the biomass in the MBR (L) is retained by membranes. Fungal relative abundance would be diversely affected in MBR respect to conventional activated sludge systems, in relation to their different tendency to settle with respect to the rest of suspended solids in the reactors. Finally the shear stress caused by more intense aeration system typical of MBR can also impact on fungal activity. In a recent study on fungi of MBR reactors present in the literature, few fungal species were reported (Maza-Márquez et al., 2016). However, this 232 233 234 235 236 237 238 239 240 241 242 243 244 245 246 247 248 249 250 251 252 253 254 255
hypothesis should be confirmed by comparing the two configuration reactors treating the same kind of influent.
Considering overall data, PERMANOVA test pointed out 93% dissimilarity between the two samples. The group of the unidentified fungi mostly contributed to this difference (10-27%), and was particularly abundant in M (Table 3). The cause of the massive presence of fast growing fungi (Mucor spp.) and mycoparasites (Trichoderma spp.) in M, determined the inhibition and/or the failure of isolation in pure culture of other fungi with lower growth rate. This caused the failure in the identification of a great number of morphotypes grown on the sample M. Mucor and Trichoderma are genera retrieved frequently in municipal WWTPs, which receive wastewaters with high concentration of organic matter (Katsivela et al., 2017). Intuitively, they are relatively less abundant in landfill leachate, in which the organic matter, although in high concentration, is characterised by highly recalcitrant molecules (Tigini et al., 2014).
Selective media with low glucose concentration (MEAp) or fungistatic components (DRBC) were used in order to solve this issue. Moreover, a medium (WA) with wastewaters as principal source of carbon was used to limit the development of fungi coming from the surrounding environment. These fungi could be present under non-dividing, but metabolically active state, in mixed liquors. These fungi could be reactivated by the nutrients of a rich cultural medium or the presence of antibiotics that inhibit autochthonous bacteria (Haruta and Kannol, 2015). The list of the species that mostly contributed to the intragroup similarity is reported in Table 3.
Considering separately the data obtained on the three media (Figure 3), the highest dissimilarity percentages between L and M were relieved on WA medium (99% and 97% at 25 °C and 15 °C, respectively). On the contrary, the lowest ones were relieved on DRBC (89% and 86% at 25 °C and 15 °C, respectively).
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Within each sample, the culture medium deeply affected the development of fungi both from quantitative and qualitative points of view (Table 2, Figure 4). PERANOVA test underlined significant intergroup (different media at the same temperature) dissimilarities. The medium that mostly determined these differences was WA: dissimilarities between this medium and the other two were in the range of 60-90% in M and 87-99% in L. The fungal species in WA, that determined these differences, are characterised by high toxicity tolerance and could have biotechnological applications in bioremediation field. They will be discussed separately in the further paragraph.
On the contrary, DRBC and MEAp showed lower dissimilarity (47-73% in M and 73-90 in L). Three species of Trichoderma (T. asperellum in M; T. asperellum, T. harzianum and T. asperelloides in L) contributed significantly to the differences (3-19%). However,
contrarily to the expectation, these species were more abundance on DRBC, with respect to MEAp. On the other hand, DRBC inhibited the genus Mucor. Actually, M. luteus (in M) and M. plumbeus (in L) were among the main contributors to MEAp-DRBC dissimilarity (11.3% and 2.5%, respectively).
The problem of fast growing fungi has been already relieved in the evaluation of fungal exposition risk in work environment (Katsivela et al., 2017; Nunes et al., 2012). Despite DRBC is generaly suggested as suitable for successful fungal isolation from food by limiting fast growing fungi (Beuchat and Mann, 2016), its lower performance towards the genus Trichoderma was already signalled (Pitt and Hocking, 2009). Trichoderma abundance in M cultured on DRBC could be at the base of the lower number of isolated taxa with respect to MEAp, in particular in M (Table 3).
These results suggest that WA can be a valid medium for the isolation of characteristic culturable mycobiota in activated sludge.
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Incubation temperature was another culture parameter that was considered for the impact of fungal colony development in mixed liquors samples. We considered two
temperatures (25 and 15 °C) representing the seasonal temperature extremes in the activated sludge tank at temperate latitudes.
Lower fungal load was achieved at lower temperature, with significant difference in four cases out of six (Table 2). PERANOVA test always pointed out significant differences in fungal biodiversity. Nevertheless, the intergroup dissimilarity was relatively low, ranging between 31% and 67% (Figure 4). This could be due to the low intragroup similarity that ranged between 55% and 77%, with the exception of M cultured on DRBC, which ranged between 86% (15 °C) and 90% (25 °C). It could be concluded that the incubation temperature influences the fungal development in lower extent with respect to the culture medium.
The effect of altitudinal location of municipal WWTPs on fungal population of activated sludge was recently assessed (Niu et al., 2017). However, a precise identification of the parameters affecting the fungal population was not recognised. Despite several natural factors change with the altitude (temperature, UV irradiation, etc.) the characteristics of the influent seem to be the main cause of variability in microorganism population (Niu et al., 2015). So, this variability is mainly attributable to anthropic factors, i.e. the load of wastewaters and the contaminant concentration due to the demographic density, local practises and diet, the recalcitrance of molecules due to facilities (hospitals, etc.) that confer wastewaters to the treatment plants, pollution of runoff waters, etc.
3.3 Identification of fungi as potential agent of degradation in activated sludge
The species present in WA that most contributed to the dissimilarity with the other two media are potentially agent of degradation in activated sludge. They showed an interesting capacity to tolerate wastewater toxicity and probably to use pollutants as main source of 305 306 307 308 309 310 311 312 313 314 315 316 317 318 319 320 321 322 323 324 325 326 327 328 329
nutrients. Actually, in WA medium there was only a minimal quantity of glucose (2 g L-1),
which was added to allow the conidial germination (phase in some way critical for fungi). However, this was not sufficient to sustain an active growth for the entire incubation period (30 days). Fungi retrieved in this medium should transforme in some extent the substrate provided with the wastewater.
In M they were Phoma medicaginis var. medicaginis (9.4-9.9%), Chaetomium globosum (5.2%), and Geotrichum candidum (4.6-5.7%). These three species typically colonise keratinous materials (Sterflinger, 2010). Hair and fibrous substances are particularly abundant in municipal wastewaters representing a serious issue both for mechanical
occlusions and their recalcitrance to bacterial oxidation (Frechen et al., 2008). Thus, the abundance of these fungi isolated with WA medium could indicate a role in the degradation of keratinous material present in municipal wastewaters. In addition to keratinolytic enzymes, G. candidum and C. globosum display a wide range of enzymes, e.g. laccases, peroxidases, monoxigenases, etc. (Varnaitė et al., 2011), active towards different recalcitrant compounds through unspecific mechanisms and could contribute to the biotransformation of most recalcitrant molecules in a synergic cooperation with other organisms of activated sludge. In particular, fungal laccases seem to be involved in the removal of micropollutants decreasing both ecotoxicity and estrogenic activity (Spina et al., 2015). Their dominance in WA and low presence in other media indicates that they are outcompeted by other fungi, when easily degraded C sources are available. This could be a consistent possibility in case of plats with low retention time, and could represent a limit in fungal application aimed to in-situ
bioaugmentation. In order to overcome a possible failure, they could be cultured in a side stream bioreactor in which the most degradable carbon sources are already consumed and the most recalcitrant molecules remain substantially unchanged.
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Other notable fungi that contribute to WA intragroup similarity, i.e. F. solani, M. ruber, M. circinelloides f. lusitanicus, M. circinelloides var. circinelloides (Table 3), represent a source of potential bioremediation agents. Also these species could have biodegradation role in activated sludge. Actually they produce different enzymes that can degrade pollutant typically present in municipal wastewaters. In particular, all of them have been reported to produce agmatine oxidase, that can oxidize histamine, putrescine, 1,3-diaminopropane and cadaverine (Isobe et al., 1982). These substances are the reaction products of the degradation of animal proteins, which are particularly abundant (37% of the dry weight) in biosolids of municipal wastewaters (Mathur, 1998). Thus, these species could cooperate with protein degrader microorganisms (fungi and bacteria) and contribute to the removal of smells. Moreover, some of them (i.e. F. solani M. circinelloides var. circinelloides) can easily compete with other species and/or display versatility in the exploitation of carbon sources, since they are frequent even in the other two media (Table 3).
In L sample, the most abundant species that showed the highest contribution to dissimilarity were Scedosporium apiospermum (24.4-25.8%), Aspergillus pseudodeflectus (6.3-6.4%), Scopulariopsis brevicaulis (4.4%), and Pseudallescheria boydii (3.8-4.2%). These species have been demonstrated to have biodegradation capabilities towards several
xenobiotics (Ishii et al., 2009; Raaman et al., 2012; Verma et al., 2017). In particular, P. boydii group complex can attack the phenolic compounds (Clauβen et al., 1998). Thus, these species could be involved in the degradation of tannins and phenols, particularly abundant in mature landfill leachates (Bardi et al., 2017). A. pseudodeflecus, on the other hand, has been reported to degrade disel fuel (Ghanem et al., 2016).
Besides the species frequently isolated on WA, other fungi retrieved with less frequency are notable. In particular, Acrostalagmus luteoalbus, Chrysosporium
keratinophilum, F. solani, S. candida, T. virens, Yunnania pennicillata. Some of these species 354 355 356 357 358 359 360 361 362 363 364 365 366 367 368 369 370 371 372 373 374 375 376 377 378
are reported in Brenda database to produce peroxidases, laccases, tannases, and nitrate
riductases potentially involved in degradation in activated sludge. On the other hand, some of these fungi (i.e. Pseudallescheria/Scedosporium species complex, Aspergillus spp.,
Scopulariopsis spp., Chrysosporium keratinophilum) can be human pathogen and require a biosafety level 2 for their manipulation. Thus, their bioaugmentation in WWTPs should be cautionally evaluated. Both biodegradation and pathoghenic capabilities are strain-specific features, and they should be explored for each isolated strain.
Considering all these results, WA medium showed to be a potential tool for the isolation of fungi with high biodegradation potential.
4 Conclusions
Viable and metabolically active mycobiota of mixed liquors from municipal
wastewaters and landfill leachates was evaluated through selective culture depending method. The WA medium was useful for restraining fungi that could be randomly present, without the capacity to exploit pollutants as the main source of nutrients. WA allowed isolating the typical fungal population of the samples, limiting fast growing or mycoparasite fungi too. Moreover, it allowed the isolation of fungi with a potential role in the degradation of pollutants specific of wastewaters. WA is recommended as a tool for the isolation of fungi with a high biodegradation potential. In the next future, the fungi isolated on this medium will be tested in contolled environment in order to evaluate their effective contribution to
wastewater bioremediation, monitoring pollutant removal and enzyme production.
Acknowledgements
This work was funded by the Ministero dell’Istruzione, dell’Università e della Ricerca (MIUR) with the FIRB project (grant number RBFR13V3CH_002).
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Figure 1. Algal growth inhibition, expressed as percentage with respect to the control (average of six repetitions), caused by filtered samples of mixed liquors selected by municipal wastewater (M) and landfill leachate (L).
Figure 2. Phytotoxicity, expressed as inhibition percentage of the root elongation with respect to the control (average of four repetitions), caused by the mixed liquors selected by municipal wastewater (M) and landfill leachate (L).
Figure 3. Similarity between the two samples (M and L) cultured on different media (MEAp, DRBC, WA) at two temperatures (25 °C and 15 °C).
Figure 4. Similarity among cultured media (MEAp, DRBC, WA) and two temperatures (25 °C and 15 °C) for the two samples (M and L).
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