Nature mining for bioactive enzymes from plants and microorganisms is highlighted in building a sustainable bio-based economy. The typical reaction of laccase is the oxidation of a phenolic compound with the concurrent reduction of molecular oxygen to water (Schaechter 2009). Laccases have very broad substrate specificities and can oxidize a variety of substrates, such as di- and polyphenols, aromatic amines, and a considerable range of other compounds. Laccase is effective in dye decolorization (Bello-Gil et al. 2018; Hao et al. 2016), and it can be combined with other enzymes in the bioremediation of reactive dye (Khan and Fulekar 2017). Laccase has the potential to be implemented in various industrial fields, such as biomass conversion, waste water treatment (Jasińska et al. 2015), polymer syntheses, and remediation of bio-based chemicals, etc. Laccase-producing fungi have found uses in a wide range of technological applications (Chitradevi and Sivakumar 2011; Forootanfar and Faramarzi 2015). However, the great potential of rhizosphere as a gold ore of laccase-producing fungi has not been fully investigated and there is no report concerning about such strains from
Currently synthetic dyes are used broadly in the textile dyeing, paper printing, color photography, pharmaceuticals, food and drink, cosmetic and leather industries. At present, more than 100 000 diverse dyes are present, with a yearly production of above 700 000 metric tons. These industries release a massive amount of colored effluents into natural water bodies, with or without treatment. The textile industry alone discharges 280 000 tons of dyes annually (Patel et al. 2015), making it the principal contributor to tinted effluent discharge.
Although a variety of treatment technologies are accessible, e.g., adsorption, chemical oxidation, precipitation, coagulation, filtration electrolysis and photo-degradation, the biological and microbiological methods using activated sludge, pure cultures, microbial consortia and degradative enzymes are cost-effective and eco-friendly alternatives. However, little information of fungi mediated decolorization is available for most dyes. For instance, the nonviable biomass of
One ml of RG19 at a concentration of 20–500 mg/l was mixed with 0.5 ml of each fungal suspension (two member consortium) or 0.333 ml of each fungal suspension (three member consortium) and 8 ml H2O. Thus, the final concentration of RG19 was 2–50 mg/l. Each experiment was performed in triplicate at 30°C for 4 days, and the dye decolorization was determined as described above. Different reactive dyes, mixed with the equal ratio, were used in the dye decolorization. COD was determined by the standard potassium dichromate method.
For high performance liquid chromatography (HPLC), the above solid residue was dissolved in a small volume of HPLC-grade methanol, and the sample was then used for analytical studies. This was the test sample. A dye solution of 20 mg/l was used as a biotic control. Agilent1200 (Palo Alto, CA) HPLC conditions: C18 column, 250 × 4.6 × 5 μm, 30°C, injection volume 5 μl, flow speed 1 ml/min, mobile phase H2O/acetonitrile, detection wavelength 632 nm. Gradient elution: 0–15 min, H2O 90%/acetonitrile 10%; 15–21 min, H2O 10%/acetonitrile 90%; 21–25 min, H2O 90%/acetonitrile 10%.
The biotransformation was monitored using FTIR (Fourier transform infrared) (Bio-Rad FTIR Model FTS 135) and compared with the control sample. The FTIR analysis was performed in the mid-IR region of 450–4000 cm−1 with 16 scan speed. C18 solid phase microextraction column (Agilent) was used to extract degradation products from the filtered supernatant, with dichloromethane as the elution solvent and the elution rate 2 ml/min. The samples were mixed with spectroscopically pure KBr in the ratio of 5:95, pellets were fixed in the sample holder, and the IR scan was performed. The dye decolorization was monitored via UV-Vis spectroscopic analysis (Hitachi U-2800, Japan), using supernatants.
The functional annotations were performed by the alignments with NCBI Nt, NR, SwissProt, KOG and KEGG databases using Blast N (Nt) or Blast X/Diamond (the rest) (Buchfink et al. 2015). BLAST2GO (default parameters;
The software RSEM (
The GO classification and KEGG pathway visualization of DEGs were performed. The pathway enrichment analysis by the phyper function of R package identified significantly enriched metabolic pathways and signal transduction pathways in DEGs compared with the reference gene background. The Q value was obtained by the Bonferroni correction of the
Proteins usually perform functions after they are combined into a complex by interaction. The interacting DEGs usually have similar functions. In this study, DEGs were mapped to STRING database (
Cells of pure
Enzyme activities were monitored spectrophotometrically (Shanghai Metash Instruments Co., Ltd., China) with a total volume of 10 ml at room temperature (25°C). For all enzymes, the supernatant and cell suspension were boiled for 20 min and used as the control inactivated enzyme. Laccase activity was determined as described above, except that the minimal amount of carbon and nitrogen sources were used in the liquid culture. Lignin peroxidase (LiP) reaction mixture in 50 mM sodium tartrate buffer (pH 3.0) contained 10 mM veratryl alcohol as substrate, 4 mM H2O2 and 1.0 ml enzyme. The reaction was monitored by measuring the change at 310 nm (Jasińska et al. 2015). Manganese peroxidase (MnP) reaction mixture in 50 mM sodium malonate buffer solution (pH 4.5) contained 20 mM DMP (2,6-dimethoxyphenol) as substrate, 20 mM MnSO4, 4 mM H2O2 and 1.0 ml enzyme. The reaction was monitored by measuring the change at 470 nm (Jasińska et al. 2015). Tyrosinase activity was determined by the formation of
NADH-dichlorophenolindophenol (DCIP) reductase activity was determined as described by Dawkar et al. (2010). The reaction mixture in 50 mM phosphate buffer solution (pH 7.4) contained 1.72 mM DCIP (2,6-dichloroindophenol sodium salt) and 0.70 mM NADH as the substrate, and 1.0 ml enzyme. The reaction was monitored by measuring the change at 620 nm. Azo reductase activity was determined according to the method described by Ramalho et al. (2005), which monitored the change of methyl orange concentration at 461 nm. Ferric reductase activity was determined according to the method described by Ramalho et al. (2005), and the absorbance at 562 nm was measured against a blank prepared similarly but with inactivated enzyme. All determinations were performed in triplicate.
The process parameters that influence laccase production were optimized. The type of the carbon/nitrogen source influenced the laccase production by all strains (Fig. 1). When the carbon source was sucrose, all four strains showed the relatively higher laccase activity (Fig. 1C); when the carbon source was starch, lactose, or glucose, only DJTU-sh7 exhibited the high laccase activity; when fructose was the sole carbon source, the laccase production was meager in all strains. On the other hand, peptone could be the best nitrogen source of four strains (Fig. 1D), followed by yeast extract and beef extract, while the other four nitrogen sources were not ideal.
The impacts of the incubation temperature, the initial pH, and dye concentration on the decolorization ability of DJTU-sh7 were investigated. Without fungi, adjusting pH alone did not decolorize any of the nine reactive dyes (data not shown). The maximal decolorization of 20 mg/l RG19 by the DJTU-sh7 suspension was obtained at pH 4.5 after 4 d reaction, and RG19 was decolorized within a broad range of pH 3.5–7. The maximal removal of RG19 was obtained at 35°C and pH 4.5 after 24 h shake flask culture. The conspicuous elimination of RG19 of 2–20 mg/l was observed at 35°C and pH 4.5 after 4 d reaction. The decreased decolorization of RG19 of 40–50 mg/l was obvious, implying the saturation effect of the decolorization process.
A
Three reactive dyes were mixed together, which simulated the industrial dye manufacturing effluent. COD was used to evaluate the mixed dye removal efficiency (Fig. 2B). Both color and COD removal efficiency was increased in the consortium mediated biotransformation, when compared with the single strain. The DJTU-sh7-containing consortium was more efficient than the one without it, and the initial COD value that could be processed by it is two orders of magnitude higher than that processed by a laccase-producing bacterial consortium for the treatment of industrial dye effluent (Patel et al. 2015).
The FTIR spectra before and after dye removal were compared. Peaks at 722 cm–1 (alkene, OH bending), 1014 and 1193 cm–1 (sulfonic acid group), and 1622 cm–1 (C-NH2) were absent after dye decolorization, suggesting the cleavage of the azo bond and the dye degradation, which was supported by the appearance of new peaks. In TLC, a few solvent systems containing 5% acetic acid were used. In all cases, an unambiguous bright blue spot, representing the decolorizing product, was observed in the organic extract of the reaction solution under UV light, while RG 19 was significantly reduced. RG19 is a naphthol type azo dye with the hydroxyl group at
In GO annotation of biological process, 6274 Unigenes belong to “metabolic process” (Fig. 3A), followed by “detoxification” (65), etc. In molecular function, 5045 Unigenes fall into “catalytic activity” (Fig. 4A), followed by “transporter activity” (727), “nucleic acid binding transcription factor (TF) activity” (687), and “antioxidant activity” (62), etc. These data indicate the great potential of
In KEGG annotation, 1342 (10.34% of annotated genes) belong to “Biosynthesis of secondary metabolites” and 20 belong to “Xenobiotics biodegradation and metabolism”. Fifty two Unigenes belong to “Mismatch repair”, followed by “Base excision repair” (52). One hundred twenty eight (0.99%) Unigenes fall into “ABC transporters”. These statistics further suggest potential of our
In the control sample, 89.3% of clean reads were mapped onto the de novo-assembled Unigenes, while 88.2% and 86.6% of reads were mapped to the Unigenes in guai and green, respectively. In total, 15 123, 17 171, and 15 884 Unigenes were expressed in ordi, guai, and green, respectively, indicating that organic compound treatments induced extensive elicitor-specific transcriptome remodeling. The most abundant Unigenes in the three conditions were distinct. These results indicate dramatic transcriptome remodeling and reprogramming in
Cluster analysis of expression patterns of 21 220 shared Unigenes among all three conditions (ordi, guai, and green) was performed with software Mfuzz. These genes were divided into 12 clusters. In clusters 3 (1310 Unigenes), 5 (2988), 8 (1042) and 12 (1240), the gene expression level in green was higher than those in ordi and guai. Most differentially expressed genes (DEGs) involved in azo dye biotransformation and degradation are included in these clusters and are highlighted in the following analyses.
Genes with similar expression patterns usually have the functional correlation. In the cluster analysis, 9795 DEGs fall into scores of unique clusters showing distinct expression patterns in the union set of ordi vs. guai, ordi vs. green, and guai vs. green (Fig. 4). Some clusters were identified, in which most Unigenes were upregulated in green when compared with ordi, and either upregulated, downregulated, or unaltered in guai as compared with ordi. These clusters might contain genes closely associated with dye biotransformation and decolorization. Many genes in these clusters are also involved in stress and defense responses.
The initial step of azo dye metabolism in many filamentous fungi involves the action of laccase (multicopper oxidase) and peroxidases (Fig. 5) (Solis et al. 2012; Jasińska et al. 2015). Iron transport multicopper oxidase fio1 (Unigene4094_All) was included in an 86-gene cluster and was significantly upregulated in green as compared with guai and ordi. Catalase and peroxidases are evolutionarily closely related. At least five Unigenes, representing catalase/peroxidase, had significantly higher expression in green than in other two conditions, as an essential response to oxidative stress triggered by RG19.
Monooxygenases, such as cytochrome p450 (CYP) and flavin-containing monooxygenase (FMO) are well known phase I xenobiotic metabolizing enzymes (Hao and Xiao 2011; Ma et al. 2017). At least 22 monooxygenase genes, e.g., benzoate 4-monooxygenase (five Unigenes), phenol 2-monooxygenase (four Unigenes), and dimethylaniline monooxygenase (three Unigenes), were upregulated significantly in green. For FAD-dependent monooxygenase (Unigene3165_All), the log2FC values in ordi vs. guai, ordi vs. green, and guai vs. green were 1.68, 3.05, and 1.38 respectively; for FMO (dimethyl-aniline monooxygenase CL1649.Contig2_All), the corresponding values were –0.93, 1.15, and 2.08 respectively. Alkane sulfonate monooxygenase (EC 1.14.14.5; Unigene302_All) catalyzes the transformation of alkane sulfonate (R-CH2-SO3H) + FMNH2 + O2 into aldehyde (R-CHO) + FMN + sulfite + H2O (Zhan et al. 2008), and thus might be indispensable in azo dye desulfonation. The log2FC values of this gene in ordi vs. guai, ordi vs. green, and guai vs. green were 0.69, 1.16, and 0.47 respectively.
Phenylacetate 2-hydroxylase catalyzes the first step of phenylacetate catabolism (Rodríguez-Sáiz et al. 2001), which generates fumarate and acetoacetate and bridges between azo dye degradation intermediate and TCA cycle. Five Unigenes of this CYP were significantly increased in green. Salicylate hydroxylase [EC:1.14.13.1], catalyzing the transformation of salicylate + NADH + 2H+ + O2 into catechol + NAD+ + H2O + CO2, might be essential in the complete degradation of both azo dye and phenolic, as three Unigenes of this monooxygenase were significantly upregulated in both green and guai. Epoxide hydrolase (Unigene8943_All), exerting its function following CYP, was upregulated only in green. These enzymes might participate in the conversion of azo dye degradation intermediates.
Induction of the activity of aerobic enzyme dioxygenase indicates its involvement in dye removal (Cirik et al. 2013; Nor et al. 2015). Aromatic-ring-hydroxylating dioxygenases (ARHD) incorporate O2 into their substrates in the dihydroxylation reaction. The product is (substituted) cis-1,2-dihydroxycyclohexadiene, which is subsequently converted to (substituted) benzene glycol by a cis-diol dehydrogenase, followed by ring opening. The substantially increased expression of ARHD (Unigene3184), dioxygenases (CL864.Contig1_All and Unigene3045), and isomerase (Unigene2274) suggests their prominent role in the
Except CYP and peroxidase, other enzymes involved in the oxidative reactions were also increased at the mRNA level. For example, tyrosinase-encoding Unigene2982_All was significantly upregulated in green (log2FC 1.81 in ordi vs. green) rather than in guai (log2FC 0.6 in ordi vs. guai) or ordi. AAO encoding Unigene7311_All had much more mRNA expression in green (log2FC 6.08 in ordi vs. green) than in guai (log2FC –1.51 in ordi vs. guai) or ordi. This enzyme was also involved in the decolorization of azo dye in
The role of reductase in the dye decolorization cannot be neglected (Song et al. 2017), especially when the oxygen supply is not sufficient. Ferric reductase-encoding Unigene558_All and Unigene7872_All were dramatically upregulated in green (log2FC1.79 and 1.2 respectively in ordi vs. green) instead of guai. In two bacterial strains, supplementation of 5 mM ferric chloride increased azo dye decolorization rate (Ng et al. 2014); ferric reductase activity was consistent with synergistic effects of ferric chloride and ferric citrate on these strains. Ferric reductase is membrane associated and involved in respiratory electron transport (Xu et al. 2007). Azo reduction by bacterial strains is coupled to the oxidation of electron donors and linked to the electron transport and energy conservation in the cell membrane (Hong and Gu 2010). Our transcriptomic and phenotypic (see below) results represent the first example of the role of ferric reductase in fungal degradation of azo dye. On the other hand, the enhanced cellular oxidation against the azo dye attack could be balanced by the upregulation of ferric reductase, which might protect cell wall integrity and mitochondrial function (Yu et al. 2014), and improve the oxidative stress tolerance (Xu et al. 2014). Correspondingly, manganese superoxide dismutase (SOD; Unigene3085_All) and SOD[Cu-Zn] (Unigene6367_All) were upregulated in green to attenuate the deleterious effect of reactive oxygen species (ROS). Unigene4933_All, representing glutathione S-transferase (GST), was upregulated to exert antioxidant activities. Both GST and N-acetyltransferase (NAT) are phase II xenobiotic metabolizing enzymes (Hao et al. 2010). NAT enables acetyl coenzyme A-dependent detoxification of aromatic amines (Cocaign et al. 2013). Five Unigenes, representing NATs, were significantly upregulated in green as compared with ordi, one of which (Unigene622_All) clustered with laccase.
Enzyme activities of
Enzyme (U/ml) | RG19 addition | No RG19 | ||
---|---|---|---|---|
Intracellular | Extracellular | Intracellular | Extracellular | |
Laccase | ND | 26.241 ± 0.760* | ND | 17.108 ± 0.895 |
Lignin peroxidase | 0.164 ± 0.006^ | 0.120 ± 0.006^ | 0.152 ± 0.017 | 0.097 ± 0.002 |
Mn peroxidase | ND | ND | ND | ND |
Tyrosinase | 45.000 ± 3.000^ | 38.667 ± 8.386^ | 42.333 ± 6.429 | 33.667 ± 2.309 |
Aryl alcohol oxidase | 14.093 ± 0.056* | 5.900 ± 0.001* | 5.244 ± 0.056 | 3.572 ± 0.056 |
Ferric reductase | 7.845 ± 0.137** | 11.788 ± 0.182* | 6.849 ± 0.068 | 9.000 ± 0.182 |
Azo reductase | 22.853 ± 0.544^ | 12.696 ± 0.670^ | 23.242 ± 0.155 | 12.204 ± 0.432 |
NADH-DCIP reductase | 3.644 ± 1.547^ | 8.926 ± 0.696^ | 2.603 ± 1.610 | 7.550 ± 2.260 |
Values are mean ± SD;
NADH-DCIP reductase was involved in cleavage of azo bonds (Song et al. 2017), which were the chromophoric groups of azo dyes. NADH-DCIP reductase was detected intracellularly and extracellularly in the presence or absence of RG19, and the extracellular enzyme activity was higher than the intracellular one. The differences of NADH-DCIP reductase activity were not statistically significant between RG19 treatment and no RG19 (Table I), so were the differences of azo reductase. These data suggest that the activity of both reductases was not induced by RG19. Rather, the activity of ferric reductase might be induced by RG19, as its intracellular and extracellular activity was significantly higher after RG19 exposure. This suggests its prominent role in decolorization and biotransformation of azo dye in
For the first time, laccase-producing fungi were isolated from
The characterization of