Biodegradation of nitro-substituted explosives by white-rot fungi: A mechanistic approach

The contamination of wastewater with textile dyes has emerged as a pressing environmental concern due to its persistent nature and harmful effects on ecosystems. Conventional dye treatment methods have proven inadequate in effectively breaking down complex dye molecules. However, a promising alternative for textile dye degradation lies in the utilization of white rot fungi, renowned for their remarkable lignin-degrading capabilities. This review provides a comprehensive analysis of the potential of white rot fungi in degrading textile dyes, with a particular focus on their ligninolytic enzymes, specifically examining the roles of lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase in the degradation of lignin and their applications in textile dye degradation. The primary objective of this paper is to elucidate the enzymatic mechanisms involved in dye degradation, with a spotlight on recent research advancements in this field. Additionally, the review explores factors influencing enzyme production, including culture conditions and genetic engineering approaches. The challenges associated with implementing white rot fungi and their ligninolytic enzymes in textile dye degradation processes are also thoroughly examined. Textile dye contamination poses a significant environmental threat due to its resistance to conventional treatment methods. White rot fungi, known for their ligninolytic capabilities, offer an innovative approach to address this issue. The review delves into the intricate mechanisms through which white rot fungi and their enzymes, including LiP, MnP, and laccase, break down complex dye molecules. These enzymes play a pivotal role in lignin degradation, a process that can be adapted for textile dye removal. The review also emphasizes recent developments in this field, shedding light on the latest findings and innovations. It discusses how culture conditions and genetic engineering techniques can influence the production of these crucial enzymes, potentially enhancing their efficiency in textile dye degradation. This highlights the potential for tailored enzyme production to address specific dye contaminants effectively. The paper also confronts the challenges associated with integrating white rot fungi and their ligninolytic enzymes into practical textile dye degradation processes. These challenges encompass issues like scalability, cost-effectiveness, and regulatory hurdles. By acknowledging these obstacles, the review aims to pave the way for practical and sustainable applications of white rot fungi in wastewater treatment. In conclusion, this comprehensive review offers valuable insights into how white rot fungi and their ligninolytic enzymes can provide a sustainable solution to the urgent problem of textile dye-contaminated wastewater. It underscores the enzymatic mechanisms at play, recent research breakthroughs, and the potential of genetic engineering to optimize enzyme production. By addressing the challenges of implementation, this review contributes to the ongoing efforts to mitigate the environmental impact of textile dye pollution. PRACTITIONER POINTS: Ligninolytic enzymes from white rot fungi, like LiP, MnP, and laccase, are crucial for degrading textile dyes. Different dyes and enzymatic mechanisms is vital for effective wastewater treatment. Combine white rot fungi-based strategies with mediator systems, co-culturing, or sequential treatment approaches to enhance overall degradation efficiency. Emphasize the broader environmental impact of textile dye pollution and position white rot fungi as a promising avenue for contributing to mitigation efforts. This aligns with the overarching goal of sustainable wastewater treatment practices and environmental conservation. Consider scalability, cost-effectiveness, and regulatory compliance to pave the way for sustainable applications that can effectively mitigate the environmental impact of textile dye pollution.

The white rot fungi technology is very different from other more well-established methods of bioremediation (e.g., bacterial systems). The differences are primarily due to the mechanisms discussed previously. The unusual mechanisms used by the fungi provide them with several advantages for pollutant degradation, but the complexity of these mechanisms has also made the technology slow to emerge as a viable method of bioremediation. One distinct advantage that white rot fungi have over bacterial systems is that they do not require preconditioning to a particular pollutant. Bacteria must be preexposed to a pollutant to allow the enzymes that degrade the pollutant to be induced. The pollutant must also be present in a significant concentration, otherwise induction of enzyme synthesis will not occur. Therefore, there is a finite level to which pollutants can be degraded by bacteria. In contrast, the degradative enzymes of white rot fungi are induced by nutrient limitation. Thus, cultivate the fungus on a nutrient that is limited in something, and the degradative process will be initiated. Also, because the induction of the lignin-degrading system is not dependent on the chemical, pollutants are degraded to near-nondetectable levels by white rot fungi. Another unique feature of pollutant degradation by white rot fungi involves kinetics. The process of chemical conversion by these fungi occurs via a free-radical process, and thus the degradation of chemicals often follows pseudo-first-order kinetics. In fact, in several studies, it has been found that the rate of mineralization or disappearance of a pollutant is proportional to the concentration of the pollutant. This makes the time required to achieve decontamination more important than the rate of degradation. Because the metabolism of chemicals by bacteria involves mostly enzymatic conversions, pollutant degradation often follows Michaelis-Menton-type kinetics. Therefore, Km values of various degradative enzymes with respect to the pollutant must be considered when using bacteria for bioremediation. Considering this, the solubility of a pollutant or a mixture of pollutants might also present a problem for bacterial degradation. In contrast, using a nonspecific free-radical-based mechanism, the fungi are able to degrade insoluble complex mixtures of pollutants, such as creosote (Aust and Bumpus 1989) and Arochlor (Bumpus and Aust 1987b). Inexpensive nutrient sources, such as sawdust, wood chips, surplus grains, and agricultural wastes, can be used to effectively cultivate white rot fungi.(ABSTRACT TRUNCATED AT 400 WORDS)

Lignin is the second most abundant polymer (after cellulose) in the biosphere, accounting for 20 to 35% of the dry weight of wood. Lignin is a complex high molecular weight aromatic polymer formed from the random condensation of three different phenylpropanoid precursors, p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol (4-hydroxy-, 3-methoxy-4-hydroxy-, and 3,5-dimethoxy-4-hydroxy-phenylpropanol, respectively). Lignin behaves as a glue providing strength to fibers, serves as a hydrophobic impermeable seal across cell walls, and as a barrier to microbial attack (34). The hydrophobic and irregular structure of lignin renders the polymer inaccessible to hydrolytic enzymes. The only mechanism that can account for the initial attack of lignin is a non-specific extracellular oxidative process (79). White rot fungi constitute the most important group of microorganisms responsible for the biodegradation of nature's most complex polymer, lignin (39, 79, 136). White rot fungi are, for the most part, higher fungi with macroscopic fruiting bodies (e.g., mushrooms, conches etc.), belonging to the order Basidiomycetes. These organisms play a vital role in earth's global ecology, by secreting extracellular ligninolytic enzymes to bring about an unique oxidative erosion of the complex aromatic structure of lignin. The lignin polymer generally does not serve as a sole source of carbon and energy, rather it is cometabolized by white rot fungi utilizing carbohydrates or other readily assimilatable compounds. The physiological purpose of lignin degradation is to provide white rot fungi with better access to hemicellulose and cellulose in wood, which are the true primary substrates of the fungi. Ligninolytic metabolism is usually triggered by nutrient limitation, typically either nitrogen or carbon starvation (79, 109). The main components of the ligninolytic enzyme system are shown in Table 6.1. The most common lignin degrading enzymes are two closely related peroxidases, manganese

ABSTRACT: One of the extracellular enzymes involved in ligninolytic is lacasse, secreted by white and brown rot fungus. The class of blue copper proteins that includes laccases consists of N-glycosylated multicopper oxidases. Ascomycetes, Deuteromycetes, and Basidiomycetes are all fungi that contain laccase; many of these are white-rot fungi that break down lignin. Due to their wide range of substrate specificity, laccases have been the focus of extensive research over the past few decades. Their latest applications include anything from the textile pulp and paper industries to culinary applications and bioremediation techniques. Laccases are also used in organic synthesis, where phenols and amines are common substrates. Dimers and oligomers are produced due to the coupling of reactive radical intermediates in these reactions. The current investigation gathered 50 white rot fungi, and the most incredible laccase-producing organisms in submerged fermentation were looked into. Ten of the 21 cultures displayed a reddish-brown color zone. Of these ten isolates (PTD 19, PTD 4, PP2J15, LKT 34, ITC 1, NRL 7, GOJ 7, PTD2, PP2J, and PKT12), only PP2J15 and GOJ 7 displayed the most reddish-brown color zone. The isolation of white rot fungus, their molecular characterization, and testing for laccase production are all covered in this Paper. Talaromyces verruculosus and Cladosporium cladosporioides were identified as the PP2J15 and GOJ 7 strains based on sequence comparison and phylogenetic analysis with reference taxa.

Many white rot fungi are able to produce de novo veratryl alcohol, which is known to be a cofactor involved in the degradation of lignin, lignin model compounds, and xenobiotic pollutants by lignin peroxidase (LiP). In this study, Mn nutrition was shown to strongly influence the endogenous veratryl alcohol levels in the culture fluids of N-deregulated and N-regulated white rot fungi Bjerkandera sp. strain BOS55 and Phanerochaete chrysosporium BKM-F-1767, respectively. Endogenous veratryl alcohol levels as high as 0.75 mM in Bjerkandera sp. strain BOS55 and 2.5 mM in P. chrysosporium were observed under Mn-deficient conditions. In contrast, veratryl alcohol production was dramatically decreased in cultures supplemented with 33 or 264 (mu)M Mn. The LiP titers, which were highest in Mn-deficient media, were shown to parallel the endogenous veratryl alcohol levels, indicating that these two parameters are related. When exogenous veratryl alcohol was added to Mn-sufficient media, high LiP titers were obtained. Consequently, we concluded that Mn does not regulate LiP expression directly. Instead, LiP titers are enhanced by the increased production of veratryl alcohol. The well-known role of veratryl alcohol in protecting LiP from inactivation by physiological levels of H(inf2)O(inf2) is postulated to be the major reason why LiP is apparently regulated by Mn. Provided that Mn was absent, LiP titers in Bjerkandera sp. strain BOS55 increased with enhanced fungal growth obtained by increasing the nutrient N concentration while veratryl alcohol levels were similar in both N-limited and N-sufficient conditions.

White-rot fungus is a common lignin-degrading fungus. However, compared with those of microorganisms that biodegrade lignin alone, synergistic systems of electro-Fenton processes and white-rot fungi are superior because of their high efficiency, mild conditions, and environmental friendliness. To investigate the details of lignin degradation by a synergistic system comprising electro-Fenton processes and white-rot fungi, lignin degradation was studied at different voltages with three lignin-degrading fungi (Phanerochaete chrysosporium, Lentinula edodes, and Trametes versicolor). The lignin degradation efficiency (82∼89%) of the synergistic systems at 4 V was higher than that of a control at 96 h post inoculation. Furthermore, the H2O2 produced and phenolic lignin converted in the system can significantly enhance the efficiency of ligninolytic enzymes, so a considerably increased enzyme activity was obtained by the synergistic action of electro-Fenton processes and white-rot fungi. 13C NMR spectroscopy revealed that aromatic structure units (103–162 ppm) were effectively degraded by the three fungi. This study shows that the combination of electro-Fenton processes and white-rot fungi treatment significantly improved the lignin degradation efficiency, which established a promising strategy for lignin degradation and valorization.

With global attention and research now focused on looking for the abatement of pollution, white-rot fungi is one of the hopes of the future. The lignin-degrading ability of these fungi have been the focus of attention for many years and have been exploited for a wide array of human benefits. This review highlights the various enzymes produced by white-rot fungi for lignin degradation, namely laccases, peroxidases, aryl alcohol oxidase, glyoxal oxidase, and pyranose oxidase. Also discussed are the various radicals and low molecular weight compounds that are being produced by white-rot fungi and its role in lignin degradation. A brief summary on the developments in research of decolorization of dyes using white-rot fungi has been made.

Effects of nitrogen source and concentration and organic carbon cosubstrates on lignin and cellulose degradation by Streptomyces badius strain 252 were examined using 14C-labeled substrates prepared from Pseudotsuga menziesii twigs. As compared with white-rot fungi, which do not degrade lignin in the absence of a readily metabolizable carbon cosubstrate, degradation of a milled-wood lignin occurred in a minimal medium, although degradation by S. badius was greatly enhanced when organic nitrogen and an organic carbon cosubstrate were added to the medium. Lignin degradation was greatest in the presence of high levels of organic nitrogen. Further enhancement of lignin and cellulose degradation occurred in a medium containing organic nitrogen supplemented with low levels of NO3-. The specific effects of inorganic nitrogen on lignocellulose degradation by S. badius in an otherwise optimal medium included both enhancement and inhibition of lignin or cellulose degradation depending on the source and concentration of inorganic nitrogen used. These effects were distinctly different from those observed with white-rot fungi and were shown to be specific ion effects on polymer degradation and not simply a salt concentration effect on cellular growth.

Lignocellulosics are the major structural component of woody and nonwoody plants and represent a major source of renewable organic matter. The plant cell wall consists of three major polymers: cellulose, hemicellulose, and lignin. Lignocellulose biomass, available in huge quantity, has attracted considerable attention as an alternate resource for pulp and paper, fuel alcohol, chemicals, and protein for food and feed using microbial bioconversion processes. The current industrial activity of lignocellulosic fermentation is limited because of the difficulty in economic bioconversion of these materials to value-added products. Lignin is degraded to different extents by variety of microorganisms including bacteria, actinomycetes, and fungi, of which wood-rotting fungi are the most effective, white-rot fungi in particular. White-rot fungi degrade wood by a simultaneous attack on the lignin, cellulose, and hemicellulose, but few of them are specific lignin degraders. The selective lignin degraders hold a potential role in economically bioconversion of plant residues into cellulose-rich materials for subsequent bioethanol and animal feed production. Different fungi adapt in accordance to conditions existing in the ecosystem and complete their task of carbon recycling of the lignified tissues, and some white-rot fungi have capability to completely mineralize it. It is known that white-rot fungi are able to perform lignin degradation by an array of extracellular oxidative enzymes, the best characterized of which are lignin peroxidase (LiP), manganese peroxidase (MnP), and laccase. However, the regulation of the production of individual enzymes and lignin degradation is a complex phenomenon. Unfortunately, even selected white-rot fungi take long in delignifying the lignocellulosic substrates. Therefore, it is necessary to improve these fungi for their ability to degrade lignin through various conventional and modern approaches. A considerable progress has been made in this direction during the past two decades; LiP, MnP, and laccase genes have been cloned, and an efficient Agrobacterium-mediated transformation system has been developed, which will eventually help in successful expression of the desired protein. This chapter presents an overview of diversity of lignin-degrading microorganisms and their enzymes especially in developing animal feed. In addition to that, advances in molecular approaches to enhance the delignification capability of microorganisms are also discussed.

The degradation of lignocellulose by fungi, especially white-rot fungi, contributes a lot to carbon cycle, bio-fuel production, and many other bio-based applications. However, the existing enzymatic and non-enzymatic degradation mechanisms cannot be unequivocally supported by in vitro simulation experiment, meaning that additional mechanisms might exist. Right now, it is still very difficult to discover new mechanisms with traditional forward genetic approaches. To disclose novel lignin degradation mechanisms in white-rot fungi, a series of fusants from wide cross by protoplast fusion between Pleurotus ostreatus, a well-known lignin-degrading fungus, and Saccharomyces cerevisiae, a well-known model organism unable to degrade lignocellulose, was investigated regarding their abilities to degrade lignin. By analyzing the activity of traditional lignin-degrading enzyme, the ability to utilize pure lignin compounds and degrade corn stalk, a fusant D1-P was screened out and proved not to contain well-recognized lignin-degrading enzyme genes by whole-genome sequencing. Further investigation with two-dimension nuclear magnetic resonance (NMR) shows that D1-P was found to be able to degrade the main lignin structure β-O-4 linkage, leading to reduced level of this structure like that of the wild-type strain P. ostreatus after a 30-day semi-solid fermentation. It was also found that D1-P shows a degradation preference to β-O-4 linkage in Aβ(S)-threo. Therefore, wide cross between white-rot fungi and S. cerevisiae provides a powerful tool to uncover novel lignocellulose degradation mechanism that will contribute to green utilization of lignocellulose to produce bio-fuel and related bio-based refinery.

Degradation of lignin in pulp mill wastewaters by white-rot fungi on biofilm

Lytic polysaccharide monooxygenase synergized with lignin-degrading enzymes for efficient lignin degradation

Of eight white-rot fungi examined, seven fungi grew on nitrogen-limited poplar wood meal medium and degraded 14C-lignin in wood meal to 14CO2. Increased oxygen enhanced both the rate and extent of degradation. However, whereas Pleurotus ostreatus, Pycnoporus cinnabarinus 115 and Pycnoporus cinnabarinus A-360 degraded 12–17% of 14C-(U)-lignin of poplar wood to 14CO2 also in an air atmosphere, Sporotrichum pulverulentum, Phlebia radiata 79 and Phanerochaete sordida 37 degraded only 1–5% under these conditions. Addition of cellulose and glucose to the poplar wood medium stimulated degradation of 14C-(RING)-lignin of poplar wood by Phlebia radiata 79 but repressed degradation by Polyporus versicolor and Pleurotus ostreatus. Cellulose added to the wood meal medium had no effect on the degradation of lignin by Phanerochaete sordida 37 and Sporotrichum pulverulentum but glucose slightly repressed lignin degradation by these fungi. Those white-rot fungi which were considered as preferentially lignin attacking fungi could degrade 14C-(RING)-lignin of poplar wood efficiently under 100% oxygen. They did not require an extra energy source in addition to wood meal polysaccharides for rapid ring cleavage and they degraded up to 50–60% of the 14C-lignin to 14CO2 in 6–7 weeks at a maximum rate of 3–4% per day.

Biodegradation of Aromatic Toxic Pollutants by White Rot Fungi

Heat treatment at relatively high temperatures (from 150 to 260°C) is an effective method to improve the durability of wood. This study investigates the reasons for the decay resistance of heat‐treated and nontreated wood with respect to the polymeric structural constituents by solid‐state cross‐polarization/magic‐angle spinning (CP–MAS) 13C‐NMR analysis before and after exposure to brown rot and white rot fungi. An industrial two‐stage heat‐treatment method under relatively mild conditions (<200°C) has been used to treat the samples. Brown rot fungi attack polymeric carbohydrates of nontreated Scots pine sapwood at C4, resulting in cleavage and eventually depolymerization of cellulose and hemicelluloses. The attack at the carbohydrate C6, which has never been observed before, is remarkable because the C6 CH2OH group has no covalent structural function but acts in fixing the three‐dimensional carbohydrate configuration just by secondary forces. The CH2OH group carries OH, which forms some of the strongest hydrogen bonds in the structure of the crystalline native cellulose. It is suggested that the fungus tries to cleave this group to open the cellulose crystalline structure into an amorphous structure to decrease its water repellency to facilitate enzymatic cellulose degradation. Considerable degradation of the hemicelluloses occurs during brown rot fungal exposure, whereas in general the attack on lignin is rather limited, being mainly demethoxylation. However, Gloeophyllum trabeum is an active brown rot fungus in the (partial) degradation of lignin because there is some indication of ring opening of the aromatic ring of lignin during fungal exposure. Aromatic ring opening has also been observed after exposure to Coriolus versicolor, a white rot fungus. The demethoxylation of lignin and some attack on wood carbohydrates are also characteristic of the attack of this white rot fungus. The CP–MAS 13C‐NMR spectra of heat‐treated Norway spruce reveal similarities but also clear differences after fungal exposure in comparison with nontreated Scots pine sapwood. Brown rot fungi seem to have a preference to attack the carbohydrates of heat‐treated wood at C4 and especially C1, cleaving the skeleton of cellulose and glucomannans. In untreated Scots pine sapwood, this attack mainly occurs at C4, the nonreducing end of the glucose unit. An attack on the out‐of‐the‐ring alcoholic group CH2OH of the carbohydrates of heat‐treated Norway spruce is less obvious than that in untreated Scots pine. The attack on C3/C5 of the carbohydrates is remarkable, indicating ring opening of the glucose units, which has not been observed in nontreated Scots pine sapwood. Lignin degradation is limited to demethoxylation, and low or no aromatic ring opening is observed, even after C. versicolor exposure. © 2006 Wiley Periodicals, Inc. J Appl Polym Sci 101: 2639–2649, 2006