Waste Management xxx (2014) xxx–xxx
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Fungal and enzymatic treatment of mature municipal landfill leachate Gabriela Kalcˇíková ⇑, Janja Babicˇ, Aleksander Pavko, Andreja Zˇgajnar Gotvajn Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškercˇeva 5, SI-1000 Ljubljana, Slovenia
a r t i c l e
i n f o
Article history: Received 7 October 2013 Accepted 19 December 2013 Available online xxxx Keywords: Biotreatability Landfill leachate Ligninolytic enzymes White rot fungi Toxicity
a b s t r a c t The aim of our study was to evaluate biotreatability of mature municipal landfill leachate by using white rot fungus and its extracellular enzymes. Leachates were collected in one active and one closed regional municipal landfill. Both chosen landfills were operating for many years and the leachates generated there 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 and as it utilizes present organic matter as a source of carbon, the results were showing 60% of DOC and COD removal and decreased toxicity to the bacterium Aliivibrio fischeri. On the other hand, growth of the fungus was inhibited in the presence of the leachate from the active landfill. However, when the leachate was introduced to a crude enzyme filtrate containing extracellular ligninolytic enzymes, removal levels of COD and DOC reached 61% and 44%, respectively. Furthermore, the treatment led to detoxification of the leachate to the bacterium Aliivibrio fischeri and to reduction of toxicity (42%) to the plant Sinapis alba. Fungal and enzymatic treatment seems to be a promising biological approach for treatment of mature landfill leachates and their application should be further investigated. Ó 2014 Published by Elsevier Ltd.
1. Introduction Biological treatment is often used due to its reliability, simplicity and high cost-effectiveness and provides many advantages in terms of biodegradable matter and nitrogen compounds removal. However, the efficiency of biological processes is strongly limited in presence of refractory or inhibitory compounds in wastewaters (Renou et al., 2008). One of the wastewaters often treated by biological processes is the landfill leachate. Landfill leachate is generated in every landfill and the leachate composition greatly varies depending mainly on the age of the landfill (Neczaj et al., 2005). In the beginning of the landfill operation, acidogenic phase takes place. This early phase of the landfill lifecycle leads to the release of a large quantity of highly biodegradable volatile fatty acids: they create as much as 95% of the organic content (Armstrong and Rowe, 1999). Such a young leachate can be successfully treated by different biological methods. After the relatively short acidogenic phase (up to 5 years after the waste placement) the efficiency of biological treatment plants slowly decreases due to changing conditions in the body of landfills. Organic matter produced during the acidogenic phase is utilized by the microorganisms presented in the landfill which reached the methanogenic phase. Thus the landfill becomes anaerobic digester by itself. During this phase, high concentrations of refractory humic and fulvic acids, which are the products of microbial ⇑ Corresponding author. Tel.: +386 1 24 19 500. E-mail address: [email protected] (G. Kalcˇíková).
degradation, appear in the methanogenic leachates (Batarseh et al., 2010). Concentration of biodegradable organic matter in an old but active landfill can be relatively stable after many years due to the continual waste filling which provides a source of carbon available for microbial growth (Armstrong and Rowe, 1999). However, the efficiency of the aerobic treatment is also affected by a high concentration of inhibitory compounds in the methanogenic leachate, such as ammonium nitrogen, which has a significant effect on slow growing nitrifying organisms. Salinity of leachate also increases with landfill age and causes sludge bulking resulting in high concentration of suspended solids in the effluent of the activated sludge system (Di Iaconi et al., 2006). Recently, fungal treatment has been intensively studied and white rot fungi have shown a great potential for removal of hazardous and toxic pollutants. White rot fungi produce various extracellular enzymes, including laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP) which are involved in the degradation of lignin and their natural lignocellulosic substrates (Wesenberg et al., 2003). Ligninolytic enzymes are even capable of degrading various pollutants as phenols, pesticides, polychlorinated biphenyls, chlorinated insecticides, dyes and a range of other compounds (Brijwani et al., 2010). White rot fungi enzymes were most often applied for treatment of textile wastewaters (Chander and Arora, 2007; Nilsson et al., 2006; Rodríguez-Couto, 2012; Wesenberg et al., 2003) due to their excellent ability of decolorization and detoxification of dyes (Erkurt et al., 2007). Ellouze et al. (2009) have reported a successful treatment of a young landfill leachate by different strains of white rot fungi. However, fungal
0956-053X/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.wasman.2013.12.017
Please cite this article in press as: Kalcˇíková, G., et al. Fungal and enzymatic treatment of mature municipal landfill leachate. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2013.12.017
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treatment of mature leachate generated in old landfills has not been investigated so far. Furthermore, treatment involving fungi and their extracellular enzymes may be beneficial during the whole landfill lifecycle, since it offers easy degradation of non-stabilized organic matter as cellulose, hemicellulose and lignin occurring in the leachate at the beginning of landfill operation, and degradation of stable, refractory organic matter as humic and fulvic acids appearing in the later years of landfill maturing (Zavarzina et al., 2004). In this context, the aim of our study was to evaluate the potential of using the white rot fungus Dichomitus squalens and its extracellular enzymes for treatment and reduction of toxicity of mature landfill leachates from active and closed landfills with different composition and content of organic matter.
2. Material and methods 2.1. Characterizations of landfill leachates Landfill leachates were sampled in two regional municipal landfills. The sampling sites were chosen to obtain leachates from landfills of different ages and thus different contents of biodegradable organic matter. First landfill selected was approximately 30 years old and it is still active with continuous waste disposal. The leachate was sampled in March 2011 (L1) at the effluent from retention basin. The second chosen landfill operated from 1960, it has been closed since 2001 and it was used for disposal of municipal waste and co-disposal of different wastes from tannery industry. The leachate was sampled in March 2012 (L2) from the open retention basin (Martic´, 2012). The sample was immediately transported in high density polyethylene containers to the laboratory. Physico-chemical parameters and toxicity were evaluated immediately; the samples for treatment experiments were frozen at 28 ± 2 °C. The quality of landfill leachates was evaluated by physicochemical parameters: pH, BOD5 (Biochemical Oxygen Demand) (ISO 5815-1, 2003), COD (Chemical Oxygen Demand) (ISO 6060, 1989), DOC (Dissolved Organic Carbon) (ISO 8245, 1999), ammonium nitrogen (ISO 7150-1, 1984), nitrite and nitrate nitrogen, orthophosphates and chlorides (Public Health Association, 2012). Each analysis was performed twice in two parallels and standard deviation (SD) was calculated. The samples were not filtered prior analyses. All measurements were validated by using reference materials. In addition to physico-chemical parameters, toxicity of the leachates was monitored during the treatment procedures by two toxicity tests using the marine bacterium Aliivibrio fischeri and the terrestrial plant Sinapis alba. These organisms belong to sensitive species, they are often used for toxicity testing and both tests require only a low amount of the sample. The marine bacterium is tolerant to high concentrations of inorganic ions in landfill leachates and thus the changes in toxicity refer to changes in organic pollutants content while the terrestrial plants present high sensitivity to both organic and inorganic components. The test using freeze-dried luminescent bacterium Aliivibrio fischeri (ISO 11348-3, 2007) is based on the measurement of inhibition of bioluminiscence in presence of a toxic sample. The bioluminescence was measured prior and after incubation (30 min) by a LUMIStox luminometer (DR. LANGE, Germany) and the inhibition (%) was calculated. The second toxicity test uses seeds of white mustard Sinapis alba. Phytotoxic effects of leachates which result in inhibition (%) of root growth in the first 3 days of the plant germination period were measured (MZˇP, 2007). Each tested leachate was diluted by growth medium to obtain test concentrations which caused more than 50% and less than 100% inhibition.
2.2. Fungal and enzymatic treatment White rot fungus Dichomitus squalens was chosen for fungal and enzymatic treatment due to its excellent ability to produce extracellular enzymes laccase (Lac) and manganese peroxidase (MnP) which degrade different pollutants. The fungus Dichomitus squalens MZKI B1233 was obtained from the MZKI culture collection (National Institute of Chemistry, Ljubljana, Slovenia). The strain was maintained on 2.5% malt agar plates at 4 °C as described previously (Pavko and Novotny´, 2008). The mycelial suspension of Dichomitus squalens was prepared by inoculation of four 1 cm diameter plugs from the fungus growing zone on malt agar, in 50 mL of nitrogen limited mineral medium (Tien and Kirk, 1988) in a 250 mL Erlenmeyer flask. This was incubated at 28 °C in an incubator (Heraeus, Function Line IP 20, Germany). After 6–7 days a dense mycelial mass was formed. To prepare the mycelium suspension for inoculation, the suspension was disrupted with an Ultra-Turrax T25 (Janke & Kunkel, IKA Labortechnik, Germany) at 9000 rpm under sterile conditions prior to inoculation. 2.2.1. Growth of D. squalens in medium with landfill leachate First, growth of fungus in landfill leachates was investigated. The treatments were prepared by inoculating 100 mL of growth medium (Babicˇ and Pavko, 2012) with 50% v/v of landfill leachate without nitrogen and carbon source, but with beech wood sawdust, in 250 mL Erlenmeyer flask with 5% v/v of mycelial suspension. The control medium was prepared and inoculated in the same way. It contained 50% v/v of deionized water with pH 4.5. For a positive control the optimized growth medium with beech wood sawdust was used. The Erlenmeyer flasks with fungus were incubated on a RVI-403 rotary shaker (Tehtnica, Slovenia) under constant temperature (28 °C ± 1 °C) and agitation of 150 rpm for 8 days. Aliquots of the liquid culture were collected for determination of extracellular laccase (Lac) and manganese peroxidase (MnP) enzymes activities; DOC, COD and N-NHþ 4 removal as well as decrease of toxicity to Aliivibrio fischeri and Sinapis alba were monitored. For the determination of biomass (with beech wood sawdust) dry weight, duplicate flasks were harvested at indicated times and filtered through Whatman No. 1 filter paper that had previously been dried at 105 °C to a constant weight. The biomass (with beech wood sawdust) retained on the filter paper was dried at 105 °C to a constant weight, and the biomass (with beech wood sawdust) weight and concentration of TOC (%) in biomass were determined. 2.2.2. Ligninolytic enzyme production in shaken cultures For production of ligninolytic enzymes of white rot fungus Dichomitus squalens, the shaken cultures were prepared by inoculating 200 mL of the optimized growth mineral media containing beech wood sawdust (Babicˇ and Pavko, 2012) in a 500 mL Erlenmeyer flask with 5% v/v of the mycelial suspension. The fungus was incubated on a RVI-403 rotary shaker under constant temperature (28 °C ± 1 °C) and agitation of 150 rpm. Dichomitus squalens biomass was separated after 7 days of cultivation from the extracellular medium with ligninolytic enzymes through Whatman filter No. 1. Crude enzyme filtrate was used for the evaluation of toxicity and biodegradability of landfill leachates. 2.2.3. Toxicity of the landfill leachates to ligninolytic enzymes The experiments for determination of toxicity of landfill leachates to Lac enzymes were prepared in 250 mL Erlenmeyer flasks with 100 mL of medium with 10% v/v of the crude enzyme filtrate and 90% v/v of properly diluted landfill leachate. Dilutions were: 10, 30, 50, 70 and 90% v/v. Deionized water with pH 4.5 was used for dilution. Aliquots of the liquid culture were collected for determination of activity of Lac enzymes.
Please cite this article in press as: Kalcˇíková, G., et al. Fungal and enzymatic treatment of mature municipal landfill leachate. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2013.12.017
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2.2.4. Biodegradation of the landfill leachates by D. squalens ligninolytic enzymes To evaluate biodegradation of landfill leachate alone, the DOC of crude enzyme filtrate was monitored for several days until fructose (source of carbon in medium for production of ligninolytic enzymes) was utilized and DOC remained constant. Afterwards, crude enzyme filtrate was used for the treatment of landfill leachate. The treatment experiments were performed on a RVI403 rotary shaker under constant temperature (28 °C ± 1 °C) and agitation of 150 rpm in the dark for 5 days. They included test mixture (landfill leachate), blank control and abiotic test. The test mixture was prepared by dilution of landfill leachate by crude enzyme filtrate (50% v/v). The blank control contained crude enzyme filtrate and instead of landfill leachate deionized water. The abiotic test was used for monitoring of possible abiotic degradation of the leachate and it contained landfill leachate and inactivated crude enzyme filtrate. Enzymes in the filtrate were inactivated by 1 mmol L1 NaN3. The pH of the landfill leachates and deionized water used in the treatment experiment was always adjusted to 4.5 ± 0.2. The Lac and MnP enzymes activity, DOC and COD, concentration of ammonium nitrogen and toxicity to Aliivibrio fischeri and Sinapis alba were monitored daily for 5 days. 2.2.5. Enzyme assays The Lac activity in all of the experiments was measured by monitoring the oxidation of 5 mM 2,2-azinobis(3-ethylbenzothiozoline-6-sulfonate) (ABTS) at 420 nm (Johannes et al., 1996) and MnP activity by monitoring the oxidation of 2,6-dimethoxyphenol (DMP) at 469 nm (Field et al., 1996). One unit of enzyme activity was determined as the amount of enzyme oxidizing 1 lmol of the corresponding substrate per minute. All spectrophotometric measurements were conducted using a Perkin-Elmer spectrophotometer, type Lambda 25 (USA). The substrates for the Lac and MnP activity assays were both purchased from Sigma (USA). 3. Results and discussion 3.1. Characterization of the landfill leachates Physico-chemical parameters of leachates (L1, L2,) are presented in Table 1. Both investigated leachates showed similar characteristics to the leachates sampled in other municipal landfills (Kulikowska and Klimiuk, 2008; Guo et al., 2010). They were considered as representative samples which can be found in any municipal landfill. The pH values of both leachates were always in alkaline range indicating that the selected landfills are already in methanogenic phase of their life cycle. The L1 leachate sampled in the active landfill was more polluted by organics than the L2 leachate sampled in the closed landfill, while the concentrations of inorganic components were comparable. The active landfill is constantly filled by fresh waste which results in increased BOD5/
Table 1 Physico-chemical parameters of raw landfill leachates (L1 and L2). Parameters are given as mean values with standard deviations (±). Parameters
L1
L2
pH (/) DOC (mg L1) COD (mg L1) BOD5 (mg L1) 1 N-NHþ 4 (mg L ) 1 N-NO 3 (mg L ) 1 N-NO 2 (mg L )
8.1 (±0.2) 1075 (±42) 3263 (±148) 1090 (±106) 519 (±7) 4.1 (±0.1) 1.0 (±0.1) 4.5 (±0.4)
8.3 (±0.2) 467 (±6) 1086 (±76) 27 (±0.1) 732 (±2) 10 (±0.4) 0.40 (±0.01) 2.3 (±0.1)
1100 (±100)
750 (±25)
1 P-PO3 4 (mg L ) Cl (mg L1)
3
COD ratio (0.3) of the L1 leachate. On the other hand, leachate from 40 years closed landfill expressed very low BOD5/COD ratio (0.02). Together with high concentrations of inorganic component and alkalic pH, leachates can be classified as mature ones (Foo and Hameed, 2009). Although not analyzed, lignin, cellulose and hemicellulose are assumed to be prevalent organics in the L1 leachate from the active landfill while stable humic and fulvic acids are expected to be a major part of the organic matter in the L2 leachate generated in the closed landfill (Williams, 2005). 3.2. Fungal and enzymatic treatment Treatment of leachate by white rot fungus Dichomitus squalens and its extracellular enzymes was investigated. The L1 leachate (sampled in old but active landfill) and the L2 leachate (generated in old, closed landfill) were introduced as 50% v/v. First, growth of fungus in landfill leachates was investigated. The L1 leachate from the active landfill did not support the growth of fungus at all. The COD and DOC did not decrease less than 2% in 4 days of the experiment and no laccase (Lac) and manganese peroxidase (MnP) enzymes were produced. The amount of the biomass determined at the end of the experiment was comparable to the amount of biomass in the control test in which a growth of fungus was not expected (test without source of carbon and nitrogen). On the other hand, a growth of fungus and, consecutively, a removal of organic matter were indicated in the L2 leachate (Table 2). Removal of 60% of DOC and COD in the test mixture with the L2 leachate was observed after 7 days of incubation. In the control test which did not contain source of carbon and nitrogen, 25% of DOC and 10% of COD was removed during the incubation period, but it was probably caused by degradation of extinct biomass due to the absence of an organic carbon source. During this time, minimal activity of both Lac and MnP enzymes was monitored in the test mixture with the L2 leachate. Generally, the ligninolytic enzymes are thought to be expressed during secondary metabolism when carbon and nitrogen become limiting (Ellouze et al., 2009). The discrepancy in Lac production was perhaps attributable to the distinct type of nitrogen source in the positive control and the test mixture, considering the fact, that the type of nitrogen source influences the Lac production (Kaal et al., 1995). After the experiment, 44% reduction of toxicity to bacterium Aliivibrio fischeri was observed (Table 2). However, toxicity of the L2 leachate to the plant Sinapis alba was not changed, probably due to high concentrations of inorganic ions (e.g. ammonium nitrogen, chlorides) which can cause osmotic stress in plants and they do not effect bacterium Aliivibrio fischeri which lives in a marine environment. After the experiments, the amount of biomass produced in the test mixture with the L2 leachate was 0.683 (±0.015) g and it was 44% higher than in the control test where the growth of fungus was not expected 0.474 (±0.012) g. Concentration of organic carbon in the biomass produced in the test mixture with the L2 leachate differed less than 1% from the positive control and, thus, the sorption of organic matter on biomass was evaluated as insignificant. The positive control proceeded well during all experiments and after 7 days, production of both Lac and MnP enzymes started and at the end of the experiment, the activity of Lac was in a normal range comparable to our other experiment; 525 (±4) U L1 Lac and 231 (±3) U L1 MnP. Thus experiments were performed under optimal condition for the fungus and they were assessed as valid. The growth of fungus Dichomitus squalens in leachates from active landfills which are still filled by fresh waste can be limited due to presence of inhibiting components affecting the fungus. Furthermore, such toxicants are probably degraded during waste stabilization in closed landfills, because the ability of the fungus to grow and to use organic matter as a carbon source was observed in
Please cite this article in press as: Kalcˇíková, G., et al. Fungal and enzymatic treatment of mature municipal landfill leachate. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2013.12.017
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Table 2 Enzymes activity and decrease of toxicity and organic matter contents (DOC, COD) in the L2 leachate during growth of fungus. Parameters are given as mean values with standard deviations (±). Parameter
Time (days)
DOC (mg L1) COD (mg L1) Lac (U L1) MnP (U L1) Inhibition Sinapis alba (%) Inhibition Aliivibrio fischeri (%) a
0
2
4
7
835 (±20) 1740 (±34) 0 0 91.9 (±1.8) 68.3 (±1.1)
448 (±7) 748 (±17) 1.2 (±0.4) 2.1 (±0.2) 80.4 (±7.4) 44.8 (±0.7)
377 (±60) 661 (±104) 2.8 (±0.6) 2.6 (±0.2) 90.5 (±1.6) 34.9 (±4.4)
327 (±11) 696a 1.2 (±0.6) 2.4 (±0.7) 90 (±3) 38.3 (±3.9)
One parallel was discarded due to unexpected increase of COD.
the leachates from the stabilized landfill (the L2). However, the production of ligninolytic enzymes, which are able to degrade many pollutants, was not observed in any of the cases. For that reason, crude filtrate containing ligninolytic enzymes produced by fungus Dichomitus squalens under optimal condition was prepared and toxicity of both leachates to Lac enzymes was evaluated (Fig. 1A and B). Lac activity decreased with addition of different concentrations of the L1 leachate (Fig. 1A), however, in lower concentrations of the leachate it has been noticed that Lac reached starting activity after 2 days. This phenomenon is not usual, but Lac activity can be enhanced under nonsterile conditions as reported by Baldrian (2004). It is hypothesized that in lower concentrations of landfill leachate Lac enzymes may be stimulated while in higher concentrations of landfill leachate their activity is inhibited by presence of high concentrations of pollutants. In the case of the L2 leachate,
no significant effect was observed (Fig. 1B). In the first 3 days, the activity of Lac enzymes was comparable in all concentrations of the L2 leachate. After this time, the activity started decreasing in the control sample and in the system containing low concentrations of the L2 leachate and thus it is assumed that the organic substrate for enzymes had probably been utilized. After 7 days of the experiment, the activity of enzymes was highest in 90% v/v of the L2 leachate indicating no effect of leachate to enzymes at high concentrations. For further degradation/treatment experiments with enzymes, a concentration of 50% v/v of leachates was chosen, because at this concentration the L1 leachate caused less than 35% inhibition of enzymes activity in 7 days and the same concentration was also used for the L2 leachate. The most intensive degradation of the L1 leachate occurred during the first 24 h of treatment (Table 3). Maximal DOC removal efficiency was 44%, while COD removal reached 61%. COD and DOC in blank control was maintained constant during the whole experiment with minimal fluctuations (less than 10%). Abiotic control was constant for the first 40 h and afterwards DOC started to decrease, probably due to the abiotic degradation of landfill leachate. The concentration of ammonium nitrogen was stable during the whole treatment experiment. The activity of enzymes in the test mixture was fairly stable for the first days of the experiment and on the third day it started to decrease, probably due to the lack of organic matter in the landfill leachate which could be used as substrate for the enzymes. The activity of enzymes in the blank test dropped rapidly after the first day of the experiment and after 5 days the enzymatic activity was nearly undetectable. The activity of enzymes in the abiotic test was also measured and it was inhibited during the whole experiment.
Fig. 1. The effect of different concentrations of the L1 leachate (A) and the L2 leachate (B) on the activity of laccase (Lac) enzymes.
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Table 3 Enzymes activity and decrease of toxicity and organic matter contents (DOC, COD) in the L1 leachate during enzymatic treatment. Parameters are given as mean values with standard deviations (±). Parameter
DOC (mg L1) COD (mg L1) Lac (U L1) MnP (U L1) Inhibition Sinapis alba (%) Inhibition Aliivibrio fischeri (%) a
Time (days) 0
1
2
3
5
588 (±13) 1635 (±52) 738 (±5) 152 (±2) 82.5 (±1.5) 84.3 (±0.3)
330 (±1) 1002 (±18) 610 (±0.2) 176 (±4) 49.8 (±4.5) 43.7 (±3.1)
329 (±5) 1055 (±35) 629 (±13) 141 (±5) 57.9 (±1.4) 37.1 (±3.0)
319 (±1) 633 (±70) 469 (±23) 113 (±6) 60.3 (±1.3) 11.1 (±7.6)
297 (±6) 791 (±18) 351 (±89) 83 (±28) 48.2 (±14) NTa
Non-Toxic (NT). Sample did not cause measurable toxic effect.
Toxicity of the landfill leachate to the plant Sinapis alba significantly decreased and enzymatic treatment led to a detoxification of the landfill leachate to the bacterium Aliivibrio fischeri (Table 3). After 7 days of the L2 leachate degradation, only 23% of organic matter (COD and DOC) was removed, however, it was comparable to the decrease of organic content in the blank control and thus it was evaluated as an insignificant difference and the L2 leachate was assessed as not biodegradable by ligninolytic enzymes. Landfill leachates are mixtures of various compounds and their composition depends on many factors, mostly on disposed waste and the phase of the landfill’s life cycle (Kulikowska and Klimiuk, 2008). The co-disposal of fresh waste to already stabilized waste is a source of organics as lignin and cellulose which are used as substrate for ligninolytic enzymes. Phenolic and other aromatic compounds were identified in active landfills (Yasuhara et al., 1997) and such compounds can also be easily degraded by Lac enzymes (Brijwani et al., 2010). The contribution of fresh waste to biodegradability of leachate is crucial for biodegradation and the concentration of organic biodegradable matter is, together with leachate toxicity, a limiting factor for fungal treatment. Fungal or enzymatic treatment may be preferable over biological treatments since it offers biodegradation of many pollutants and thus reduction of COD and toxicity of landfill leachates. For a large-scale application, a reactor system with immobilized fungus can be employed. A packed-bed reactor with wood chips or a reactor with silica-based porous support with immobilized white rot fungi has been reported to be much more efficient for treatment of 2-chlorphenol than suspended growth reactor (Lewandowski et al., 1990). Ehlers and Rose (2005) have demonstrated high efficiency of immobilized white rot fungi in a trickling packed-bed reactor with wood (pine) chips and glass beads for biodegradation of phenol and chlorinated phenol. A large-scale application of ligninolytic enzymes for treatment and detoxification of wastewaters is also possible. Chemically modified silica with imidazol groups appeared as an efficient support for immobilization of Lac and such system showed high efficiency in decolorization of dyes (Peralta-Zamora et al., 2003). Dodor et al. (2004) have investigated immobilized Lac enzymes for degradation of PAHs and the results indicated that immobilization improves stability of Lac to temperature, pH and inhibitors compared to free enzymes. However, fungal and enzymatic treatment may include some disadvantages as the requirements of nutrients, low pH, production of mycelia and an additional treatment step to remove ammonium nitrogen. 4. Conclusion Mature landfill leachates generated in old landfills cannot be treated by the conventional biological treatment methods because of the leachates’ low biodegradability and the high concentrations of inhibitory compounds. As no further study was identified that addresses successful biological treatment of a mature municipal landfill leachate, our study for the first time provided a clear
evidence that even an extremely mature leachate can be successfully biologically treated by the white rot fungi. By application of the fungal and enzymatic treatment, organic matter content and toxicity can be considerably reduced. Since the fungus was able to grow and partially degrade the mature leachate from the closed landfill, it could be employed for mature leachate treatment at the landfill site. After the landfill closure, the amount of the leachate and organic matter decreases with time and thus it is supposed that the fungal treatment could be a promising method for a post-closure landfill care. Ligninolytic enzymes produced by the fungus were responsible for the removal of major part of the organic matter and the reduction of toxicity in the leachate from the active landfill. Immobilized enzymes could be practically used for landfill leachate treatment, but because of the high pollution and toxicity of leachates from active landfills it should be also accompanied by some other treatment methods, e.g. physico-chemical ones because the enzymatic treatment alone cannot assure a sufficient treatment efficiency.
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ISO 5815-1, 2003. Water Quality – Determination of biochemical oxygen demand after n days (BODn), Part 1: Dilution and seeding method with allylthiourea addition, first ed., Geneva. ISO 6060, 1989. Water Quality – Determination of the chemical oxygen demand, second ed., Geneva. ISO 7150-1, 1984. Water Quality – Determination of ammonium, Part 1: Manual spectrometric method, first ed., Geneva. ISO 8245, 1999. Water Quality – Guidelines for the determination of total organic carbon (TOC) and dissolved organic carbon (DOC), second ed., Geneva. ISO 11348-3, 2007. Water Quality – Determination of the inhibitory effect of water samples on the light emission of Vibrio fischeri (Luminescent Bacteria Test), Part 3: Method using freezed-dried bacteria, second ed., Geneva. Johannes, C., Majcherczyk, A., Hüttermann, A., 1996. Degradation of anthracene by laccase of Trametes versicolor in the presence of different mediator compounds. Appl. Microb. Biotechnol. 46, 313–317. Kaal, E.E., Field, J.A., Joyce, T.W., 1995. Increasing ligninolytic enzyme activities in several white-rot basidiomycetes by nitrogen sufficient media. Bioresour. Technol. 53, 133–139. Kulikowska, D., Klimiuk, E., 2008. The effect of landfill age on municipal leachate composition. Bioresour. Technol. 99, 5981–5985. Lewandowski, G.A., Armenante, P.M., Pak, D., 1990. Reactor design for hazardous waste treatment using a white rot fungus. Water Res. 24, 75–82. Martic´, M., 2012. Assessment of the impact of municipal landfill leachate to higher plants. Diploma thesis. University of Ljubljana, Ljubljana. MZˇP, 2007. The Guideline of the Department of waste management to determine the ecotoxicity of waste. Ministry of the Environment of the Czech Republic, Prague (In Czech). Neczaj, E., Okoniewska, E., Kacprzak, M., 2005. Treatment of landfill leachate by sequencing batch reactor. Desalination 185, 357–362. Nilsson, I., Möller, A., Mattiasson, B., Rubindamayugi, M.S.T., Welander, U., 2006. Decolorization of synthetic and real textile wastewater by the use of white-rot fungi. Enzym. Microb. Technol. 38, 94–100.
Pavko, A., Novotny´, Cˇ., 2008. Induction of ligninolytic enzyme production by Dichomitus squalens on various types of immobilization support. Acta Chim. Slov. 55, 648–652. Peralta-Zamora, P., Pereira, C.M., Tiburtius, E.R.L., Moraes, S.G., Rosa, M.A., Minussi, R.C., Durán, N., 2003. Decolorization of reactive dyes by immobilized laccase. Appl. Catal. B: Environ. 42, 131–144. Public Health Association, 2012. American Water Works Association, Water Environmental Federation. Standard Methods for the Examination of Water and Wastewater, 22 ed., Washington, DC. Renou, S., Givaudan, J.G., Poulain, S., Dirassouyan, F., Moulin, P., 2008. Landfill leachate treatment: review and opportunity. J. Hazard. Mater. 150, 468–493. Rodríguez-Couto, S., 2012. A promising inert support for laccase production and decolouration of textile wastewater by the white-rot fungus Trametes pubescesns. J. Hazard. Mater. 233–234, 158–162. Tien, M., Kirk, T.K., 1988. Lignin peroxidase of Phanerochaete chrysosporium. In: Willis, A., Kellogg, S.T. (Eds.), Wood. Academic Press, San Diego, pp. 238–249. Wesenberg, D., Kyriakides, I., Agathos, S.N., 2003. White-rot fungi and their enzymes for the treatment of industrial dye effluents. Biotechnol. Adv. 22, 161–187. Williams, P.T., 2005. Waste Treatment and Disposal. John Wiley & Sons, Chichester. Yasuhara, A., Shiraishi, H., Nishikawa, M., Yamamoto, T., Uehiro, T., Nakasugi, O., Okumura, T., Kenmotsu, K., Fukui, H., Nagase, M., Ono, Y., Kawagoshi, Y., Baba, K., Noma, Y., 1997. Determination of organic components in leachates from hazardous waste disposal sites in Japan by gas chromatography–mass spectrometry. J. Chromatogr. A 774, 321–332. Zavarzina, A.G., Leontievsky, A.A., Golovleva, L.A., Trofimov, S.Y., 2004. Biotransformation of soil humic acids by blue laccase of Panus tigrinus 8/18: an in vitro study. Soil Biol. Biochem. 36, 359–369.
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Fungal and enzymatic treatment of mature municipal landfill leachate Gabriela Kalcˇíková ⇑, Janja Babicˇ, Aleksander Pavko, Andreja Zˇgajnar Gotvajn Faculty of Chemistry and Chemical Technology, University of Ljubljana, Aškercˇeva 5, SI-1000 Ljubljana, Slovenia
a r t i c l e
i n f o
Article history: Received 7 October 2013 Accepted 19 December 2013 Available online xxxx Keywords: Biotreatability Landfill leachate Ligninolytic enzymes White rot fungi Toxicity
a b s t r a c t The aim of our study was to evaluate biotreatability of mature municipal landfill leachate by using white rot fungus and its extracellular enzymes. Leachates were collected in one active and one closed regional municipal landfill. Both chosen landfills were operating for many years and the leachates generated there 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 and as it utilizes present organic matter as a source of carbon, the results were showing 60% of DOC and COD removal and decreased toxicity to the bacterium Aliivibrio fischeri. On the other hand, growth of the fungus was inhibited in the presence of the leachate from the active landfill. However, when the leachate was introduced to a crude enzyme filtrate containing extracellular ligninolytic enzymes, removal levels of COD and DOC reached 61% and 44%, respectively. Furthermore, the treatment led to detoxification of the leachate to the bacterium Aliivibrio fischeri and to reduction of toxicity (42%) to the plant Sinapis alba. Fungal and enzymatic treatment seems to be a promising biological approach for treatment of mature landfill leachates and their application should be further investigated. Ó 2014 Published by Elsevier Ltd.
1. Introduction Biological treatment is often used due to its reliability, simplicity and high cost-effectiveness and provides many advantages in terms of biodegradable matter and nitrogen compounds removal. However, the efficiency of biological processes is strongly limited in presence of refractory or inhibitory compounds in wastewaters (Renou et al., 2008). One of the wastewaters often treated by biological processes is the landfill leachate. Landfill leachate is generated in every landfill and the leachate composition greatly varies depending mainly on the age of the landfill (Neczaj et al., 2005). In the beginning of the landfill operation, acidogenic phase takes place. This early phase of the landfill lifecycle leads to the release of a large quantity of highly biodegradable volatile fatty acids: they create as much as 95% of the organic content (Armstrong and Rowe, 1999). Such a young leachate can be successfully treated by different biological methods. After the relatively short acidogenic phase (up to 5 years after the waste placement) the efficiency of biological treatment plants slowly decreases due to changing conditions in the body of landfills. Organic matter produced during the acidogenic phase is utilized by the microorganisms presented in the landfill which reached the methanogenic phase. Thus the landfill becomes anaerobic digester by itself. During this phase, high concentrations of refractory humic and fulvic acids, which are the products of microbial ⇑ Corresponding author. Tel.: +386 1 24 19 500. E-mail address: [email protected] (G. Kalcˇíková).
degradation, appear in the methanogenic leachates (Batarseh et al., 2010). Concentration of biodegradable organic matter in an old but active landfill can be relatively stable after many years due to the continual waste filling which provides a source of carbon available for microbial growth (Armstrong and Rowe, 1999). However, the efficiency of the aerobic treatment is also affected by a high concentration of inhibitory compounds in the methanogenic leachate, such as ammonium nitrogen, which has a significant effect on slow growing nitrifying organisms. Salinity of leachate also increases with landfill age and causes sludge bulking resulting in high concentration of suspended solids in the effluent of the activated sludge system (Di Iaconi et al., 2006). Recently, fungal treatment has been intensively studied and white rot fungi have shown a great potential for removal of hazardous and toxic pollutants. White rot fungi produce various extracellular enzymes, including laccase (Lac), lignin peroxidase (LiP) and manganese peroxidase (MnP) which are involved in the degradation of lignin and their natural lignocellulosic substrates (Wesenberg et al., 2003). Ligninolytic enzymes are even capable of degrading various pollutants as phenols, pesticides, polychlorinated biphenyls, chlorinated insecticides, dyes and a range of other compounds (Brijwani et al., 2010). White rot fungi enzymes were most often applied for treatment of textile wastewaters (Chander and Arora, 2007; Nilsson et al., 2006; Rodríguez-Couto, 2012; Wesenberg et al., 2003) due to their excellent ability of decolorization and detoxification of dyes (Erkurt et al., 2007). Ellouze et al. (2009) have reported a successful treatment of a young landfill leachate by different strains of white rot fungi. However, fungal
0956-053X/$ - see front matter Ó 2014 Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.wasman.2013.12.017
Please cite this article in press as: Kalcˇíková, G., et al. Fungal and enzymatic treatment of mature municipal landfill leachate. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2013.12.017
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treatment of mature leachate generated in old landfills has not been investigated so far. Furthermore, treatment involving fungi and their extracellular enzymes may be beneficial during the whole landfill lifecycle, since it offers easy degradation of non-stabilized organic matter as cellulose, hemicellulose and lignin occurring in the leachate at the beginning of landfill operation, and degradation of stable, refractory organic matter as humic and fulvic acids appearing in the later years of landfill maturing (Zavarzina et al., 2004). In this context, the aim of our study was to evaluate the potential of using the white rot fungus Dichomitus squalens and its extracellular enzymes for treatment and reduction of toxicity of mature landfill leachates from active and closed landfills with different composition and content of organic matter.
2. Material and methods 2.1. Characterizations of landfill leachates Landfill leachates were sampled in two regional municipal landfills. The sampling sites were chosen to obtain leachates from landfills of different ages and thus different contents of biodegradable organic matter. First landfill selected was approximately 30 years old and it is still active with continuous waste disposal. The leachate was sampled in March 2011 (L1) at the effluent from retention basin. The second chosen landfill operated from 1960, it has been closed since 2001 and it was used for disposal of municipal waste and co-disposal of different wastes from tannery industry. The leachate was sampled in March 2012 (L2) from the open retention basin (Martic´, 2012). The sample was immediately transported in high density polyethylene containers to the laboratory. Physico-chemical parameters and toxicity were evaluated immediately; the samples for treatment experiments were frozen at 28 ± 2 °C. The quality of landfill leachates was evaluated by physicochemical parameters: pH, BOD5 (Biochemical Oxygen Demand) (ISO 5815-1, 2003), COD (Chemical Oxygen Demand) (ISO 6060, 1989), DOC (Dissolved Organic Carbon) (ISO 8245, 1999), ammonium nitrogen (ISO 7150-1, 1984), nitrite and nitrate nitrogen, orthophosphates and chlorides (Public Health Association, 2012). Each analysis was performed twice in two parallels and standard deviation (SD) was calculated. The samples were not filtered prior analyses. All measurements were validated by using reference materials. In addition to physico-chemical parameters, toxicity of the leachates was monitored during the treatment procedures by two toxicity tests using the marine bacterium Aliivibrio fischeri and the terrestrial plant Sinapis alba. These organisms belong to sensitive species, they are often used for toxicity testing and both tests require only a low amount of the sample. The marine bacterium is tolerant to high concentrations of inorganic ions in landfill leachates and thus the changes in toxicity refer to changes in organic pollutants content while the terrestrial plants present high sensitivity to both organic and inorganic components. The test using freeze-dried luminescent bacterium Aliivibrio fischeri (ISO 11348-3, 2007) is based on the measurement of inhibition of bioluminiscence in presence of a toxic sample. The bioluminescence was measured prior and after incubation (30 min) by a LUMIStox luminometer (DR. LANGE, Germany) and the inhibition (%) was calculated. The second toxicity test uses seeds of white mustard Sinapis alba. Phytotoxic effects of leachates which result in inhibition (%) of root growth in the first 3 days of the plant germination period were measured (MZˇP, 2007). Each tested leachate was diluted by growth medium to obtain test concentrations which caused more than 50% and less than 100% inhibition.
2.2. Fungal and enzymatic treatment White rot fungus Dichomitus squalens was chosen for fungal and enzymatic treatment due to its excellent ability to produce extracellular enzymes laccase (Lac) and manganese peroxidase (MnP) which degrade different pollutants. The fungus Dichomitus squalens MZKI B1233 was obtained from the MZKI culture collection (National Institute of Chemistry, Ljubljana, Slovenia). The strain was maintained on 2.5% malt agar plates at 4 °C as described previously (Pavko and Novotny´, 2008). The mycelial suspension of Dichomitus squalens was prepared by inoculation of four 1 cm diameter plugs from the fungus growing zone on malt agar, in 50 mL of nitrogen limited mineral medium (Tien and Kirk, 1988) in a 250 mL Erlenmeyer flask. This was incubated at 28 °C in an incubator (Heraeus, Function Line IP 20, Germany). After 6–7 days a dense mycelial mass was formed. To prepare the mycelium suspension for inoculation, the suspension was disrupted with an Ultra-Turrax T25 (Janke & Kunkel, IKA Labortechnik, Germany) at 9000 rpm under sterile conditions prior to inoculation. 2.2.1. Growth of D. squalens in medium with landfill leachate First, growth of fungus in landfill leachates was investigated. The treatments were prepared by inoculating 100 mL of growth medium (Babicˇ and Pavko, 2012) with 50% v/v of landfill leachate without nitrogen and carbon source, but with beech wood sawdust, in 250 mL Erlenmeyer flask with 5% v/v of mycelial suspension. The control medium was prepared and inoculated in the same way. It contained 50% v/v of deionized water with pH 4.5. For a positive control the optimized growth medium with beech wood sawdust was used. The Erlenmeyer flasks with fungus were incubated on a RVI-403 rotary shaker (Tehtnica, Slovenia) under constant temperature (28 °C ± 1 °C) and agitation of 150 rpm for 8 days. Aliquots of the liquid culture were collected for determination of extracellular laccase (Lac) and manganese peroxidase (MnP) enzymes activities; DOC, COD and N-NHþ 4 removal as well as decrease of toxicity to Aliivibrio fischeri and Sinapis alba were monitored. For the determination of biomass (with beech wood sawdust) dry weight, duplicate flasks were harvested at indicated times and filtered through Whatman No. 1 filter paper that had previously been dried at 105 °C to a constant weight. The biomass (with beech wood sawdust) retained on the filter paper was dried at 105 °C to a constant weight, and the biomass (with beech wood sawdust) weight and concentration of TOC (%) in biomass were determined. 2.2.2. Ligninolytic enzyme production in shaken cultures For production of ligninolytic enzymes of white rot fungus Dichomitus squalens, the shaken cultures were prepared by inoculating 200 mL of the optimized growth mineral media containing beech wood sawdust (Babicˇ and Pavko, 2012) in a 500 mL Erlenmeyer flask with 5% v/v of the mycelial suspension. The fungus was incubated on a RVI-403 rotary shaker under constant temperature (28 °C ± 1 °C) and agitation of 150 rpm. Dichomitus squalens biomass was separated after 7 days of cultivation from the extracellular medium with ligninolytic enzymes through Whatman filter No. 1. Crude enzyme filtrate was used for the evaluation of toxicity and biodegradability of landfill leachates. 2.2.3. Toxicity of the landfill leachates to ligninolytic enzymes The experiments for determination of toxicity of landfill leachates to Lac enzymes were prepared in 250 mL Erlenmeyer flasks with 100 mL of medium with 10% v/v of the crude enzyme filtrate and 90% v/v of properly diluted landfill leachate. Dilutions were: 10, 30, 50, 70 and 90% v/v. Deionized water with pH 4.5 was used for dilution. Aliquots of the liquid culture were collected for determination of activity of Lac enzymes.
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2.2.4. Biodegradation of the landfill leachates by D. squalens ligninolytic enzymes To evaluate biodegradation of landfill leachate alone, the DOC of crude enzyme filtrate was monitored for several days until fructose (source of carbon in medium for production of ligninolytic enzymes) was utilized and DOC remained constant. Afterwards, crude enzyme filtrate was used for the treatment of landfill leachate. The treatment experiments were performed on a RVI403 rotary shaker under constant temperature (28 °C ± 1 °C) and agitation of 150 rpm in the dark for 5 days. They included test mixture (landfill leachate), blank control and abiotic test. The test mixture was prepared by dilution of landfill leachate by crude enzyme filtrate (50% v/v). The blank control contained crude enzyme filtrate and instead of landfill leachate deionized water. The abiotic test was used for monitoring of possible abiotic degradation of the leachate and it contained landfill leachate and inactivated crude enzyme filtrate. Enzymes in the filtrate were inactivated by 1 mmol L1 NaN3. The pH of the landfill leachates and deionized water used in the treatment experiment was always adjusted to 4.5 ± 0.2. The Lac and MnP enzymes activity, DOC and COD, concentration of ammonium nitrogen and toxicity to Aliivibrio fischeri and Sinapis alba were monitored daily for 5 days. 2.2.5. Enzyme assays The Lac activity in all of the experiments was measured by monitoring the oxidation of 5 mM 2,2-azinobis(3-ethylbenzothiozoline-6-sulfonate) (ABTS) at 420 nm (Johannes et al., 1996) and MnP activity by monitoring the oxidation of 2,6-dimethoxyphenol (DMP) at 469 nm (Field et al., 1996). One unit of enzyme activity was determined as the amount of enzyme oxidizing 1 lmol of the corresponding substrate per minute. All spectrophotometric measurements were conducted using a Perkin-Elmer spectrophotometer, type Lambda 25 (USA). The substrates for the Lac and MnP activity assays were both purchased from Sigma (USA). 3. Results and discussion 3.1. Characterization of the landfill leachates Physico-chemical parameters of leachates (L1, L2,) are presented in Table 1. Both investigated leachates showed similar characteristics to the leachates sampled in other municipal landfills (Kulikowska and Klimiuk, 2008; Guo et al., 2010). They were considered as representative samples which can be found in any municipal landfill. The pH values of both leachates were always in alkaline range indicating that the selected landfills are already in methanogenic phase of their life cycle. The L1 leachate sampled in the active landfill was more polluted by organics than the L2 leachate sampled in the closed landfill, while the concentrations of inorganic components were comparable. The active landfill is constantly filled by fresh waste which results in increased BOD5/
Table 1 Physico-chemical parameters of raw landfill leachates (L1 and L2). Parameters are given as mean values with standard deviations (±). Parameters
L1
L2
pH (/) DOC (mg L1) COD (mg L1) BOD5 (mg L1) 1 N-NHþ 4 (mg L ) 1 N-NO 3 (mg L ) 1 N-NO 2 (mg L )
8.1 (±0.2) 1075 (±42) 3263 (±148) 1090 (±106) 519 (±7) 4.1 (±0.1) 1.0 (±0.1) 4.5 (±0.4)
8.3 (±0.2) 467 (±6) 1086 (±76) 27 (±0.1) 732 (±2) 10 (±0.4) 0.40 (±0.01) 2.3 (±0.1)
1100 (±100)
750 (±25)
1 P-PO3 4 (mg L ) Cl (mg L1)
3
COD ratio (0.3) of the L1 leachate. On the other hand, leachate from 40 years closed landfill expressed very low BOD5/COD ratio (0.02). Together with high concentrations of inorganic component and alkalic pH, leachates can be classified as mature ones (Foo and Hameed, 2009). Although not analyzed, lignin, cellulose and hemicellulose are assumed to be prevalent organics in the L1 leachate from the active landfill while stable humic and fulvic acids are expected to be a major part of the organic matter in the L2 leachate generated in the closed landfill (Williams, 2005). 3.2. Fungal and enzymatic treatment Treatment of leachate by white rot fungus Dichomitus squalens and its extracellular enzymes was investigated. The L1 leachate (sampled in old but active landfill) and the L2 leachate (generated in old, closed landfill) were introduced as 50% v/v. First, growth of fungus in landfill leachates was investigated. The L1 leachate from the active landfill did not support the growth of fungus at all. The COD and DOC did not decrease less than 2% in 4 days of the experiment and no laccase (Lac) and manganese peroxidase (MnP) enzymes were produced. The amount of the biomass determined at the end of the experiment was comparable to the amount of biomass in the control test in which a growth of fungus was not expected (test without source of carbon and nitrogen). On the other hand, a growth of fungus and, consecutively, a removal of organic matter were indicated in the L2 leachate (Table 2). Removal of 60% of DOC and COD in the test mixture with the L2 leachate was observed after 7 days of incubation. In the control test which did not contain source of carbon and nitrogen, 25% of DOC and 10% of COD was removed during the incubation period, but it was probably caused by degradation of extinct biomass due to the absence of an organic carbon source. During this time, minimal activity of both Lac and MnP enzymes was monitored in the test mixture with the L2 leachate. Generally, the ligninolytic enzymes are thought to be expressed during secondary metabolism when carbon and nitrogen become limiting (Ellouze et al., 2009). The discrepancy in Lac production was perhaps attributable to the distinct type of nitrogen source in the positive control and the test mixture, considering the fact, that the type of nitrogen source influences the Lac production (Kaal et al., 1995). After the experiment, 44% reduction of toxicity to bacterium Aliivibrio fischeri was observed (Table 2). However, toxicity of the L2 leachate to the plant Sinapis alba was not changed, probably due to high concentrations of inorganic ions (e.g. ammonium nitrogen, chlorides) which can cause osmotic stress in plants and they do not effect bacterium Aliivibrio fischeri which lives in a marine environment. After the experiments, the amount of biomass produced in the test mixture with the L2 leachate was 0.683 (±0.015) g and it was 44% higher than in the control test where the growth of fungus was not expected 0.474 (±0.012) g. Concentration of organic carbon in the biomass produced in the test mixture with the L2 leachate differed less than 1% from the positive control and, thus, the sorption of organic matter on biomass was evaluated as insignificant. The positive control proceeded well during all experiments and after 7 days, production of both Lac and MnP enzymes started and at the end of the experiment, the activity of Lac was in a normal range comparable to our other experiment; 525 (±4) U L1 Lac and 231 (±3) U L1 MnP. Thus experiments were performed under optimal condition for the fungus and they were assessed as valid. The growth of fungus Dichomitus squalens in leachates from active landfills which are still filled by fresh waste can be limited due to presence of inhibiting components affecting the fungus. Furthermore, such toxicants are probably degraded during waste stabilization in closed landfills, because the ability of the fungus to grow and to use organic matter as a carbon source was observed in
Please cite this article in press as: Kalcˇíková, G., et al. Fungal and enzymatic treatment of mature municipal landfill leachate. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2013.12.017
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Table 2 Enzymes activity and decrease of toxicity and organic matter contents (DOC, COD) in the L2 leachate during growth of fungus. Parameters are given as mean values with standard deviations (±). Parameter
Time (days)
DOC (mg L1) COD (mg L1) Lac (U L1) MnP (U L1) Inhibition Sinapis alba (%) Inhibition Aliivibrio fischeri (%) a
0
2
4
7
835 (±20) 1740 (±34) 0 0 91.9 (±1.8) 68.3 (±1.1)
448 (±7) 748 (±17) 1.2 (±0.4) 2.1 (±0.2) 80.4 (±7.4) 44.8 (±0.7)
377 (±60) 661 (±104) 2.8 (±0.6) 2.6 (±0.2) 90.5 (±1.6) 34.9 (±4.4)
327 (±11) 696a 1.2 (±0.6) 2.4 (±0.7) 90 (±3) 38.3 (±3.9)
One parallel was discarded due to unexpected increase of COD.
the leachates from the stabilized landfill (the L2). However, the production of ligninolytic enzymes, which are able to degrade many pollutants, was not observed in any of the cases. For that reason, crude filtrate containing ligninolytic enzymes produced by fungus Dichomitus squalens under optimal condition was prepared and toxicity of both leachates to Lac enzymes was evaluated (Fig. 1A and B). Lac activity decreased with addition of different concentrations of the L1 leachate (Fig. 1A), however, in lower concentrations of the leachate it has been noticed that Lac reached starting activity after 2 days. This phenomenon is not usual, but Lac activity can be enhanced under nonsterile conditions as reported by Baldrian (2004). It is hypothesized that in lower concentrations of landfill leachate Lac enzymes may be stimulated while in higher concentrations of landfill leachate their activity is inhibited by presence of high concentrations of pollutants. In the case of the L2 leachate,
no significant effect was observed (Fig. 1B). In the first 3 days, the activity of Lac enzymes was comparable in all concentrations of the L2 leachate. After this time, the activity started decreasing in the control sample and in the system containing low concentrations of the L2 leachate and thus it is assumed that the organic substrate for enzymes had probably been utilized. After 7 days of the experiment, the activity of enzymes was highest in 90% v/v of the L2 leachate indicating no effect of leachate to enzymes at high concentrations. For further degradation/treatment experiments with enzymes, a concentration of 50% v/v of leachates was chosen, because at this concentration the L1 leachate caused less than 35% inhibition of enzymes activity in 7 days and the same concentration was also used for the L2 leachate. The most intensive degradation of the L1 leachate occurred during the first 24 h of treatment (Table 3). Maximal DOC removal efficiency was 44%, while COD removal reached 61%. COD and DOC in blank control was maintained constant during the whole experiment with minimal fluctuations (less than 10%). Abiotic control was constant for the first 40 h and afterwards DOC started to decrease, probably due to the abiotic degradation of landfill leachate. The concentration of ammonium nitrogen was stable during the whole treatment experiment. The activity of enzymes in the test mixture was fairly stable for the first days of the experiment and on the third day it started to decrease, probably due to the lack of organic matter in the landfill leachate which could be used as substrate for the enzymes. The activity of enzymes in the blank test dropped rapidly after the first day of the experiment and after 5 days the enzymatic activity was nearly undetectable. The activity of enzymes in the abiotic test was also measured and it was inhibited during the whole experiment.
Fig. 1. The effect of different concentrations of the L1 leachate (A) and the L2 leachate (B) on the activity of laccase (Lac) enzymes.
Please cite this article in press as: Kalcˇíková, G., et al. Fungal and enzymatic treatment of mature municipal landfill leachate. Waste Management (2014), http://dx.doi.org/10.1016/j.wasman.2013.12.017
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Table 3 Enzymes activity and decrease of toxicity and organic matter contents (DOC, COD) in the L1 leachate during enzymatic treatment. Parameters are given as mean values with standard deviations (±). Parameter
DOC (mg L1) COD (mg L1) Lac (U L1) MnP (U L1) Inhibition Sinapis alba (%) Inhibition Aliivibrio fischeri (%) a
Time (days) 0
1
2
3
5
588 (±13) 1635 (±52) 738 (±5) 152 (±2) 82.5 (±1.5) 84.3 (±0.3)
330 (±1) 1002 (±18) 610 (±0.2) 176 (±4) 49.8 (±4.5) 43.7 (±3.1)
329 (±5) 1055 (±35) 629 (±13) 141 (±5) 57.9 (±1.4) 37.1 (±3.0)
319 (±1) 633 (±70) 469 (±23) 113 (±6) 60.3 (±1.3) 11.1 (±7.6)
297 (±6) 791 (±18) 351 (±89) 83 (±28) 48.2 (±14) NTa
Non-Toxic (NT). Sample did not cause measurable toxic effect.
Toxicity of the landfill leachate to the plant Sinapis alba significantly decreased and enzymatic treatment led to a detoxification of the landfill leachate to the bacterium Aliivibrio fischeri (Table 3). After 7 days of the L2 leachate degradation, only 23% of organic matter (COD and DOC) was removed, however, it was comparable to the decrease of organic content in the blank control and thus it was evaluated as an insignificant difference and the L2 leachate was assessed as not biodegradable by ligninolytic enzymes. Landfill leachates are mixtures of various compounds and their composition depends on many factors, mostly on disposed waste and the phase of the landfill’s life cycle (Kulikowska and Klimiuk, 2008). The co-disposal of fresh waste to already stabilized waste is a source of organics as lignin and cellulose which are used as substrate for ligninolytic enzymes. Phenolic and other aromatic compounds were identified in active landfills (Yasuhara et al., 1997) and such compounds can also be easily degraded by Lac enzymes (Brijwani et al., 2010). The contribution of fresh waste to biodegradability of leachate is crucial for biodegradation and the concentration of organic biodegradable matter is, together with leachate toxicity, a limiting factor for fungal treatment. Fungal or enzymatic treatment may be preferable over biological treatments since it offers biodegradation of many pollutants and thus reduction of COD and toxicity of landfill leachates. For a large-scale application, a reactor system with immobilized fungus can be employed. A packed-bed reactor with wood chips or a reactor with silica-based porous support with immobilized white rot fungi has been reported to be much more efficient for treatment of 2-chlorphenol than suspended growth reactor (Lewandowski et al., 1990). Ehlers and Rose (2005) have demonstrated high efficiency of immobilized white rot fungi in a trickling packed-bed reactor with wood (pine) chips and glass beads for biodegradation of phenol and chlorinated phenol. A large-scale application of ligninolytic enzymes for treatment and detoxification of wastewaters is also possible. Chemically modified silica with imidazol groups appeared as an efficient support for immobilization of Lac and such system showed high efficiency in decolorization of dyes (Peralta-Zamora et al., 2003). Dodor et al. (2004) have investigated immobilized Lac enzymes for degradation of PAHs and the results indicated that immobilization improves stability of Lac to temperature, pH and inhibitors compared to free enzymes. However, fungal and enzymatic treatment may include some disadvantages as the requirements of nutrients, low pH, production of mycelia and an additional treatment step to remove ammonium nitrogen. 4. Conclusion Mature landfill leachates generated in old landfills cannot be treated by the conventional biological treatment methods because of the leachates’ low biodegradability and the high concentrations of inhibitory compounds. As no further study was identified that addresses successful biological treatment of a mature municipal landfill leachate, our study for the first time provided a clear
evidence that even an extremely mature leachate can be successfully biologically treated by the white rot fungi. By application of the fungal and enzymatic treatment, organic matter content and toxicity can be considerably reduced. Since the fungus was able to grow and partially degrade the mature leachate from the closed landfill, it could be employed for mature leachate treatment at the landfill site. After the landfill closure, the amount of the leachate and organic matter decreases with time and thus it is supposed that the fungal treatment could be a promising method for a post-closure landfill care. Ligninolytic enzymes produced by the fungus were responsible for the removal of major part of the organic matter and the reduction of toxicity in the leachate from the active landfill. Immobilized enzymes could be practically used for landfill leachate treatment, but because of the high pollution and toxicity of leachates from active landfills it should be also accompanied by some other treatment methods, e.g. physico-chemical ones because the enzymatic treatment alone cannot assure a sufficient treatment efficiency.
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