Tannery Wastewater Recalcitrant Compounds Foster the Selection of Fungi in Non-Sterile Conditions: A Pilot Scale Long-Term Test.

This study demonstrated that a microbial community dominated by fungi can be selected and maintained in the long-term under non-sterile conditions, in a pilot-scale packed-bed reactor fed with tannery wastewater. During the start-up phase, the reactor, filled with 0.6 m3 of polyurethane foam cubes, was inoculated with a pure culture of Aspergillus tubingensis and Quebracho tannin, a recalcitrant compound widely used by tannery industry, was used as sole carbon source in the feeding. During the start-up, fungi grew attached as biofilm in carriers that filled the packed-bed reactor. Subsequently, the reactor was tested for the removal of chemical oxygen demand (COD) from an exhaust tanning bath collected from tanneries. The entire experiment lasted 121 days and average removals of 29% and 23% of COD and dissolved organic carbon (DOC) from the tannins bath were achieved, respectively. The evolution of the microbial consortium (bacteria and fungi) was described through biomolecular analyses along the experiment and also developed as a function of the size of the support media.

The role of cosubstrate and mixing on fungal biofilm efficiency in the removal of tannins.

Tannins are polyphenolic compounds produced by plants and they are used in industrial vegetable tanning of leather. Tannins represent one of the low biodegradability substances in tannery wastewaters with high recalcitrant soluble chemical oxygen demand, furthermore high concentration of tannins can inhibit biological treatment. In the present study, four novel rotating submerged packed bed reactors were inoculated with a selected fungal strain to reach a biological degradation of tannins in non-sterile conditions. The selected fungal strain, Aspergillus tubingensis MUT 990, was immobilised in polyurethane foam cubes carriers and inserted inside a submerged rotating cage reactors. The reactors were feed with a solution composed of four tannins: Quebracho (Schinopsis spp.), Wattle (Mimosa spp.), Chestnut (Castanea spp.) and Tara (Caesalpinia spp.). Four reactors with a volume of 4 L each were used, the co-substrate was pure malt extract, the hydraulic retention time was 24 h and the pH setpoint was 5.5. The reactors configuration was chosen to allow the study of the effect of rotation and the co-substrate addition on tannins removal. The experiment lasted two months and it was achieved 80% of chemical oxygen demand and up to 90% dissolved organic carbon removal, furthermore it was detected an important tannase activity.

Wastewater-Agar as a selection environment: A first step towards a fungal in-situ bioaugmentation strategy.

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.

Biotransformation of industrial tannins by filamentous fungi.

Tannins are secondary metabolites that are widely distributed in the plant kingdom. They act as growth inhibitors for many microorganisms: they are released upon microbial attack, helping to fight infection in plant tissues. Extraction of tannins from plants is an active industrial sector with several applications, including oenology, animal feeding, mining, the chemical industry, and, in particular, the tanning industry. However, tannins are also considered very recalcitrant pollutants in wastewater of diverse origin. The ability to grow on plant substrates rich in tannins and on industrial tannin preparations is usually considered typical of some species of fungi. These organisms are able to tolerate the toxicity of tannins thanks to the production of enzymes that transform or degrade these substrates, mainly through hydrolysis and oxidation. Filamentous fungi capable of degrading tannins could have a strong environmental impact as bioremediation agents, in particular in the treatment of tanning wastewaters.

Bioremediation of Landfill Leachate with Fungi: Autochthonous vs. Allochthonous Strains.

Autochthonous fungi from contaminated wastewater are potential successful agents bioremediation thanks to their adaptation to pollutant toxicity and to competition with other microorganisms present in wastewater treatment plant. Biological treatment by means of selected fungal strains could be a potential tool to integrate the leachate depuration process, thanks to their fungal extracellular enzymes with non-selective catalytical activity. In the present work, the treatability of two real samples (a crude landfill leachate and the effluent coming from a traditional wastewater treatment plant) was investigated in decolorization experiments with fungal biomasses. Five autochthonous fungi, Penicillium brevicompactum MUT 793, Pseudallescheria boydii MUT 721, P. boydii MUT 1269, Phanerochaete sanguinea MUT 1284, and Flammulina velutipes MUT 1275, were selected in a previous miniaturized decolorization screening. Their effectiveness in terms of decolorization, enzymatic activity (laccases and peroxidases), biomass growth and ecotoxicity removal was compared with that of five allochthonous fungal strains, Pleurotus ostreatus MUT 2976, Porostereum spadiceum MUT 1585, Trametespubescens MUT 2400, Bjerkanderaadusta MUT 3060 and B. adusta MUT 2295, selected for their well known capability to degrade recalcitrant pollutants. Moreover, the effect of biomass immobilization on polyurethane foam (PUF) cube was assessed. The best decolorization (60%) was achieved by P. spadiceum MUT 1585, P. boydii MUT 721 and MUT 1269. In the first case, the DP was achieved gradually, suggesting a biodegradation process with the involvement of peroxidases. On the contrary, the two autochthonous fungi seem to bioremediate the effluent mainly by biosorption, with the abatement of the toxicity (up to 100%). The biomass immobilization enhanced enzymatic activity, but not the DP. Moreover, it limited the biomass growth for the fast growing fungi, MUT 721 and MUT 1269. In conclusion, robust and versatile strains coming from well-characterized collections of microorganisms can obtain excellent results comparing and even exceeding the bioremediation yields of strains already adapted to pollutants.

Tannery mixed liquors from an ecotoxicological and mycological point of view: Risks vs potential biodegradation application.

Fungi are known to be present in the activated sludge of wastewater treatment plants (WWTP). Their study should be at the base of an overall vision of the plant effectiveness and of effluents sanitary impact. Moreover, it could be fundamental for the implementation of successful bioaugmentation strategies aimed at the removal of recalcitrant or toxic compounds. This is one of the first studies on the cultivable autochthonous mycoflora present in the mixed liquors of two WWTP treating either vegetable or chromium tannery effluents. All samples showed a risk associated with potential pathogens or toxigenic species and high ecotoxicity (Lepidium sativum and Raphidocelis subcapitata were the most sensitive organisms). Diverse fungal populations developed, depending on the origin of the samples (63% of the 102 identified taxa were sample-specific). The use of a fungistatic was determinant for the isolation and, thus, for the identification of sample-specific species with a lower growth rate. The incubation temperature also affected the mycoflora composition, even though at lower extent. A selective medium, consisting of agarised wastewater, allowed isolating fungi with a biodegradation potential. Pseudallescheria boydii/Scedosporium apiospermum species complex was ubiquitously dominant, indicating a possible role in the degradation of pollutants in both WWTP. Other species, i.e. Trichoderma spp., Trematosphaeria grisea, Geotrichum candidum, Lichtheimia corymbifera, Acremonium furcatum, Penicillium simplicissimum, Penicillium dangeardii, Fusarium solani, Scopulariopsis brevicaulis potentially could be involved in the degradation of specific pollutants of vegetable or chromium tannery wastewaters. However, several of these fungi are potential pathogens and their application, for an in situ treatment, must be carefully evaluated.

Microalgae treatment removes nutrients and reduces ecotoxicity of diluted piggery digestate.

Liquid digestate is considered as an important by-product of anaerobic digestion of agriculture wastes. Currently, it is very often directly spread on local agricultural land. Yet recently concerns on its environmental risk of this processing has begun to rise. On the other hand, investigations on the effectiveness of microalgae for wastewater treatment have started to consider also this complex matrix. In this study, we cultured the green alga Chlorella vulgaris in diluted digestate coming from the anaerobic digestion of pig slurry and corn, with the aim to significantly reduce its toxicity and its very high nutrient concentration. For this purpose, a battery of toxicity tests composed of four acute and two chronic bioassays was applied after the alga cultivation. Results were compared with those obtained in the initial characterization of the digestate. Results show that highly diluted piggery digestate can be a suitable medium for culturing microalgae, as we obtained a high removal efficiency (>90%) for ammonia, total nitrogen and phosphate, though after a few days phosphorus limitation occurred. Toxicity was significantly reduced for all the organisms tested. Possible solutions for optimizing this approach avoiding high dilution rates are discussed.

Is digestate safe? A study on its ecotoxicity and environmental risk on a pig manure.

Digestate represents a precious by-product in particular in agriculture, however its impact on the environment and human health is still unexplored. In this work, the toxicity of a pig slurry digestate was assessed through 7 ecotoxicity tests and considering 10 different endpoints. Besides, a synthetic index was applied to the outputs of the battery of tests for the environmental risk assessment, in order to evaluate the opportunity to use directly this kind of digestate in agriculture or to introduce an additional treatment. All the organisms were sensitive to digestate toxicity (EC50 ranged from 14.22% for Cucumissativus to 0.77% for Raphidocelis subcapitata). The physical-chemical features at the base of this toxicity seem to be the high content of ammonium, salinity, COD, phosphate and colour. The synthetic index showed that the digestate was very toxic and associated to an extremely high environmental risk. Thus, a pre-treatment is needed to reduce its toxicity and environmental impact, whatever could be its exploitation.

Evaluation of an eventual ecotoxicity induced by textile effluents using a battery of biotests.

Textile industry is considered as one of the important factors of the economic growth in Tunisia. However, this prominent role has certainly some drawbacks mainly represented by the huge amounts of textile wastewaters generated that become a real menace to nature. Many previous studies showed the purifying potential of some activated sludge and bacteria (Pseudomonas putida) to decolourize textile effluents. However, in many cases, decolourization of wastewaters is not necessary associated with detoxification, generating a real risk for the ecosystem in general. We evaluated in this work the induced toxicity of a textile effluent before and after its treatment with activated sludge followed by P. putida, using a battery of biotests. This study proved the detoxifying power of the activated sludge according to most of ecotoxicity tests. The treatment with P. putida did not improve the quality of the effluent; on the contrary, it could increase its toxicity. Daphnia magna and Raphidocelis subcapitata appear to be the most sensitive organisms in assessing eventual toxicity caused by this kind of wastewaters.

Mycological and ecotoxicological characterisation of landfill leachate before and after traditional treatments.

Pollution caused by landfill leachates is one of the main problems of urbanised areas, on account of their chemical composition, which turn in an ineffective treatment. A characterisation of leachates, which takes into account chemical, ecotoxicological and mycological aspects, is basilar for the evaluation of environmental impact of leachate and the development of suitable treatment techniques. In this study, the toxicity of a raw leachate and an effluent coming from traditional wastewater treatment plant was assessed by means of 4 ecotoxicological assays. Both the samples exceed the legal threshold value according to all the tested organisms, indicating the ineffectiveness of activated sludge treatment in the reduction of toxicity. The autochthonous mycoflora of the two samples was evaluated by filtration. The fungal load was 73 CFU for leachate and 102 CFU for the effluent. Ascomycetes were the dominant fraction (81% and 61%, for leachate and effluent respectively), followed by basidiomycetes (19% and 39%, respectively). Most of them were potential emerging pathogens. A decolourisation screening with autochthonous fungi was set up towards both samples in the presence or absence of glucose. Eleven fungi (basidiomycetes and ascomycetes) achieved up to 38% decolourisation yields, showing to be promising fungi for the bioremediation of leachates. Further experiment will be aimed to the study of decolourisation mechanism and toxicity reduction.

Integrated fungal biomass and activated sludge treatment for textile wastewaters bioremediation.

A combined biological process was investigated for effective textile wastewater treatment. The process consisted of a first step performed by selected fungal biomasses, mainly devoted to the effluent decolourisation, and of a subsequent stage by means of activated sludge, in order to reduce the remaining COD and toxicity. In particular, the treatment with Trametes pubescens MUT 2400, selected over nine strains, achieved very good results in respect to all parameters. The final scale-up phase in a moving bed bioreactor with the supported biomass of the fungus allowed to verify the effectiveness of the treatment with high volumes. Despite promising results, further steps must be taken in order to optimize the use of these biomasses for a full exploitation of their oxidative potential in textile wastewater treatment.

Influence of culture medium on fungal biomass composition and biosorption effectiveness.

Zygomycetes such as Cunninghamella elegans seem to be promising biosorbents for pollutants removal from wastewaters because of their particular cell wall characteristics. In this article the effect of ten culture media on C. elegans biomass composition was investigated by means of Fourier transform infra red spectroscopy (FTIR). Biomasses grown on starches from potatoes and cereals were characterised by high amount of chitin and polysaccharides, the glucose gave rise to a biomass rich in acidic polysaccharides and lipids. By contrast, biomasses grown on corn steep liquor were poor in acidic polysaccharides and, when N sources and micronutrients were added, rich in proteins. The lipid content of the biomass generally increased by halving nutrients. Biosorption yields of these biomasses towards four wastewater models were assessed in terms of colour, salts and toxicity reduction. The biomasses rich in proteins and acid polysaccharides were less effective in removing reactive and direct dyes, whereas those rich in cationic polysaccharides showed a higher affinity for these dyes. Both chromatography and FTIR analyses showed that biomasses cultured in halved C and N had the highest affinity for salts. The wastewaters detoxification was quite always achieved, with values often lower that the Italian legal threshold limit.

Decolourisation and detoxification in the fungal treatment of textile wastewaters from dyeing processes.

In this study a selected fungal strain, Bjerkandera adusta (Willdenow) P. Karsten MUT 3060, was tested in different culture conditions to assess its real potential for bioremediation of textile wastewaters in terms of both decolourisation and detoxification. The fungus efficiently decolourised (colour removal up to 96%) four simulated wastewaters that mimic the recalcitrance of real ones for pH values, concentration of dyes, additives and salts. In the culture condition with the lowest N content, the decolourisation was coupled with an important detoxification of two simulated effluents, underlining the important influence of the cultural medium composition not only on the degradation but also on the detoxification of industrial wastes. In the other cases, despite an extensive decolourisation, no detoxification was observed. The fungus was further tested against a real effluent, collected from a wastewater treatment plant before and after the tertiary treatment (ozonation) to compare the two technologies in terms of chemical and toxicological parameters. The fungal treatment, although less efficient than ozonation, caused a good decolourisation of the effluent, with colour values within the threshold limits of the Italian law; both the fungal and the ozone treatment caused a detoxification, but only towards one of the three organisms used for the ecotoxicological tests. These results underline the crucial importance of the ecotoxicological analysis in assessing the applicability of a wastewater treatment.

Cunninghamella elegans biomass optimisation for textile wastewater biosorption treatment: an analytical and ecotoxicological approach.

The effect of pre-treatments on the composition of Cunninghamella elegans biomass and on its biosorption yields in the treatment of simulated textile wastewaters was investigated. The inactivated biomass was subjected to physical treatments, such as oven drying and lyophilisation, and chemical treatments using acid or alkali. The wastewater colour, COD and toxicity variations were evaluated. The lyophilisation sped up the biosorption process, whereas the chemical pre-treatment changed the affinity of biomass for different dyes. The alkali per-treated biomass achieved the highest COD reduction in the treatment of alkali wastewaters, probably because no release of alkali-soluble biomass components occurred under the alkaline pH conditions. Accordingly, only the acid pre-treated biomass decreased the COD of the acidic effluent. The ecotoxicity test showed significant toxicity reduction after biosorption treatments, indicating that decolourisation corresponds to an actual detoxification of the treated wastewaters. Fourier transform infrared spectroscopy, differential scanning calorimetry and thermogravimetric analyses of biomasses allowed highlighting their main chemical and physical properties and the changes induced by the different pre-treatments, as well as the effect of the chemical species adsorbed from wastewaters.

Evaluation of toxicity, genotoxicity and environmental risk of simulated textile and tannery wastewaters with a battery of biotests.

Textile and tannery wastewaters are complex mixtures of toxic pollutants and only a battery of ecotoxicity tests can assess their potential environmental impact and the actual effectiveness of alternative treatments. In this work the toxicity of four simulated textile and tannery wastewaters was evaluated by means of a battery of seven bioassays, using organisms that belong to different trophic levels. Moreover, since the outputs of the bioassay battery were quite difficult to compare, a novel synthetic index for environmental risk assessment was applied to the outputs of the test battery. All four simulated wastewaters were very toxic but they showed no mutagenic effect. The alga Pseudokirchneriella subcapitata was the most sensitive organism. In addition, the use of two mathematical models pointed out the interaction effect between dyes and salts, which resulted in a synergistic effect of wastewater toxicity.

Scale-up of a bioprocess for textile wastewater treatment using Bjerkandera adusta.

Twelve basidiomycetes were investigated for their ability to degrade 13 industrial dyes and to treat four model wastewaters from textile and tannery industry, defined on the basis of discharged amounts, economic relevance and representativeness of chemical structures of the contained dyes. The best degradation yields were recorded for one strain of Bjerkandera adusta able to completely decolourise most of the dyes and to decolourise and detoxify three simulated wastewaters, showing a significant physiological versatility which is very useful for application purposes. The effects of different nutrient sources were investigated in order to optimize the yields of decolourisation and detoxification. Manganese-peroxidase and manganese-independent peroxidase were the only recorded enzymatic activities. In order to evaluate its true bioremediation potential, this strain was packed in a fixed-bed bioreactor, for treatment of large volumes of a real wastewater. The fungus resulted effective during 10 cycles of decolourisation, remaining active for a very long period, in non-sterile conditions.

Chromium removal from a real tanning effluent by autochthonous and allochthonous fungi.

Heavy metals represent an important ecological and health hazard due to their toxic effects and their accumulation throughout the food chain. Conventional techniques commonly applied to recover chromium from tanning wastewaters have several disadvantages whereas biosorption has good metal removal performance from large volume of effluents. To date most studies about chromium biosorption have been performed on simulated effluents bypassing the problems due to organic or inorganic ligands present in real industrial wastewaters that may sequestrate the Cr(III) ions. In the present study a tanning effluent was characterized from a mycological point of view and different fungal biomasses were tested for the removal of Cr(III) from the same tanning effluent in which, after the conventional treatments, Cr(III) amount was very low but not enough to guarantee the good quality of the receptor water river. The experiments gave rise to promising results with a percentage of removed Cr(III) up to 40%. Moreover, to elucidate the mechanisms involved in biosorption process, the same biomasses were tested for Cr(III) removal from synthetic aqueous solutions at different Cr(III) concentrations.

Isolation and characterisation of polychlorinated biphenyl (PCB) degrading fungi from a historically contaminated soil.

BACKGROUND: Polychlorinated biphenyls (PCBs) are widespread toxic pollutants. Bioremediation might be an effective, cost competitive and environment-friendly solution for remediating environmental matrices contaminated by PCBs but it is still unsatisfactory, mostly for the limited biodegradation potential of bacteria involved in the processes. Very little is known about mitosporic fungi potential in PCB bioremediation and their occurrence in actual site historically contaminated soils. In the present study, we characterised the native mycoflora of an aged dump site soil contaminated by about 0.9 g kg-1 of Aroclor 1260 PCBs and its changing after aerobic biotreatment with a commercial complex source of bacteria and fungi. Fungi isolated from the soil resulting from 120 days of treatment were screened for their ability to adsorb or metabolise 3 target PCBs. RESULTS: The original contaminated soil contained low loads of few fungal species mostly belonging to the Scedosporium, Penicillium and Aspergillus genera. The fungal load and biodiversity generally decreased throughout the aerobic treatment. None of the 21 strains isolated from the treated soil were able to grow on biphenyl (200 mg L-1) or a mixture of 2-chlorobiphenyl, 4,4'-dichlorobiphenyl and 2,2',5,5'-tetrachlorobiphenyl (20 mg L-1 each) as sole carbon sources. However, 16 of them grew in a mineral medium containing the same PCBs mixture and glucose (10 g L-1). Five of the 6 isolates, which displayed the faster and more extensive growth under the latter conditions, were found to degrade the 3 PCBs apparently without the involvement of ligninolytic enzymes; they were identified as Penicillium chrysogenum, Scedosporium apiospermum, Penicillium digitatum and Fusarium solani. They are the first PCB degrading strains of such species reported so far in the literature. CONCLUSION: The native mycoflora of the actual site aged heavily contaminated soil was mainly constituted by genera often reported as able to biodegrade organopollutants. It was generally remarkably reduced after the biotreatment, which however resulted in the selection of few mitosporic fungal species able to biodegrade PCBs. This is the first study in which an extensive characterisation of the cultivable indigenous mycoflora of an actual site aged PCB contaminated soil, as well as its changes upon soil bioremediation treatment, was conducted. Moreover, this is the first paper in which 5 strains ascribable to 4 mitosporic species able to biodegrade PCB are reported in the literature.

Decolourisation and detoxification of textile effluents by fungal biosorption.

Textile effluents, in addition to high COD, display several problems mainly due to toxicity and recalcitrance of dyestuffs. Innovative technologies effective in removing dyes from large volumes of effluents at low cost and in a timely fashion are needed. Fungi are among the most promising organisms for dye biosorption. In this study dye decolourisation, COD and toxicity decrease of three wastewater models after the treatment with inactivated biomasses of three Mucorales fungi cultured on two different media were evaluated. Fungal biomasses displayed good sorption capabilities giving rise to decolourisation percentages up to 94% and decrease in COD up to 58%. The Lemna minor toxicity test showed a significant reduction of toxicity after biosorption treatments, indicating that decolourisation corresponds to an actual detoxification of the treated wastewaters.