Sago palm (Metroxylon sagu) is an important starch-producing crop in Southeast Asia, where its trunk is processed to extract sago flour (Allaily et al., 2024). This extraction process generates significant volumes of agro-industrial residues, primarily sago pith waste (SPW) and bark (Tiro et al., 2018; Susanto et al., 2024). These residues are typically discarded or dumped near water systems, contributing to localized pollution and biomass accumulation (Hung et al., 2018; Saleh et al., 2020). SPW is composed of a starchy-lignocellulosic matrix containing starch, cellulose, hemicellulose and lignin (Amin et al., 2019), making it an abundant but underutilized substrate with potential value in feed and biotechnological applications (Abosiada et al., 2017). However, its direct use in livestock diets remains limited due to low crude protein (CP) and high crude fiber (CF), which reduce digestibility, nutrient uptake and overall feeding value (Wardono et al., 2021). The recalcitrant lignocellulosic structure further impairs palatability and microbial degradation, constraining both ruminant and monogastric utilization (Parmar et al., 2019). Although SPW contains appreciable starch, its nutrient profile is insufficient to support desirable production performance when fed untreated (Lai et al., 2013).

Solid-state fermentation (SSF) has emerged as an effective strategy to enhance the nutritional value of agro-industrial residues. Operating under low-moisture conditions and mimicking natural fungal growth, SSF enables filamentous fungi to secrete cellulases, xylanases and amylases that hydrolyze complex polysaccharides into more digestible components (Thomas et al., 2013; Conceição et al.,  2022; Perwez and Al Asheh, 2025). Beyond improving CP and reducing CF, SSF offers economic and environmental advantages such as minimal energy input, lower water consumption and reduced wastewater generation compared with submerged fermentation (Krishna, 2005; Bhargava et al., 2008; Prabhu et al., 2022). Filamentous fungi, particularly Aspergillus niger and Trichoderma harzianum, are widely used in SSF for their strong enzymatic capabilities and proven efficacy in upgrading low-value biomass (Fiedler et al., 2018). Their action is mediated through the extracellular secretion of hydrolytic enzymes, which break down complex polymers into simpler molecules that can be absorbed and utilized (Wikandari et al., 2022; Zhou et al., 2024). A. niger is known for producing amylases, glucoamylases, pectinases, xylanases and β-glucanases that contribute to carbohydrate depolymerization and improvement of crude protein content through fungal biomass accumulation (Krijgsheld et al., 2012; Fiedler et al., 2018). Its strong fiber-degrading activity and proven efficacy in SSF make it suitable for improving the nutritional profile of agro-waste, with reported increases in crude protein and ash and significant reductions in fiber content (Sadh et al., 2018). Moreover, T. harzianum produces an array of lignocellulolytic enzymes cellulases and hemicellulases that facilitate fiber degradation and nutrient release from complex substrates (Abosiada et al., 2018; Zhang et al., 2023). Prior studies report its effectiveness in improving the nutritional quality of fibrous materials such as rice straw and duckweed through increase in protein and reductions in fiber (Kocher et al., 2007; Setiyatwan et al., 2018). Although co-culturing fungi can sometimes yield synergistic effects, outcomes depend on species compatibility and fermentation conditions, as antagonism or competition may limit performance (Setiyatwan et al., 2019). Recent evidence further supports the efficiency of SSF in upgrading agro-industrial feed resources. For instance, SSF of cottonseed meal using Saccharomyces cerevisiae reduced anti-nutritional gossypol while improving nutrient quality, demonstrating the dual functionality of fermentation in detoxification and enrichment (Dharmakar et al., 2023). Similarly, fungal bioconversion of palm oil sludge using Neurospora sp. significantly enhanced nutritive value and bioactive components (Hasanuddin et al., 2025).

Although fungal SSF has been applied to various agro-industrial residues, SPW remains poorly studied and no work has compared the species-specific and time-dependent effects of A. niger, T. harzianum and their co-culture on this substrate. Aflatoxin behavior during SPW fermentation has likewise not been examined. The present study addresses these gaps by evaluating the three fungal treatments across five incubation periods and assessing changes in proximate composition and aflatoxin levels. Through this approach, we provide essential baseline evidence to guide the safe and effective bioconversion of SPW.

Microorganisms
 
Pure strains of A. niger and T. harzianum were obtained from the culture bank of the Philippine National Collection of Microorganisms (University of the Philippines Los Baños). Cultures were maintained on potato dextrose agar (PDA) at 5^oC and subcultured on fresh PDA plates prior to use. Plates were incubated at 28oC for 5 days to ensure active mycelial growth. All handling was conducted under aseptic conditions.
 
Sample preparation and preservation
 
The experiment was conducted at the Department of Animal Science, College of Agriculture and Agri-Industries, Caraga State University, Butuan City, Philippines, from August 2022 to July 2023. SPW samples were sourced from smallholder sago processors in the Caraga Region, Philippines. Fresh SPW was sun-dried for 3-5 days, followed by oven-drying at 60^oC for 6 h to achieve uniform moisture reduction. The dried material was ground, passed through a 1.0-mm sieve and stored in airtight containers at room temperature until analysis.
 
Preparation of fungal inocula
 
PDA was prepared, sterilized at 121^oC for 15 minutes and supplemented with streptomycin (250 μL per liter) to prevent bacterial contamination. A. niger was transferred onto PDA plates using sterile streaking, while a small block of T. harzianum mycelium was aseptically placed onto fresh plates. All plates were sealed, incubated at 28^oC for 7 days and used as inoculum sources.
 
Solid-state fermentation (SSF) of SPW
 
SSF was conducted following Evans et al., (2013) with minor modifications. Ninety grams of sieved SPW were dispensed into each of forty-eight 500-mL beakers, covered with aluminum foil and sterilized at 121^oC for 15 minutes. After cooling, substrates were aseptically transferred into sterile foil trays and inoculated with fungal suspensions inside a laminar flow cabinet. Three fermentation treatments were established: (1) A. niger monoculture inoculated with 15 mL of a 2% spore suspension, (2) T. harzianum monoculture inoculated with the same volume and concentration and (3) a combined culture composed of A. niger and T. harzianum mixed at a 1:1 inoculum ratio. An uninoculated control was included for baseline comparison. Fermentation was conducted at ambient room temperature (28±2^oC) under laboratory conditions. Triplicate samples were collected at 48, 72, 96, 120 and 144 hours. To halt fungal activity, samples were oven-dried at 60^oC for 72 hours, homogenized, milled and sealed in airtight bags for analysis.
 
Proximate analysis of [un]fermented SPW
 
Crude protein (CP), crude fiber (CF), ash and moisture content were analyzed from triplicate samples at each incubation interval. All analyses were performed at the Regional Feed Chemical Analysis Laboratories (Department of Agriculture, Regions VII and XIII) using standard AOAC (2005) procedures.
 
Aflatoxin determination
 
Total aflatoxins (B1, B2, G1, G2) were quantified using AOAC Official Method 991.31. Samples underwent immunoaffinity column cleanup followed by fluorometric detection and results were compared against the regulatory safety limit of 20 ppb for livestock feed established by the United States Food and Drug Administration (FDA, 2020) and the Codex Alimentarius Commission (2003).
 
Statistical analysis
 
A two-way ANOVA tested the effects of fungal treatment, incubation time and their interaction on CP, moisture and ash. Normality (Shapiro-Wilk) and homogeneity of variance (Levene’s test) were confirmed prior to analysis. Tukey’s HSD was used for post-hoc comparisons. Because CF did not meet parametric assumptions, treatment effects within each time point were analyzed using the Kruskal–Wallis test. The uninoculated control, lacking time-based replication, was excluded from inferential statistical analysis. However, it was retained as a descriptive baseline to facilitate interpretation of treatment-induced changes relative to the original substrate. All analyses used a significance level of P<0.05.

Crude protein enrichment
 
As shown in Table 1, crude protein values under all fungal treatments were numerically higher than the uninoculated SPW baseline (2.56±0.07%). The overall fungal treatment effect was significant (P = 0.0043). Although time alone did not significantly influence CP (P = 0.0887), T. harzianum consistently supported higher CP values at later stages, reaching the maximum at 144 h (2.89±0.23%). This represents a 12.9% increase over baseline and supports previous findings that Trichoderma spp. effectively convert lignocellulosic substrates into protein-enriched biomass through rapid mycelial proliferation and extracellular enzyme secretion. Similar protein enrichment has been reported in fungal fermentation of agro-residues. Trichoderma spp. fermentation of cacao seed waste increased protein and reduced crude fiber, resulting in higher feed intake and weight gain in sheep (Sujono et al., 2020). The CP increase likely reflects fungal biomass accumulation and partial hydrolysis of structural carbohydrates, releasing assimilable carbon and nitrogen for growth, as reported for pangola grass and cassava peel fermentations (Hu et al., 2012; Tonukari et al., 2016). In contrast, the co-culture showed reduced CP at early time points (2.55±0.11% at 48 h), suggesting metabolic interference or resource competition between A. niger and T. harzianum. Antagonistic interactions of this type have been observed in other dual-fungus systems and are often associated with inhibitory metabolites, overlapping enzyme systems, or differential growth rates limiting synergistic expression (Boddy, 2016; Reksiana et al., 2024). Across treatments, T. harzianum demonstrated the most consistent protein-enriching capacity, likely due to its strong cellulolytic activity, enabling rapid access to fermentable carbohydrates and sustained biomass accumulation.



Crude fiber degradation
 
Crude fiber (CF) decreased under all treatments, but significant differences emerged only at 144 h (P = 0.0273) (Fig 1). At this time point, A. niger achieved the greatest reduction (8.07±0.43%), followed by the co-culture, whereas T. harzianum retained substantially higher fiber (15.02±0.62%). The greater CF reduction by A. niger reflects its known secretion of high-activity cellulases, xylanases and β-glucosidases capable of depolymerizing both amorphous and crystalline regions of lignocellulose (Park et al., 2017). Similar patterns were reported during the fermentation of cassava peel, pineapple waste and other agro-residues, where A. niger showed strong fiber solubilization and carbohydrate depolymerization due to its high cellulase and hemicellulase activity (Evans et al., 2013; Tonukari et al., 2016; Laothanachareon et al., 2022). By contrast, the weaker CF reduction under T. harzianum may reflect substrate-specific variability in enzyme induction, as SPW contains a heterogeneous fiber matrix dominated by lignified tissues (Lima et al., 2024). The intermediate performance of the co-culture again suggests interference rather than synergy. Similar inhibitory or antagonistic interactions have been reported in other co-inoculated studies, where overlapping metabolic demands constrain cellulolytic activity (Lani et al., 2021). From these observations, it can be inferred that A. niger is the more efficient degrader of SPW lignocellulose, especially during extended SSF.


 
Moisture dynamics
 
Moisture content (MC) was influenced primarily by fermentation time (P = 0.0002), following a typical SSF pattern of early moisture elevation due to metabolic water release, followed by gradual decline from evaporative losses and substrate heating. Differences among treatments were not significant (P = 0.1469), although early variations were observed at 48 h, when A. niger and T. harzianum exhibited higher MC (14.75±3.73% and 15.28±1.70%) than the co-culture (10.93±1.36%). The lower MC in the co-culture may indicate accelerated substrate respiration or higher localized heat generation due to fungal competition (Raimbault, 1998). After 72 h, moisture content stabilized across treatments as the substrate reached physicochemical equilibrium, a trend consistent with earlier observations in SSF of soybean hulls (Egwumah et al., 2023) and sago hampas (Lani et al., 2021). For field-scale application, the moisture fluctuations observed reflect typical SSF physicochemical dynamics rather than treatment-specific effects.
 
Ash content variability
 
 Ash content differed among fungal treatments (P = 0.0085) but did not vary significantly across incubation time (P = 0.0817). Values remained within a narrow range (7.56-8.55%), with monocultures generally exhibiting higher ash than the co-culture. Increased ash during SSF typically results from microbial degradation of organic matter, which concentrates the mineral fraction rather than increasing total mineral content. This pattern is consistent with previous observations in soybean hull fermentation with S. cerevisiae (Egwumah et al., 2023) and mixed-culture sago waste fermentation (Lani et al., 2021). Similarly, Wardono et al., (2022) emphasized that ash elevation during the fermentation of sago hampas reflects relative mineral concentration as carbohydrates are metabolized. While moderate ash increases may indicate effective organic matter breakdown, excessively high ash can impair palatability and nutrient utilization, as noted by Korombé et al.  (2024). In the present study, ash values remained within acceptable limits, suggesting improved substrate degradation without compromising feed quality.
 
Co-culture interactions
 
The combined culture of A. niger and T. harzianum displayed inconsistent effects across proximate components, particularly during early fermentation. Suppressed CP at 48-72 h and limited CF reduction at 144 h indicate antagonistic interactions rather than synergistic activity. Reksiana et al., (2024) reported that these effects could arise from competition for oxygen, carbon sources, or micronutrients, differences in growth kinetics, or the production of antagonistic secondary metabolites. Comparable inhibitory responses have been documented in A. niger-Trichoderma co-cultures on Indigofera and other agro-residues where overlapping enzyme pathways reduced overall efficiency (Puspitasari et al., 2019; Prabhu et al., 2022). Late-stage partial recovery (120-144 h) suggests metabolic adjustment over time, but the co-culture remained less consistent than monocultures. These results demonstrate that strain compatibility is critical when designing mixed-fungal SSF systems for SPW.
 
Aflatoxin dynamics in fermented SPW
 
Total aflatoxins remained well below the 20 ppb regulatory limit across all treatments and incubation periods, confirming the safety of SSF under the conditions applied (Fig 2). The lowest concentration occurred in A. niger at 72 h (0.70 ± 0.00 ppb), while the highest values were observed in the co-culture at 120 h (6.07±0.07 ppb). T. harzianum produced consistently low levels (<3.2 ppb). The absence of aflatoxin build-up indicates that the A. niger strain used is non-aflatoxigenic and that the fermentation conditions did not promote toxin synthesis. This finding is particularly important for field-scale applications, as the genus Aspergillus includes aflatoxin-producing species, necessitating strict monitoring during feed processing (Park et al., 2017). Moreover, preventing aflatoxin contamination is critical for animal health, given the well-documented hepatotoxic and carcinogenic risks associated with these compounds (Coppock et al., 2018). The low aflatoxin levels observed across all treatments validate SPW as a safe substrate for fungal bioconversion and support its potential integration into livestock feeding systems.

This study shows that fungal solid-state fermentation can improve the nutritional quality of sago pith waste while maintaining aflatoxin safety. From a practical standpoint, T. harzianum at 144 h can be recommended when protein enrichment is the primary objective, whereas A. niger at 144 h is more effective for fiber reduction. Aflatoxin levels remained well below regulatory limits across all treatments. Thus, the choice of fungal treatment and incubation time should be guided by the targeted nutritional objective. In summary, fungal SSF offers a practical and safe way to upgrade SPW into a more valuable feed ingredient. Although the study focused on basic proximate and aflatoxin indicators, the findings offer practical relevance for small-scale and industrial feed operations, where low-cost improvements in local feed resources are prioritized.

The authors acknowledge the support of the Philippine Government through the Department of Budget and Management (DBM), under the Regular Agency Fund allocated to Caraga State University, which made this research possible. Appreciation is also extended to the Regional Feed Chemical Analysis Laboratory of the Department of Agriculture - Regional Field Offices VII and XIII, whose expertise and resources were instrumental in the thorough analysis of the samples.
 
Disclaimers
 
The views and conclusions expressed in this article are solely those of the authors and do not necessarily reflect the views of their affiliated institutions. The authors are responsible for the accuracy and completeness of the information provided but do not accept any liability for any direct or indirect losses resulting from the use of this content.

The authors declare that there are no conflicts of interest regarding the publication of this article. No funding or sponsorship influenced the design of the study, data collection, analysis, decision to publish, or preparation of the manuscript.