In vitro evaluation of three white rot fungi for their lignolytic potential and biopulping action on Eucalyptus and Bamboo

doi:10.21203/rs.3.rs-4374335/v1

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In vitro evaluation of three white rot fungi for their lignolytic potential and biopulping action on Eucalyptus and Bamboo

https://doi.org/10.21203/rs.3.rs-4374335/v1

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The study aimed to assess the lignolytic enzyme production in three white rot fungi (WRF) upon stimulation with metal ion (FeSO₄.7H₂O) and thereby develop an optimal fungal consortium for biopulping applications. Invitro wood decay experiments conducted with Eucalyptus and Bamboo demonstrated significant weight loss following inoculation with Trametes versicolor (TV), Ganoderma lucidum (GL), and Schizophyllum commune (SC). Over time (45–90 days), the weight loss gradually increased in all inoculations, ranging from 6.5–16.9% for Eucalyptus and 7.9–17.7% for Bamboo. Qualitative plate assays revealed TV’s positive ligninolytic potential in all tests, SC in three, and GL in only two tests. Secondary screening confirmed laccase, manganese peroxidase, and lignin peroxidase activities for all fungi, with SC consistently exhibiting higher activity. Fourier Transform Infrared Spectroscopy analysis of Eucalyptus sawdust treated with individual fungi and the consortium (TV, GL, and SC) showed significant lignin degradation compared to the control. While individual strains displayed a decrease in lignin-associated bands, the consortium exhibited a more pronounced decrease (98.55% and 84.21% at I₁₅₁₁/I₁₇₃₉ and I₁₂₄₈/I₁₇₃₉), indicating synergistic effects. Conversely, cellulose and hemicellulose associated band intensities increased, suggesting preferential lignin degradation by the fungal consortium. Overall, the study highlights the potential of all three fungal isolates to produce lignolytic enzymes with industrial applications.

Trametes versicolor

Ganoderma lucidum

Schizophyllum commune

delignification

fungal consortium

Biopulping

FTIR

Lignocellulose constitutes the most prevalent biomass on earth and is found abundantly in wood and various agricultural residues [1]. The biological breakdown of lignocellulose has emerged as a crucial area of research, particularly concerning future bio-economics reliant on biomass as a raw material for various bioproducts and bioenergy applications[2].The fundamental constituents of lignocellulose are cellulose, hemicellulose, and lignin, although the primary composition varies based on the plant species and origin. Generally, hardwood-derived lignocellulosic materials comprise about 40–55% cellulose, 24–40% hemicellulose, and 18–25% lignin, whereas softwood-derived materials typically contain approximately 45–50% cellulose, 25–35% hemicellulose, and 25–35% lignin[3].

Cellulose, tightly bound to lignin in plants, poses challenges in separating lignin from the lignocellulosic structure, necessitating a delignification process to isolate high-purity cellulose. Current pretreatment methods encompass physical, chemical, physicochemical, and biological approaches. Presently, physicochemical methods such as steam-explosion, ammonia fiber explosion, and wet oxidation dominate, albeit often demanding high energy and capital investments. These methods may lead to sugar degradation and the formation of inhibitor compounds, hampering subsequent processes. Moreover, the chemical usage adds to expenses and environmental concerns due to toxic waste generation[4]. Hence, the biological pretreatment, known as biodelignification, emerges as a solution, employing suitable microbes which express appropriate enzymes. Various microorganisms, notably fungi and bacteria, possess the ability to degrade lignin, by producing enzymes for polymer breakdown within lignocellulosic substrates. Among these, White Rot Fungi (WRF) are renowned for their effectiveness in lignin decomposition[5].

WRF exhibit efficient lignin degradation through the production of various extracellular ligninolytic enzymes like laccase (Lac), manganese peroxidase (MnP), and lignin peroxidase (LiP). The extent of delignification by these fungi varies, and is determined by the fungal species and the carbohydrate composition in the lignocellulosic substrate. Certain WRFs demonstrate the capability to generate all lignolytic enzymes, while others display selectivity[6]. There is paucity of scientific literature on the utilization of pretreatment processes utilizing ligninolytic enzymes for biomass degradation and deconstruction. Further, some species exhibit synergistic interactions through multiple enzyme activities, thereby significantly enhancing lignocellulose material degradation. To this end, it was hypothesized that co-cultures of suitable microorganisms may lead to higher delignification, ultimately improving the invitro fermentation index compared to monoculture[7].There is a growing interest in utilizing microbial consortia instead of single-strain systems in industrial and environmental biotechnology applications due to their potential to leverage diverse metabolic capabilities and synergies[8, 9]. Consequently, naturally occurring or engineered microbial consortia containing WRF may prove advantageous in bioprocessing.

Comprehending the mechanisms behind the activities of wood-degrading fungi is crucial, not only for advancing our knowledge of fungal ecology and physiology, but also for unleashing their potential in various applications. Consequently, integrating environmentally friendly biotechnological approaches utilizing wood-degrading fungi for lignocellulose breakdown holds promise for driving substantial progress. The current study was conducted with the objective of developing a consortium of lignolytic enzymes sourced from wood decay white-rot fungi, tailored specifically to efficiently break down lignin in biopulping applications.

Collection and Isolation of Fungi

Three WRF were collected from the natural forests and growing on wood and identified based on the morphology of fruiting bodies. The three fungi were identified as Trametes versicolor (TV), Ganoderma lucidum (GL), and Schizophyllum commune (SC). The collected fruiting bodies were brought into the Plant Pathology laboratory, FCRI, and isolated on to Potato Dextrose Agar (PDA) by following standard protocol[10]. Pure cultures were then derived by sub culturing and stored at 4°C. Subsequent experiments were conducted using either the individual cultures of these three fungal species or all the three species together as a consortium.

In vitro wood decay test

Apparently healthy wood disks ofEucalyptus tereticornis Sm. and Dendrocalamusstrictus (Roxb.) Nees were cut into 2 x 0.5 cm blocks, for in vitro decay tests to study the degradation capacity of the fungal species. Each block was marked, weighed, soaked overnight for optimal moisture, autoclaved, and surface-sterilized. These blocks were then placed in Petri plates containing PDA and inoculated with one of three fungal species. The wood blocks were incubated for 45 to90 days at 25 ± 1°C, with four replications for each period. The study designconsidered each fungus as a distinct group, resulting in four treatmentsincludingcontrol. After incubation, blocks were cleaned, oven-dried for 48 hours, and reweighed to calculate the percentage weight loss using the equation[11].

% weight loss = \(\frac{(\text{O}\text{r}\text{i}\text{g}\text{i}\text{n}\text{a}\text{l} \text{d}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t} - \text{F}\text{i}\text{n}\text{a}\text{l} \text{d}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t})}{\left(\text{O}\text{r}\text{i}\text{g}\text{i}\text{n}\text{a}\text{l} \text{d}\text{r}\text{y} \text{w}\text{e}\text{i}\text{g}\text{h}\text{t}\right) }*100\)

Primary Screening

The fungal isolates wereassessed for their lignolytic capabilities employing the qualitative plate assays viz a viz., Methyl Orange Dye Decolorization Plate Assay, Guaiacol Plate Assay, Azure B Dye Decolorization Plate Assay and Tannic Acid Plate Assay. In these assays, various indicators such as methyl orange, azure B, guaiacol, and tannic acid were utilized as substrates to detect the lignolytic potential of the isolates, as detailed below.

For each assay, the cultures were inoculated onto Petri plates containing PDA and either of the indicators such as 0.5% methyl orange (MO), 0.01% guaiacol (GL), 0.01% azure B (AB), and 0.2% tannic acid (TA). These plates were incubated at 25^oC and monitored for a period of two weeks. Positive reaction is indicated by the formation of a clear zone, brown zone, clear zone and yellow to light brown zone around the colony for each of the methyl orange (MO), 0.01% guaiacol (GL), 0.01% azure B (AB), and 0.2% tannic acid (TA) indicators, respectively. Positive results indicate the production of lignin degrading enzymes which decolorize polymeric dyes (MO and AB) and oxidize synthetic substrates (GL and TA)[12].

Secondary screening

After primary screening, fungal isolates underwent secondary screening to assess ligninolytic enzyme production capacity. This process involved cultivating the fungi in Potato Dextrose Broth (PDB), allowing the fungi to release the extracellular lignolytic (Lac, MnP, and LiP) enzymes into the culture as they grow.Fungal inoculation was done with a 7-mm disc in conical flasks containing 300 ml PDB, incubated at 25°C. Enzymatic activities in the culture medium were measured using a UV-Vis spectrophotometer (RAY LEIGH UV-2601) at specific wavelengths.

Induction of lignolytic enzyme activity and enzyme assay

Certain heavy metals, including FeSO₄·7H₂O enhance lignolytic enzyme activity in fungi[13, 14]. For evaluation, the cultures were treated with increasing concentrations of FeSO₄·7H₂O for 7 days and the induction of various lignolytic enzymes in response to varying concentrations of heavy metal was studied to choose the optimal concentration.Two fungal plugs were inoculated into conical flasks with 300 ml PDB and were placed on a rotary shaker (120 rpm) for two weeks. In the first week after incubation,crude extract was obtained to assessthe basal activity of ligninolytic enzymes. After the first week, different concentrations of metal ion (FeSO₄·7H₂O), including T1 – Control (0.0 mM), T2 − 0.25 mM, T3–0.50 mM, T4–1.00 mM, and T5–2.00 mM, were added to determine the effect of increasing concentrations of FeSO₄·7H₂O on enzyme activity.Each treatment was replicated thrice. Enzyme activity assays were performed with 20 ml samples extracted from all flasks after second week.Samples were filtered through muslin cloth to eliminate solid residues and impurities. The clarified liquid medium underwent centrifugation at 10,000×g for 30 minutes at 4°C. The resulting supernatant, containing the enzyme extract wasused to measure the activity of Lac, LiP, and MnP.

Lac enzyme activity was evaluated using 2, 2’-azino–bis (3-ethylbenzothiazoline)-6-sulphonic acid as substrate[15].MnP activity was evaluated spectrophotometrically using 3-(dimethylamino) benzoic acidand 3-methyl-2-benzothiazolinone-hydrazone as substrates (Ɛ = 53000 cm^− 1M^− 1) [16].LiPactivity was determined using Azure B dye (Ɛ= 48,800 cm^− 1M^− 1)[17]. Enzyme activity was quantified using the molar extinction coefficients of Lac, MnP, and LiP. One unit (IU)ofenzyme activity represented the amount needed to oxidize 1 µM of substrate per minute and calculated using the following equation[18].

𝐄𝐧𝐳𝐲𝐦𝐞𝐚𝐜𝐭𝐢𝐯𝐢𝐭𝐲 (𝐼𝑈 /𝑚𝑙) = \(\frac{{\Delta }\text{A}\text{*}\text{V}}{{\Delta }\text{E}\text{*} {\epsilon }\text{*}{\Delta }\text{t}}\)* 10⁶ units/ml or µmoles/ml/minute

ΔA: Optical density at λ_max; V: Volume of total assay; ΔE: Volume of Enzyme Used; ε: Extinction Coefficient; Δt: Incubation Time (minutes)

Testing the synergistic effects of the fungal consortium

An attempt was made to study the synergistic effects, if any of the fungal consortium with optimal lignolytic enzyme activity forbiopulping applications. Fungi were inoculated individually and as a consortium into eucalyptus sawdust submerged in PDB. Each flask contained 25g sterilized sawdust in 100ml PDB. After inoculation, flasks were left for 10 days, and then treated with the optimal metal ion concentration, i.e., 0.5mM FeSO₄.7H₂Ofor increasing the lignolytic enzyme activity. After 20 days, the treated sawdust was collected, dried, powdered, and subjected to FTIR spectroscopic analysis at Osmania University-Central Facility for Research and Development to assess the changes in wood composition i.e. lignin degradation. The FTIR spectra in the fingerprint region (1800 to 550 cm^− 1) were examined for characteristic lignin and carbohydrate bands.

Statistical analysis

A completely randomized design was employed for invitro wood decay experiments. These assays with each fungal species were performed in quadruplicate to obtain the mean ± SE. Lignolytic activities among the different fungal species were tested using one-way analysis of variance (ANOVA) at p < 0.05.

In vitro wood decay tests using E. tereticornis

Preliminary screening was conducted using invitro wood decay tests to compare the weight loss of treated (with fungi) and non-treated E. tereticornisand D. strictus wood blocks. After 45 days of observation, each fungal treatment resulted in significant weight loss in the E. tereticornis blocks. The highest weight loss of 9.492% was observed in the blocks treated with G. lucidum, while the blocks treated with T. versicolor and S. commune showed nearly identical percentages of mass losswith 7.412 and 6.535 percent loss (Fig. 1a). All three species demonstrated a noteworthy ability for E. tereticornis decay when compared to the control group.

Following a 90-day treatment, each fungus used in the experiment exhibited similar mean weight loss percentages, with a gradual increase in weight loss from 45 days to 90 days. The percentage of weight loss for blocks treated with T. versicolor,G. lucidum, and S. commune after 90 days is 16.9%, 15.3%, and 15.1%, respectively (Fig. 1b). Statistically, there were no significant differences in weight loss observed among the fungal treatments. Nevertheless, three treatments demonstrated a significant difference from the control group.

In vitro wood decay tests using D. strictus

Following 45 days of fungal treatment, D. strictus wood blocks displayed a significant weight loss, indicating the colonization and decay of the wood by the fungi. The blocks treated with each of the fungi exhibited nearly identical levels of weight loss, categorizing them into the same group (Fig. 1a). Nevertheless, the maximum weight loss was observed in T. versicolor-treated blocks at 8.92%, followed closely by G. lucidum-treated blocks with a weight loss of 8.48%, whereas S. commune treated blocks showed a percent loss of 7.893. These blocks were meticulously observed for unique characteristics that revealed the fungal impact on the wood. Approximately between days 20 and 30, the blocks treated with S. commune displayed the presence of observable color-changing compounds. Furthermore, G. lucidum underwent a change in its growth pattern when approaching the blocks, transitioning from the growth phase to the reproductive phase and producing numerous structures resembling spores in the culture.

Following 90 days of treatment, both G. lucidum and T. versicolor-treated blocks exhibited similar mean mass loss percentages of 17.73% and 16.97% respectively. There was a gradual increase in weight loss from 45 days to 90 days in both the fungi. On the other hand, S. commune-colonized blocks reported the lowest percentage of mass loss at 12.80% (Fig. 1b), indicating that these blocks were less affected by S. commune compared to the other two fungal species.

Our results for E. tereticornis are in line with the previous findings reported by Negrao et al.[19], with a weight loss range of 15–17%after 120 days of biodegradation.Similar mass losses in Eucalyptus wood were reported by Oliveira et al.[20] and Aguiar et al. [21]using Gloeophyllumtrabeum under conditions comparable to our study.Similarly, Sudha et al. [22] reported lower weight losses in Teak blocks treated with TV, GL, and SC (7.50%, 6.60%, and 4.90%, respectively) after 45 days, increasing to 12%, 8.88%, and 6.99% after 60 days.

Primary Screening

Primary screening of the three fungal species in demonstrated significant lignolytic potential by degrading methyl orange by formingclear zone around the fungal disc. This underscores the robust lignolytic activity of all the three fungi (Fig. 2). Sharma et al. [12] reported that in methyl orange assays, clear zones around fungal colonies are indicative of their lignolytic process.Three fungal species displayed clear zone around the disc indicating their ability to break down azure B (Fig. 2). Chaudhary et al. [23]quoted that the azure B undergoes a visible color change upon LiP action, aiding in its identification. Arantes and Milagres [24] studied azure B degradation by MnP and Lac from white rot fungi.Furthermore,in theguaiacol plate assay, among the three fungal species, only TV exhibited a positive result by forming brown coloration around the fungal disc (Fig. 3). However, GL and SC did not respond positively to guaiacol. According to Sahni & Phutela[25], guaiacol is a common substrate for qualitative and quantitative Lac determination, and frequently employed in screening of fungal strains for their ligninolytic activity. However, in case of SC, our results are in contrast with Goud et al. [26], where it was reported that Schizophyllum sp., exhibited the highest activity zone. In case of tannic acid plate assay, TV, and SC both displayed positive results by forming yellow to brown coloration zone around the disc (Fig. 4).TV exhibited notable lignin-degrading activity with tannic acid, evidenced by the distinct color change attributed to lignolytic enzyme production. This assay, a variant of the Bavendamm test (1928) utilizing tannic acid or gallic acid, is widely employed. It involves agar supplemented with tannic acid to observe a brown zone around fungal colonies, indicative of lignolytic activity. The extent of oxidation zone serves as a measure for lignolytic activity quantification, with the appearance of the brown zone reflecting the overall Lac oxidation activity of the fungi[27]. Lignin modification with tannic acid has been observed in various studies of white rot fungi yielding positive outcomes[28].

Secondary screening by enzyme assay

Three fungal species were used to produce the lignolytic enzyme extracts in the PDB liquid cultures. Initially, enzyme activities were assessed without addition of metal ions i.e., control (0.0 mM), showing laccase ranging from 12 to 17 IU/ml, MnP from 15 to 19 IU/ml, and LiP from 8 to 10 IU/ml among three fungal isolates. Subsequent testing aimed to identify optimal concentration of FeSO₄.7H₂O for enhanced lignolytic activity. FeSO₄.7H₂O significantly enhanced Lac, MnP, and LiP activity, two to three times higher than the control. All three fungi exhibited maximum activity at 0.5mM of FeSO₄.7H₂O, confirming it as the optimal concentration for inducing lignolytic enzyme activity. FeSO₄ increased Lac in TV [29], while, Fe²⁺ inhibited enzyme activity in GL[30].

In the case of TV, Lac (32.320 IU/ml), MnP (22.21 IU/ml), and LiP (13.90 IU/ml) enzyme activity was observed (Fig. 5a). On the other hand, for GL, 24.38 IU/ml for Lac, 29.30 IU/ml for MnP, and 14.150 IU/ml for LiP were observed (Fig. 5b). SC inoculated PDB extracts showed maximum lignolytic activity (35.85 IU/ml for Lac, 45.90 IU/ml for MnP, and 14.10 IU/ml for LiP) following the addition of FeSO₄.7H₂O (Fig. 5c). All enzyme activities peaked with SC inoculated PDB extracts, demonstrating its effectiveness against FeSO₄.7H₂O. Irshad and Asgher [31] reported enzyme activities of SC with MnP at 3745 IU/ml, LiP at 2700 IU/ml, and lac at 345 IU/ml after adding metal ion mediators. Yasmeen et al. [32] noted SC with highest production of LiP (915.3 IU/ml), MnP (590.5 IU/ml), and Laccase (85.92 IU/ml). In their study, GL exhibited Laccase (76.77 IU/ml), MnP (458.6 IU/ml), and LiP (418.5 IU/ml). Bhatt [18] reported T. hirsute withMnP at 168.69 IU/ml, Laccase at 221.27 IU/ml, and LiP at 58.44 IU/ml, while TV displayed MnP (171.91 IU/ml), Laccase (56.39 IU/ml), and LiP (203.61 IU/ml). Enzyme activities varied based on substrate used for lignolytic enzyme production.

Testing the synergistic effects of the fungal consortium

To establish a fungal consortium comprising all three fungi employed in the study, eucalyptus sawdust was inoculated both individually and as a consortium. Following the test period, the samples were analyzed using FTIR spectroscopy.

FTIR analysis

The findings from the FTIR study on decayed eucalyptus sawdust samples treated with individual fungi as well as the mixture of fungi (consortium)are presented here. The spectra were compared with those of non-decayed eucalyptus sawdust (control) samples soaked in the medium for the same duration as that of the fungal treatment. This comparison aimed to explore the specific changes in chemical composition and spectral bands that occurred in the eucalyptus saw dust because of fungal biodegradation activity.In FTIR analysis for wood samples, the spectral region between 1800–550 cm⁻¹ is commonly referred to as the "fingerprint region," which is used to study chemical modifications in cellulose, hemicellulose, and lignin. The most representative FTIR bands investigated in the spectral region of 1800–550 cm^− 1 aresummarizedin Table 1[33].FTIR spectra showed significant changes post-fungal decay compared to the control. The 1511 cm⁻¹ peak, linked to lignin, indicated aromatic skeletal vibration (C = C). At 1248 cm⁻¹, stretching in phenol-ether bonds within lignin occurred. Peaks at 1738, 1375, 1158, and 898 cm⁻¹ reflected cellulose and hemicellulose components. Rodrigues et al. [34] proposed a calibration fit, employing the 1511 and 1158 cm⁻¹ peak heights for lignin and carbohydrates, respectively. The average relative intensities of lignin (1511 cm⁻¹) to carbohydrates (1739, 1375, 1158, 898 cm⁻¹) were computed, providing insights into the compositional changes induced by fungal decay (Table 2).

Table 1

Characteristic bands of FTIR spectra in the fingerprint region
Wave number (cm^− 1)	Functional group	Assignment	References
1737	–COOH (C = O)	free carbonyl group, stretching of acetyl or carboxylic acid in hemicelluloses	[35, 36]
1650	C = O	quinines and quinine methides, adsorbed water
1601	C = C	Aromatic ring (lignin)
1511	C = C	Aromatic ring (lignin), stronger guaiacyl element than syringyl
1460	C–H	CH₃ deformation in lignin	[37, 38]
1425	CH₂	Aromatic skeletal vibrations of lignin,and C–Hdeformation in plane (cellulose)
1375	CH	C–H deformation in cellulose and hemicelluloses
1328	O–H	Phenol group (cellulose)
1248	CO	Guaiacyl ring breathing with CO-stretching (lignin and hemicelluloses), esters
1163	C–O–C	Carbohydrate	[39, 40]
1120	C–H	Guaiacyl and syringyl (lignin)
1029	C–O, C–H	Primary alcohol, guaiacyl(lignin)
898	C–H	C–H deformation in cellulose

After exposure to various WRF individually and in combination, absorption bands at 1601 and 1425 cm⁻¹, linked to C = C and CH₂ deformation in lignin, notably decreased, indicating selective degradation or removal of lignin compounds [41]. Conversely, carbonyl bands associated with xylan at 1737 and 896 cm⁻¹ intensified. The 1650 cm⁻¹ bands, indicative of lignin effects, decreased post-fungal exposure, primarily affecting quinines and quinine methides. This selective removal of structural lignin components in eucalyptus sawdust was noted, while carbohydrate bands remained stable, as WRF primarily targeted lignin degradation [42].

Table 2

Average ratios of the intensity of lignin associated band with carbohydrate bands for decayed and non-decayed samples
	I₁₇₃₉/I₁₅₁₁	I₁₃₇₅/I₁₅₁₁	I₁₁₅₈/I₁₅₁₁	I₈₉₈/I₁₅₁₁	I₁₅₁₁/I₁₇₃₉	I₁₂₄₈/I₁₇₃₉
Control	2.881	2.697	2.146	1.847	0.347	0.849
TV	9.948	6.295	2.524	2.124	0.100	0.249
GL	10.159	6.640	2.174	1.929	0.098	0.245
SC	26.021	16.797	3.754	2.275	0.038	0.173
Consortium	181.35	121.61	19.471	17.546	0.005	0.134

ForTV, GL, and SC, the ratios of I₁₅₁₁/I₁₇₃₉ were observed to decrease from 0.347 (Control) to 0.100, 0.098, and 0.038, respectively. Similarly, the ratios of I₁₂₄₈/I₁₇₃₉ showed a decrease from 0.849 (Control) to 0.249, 0.245, and 0.173 for these fungi in the same sequence. This decline in intensity ratios signifies significant lignin degradation in Eucalyptus under decay by WRF. Notably, the ratios exhibited a more pronounced decrease with the consortium, registering values of 0.005 and 0.134 at I₁₅₁₁/I₁₇₃₉ and I₁₂₄₈/I₁₇₃₉. The FTIR study revealed that compared to the control, TV, GL, and SC caused a reduction in intensity ratio at I₁₅₁₁/I₁₇₃₉ by 70.58%, 71.75%, and 89.04%, respectively. When used together as a consortium, this reduction was even more pronounced, reaching 98.55%. Similarly, at I₁₂₄₈/I₁₇₃₉, the reduction percentages were 70.67%, 71.14%, and 79.62% in the same sequence, while with the consortium, it amounted to 84.21%. This implies a synergistic and cumulative effect of all three fungal species, enhancing their degradative efficiency beyond individual strains. The I₁₇₃₉/I₁₅₁₁, I₁₃₇₅/I₁₅₁₁, I₁₁₅₈/I₁₅₁₁, and I₈₉₈/I₁₅₁₁ values exhibited a significant and sharp increase in all Eucalyptus sawdust samples subjected to various fungal treatments compared to the control. This substantial increase strongly indicates the lesser efficacy of WRF in degrading hemicellulose and cellulose compared to lignin. This observation aligns with findings from a prior study on decayed wood by Pandey and Pitman [35], wherein white rot fungi were found to primarily target lignin over the carbohydrate fraction (cellulose and hemicellulose). All observed changes in FTIR spectra collectively suggest that all three fungal isolates exhibited a preference for degrading lignin over hemicellulose and cellulose.

Castoldi et al. [43] showcased the effectiveness of GL, and various Trametes spp. in pretreatedEucalyptus grandis sawdust biologically. Their assessment, conducted using FTIR spectroscopy, revealed decreases in specific bands, indicating lignin degradation, a phenomenon also observed in our experiments. Singhal et al. [44] investigated Cryptococcus albidus for biopulpingof Eucalyptus, confirming lignin reduction post-pretreatment via FTIR spectroscopy, validating fungal pretreatment is effective in biopulping. Xu et al. [36] reported lignin degradation in P. edulis bamboo through reduced lignin to carbohydrate peak intensity ratios, aligning with our research findings. Similarly, Xie et al. [45] utilized a co-fungal approach in poplar sawdust delignification, noting enhanced delignification compared to individual strains, corroborating our co-culturing strategy efficacy. Kelkar and Shukla [41] demonstrated white rot fungal ability to degrade bamboo lignin, confirmed by FTIR spectroscopy peak intensity ratio analysis. Chi et al. [46] explored co-cultivation potential in biopulping, observing increased enzyme activities with consortium, indicating superior biopulping outcomes. This study presents the first evidence of the co-culturing impact of three white rot fungi, enhancing lignolytic activity with metal ions and suggesting potential biopulping applications in Telangana.

The study aimed to isolate and assess fungi with lignolytic abilities to create an optimal consortium for biopulping. Three fungi were identified based on morphology and tested for wood decay, demonstrating significant weight reduction. In Primary screening, TV showed positive results in all tests, while SC and GL performed well in three and two tests, respectively, confirming their lignin degradation capabilities. Secondary screening revealed varying enzyme activities, with SC consistently displaying the highest levels. Metal ion induction, particularly 0.5 mM FeSO₄.7H₂O, enhanced enzyme activity. Biological pretreatment of eucalyptus sawdust with the fungi selectively degraded lignin over hemicellulose and cellulose, supported by FTIR spectroscopy. Consortium treatment showed pronounced lignin degradation compared to individual cultures, suggesting potential for industrial applications. However, challenges like optimizing enzyme efficiency, scalability, and economic viability must be addressed for widespread commercial adoption of biopulping. Further research is essential to refine the process for large-scale implementation in the industry.

Author Contributions

The study framework was developed by SC, SB, and JB. SC was responsible for designing and conducting the experiments, as well as analyzing the samples. KS provided the necessary wood materials for the experiments. MKD aided in conducting the experiments and analyzing samples in the laboratory. SC, SB, and JB collectively interpreted the results. SC drafted the initial manuscript, with SB and JB contributing to its final development. All authors reviewed and approved the final manuscript version.

Funding Declaration

No funding

Conflict of interest

The authors do not have any financial or non-financial interests to disclose.

Data availability

The data generated and analyzed during this study are available from the corresponding author upon reasonable request.

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In vitro evaluation of three white rot fungi for their lignolytic potential and biopulping action on Eucalyptus and Bamboo

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Abstract

Figures

INTRODUCTION

MATERIALS AND METHODS

Collection and Isolation of Fungi

Primary Screening

Secondary screening

Induction of lignolytic enzyme activity and enzyme assay

Testing the synergistic effects of the fungal consortium

Statistical analysis

RESULTS AND DISCUSSION

Primary Screening

Secondary screening by enzyme assay

Testing the synergistic effects of the fungal consortium

FTIR analysis

Conclusion

Declarations

References

Additional Declarations

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