The economic expansion of the three southern border provinces and farmer employment will influence the 20-year national strategy plan and the sufficiency of the economy’s vision of security and development. Promoting animal husbandry can boost local income. Animal breeds and feed quality must improve to boost livestock productivity. The cost of producing animal feed will boost farmer profits. We need to discover recipes that improve animal feed, including using microbes to ferment and increase protein and nutrients. Using microbes, such as Saccharomyces cerevisiae, Rhizopus oryzae, Aspergillus niger, Lactobacillusdelbruckii, and Bacillus spp., may increase protein and nutritional contents, enhancing animal feed use. Thai farmers in the southern border regions can use Trichoderma harzianum and Rhizopus spp. on fibrous feed sources to produce high-quality and value-added livestock products. This is essential for local economic growth.

(Ullah et al., 2023), (Dabul et al., 2025), and (Evangelista et al., 2025) found that yeast-fermented cassava chips (S. cerevisiae) increased protein content by 47.5%, improving animal feed quality and protein nutritional value. The fermentation of cassava peels with S. cerevisiae over three days raised protein content from 4.4% to 10.9% and lowered cyanide toxin levels from 21.3 to 9.5 mg/kg, which is significant given that 50–100 mg can kill or sicken animals. As a high-quality protein source rich in amino acids like lysine and histidine, yeast has 40%–60% protein in its cells. Animals get vitamins, minerals, and proteins via yeast cell decomposition. Mixtures of microorganisms improve animal feed protein (Rodríguez Madrera et al., 2015); (Murata et al., 2021); the protein content of soybean meal increased from 48.6% to 70.6% after three days of co-fermentation with yeast and lactic acid bacteria. Additive combinations affect the quality of rice straw silage; 5 treatment approaches were evaluated across 4 silo density levels (200, 300, 400, and 500 kg/m³). The treatments included lactic acid bacteria (LAB) inoculants, molasses (M), cellulosic enzymes (E), and two combination treatments (M+LAB and E+LAB). The research revealed that single additives (molasses or crude enzymes) increased dry matter losses at lower densities, presenting risks for silage quality. However, the LAB related to treatments, particularly combination approaches, demonstrated superior performance. At a silo density of 300 kg/m³, acetic acid produced by LAB treatments effectively inhibited harmful yeast and mold populations (Du et al., 2025). The fermentation of palm oil cakes investigated using a mixed culture of S. cerevisiae and Bacillus subtilis (50:50 ratio) over periods of 0, 15, 30, and 45 days was found that the fermentation of palm kernel meal with yeast at a fermentation period of 45 days increased the protein content from 7.75% to 12.36%, representing an increase of 22.92% compared to the group not prepared with S. cerevisiae and B. subtilis(Nopparatmaitree et al., 2014). This treatment was particularly intended to improve the nutritive value of palm oil by-products as feed for ruminant animals such as cattle, goats, and sheep (Tefera et al., 2014). (Gunun et al., 2023) investigated the nutritional value of cassava peel by fermentation with yeast (S. cerevisiae) or effective microorganisms (EM) for use as a concentrate replacement in goat diets. After 14 days, crude protein increased from 2.1% to 13.8%. Feeding trials showed no significant differences in intake, digestibility, rumen fermentation, or growth performance among treatments. However, replacing 50% of the concentrate with yeast-fermented cassava peel reduced feed cost per gain by up to 32%. Fermenting cassava with yeast (Y), effective microorganisms (EM), and EMY increases its protein content to 42.1%, 44.2%, and 45.3% of dry matter (Polyorach et al., 2017). Microorganisms use carbohydrates as a carbon source for development during rumen fermentation, boosting protein content in animal feed. Ruminants consume fibrous feeds, mostly cellulose, hemicellulose, and lignin. Animal feed has strong cell walls that rumen bacteria cannot penetrate. A typical method is to use microbes to change animal feed fibrous materials to boost cellulolytic bacteria. These microorganisms include fungi and bacteria (Polyorach et al., 2013); (Tefera et al., 2014). The yeast medium solution (YMS) applied for twenty-one days on fermenting rice straw of various lengths lowered NDF and ADF concentrations (Djunu, 2022).

The findings suggest that supplementing animal feed with yeast and beneficial bacteria can enhance the protein content of cassava, thereby improving its value as cattle feed. Fermentation of boiled cassava root with teff flour provides a suitable substrate for microbial activity, while the microorganisms involved in cassava fermentation contribute to improved nutritional quality and facilitate biochemical synthesis (Halake & Chinthapalli, 2020). Although several studies have examined microbial fermentation for feed improvement, limited research has focused on exploring indigenous fungal strains from unique ecosystems, such as the Hala-bala forest, which may harbor novel cellulolytic and xylanolytic species with superior enzymatic efficiency. The objective of this study was to isolate and screen fungi from soil and decayed wood samples collected from Hala-bala forest for their cellulase and xylanase production capabilities. The primary objective was to identify fungal strains with superior enzymatic efficiency for rice straw decomposition. Subsequently, the selected high-performance fungal isolates were evaluated for their potential in rice straw fermentation as a strategy for enhancing feed quality for ruminant nutrition.

Fungal isolation from soil and rotten wood in Hala-Bala forest, Yala Province, obtained 15 isolates as single colonies, as shown in Figure 1. In isolate 1 (A), the fungus grows in all directions from a spot in the petri dish. Fungal hyphae are dense in the core and thin out toward the edges. Some dark green hyphae include white rings, possibly suggesting spore production or growth phases. The fine hyphal surface spreads uniformly around the dish. The thick central hyphae have black patches, and the growth boundary is smooth and symmetrical. This fungus may be Trichoderma. Isolate 2 (B), the fungus fills virtually the entire petri dish, spreading outward from a center point. The white spot in the middle indicates beginning growth. Dark brown to black hyphae may vary due to environmental changes. A fine hyphal surface has high density in the center and decreasing density at the edges. Smooth growth boundaries with clear growth rings indicate phases. Due to its extensive growth, black hyphae, and copious spore generation, this fungus is likely Aspergillus. Isolate 3 (C), multiple hyphal masses arise as the fungus clusters over the petri dish. Brown to dark brown hyphae indicate spore or fungal development and white rings indicate early growth. Dense hyphal masses have fine and dense surfaces with varied heights. The growth limit is patchy. Due to their clustered growth and abundant hyphal production, Rhizopus spp., Mucor, or Chaetomium are likely candidates.

Isolate 4 (D), the fungus spreads in places and forms several clusters in the petri dish. These clusters contain dense, green to dark green hyphae from various fungus species. Green indicates metabolite or photosynthetic synthesis. Fine, dense hyphal surfaces are common in clusters. Smooth but spotty development borders form clusters. Clustered growth, green hyphae, and profuse spore generation suggest Penicillium spp. Isolate 5 (E), the fungus spreads across the petri dish in rings or layers, signifying different growth phases. Many fungi have dark brown or black hyphae that transition from the center to the edge. The hyphal surface is fine and dense, with peak density at the center and decreasing outward. Smooth growth boundaries with clear growth rings indicate phases. This fungus’s extensive growth, black hyphae, and copious spore generation suggest Cladosporium spp. Isolate 6 (F), the fungus creates a thick mass in the petri dish center with extensive hyphal dissemination to the periphery. The hyphae are yellow to light brown, shifting color from center to edge, suggesting spore or metabolite accumulation. Hyphae have fine, dense surfaces, especially in the core, which generate dense masses. The growth border is smooth, and the hyphae spread widely. This fungus is reckoned to be Aspergillus or Penicillium based on its thick, clustered growth and similar hue.

Isolate 7 (G), the fungus spreads across the petri plate in clusters. The light brown to dark brown hyphae indicate spore or metabolite buildup, with color variation related to growth phases. In clusters, the hyphal surface is fine and thick, with various heights. The smooth growth boundary spans the dish. This fungus’s clustered growth and abundant hyphal production suggest Rhizopus spp. or Mucor. Isolate 8 (H), This isolate exhibited concentric layers across the Petri plate, with distinct color variations ranging from golden yellow to dark brown. Such pigmentation likely reflects the accumulation of spores or secondary metabolites within the hyphae. The central zone presented a dense and fine hyphal texture, while the colony margin remained smooth, with uniform hyphal distribution. The colony’s broad spread and characteristic yellow-to-brown hues suggest affinity to Aspergillus flavus or Penicillium spp. Isolate 9 (I), growth initiated from the colony center and expanded radially. The mycelium displayed a darkened central area with paler margins, giving the colony a gradient of dark gray to black. The central hyphal surface appeared smooth, and the growth boundary was gentle and uniform. Based on its extensive growth and dark pigmentation, the isolate is likely a member of the genus Aspergillus.

Isolate 10 (J), this isolate colonized the Petri plate from the center outward, with hyphae exhibiting dark gray to black pigmentation. A pronounced dark center contrasted with lighter margins. The colony margin was smooth and extended across the plate. The morphological traits, including filamentous growth and consistent black pigmentation, suggest possible identification as Aspergillus niger or Cladosporium spp. Isolate 11 (K), the Petri dish was covered in stratified layers of fungal growth. Hyphae ranged from white to light gray, with scattered darker or brown patches indicative of sporulation or metabolite accumulation. Hyphal density was especially evident in the darker regions. The colony margin was smooth, uniformly extending across the plate. The combination of abundant growth and white-to-light-gray pigmentation suggests probable classification as Trichoderma. Isolate 12 (L), colonies displayed irregular growth patterns with grouped hyphae extending unevenly across the Petri dish. Hyphae were predominantly whitish to light gray, with darker patches indicating areas of spore or metabolite buildup. Compact and fine hyphal surfaces were observed in zones of denser growth. Margins were typically smooth and extended to the dish edge, although uneven distribution was evident. The morphological profile is consistent with Rhizopus or Mucor spp.

Isolate 13 (M) uniformly colonized the plate, with dense, smooth, and fine hyphae distributed evenly throughout. The colony appeared completely white, lacking observable pigmentation. The growth margins were smooth and continuous. Based on its dense, pure white hyphae with uniform coverage, this isolate is likely attributable to Penicillium or Geotrichum, both known for producing compact, unpigmented colonies. Isolate 14 (N), growth was radial, covering the plate from the center outward. White speckling was observed across the colony, indicative of spore formation. Hyphae exhibited green to dark green pigmentation, while the central region contained densely packed, fine hyphae. The smooth margin extended to the edge of the dish. The characteristic green pigmentation and morphology are strongly indicative of Penicillium spp. Isolate 15 (O), colonies formed multiple concentric clusters, producing layered or ring-like patterns across the plate. Hyphae displayed a striking emerald to dark green coloration, interspersed with white dots associated with spore or metabolite accumulation. The hyphal surface appeared compact and finely textured, especially along the concentric rings. Margins were smooth and extended across the entire Petri dish. Given the broad colony spread and distinctive green pigmentation, the isolate is most consistent with Penicillium or Aspergillus spp.

The diversity of fungi with varying capacities for cellulase and xylanase enzyme production aligns with the ecological characteristics of the Hala-Bala forest, where high moisture levels and the continuous accumulation of organic matter promote the development of a rich microbial community (Singh et al., 2018). The identification of well-recognized fungal genera – namely Trichoderma, Aspergillus, Penicillium, Fusarium, Chaetomium, and Geotrichum – was achieved through both morphological examination and molecular analysis of the 15 isolates. Their frequent association with lignocellulose decomposition highlights their ecological role in degrading plant residues in forest ecosystems (Qu et al., 2025). T. reesei and T. viride were widely reported for high cellulase production in both natural habitats and industrial contexts (Cano et al., 2021). High enzymatic activity, especially from T. reesei and A. niger, was consistent with earlier findings (Zhong et al., 2021), with the efficiency of T. reesei linked to its well-studied cellulase gene clusters. Conversely, F. oxysporum displayed moderate activity, which may result from substrate specificity or enzyme repression. These results underscore the potential of native fungal strains for improving biomass utilization as ruminant feed.

Enzyme activity assays revealed significant differences (p<0.05) in cellulase and xylanase production among the isolates. T. reesei exhibited the highest activity for both enzymes, with clear zone diameters of 5.2 cm for cellulase and 6.8 cm for xylanase, demonstrating its superior lignocellulose-degrading potential. These observations reinforce prior reports that designate T. reesei as a benchmark species for industrial enzyme production due to its efficient regulation of gene expression and high secretion capacity (Shrestha et al., 2022). Other noteworthy producers, including A. niger, A. flavus, and F. oxysporum, are valued for their diverse metabolic repertoire, while T. viride emerged as a promising candidate for hemicellulose degradation, with notable xylanase activity (6.5 cm). By contrast, Cladosporium cladosporioides and C. sphaerospermum displayed relatively weak activity, possibly constrained by genetic factors or unsuitable culture conditions. Despite their natural abundance, these species may not strongly express lignocellulolytic enzymes, reaffirming the importance of strain-specific selection for biotechnology. The co-production of cellulase and xylanase by Trichoderma and Aspergillus illustrates their robust enzymatic systems capable of hydrolyzing cellulose and hemicellulose simultaneously. Meanwhile, moderate activity in Penicillium expansum and Geotrichum candidum suggests opportunities for optimization through strain improvement or fermentation control. These results carry industrial implications, as species such as T. reesei, A. niger, and P. oxalicum hold promise for waste management, composting, feed additive production, and biofuel generation (Wu et al., 2022). Moreover, the study highlights the significance of targeted screening of strains with desirable enzymatic traits and emphasizes the unexplored potential of ecosystems like the Hala-Bala forest for uncovering novel resources.

The potential of selected fungi was further validated by the fermentation trials with rice straw. Rice straw treated with Aspergillus niger, Aspergillus flavus, and Trichoderma viride exhibited favorable color, aroma, and texture, indicating high-quality fermentation suitable for animal feed, consistent with earlier descriptions (McDonald et al., 1991). Recent research by (Chen et al., 2019) similarly demonstrated that T. viride improved the nutritive value, and lignin and cellulose degradation ratios were 26.38% and 33.29%. In addition, A. niger exhibited higher enzyme activity on pretreated biomass when moistened with modified basal salt media (BSM) (Sharma et al., 2012) By contrast, rice straw fermented with T. reesei exhibited inferior physical qualities, likely due to over-degradation that damaged its structural properties (Wanapat et al., 2009). These outcomes stress the importance of carefully selecting fungal species to achieve the best compromise between nutritional enrichment and structural preservation in rice straw treatment. Concerning silage quality, previous guidelines recommend an optimal pH between 3.8 and 4.5 for long-term preservation (McDonald et al., 1991). In this study, however, all treatments exceeded this threshold, potentially compromising stability. The closest values to the desired range were recorded for the control (4.50) and C. globosum-treated silage (4.53). Achieving sufficiently low pH is critical to inhibit undesirable bacterial growth (Cheli et al., 2013). Interestingly, the untreated rice straw contained the highest lactic and butyric acid levels, which are key in reducing pH and suppressing spoilage organisms, thereby safeguarding silage quality (Weinberg & Muck, 1996). However, the control group’s elevated butyric acid level could suggest Clostridium bacteria contamination or inadequate fermentation (McDonald et al., 1991)

The rice straw fermented with C. globosum had the second greatest lactic acid content (1.67) and the highest acetic acid level (0.095), which has the quality of suppressing the growth of yeast and fungus. Exposure to air helps the silage be more stable (Danner et al., 2003). Rice straw fermented with A. flavus had the least butyric acid concentration (0.18), which is beneficial as butyric acid is usually linked with off-odor and silage degradation (McDonald et al., 1991) On the other hand, one should consider A. flavus’s possibility of producing aflatoxins, which could endanger animals (Pitt & Hocking, 2009).

This work supports (Ike & Tokuyasu, 2018), who indicated that T. reesei, the filamentous fungus, an anamorph of Hypocrea jecorina, is a well-known producer of hydrolytic enzymes for lignocelluloses, i.e., cellulases and hemicellulases. The work by (Singh et al., 2018) also shows T. reesei is quite good at generating xylanase and cellulase enzymes. Current studies support the findings of (Goda et al., 2024), who indicated that A. niger and A. flavus also have notable capacity for generating cellulase-degrading enzymes, hence corroborating the findings that these fungi display high enzyme production rates. High-efficiency fungi for several industrial uses, such as biofuel generation, environmental organic matter decomposition, and application in animal feed industries, will be chosen and developed using this study as a foundation. Examining the enzyme production potential of various fungi can help academics and business professionals select appropriate fungi and improve enzyme production efficiency for industrial use.

The rise in crude protein content in rice straw fermented with T. viride and T. reesei (7.70% and 7.63%, respectively) over the control (7.04%) could be caused by the fungus generating single-cell proteins throughout their development (Sharma & Arora, 2010). Trichoderma fungi can also create effective lignocellulose-degrading enzymes, which could free proteins attached to the lignocellulosic structure (Jafari et al., 2007). Consistent with (Cano et al., 2021), the increase in crude protein in rice straw fermented with Trichoderma spp. was brought on by the formation of single-cell protein during the fungal growth, whose study suggested that solid fermentation with T. fungus could improve the nutritional profile of agricultural waste by successfully increasing the protein content. Less lignin was visible in rice straw fermented with F. oxysporum, suggesting it can produce efficient lignin-degrading enzymes.

The fermentation with F. oxysporum produced the highest levels of NDF and ADF (71.33% and 39.52%, respectively) in rice straw, even though it produced the lowest lignin content (ADL) (6.08%), indicating that this fungus can degrade lignin. The results are consistent with the study that was carried out by (Gupta & Chundawat, 2020), who identified genes associated with lignin degradation through genomic analysis of F. oxysporum. Lignin is a tough component to decompose, and it impairs fiber digestion by ruminants. Therefore, its reduction is noteworthy (Zhong et al., 2021). T. viride and A. niger produced the least NDF and hemicellulose levels in rice straw; in the meantime, supporting the findings of (Rahman et al., 2022), who discovered that this group of fungi may generate very effective cellulase and xylanase enzymes. It lowers the cell wall makeup of rice straw. Xing et al. (2020) also found that these fungi may generate potent hemicellulose-degrading enzymes capable of efficiently destroying the fibrous structure of rice straw. Especially in fermentations with F. oxysporum, T. viride, and T. reesei, which had the lowest NFE content, the fungi may be using soluble carbohydrates as an energy source for growth, causing a decline in NFE content in rice straw fermented with fungi compared to the control group (Kogo et al., 2017). (Sukkaew, 2021) found that before breaking more complicated polysaccharides, bacteria use easily degradable carbon sources, such as sugars and starch, which are the components of NFE, during the solid phase fermentation process. The rise in lipid content in rice straw fermented with A. niger (1.83%) and C. globosum (1.63%) relative to the control group (1.41%) could be caused by either the buildup of lipids in fungal cells or the generation of fatty acids during the fermentation process. This agrees with (Andlar et al., 2018), who discovered that various fungal strains can generate and accumulate intracellular lipids during the breakdown of lignocellulosic materials. According to (Patel et al., 2020), the rise in fermented materials’ lipids could be caused by the generation of fungal lipase enzymes, which alter the lipid profile throughout the fermentation process. Despite extensive research on the application of fungal enzymes for lignocellulosic biomass degradation, few studies have systematically screened native fungal strains from unique ecological niches such as the Hala-Bala Forest, a biodiverse tropical rainforest in southern Thailand. Most previous works have focused on commercial or model strains under laboratory conditions, which may not reflect the adaptive capabilities of indigenous fungi in real-world fermentation systems. This study introduces a novel approach by isolating cellulase- and xylanase-producing fungi from undisturbed forest environments and evaluating their direct application in rice straw fermentation for ruminant feed. Unlike prior studies, this work integrates enzymatic activity screening, fermentation performance, and nutritional improvement analysis, providing a holistic assessment of fungal potential. Therefore, the novelty of this research lies in the use of native fungal biodiversity for agricultural bioconversion, which contributes to both sustainable livestock production and circular bioeconomy development.

This study successfully isolated and characterized 15 fungal species from soil and decaying wood samples in Thailand’s Hala-Bala forest, with Trichoderma reesei demonstrating the highest cellulase and xylanase production activities (5.2 and 6.8 cm clear zone diameters, respectively), followed by Aspergillus flavus (4.0 and 5.8 cm, respectively). Rice straw fermentation trials revealed that A. niger, A. flavus, and T. viride produced superior fermented substrates with optimal organoleptic properties, favorable pH levels, and enhanced chemical composition suitable for ruminant nutrition. These indigenous tropical fungi demonstrate significant potential for improving livestock feed formulation through sustainable agricultural waste valorization, effectively converting rice straw into high-quality animal feed with improved nutritional value.

We certify that there is no conflict of interest with any financial, personal, or other relationships with other people or organizations related to the material discussed in the manuscript.

The authors acknowledge the financial support provided by Yala Rajabhat University. They also express their gratitude to the Southern Border Researchers Association for supporting the research facility and to the staff of the Faculty of Science, Technology, and Agriculture, Yala Rajabhat University, for their continuous encouragement, support, and assistance.

During the preparation of this article, the authors used ChatGPT to improve language and readability, and subsequently reviewed and edited the content to ensure accuracy and academic standards.