Removal of a textile dye (RBBR) from the water environment by fungi isolated from lignocellulosic composts

A representative group of hydrophilic fungi from the genus Trichoderma isolated from lignocellulose composts with varying degrees of maturity was analyzed for their ability to biodegrade a harmful anthraquinone dye, i.e. Remazol Brilliant Blue R (RBBR). In RBBR-containing post-culture liquids, there were determined the degree of RBBR decolorization, horseradish peroxidase-like, superoxide dismutase-like, and xylanase activities, and the concentrations of low-molecular phenolic compounds. The study results demonstrated that Trichoderma asperellum, T. harzianum, and T. lixii strains isolated from compost containing larger amounts of easily available lignocellulose fractions, i.e. grasses, exhibit higher RBBR decolorization effi ciency ranging from 0.3 to 62% than T. citrinoviride strains isolated from compost II, which contained greater quantities of hardly degradable lignocellulose. The decolorization of remazol blue R by the investigated Trichoderma strains intensifi ed signifi cantly with the increase in peroxidase activity and it was correlated with a decline in the content of low-molecular phenolic compounds. The dynamics of changes in the horseradish peroxidase-like, superoxide dismutase, and xylanase activities in the aqueous post-culture liquids of the investigated fungal strains depended largely on the duration of the culture. Given their ability to adapt to water environments, e.g. wastewater, and to decolorize and detoxify the RBBR anthraquinone dye, Trichoderma fungi can be used for bioremediation of such environments. Removal of a textile dye (RBBR) from the water environment by fungi isolated from lignocellulosic composts 13 (2012). The ability to biodegrade RBBR is also tested in micromycetes, as reported by Jasińska et al. (2012) and Noman et al. (2019), in Staphylococcus sp. bacteria (Velayutham et al. 2018), and in bacterial and/or fungal consortia (Khudhair et al. 2015; Lade et al. 2016). Microorganisms that grow well in the water environment are especially valuable organisms playing an important role in the biodetoxifi cation of colored industrial wastewater. They include representatives of the Trichoderma genus, which were shown by Grabińska-Łoniewska (2004) as hydrophilic well-growing and well-sporulating fungi in water environments including wastewater. Among them, fungi isolated from natural environments that are rich in lignocellulosic organic matter seem to be very promising in this regard. There is a relationship between the ligninolytic abilities of fungi and the abilities to biodegrade other aromatic compounds, including colored ones, as reported by Ulmer et al. (1984). López et al. (2006) and Chamuris et al. (2000) suggest that micromycetes isolated from composts can be potential wood lignocellulose degraders. In turn, Ryazanova et al. (2015) have demonstrated that, besides their ability to decompose cellulose, Trichoderma fungi have the ability to degrade lignin. The involvement of Trichoderma in decolorization and biodegradation of post-industrial lignin has been reported by Rybczyńska-Tkaczyk and Korniłłowicz-Kowalska (2017). Given some reports on the involvement of micromycetes in the biological modifi cation of colored contaminants in wastewater and the potential ligninolytic abilities of Trichoderma fungi, the aim of the study was to assess the possibility of modifi cation of structurally lignin-related Remazol Brilliant Blue R (RBBR) by Trichoderma strains isolated from composts with varying maturity. This is a new approach to look for microbial strains that can be useful for elimination of post-industrial dyes present in wastewater before introduction thereof into waters. Materials and methods Composts as a source of potentially ligninolytic fungi Trichoderma fungi were isolated from two lignocellulose composts with a varied maturation degree composted for 10, 20, and 30 weeks. Compost I (PGSF) contained 42.86% of pine bark, 34.28% of grass, 20.00% of sawdust, and 2.86% of broiler chicken feathers, C/N=25. Compost II (PSSF) contained 25.54% of pine bark, 10.63% of wheat straw, 51.07% of sawdust, and 12.76% of broiler chicken feathers, C/N=25. A detailed characterization of the composting process is presented in a study conducted by Bohacz (2017). Fungi with potential ability to biodegrade lignocellulose, including fungi of the genus Trichoderma, were isolated on Petri dishes where 5 g of each type of compost was sprinkled with powdered industrial lignin (9 dishes for each type of the compost). After a 10-week incubation at 26°C, mycelia were transferred onto agar medium containing 0.02% RBBR and 0.25% glucose as described by Korniłłowicz-Kowalska et al. (2008). Strains that exhibited RBBR decolorization abilities on Petri dishes were checked for their ability to decolorize RBBR and release ligninolytic enzymes as well as low-molecular compounds in liquid cultures of these fungi. Trichoderma strains were selected for the study of ligninolytic activity, as they were most abundant among all the isolated micromycetes and constituted over 30% in compost I and approx. 50% in compost II (unpublished data). Identifi cation of Trichoderma strains Preliminary identifi cation of six fungal strains (designated VII, XII, XIX, VI, VIII, and XXV) was based on macroand microscopic features, i.e. the colony morphology, the size and shape of spores, and the structure of the conidiophore. The observations were carried out using an Olympus BX-41 microscope with a CVIII4 digital camera integrated with a computer and the Cell-A program for archiving and documenting photographs. Final verifi cation was based on systematic keys (Domsch et al. 2007). To confi rm species affi liation, the fungi were identifi ed by means of PCR using specifi c primers and sequencing of PCR templates. The sequences were deposited in the GenBank under the following accession numbers: MH571704.1 Trichoderma harzianum Rifai, MH 602297.1 Trichoderma lixii (Pat.) P. Chaverri, MH 602236.1 Trichoderma asperellum Samuels, Lieckf & Nirenberg, MH 602423.1 Trichoderma citrinoviride Bissett, MH 602287.1 Trichoderma citrinoviride Bissett and MH 602289.1 Trichoderma citrinoviridae Bissett. Strains VII, XII, and XIX were isolated from compost I (PGSF) in composting week 10, 20, and 30, respectively. Strains VI, VIII, and XXV originated from compost II (PSSF) and were isolated in composting week 10, 20, and 30, respectively. Strain cultures Static fungal cultures were run on the modifi ed medium described in a paper by Korniłłowicz-Kowalska et al. (2008) with the addition of 0.02% RBBR and 0.25% glucose. The media were inoculated with 1 agar disk containing 7-day mycelium. The cultures were incubated at 28°C for 32 days. The cultures of each strain were run in triplicate. Analytical methods The biodegradation ability of the tested Trichoderma strains was determined in aqueous solutions obtained from cultures supplemented with 0.02% Remazol Brilliant Blue R (RBBR, C22H16N2Na2O11S3) as a substrate. After 4, 8, 12, 16, 20, 24, 28, and 32 days of culture, the percentage decolorization of Remazol Brilliant Blue R, enzyme activity, and content of low-molecular phenolic compounds were determined in the post-culture liquids, which were separated from the mycelium by fi ltration on Millipore fi lters with a pore diameter of 0.22 μm, PVDF, and diam. 33 mm. Decolorization of RBBR as a reliable indicator of detoxifi cation of anthraquinone dyes was determined spectrophotometrically at 595 nm. The percentage of decolorization (DEC) was calculated using the formula proposed by López et al. (2006). Horseradish peroxidase-like (HRP-like) activity was determined using o-dianisidine as a substrate in acetate buff er pH = 5.5 as shown by Claiborne and Fridovich (1979). Superoxide dismutase (SOD-like) activity was determined as proposed by Marklund and Marklund (1974) and modifi ed by Paździoch-Czochra et al. (2003) using pyrogallol as a substrate and in 0.5 M TRIS-HCl buff er, pH 8.5,