Bioresource Technology 96 (2005) 1936–1942
Fungal treatment of a delignification effluent from a nitrocellulose industry Joa˜o V.B. Souza, E´rica S. da Silva, Fla´vio T. da Silva, Teresa C.B. Paiva
*
Departamento de Biotecnologia, Faculdade de Engenharia Quı´mica de Lorena, Rodovia Itajuba´ Lorena, Km 74,5, Bairro Campinho, CEP 12600-970, C.P. 116, Lorena, SP, Brazil Received 24 April 2003; received in revised form 18 January 2005; accepted 18 January 2005 Available online 29 March 2005
Abstract Twelve strains of filamentous fungi, most of them belonging to the Deuteromycetes class, were isolated from activated sludge adapted to the delignification effluent from a nitrocellulose industry and screened to be used in the treatment of the effluent. The screening experiment was carried out using the effluent without co-substrate, treated for 120 h and pH 5. Aspergillus 2BNL1, Aspergillus 1AAL1 and Lentinus edodes UEC 2019 showed the highest effluent color reduction rates between 83% and 95%. The white-rot fungus L. edodes UEC 2019 was used as the control for the decolorization. In addition to color reduction, total phenol was also reduced in 56% and 79% by Aspergillus 2BNL1 and L. edodes UEC 2019, respectively. A kinetic experiment showed that Aspergillus 2BNL1 and Aspergillus 1AAL1 reduced the effluent color in the range of 81–95% at the first 24 h while L. edodes required 72 h to achieve a similar result. UV/Visible spectra revealed that all fungi treatments were able to decrease the chromophore compounds present in the effluent, except Aspergillus 1AAL1 that increased the UV absorptions. The molar weight distribution analysis showed that the three fungi were able to change the pattern of the effluent chromatogram, probably by degradation of the high molecular weight compounds. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Fungi screening; White-rot fungi; Deuteromycetes; Zigomycetes; Effluent treatment; Cotton delignification effluent; Nitrocellulose industry
1. Introduction During the production of nitrocellulose, an important polymer in the industry of inks, lacs and explosives, effluents are also produced. Delignification effluent results from the alkaline treatment of cotton fibers. This effluent has dark color, a large amount of organic matter and a high degree of toxicity (Paiva et al., 2001). The conventional biological treatment of this kind of effluent by activated sludge has not shown effectiveness, because its microorganisms do not have a metabolism able to *
Corresponding author. Tel.: +55 12 3159 5123; fax: +55 12 553 3165. E-mail address:
[email protected] (T.C.B. Paiva). 0960-8524/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2005.01.027
degrade high molecular weight lignin derivatives (Mittar et al., 1992). Since 1977, many studies dealing with the treatment of effluents coming from cellulose industry have utilized fungi as an alternative method, since they are able not only to metabolize lignin and its derivatives, but also to reduce the effluentÕs color, organic matter and toxicity (Eaton and Chang, 1980; Sundman et al., 1981; Mehna et al., 1995). However, application of this knowledge on an industrial scale, has been minimal. For the biological treatment of cellulose industry effluents to be successful, the microorganism must be able to grow rapidly, to predominate in the environment with competition and to degrade low and high molar weight lignin derivatives. These requirements are
J.V.B. Souza et al. / Bioresource Technology 96 (2005) 1936–1942
fulfilled by the white-rot fungi, microorganisms that have the capability to degrade lignin in the environment (Archibald et al., 1990; Eriksson et al., 1990). However, soft-rot fungi, Deuteromycetes and Ascomycetes, have also given optimal results in the biological treatment of effluents (Prasad and Joyce, 1991; Malarczyk et al., 1998; Nagarathnamma and Bajpai, 1999). In order to identify the most appropriated fungal species for new biotechnological processes, a number of screening studies have been carried out (Galeno and Agosin, 1990; Esposito et al., 1991; Sayadi and Ellouz, 1993). However, research on the utilization of fungi, isolated from induced conditions are still required, to find most suitable strains for the biological treatment. The aim of this work was to isolate filamentous fungi from activated sludge previously adapted to a delignification effluent, as well as to identify and employ these fungi in the biological treatment of the effluent. The strains having the highest ability to reduce the effluent color were more deeply investigated in this work.
2. Methods 2.1. Effluent: origin and characteristics
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Agar, pH 5) slants supplemented with chlorafenicol (400 mg/L). After 72 h, the fungi colonies that grew were quantified as CFU/mL (Colony Formation Units per milliliter) and isolated. The genera of isolated fungi were identified using their macro and microscopic morphological characteristics, as described by Silveira (1995) and Teixeira et al. (1999). 2.3. Effluent treatment Three mycelium agar plugs with 5 mm in diameter, obtained along a uniform circumference from the malt-extract-agar plates for Basidiomycetes and 105 spores/mL for Deuteromycetes and Zigomycetes were inoculated into a 125 mL Erlenmeyer containing 25 mL of a growth medium (Potato Dextrose Broth) at the temperature of 25 °C under 100 rpm. After 120 h the pre-inoculum medium was replaced by 25 mL of effluent obtained previously (item 2.1) without extra carbon source and then the effluent was treated for 120 h under the same conditions. The experiments were carried out in triplicate. The results of effluent treatment by fungi were analyzed by the ANOVA variance test complemented by Dunnet test using GraphPad InStat software.
The effluent produced in the delignification stage of cotton from a nitrocellulose industry was employed in all the experiments. Its physical and chemical characteristics were determined: color = 48,460 ± 598 CU; pH = 12; total phenol = 524 ± 14 mg/L; COD = 14,060 ± 253 mg/L; total solid = 19,300 ± 300 mg/L (Souza et al., 2002). The original cotton delignification effluent was always diluted (10% v/v) in distilled water and the pH value was adjusted from 12 to 5 using a 72% H2SO4 solution (approx. 1.5 mL/L of the effluent) before being used in the treatment tests.
2.4. Analytical assays
2.2. Isolation and identification of fungi from activated sludge adapted to the effluent
2.4.2. Total phenol Total phenol was determined by following the standard procedure as previously described by APHA (1989). A volume of 250 lL of a carbonate–tartarate solution (12 g/L) and 25 lL of Folin–Ciocalteu reagent were added separately to 1000 lL of treated or untreated effluent and its absorbance was measured at 700 nm in a UV/visible U-2000 Hitachi—Japan spectrophotometer using phenol as standard.
2.2.1. Activated sludge acclimatization Decanted activated sludge from wastewater treatment conducted at a paper and pulp industry was used for acclimatization of the fungi according to conventional methodology (Cammarota, 1991). A synthetic medium (sucrose 1 g/L, urea 0.068 g/L and K2HPO2 0.027 g/L; pH 7), used to maintain the biomass, was gradually replaced by the effluent. The protozoan and metazoan were quantified by microscopy (microscopy Will Wetzlar—Netherlands) in a hemacitometer. 2.2.2. Fungi isolation and identification The fungi isolation, from the adapted activated sludge, was performed three times, using the conventional dilution technique in PDA (Potato Dextrose
2.4.1. Color The effluent color, before and after treatment was determined according to the CPPA standard method (CPPA, 1975). The effluent samples were centrifuged (centrifuge Excelsa baby—Brazil) at 10,500g for 30 min to remove suspended solids. The pH of the supernatant was then adjusted to 7.6 using sodium phosphate buffer and its absorbance was determined at 465 nm in a UV/ visible U-2000 Hitachi—Japan spectrophotometer.
2.4.3. Enzyme assays The phenoloxidases activity was determined using syringaldazine as substrate, as described by Szklarz et al. (1989). Laccase was determined by mixing 0.5 mL of the treated effluent with 0.3 mL citrate–phosphate buffer (0.2 M, pH 5.0), 0.1 mL syringaldazine (1 mM) and 0.1 mL H2O. The total phenoloxidases activity was determined following the same procedure, but
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substituting H2O for H2O2 (2 mM). The peroxidase activity was determined by the difference between the total phenoloxidase and laccase activity. The activities were analyzed in a UV/visible U-2000 Hitachi—Japan spectrophotometer at 525 nm. One unit of enzyme activity (IU) was defined as the amount of enzyme that oxidizes 1 lmol of the syringaldazine by minute. 2.4.4. UV/visible spectrometry The spectrum of the treated and untreated effluent, samples 10 times diluted, were carried out in the range of 200–500 nm with an interval of 5 nm using a UV/visible U-2000 Hitachi—Japan spectrophotometer. 2.4.5. Molecular weight distribution Molecular weight distribution of effluent compounds was determined using by High Performance Size Exclusion Chromatography (HPSEC) using a Shimadzu— Japan chromatograph with an Ohpak SB-803 HQ column eluted with degassed water at a flow rate of 1.0 mL/min and 25 °C. The peaks of the compounds were detected by a LC-10 refractive index detector. The chromatographic column was calibrated with polyethylene glycol (PEG) from 35,000 to 300 g/mol and ethylene glycol (62.07 g/mol).
3. Results and discussion 3.1. Isolation and identification of fungi from activated sludge adapted to the effluent To identify the best fungi strains for effluent treatment, activated sludge was acclimated to the delignification effluent during 9 days in a semi-continuous process using a retention time of 72 h. During the fungi isolation procedures the number of protozoa ranged from 103 to 104 organisms/L, which is adequate for treating domestic wastewater as predicted by Vazolle´r and Garcia (1989). This result showed that the sludge during the fungi isolation at fourth, sixth and eighth days presented optimal biological activity. The CFU/mL during the isolation process of the fungi ranged from 1400 to 3600, however the number of different strains was limited to 12. These strains were morphologically identified as Deuteromycetes and Zigomycetes, two classes of fungi which were previously reported by Vazolle´r and Garcia (1989) in the wastewater treatments using activated sludge. These strains were identified as Aspergillus, Penicillium, Paecilomyces and Mucor in the amount of 7, 2, 2 and 1, respectively. All these microorganisms genera were previously reported for the production of organic acids, hydrolytic enzymes (pectinases, amylases, proteases and cellulases) and oxidative enzymes as phenoloxidases (Bononi, 1998). These fungi are well distributed in the environment (soil, air and decaying of wood) and
some Aspergillus species have already been used in the treatment of effluents from pulp and paper industry (Dutta et al., 1985; Esposito, 1995). 3.2. Effluent treatment The industrial use of fungi treatment requires to find a strain able to adapt to high effluent concentrations, to treat the effluent in a short hydraulic retention time and to reuse of the biomass. In this context, Lentinus edodes UEC 2019, Trametes versicolor and Trametes vilosa CCT 5567 have been reported by Esposito (1992), Bajpai et al. (1993) and Silva et al. (2002), respectively, as the most effective fungi for color removal from pulp and paper industry effluent and as a result of this they were employed as a reference to evaluate the efficiency of the isolated strains. The effectiveness of the effluent treatment employing 15 strains was determined by color and total phenol reduction and phenoloxidases production at 120 h of treatment and the results are shown in Table 1. All the strains were able to reduce the effluent color, but the highest reduction rates were attained with Aspergillus 2BNL1 (95% ± 3%), Aspergillus 1AAL1 (81% ± 3%), that the means reported according to Dunnet test indicated no significance difference at 0.05% level of probability. The result obtained by the use of Aspergillus 1AAL1 and Aspergillus 2AAL2 were similar to those reached by and L. edodes employed as a reference strain. L. edodes UEC 2019, T. versicolor and T. vilosa CCT 5567, fungi well described for the lignin degradation (Archibald et al., 1990; Eriksson et al., 1990) and that were used as reference (Table 1) produced the highest total phenol reduction rates (63–79%) and the results produced by them indicated no significance difference was observed at the 0.05% level of probability (Table 1). Almost all the fungi isolated from the activated sludge (9) were also able to reduce total phenol and only 3 increased (Paecilomyces 1AAL2, Aspergillus 2ABL3, Aspergillus 2AAL2). This increase was probably due to the depolymerization of lignin fragments, as well to the compounds secreted by microorganism anabolism and to the non-specific reaction of unknown substances with the Folin–Ciocalteu reagent (Esposito, 1995; Paiva, 1999). The laccase activity determined after the effluent treatments was low except for T. versicolor (Table 1). However, the laccase activity produced by studied strains in this work were similar to that reported by Esposito (1992) and Paiva (1999) who showed that the fungi with high enzymatic activity in the culture media produce low activity during the effluent treatment. The peroxidase activity was detected only in the treatment with L. edodes, which produced 1 IU/L of enzyme. This activity was described by Esposito (1995), who demonstrated that this strain produces MnP (manganese
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Table 1 Color and total phenol reduction and phenoloxidase production during the treatment of a delignification effluent, lasting 120 h Microorganism
Aspergillus 2BNL1 Aspergillus 1AAL1 L. edodes (UEC 2019) Aspergillus 2AAL2 T. vilosa (CCT 5567) Penicillium 2AVL1 T. versicolor Penicillium 2CVL1 Aspergillus 2DVL1 Aspergillus 1AVL3 Aspergillus 2ABL3 Mucor 1BBL1 Paecilomyces 3AAL1 Paecilomyces 1AAL2 Aspergillus 1CML1
Color
Total phenol a
CU ± SE
Reduction (%) ± SE
mg/L ± SE
242 ± 122 782 ± 144 813 ± 63 970 ± 274 1415 ± 222 1578 ± 120 1695 ± 165 2366 ± 106 2424 ± 102 2483 ± 169 3315 ± 150 3324 ± 108 3544 ± 265 3907 ± 417 4031 ± 416
95 ± 3(a) 84 ± 3(b) 83 ± 2(b) 80 ± 6(b, c) 71 ± 5(c, d) 67 ± 3(d) 65 ± 4(d) 51 ± 3(e) 50 ± 3(e) 49 ± 4(e) 32 ± 3(f) 31 ± 3(f) 27 ± 6(f, g) 19 ± 9(g) 17 ± 9(g)
23 ± 8 58 ± 34 11 ± 2 69 ± 25 19 ± 4 35 ± 7 19 ± 4 25 ± 7 40 ± 11 39 ± 15 53 ± 8 32 ± 8 28 ± 2 58 ± 4 46 ± 8
Phenoloxidases a
Reduction (%) ± SE 56 ± 11(b, c, d) 28 ± 5(f, g) 79 ± 5(a) 56 ± 28b 63 ± 8(a, b, c) 42 ± 3(d, e, f) 65 ± 8(a, b) 45 ± 4(c, d, e, f) 36 ± 6(e, f, g) 42 ± 3(d, e, f) 9 ± 7b 49 ± 3(d, c, b, e) 47 ± 6(c, d, e) 7 ± 3b 21 ± 3(g)
Laccase (IU/L) ± SE
Peroxidases (IU/L) ± SE
1.5 ± 0.7 1.5 ± 0.2 0.8 ± 0.2 0.9 ± 0.5 1.4 ± 0.4 1.8 ± 1.6 105 ± 7 1.1 ± 0.3 1.7 ± 0.8 1.3 ± 0.1 1.4 ± 0.8 3.0 ± 0.8 1.0 ± 0.1 0.7 ± 0.5 1.0 ± 0.2
0 0 1±1 0 0 0 0 0 0 0 0 0 0 0 0
24
72
120
Laccase (IU/L) Peroxidases (IU/L)
Color Total phenol Laccase Peroxidase
45 40 35 30 25 20 15 10 5 0 168
Time (h) 100 90 80 70 60 50 40 30 20 10 0
30
(b)
25 20 15 Color Total phenol Laccase Peroxidase
0
24
72
120
10 5
Laccase (IU/L) Peroxidases (IU/L)
Color Removal (%) Total phenol removal (%)
0 168
Time (h)
(c)
100 80 60 40 20 0 -20 -40 -60 -80 -100 -120 -140 -160
30 25 Color Total phenol Laccase Peroxidase
20 15 10 5
0
24
72
120
Laccase (IU/L) Peroxidases (IU/L)
3.2.1. Treatment with Aspergillus 2BNL1, Aspergillus 1AAL1, and L. edodes UEC 2019 Aspergillus 2BNL1, Aspergillus 1AAL1 and L. edodes (reference strain) exhibited the greatest ability to remove the effluent color and they were subsequently used in a kinetic study. Color and total phenol reductions and phenoloxidase production were employed as parameters in 168 h of effluent treatment (Fig. 1). Aspergillus 2BNL1 and Aspergillus 1AAL1 provided the maximum color reduction after 24 h while L. edodes, the control microorganism, gave the similar result only after 72 h. Esposito (1995) also observed a maximum color reduction after 72 h of treatment of an E1 effluent from kraft pulp with L. edodes UEC 2019. L. edodes produced the highest total phenol reduction (82%) at 168 h of treatment. Aspergillus 2BNL1 also demonstrated some ability to reduce total phenol. For both fungi there was a direct correlation between total phenol reduction and color reduction, as earlier
(a)
100 90 80 70 60 50 40 30 20 10 0 0
Color removal (%) Total phenol removal (%)
peroxidase) and LiP (lignin peroxidase). The low production of peroxidase by the isolated strains tested was expected, since the fungi used in the treatment were mostly Deutoromycetes, a class of microorganisms that are not described in the literature as peroxidase like phenoloxidase producers (Duran and Esposito, 1998). There was no correlation between the enzymatic activity and effluent decolorization, since the high phenoloxidases activities were not correlated with high decolorization. It could be observed with T. versicolor, microorganism with high laccase activity as described by Addleman and Archibald (1993) and Addleman et al. (1995), that in the experimental conditions produced 105 ± 7 U/L and reduced color by only 65 ± 4%.
Color removal (%) Total phenol removal (%)
Initial color (4846 ± 60 CU); initial total phenol (52.3 ± 1.4 mg/L); number of replicates (treatment) = 3; ±SE (standard error). a The means reported with the same letter indicate that no significant difference was observed at 0.05% level of probability. b Rate of increase in the total phenol content. Ex. 9% = increase of 9% in the total phenol content.
0 168
Time (h)
Fig. 1. Color and total phenol reduction and phenoloxidase production during the treatment of a delignification effluent with L. edodes UEC2019 (a), Aspergillus 2BNL1 (b) and Aspergillus 1AAL1 (c). Replicates n = 3; the vertical bars shows standard error.
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. . .
. .
. .
.
.
(a)
(b)
Fig. 2. UV/visible spectra of the effluent treated for 72 h by the fungi L. edodes UEC 2019, Aspergillus 1AAL1 and Aspergillus 2BNL1.
2BNL1 is exhibited in Fig. 3 and showed that all fungi were able to modify the effluent. L. edodes probably promoted the polymerization of the compounds present in the effluent, as revealed by the peak eluted from 4 to 5 mL. It also can be observed the peak disappearance from 6.9 to 7.2 mL and the intensity decrease of the peak at 6.4 mL elution volumes. On the other hand, an increase in the low molecular weight compounds was observed by the Aspergillus 1AAL1 and Aspergillus 2BNL1 treatment, as observed by the appearance of the peaks eluted from 9 to 11 mL. It was probably due to the depolymerization of high molecular weight effluent constituents. All fungi were able to reduce the color of the effluent significantly but the patterns of molecular mass distribution were different. The high molecular mass compounds, which are mainly responsible for the color of the effluent (Herna´ndes et al., 1994), were degraded in all treatments. This response was also detected by Sundman et al. (1981), who affirmed that the decolorization of the effluent is a combination of chromophores destruction and decomposition of the polymer to low
40000
35 Kg/mol
4 Kg/mol
600 g/mol
62 g/mol
I
I
I
I
Untreated effluent
35000
Treated by Aspergillus 2BNL1 (24h)
30000
mV (RI)
described by Esposito (1992); Cammarota (1991) and Silva et al. (2002) for L. edodes UEC 2019, Phanerochatea chrysosporium and T. versicolor, respectively. However, the fungus Aspergillus 1AAL1 increased the total phenol content at the first 24 h and then reduced it. All experiments showed that although belonging to the same genus, Aspergillus 2BNL1 and Aspergillus 1AAL1 exhibited different behavior. L. edodes produced the highest peroxidase activity (35 ± 5 IU/L) at 24 h of the treatment. Both Aspergillus fungi showed their highest activity between 24 and 72 h, but their activities were 10 times lower than the L. edodes activity. At 120 h of treatment no significant peroxidase activity was detected for these fungi. Laccase activity which was detected for Aspergillus, ranged from 0.1 to 1.9 IU/L. UV/visible spectra of the effluent treated by the fungi L. edodes, Aspergillus 1AAL1 and Aspergillus 2BNL1 is shown in Fig. 2. The UV wavelength (Fig. 2a) showed that the absorption increased after the treatment with Aspergillus 1AAL1. The increase in the UV wavelength by Aspergillus 1AAL1 can be attributed to the depolymerization of lignin fragments, as well as to the compounds secreted by microorganism anabolism and was correlated to the results of total phenol content during the treatment, as shown in Fig. 1c. On the other hand, the UV wavelength absorption decreased after the treatment with Aspergillus 2BNL1 and L. edodes. Ferraz (1991) described that the reductions in 280 nm and 310 nm wavelength can be explained by the degradation of aromatic rings and by the degradation of lignin a–b insaturation and a-keto groups of lignin, respectively. The visible wavelength (Fig. 2b) revealed a decrease in absorption in the treated effluent. Aspergillus 2BNL1 was the fungi that provided the best reduction rates followed by L. edodes and Aspergillus 1AAL1. This result was correlated to color reduction shown in Fig. 1. The molecular weight distribution of the compounds presented in the untreated and treated effluent with the fungi L. edodes, Aspergillus 1AAL1 and Aspergillus
Treated by Aspergillus 1AAL1 (24h) Treated by Lentinus edodes (72h)
25000 20000 15000 10000 5000 0
3
4
5
6 7 8 Retention volume (mL)
9
10
11
Fig. 3. Molecular weight distribution determined by HPSEC of the compounds present in the effluent untreated and treated with the fungi L. edodes, Aspergillus 1AAL1 and Aspergillus 2BNL1.
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molecular weight colorless, soluble or/and volatile products.
4. Conclusions The technique used to isolate fungi from the activated sludge, adapted to the delignification effluent resulted in 12 strains of filamentous fungi mostly belonging to the Deuteromycetes class. During the screening for the effluent treatment, the fungi Aspergillus 2BNL1, Aspergillus 2AAL1 and L. edodes provided the highest rates of effluent color reduction. Aspergillus 2BNL1 and Aspergillus 1AAL1, the microorganisms which were isolated from activated sludge, were faster in the color reduction than L. edodes, which was used as the control. The molar mass distribution analysis showed that the three fungi were able to change the pattern of the untreated chromatogram, degrading high molecular mass compounds in few hours of treatment.
Acknowledgements We wish to thank M. Eunice Machado for critical reading of this manuscript. The technical assistance of Lu´cia A.B.A. Castro is also acknowledged. This research has been supported by FAPESP CAPES/PICDT, and SCTDE/SP. J. Souza thanks a student fellowship supported by FAPESP under contract number 03/ 00653-2.
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