*4.2.5. Review of landfill leachate treatment studies with fungi*

Various experiments were done in different types of cultivation vessels. Laboratory equip‐ ment, such as multiwell plates and Erlenmeyer flasks, as well as packed bed aerated columns with immobilized culture, was used in the experiments. The aim was mainly to reduce COD, color, and toxicity with various fungal cultures, mostly under sterile conditions. However, the experiments were done with pure and diluted landfill leachates, while the effects of ammoni‐ um-N and additional substrate were also investigated. The experiments and their results are presented in Table 8 and briefly discussed in the paragraphs that follow.

Four ecotoxicological assays were used to asses the toxicity of a raw landfill leachate and a mixture of a raw landfill leachate and effluent coming from traditional wastewater treatment plant. All tested organism indicated that both samples exceeded the legal threshold value, showing the ineffectiveness of activated sludge treatment in the reduction of toxicity. To investigate the potential of fungal enzymes for bioremediation, the autochthonous mycoflora of the two samples was evaluated by filtration, because they are already acclimatized to their toxic environment. Ascomycetes showed to be the dominant fraction in both samples, followed by basidiomycetes. However, the samples may represent a risk for human health, since some emerging pathogens were present. A decolorization screening with autochthonous fungi was done with both samples in the presence or absence of glucose. Eleven fungi basidiomycetes and ascomycetes gave promising mycoremediation results by achieving up to 38% decolori‐ zation yields [98].

A biological process of landfill leachate treatment with the selected fungal strains on the basis of experiments under sterile conditions in Erlenmeyer flasks was developed. Samples with different concentrations (10%-100%) of young landfill leachates (LFL) with high contents of organic matter, ammonia, salts, heavy metals, phenols and hydrocarbons were treated with *Trametes trogii*, *Phanerochaete chrysosporium*, *Lentinus tigrinus* and *Aspergillus niger*. COD removal efficiencies for *P. chrysosporium*, *T. trogii* and *L. tigrinus* were of 68%, 79% and 90%, respectively, with a two-fold diluted LFL. COD reductions were accompanied by high-enzyme secretion and toxicity reduction, expressed as percent bioluminescence inhibition (< 20%). The effluent was toxic to these strains at LFL concentrations higher than 50% and caused growth inhibition. On the other hand, *A. niger* showed to tolerate raw LFL, since it grew in this media, compared to other tested strains. However, this strain is inefficient in removing phenols and hydrocarbons since toxicity reduction was very low [15].

Landfill leachates, containing high concentrations of phenols and hydrocarbons as well as ammonium nitrogen, and exhibiting high COD and high toxicity, were treated in laboratory experiments by selected strains of white rot fungi (*Trametes trogii* and *Phanerochaete chrysospo‐ rium*), with the aim of landfill leachate detoxification. Since high amounts of ammonium nitrogen are problematic for microorganisms, its effect on mycelia growth and enzyme secretion was studied first by adding NH4Cl to the growth media. Results obtained during the 14 days of cultivation showed that ammonium chloride was not problematic below 2 g L-1, while at 5 g L-1, inhibition of growth and enzyme secretion occurred. Experiments with *T. trogii* and *P. chrysosporium* in 50 v/v.% of the leachate showed 79% and 68% COD removal efficiencies. High enzyme secretion for each strain and a reduction in phenols and hydrocar‐ bons concentrations occurred and consequently, important reduction in the toxicity was achieved [99].

Biotreatability of mature municipal landfill leachate with white rot fungus and its extracellular ligninolytic enzymes was studied in Erlenmeyer flasks. Leachates were obtained from one active and one closed regional municipal landfill, which operated for many years. Both leachates were polluted by organic and inorganic compounds. The white rot fungus *Dichomitus squalens* was able to grow in the mature leachate from the closed landfill. Since it utilized present organic matter as a carbon source, the results showed 60% of DOC and COD removal and decreased toxicity to the bacterium *Aliivibrio fischeri*. In the leachate from the active landfill, the growth of the fungus was inhibited. However, when the leachate was treated with crude enzyme filtrate containing extracellular ligninolytic enzymes, 61% and 44% removal of COD and DOC was reached. In addition, a significant detoxification was proved with the bacterium *Aliivibrio fischeri* and plant *Sinapis alba*. Results showed that the fungal and enzymatic treatment is a promising biological approach for the treatment of mature landfill leachates, so this type of research should continue [70].

Color removal of a landfill leachate in a continuous experiment by immobilized *Trametes versicolor* on polyurethane foam was studied in a 3 L aerated column. Initially, batch experi‐ ments in Erlenmeyer flasks under sterile conditions were conducted to find the optimum, pH, optimum co-substrate dose, leachate dilution and contact time for microbial growth, and color removal. The same immobilized fungi under optimal conditions were used after 4 and 15 days of initial growth in several 5-day cycles to test the reuse of fungi. Experiments with the diluted leachate were done to check the effect of organic loading on color removal. Glucose was used as a co-substrate. Results show that that the same immobilized fungi can be reused for at least four cycles, each 5 days long. By using 4-day initially grown biomass, the dilution of leachate did not significantly increase the color removal efficiency without additional glucose. How‐ ever, about 50% better results in color, BOD and COD removal were obtained by addition of 3 g L-1 glucose to the concentrated leachate. Using 15-day initially grown biomass, slightly better color removal was achieved with concentrated leachate. About a twofold increase in color removal in five times diluted leachate was achieved with this biomass, when glucose was added. Results show that *T. versicolor* is capable of treating a highly contaminated landfill leachate and can be considered as a potentially useful microorganism [100].

A biological process of landfill leachate treatment with the selected fungal strains on the basis of experiments under sterile conditions in Erlenmeyer flasks was developed. Samples with different concentrations (10%-100%) of young landfill leachates (LFL) with high contents of organic matter, ammonia, salts, heavy metals, phenols and hydrocarbons were treated with *Trametes trogii*, *Phanerochaete chrysosporium*, *Lentinus tigrinus* and *Aspergillus niger*. COD removal efficiencies for *P. chrysosporium*, *T. trogii* and *L. tigrinus* were of 68%, 79% and 90%, respectively, with a two-fold diluted LFL. COD reductions were accompanied by high-enzyme secretion and toxicity reduction, expressed as percent bioluminescence inhibition (< 20%). The effluent was toxic to these strains at LFL concentrations higher than 50% and caused growth inhibition. On the other hand, *A. niger* showed to tolerate raw LFL, since it grew in this media, compared to other tested strains. However, this strain is inefficient in removing phenols and

Landfill leachates, containing high concentrations of phenols and hydrocarbons as well as ammonium nitrogen, and exhibiting high COD and high toxicity, were treated in laboratory experiments by selected strains of white rot fungi (*Trametes trogii* and *Phanerochaete chrysospo‐ rium*), with the aim of landfill leachate detoxification. Since high amounts of ammonium nitrogen are problematic for microorganisms, its effect on mycelia growth and enzyme secretion was studied first by adding NH4Cl to the growth media. Results obtained during the 14 days of cultivation showed that ammonium chloride was not problematic below 2 g L-1, while at 5 g L-1, inhibition of growth and enzyme secretion occurred. Experiments with *T. trogii* and *P. chrysosporium* in 50 v/v.% of the leachate showed 79% and 68% COD removal efficiencies. High enzyme secretion for each strain and a reduction in phenols and hydrocar‐ bons concentrations occurred and consequently, important reduction in the toxicity was

Biotreatability of mature municipal landfill leachate with white rot fungus and its extracellular ligninolytic enzymes was studied in Erlenmeyer flasks. Leachates were obtained from one active and one closed regional municipal landfill, which operated for many years. Both leachates were polluted by organic and inorganic compounds. The white rot fungus *Dichomitus squalens* was able to grow in the mature leachate from the closed landfill. Since it utilized present organic matter as a carbon source, the results showed 60% of DOC and COD removal and decreased toxicity to the bacterium *Aliivibrio fischeri*. In the leachate from the active landfill, the growth of the fungus was inhibited. However, when the leachate was treated with crude enzyme filtrate containing extracellular ligninolytic enzymes, 61% and 44% removal of COD and DOC was reached. In addition, a significant detoxification was proved with the bacterium *Aliivibrio fischeri* and plant *Sinapis alba*. Results showed that the fungal and enzymatic treatment is a promising biological approach for the treatment of mature landfill leachates, so this type

Color removal of a landfill leachate in a continuous experiment by immobilized *Trametes versicolor* on polyurethane foam was studied in a 3 L aerated column. Initially, batch experi‐ ments in Erlenmeyer flasks under sterile conditions were conducted to find the optimum, pH, optimum co-substrate dose, leachate dilution and contact time for microbial growth, and color removal. The same immobilized fungi under optimal conditions were used after 4 and 15 days

hydrocarbons since toxicity reduction was very low [15].

achieved [99].

140 Wastewater Treatment Engineering

of research should continue [70].

*Trametes versicolor* and *Flavodon flavus* immobilized on PUF cubes were used for treatment of landfill leachate. Experiments were done in shaken flasks at room temperature. Effects of pH and co-substrates (glucose, corn starch, and cassava) were investigated at different contact times. Treatment efficiency was evaluated based on color, BOD, and COD removal. For both types of fungi, the optimum pH was 4, the optimum co-substrate concentration was 3 g L-1 and the optimum contact time was 10 days. Depending on co-substrate and fungus, the addition of co-substrate at optimum conditions could remove 60-78% of color and reduce 52%-69% of BOD and COD. Promising results prove a potential usefulness of the white rot fungi in the treatment of landfill leachate [101].

Treatment of the landfill leachate by mycelia of *Ganodermaaustrale* immobilized on Ecomat (organic fibers made from oil palm empty fruit brunches), packed in a 0.63 L column, was investigated. Continuous recycling of 1 L of raw leachate and 50 v/v.% diluted leachate at a constant flow (20 mL min-1) was operated for 10 cycles. The results were evaluated on the basis of pH and COD, BOD and ammonium nitrogen removal. Only slight BOD removal was achieved for the raw leachate, while no effect was observed for the diluted sample. COD removal occurred after each cycle with the diluted leachate. Higher COD removal (51%) was observed with the diluted leachate, compared to the raw leachate (23%), after the tenth cycle. Ammonium nitrogen was also reduced after cycle 8 for the diluted (46%) and the raw leachate (31%). The results suggest that the white rot fungus *G. australe* can be considered as a potential candidate in the landfill leachate treatment [102].

Combined fungal and bacterial treatment was also studied. A sequential process in two 10 L laboratory reactors was carried out using a fungal sp. (*Phanerochaete sp*.), followed by a bacterial sp. (*Pseudomonas sp*.) for the degradation and detoxification of contaminants in the landfill leachate. The optimization of cultivation and process parameters for individual fungal and bacterial isolates was done with Box-Behnken design (BBD) and response surface methodology (RSM) for three variables (C source, N source and duration), considering two responses (% of COD and color removal). Treatment in the sequential bioreactor under optimized conditions removed 76.9% of COD and 45.4% of color. In addition, no statistically significant DNA damage of the cells at the end of the treatment was observed, allowing the effluent to be discharged [103].


**Table 8.** Overview of some fungal landfill leachate treatment studies [15, 70, 98-102].
