White Rot Fungi

The growth of the culture took place on the PUF pieces and the mycelia were easily attached to the carrier. Seven days old culture, in general, has been recommended for decolorization and COD reduction of pulp and paper mill effluents (Singh, 1993). The white rot fungi in immobilized form have been reported as efficient producer of extra-cellular lignin peroxidase 18 .

Filamentous cultures of 26 strains of wood-destroying white-rot fungi, izolated from Tugay forests of Azerbaijan, were used as an object of research: Bjerkandera fumosa (Pers.:Fr.)P.Karst, Fomes fomentarius (L.:Fr.)Gill., Ganoderma lipsiense (Batsch.)G.F., Lenzites betulina(Fr.)Fr. , Panus tiqrinus (Fr.)Sing., Phellinus igniarius ( L.:Fr.)Quel., Pleurotus ostreatus Jac.:Fr.)Kumm.,Schizophillum commune Fr.,Trametes cervina (Schwein.) Bress., T. qibbosa (Pres.)F.r, T. hirsuta (Wulfen:Fr.) Pilat, T. ochracea (Pers.) Gibb .et . Ryvarden,T. versicolor (L.:Fr.)Pilat. Fungal cultures were kept in wort-agar medium ( 4-5 points), pH 5,0 and replanted once in six months. In order to study enzymatic activity fungi were cultivated in submerged culture on shaker 180 rpm during 96 hours at 28˚ and 37˚С in the medium with the following composition (q/l ): wheat bran -15; NН 4 NO 3 -1,5; KH 2 PO 4 – 0.4; NaCl-0,5; MgSO 4 -

The degradation of wood by white-rot fungi has been reported [21–23]. Different methods have been applied to investigate wood decay, including microscopy techniques [24–26]; differential scanning calorimetric [27]; X-ray diffrac- tion [28]; gas chromatography—mass spectrometry (GC–MS) spectroscopy, chemical analysis [29, 30]; Nuclear magnetic resonance (NMR); and Fourier transform infrared spectros- copy [31–33]. Two-dimensional (2D) NMR techniques in the cell wall and lignin research have improved over the past dec- ade [34]. Among various 2D NMR spectroscopic techniques available, Heteronuclear Single-Quantum Coherence (HSQC) is the most common. Solution-state 2D NMR provided an interpretable structural fingerprint of the lignin and carbohy- drates of the cell wall, without further structural modification applied during the ball milling and ultra-sonication step [33, 35].

twenty two basidiomycetous fungi were screened for laccase activity using the chromogenic screening method on PDA-guaiacol medium. Table 1 summarizes the results of primary screening for the laccase producing fungal isolates. Of the twenty two isolates, ten isolates were laccase positive. On performing qualitative screening by using ABTS and guaiacol as a substrate the fungal isolates formed deep green and brown zone respectively around and above the colony which is a positive confirmation for laccase activity. Similarly, Arora and Sandhu (1985) used lignin guaiacol agar medium for screening such fungi and Kiiskinen et al. (2004) cultivated fungi on agar media containing indicator compounds (such as RBBR, poly R-478, guaiacol, and tannic acid). Apart from this, indicators like 2, 2´-Azino-bis-(3- ethylbenzthiazoline-6-sulphonic acid) (ABTS) is also suitable for screening because its one electron oxidation product is soluble in water, stable, and intensely green. Field et al. (1992) demonstrated that a polymeric dye could be used for screening white rot fungi. After primary screening, cultures were subjected to secondary screening. The isolates LCJ155, LCJ164 and LCJ169 were found to produce high activities of laccase during submerged fermentation. Quantitative experiments in shake flask culture showed a laccase activity of 0.807, 0.777 and 0.668 U/mL on the eighth day (Figure 1). Hence, cultures LCJ155, LCJ164 and LCJ169 were selected for further investigation.

The production of extracellular MnP in each organism was followed in two parallel cultivations in 1 L shake flasks containing 300 mL of liquid media with 90 rpm agitation. Cultivation temperatures were 37°C for P. chrysosporium, 25°C for Phlebia sp. Nf b19 and 28°C for all other white- rot fungi strains. P. radiata was grown in low-nitrogen ADMS medium pH 4.5 (Hatakka & Uusi-Rauva 1983) with 1% glucose, 0.05% (w/v) Tween 80 and 1 mM veratryl alcohol (VA, 3,4-dimethoxybenzyl alcohol). Supplements 180 μM Mn2+ and 10mM Sodium malonate were added on the 4th day. P. rivulosus was grown in low-nitrogen ADMS medium pH 4.5 (Hatakka & Uusi-Rauva 1983) with 1% glucose and 0.05% (w/v) Tween 20. Supplements 24 μM Mn2+ and 0.36 mM VA were added on the 4th day. P. chrysosporium was grown in Kirk’s medium pH 4.5 (Urek & Pazarlioglu 2004; Tien & Kirk 1988 and Bonnarme & Jeffries 1990) with 1% glucose and 0.05% (w/v) Tween 80. Supplements 728 μM Mn2+ and 0.36 mM VA were added on the 4th day. Bjerkandera sp. was grown in Kirk’s medium pH 4.5 (Palma et al. 2000) with 1% glucose and 0.05% (w/v) Tween 80, 235 μM Mn2+ and 0.36 mM VA. Phlebia sp. Nf b19 was grown in a glucose-yeast extract medium pH 4.5 (Nuske et al. 2002) with 0.5% glucose, 0.03% yeast extract and 200 μM Mn2+. Cultivations were continued until extracellular MnP activity was detected and it started to decline.

Several species of white-rot fungi were investigated for their utility in prolonged decolouration of the recalcitrant sulfonated azo dye, amaranth. Trametes pubescens, T. multicolor, T. meyenii and T. versicolor decoloured amaranth azo-dye best on low-nitrogen agar-solidified media whereas Bjerkandera adusta and Phlebia radiata were most effective in low nitrogen medium supplemented with manganese. Trametes cotonea did not decolour effectively under any condition. The decolouring Trametes species were also effective in liquid culture whereas B. adusta and P. radiata were not. Trametes meyenii, T. pubescens and T. multicolor were equal to or better than commonly employed T. versicolor at decolouring amaranth. This is the first study to show the dye decolouration potential of T. meyenii, T. pubescens, and T. multicolor. Supplementing with Mn(II) increased assayable manganese peroxidase activity, but not long-term decolouration, indicating that laccase is the main decolourizing enzyme in these Trametes species. This appears to be because of inadequate Mn 3+ chelation required by manganese peroxidase because adding relatively low amounts of malonate enhanced decolouration rates. The ability of Trametes meyenii to simultaneously decolour dye over prolonged periods of time while growing in relatively nutrient-rich medium appears to be unique amongst white-rot fungi, indicating its potential in wastewater bioremediation.

dehydration and environmental injuries can be considered a general function of extracellular polysaccharides. The EPS by nature of their glucan content that is composed mainly of glucose, mannose, galactose, xylose and fucose [37] [38], could also be considered as storage compounds, which are consumed by the fungus when ex- ogenous carbon sources are limited [21]. Therefore, the observed decrease in EPS seen in most tested WRF after two weeks of incubation, is a result of degradation and utilization of the sugar monomers in the EPS. Also, some of the sugars detected could actually be by-products from the degradation of the EPS formed in the earlier stages of growth of the fungus on the substrate. Other roles of the extracellular polysaccharides are to facilitate the dif- fusion and concentration of biodegradation enzymes, and also provide a medium with suitable ionic and pH conditions for degradation activities [21]. McCue and Shetty [39] investigated the involvement of lignin degra- dation activities in SSB of soybean by Lentinus edodes and they observed that decreased antioxidant activity was associated with total peroxidase and laccase activity. There are many reports on studies of EPS production by white rot fungi in submerged fermentation [40]-[42]. Their application is in nutraceuticals, especially β -glucans [43] and environmental remediation [44]. However, studies on exopolysaccharide production in lig- nocellulosic substrates under SSF have been few [16] [36].

There are only a few reports on the use of OPF fiber for production of bioethanol. To the best of our knowledge, there are only two reports on the use of oil palm frond fiber for bioethanol production with emphasis on the effects of hot compressed water pretreatment [7-8]. These methods of pretreatment have resulted in quite high glucose yield, based on the potential glucose in OPF. Another interesting report is the microscopic studies of oil palm frond after various kinds of pretreatments, that are chemical (acid and alkaline), liquid hot water (autoclaving at 121 °C for 15 minutes), and microbial pretreatment using Aspergillus niger [9]. This study suggests that the most effective pretreatment is liquid hot water. Biodegradation of lignin in OPF by white rot fungi is reported by Namoolnoy et al. [10]; however, the species of the isolates of white rot fungi used is not clear. White rot fungi are known as an effective lignin degrading microorganisms, so that they can help in the pretreatment of lignocellulosic feedstock for bioethanol production. The fungi produce three types of ligninolytic enzymes, lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase. The process takes quite a long time; however, it uses less energy and is more environmentally friendlier than physical, mechanical or chemical processes. The species Phanerochaete chrysosporium and Trametes versicolor are the most commonly used white rot fungi in lignin biodegradation studies, because these two fungi have good ligninolytic properties, can grow fast, and are easy to handle in the culture [11]. This research is aimed at studying the effects of two species of white rot fungi, P. chrysosporium and T. versicolor for OPF pretreatment on the sugar obtained after saccharification.

The secretome from 21-d I. lacteus SSF cultures was com- pared to those from two white-rot fungi, P. chrysosporium and P. ostreatus. The growth of these species on wheat straw produced different degradation patterns, and the biotreated material gave sugar yields lower than those attained for I. lacteus after enzymatic hydrolysis (Table 1). The MS/MS data from their whole EPP were searched against JGI and Uniprot (Additional file 1: Tables S6-S9). In all cases, the JGI database returned more hits than Uniprot (Figure 2). This is because many hypothetical proteins, deduced from genomic sequences already available, are de- posited in that database. Moreover, the percentages yielded for some protein groups were quite different when the in- puts from both databases were compared. This is probably due to the fact that many proteins from the JGI have not yet been annotated and may need to be corrected. Even higher differences were found when the number of proteins identified was compared to those predicted from genomes. A total of 769 proteins have been predicted to be part of P. chrysosporium secretome [33]. However, in the current work, 4-fold fewer proteins were detected (around 191). This finding highlights the need of studying secretomes from cultures and not by computational predictions, since the protein set released to the extracellular medium is vari- able and strongly depends on the environment.

The efficiency of three white rot fungi Trametes versicolor, Lenzites betulina and Polyporus elegans in bringing about decolorization and enhancement of xylanase production was studied using sawdust liquid extract, a crude source of xylan at two different concentrations of 12% and 20%. The selected cultures were proved to be efficient in both decolorization of xylan and production of xylanases. A clear linear correlation between the enzyme production and decolorization was observed. The decolorization percentage increased with increase in enzyme production from 6 th to 12 th days of incubation. Trametes versicolor showed maximum decolorization and enzyme production at both the concentrations. It showed 100% of decolorization and 900ug/ml enzyme production at 12% and 95% decolorization and 750ug/ml enzyme at 20%. Lenzites betulina and Polyporus elegans showed 90% and 85% of decolorization and 700ug/ml, 650ug/ml enzyme production at 12% while 85% and 80% decolorization with 650ug/ml, 583ug/ml enzyme production at 20 % concentration of saw dust extract respectively. The study of the bleaching and delignifying (kappa number reduction) ability of wood degrading fungi on pulp were also studied. The results obtained revealed that the selected cultures were able to reduce kappa number. Trametes versicolor was able to reduce kappa number up to 10 points, Lenzites betulina and Polyporus elegans upto 6 and 2 points respectively. Xylanase enzyme was secreted during bleaching by all the three white rot fungi. Based on the results it can be interpreted that xylanases enhance the delignification process and therefore the bleaching of pulp.

Enzymes secreted by microorganisms play a key role in biodegradation, bioconversion and bioremediation (Singh et al., 2007 a & b). Several microorganisms including bacteria, actinomycetes, fungi, yeast and algae have been reported for the production of enzymes. The capability of white rot fungi to secret extracellular enzymes which have low or no substrate specificity has attracted the attention of scientist around the globe. This property can be exploited for various applications particularly the degradation of vast variety of recalcitrant compounds resembling lignin structure. According to Erikkson (1993), white rot fungus are the only microorganisms which degrade lignin to any substantial degree.

Water hyacinth is a common type of lignocellulosic plant with high breeding rate, which can survive and give advantage to sewage treatment plant such as waste stabilization pond. While enhancing the effectiveness of the treatment, the plant can produce ethanol that can use as car fuel. The characteristic of rapid growth of this plant is very important which it is possible to fulfill the demand of the daily usage in Malaysia. White rot fungi also has an abundance source that is good in lignin biodegradation. Biodegradation of lignin is very crucial in the process of maximizing the production of bioethanol. Production ethanol from water hyacinth will not disturb food production because it is not food resources to human. Besides that, using ethanol as car fuel will give good impact to environment by reducing carbon emission.

White rot fungi (WRF) are characterized by a number of benefits that may be exploited in the bioremediation techniques. As key components of their lignin-degrading structures are extracellular, these fungi can degrade insoluble chemicals sub- stances such as lignin or a markedly diverse range of extremely steady or toxic ecological pollutants [Asgher et al., 2008]. Additionally, the substrates are fast colonized by the mycelial development and hyphal extension allows soil penetration, ap- proaching the contaminants more efficiently than other microbes [Reddy and Mathew, 2001; Fra- goeiro and Magan, 2008]. The aforementioned qualities may improve the natural, mechanical and enzymatic influence with the surroundings [Maloney, 2001]. In order to comprehend the de- grading capability of WRF, it is essential to ex- amine their ecological specialization. The above- mentioned fungi, including the effectively de- grading lignin species, constitute a physiological, rather than a taxonomic group [Pointing, 2001].

The White rot fungi are chief agents of biodegradation of lignin-cellulosic material in nature which contribute in the global carbon recycling [9]. These fungi considered as a key group in forest ecosystem maintenance and serve as decomposer, bioremediation, waste management agent, pathogen and also have medicinal value and hence recommended for decolourisation and bioremediation [7]. Phanerochaetec chysosporium, Trametes versicolor, Bjerkandera adjusta and Pleurotus are few important species that had demonstrated the bioremediation potential by virtue of different lignolytic enzymes such as laccases and peroxidises [10]. Many enzymes are involved in the oxidative degradation of dyes, including lignin peroxidase (LiP), manganese peroxidase (MnP) and laccase [11]. These oxidative enzymes are unspecific in nature thus cause the degradation of recalcitrant compounds such as single aromatic molecules, other xenobiotics and dyes [12]. White rot fungi are known to have ligninotytic enzymes which are no specific in nature and used in decolorization of synthetic dyes by the ligninolytic activity. Phanerochaete chrysosporium, Trametes versicolor, Schizophyllum commune and Lenzite seximia are few white rot fungi reported to have dye degradation ability [13]. In the present study wild mushrooms collected from Himachal Pradesh were evaluated for decolourization and degradation potential against two synthetic dyes sunset yellow-FCF and Acid orange-7.

Varying pH (2.5-6.5) indecolorization of RBBR by both extracts of Coriolus versicolor and Pleurotus ostreatus (Fig 1 and 2) shows that the maximum decolorization occurs at pH 4. At this optimum, maximum decolorizations were 58% and 47% by C. versicolor and P. ostreatus respectively. The result obtained was in agreement with previous findings which showed optimum decolorization by white rot fungi in acidic condition (Mansur et al., 2003; Palmieri et al., 2005; Kaushik and Malik, 2009; Asgher et al., 2009).

(Received 22 August, revised 30 November, accepted 2 December 2019) Abstract: This research focused on the degradation of chlorpyrifos via immo- bilized white rot fungi in soil, with the aim to select excellent degrading strains and an optimal carrier of white rot fungi. Immobilization of white rot fungi was assessed on corn stover, wheat straw, peanut shells, wood chip, and corn cobs. Phlebia sp., Lenzites betulinus and Irpex lacteus were grown in defined nutri- ent media for the remediation of pesticide-contaminated soils. The carrier of the biomass was determined by observing the growth of white rot fungi. The results showed that corn stover and wheat straw are suitable carriers of the immobilized white rot fungi and that Phlebia sp. and Lenzites betulinus have a positive effect on the degradation of chlorpyrifos. At 30 °C and neutral pH, the degradation rate of chlorpyrifos was 74.35 %, Phlebia sp. being immobilized by corn stover in 7 days, which was the best result compared to other combin- ations of strains and carriers. The orthogonal experiment showed that the pH value and temperature affected the pollutant degradability more than the initial concentration and the biomass dosage.

White rot fungi is commonly known as wood decaying fungi which grows on moist wood and digests converts it to rot. Rice straw degradation by white rot fungi has potential to increase the digestibility of animal feed and its nutritional value for the production of lignolytic enzymes. Twenty white rot fungal isolates were obtained from wood samples. These fungal isolates were subjected to qualitative screening tests for lignolytic activity on solid media containing several polymeric dyes like poly R-478, Tannic acid, Azure-B, Remazol brilliant blue R. Out of twenty isolates four isolates with appreciable enzyme activity were selected based on the dye decolourisation property. The optimized conditions resulted in high lignin modified enzymes (LME) production with the 745 Uml -1 of Laccase, 265 Uml -1 of MnPase and 142 Uml -1 of Peroxidase within 12 days of incubation, at the pH - 6.5 and

Rajrathanam et al. (1979) reported that hemicellulase activity of P. florida cultivated on rice straw showed gradual increase during mycelial growth and a steep increase during fruit body development. Ortega et al. (1993) reported that xylanase activity of P. ostreatus developed rapidly on sugarcane bagasse in a solid state fermentation. Vladimir et al. (2003) reported fluctuations in xylanase activity in the surface layer and found maximal enzyme activities during fruiting stage of P. ostreatus. Ahalawat et al. (2005) reported Volvariella volvacia secreted maximum xylanase enzyme at 10 or 13 days of growth on paddy straw. Singh et al (2008) reported the production of appreciable amount of xylanase by white rot fungi on wheat straw.

cMnP could brighten UKP about 10 points and 20 U of cMnP could brighten UKP about 13 points; in contrast, the MnP of P. chrysosporium could brighten UKP only about 5 and 6 points under the same conditions. These results indicated that the cMnP had better bleaching ability when Milli-Q water was replaced by the organic-free model white-water; when cMnP was used to bleach UKP, the MnP reaction was enhanced by salts in the organic-free model Table 2. Effect of Mn 2⫹ concentration on brightness increase of UKP

This indicates that the white- rot fungus decomposed lignin somewhat preferentially during the decay of acetylated wood and nonacetylated wood.. X-ray diffraction curves o[r]